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Clinical significance of altered collagen-receptor functioning in platelets with emphasis on glycoprotein VI
Accepted Manuscript Clinical significance of altered collagen-receptor functioning in platelets with emphasis on glycoprotein VI
Alan T. Nurden PII: DOI: Article Number: Reference:
S0268-960X(19)30069-4 https://doi.org/10.1016/j.blre.2019.100592 100592 YBLRE 100592
To appear in:
Blood Reviews
Please cite this article as: A.T. Nurden, Clinical significance of altered collagen-receptor functioning in platelets with emphasis on glycoprotein VI, Blood Reviews, https://doi.org/ 10.1016/j.blre.2019.100592
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ACCEPTED MANUSCRIPT Clinical Significance of Altered Collagen-Receptor Functioning in Platelets with Emphasis on Glycoprotein VI Alan T Nurden
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Institut de Rhythmologie et de Modélisation Cardiaque, Hôpital Xavier Arnozan, Pessac, France
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Correspondence: Alan T Nurden
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IHU-LIRYC, PTIB, Hôpital Xavier Arnozan, 33600 Pessac, France Tel: +33.5.56.55.19.52
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Fax: +33.5.57.10.28.64
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E-mail: [email protected]
ACCEPTED MANUSCRIPT Abstract Much interest surrounds the receptors α2β1 and glycoprotein VI (GPVI) whose synchronized action mediates the attachment and activation of platelets on collagen, essential for preventing blood loss but also the most thrombogenic component of the vessel wall. Subject to density variations on platelets through natural polymorphisms, the absence of α2β1 or GPVI uniquely
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leads to a substantial block of hemostasis without causing major bleeding. Specific to the
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megakaryocyte lineage, GPVI and its signaling pathways are most promising targets for antithrombotic therapy. This review looks at the clinical consequences of the loss of collagen receptor function with emphasis on both the inherited and acquired loss of GPVI with brief
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mention of mouse models when necessary. A detailed survey of rare case reports of patients with inherited disease-causing variants of the GP6 gene is followed by an assessment of the
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causes and clinical consequences of acquired GPVI deficiency, a more frequent finding most often due to antibody-induced platelet GPVI shedding. Release of soluble GPVI is brought
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about by platelet metalloproteinases; a process induced by ligand or antibody binding to GPVI or even high shear forces. Also included is an assessment of the clinical importance of GPVI-
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mediated platelet interactions with fibrin and of the promise shown by the pharmacological
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inhibition of GPVI in a cardiovascular context. The role for GPVI in platelet function in inflammation and in the evolution and treatment of major illnesses such as rheumatoid arthritis,
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cancer and sepsis is also discussed.
Keywords: Glycoprotein VI; inherited defects; acquired defects, polymorphisms; soluble GPVI shedding; mouse models; inflammation; anti-thrombotic therapy.
ACCEPTED MANUSCRIPT 1. Introduction Platelets maintain blood circulation by preventing bleeding through hemostatic plug formation at sites of vessel injury and by preserving blood vessel integrity [1,2]. A source of active metabolites, cytokines and growth factors, platelets also promote angiogenesis and tissue repair [3]. Collagen is the main substrate for platelets in subendothelium and its exposure in
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atherosclerotic plaques initiates occlusion in myocardial infarction and stroke [4-6]. Under
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flow, platelets first interact through glycoprotein (GP) Ibα which binds to von Willebrand factor (VWF) multimers pre-adsorbed to collagen; the platelets then translocate along the collagen fibers favoring the interactions that permit firm adhesion [7,8]. Platelet interaction
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with collagen is a multistep process that principally involves two receptors, integrin α2β1 and the immunoglobulin superfamily member, GPVI that act in synergy in a density-dependent
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manner with GPVI principally governing the signaling responses [9-12]. GPVI (58-62 kDa) has a single chain and is co-expressed with the promiscuous Fc
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receptor chain (FcR). Formed by non-covalent transmembrane contact, this complex is exclusive to platelets and megakaryocytes (MKs). Three groups independently cloned the GP6
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gene some 20 years ago [13-15]. Located on the long arm of chromosome 19 (19q.13.42), GP6
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has 8 exons that encode 2 terminal disulfide- linked immunoglobulin (Ig)-homology loops (IgC1 and Ig-C2), a short glycosylated stalk, a transmembrane domain and a 51 amino acid (aa)
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cytoplasmic tail in the principally located splice form (Fig. 1). The protein has two extracellular N- and many O-glycosylation sites that are important for function [16,17]. The Ig domains contain distinct but overlapping binding sites for collagen, collagen-related peptide (CRP) and the snake venom protein convulxin (Cvx), two ligands used to mimic collagen activation of platelets in diagnostic and research [18]. The GPVI cytoplasmic tail includes binding sites for calmodulin and the tyrosine kinases Fyn/Lyn [19,20]. Ligand binding to GPVI activates Fyn or Lyn allowing them to mediate phosphorylation of the two FcR cytoplasmic tail immuno-
ACCEPTED MANUSCRIPT tyrosine based activation motifs (ITAMs). This favors the phosphorylation of Syk and the formation of a signaling complex containing adaptor proteins such as Linker of Activation of T cells (LAT) and SLP76 with the involvement of Src, phosphatidylinositol 3-kinases (PI3kinases), phospholipase C2 (PLC2) and Ca2+-mobilization; steps that initiate platelet spreading and aggregation as well as other functional responses (reviewed in [21]). ITAM-
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mediated signaling is essential for collagen- induced activation of αIIbβ3 itself exhibiting
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functional interplay with α2β1 [22-24]. At the same time negative regulation of GPVI signaling is mediated by G6b-B, PECAM and the protein tyrosine phosphatase, CD148 [25-27]. The relative role of monomeric or dimeric forms of GPVI in mediating the platelet
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response was the object of early debate [28-31]. Clustering and association with lipid rafts increases GPVI avidity for collagen and promotes downstream signaling. GPVI is promiscuous
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with laminin, extracellular matrix metalloproteinase inducer (EMMPRIN), fibronectin (Fn) and vitronectin (Vn) other potential ligands in the vessel wall [32-36] (Fig. 1). The hormone
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adiponectin and histones are plasma ligands [37,38]. Then the role of GPVI was transformed by the late discovery that GPVI also mediates platelet interactions with fibrin [39,40]. Binding of
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GPVI to fibrin also leads to platelet activation with phosphatidylserine (PS) exposure
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promoting more thrombin generation in a feedback loop. But controversy persists with regard to the relative roles of monomeric versus dimeric GPVI in mediating GP function. Thus, while
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Onselaer et al [41] showed that monomeric GPVI binds to the fibrin D-dimer domain and that soluble D-dimer blocked platelet activation by both fibrin and collagen, Ebrahim et al [42] using recombinant technology found that dimeric GPVI binds to collagen but not fibrin under static conditions and arterial flow. Then, Induruwa et al [43] confirmed that collagen-binding dimeric GPVI recognized soluble D-dimer, monomeric and polymerized fibrin and extended the results to Fg. Mangin et al [44] extended these findings by showing that binding of GPVI to immobilized fibrinogen (Fg) under flow is followed by a time-dependent formation of
ACCEPTED MANUSCRIPT lamellipodial sheets and stress fibers. In a recent review, Slater et al [45] show how technical issues related to the use of recombinant GPVI protein may account for some of the above discrepancies. In summary, GPVI is now recognized as a pluripotent receptor whose influence extends beyond the initial platelet-collagen interaction to thrombus build up and coagulation. Such observations suggest that it is a player not only in thrombosis but also in inflammatory
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states including sepsis. GPVI deficiency therefore has implications far beyond that of a
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potential bleeding diathesis. The object of this review is to assess the clinical effects of inherited and acquired deficiencies of GPVI in human disease with brief mention of mouse
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models and when appropriate of the α2β1 integrin, its companion receptor for collagen.
2. Natural polymorphis ms and variations in collagen receptor expression
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Early studies established that the platelet response to collagen was linked to the surface density of platelet collagen receptors. Historically, findings were first reported for α2β1,
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polymorphisms in the coding regions of the ITGA2 gene (5q23-31) being linked to a 5-fold variation in platelet α2β1 density in a control human population [46]. Three dimorphic allelic
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coding variants were highlighted: A1 (807T/1648G) associated with high, A2 (807C/1648G)
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and A3 (807C/1648A) with lower α2β1 densities. Importantly, the rate of attachment of platelets to collagen at high shear (1,500/sec) in a flow chamber correlated with the α2β1 copy
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number. The genetics of α2β1 expression were extended when further polymorphisms within the ITGA2 proximal 5’-regulatory regions and linked to decreased binding of the Sp1 transcription factor were also shown to influence α2β1density [47]. At this time, Furihata et al [48] in the Kunicki laboratory described a 5-fold range in GPVI expression in platelets of normal donors and linked Cvx or CRP-induced prothrombinase activity (and therefore thrombin generation) to GPVI density. A series of 10 SNPs (5 synonymous and 5 nonsynonymous affecting coding regions) were found in the GP6 gene [49,50]. Highlighted were 2
ACCEPTED MANUSCRIPT common haplotypes affecting 5 aa, 3 in the extracellular domain and two in the cytoplasmic tail of GPVI (“a” allele, SKTQH; “b” allele PEALN) with allelic frequencies of 0.85 and 0.13 respectively (Fig. 2). The “b” allele associated with lower GPVI density on platelets and reduced platelet functional responses to collagen or CRP [49,50]. However, recent studies on larger cohorts underscore a somewhat lower variability. For example, Best et al [51] showed
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GPVI levels to vary 1.5 fold and α2β1 levels 3-fold among 102 healthy donors. Platelets of
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donors with the “b” allele of GPVI representing 22% of the population had 10% lower surface expression of GPVI. Later, Watkins et al [52] generated a high resolution SNP map by sequencing the promoter, exons and splice sites of GP6 for 96 unrelated Caucasian individuals.
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The SKTQH and PEALN isoforms were confirmed as major alleles. Among 18 SNPs was a rare non-synonymous SNP (103Leu>Val) (MAF=0.005) giving reduced ligand binding. Their
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study population was then extended to 2000 individuals with the identification of 12 additional isoforms and significant changes in GP6 haplotype expression between people of different
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geographical and ethnic backgrounds.
The degree by which the above haplotypes controlled GPVI density and platelet
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function continued to be less striking in larger cohorts. Jones et al [53] studied 506 subjects
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identifying individuals who were hyper- or hypo-responders to CRP; sequence variations at the GP6 locus, primarily the SKTQH and PEALN haplotypes now accounted for only 35% of the
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variation. The Kunicki group turned to expression studies and showed that the presence of the cytoplasmic domain LN substitutions in the “b” allele significantly diminished signal transduction, an effect that over-rode ligand binding or GPVI copy number [54]. As discussed earlier (1. Introduction), the GPVI cytoplasmic domain binds calmodulin and the tyrosine kinases Fyn/Lyn. Trifiro et al [54] showed that L317 conferred an increased affinity for calmodulin while N322 attenuated binding to Fyn/Lyn and consequently Syk phosphorylation.
ACCEPTED MANUSCRIPT Do variations in GPVI expression and haplotype-related functional differences of platelets have clinical consequences? Kunicki et al [55] compared secondary platelet candidate gene polymorphisms with bleeding severity in pedigrees of 14 index cases with type I von Willebrand disease (VWD) where patients have low VWF levels. Increased bleeding scores were linked to ITGA2 (807C), ITGA2B (Ile843) and GP6 (PEALN) haplotypes but no
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associations were found for 6 other candidate genes. When extended to an analysis of pedigrees
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with VWD type 2A, 2B and 2M, of 5 selected platelet genes only an ITGA2 promoter haplotype (-52T) was associated with an increased bleeding severity and no correlations were seen for GP6 [56]. In fact, the 807C and -52T variants are in strong linkage disequilibrium and
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both associate with low α2β1density. Finally, Cheli et al [57] performed a genome-wide SNP scan using Gp6-/- mice, and revealed significant linkage of animals with a prolonged tail
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bleeding time to a locus on chromosome 4 designated as modifier of hemostasis 4 (Mh4). The authors concluded that one or more genes at this locus control the extent to which thrombus
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formation is disrupted in the absence of GPVI. No follow-up studies on Mh4 have been
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reported.
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3. Inherited defects of GPVI
The first report of a possible inherited deficiency of GPVI (OMIM:614201) came when Moroi
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et al [58] described a Japanese woman with lifelong mild mucocutaneous bleeding and platelets that failed to adhere, secrete or aggregate with type I and type III collagen despite a full response to ADP, epinephrine, arachidonic acid (AA), thrombin and ristocetin (Table 1). Significantly, clot retraction was normal and her bleeding time only slightly prolonged. Radiolabeling of surface GPs showed a deficit of GPVI but a normal presence of other membrane receptors. Her platelets failed to bind a human antibody to GPVI (see also Sugiyama et al. [59] and Section 6). Platelets of her non-consanguineous parents had 50% GPVI
ACCEPTED MANUSCRIPT expression and responded well to collagen. Arai et al [60] then reported a Japanese woman with a congenital mild bleeding syndrome whose severity decreased with age. Her bleeding time was only moderately prolonged. Nevertheless, extensive bleeding accompanied hysterectomy. Her platelet count was moderately reduced (Table 1). While her platelets were refractory with collagen they responded well to other agonists. Platelet adhesion to collagen under static
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conditions was mildly reduced. Clot retraction was normal. Western blotting (WB) using
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human antibody to GPVI (see Sugiyama et al. [59]) revealed about 10% residual GPVI in her platelets. Other membrane receptors including integrin α2β1 were normally present. Thus 10% residual GPVI was insufficient to support platelet activation by collagen. Enigmatic, was the
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report from Goto et al [61] describing how platelets from the above patients also showed less firm adhesion on vWF.
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The first reported GP6 gene mutations came from Dumont et al [62]. The patient was a young girl with mild bleeding since infancy, a PFA-100 closure time prolonged to 210 seconds
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for collagen/epinephrine and poorly responsive to collagen and CRP (Table 1). Noteworthy is that secretion in response to collagen and collagen- induced thrombin generation was also
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impaired. Flow cytometry showed GPVI at 540 copies per platelet versus 3076 for the controls;
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WB confirmed the incomplete deficiency with a smeared migration of the residual protein. FcR was normally present. DNA sequencing revealed compound heterozygozity for two
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predicted disease-causing GP6 mutations (Fig. 2): (i) a c.172C>T transition giving an Arg38Cys (R38C) mutation within exon 3 and affecting the first Ig loop of GPVI and (ii) a duplication of 5 nucleotides (c.356_360) in exon 4 resulting in a frameshift (fs), a premature stop 30 codons downstream, an absence of the corresponding mRNA probably indicated mRNA decay. Introduction of Arg38Cys into recombinant GPVI reproduced the abnormal migration as seen by WB and a marked loss of collagen binding. The smeared migration was hypothesized to result from mismatched disulfides caused by the extra Cys residue. The ability
ACCEPTED MANUSCRIPT of the patient’s platelets to form thrombi on collagen under flow was reduced by 43%. In a parallel report, Hermans et al [63] described a young woman with moderate lifelong bleeding marked by several episodes of post-traumatic and post-surgery bleeding. Reduced platelet function responses were selective to collagen and its mimetics. Although thrombus formation on collagen under flow was poor, more single platelets attached. Enigmatically, the PFA-100
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closure time with the collagen/ADP membrane was shortened while that for the
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collagen/epinephrine cartridge was unchanged. Her platelet count and platelet ultrastructure were normal. GPVI expression was much reduced with residual protein normally migrating on WB. DNA sequencing revealed two heterozygous GP6 mutations: (i) an out-of frame 16bp del
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predicting a stop codon after 56 aa and (ii) a missense Ser176Asn substitution within the second Ig domain of GPVI (Fig. 2). Ser176 is highly conserved between species and its
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substitution was predicted to disrupt H-bonding; the second short truncated variant was not expressed. Both parents were heterozygous carriers and asymptomatic; in fact the mother with
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GPVIAsn176 unexpectedly had platelets normally expressing GPVI. Studies in HEK293 cells confirmed a normal posttranslational processing of GPVIAsn176 suggesting a functional
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defect.
