Platelet Inhibitory Receptors

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Platelet Inhibitory Receptors Zoltan Nagy and Yotis A. Senis Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom

INTRODUCTION 279 BACKGROUND 279 PROSTACYCLIN RECEPTOR: BROAD-SPECTRUM INHIBITOR OF PLATELET ACTIVATION 280 SOLUBLE GUANYLATE CYCLASE: BROAD-SPECTRUM INHIBITOR OF PLATELET ACTIVATION 281 ITIM-CONTAINING RECEPTORS 282 PECAM-1: Selective Inhibitor of Platelet Activation 282 PECAM-1 Function 284 PECAM-1 Signaling 284 G6b-B: Critical Regulator of Platelet Homeostasis 285 G6b-B Function 285 G6b-B Signaling 286 OTHER PLATELET ITIM-CONTAINING RECEPTORS 287 LAIR-1 288 TLT-1 288 CEACAM1 and CEACAM2 288 PIR-B 288 CONCLUSION 289 REFERENCES 289