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My group in Bordeaux set up sequencing of the GP6 gene in response to a request from Dr. Diego Mezzano who was investigating unrelated patients (2 female and 2 male) belonging
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to 4 non-consanguineous Chilean families [64]. All propositi presented with lifelong mild mucocutaneous bleeding (Table 1). Platelets of each were specifically refractory to collagen or, when tested, CRP or Cvx. When measured, the bleeding time was moderately prolonged. Flow cytometry failed to locate GPVI on the platelets of each patient. Sequencing of GP6 revealed an adenine insertion in exon 6 (c.711_712insA) causing a frameshift and generating a stop codon at aa242 of the mature protein; this homozygous variant was common to all patients who all expressed the high density “a” allele for GP6 (Fig. 2). There was no family history of
ACCEPTED MANUSCRIPT bleeding and the families are from different rural regions of Chile and while a founder effect can be hypothesized there is currently no clear explanation. The mutation predicts the synthesis of a soluble form of GPVI truncated extracellular to the trans-membrane domain. Indeed, WB detected a limited presence of truncated protein (around 49 kDa) in the platelets of each patient but no full-length GPVI. Obligate heterozygotes were asymptomatic and aggregated well with
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collagen. Intriguingly a sister of one of the patients was also homozygous for the mutation but
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has never shown signs of bleeding despite platelets unresponsive to collagen and lacking surface GPVI. Later, Onselaer et al [41] reported two more patients and showed that platelet adhesion to fibrin was also reduced by 40%, with αIIbβ3 the obvious candidate as the second
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fibrin receptor. Those platelets that did attach showed markedly reduced spreading, only occasional platelets showed signs of activation. Then, Mangin et al [44] studying platelets from
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two of the patients confirmed reduced adhesion on immobilized Fg, the platelets failing to form lamellipodal sheets and stress fibres. Blocking αIIbβ3 of GPVI negative platelets resulted in a
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total absence of adhesion on Fg. They also confirmed that clot retraction was normal, presumably mediated by αIIbβ3.
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The results so far accumulated from inherited GPVI deficiency support the conclusion
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that the hemorrhagic tendency is mostly moderate, contrasting for example, with the more severe bleeding that accompanies GPIb-IX deficiency in Bernard-Soulier syndrome and αIIbβ3
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deficiency in Glanzmann thrombasthenia (GT) [65,66]. Notwithstanding, the number of patients is small and care is needed not to over-interpret the available data for more information is required – especially for situations such as childbirth and trauma-related bleeding including surgery where two of the above patients did bleed excessively (Table 1).
4. Mouse models for α2β1 and GPVI deficiency
ACCEPTED MANUSCRIPT Mouse models have proved essential in understating the dynamics of the platelet collagen interaction and the roles of α2β1 and GPVI. First, mice genetically depleted of β1 integrins were shown to retain a normal bleeding time despite delayed but substantial thrombus formation on collagen fibrils and a lack of platelet aggregation with soluble collagen [67]. As these mice also lack α5β1 and α6β1 receptors for Fn and laminin, efforts turned to generating
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α2-deficient mice [68,69]. Höltkotter et al [68] showed that the mice developed normally with
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no obvious fertility or other physiological defects and that their platelets showed the specific defects in the collagen response seen for β1 knockout. Again there was no bleeding phenotype and the tail bleeding time was unchanged. Chen et al [69] confirmed that α2β1 knockout mice
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were viable with normal wound healing but detected a multifaceted phenotype with defects extending to epithelial cells and branching morphogenesis. Significantly, a blocking
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monoclonal antibody (MoAb) to GPVI totally abrogated the adhesion of the α2β1-deficient platelets to collagen [68]. Mice whose platelets were immunologically depleted of GPVI
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(detailed in Section 5) also only had a mild disruption of primary hemostasis despite the platelets showing reduced thrombus formation on collagen under flow [70,71]. It was
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significant then when platelets from mice genetically engineered to lack GPVI or FcR (also
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leading to GPVI deficiency) showed a severe loss of platelet adhesion, spreading and thrombus formation on collagen but again showed little bleeding [72]. Interestingly, mouse platelets
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genetically modified to express 5-fold lower levels of GPVI showed comparable thrombus formation on a high-density collagen coated surface to controls in so doing raising questions about the clinical significance of human GP6 polymorphisms [51]. In summary, α2β1 and GPVI clearly act in synergy and have complementary roles.
5. Shedding of GPVI from mice and human platelets
ACCEPTED MANUSCRIPT Shedding of the bulk of the soluble extracellular domain of GPVI (sGPVI) from circulating platelets causes loss of the collagen- interacting domains thereby regulating platelet function. Recognizing and understanding the mechanisms behind GPVI shedding is key to understanding acquired GPVI pathology. a) Antibody and ligand-induced release of sGPVI. Pioneering studies from Bernhard Nieswandt
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and his group showed that injecting rat MoAbs to GPVI (the JAQ series) into mice led not only
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to a transient thrombocytopenia but also to a long-term (at least 10 days) immune-depletion of GPVI from platelets with thromboembolic protection [67,70,71,73,74]. Studies performed with human platelet-rich plasma showed that adding mouse MoAbs against GPVI not only resulted
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in sGPVI shedding, the majority also induced platelet aggregation [75]. Release of sGPVI also occurred with monovalent Fab fragments (showing that Fc-receptor interactions were not
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necessary) and also occurred with MoAbs against both ligand and non-ligand-binding domains of GPVI. Enigmatically, no loss of GPVI occurred when JAQ antibodies were incubated with
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mouse platelets in vitro. But in vivo in mice both antibody-induced GPVI shedding and the thrombocytopenia are rapid (maximum at 30 min with return of the platelet count to normal
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after 48h); it is interesting to ask whether within this short time scale the bleeding risk was
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more severe [71]. Intracellular signaling is required for GPVI loss; shedding was prevented when elements of FcR-mediated ITAM signaling were interfered with. For example, GPVI
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shedding did not occur in LAT-/- mice (LAT is part of the GPVI signalosome); instead JAQ1GPVI complexes were internalized by the platelets followed by a slow intracellular degradation. The LAT-/- platelets failed to respond to collagen, but increases in bleeding time were modest [76]. Boylan et al [77] introduced human platelets into the circulation of non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice; Fab fragments of a MoAb to human GPVI were then injected. Shedding of sGPVI occurred from the human but not the
ACCEPTED MANUSCRIPT mouse platelets. But dampened platelet responses to ADP and thrombin were also seen. In a more classic mouse model, others also noted that JAQ1-induced GPVI shedding was accompanied by a short transient decrease in the platelet response to thrombin [78]. Such a loss would also accentuate bleeding risk. Important but enigmatic is that not all MoAbs to GPVI cause in vivo GPVI shedding or depletion although they may block function [79,80]. Clearly
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antibodies to GPVI in patients have to be tested individually. As GPVI also contributes to
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platelet aggregate formation under flow by interacting with platelet-bound Fg or fibrin, thrombus formation is also affected by GPVI loss [43,44]. One study reports that newly formed fibrin promotes shedding of GPVI from human platelets independently of ITAM signaling and
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that this occurs in pathology, for example in patients with severe burns [81]. b) Mechanisms for GPVI loss. Shedding of sGPVI is by proteolysis through the action
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of metalloproteinases with the release of a large 55 kDa fragment leaving a short transmembrane and intracellular fragment attached to the platelet (Fig. 1). Most studies have
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identified “A Disintegrin and Metalloproteinase Domain-containing protein 10” (ADAM10) as the protease that cleaves GPVI although ADAM17 and other metalloproteinases may also
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intervene [74,82,83]. The process appears under intracellular control, agents blocking the
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interaction of calmodulin with the GPVI cytoplasmic tail stimulate GPVI cleavage. GPVI can also be removed from the platelet surface by intracellular clearing and both shedding and
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internalization are abrogated in mice with a point mutation in the FcR ITAM domain again showing a role for intracellular signaling [76,84]. Release of sGPVI also occurs during coagulation by a process highly dependent on FXa that was speculated to activate ADAM10 directly and, as explained above, through direct interaction with fibrin [81,85]. A key question is how ADAM10 or other proteases in platelets find and cleave ligandor antibody-bound GPVI so rapidly. Localization using fluorochrome- labeled antibodies showed that the principle ADAM10 pools of platelets are intracellular but are organized
ACCEPTED MANUSCRIPT independently of P-selectin and α-granules (see Fig. 4 of ref [86]). So presumably a specific surface translocation mechanism is necessary. While beyond the scope of this review, recent studies suggest that several mechanisms can lead to sGPVI release and metalloproteinase activation with a role for membrane tetraspanin family as mediators of GPVI lateral diffusion and/or carriers or activators of metalloproteinases [87,88].
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c) Importance of shear. As well as ligand binding, transient exposure to mechanical
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force can lead to metalloproteinase activation and sGPVI release [89]. This is very relevant as high shear occurs in atherosclerosed coronary vessels or after ischemic events, and indeed high levels of circulating sGPVI have been detected in pilot studies on patients with stable angina
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pectoris and stroke [89,90]. Similar situations occur in acquired von Willebrand disease in a cardiovascular context and with the use of ventricular assist devices and extracorporeal
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membrane oxygenation where loss of platelet receptors (GPIbα and GPVI) and loss of platelet
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function can be a cause of bleeding [91].
6. Pathology of α2β1 deficiency and less well-known collagen receptors on platelets
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Early reports described inherited defects of α2β1 (originally known as Very Late Antigen
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(VLA)-2 and then GPIa-IIa) in a mild to severe bleeding disorder linked to a much reduced platelet response to collagen [92,93]. The patients had excessive post-traumatic bleeding and
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for women, menorrhagia. Other reports described GPIa deficiency associated with a lack of thrombospondin or in myeloproliferative disorders [51,94,95]. The relatively severe bleeding experienced by the patients reported by Nieuwenhuis et al [92,93] contrasts to the absence of bleeding in mice with platelets genetically modified to lack α2β1 (see Section 4). The genetic defect in these families needs elucidating. Interestingly, Noris et al [96] described inherited α2β1deficiency but in the context of autosomal dominant thrombocytopenia and mild bleeding later shown to be associated with variants in the ANKRD26 gene [97]. Another purported
ACCEPTED MANUSCRIPT collagen receptor on platelets is CD36 (GPIV) [98]. However, patients genetically lacking CD36 respond normally to collagen and at best it has a minor role [99]. Another purported collagen receptor is leukocyte-associated immunoglobulin- like receptor (LAIR-1) but it has not been linked to a specific pathology [100]. Autoantibodies to α2β1 are a cause of ITP [101,102] but little is known of their potential blocking of the platelet-collagen response. One exception is
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an inhibitory IgM antibody cloned and sequenced by Deckmyn et al [103]. A MoAb binding to
integrin in the signaling response to collagen [104].
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αIIb also specifically blocks the platelet/collagen interaction underlining a potential role for this
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7. Acquired defects associated with antibodies to GPVI in human pathology Historically, Sugiyama et al [59] reported the first acquired defect of GPVI in a Japanese
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woman with ITP associated with Grave’s disease (Table 2). Thrombocytopenia was severe and associated with spontaneous mucocutaneous bleeding and the patient received platelet
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transfusions. Corticotherapy restored her platelet count to near normal but mild bleeding was still occasionally seen. Her platelets aggregated well to ADP, epinephrine, AA, thrombin and
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ristocetin but were refractory to collagen and this included dense granule secretion and
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thromboxane B2 synthesis. Although platelet adhesion to collagen fibrils was reduced, some platelets did attach. Significantly, both her plasma and isolated IgG induced the aggregation of
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control platelets, a response shown to depend on dense granule secretion. Her antibody primarily immunoprecipitated a membrane GP in the 50-60 kDa range from detergent solubilized control platelets later shown to be GPVI. Takahashi & Moroi [105] described a middle-aged Japanese woman with systemic lupus erythematosus (SLE), chronic thyroiditis and mild bleeding whose platelets showed similar characteristics. Her thrombocytopenia was initially severe but her platelet count normalized after corticosteroid therapy but again bleeding continued. Platelet-associated IgG was elevated and an antibody to GPVI reactive in WB
ACCEPTED MANUSCRIPT present in her serum; platelet GPVI was much reduced. Her antibody aggregated control platelets; cross-linking GPVI in the platelet membrane was a proposed mechanism. Significantly, F(ab’)2 fragments of the antibody of Sugiyama et al [59], now identified as an anti-GPVI, induced signaling pathways identical to those used by collagen and its mimetics Cvx and CRP, with tyrosine phosphorylation of Syk and Src [106]. Platelets from the Japanese
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patients deficient in GPVI responded to collagen with a normal α2β1-dependent tyrosine
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phosphorylation of Src but GPVI-dependent phosphorylation of Syk and activation of PLC2, Vav and FAK (focal adhesion kinase) was severely reduced [107].
Boylan et al [108] then reported a young woman in the USA with ITP and mild bleeding
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and moderate thrombocytopenia that reproduced the findings of the Japanese patients. Adherence and thrombus formation on collagen under flow were shown to be defective.
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Despite a much-reduced platelet expression of GPVI and FcRher platelets contained mRNA encoding GPVI and no mutations were found in the GP6 gene. The patient’s plasma possessed
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an IgG antibody to GPVI speculated to cause the rapid clearance of sGPVI from the plasma.
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An unusual case was an elderly woman from Bordeaux with gray platelet syndrome (GPS) and an antibody to GPVI [108]. GPS is an inherited platelet disorder caused by mutations in
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NBEAL2, a gene essential for the packaging of proteins during -granule synthesis in MKs [110]. GPS is associated with mild to severe mucocutaneous bleeding, macrothrombocytopenia
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an enlarged spleen and myelofibrosis that was strongly suspected although a confirmatory bone marrow biopsy was not performed [111]. Unlike typical GPS, platelet aggregation with collagen or Cvx was virtually absent and there was a severe reduction of GPVI in her platelets. Autoantibodies to GPVI were not detected either on her platelets or in her serum; sequencing GP6 revealed no potential disease-causing mutations (the patient had the high density “a” allele). It was speculated but not proven that endogenous metalloprote inase activity in the marrow was responsible for GPVI loss.
ACCEPTED MANUSCRIPT Studies continued with two more unrelated female Japanese patients with a mild bleeding diathesis, moderate thrombocytopenia and platelet function typical of acquired GPVI deficiency (cases 5 and 6, Table 2). For both, the platelet count gradually recovered but occasional mild bleeding continued; for the first platelets attached poorly to collagen under static conditions with spreading profoundly affected [112,113]. In both cases, plasma antibody
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to GPVI was not detected but platelet-associated IgG was elevated and eluates contained
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antibodies to GPVI as shown in a dot-blot assay using recombinant protein. Testing against transfected αIIbβ3-expressing heterologous cells also detected antibodies to αIIbβ3 in the second patient [113]. Significantly, as the patient’s platelet count increased, platelet expression
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of GPVI improved and the bleeding tendency diminished as antibody titers in platelet eluates decreased. Although GPVI shedding is a tempting hypothesis for these patients, sGPVI was not
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elevated and neither was the 10 kDa membrane-bound remnant of GPVI observed in WB. Not least, these cases show the importance of careful antibody testing.
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Gardiner et al [114] continued the reports of severe thrombocytopenia with ITP linked to anti-GPVI antibody. Corticotherapy again restored her platelet count but spontaneous
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pathological bruising continued. The collagen/epinephrine PFA-100 closure time was markedly
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prolonged. Here, platelet-associated IgG was elevated and plasma autoantibody to GPVI present. The antibody aggregated control platelets, an activity blocked by sGPVI and in part by
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a MoAb (IV.3) binding
. Decreased GPVI on her platelets and analyses for sGPVI
and of the residual transmembrane domain and cytoplasmic tail of GPVI strongly suggested shedding of GPVI, and this was confirmed when her plasma caused GPVI loss from control platelets. Interestingly, the authors later showed that 6-month treatment with the TPO receptor mimetic, romiplostim, restored both the platelet response to collagen and GPVI expression on her platelets but enigmatically, sGPVI in her plasma remained elevated and plasma autoantibody to GPVI continued to be detected [115]. How these results are explained is
ACCEPTED MANUSCRIPT unclear although previous studies have shown that TPO up-regulates GPVI expression in MKs by c-Mpl-mediated demethylation of a cytosine phosphate-guanosine-rich island within the GP6 promoter [116]. A young French woman with an ITP-like condition came to our attention in 2005 with severe thrombocytopenia (case 8, Table 2); corticotherapy restored her platelet count but within
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a year a lupus nephropathy led to the diagnosis of SLE with antibody to DNA [117]. She
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improved after treatment but later her condition again worsened and she received transfusions as a precaution prior to biopsies. In 2006, platelet function testing revealed a selective absence of platelet responses to collagen and Cvx and a specific platelet deficiency of GVI and FcR;
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her PFA-100 closure time (collagen + epinephrine) was markedly prolonged. Testing throughout subsequent treatment revealed a progressive restoration of the platelet responses to
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collagen and increases in platelet GPVI expression and her PFA-100 closure time normalized. Plasma samples analyzed for anti-GPVI antibodies employing a specially developed ELISA
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with recombinant sGPVI showed that antibody to GPVI was initially strong but that its titer fell as platelet GPVI levels improved. Plasma sGPVI remained low. DNA sequencing showed no
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potential disease-causing mutations and that the patient’s GP6 possessed the “a” allele
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associated with higher GPVI levels. The plasma antibody was of the IgG3 subtype and when titers were strong it activated control platelets with long incubations resulting in sGPVI release
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and a loss of the collagen response. Significantly, restoration of platelet GPVI levels and the collagen response was associated with loss of her nephrotic syndrome. An unusual case reported by Sanchez-Guiu et al [118] concerned an atypical plateletreacting IgM class cold agglutinin in a woman with a lifelong history of mild bleeding and chronic moderate thrombocytopenia. A unique diagnostic feature was pseudothrombocytopenia that was temperature-dependent but EDTA-independent. Her IgM bound GPVI and aggregated control platelets. Her own platelets had elevated bound IgM and showed signs of activation
ACCEPTED MANUSCRIPT (increased TXA2 production and surface P-selectin). Low GPVI expression and increased levels of sGPVI in her plasma was suggestive of shedding. The patient had prolonged closure times in the PFA-100 with both cartridges with platelet plugs forming more quickly when the test was performed immediately after the blood was taken (due to the spontaneous agglutination). Platelet function testing was abandoned for this reason. Platelet activation was
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blocked by a GPVI-Fc fusion protein and by inhibitors of the tyrosine kinase Src family
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and of Syk . Her IgM agglutinin has persisted over a 5-yr period. No abnormalities were seen on sequencing of the GP6 coding sequences. No other family member was affected. Finally in this Section, Rabbolini et al [119] described a middle-aged woman with a
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long-standing history of ITP and easy bruising. Severe thrombocytopenia improved with immunosuppressive therapy but a relapse required treatment with IVIgG given with antibiotics.
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Tests performed prior to hip surgery revealed a specifically defective collagen response and severe platelet deficiencies of GPVI. Next generation sequencing failed to reveal potential
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disease-causing mutations. Her serum aggregated control platelets; a response prevented by
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prior cleavage (and loss) of GPVI or by blocking the platelet FcRIIA receptor. An important feature as seen with other cases is that bleeding continued despite a platelet count well above
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the 30x109 /L threshold normally considered to merit special precautions in ITP. Quite remarkably, this section deals with 10 case reports describing patients with
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acquired GPVI deficiency due mainly to the presence of anti-GPVI antibodies - and all are women (Table 2). It will be interesting to see if new case reports continue this trend. This section also proves that anti-GPVI antibodies can give rise to ITP and I agree with Rabbolini et al [119] that they may well be an under-recognized cause. Also, the continued occurrence of bleeding in some patients after platelet count (and often GPVI expression) improves on treatment suggests that some antibodies may be function blocking although this has rarely been
ACCEPTED MANUSCRIPT proven. While antibodies clearly provoke sGPVI shedding for some internalization and intracellular processing of antibody-GPVI complexes is a possible alternative pathway.
8. GPVI and megakaryocytopoiesis In a companion paper in Blood Reviews [120] I have assessed the roles of autoantibodies to
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αIIbβ3 in acquired GT. Some marked differences can be observed between antibodies to GPVI
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and those directed against αIIbβ3. First, while autoantibodies to GPVI may induce receptor shedding those to αIIbβ3 do not. Secondly αIIbβ3 appears early in MK differentiation (being present in stem cells) and autoantibodies to the integrin can directly influence proplatelet
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production resulting in altered platelet biogenesis [discussed in 120]. In contrast, the expression of GPVI increases late in MK maturation and while the interaction of both GPVI and α2β1 with
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collagen combine to negatively regulate proplatelet production, this is part of a natural process to prevent premature platelet release in the bone marrow [121,122]. So far, inherited diseases of
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GPVI do not lead to platelet size increases contrasting also with acquired antibodies to GPIbIX-V and the inherited loss of GPIb-IX-V in BSS [65,123]. Still to be resolved, however, is the
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reason why platelet GPVI levels in mice return to normal much later than the platelet count
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after antibody-induced GPVI loss [71]. Does this have a parallel in human acquired GPVI loss?
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9. Bleeding syndromes associated with acquired defects in GPVI-dependent intracellular signaling pathways
Rare reports also describe patients for whom the molecular basis for loss of GPVI function is intra-cellualr. Bellucci et al [124] described defective collagen- induced platelet activation associated with bleeding in two patients with malignant hemopathies (cases 11 and 12, Table 2). A man with myelodysplasia, anemia and low white blood cell numbers suddenly developed a bleeding syndrome. His platelet count was normal but his bleeding time was very long. He
ACCEPTED MANUSCRIPT gradually developed severe aplasia and severe thrombocytopenia with an eventual transformation to myelomonoblastic leukemia from which he died. An elderly woman with eventually fatal chronic lymphocytic leukemia started bleeding after treatment with heparin for a pulmonary embolism. Her PFA-100 closure time was prolonged for collagen/epinephrine. Both patients had a severely impaired platelet response to collagen, CRP and Cvx whereas that
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to other agonists including ADP, TRAP, AA and ristocetin was normal or only partially
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decreased. GPVI was fully expressed in the platelets of both patients and bound Cvx yet neither Cvx nor specific activating MoAbs to GPVI induced secretion or Ca2+-mobilization. Perfusiondependent platelet adhesion and thrombus formation on collagen were both highly impaired.
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Inhibitory antibodies were not present in the plasma of the patients and a signaling defect proposed but not resolved. Best et al [51] also showed that as well as a low α2β1 content some
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patients with myeloproliferative syndromes also had decreased GPVI function. The case histories in this review conclude with a report that illustrates the difficulties of
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working up a suspected defect of GPVI [125]. A woman with a lifelong experience of mild bleeding gave birth to 2 children without excessive bleeding. There was no family history of
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bleeding and platelet counts were normal. When first tested, her platelets failed to aggregate
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with collagen; but the response later improved and while remaining delayed and low to Cvx was normal to all other agonists. Collagen- induced release from alpha-granules and dense-
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granules was variable. Anti-platelet antibodies were not detected in her serum or on her platelets and plasma levels of sGPVI matched those of controls. The PFA-100 closure time was not prolonged for either cartridge. Sequencing of the GP6 gene showed no disease-causing mutations. A rare heterozygous c.1038C>T variant in the 3’untranslated region had no effect on GPVI mRNA expression and GPVI was normally expressed in her platelets. The fact that collagen- induced platelet responses improved over time suggests an acquired defect (perhaps drug-induced) but the molecular cause remains an enigma.
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10. Platelets and GPVI in inflammation and other non-hemostatic events By adhering to foreign surfaces and by secreting metabolites and many biologically active proteins platelets promote not only tissue repair and wound healing but are vital for inflammation and innate immunity (reviewed in [3]). Clearly, as a collagen and fibrin receptor,
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GPVI is an important player in these processes. In addition, the structurally related ITAM
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receptor CLEC-2 acting through podoplanin expressed on vascular cells also exerts a major and multifaceted role in inflammation and is key for the development of the vascular and lymphatic systems [6,126,127]. While altered development of the lymphatic system is seen with CLEC-2
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deficiency [128]; ductus arteriosus is not a feature of GPVI deficiency in mice or man. During inflammation, platelets not only facilitate leukocyte infiltration at endothelial cell junctions
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through expression of P-selectin they also assure hemostasis by sealing breaks in the endothelium (reviewed in [129]). While deficiencies of GPVI or CLEC-2 in mice do not
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compromise wound healing; their double deficiency actually enhanced it probably by allowing more bleeding into wound sites by severely interfering with hemostasis in mice [130,131].
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How deficiencies of GPVI in man influence inflammation is an important area for future
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research.
Pioneering studies by Goerge et al [132], showed how thrombocytopenia promoted
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intra-alveolar bleeding and respiratory distress during endotoxin-induced lung inflammation. They also showed bleeding from venules during the cutaneous Arthus reaction where loss of vascular integrity is linked to immune complex deposition. Studies in mice showed that single platelets seal neutrophil- induced vascular breaches via GPVI during immune-complexmediated dermatitis [133]. The absence of GPVI (and a fall in platelet count) favored neutrophil- induced inflammatory bleeding. Another mouse model showed that platelet recruitment to the inflamed glomerulus also occurs in part by a GPVI-mediated mechanism
ACCEPTED MANUSCRIPT with P-selectin crucial [134]. Gros et al [133] speculated such interactions might involve EMMPRIN, a counter-receptor for GPVI expressed by leukocytes although other proteins potentially exposed in the vessel wall and able to bind GPVI include laminin, Fn and Vn (Fig. 1); fibrin is another possibility as are histones if DNA nets are present. A pathological role for GPVI and the platelet-collagen interaction in the release of biologically active microparticles
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(MPs) from platelets was shown in a mouse model of rheumatoid arthritis suggesting GPVI as
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a therapeutic target in this condition [135]. However, later studies by the same group showed that rather than MPs, lipid derivatives are the active platelet-released components [136,137]. Platelets are necessary for host defense during bacterial infection and limit bacterial
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growth and dissemination [138,139]. They also help prevent hemorrhage during sepsis and limit cytokine responses. A role for GPVI is illustrated by increased bacterial growth in the
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lungs and in distant body sites when GPVI-/- mice (or mice depleted of GPVI by antibodyinduced shedding) were infected with klebsiella pneumonia bacteria to induce pneumonia-
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induced sepsis [140]. Fewer platelets and platelet-leukocyte aggregates were observed in the bronchoalveolar space. GPVI on platelets reacting with its ligands collagen, fibrin and histones
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at sites of inflammation leads to up-regulated leukocyte function; in parallel changes in
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cytokine levels suggest that GPVI has an anti-inflammatory effect. The net effect will depend on the site and the nature of the inflammation. For example, in mouse models of cutaneous
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inflammation, GPVI has a pro-inflammatory effect. its absence leading to decreased platelet interplay with macrophages and increased anti-inflammatory markers and therefore a lower vasculo-protective effect [141]. On a wider scale, elevated sGPVI levels in many acute and inflammatory conditions including patients with severe burns, inflammatory bowel and kidney disease, elective cardiac surgery, trauma, acute brain injury or prolonged ventilation suggest ongoing GPVI loss and this was particularly so in sepsis [81]. For further information in this field I invite the reader to consult an excellent recent review by Boulaftali et al [142].
ACCEPTED MANUSCRIPT Clearly the role of GPVI is complex with GPVI loss favoring bleeding at some inflamed sites particularly when thrombocytopenia is also a factor. But much research is needed in this field and it is already apparent that the role of GPVI (and platelets) may vary considerably with respect to the site and cause of different clinical inflammatory states. The influence of administered therapeutics on bleeding (for example, anti-inflammatatory drugs) and to combat
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thrombocytopenia (for example, in sepsis) must also be taken into account.
11. Thrombosis and the potential therapeutic use of anti-GPVI agents Platelet receptor polymorphisms emerged as risk factors for arterial thrombotic disease and this
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has included those influencing α2β1 and GPVI expression (see above Section 2). More importantly, GPVI has become a novel pharmacological target for drugs effective in thrombotic
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therapy but with preserved hemostatic risk (reviewed in [143]). a) Collagen receptor polymorphisms as thrombotic risk factors. Early studies suggested
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that variations in collagen receptor density helped determine risk in ischemic or cardiac disease and in thrombo-inflammatory conditions such as stroke (reviewed in [144]). For example, the
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dimorphic α2β1 C807/T873 allele associated with high α2β1 density was linked to myocardial
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infarction or stroke in younger patients, findings that were not confirmed in Japan suggesting race variations [145-147]. For GPVI, early studies suggested that high expression with
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polymorphic variation at the GP6 locus gave increased risk of coronary artery disease and myocardial infarction but the study groups were small [49,148-151]. Interestingly, a recent study associated the GPVI “a” allele with a two-fold higher level of platelet MPs release and with increased sepsis severity in a pediatric population [152]. Hopefully, the use of modern genomics will put such studies on a different level and enable a more personalized risk assessment than at present [153]. So far, diagnostic or treatment changing data has not been obtained.
ACCEPTED MANUSCRIPT b) Sticky platelet syndrome. The so-called “sticky platelet syndrome” refers to a loosely defined group of patients with increased thrombotic tendency usually defined as an increased sensitivity of platelets to aggregate with low doses of ADP and/or epinephrine (reviewed in [154]). The syndrome is claimed to have an autosomal dominant trait linked to variants in a wide range of genes. The Kubisz group [154] have linked polymorphisms in the GP6 gene to
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its pathogenicity describing over-expression of GP6 haplotypes; however, much more stringent
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evidence is required to prove the existence of sticky platelet syndrome as a distinct entity and to establish a molecular basis for the role of GPVI. One of the associated symptoms is fetal loss (and fetal growth retardation) and perhaps the most convincing finding is the association found
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by Skerenova et al. [155] between fetal loss and the presence of the low frequency GP6 “b” allele (PEAN). A reduced GPVI signaling was a suggested cause.
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c) Direct targeting of platelet GPVI in arterial thrombosis and stroke. The results obtained for genetically modified mice or mice with antibody-induced GPVI depletion showed
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that loss of GPVI occurred without major disruption of hemostasis (Sections 4 and 5). Most patients with inherited or acquired GPVI deficiency have mild bleeding while no thrombo-
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embolic events have been reported (Table 1). It is therefore not surprising that research into
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GPVI as a potential target for safe antithrombotic therapy including stroke has attracted much attention. Collagen fibrils have multiple partners during platelet adhesion and thrombus
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formation following atherosclerotic plaque rupture [156-158], and the continued functioning of other platelet membrane receptors including GPIbα, α2β1 and αIIbβ3 may limit bleeding. Diverging concepts for abrogating GPVI-dependent platelet signaling have emerged, three of which are now briefly mentioned. The first is the intravenous infusion of a recombinant soluble dimeric GPVI-Fc fusion protein (Revacept) blocking GPVI-reactive sites in collagen exposed in atherosclerotic plaques. Animal studies showed that Revacept both reduces thrombus formation and improves vascular dysfunction [159,160]. It is about to enter a phase 2 trial in
ACCEPTED MANUSCRIPT patients undergoing elective percutaneous coronary intervention [161]. A second approach is to therapeutically induce GPVI shedding [74]. This, however, remains largely unproven despite increases in our knowledge of how intracellular mechanisms can regulate sGPVI release, as for example through the production of reactive oxygen species [162]. Large problems remain however, for example, with regard to controlling the specificity, extent and duration of GPVI
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shedding. The third and currently the most promising approach is by direct pharmacological
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inhibition of GPVI using an antibody or with peptides derived from snake venom proteins [163-167]. Fab fragments of a MoAb to GPVI, ACT017, have been recently shown to have excellent safety and tolerability profiles in a phase 1 study in man while blocking collagen-
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induced platelet aggregation in a dose-dependent manner [168]. As well as arterial thrombosis and stroke, GPVI inhibitors have potential to prevent cardiac ischemia perfusion injury
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[158,169]. The results of phase 2 and possibly phase 3 clinical trials with these new drugs will be anxiously awaited. Whether patients with deficiencies of GPVI are protected against
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cardiovascular disease is an interesting question to answer in coming years as is the question of whether collagen receptor polymorphisms (see Section 2) will prove useful in predicting those
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patients most susceptible to benefit from these drugs.
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d) Soluble fibrin as a regulator of GPVI. Interestingly, difference in the way that GPVI interacts with collagen and fibrin or Fg provides the opportunity to developing inhibitors
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specific for each ligand [39-44]. Interestingly, soluble fibrin formed by restricted thrombin generation acts as a GPVI antagonist sterically blocking access to its ligands and the functional response [170]. Thrombin generation in response to trauma, cancer and disseminated intravascular coagulation (DIC) and plasmin generation may lead to circulating fibrin monomers or soluble protofibrils [171]. Soluble fibrin monomers can be maintained for several hours and render platelets non-responsive to collagen, a finding that would extend to donor platelets in the event of transfusion. According to Lee et al [170] 1% conversion of circulating
ACCEPTED MANUSCRIPT Fg would lead to 90 nM soluble fibrin – enough to overwhelm platelet GPVI and lead to loss of function of endogenous or transfused platelets. However, Onselaer et al [41] reached more conservative conclusions. Whether the soluble fibrin simply blocks GPVI or additionally desensitizes its signaling mechanism requires further research. The interaction between fibrin and/or Fg with GPVI explains early observations where mice deficient in GPVI failed to form
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an occlusive thrombus in a ferric chloride injury model, even though the onset of thrombosis
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was unaltered [172].
e) Deep vein thrombosis. An exciting new development is the concept that GPVI may mediate, at least in part, platelet involvement in deep vein thrombosis. Using a microfluidic
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model mimicking the low stasis conditions seen in human venous valves, Loyau et al [173] showed that tissue factor-dependent fibrin formation was followed by platelet accumulation
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that was blocked by a Fab fragment to GPVI or by D-dimer. Platelet accumulation occurred in a red cell-dependent manner and platelet activation with PS expression favored thrombin
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generation and thrombus growth. This opens the possibility of using anti-GPVI blockers in combination with tissue-type plasminogen activator in venous thrombosis. Whether patients
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lacking GPVI are protected against deep vein thrombosis is unknown.
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e) GPVI and cancer. Platelets and platelet-derived MPs exert important roles in cancer dissemination and metastasis and GPVI is one of the receptors involved [174,175]. This opens
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a particularly exciting field for anti-GPVI drugs, particularly as blocking GPVI in mouse models increases bleeding within newly formed tumors increasing the efficacy of chemotherapy [176].
12. Conclusions and future directions The data in this review mostly confirm the often-cited tendency of a low hemorrhagic tendency for patients with inherited deficiencies of GPVI and only moderately prolonged bleeding times.
ACCEPTED MANUSCRIPT However, the number of patients with disease-causing GP6 variants remains very low and there is already evidence for two subjects of excessive trauma-related bleeding (Table 1). Dominant are the families living in rural areas of Chile with a common 2bp insertion followed by a stop codon suggestive of a founder effect [64]. A much larger cohort of patients of wider geographical distribution is required before definite conclusions can be made on a modest
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bleeding severity. Notwithstanding, the studies on GP6 knockout mouse models also point in
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this direction. Acquired loss of GPVI function due to metalloproteinase- induced shedding is more common and again in most case reports results in little bleeding. However, acquired GPVI loss is often associated with thrombocytopenia that on occasion is severe; anti-GPVI
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antibodies clearly are a cause of ITP [119]. Thus platelet count is another possible factor influencing phenotype, although guidelines for ITP suggest that platelet counts >50 x 109 /L are
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unlikely alone to cause major bleeding [177]. However blocking GPVI function with antibodies acting as inhibitors would amplify loss of the GPVI/collagen interaction. Acquired GPVI
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deficiency may also be underestimated as testing for antibody against GPVI in ITP is not generalized while in some subjects their presence may be asymptomatic as indeed was the case
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for a Chilean subject with inherited GPVI deficiency (Table 1). In rare patients antibody to
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GPVI was only shown after elution from the patients’ platelets. While these may be of low titer but high affinity this has yet to be proven. GPVI shedding itself could result in rapid antibody
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clearance from plasma by immunoadsorption. Another explanation for negative testing is endocytosis of occupied GPVI with bound antibodies transported to intra-cellular sites. Striking is the high proportion of women featured in all case reports for GPVI deficiency and in acquired causes only women were identified. While this could suggest the presence of unrecognized sex-linked factors in controlling bleeding or antibody production, why should this be a unique feature of GPVI?
ACCEPTED MANUSCRIPT Perhaps the most important finding seen for GPVI in recent years is that it in part mediates platelet interaction with fibrin and receptor-bound Fg. Thus not only does GPVI help bring about platelet binding to the vessel wall, it also intervenes in thrombus build up. Historically, αIIbβ3 was thought to be the platelet receptor for fibrin; however, GT platelets that lack this integrin bind to fibrin polymers even though they are not activated as a result
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(reviewed in [120]). Similarly, while GT platelets attach to collagen or exposed
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subendothelium, they do not spread [178] and this is despite the normal presence of α2β1 and GPVI. Thus intracellular signaling in response to collagen requires receptor inter-play and is quite complex and it is interesting to note that GT platelets lacking αIIbβ3 fail to retract a clot
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whereas platelets deficient in GPVI retain clot retraction. Further research is needed here. The emergence of agents blocking GPVI or GPVI signaling for anti-thrombotic therapy
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is highly encouraging. Nonetheless, the complexity of the platelet/collagen interactions and of their signaling pathways will necessarily require the most careful evaluation of the situations
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where drugs to GPVI are used. Not to be forgotten are the possible consequences of their longterm use on platelet production in the marrow for GPVI has been shown to regulate platelet
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biogenesis by mediating the MK interaction with collagen [121,122]. High trauma situations
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including childbirth still need to be evaluated for these drugs, as does their use in combination with specialized medications in cardiovascular disease, inflammation and major illnesses such
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as sepsis and rheumatoid arthritis. Studies on their potential for wider use in cancer are highly promising but in their infancy and offer much scope for future use as an adjunct to chemotherapy. Finally, a major requirement is a rapid and more adept clinical test to assess the clinical risk of bleeding after GPVI loss or therapeutic blockade. Results obtained with the PFA-100 were highly variable for patients with inherited and acquired GPVI loss although the collagen/epinephrine cartridge was generally more sensitive, perhaps better showing the
ACCEPTED MANUSCRIPT importance of collagen-induced ADP release from platelets. Use of computer-derived analysis of platelet adhesion and thrombus formation under shear or under static conditions on collagen and other surfaces would be a great improvement. Potential procedures have been reviewed by
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Brouns et al [179]
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Practice points
Patients with inherited or acquired collagen or receptor function mostly have conserved hemostasis with little bleeding although trauma and some inflammatory
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states may represent an exception. The use of anti-GPVI drugs in antithrombotic therapy is promising but their long-term use has yet to be tested. The detection and characterization of anti-GPVI antibody is vital in acquired disorders
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-
and testing should take into account low-titre antibodies that may be in large part
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adsorbed onto the platelets.
Autoantibodies to GPVI may induce sGPVI release through metalloproteinase-
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Research agenda
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induced shedding and testing for the soluble form is recommended.
Improvements in identifying autoantibodies to GPVI with rapid and easy to perform tests to identify both epitope targets and inhibitor activity. Large studies should investigate any possible sex linkage.
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Studies to increase our knowledge of the active sites on GPVI whose loss leads to defective function, to better understand the role of dimerization and to design a test that better measures the effect of GPVI loss on bleeding risk.
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The application of high-throughput screening to identify platelet, blood cell, plasma and vascular gene variants that will influence bleeding and will help choose the use of anti-GPVI drugs.
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Inaugurating active international databases to clearly establish the frequency of GPVI deficiencies (inherited or acquired) and to evaluate the effectiveness of different
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treatments blocking collagen receptor function in a cardiovascular disease or other
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context.
Conflicts of Interest
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Acknowledgements and Addendum
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The author declares no conflict of interest.
The author thanks Paquita Nurden for her help and for her support during the writing of this
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review. After this manuscript was submitted, Martine Jandrot-Perrus and her colleagues
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published a short review on this subject that gives an alternative view of some of the data that I covered (Jandrot-Perrus M, Hermans C, Mezzano D. Platelet glycoprotein VI genetic and
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qualitative defects. Platelets 2019;30:708-713).
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Platelets 2018;29:662-669.
ACCEPTED MANUSCRIPT Figure Legends
Figure 1. Cartoon showing the purported structure of the GPVI-FcR complex with essential information on its ligands, signaling pathways and its modification in inherited and acquired
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disease. Note the presence of negative (-ve) and positive (+ve) signaling pathways.
Figure 2. Cartoon showing the organization of the GP6 gene as it would encode the common GPVI type I splice form. Featured are the published disease-causing mutations, those giving rise to compound heterozgous expression are in the same color. *A homozygous mutation
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common to 6 unrelated families in Chile. Common high density “a” (SKTQH) and low
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variants are fully referenced in the text.
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density “b” (PEALN) alleles are also shown. The gene structure and the mutations or gene
ACCEPTED MANUSCRIPT Table 1: Clinical and biological data for cases with inherited defects of GP6
1
2
F
F
Japan
Normal
Moderate thrombocyto penia
Japan
(110140x109 /L)
F
France
Normal
Absent
Specifi c severe reducti on to collage n
10%
Specifi c absenc e with collage n and CRP
<20%; residual GPVI, smeared on WB
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3
Specifi c absenc e to collage n
Belgiu m
511
Normal
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Sever al cases (M and F)
Chile
Severe reducti on for collage n, Cvx and CRP
Much reduced
Specifi c absenc e for collage n, CRP and Cvx.
No surface GPVI. Residual truncated protein in cytoplas m
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F
CE
4
Normal for all patients
GP6 variant
Bleeding symptom s
Family
Refere nce
ND
Mild bleeding (purpura, menorrha gia and epistaxis)
Parents nonsymptomat ic with 50% expression of GPVI
Moroi et al, 1989
Lifelong mild bleeding. Bleeding after surgery required transfusio n
No family history of bleeding
Arai et al, 1995
Comp Het Arg38Cys + c.356_560 dup + fs + stop
Mild lifelong bleeding (ecchymo ses)
Parents heterozygo us for one mutation
Dumon t et al, 2009
Comp Het Ser176Asn + 16bp out-offrame del + stop
Moderate life-long bleeding (ecchymo ses, epistaxis, menorrha gia) + posttrauma bleeding
Parents heterozygo tes and asymptom atic.
Herman s et al, 2009
Homozygo us c.711_712i nsA + fs + stop
Mild bleeding (easy bruising, epistaxis, gum bleeding), one sister asymptom atic
All cases in unrelated families from different parts of Chile. Founder effect suspected.
Matus et al., 2013; Onselae r et al, 2017; Mangin et al, 2018
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1
Platelet GPVI Platelet count aggregati expressi on on2
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Count ry of origin
ND
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Gend er
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Ca se
Country of residence/origin; 2GPVI expression determined by flow cytometry or western blot (WB); Cvx, convulxin; CRP, collagen-related peptide; ND, not determined; M, male; F, female; ins, insert; fs, frameshift; Comp het, compound heterozygote 1
ACCEPTED MANUSCRIPT Table 2: Clinical and biological data for cases with acquired defects in GPVI and defective platelet/collagen interactions
F
Japan
USA
Bleeding symptoms
Severe (15x109 /L) improved with corticotherapy (140x109 / L)
Specif ic absen ce to collag en
Bleeding late in life (skin bruising, gum bleeding, epistaxis)
Severe (20x109 /L) normalizing after corticotherapy
Specif ic reduct ion to collag en
<10%
Moderate (105135x109 /L)
Specif ic absen ce with collag en and CRP
Reduc ed to 1015% of normal
Striki ng reduct ion to collag en and Cvx
Much reduce d
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France
CE
F
Macrothrombo cytopenia (1835x109 /L)
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4
Not evaluat ed
5
F
Japan
Moderate (48x109 /L) later normalizing
6
F
Japan
Moderate (80133x109 /L) then
Antibo dy to GPVI
Yes. Platele t activat ing
Refer Clinical condition ence
ITP-like syndrome + Graves disease
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Platelet GPVI aggrega expres tion sion3
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3
F
Japan
Thrombocytop enia (platelet count2 )
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2
F
Origin1
Sugiy ama et al, 1987; Ichino he et al, 1995
Yes. Platele t activat ing
ITP + SLE + thyroiditis
Takah ashi & Moroi , 2001; Ichino he et al, 1995
Moderate bleeding (petechia, easy bruising)
Yes
ITP-like syndrome
Boyla n et al, 2004
Lifelong moderate bleeding
Not detect ed
Gray platelet syndrome and suspected myelofibrosis
Nurde n et al, 2004
Initiall y no but later detect ed in platele t eluates
ITP-like syndrome suspected
Kojim a et al, 2006; Akiya ma et al, 2009
Detect ed in platele t
ITP-like syndrome with progressive loss of
Akiya ma et al,
Mild subcutaneo uss bleeding
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1
Gen der
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Ca se
Specif ic absen ce with collag en, CRP and Cvx
Absent
Mild bleeding (petechia, gum bleeding)
Specif ic absen ce
Absent then progre ssive
Mild bleeding (easy bruising,
ACCEPTED MANUSCRIPT
F
Australia
ITP, romiplostim treatment was curative
Gardi ner et al, 2008; 2010
No severe bleeding
Yes. In plasm a and on platele ts, activat ing when strong
SLE and ITPlike conditions responsive to treatment
Nurde n et al, 2009
Lifelong bleeding
IgM cold aggluti nin
Pseudothrombo cytopenia (5x109 /L in citrated blood, 100x109 /L in EDTA)
Sanch ezGuiu et al, 2015
Severe deficie ncy.
Exagerated bruising
Plasm a antibo dy that aggreg ates contro l platele ts
ITP-like syndrome. Platelet count improved under treatment
Rabbo lini et al, 2017
None. GPVIsignali ng defcet
Myelodysplasi a and fatal myelomonobla stic leukemia
Belluc ci et al, 2004
None.
Chronic
Belluc
1020% of normal
Major deficie ncy, then recove ry
Low
Specif ic absen ce with collag en, Cvx and CRP
Norma l
Purpura in lower limbs
Specif
Norma
Extensive
11
M
France
Severe (10x109 /L) progressing to severe thrombocytope nia
12
F
France
Normal then
2009
Yes. Activa ting, presen t in plasm a and in platele t eluates
Specif ic absen ce with collag en
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F
bleeding symptoms
Epistaxis and lower limb purpura; symptoms continued despite corticother apy
Severe ITP when first studied. Moderate (91x109 /L) at time of study
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10
eluates but not in plasm a
T
Not able to be assess ed
epistaxis)
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Spain
Chronic moderate thrombocytope nia
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9
F
France/M orocco
Severe (4x109 /L) normalizing with immunosuppre ssive and corticoid therapy
Specif ic absen ce with collag en and Cvx
Australia
recove ry
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8
F
Severe (2x109 /L) normalizing with corticotherapy
Specif ic absen ce with collag en and Cvx
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7
with collag en, Cvx
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normalizing
ACCEPTED MANUSCRIPT
F
Germany
Normal
bleeding (also heparindependent thrombocyt openia)
Norma l GPVI expres sion
Mild lifelong skin hematomas + menorrhagi a
GPVI signali ng defect
lymphocytic leukemia
ci et al, 2004
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Specif ic reduct ion with collag en and Cvx, impro ving with time
l
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13
ic absen ce with collag en, Cvx and CRP
Norma l GPVI expres sion
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progressive thrombocytope nia
Unknown cause. No autoantibody to GPVI
Klüm pers et al, 2018
Country of residence/origin; 2Platelet count at its nadir; 3GPVI expression determined by flow cytometry or western blot (WB); M, male; F, female; Cvx, convulxin; CRP, collagen-related peptide
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CE
PT
ED
MA
1
Figure 1
Figure 2
Alan T. Nurden PII: DOI: Article Number: Reference:
S0268-960X(19)30069-4 https://doi.org/10.1016/j.blre.2019.100592 100592 YBLRE 100592
To appear in:
Blood Reviews
Please cite this article as: A.T. Nurden, Clinical significance of altered collagen-receptor functioning in platelets with emphasis on glycoprotein VI, Blood Reviews, https://doi.org/ 10.1016/j.blre.2019.100592
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ACCEPTED MANUSCRIPT Clinical Significance of Altered Collagen-Receptor Functioning in Platelets with Emphasis on Glycoprotein VI Alan T Nurden
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Institut de Rhythmologie et de Modélisation Cardiaque, Hôpital Xavier Arnozan, Pessac, France
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Correspondence: Alan T Nurden
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IHU-LIRYC, PTIB, Hôpital Xavier Arnozan, 33600 Pessac, France Tel: +33.5.56.55.19.52
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Fax: +33.5.57.10.28.64
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E-mail: [email protected]
ACCEPTED MANUSCRIPT Abstract Much interest surrounds the receptors α2β1 and glycoprotein VI (GPVI) whose synchronized action mediates the attachment and activation of platelets on collagen, essential for preventing blood loss but also the most thrombogenic component of the vessel wall. Subject to density variations on platelets through natural polymorphisms, the absence of α2β1 or GPVI uniquely
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leads to a substantial block of hemostasis without causing major bleeding. Specific to the
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megakaryocyte lineage, GPVI and its signaling pathways are most promising targets for antithrombotic therapy. This review looks at the clinical consequences of the loss of collagen receptor function with emphasis on both the inherited and acquired loss of GPVI with brief
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mention of mouse models when necessary. A detailed survey of rare case reports of patients with inherited disease-causing variants of the GP6 gene is followed by an assessment of the
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causes and clinical consequences of acquired GPVI deficiency, a more frequent finding most often due to antibody-induced platelet GPVI shedding. Release of soluble GPVI is brought
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about by platelet metalloproteinases; a process induced by ligand or antibody binding to GPVI or even high shear forces. Also included is an assessment of the clinical importance of GPVI-
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mediated platelet interactions with fibrin and of the promise shown by the pharmacological
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inhibition of GPVI in a cardiovascular context. The role for GPVI in platelet function in inflammation and in the evolution and treatment of major illnesses such as rheumatoid arthritis,
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cancer and sepsis is also discussed.
Keywords: Glycoprotein VI; inherited defects; acquired defects, polymorphisms; soluble GPVI shedding; mouse models; inflammation; anti-thrombotic therapy.
ACCEPTED MANUSCRIPT 1. Introduction Platelets maintain blood circulation by preventing bleeding through hemostatic plug formation at sites of vessel injury and by preserving blood vessel integrity [1,2]. A source of active metabolites, cytokines and growth factors, platelets also promote angiogenesis and tissue repair [3]. Collagen is the main substrate for platelets in subendothelium and its exposure in
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atherosclerotic plaques initiates occlusion in myocardial infarction and stroke [4-6]. Under
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flow, platelets first interact through glycoprotein (GP) Ibα which binds to von Willebrand factor (VWF) multimers pre-adsorbed to collagen; the platelets then translocate along the collagen fibers favoring the interactions that permit firm adhesion [7,8]. Platelet interaction
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with collagen is a multistep process that principally involves two receptors, integrin α2β1 and the immunoglobulin superfamily member, GPVI that act in synergy in a density-dependent
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manner with GPVI principally governing the signaling responses [9-12]. GPVI (58-62 kDa) has a single chain and is co-expressed with the promiscuous Fc
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receptor chain (FcR). Formed by non-covalent transmembrane contact, this complex is exclusive to platelets and megakaryocytes (MKs). Three groups independently cloned the GP6
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gene some 20 years ago [13-15]. Located on the long arm of chromosome 19 (19q.13.42), GP6
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has 8 exons that encode 2 terminal disulfide- linked immunoglobulin (Ig)-homology loops (IgC1 and Ig-C2), a short glycosylated stalk, a transmembrane domain and a 51 amino acid (aa)
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cytoplasmic tail in the principally located splice form (Fig. 1). The protein has two extracellular N- and many O-glycosylation sites that are important for function [16,17]. The Ig domains contain distinct but overlapping binding sites for collagen, collagen-related peptide (CRP) and the snake venom protein convulxin (Cvx), two ligands used to mimic collagen activation of platelets in diagnostic and research [18]. The GPVI cytoplasmic tail includes binding sites for calmodulin and the tyrosine kinases Fyn/Lyn [19,20]. Ligand binding to GPVI activates Fyn or Lyn allowing them to mediate phosphorylation of the two FcR cytoplasmic tail immuno-
ACCEPTED MANUSCRIPT tyrosine based activation motifs (ITAMs). This favors the phosphorylation of Syk and the formation of a signaling complex containing adaptor proteins such as Linker of Activation of T cells (LAT) and SLP76 with the involvement of Src, phosphatidylinositol 3-kinases (PI3kinases), phospholipase C2 (PLC2) and Ca2+-mobilization; steps that initiate platelet spreading and aggregation as well as other functional responses (reviewed in [21]). ITAM-
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mediated signaling is essential for collagen- induced activation of αIIbβ3 itself exhibiting
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functional interplay with α2β1 [22-24]. At the same time negative regulation of GPVI signaling is mediated by G6b-B, PECAM and the protein tyrosine phosphatase, CD148 [25-27]. The relative role of monomeric or dimeric forms of GPVI in mediating the platelet
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response was the object of early debate [28-31]. Clustering and association with lipid rafts increases GPVI avidity for collagen and promotes downstream signaling. GPVI is promiscuous
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with laminin, extracellular matrix metalloproteinase inducer (EMMPRIN), fibronectin (Fn) and vitronectin (Vn) other potential ligands in the vessel wall [32-36] (Fig. 1). The hormone
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adiponectin and histones are plasma ligands [37,38]. Then the role of GPVI was transformed by the late discovery that GPVI also mediates platelet interactions with fibrin [39,40]. Binding of
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GPVI to fibrin also leads to platelet activation with phosphatidylserine (PS) exposure
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promoting more thrombin generation in a feedback loop. But controversy persists with regard to the relative roles of monomeric versus dimeric GPVI in mediating GP function. Thus, while
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Onselaer et al [41] showed that monomeric GPVI binds to the fibrin D-dimer domain and that soluble D-dimer blocked platelet activation by both fibrin and collagen, Ebrahim et al [42] using recombinant technology found that dimeric GPVI binds to collagen but not fibrin under static conditions and arterial flow. Then, Induruwa et al [43] confirmed that collagen-binding dimeric GPVI recognized soluble D-dimer, monomeric and polymerized fibrin and extended the results to Fg. Mangin et al [44] extended these findings by showing that binding of GPVI to immobilized fibrinogen (Fg) under flow is followed by a time-dependent formation of
ACCEPTED MANUSCRIPT lamellipodial sheets and stress fibers. In a recent review, Slater et al [45] show how technical issues related to the use of recombinant GPVI protein may account for some of the above discrepancies. In summary, GPVI is now recognized as a pluripotent receptor whose influence extends beyond the initial platelet-collagen interaction to thrombus build up and coagulation. Such observations suggest that it is a player not only in thrombosis but also in inflammatory
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states including sepsis. GPVI deficiency therefore has implications far beyond that of a
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potential bleeding diathesis. The object of this review is to assess the clinical effects of inherited and acquired deficiencies of GPVI in human disease with brief mention of mouse
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models and when appropriate of the α2β1 integrin, its companion receptor for collagen.
2. Natural polymorphis ms and variations in collagen receptor expression
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Early studies established that the platelet response to collagen was linked to the surface density of platelet collagen receptors. Historically, findings were first reported for α2β1,
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polymorphisms in the coding regions of the ITGA2 gene (5q23-31) being linked to a 5-fold variation in platelet α2β1 density in a control human population [46]. Three dimorphic allelic
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coding variants were highlighted: A1 (807T/1648G) associated with high, A2 (807C/1648G)
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and A3 (807C/1648A) with lower α2β1 densities. Importantly, the rate of attachment of platelets to collagen at high shear (1,500/sec) in a flow chamber correlated with the α2β1 copy
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number. The genetics of α2β1 expression were extended when further polymorphisms within the ITGA2 proximal 5’-regulatory regions and linked to decreased binding of the Sp1 transcription factor were also shown to influence α2β1density [47]. At this time, Furihata et al [48] in the Kunicki laboratory described a 5-fold range in GPVI expression in platelets of normal donors and linked Cvx or CRP-induced prothrombinase activity (and therefore thrombin generation) to GPVI density. A series of 10 SNPs (5 synonymous and 5 nonsynonymous affecting coding regions) were found in the GP6 gene [49,50]. Highlighted were 2
ACCEPTED MANUSCRIPT common haplotypes affecting 5 aa, 3 in the extracellular domain and two in the cytoplasmic tail of GPVI (“a” allele, SKTQH; “b” allele PEALN) with allelic frequencies of 0.85 and 0.13 respectively (Fig. 2). The “b” allele associated with lower GPVI density on platelets and reduced platelet functional responses to collagen or CRP [49,50]. However, recent studies on larger cohorts underscore a somewhat lower variability. For example, Best et al [51] showed
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GPVI levels to vary 1.5 fold and α2β1 levels 3-fold among 102 healthy donors. Platelets of
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donors with the “b” allele of GPVI representing 22% of the population had 10% lower surface expression of GPVI. Later, Watkins et al [52] generated a high resolution SNP map by sequencing the promoter, exons and splice sites of GP6 for 96 unrelated Caucasian individuals.
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The SKTQH and PEALN isoforms were confirmed as major alleles. Among 18 SNPs was a rare non-synonymous SNP (103Leu>Val) (MAF=0.005) giving reduced ligand binding. Their
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study population was then extended to 2000 individuals with the identification of 12 additional isoforms and significant changes in GP6 haplotype expression between people of different
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geographical and ethnic backgrounds.
The degree by which the above haplotypes controlled GPVI density and platelet
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function continued to be less striking in larger cohorts. Jones et al [53] studied 506 subjects
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identifying individuals who were hyper- or hypo-responders to CRP; sequence variations at the GP6 locus, primarily the SKTQH and PEALN haplotypes now accounted for only 35% of the
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variation. The Kunicki group turned to expression studies and showed that the presence of the cytoplasmic domain LN substitutions in the “b” allele significantly diminished signal transduction, an effect that over-rode ligand binding or GPVI copy number [54]. As discussed earlier (1. Introduction), the GPVI cytoplasmic domain binds calmodulin and the tyrosine kinases Fyn/Lyn. Trifiro et al [54] showed that L317 conferred an increased affinity for calmodulin while N322 attenuated binding to Fyn/Lyn and consequently Syk phosphorylation.
ACCEPTED MANUSCRIPT Do variations in GPVI expression and haplotype-related functional differences of platelets have clinical consequences? Kunicki et al [55] compared secondary platelet candidate gene polymorphisms with bleeding severity in pedigrees of 14 index cases with type I von Willebrand disease (VWD) where patients have low VWF levels. Increased bleeding scores were linked to ITGA2 (807C), ITGA2B (Ile843) and GP6 (PEALN) haplotypes but no
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associations were found for 6 other candidate genes. When extended to an analysis of pedigrees
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with VWD type 2A, 2B and 2M, of 5 selected platelet genes only an ITGA2 promoter haplotype (-52T) was associated with an increased bleeding severity and no correlations were seen for GP6 [56]. In fact, the 807C and -52T variants are in strong linkage disequilibrium and
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both associate with low α2β1density. Finally, Cheli et al [57] performed a genome-wide SNP scan using Gp6-/- mice, and revealed significant linkage of animals with a prolonged tail
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bleeding time to a locus on chromosome 4 designated as modifier of hemostasis 4 (Mh4). The authors concluded that one or more genes at this locus control the extent to which thrombus
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formation is disrupted in the absence of GPVI. No follow-up studies on Mh4 have been
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reported.
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3. Inherited defects of GPVI
The first report of a possible inherited deficiency of GPVI (OMIM:614201) came when Moroi
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et al [58] described a Japanese woman with lifelong mild mucocutaneous bleeding and platelets that failed to adhere, secrete or aggregate with type I and type III collagen despite a full response to ADP, epinephrine, arachidonic acid (AA), thrombin and ristocetin (Table 1). Significantly, clot retraction was normal and her bleeding time only slightly prolonged. Radiolabeling of surface GPs showed a deficit of GPVI but a normal presence of other membrane receptors. Her platelets failed to bind a human antibody to GPVI (see also Sugiyama et al. [59] and Section 6). Platelets of her non-consanguineous parents had 50% GPVI
ACCEPTED MANUSCRIPT expression and responded well to collagen. Arai et al [60] then reported a Japanese woman with a congenital mild bleeding syndrome whose severity decreased with age. Her bleeding time was only moderately prolonged. Nevertheless, extensive bleeding accompanied hysterectomy. Her platelet count was moderately reduced (Table 1). While her platelets were refractory with collagen they responded well to other agonists. Platelet adhesion to collagen under static
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conditions was mildly reduced. Clot retraction was normal. Western blotting (WB) using
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human antibody to GPVI (see Sugiyama et al. [59]) revealed about 10% residual GPVI in her platelets. Other membrane receptors including integrin α2β1 were normally present. Thus 10% residual GPVI was insufficient to support platelet activation by collagen. Enigmatic, was the
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report from Goto et al [61] describing how platelets from the above patients also showed less firm adhesion on vWF.
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The first reported GP6 gene mutations came from Dumont et al [62]. The patient was a young girl with mild bleeding since infancy, a PFA-100 closure time prolonged to 210 seconds
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for collagen/epinephrine and poorly responsive to collagen and CRP (Table 1). Noteworthy is that secretion in response to collagen and collagen- induced thrombin generation was also
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impaired. Flow cytometry showed GPVI at 540 copies per platelet versus 3076 for the controls;
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WB confirmed the incomplete deficiency with a smeared migration of the residual protein. FcR was normally present. DNA sequencing revealed compound heterozygozity for two
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predicted disease-causing GP6 mutations (Fig. 2): (i) a c.172C>T transition giving an Arg38Cys (R38C) mutation within exon 3 and affecting the first Ig loop of GPVI and (ii) a duplication of 5 nucleotides (c.356_360) in exon 4 resulting in a frameshift (fs), a premature stop 30 codons downstream, an absence of the corresponding mRNA probably indicated mRNA decay. Introduction of Arg38Cys into recombinant GPVI reproduced the abnormal migration as seen by WB and a marked loss of collagen binding. The smeared migration was hypothesized to result from mismatched disulfides caused by the extra Cys residue. The ability
ACCEPTED MANUSCRIPT of the patient’s platelets to form thrombi on collagen under flow was reduced by 43%. In a parallel report, Hermans et al [63] described a young woman with moderate lifelong bleeding marked by several episodes of post-traumatic and post-surgery bleeding. Reduced platelet function responses were selective to collagen and its mimetics. Although thrombus formation on collagen under flow was poor, more single platelets attached. Enigmatically, the PFA-100
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closure time with the collagen/ADP membrane was shortened while that for the
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collagen/epinephrine cartridge was unchanged. Her platelet count and platelet ultrastructure were normal. GPVI expression was much reduced with residual protein normally migrating on WB. DNA sequencing revealed two heterozygous GP6 mutations: (i) an out-of frame 16bp del
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predicting a stop codon after 56 aa and (ii) a missense Ser176Asn substitution within the second Ig domain of GPVI (Fig. 2). Ser176 is highly conserved between species and its
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substitution was predicted to disrupt H-bonding; the second short truncated variant was not expressed. Both parents were heterozygous carriers and asymptomatic; in fact the mother with
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GPVIAsn176 unexpectedly had platelets normally expressing GPVI. Studies in HEK293 cells confirmed a normal posttranslational processing of GPVIAsn176 suggesting a functional
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defect.
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My group in Bordeaux set up sequencing of the GP6 gene in response to a request from Dr. Diego Mezzano who was investigating unrelated patients (2 female and 2 male) belonging
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to 4 non-consanguineous Chilean families [64]. All propositi presented with lifelong mild mucocutaneous bleeding (Table 1). Platelets of each were specifically refractory to collagen or, when tested, CRP or Cvx. When measured, the bleeding time was moderately prolonged. Flow cytometry failed to locate GPVI on the platelets of each patient. Sequencing of GP6 revealed an adenine insertion in exon 6 (c.711_712insA) causing a frameshift and generating a stop codon at aa242 of the mature protein; this homozygous variant was common to all patients who all expressed the high density “a” allele for GP6 (Fig. 2). There was no family history of
ACCEPTED MANUSCRIPT bleeding and the families are from different rural regions of Chile and while a founder effect can be hypothesized there is currently no clear explanation. The mutation predicts the synthesis of a soluble form of GPVI truncated extracellular to the trans-membrane domain. Indeed, WB detected a limited presence of truncated protein (around 49 kDa) in the platelets of each patient but no full-length GPVI. Obligate heterozygotes were asymptomatic and aggregated well with
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collagen. Intriguingly a sister of one of the patients was also homozygous for the mutation but
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has never shown signs of bleeding despite platelets unresponsive to collagen and lacking surface GPVI. Later, Onselaer et al [41] reported two more patients and showed that platelet adhesion to fibrin was also reduced by 40%, with αIIbβ3 the obvious candidate as the second
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fibrin receptor. Those platelets that did attach showed markedly reduced spreading, only occasional platelets showed signs of activation. Then, Mangin et al [44] studying platelets from
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two of the patients confirmed reduced adhesion on immobilized Fg, the platelets failing to form lamellipodal sheets and stress fibres. Blocking αIIbβ3 of GPVI negative platelets resulted in a
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total absence of adhesion on Fg. They also confirmed that clot retraction was normal, presumably mediated by αIIbβ3.
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The results so far accumulated from inherited GPVI deficiency support the conclusion
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that the hemorrhagic tendency is mostly moderate, contrasting for example, with the more severe bleeding that accompanies GPIb-IX deficiency in Bernard-Soulier syndrome and αIIbβ3
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deficiency in Glanzmann thrombasthenia (GT) [65,66]. Notwithstanding, the number of patients is small and care is needed not to over-interpret the available data for more information is required – especially for situations such as childbirth and trauma-related bleeding including surgery where two of the above patients did bleed excessively (Table 1).
4. Mouse models for α2β1 and GPVI deficiency
ACCEPTED MANUSCRIPT Mouse models have proved essential in understating the dynamics of the platelet collagen interaction and the roles of α2β1 and GPVI. First, mice genetically depleted of β1 integrins were shown to retain a normal bleeding time despite delayed but substantial thrombus formation on collagen fibrils and a lack of platelet aggregation with soluble collagen [67]. As these mice also lack α5β1 and α6β1 receptors for Fn and laminin, efforts turned to generating
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α2-deficient mice [68,69]. Höltkotter et al [68] showed that the mice developed normally with
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no obvious fertility or other physiological defects and that their platelets showed the specific defects in the collagen response seen for β1 knockout. Again there was no bleeding phenotype and the tail bleeding time was unchanged. Chen et al [69] confirmed that α2β1 knockout mice
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were viable with normal wound healing but detected a multifaceted phenotype with defects extending to epithelial cells and branching morphogenesis. Significantly, a blocking
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monoclonal antibody (MoAb) to GPVI totally abrogated the adhesion of the α2β1-deficient platelets to collagen [68]. Mice whose platelets were immunologically depleted of GPVI
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(detailed in Section 5) also only had a mild disruption of primary hemostasis despite the platelets showing reduced thrombus formation on collagen under flow [70,71]. It was
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significant then when platelets from mice genetically engineered to lack GPVI or FcR (also
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leading to GPVI deficiency) showed a severe loss of platelet adhesion, spreading and thrombus formation on collagen but again showed little bleeding [72]. Interestingly, mouse platelets
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genetically modified to express 5-fold lower levels of GPVI showed comparable thrombus formation on a high-density collagen coated surface to controls in so doing raising questions about the clinical significance of human GP6 polymorphisms [51]. In summary, α2β1 and GPVI clearly act in synergy and have complementary roles.
5. Shedding of GPVI from mice and human platelets
ACCEPTED MANUSCRIPT Shedding of the bulk of the soluble extracellular domain of GPVI (sGPVI) from circulating platelets causes loss of the collagen- interacting domains thereby regulating platelet function. Recognizing and understanding the mechanisms behind GPVI shedding is key to understanding acquired GPVI pathology. a) Antibody and ligand-induced release of sGPVI. Pioneering studies from Bernhard Nieswandt
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and his group showed that injecting rat MoAbs to GPVI (the JAQ series) into mice led not only
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to a transient thrombocytopenia but also to a long-term (at least 10 days) immune-depletion of GPVI from platelets with thromboembolic protection [67,70,71,73,74]. Studies performed with human platelet-rich plasma showed that adding mouse MoAbs against GPVI not only resulted
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in sGPVI shedding, the majority also induced platelet aggregation [75]. Release of sGPVI also occurred with monovalent Fab fragments (showing that Fc-receptor interactions were not
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necessary) and also occurred with MoAbs against both ligand and non-ligand-binding domains of GPVI. Enigmatically, no loss of GPVI occurred when JAQ antibodies were incubated with
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mouse platelets in vitro. But in vivo in mice both antibody-induced GPVI shedding and the thrombocytopenia are rapid (maximum at 30 min with return of the platelet count to normal
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after 48h); it is interesting to ask whether within this short time scale the bleeding risk was
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more severe [71]. Intracellular signaling is required for GPVI loss; shedding was prevented when elements of FcR-mediated ITAM signaling were interfered with. For example, GPVI
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shedding did not occur in LAT-/- mice (LAT is part of the GPVI signalosome); instead JAQ1GPVI complexes were internalized by the platelets followed by a slow intracellular degradation. The LAT-/- platelets failed to respond to collagen, but increases in bleeding time were modest [76]. Boylan et al [77] introduced human platelets into the circulation of non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice; Fab fragments of a MoAb to human GPVI were then injected. Shedding of sGPVI occurred from the human but not the
ACCEPTED MANUSCRIPT mouse platelets. But dampened platelet responses to ADP and thrombin were also seen. In a more classic mouse model, others also noted that JAQ1-induced GPVI shedding was accompanied by a short transient decrease in the platelet response to thrombin [78]. Such a loss would also accentuate bleeding risk. Important but enigmatic is that not all MoAbs to GPVI cause in vivo GPVI shedding or depletion although they may block function [79,80]. Clearly
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antibodies to GPVI in patients have to be tested individually. As GPVI also contributes to
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platelet aggregate formation under flow by interacting with platelet-bound Fg or fibrin, thrombus formation is also affected by GPVI loss [43,44]. One study reports that newly formed fibrin promotes shedding of GPVI from human platelets independently of ITAM signaling and
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that this occurs in pathology, for example in patients with severe burns [81]. b) Mechanisms for GPVI loss. Shedding of sGPVI is by proteolysis through the action
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of metalloproteinases with the release of a large 55 kDa fragment leaving a short transmembrane and intracellular fragment attached to the platelet (Fig. 1). Most studies have
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identified “A Disintegrin and Metalloproteinase Domain-containing protein 10” (ADAM10) as the protease that cleaves GPVI although ADAM17 and other metalloproteinases may also
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intervene [74,82,83]. The process appears under intracellular control, agents blocking the
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interaction of calmodulin with the GPVI cytoplasmic tail stimulate GPVI cleavage. GPVI can also be removed from the platelet surface by intracellular clearing and both shedding and
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internalization are abrogated in mice with a point mutation in the FcR ITAM domain again showing a role for intracellular signaling [76,84]. Release of sGPVI also occurs during coagulation by a process highly dependent on FXa that was speculated to activate ADAM10 directly and, as explained above, through direct interaction with fibrin [81,85]. A key question is how ADAM10 or other proteases in platelets find and cleave ligandor antibody-bound GPVI so rapidly. Localization using fluorochrome- labeled antibodies showed that the principle ADAM10 pools of platelets are intracellular but are organized
ACCEPTED MANUSCRIPT independently of P-selectin and α-granules (see Fig. 4 of ref [86]). So presumably a specific surface translocation mechanism is necessary. While beyond the scope of this review, recent studies suggest that several mechanisms can lead to sGPVI release and metalloproteinase activation with a role for membrane tetraspanin family as mediators of GPVI lateral diffusion and/or carriers or activators of metalloproteinases [87,88].
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c) Importance of shear. As well as ligand binding, transient exposure to mechanical
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force can lead to metalloproteinase activation and sGPVI release [89]. This is very relevant as high shear occurs in atherosclerosed coronary vessels or after ischemic events, and indeed high levels of circulating sGPVI have been detected in pilot studies on patients with stable angina
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pectoris and stroke [89,90]. Similar situations occur in acquired von Willebrand disease in a cardiovascular context and with the use of ventricular assist devices and extracorporeal
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membrane oxygenation where loss of platelet receptors (GPIbα and GPVI) and loss of platelet
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function can be a cause of bleeding [91].
6. Pathology of α2β1 deficiency and less well-known collagen receptors on platelets
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Early reports described inherited defects of α2β1 (originally known as Very Late Antigen
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(VLA)-2 and then GPIa-IIa) in a mild to severe bleeding disorder linked to a much reduced platelet response to collagen [92,93]. The patients had excessive post-traumatic bleeding and
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for women, menorrhagia. Other reports described GPIa deficiency associated with a lack of thrombospondin or in myeloproliferative disorders [51,94,95]. The relatively severe bleeding experienced by the patients reported by Nieuwenhuis et al [92,93] contrasts to the absence of bleeding in mice with platelets genetically modified to lack α2β1 (see Section 4). The genetic defect in these families needs elucidating. Interestingly, Noris et al [96] described inherited α2β1deficiency but in the context of autosomal dominant thrombocytopenia and mild bleeding later shown to be associated with variants in the ANKRD26 gene [97]. Another purported
ACCEPTED MANUSCRIPT collagen receptor on platelets is CD36 (GPIV) [98]. However, patients genetically lacking CD36 respond normally to collagen and at best it has a minor role [99]. Another purported collagen receptor is leukocyte-associated immunoglobulin- like receptor (LAIR-1) but it has not been linked to a specific pathology [100]. Autoantibodies to α2β1 are a cause of ITP [101,102] but little is known of their potential blocking of the platelet-collagen response. One exception is
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an inhibitory IgM antibody cloned and sequenced by Deckmyn et al [103]. A MoAb binding to
integrin in the signaling response to collagen [104].
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αIIb also specifically blocks the platelet/collagen interaction underlining a potential role for this
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7. Acquired defects associated with antibodies to GPVI in human pathology Historically, Sugiyama et al [59] reported the first acquired defect of GPVI in a Japanese
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woman with ITP associated with Grave’s disease (Table 2). Thrombocytopenia was severe and associated with spontaneous mucocutaneous bleeding and the patient received platelet
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transfusions. Corticotherapy restored her platelet count to near normal but mild bleeding was still occasionally seen. Her platelets aggregated well to ADP, epinephrine, AA, thrombin and
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ristocetin but were refractory to collagen and this included dense granule secretion and
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thromboxane B2 synthesis. Although platelet adhesion to collagen fibrils was reduced, some platelets did attach. Significantly, both her plasma and isolated IgG induced the aggregation of
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control platelets, a response shown to depend on dense granule secretion. Her antibody primarily immunoprecipitated a membrane GP in the 50-60 kDa range from detergent solubilized control platelets later shown to be GPVI. Takahashi & Moroi [105] described a middle-aged Japanese woman with systemic lupus erythematosus (SLE), chronic thyroiditis and mild bleeding whose platelets showed similar characteristics. Her thrombocytopenia was initially severe but her platelet count normalized after corticosteroid therapy but again bleeding continued. Platelet-associated IgG was elevated and an antibody to GPVI reactive in WB
ACCEPTED MANUSCRIPT present in her serum; platelet GPVI was much reduced. Her antibody aggregated control platelets; cross-linking GPVI in the platelet membrane was a proposed mechanism. Significantly, F(ab’)2 fragments of the antibody of Sugiyama et al [59], now identified as an anti-GPVI, induced signaling pathways identical to those used by collagen and its mimetics Cvx and CRP, with tyrosine phosphorylation of Syk and Src [106]. Platelets from the Japanese
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patients deficient in GPVI responded to collagen with a normal α2β1-dependent tyrosine
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phosphorylation of Src but GPVI-dependent phosphorylation of Syk and activation of PLC2, Vav and FAK (focal adhesion kinase) was severely reduced [107].
Boylan et al [108] then reported a young woman in the USA with ITP and mild bleeding
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and moderate thrombocytopenia that reproduced the findings of the Japanese patients. Adherence and thrombus formation on collagen under flow were shown to be defective.
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Despite a much-reduced platelet expression of GPVI and FcRher platelets contained mRNA encoding GPVI and no mutations were found in the GP6 gene. The patient’s plasma possessed
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an IgG antibody to GPVI speculated to cause the rapid clearance of sGPVI from the plasma.
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An unusual case was an elderly woman from Bordeaux with gray platelet syndrome (GPS) and an antibody to GPVI [108]. GPS is an inherited platelet disorder caused by mutations in
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NBEAL2, a gene essential for the packaging of proteins during -granule synthesis in MKs [110]. GPS is associated with mild to severe mucocutaneous bleeding, macrothrombocytopenia
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an enlarged spleen and myelofibrosis that was strongly suspected although a confirmatory bone marrow biopsy was not performed [111]. Unlike typical GPS, platelet aggregation with collagen or Cvx was virtually absent and there was a severe reduction of GPVI in her platelets. Autoantibodies to GPVI were not detected either on her platelets or in her serum; sequencing GP6 revealed no potential disease-causing mutations (the patient had the high density “a” allele). It was speculated but not proven that endogenous metalloprote inase activity in the marrow was responsible for GPVI loss.
ACCEPTED MANUSCRIPT Studies continued with two more unrelated female Japanese patients with a mild bleeding diathesis, moderate thrombocytopenia and platelet function typical of acquired GPVI deficiency (cases 5 and 6, Table 2). For both, the platelet count gradually recovered but occasional mild bleeding continued; for the first platelets attached poorly to collagen under static conditions with spreading profoundly affected [112,113]. In both cases, plasma antibody
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to GPVI was not detected but platelet-associated IgG was elevated and eluates contained
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antibodies to GPVI as shown in a dot-blot assay using recombinant protein. Testing against transfected αIIbβ3-expressing heterologous cells also detected antibodies to αIIbβ3 in the second patient [113]. Significantly, as the patient’s platelet count increased, platelet expression
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of GPVI improved and the bleeding tendency diminished as antibody titers in platelet eluates decreased. Although GPVI shedding is a tempting hypothesis for these patients, sGPVI was not
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elevated and neither was the 10 kDa membrane-bound remnant of GPVI observed in WB. Not least, these cases show the importance of careful antibody testing.
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Gardiner et al [114] continued the reports of severe thrombocytopenia with ITP linked to anti-GPVI antibody. Corticotherapy again restored her platelet count but spontaneous
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pathological bruising continued. The collagen/epinephrine PFA-100 closure time was markedly
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prolonged. Here, platelet-associated IgG was elevated and plasma autoantibody to GPVI present. The antibody aggregated control platelets, an activity blocked by sGPVI and in part by
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a MoAb (IV.3) binding
. Decreased GPVI on her platelets and analyses for sGPVI
and of the residual transmembrane domain and cytoplasmic tail of GPVI strongly suggested shedding of GPVI, and this was confirmed when her plasma caused GPVI loss from control platelets. Interestingly, the authors later showed that 6-month treatment with the TPO receptor mimetic, romiplostim, restored both the platelet response to collagen and GPVI expression on her platelets but enigmatically, sGPVI in her plasma remained elevated and plasma autoantibody to GPVI continued to be detected [115]. How these results are explained is
ACCEPTED MANUSCRIPT unclear although previous studies have shown that TPO up-regulates GPVI expression in MKs by c-Mpl-mediated demethylation of a cytosine phosphate-guanosine-rich island within the GP6 promoter [116]. A young French woman with an ITP-like condition came to our attention in 2005 with severe thrombocytopenia (case 8, Table 2); corticotherapy restored her platelet count but within
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a year a lupus nephropathy led to the diagnosis of SLE with antibody to DNA [117]. She
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improved after treatment but later her condition again worsened and she received transfusions as a precaution prior to biopsies. In 2006, platelet function testing revealed a selective absence of platelet responses to collagen and Cvx and a specific platelet deficiency of GVI and FcR;
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her PFA-100 closure time (collagen + epinephrine) was markedly prolonged. Testing throughout subsequent treatment revealed a progressive restoration of the platelet responses to
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collagen and increases in platelet GPVI expression and her PFA-100 closure time normalized. Plasma samples analyzed for anti-GPVI antibodies employing a specially developed ELISA
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with recombinant sGPVI showed that antibody to GPVI was initially strong but that its titer fell as platelet GPVI levels improved. Plasma sGPVI remained low. DNA sequencing showed no
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potential disease-causing mutations and that the patient’s GP6 possessed the “a” allele
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associated with higher GPVI levels. The plasma antibody was of the IgG3 subtype and when titers were strong it activated control platelets with long incubations resulting in sGPVI release
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and a loss of the collagen response. Significantly, restoration of platelet GPVI levels and the collagen response was associated with loss of her nephrotic syndrome. An unusual case reported by Sanchez-Guiu et al [118] concerned an atypical plateletreacting IgM class cold agglutinin in a woman with a lifelong history of mild bleeding and chronic moderate thrombocytopenia. A unique diagnostic feature was pseudothrombocytopenia that was temperature-dependent but EDTA-independent. Her IgM bound GPVI and aggregated control platelets. Her own platelets had elevated bound IgM and showed signs of activation
ACCEPTED MANUSCRIPT (increased TXA2 production and surface P-selectin). Low GPVI expression and increased levels of sGPVI in her plasma was suggestive of shedding. The patient had prolonged closure times in the PFA-100 with both cartridges with platelet plugs forming more quickly when the test was performed immediately after the blood was taken (due to the spontaneous agglutination). Platelet function testing was abandoned for this reason. Platelet activation was
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blocked by a GPVI-Fc fusion protein and by inhibitors of the tyrosine kinase Src family
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and of Syk . Her IgM agglutinin has persisted over a 5-yr period. No abnormalities were seen on sequencing of the GP6 coding sequences. No other family member was affected. Finally in this Section, Rabbolini et al [119] described a middle-aged woman with a
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long-standing history of ITP and easy bruising. Severe thrombocytopenia improved with immunosuppressive therapy but a relapse required treatment with IVIgG given with antibiotics.
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Tests performed prior to hip surgery revealed a specifically defective collagen response and severe platelet deficiencies of GPVI. Next generation sequencing failed to reveal potential
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disease-causing mutations. Her serum aggregated control platelets; a response prevented by
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prior cleavage (and loss) of GPVI or by blocking the platelet FcRIIA receptor. An important feature as seen with other cases is that bleeding continued despite a platelet count well above
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the 30x109 /L threshold normally considered to merit special precautions in ITP. Quite remarkably, this section deals with 10 case reports describing patients with
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acquired GPVI deficiency due mainly to the presence of anti-GPVI antibodies - and all are women (Table 2). It will be interesting to see if new case reports continue this trend. This section also proves that anti-GPVI antibodies can give rise to ITP and I agree with Rabbolini et al [119] that they may well be an under-recognized cause. Also, the continued occurrence of bleeding in some patients after platelet count (and often GPVI expression) improves on treatment suggests that some antibodies may be function blocking although this has rarely been
ACCEPTED MANUSCRIPT proven. While antibodies clearly provoke sGPVI shedding for some internalization and intracellular processing of antibody-GPVI complexes is a possible alternative pathway.
8. GPVI and megakaryocytopoiesis In a companion paper in Blood Reviews [120] I have assessed the roles of autoantibodies to
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αIIbβ3 in acquired GT. Some marked differences can be observed between antibodies to GPVI
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and those directed against αIIbβ3. First, while autoantibodies to GPVI may induce receptor shedding those to αIIbβ3 do not. Secondly αIIbβ3 appears early in MK differentiation (being present in stem cells) and autoantibodies to the integrin can directly influence proplatelet
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production resulting in altered platelet biogenesis [discussed in 120]. In contrast, the expression of GPVI increases late in MK maturation and while the interaction of both GPVI and α2β1 with
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collagen combine to negatively regulate proplatelet production, this is part of a natural process to prevent premature platelet release in the bone marrow [121,122]. So far, inherited diseases of
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GPVI do not lead to platelet size increases contrasting also with acquired antibodies to GPIbIX-V and the inherited loss of GPIb-IX-V in BSS [65,123]. Still to be resolved, however, is the
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reason why platelet GPVI levels in mice return to normal much later than the platelet count
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after antibody-induced GPVI loss [71]. Does this have a parallel in human acquired GPVI loss?
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9. Bleeding syndromes associated with acquired defects in GPVI-dependent intracellular signaling pathways
Rare reports also describe patients for whom the molecular basis for loss of GPVI function is intra-cellualr. Bellucci et al [124] described defective collagen- induced platelet activation associated with bleeding in two patients with malignant hemopathies (cases 11 and 12, Table 2). A man with myelodysplasia, anemia and low white blood cell numbers suddenly developed a bleeding syndrome. His platelet count was normal but his bleeding time was very long. He
ACCEPTED MANUSCRIPT gradually developed severe aplasia and severe thrombocytopenia with an eventual transformation to myelomonoblastic leukemia from which he died. An elderly woman with eventually fatal chronic lymphocytic leukemia started bleeding after treatment with heparin for a pulmonary embolism. Her PFA-100 closure time was prolonged for collagen/epinephrine. Both patients had a severely impaired platelet response to collagen, CRP and Cvx whereas that
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to other agonists including ADP, TRAP, AA and ristocetin was normal or only partially
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decreased. GPVI was fully expressed in the platelets of both patients and bound Cvx yet neither Cvx nor specific activating MoAbs to GPVI induced secretion or Ca2+-mobilization. Perfusiondependent platelet adhesion and thrombus formation on collagen were both highly impaired.
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Inhibitory antibodies were not present in the plasma of the patients and a signaling defect proposed but not resolved. Best et al [51] also showed that as well as a low α2β1 content some
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patients with myeloproliferative syndromes also had decreased GPVI function. The case histories in this review conclude with a report that illustrates the difficulties of
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working up a suspected defect of GPVI [125]. A woman with a lifelong experience of mild bleeding gave birth to 2 children without excessive bleeding. There was no family history of
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bleeding and platelet counts were normal. When first tested, her platelets failed to aggregate
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with collagen; but the response later improved and while remaining delayed and low to Cvx was normal to all other agonists. Collagen- induced release from alpha-granules and dense-
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granules was variable. Anti-platelet antibodies were not detected in her serum or on her platelets and plasma levels of sGPVI matched those of controls. The PFA-100 closure time was not prolonged for either cartridge. Sequencing of the GP6 gene showed no disease-causing mutations. A rare heterozygous c.1038C>T variant in the 3’untranslated region had no effect on GPVI mRNA expression and GPVI was normally expressed in her platelets. The fact that collagen- induced platelet responses improved over time suggests an acquired defect (perhaps drug-induced) but the molecular cause remains an enigma.
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10. Platelets and GPVI in inflammation and other non-hemostatic events By adhering to foreign surfaces and by secreting metabolites and many biologically active proteins platelets promote not only tissue repair and wound healing but are vital for inflammation and innate immunity (reviewed in [3]). Clearly, as a collagen and fibrin receptor,
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GPVI is an important player in these processes. In addition, the structurally related ITAM
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receptor CLEC-2 acting through podoplanin expressed on vascular cells also exerts a major and multifaceted role in inflammation and is key for the development of the vascular and lymphatic systems [6,126,127]. While altered development of the lymphatic system is seen with CLEC-2
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deficiency [128]; ductus arteriosus is not a feature of GPVI deficiency in mice or man. During inflammation, platelets not only facilitate leukocyte infiltration at endothelial cell junctions
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through expression of P-selectin they also assure hemostasis by sealing breaks in the endothelium (reviewed in [129]). While deficiencies of GPVI or CLEC-2 in mice do not
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compromise wound healing; their double deficiency actually enhanced it probably by allowing more bleeding into wound sites by severely interfering with hemostasis in mice [130,131].
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How deficiencies of GPVI in man influence inflammation is an important area for future
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research.
Pioneering studies by Goerge et al [132], showed how thrombocytopenia promoted
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intra-alveolar bleeding and respiratory distress during endotoxin-induced lung inflammation. They also showed bleeding from venules during the cutaneous Arthus reaction where loss of vascular integrity is linked to immune complex deposition. Studies in mice showed that single platelets seal neutrophil- induced vascular breaches via GPVI during immune-complexmediated dermatitis [133]. The absence of GPVI (and a fall in platelet count) favored neutrophil- induced inflammatory bleeding. Another mouse model showed that platelet recruitment to the inflamed glomerulus also occurs in part by a GPVI-mediated mechanism
ACCEPTED MANUSCRIPT with P-selectin crucial [134]. Gros et al [133] speculated such interactions might involve EMMPRIN, a counter-receptor for GPVI expressed by leukocytes although other proteins potentially exposed in the vessel wall and able to bind GPVI include laminin, Fn and Vn (Fig. 1); fibrin is another possibility as are histones if DNA nets are present. A pathological role for GPVI and the platelet-collagen interaction in the release of biologically active microparticles
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(MPs) from platelets was shown in a mouse model of rheumatoid arthritis suggesting GPVI as
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a therapeutic target in this condition [135]. However, later studies by the same group showed that rather than MPs, lipid derivatives are the active platelet-released components [136,137]. Platelets are necessary for host defense during bacterial infection and limit bacterial
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growth and dissemination [138,139]. They also help prevent hemorrhage during sepsis and limit cytokine responses. A role for GPVI is illustrated by increased bacterial growth in the
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lungs and in distant body sites when GPVI-/- mice (or mice depleted of GPVI by antibodyinduced shedding) were infected with klebsiella pneumonia bacteria to induce pneumonia-
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induced sepsis [140]. Fewer platelets and platelet-leukocyte aggregates were observed in the bronchoalveolar space. GPVI on platelets reacting with its ligands collagen, fibrin and histones
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at sites of inflammation leads to up-regulated leukocyte function; in parallel changes in
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cytokine levels suggest that GPVI has an anti-inflammatory effect. The net effect will depend on the site and the nature of the inflammation. For example, in mouse models of cutaneous
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inflammation, GPVI has a pro-inflammatory effect. its absence leading to decreased platelet interplay with macrophages and increased anti-inflammatory markers and therefore a lower vasculo-protective effect [141]. On a wider scale, elevated sGPVI levels in many acute and inflammatory conditions including patients with severe burns, inflammatory bowel and kidney disease, elective cardiac surgery, trauma, acute brain injury or prolonged ventilation suggest ongoing GPVI loss and this was particularly so in sepsis [81]. For further information in this field I invite the reader to consult an excellent recent review by Boulaftali et al [142].
ACCEPTED MANUSCRIPT Clearly the role of GPVI is complex with GPVI loss favoring bleeding at some inflamed sites particularly when thrombocytopenia is also a factor. But much research is needed in this field and it is already apparent that the role of GPVI (and platelets) may vary considerably with respect to the site and cause of different clinical inflammatory states. The influence of administered therapeutics on bleeding (for example, anti-inflammatatory drugs) and to combat
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thrombocytopenia (for example, in sepsis) must also be taken into account.
11. Thrombosis and the potential therapeutic use of anti-GPVI agents Platelet receptor polymorphisms emerged as risk factors for arterial thrombotic disease and this
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has included those influencing α2β1 and GPVI expression (see above Section 2). More importantly, GPVI has become a novel pharmacological target for drugs effective in thrombotic
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therapy but with preserved hemostatic risk (reviewed in [143]). a) Collagen receptor polymorphisms as thrombotic risk factors. Early studies suggested
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that variations in collagen receptor density helped determine risk in ischemic or cardiac disease and in thrombo-inflammatory conditions such as stroke (reviewed in [144]). For example, the
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dimorphic α2β1 C807/T873 allele associated with high α2β1 density was linked to myocardial
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infarction or stroke in younger patients, findings that were not confirmed in Japan suggesting race variations [145-147]. For GPVI, early studies suggested that high expression with
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polymorphic variation at the GP6 locus gave increased risk of coronary artery disease and myocardial infarction but the study groups were small [49,148-151]. Interestingly, a recent study associated the GPVI “a” allele with a two-fold higher level of platelet MPs release and with increased sepsis severity in a pediatric population [152]. Hopefully, the use of modern genomics will put such studies on a different level and enable a more personalized risk assessment than at present [153]. So far, diagnostic or treatment changing data has not been obtained.
ACCEPTED MANUSCRIPT b) Sticky platelet syndrome. The so-called “sticky platelet syndrome” refers to a loosely defined group of patients with increased thrombotic tendency usually defined as an increased sensitivity of platelets to aggregate with low doses of ADP and/or epinephrine (reviewed in [154]). The syndrome is claimed to have an autosomal dominant trait linked to variants in a wide range of genes. The Kubisz group [154] have linked polymorphisms in the GP6 gene to
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its pathogenicity describing over-expression of GP6 haplotypes; however, much more stringent
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evidence is required to prove the existence of sticky platelet syndrome as a distinct entity and to establish a molecular basis for the role of GPVI. One of the associated symptoms is fetal loss (and fetal growth retardation) and perhaps the most convincing finding is the association found
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by Skerenova et al. [155] between fetal loss and the presence of the low frequency GP6 “b” allele (PEAN). A reduced GPVI signaling was a suggested cause.
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c) Direct targeting of platelet GPVI in arterial thrombosis and stroke. The results obtained for genetically modified mice or mice with antibody-induced GPVI depletion showed
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that loss of GPVI occurred without major disruption of hemostasis (Sections 4 and 5). Most patients with inherited or acquired GPVI deficiency have mild bleeding while no thrombo-
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embolic events have been reported (Table 1). It is therefore not surprising that research into
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GPVI as a potential target for safe antithrombotic therapy including stroke has attracted much attention. Collagen fibrils have multiple partners during platelet adhesion and thrombus
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formation following atherosclerotic plaque rupture [156-158], and the continued functioning of other platelet membrane receptors including GPIbα, α2β1 and αIIbβ3 may limit bleeding. Diverging concepts for abrogating GPVI-dependent platelet signaling have emerged, three of which are now briefly mentioned. The first is the intravenous infusion of a recombinant soluble dimeric GPVI-Fc fusion protein (Revacept) blocking GPVI-reactive sites in collagen exposed in atherosclerotic plaques. Animal studies showed that Revacept both reduces thrombus formation and improves vascular dysfunction [159,160]. It is about to enter a phase 2 trial in
ACCEPTED MANUSCRIPT patients undergoing elective percutaneous coronary intervention [161]. A second approach is to therapeutically induce GPVI shedding [74]. This, however, remains largely unproven despite increases in our knowledge of how intracellular mechanisms can regulate sGPVI release, as for example through the production of reactive oxygen species [162]. Large problems remain however, for example, with regard to controlling the specificity, extent and duration of GPVI
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shedding. The third and currently the most promising approach is by direct pharmacological
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inhibition of GPVI using an antibody or with peptides derived from snake venom proteins [163-167]. Fab fragments of a MoAb to GPVI, ACT017, have been recently shown to have excellent safety and tolerability profiles in a phase 1 study in man while blocking collagen-
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induced platelet aggregation in a dose-dependent manner [168]. As well as arterial thrombosis and stroke, GPVI inhibitors have potential to prevent cardiac ischemia perfusion injury
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[158,169]. The results of phase 2 and possibly phase 3 clinical trials with these new drugs will be anxiously awaited. Whether patients with deficiencies of GPVI are protected against
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cardiovascular disease is an interesting question to answer in coming years as is the question of whether collagen receptor polymorphisms (see Section 2) will prove useful in predicting those
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patients most susceptible to benefit from these drugs.
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d) Soluble fibrin as a regulator of GPVI. Interestingly, difference in the way that GPVI interacts with collagen and fibrin or Fg provides the opportunity to developing inhibitors
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specific for each ligand [39-44]. Interestingly, soluble fibrin formed by restricted thrombin generation acts as a GPVI antagonist sterically blocking access to its ligands and the functional response [170]. Thrombin generation in response to trauma, cancer and disseminated intravascular coagulation (DIC) and plasmin generation may lead to circulating fibrin monomers or soluble protofibrils [171]. Soluble fibrin monomers can be maintained for several hours and render platelets non-responsive to collagen, a finding that would extend to donor platelets in the event of transfusion. According to Lee et al [170] 1% conversion of circulating
ACCEPTED MANUSCRIPT Fg would lead to 90 nM soluble fibrin – enough to overwhelm platelet GPVI and lead to loss of function of endogenous or transfused platelets. However, Onselaer et al [41] reached more conservative conclusions. Whether the soluble fibrin simply blocks GPVI or additionally desensitizes its signaling mechanism requires further research. The interaction between fibrin and/or Fg with GPVI explains early observations where mice deficient in GPVI failed to form
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an occlusive thrombus in a ferric chloride injury model, even though the onset of thrombosis
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was unaltered [172].
e) Deep vein thrombosis. An exciting new development is the concept that GPVI may mediate, at least in part, platelet involvement in deep vein thrombosis. Using a microfluidic
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model mimicking the low stasis conditions seen in human venous valves, Loyau et al [173] showed that tissue factor-dependent fibrin formation was followed by platelet accumulation
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that was blocked by a Fab fragment to GPVI or by D-dimer. Platelet accumulation occurred in a red cell-dependent manner and platelet activation with PS expression favored thrombin
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generation and thrombus growth. This opens the possibility of using anti-GPVI blockers in combination with tissue-type plasminogen activator in venous thrombosis. Whether patients
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lacking GPVI are protected against deep vein thrombosis is unknown.
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e) GPVI and cancer. Platelets and platelet-derived MPs exert important roles in cancer dissemination and metastasis and GPVI is one of the receptors involved [174,175]. This opens
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a particularly exciting field for anti-GPVI drugs, particularly as blocking GPVI in mouse models increases bleeding within newly formed tumors increasing the efficacy of chemotherapy [176].
12. Conclusions and future directions The data in this review mostly confirm the often-cited tendency of a low hemorrhagic tendency for patients with inherited deficiencies of GPVI and only moderately prolonged bleeding times.
ACCEPTED MANUSCRIPT However, the number of patients with disease-causing GP6 variants remains very low and there is already evidence for two subjects of excessive trauma-related bleeding (Table 1). Dominant are the families living in rural areas of Chile with a common 2bp insertion followed by a stop codon suggestive of a founder effect [64]. A much larger cohort of patients of wider geographical distribution is required before definite conclusions can be made on a modest
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bleeding severity. Notwithstanding, the studies on GP6 knockout mouse models also point in
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this direction. Acquired loss of GPVI function due to metalloproteinase- induced shedding is more common and again in most case reports results in little bleeding. However, acquired GPVI loss is often associated with thrombocytopenia that on occasion is severe; anti-GPVI
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antibodies clearly are a cause of ITP [119]. Thus platelet count is another possible factor influencing phenotype, although guidelines for ITP suggest that platelet counts >50 x 109 /L are
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unlikely alone to cause major bleeding [177]. However blocking GPVI function with antibodies acting as inhibitors would amplify loss of the GPVI/collagen interaction. Acquired GPVI
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deficiency may also be underestimated as testing for antibody against GPVI in ITP is not generalized while in some subjects their presence may be asymptomatic as indeed was the case
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for a Chilean subject with inherited GPVI deficiency (Table 1). In rare patients antibody to
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GPVI was only shown after elution from the patients’ platelets. While these may be of low titer but high affinity this has yet to be proven. GPVI shedding itself could result in rapid antibody
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clearance from plasma by immunoadsorption. Another explanation for negative testing is endocytosis of occupied GPVI with bound antibodies transported to intra-cellular sites. Striking is the high proportion of women featured in all case reports for GPVI deficiency and in acquired causes only women were identified. While this could suggest the presence of unrecognized sex-linked factors in controlling bleeding or antibody production, why should this be a unique feature of GPVI?
ACCEPTED MANUSCRIPT Perhaps the most important finding seen for GPVI in recent years is that it in part mediates platelet interaction with fibrin and receptor-bound Fg. Thus not only does GPVI help bring about platelet binding to the vessel wall, it also intervenes in thrombus build up. Historically, αIIbβ3 was thought to be the platelet receptor for fibrin; however, GT platelets that lack this integrin bind to fibrin polymers even though they are not activated as a result
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(reviewed in [120]). Similarly, while GT platelets attach to collagen or exposed
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subendothelium, they do not spread [178] and this is despite the normal presence of α2β1 and GPVI. Thus intracellular signaling in response to collagen requires receptor inter-play and is quite complex and it is interesting to note that GT platelets lacking αIIbβ3 fail to retract a clot
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whereas platelets deficient in GPVI retain clot retraction. Further research is needed here. The emergence of agents blocking GPVI or GPVI signaling for anti-thrombotic therapy
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is highly encouraging. Nonetheless, the complexity of the platelet/collagen interactions and of their signaling pathways will necessarily require the most careful evaluation of the situations
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where drugs to GPVI are used. Not to be forgotten are the possible consequences of their longterm use on platelet production in the marrow for GPVI has been shown to regulate platelet
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biogenesis by mediating the MK interaction with collagen [121,122]. High trauma situations
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including childbirth still need to be evaluated for these drugs, as does their use in combination with specialized medications in cardiovascular disease, inflammation and major illnesses such
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as sepsis and rheumatoid arthritis. Studies on their potential for wider use in cancer are highly promising but in their infancy and offer much scope for future use as an adjunct to chemotherapy. Finally, a major requirement is a rapid and more adept clinical test to assess the clinical risk of bleeding after GPVI loss or therapeutic blockade. Results obtained with the PFA-100 were highly variable for patients with inherited and acquired GPVI loss although the collagen/epinephrine cartridge was generally more sensitive, perhaps better showing the
ACCEPTED MANUSCRIPT importance of collagen-induced ADP release from platelets. Use of computer-derived analysis of platelet adhesion and thrombus formation under shear or under static conditions on collagen and other surfaces would be a great improvement. Potential procedures have been reviewed by
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Brouns et al [179]
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Practice points
Patients with inherited or acquired collagen or receptor function mostly have conserved hemostasis with little bleeding although trauma and some inflammatory
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states may represent an exception. The use of anti-GPVI drugs in antithrombotic therapy is promising but their long-term use has yet to be tested. The detection and characterization of anti-GPVI antibody is vital in acquired disorders
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-
and testing should take into account low-titre antibodies that may be in large part
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adsorbed onto the platelets.
Autoantibodies to GPVI may induce sGPVI release through metalloproteinase-
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Research agenda
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induced shedding and testing for the soluble form is recommended.
Improvements in identifying autoantibodies to GPVI with rapid and easy to perform tests to identify both epitope targets and inhibitor activity. Large studies should investigate any possible sex linkage.
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Studies to increase our knowledge of the active sites on GPVI whose loss leads to defective function, to better understand the role of dimerization and to design a test that better measures the effect of GPVI loss on bleeding risk.
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The application of high-throughput screening to identify platelet, blood cell, plasma and vascular gene variants that will influence bleeding and will help choose the use of anti-GPVI drugs.
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Inaugurating active international databases to clearly establish the frequency of GPVI deficiencies (inherited or acquired) and to evaluate the effectiveness of different
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treatments blocking collagen receptor function in a cardiovascular disease or other
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context.
Conflicts of Interest
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Acknowledgements and Addendum
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The author declares no conflict of interest.
The author thanks Paquita Nurden for her help and for her support during the writing of this
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review. After this manuscript was submitted, Martine Jandrot-Perrus and her colleagues
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published a short review on this subject that gives an alternative view of some of the data that I covered (Jandrot-Perrus M, Hermans C, Mezzano D. Platelet glycoprotein VI genetic and
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qualitative defects. Platelets 2019;30:708-713).
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155. Skerenova M, Sokol J, Biringer K, Ivankova J, Stasko J, Kubisz P, Lasabova Z. GP6 haplotype of missense variants is associated with sticky platelet syndrome manifested by fetal loss. Clin Appl Thromb Hemost 2018;24:63-69. 156. Massberg S, Gawaz M, Grüner S, Schulte V, Konrad I, Zohlinhöfer D, Heinzmann U, Nieswandt B. A crucial role of glycoprotein VI for platelet recruitment to the injured artery wall in vivo. J Exp Med 2003;197:41-49.
ACCEPTED MANUSCRIPT 157. Konishi H, Kato Y, Takaya N, Kashiwakura Y, Itoh S, Ra C, Daida H. Platelets activated by collagen through immunoreceptor tyrosine-based activation motif play pivotal role in initiation and generation of neointimal hyperplasia after vascular injury. Circulation 2002;105:912-916. 158. Takaya H, Katch Y, Iwabuchi K, Hayashi I, Konishi H, Itoh S, Okumura K, Ra C,
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160. Ungerer M, Li Z, Baumgartner C, Goebel S, Vogelmann J, Holthoff HP, Gawaz M, Münch G. The GPVI fusion protein Revacept reduces thrombus formation and
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Platelets 2018;29:662-669.
ACCEPTED MANUSCRIPT Figure Legends
Figure 1. Cartoon showing the purported structure of the GPVI-FcR complex with essential information on its ligands, signaling pathways and its modification in inherited and acquired
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disease. Note the presence of negative (-ve) and positive (+ve) signaling pathways.
Figure 2. Cartoon showing the organization of the GP6 gene as it would encode the common GPVI type I splice form. Featured are the published disease-causing mutations, those giving rise to compound heterozgous expression are in the same color. *A homozygous mutation
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common to 6 unrelated families in Chile. Common high density “a” (SKTQH) and low
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variants are fully referenced in the text.
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density “b” (PEALN) alleles are also shown. The gene structure and the mutations or gene
ACCEPTED MANUSCRIPT Table 1: Clinical and biological data for cases with inherited defects of GP6
1
2
F
F
Japan
Normal
Moderate thrombocyto penia
Japan
(110140x109 /L)
F
France
Normal
Absent
Specifi c severe reducti on to collage n
10%
Specifi c absenc e with collage n and CRP
<20%; residual GPVI, smeared on WB
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3
Specifi c absenc e to collage n
Belgiu m
511
Normal
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Sever al cases (M and F)
Chile
Severe reducti on for collage n, Cvx and CRP
Much reduced
Specifi c absenc e for collage n, CRP and Cvx.
No surface GPVI. Residual truncated protein in cytoplas m
PT
F
CE
4
Normal for all patients
GP6 variant
Bleeding symptom s
Family
Refere nce
ND
Mild bleeding (purpura, menorrha gia and epistaxis)
Parents nonsymptomat ic with 50% expression of GPVI
Moroi et al, 1989
Lifelong mild bleeding. Bleeding after surgery required transfusio n
No family history of bleeding
Arai et al, 1995
Comp Het Arg38Cys + c.356_560 dup + fs + stop
Mild lifelong bleeding (ecchymo ses)
Parents heterozygo us for one mutation
Dumon t et al, 2009
Comp Het Ser176Asn + 16bp out-offrame del + stop
Moderate life-long bleeding (ecchymo ses, epistaxis, menorrha gia) + posttrauma bleeding
Parents heterozygo tes and asymptom atic.
Herman s et al, 2009
Homozygo us c.711_712i nsA + fs + stop
Mild bleeding (easy bruising, epistaxis, gum bleeding), one sister asymptom atic
All cases in unrelated families from different parts of Chile. Founder effect suspected.
Matus et al., 2013; Onselae r et al, 2017; Mangin et al, 2018
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1
Platelet GPVI Platelet count aggregati expressi on on2
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Count ry of origin
ND
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Gend er
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Ca se
Country of residence/origin; 2GPVI expression determined by flow cytometry or western blot (WB); Cvx, convulxin; CRP, collagen-related peptide; ND, not determined; M, male; F, female; ins, insert; fs, frameshift; Comp het, compound heterozygote 1
ACCEPTED MANUSCRIPT Table 2: Clinical and biological data for cases with acquired defects in GPVI and defective platelet/collagen interactions
F
Japan
USA
Bleeding symptoms
Severe (15x109 /L) improved with corticotherapy (140x109 / L)
Specif ic absen ce to collag en
Bleeding late in life (skin bruising, gum bleeding, epistaxis)
Severe (20x109 /L) normalizing after corticotherapy
Specif ic reduct ion to collag en
<10%
Moderate (105135x109 /L)
Specif ic absen ce with collag en and CRP
Reduc ed to 1015% of normal
Striki ng reduct ion to collag en and Cvx
Much reduce d
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France
CE
F
Macrothrombo cytopenia (1835x109 /L)
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4
Not evaluat ed
5
F
Japan
Moderate (48x109 /L) later normalizing
6
F
Japan
Moderate (80133x109 /L) then
Antibo dy to GPVI
Yes. Platele t activat ing
Refer Clinical condition ence
ITP-like syndrome + Graves disease
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Platelet GPVI aggrega expres tion sion3
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3
F
Japan
Thrombocytop enia (platelet count2 )
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2
F
Origin1
Sugiy ama et al, 1987; Ichino he et al, 1995
Yes. Platele t activat ing
ITP + SLE + thyroiditis
Takah ashi & Moroi , 2001; Ichino he et al, 1995
Moderate bleeding (petechia, easy bruising)
Yes
ITP-like syndrome
Boyla n et al, 2004
Lifelong moderate bleeding
Not detect ed
Gray platelet syndrome and suspected myelofibrosis
Nurde n et al, 2004
Initiall y no but later detect ed in platele t eluates
ITP-like syndrome suspected
Kojim a et al, 2006; Akiya ma et al, 2009
Detect ed in platele t
ITP-like syndrome with progressive loss of
Akiya ma et al,
Mild subcutaneo uss bleeding
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1
Gen der
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Ca se
Specif ic absen ce with collag en, CRP and Cvx
Absent
Mild bleeding (petechia, gum bleeding)
Specif ic absen ce
Absent then progre ssive
Mild bleeding (easy bruising,
ACCEPTED MANUSCRIPT
F
Australia
ITP, romiplostim treatment was curative
Gardi ner et al, 2008; 2010
No severe bleeding
Yes. In plasm a and on platele ts, activat ing when strong
SLE and ITPlike conditions responsive to treatment
Nurde n et al, 2009
Lifelong bleeding
IgM cold aggluti nin
Pseudothrombo cytopenia (5x109 /L in citrated blood, 100x109 /L in EDTA)
Sanch ezGuiu et al, 2015
Severe deficie ncy.
Exagerated bruising
Plasm a antibo dy that aggreg ates contro l platele ts
ITP-like syndrome. Platelet count improved under treatment
Rabbo lini et al, 2017
None. GPVIsignali ng defcet
Myelodysplasi a and fatal myelomonobla stic leukemia
Belluc ci et al, 2004
None.
Chronic
Belluc
1020% of normal
Major deficie ncy, then recove ry
Low
Specif ic absen ce with collag en, Cvx and CRP
Norma l
Purpura in lower limbs
Specif
Norma
Extensive
11
M
France
Severe (10x109 /L) progressing to severe thrombocytope nia
12
F
France
Normal then
2009
Yes. Activa ting, presen t in plasm a and in platele t eluates
Specif ic absen ce with collag en
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F
bleeding symptoms
Epistaxis and lower limb purpura; symptoms continued despite corticother apy
Severe ITP when first studied. Moderate (91x109 /L) at time of study
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10
eluates but not in plasm a
T
Not able to be assess ed
epistaxis)
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Spain
Chronic moderate thrombocytope nia
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9
F
France/M orocco
Severe (4x109 /L) normalizing with immunosuppre ssive and corticoid therapy
Specif ic absen ce with collag en and Cvx
Australia
recove ry
MA
8
F
Severe (2x109 /L) normalizing with corticotherapy
Specif ic absen ce with collag en and Cvx
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7
with collag en, Cvx
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normalizing
ACCEPTED MANUSCRIPT
F
Germany
Normal
bleeding (also heparindependent thrombocyt openia)
Norma l GPVI expres sion
Mild lifelong skin hematomas + menorrhagi a
GPVI signali ng defect
lymphocytic leukemia
ci et al, 2004
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Specif ic reduct ion with collag en and Cvx, impro ving with time
l
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13
ic absen ce with collag en, Cvx and CRP
Norma l GPVI expres sion
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progressive thrombocytope nia
Unknown cause. No autoantibody to GPVI
Klüm pers et al, 2018
Country of residence/origin; 2Platelet count at its nadir; 3GPVI expression determined by flow cytometry or western blot (WB); M, male; F, female; Cvx, convulxin; CRP, collagen-related peptide
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CE
PT
ED
MA
1
Figure 1
Figure 2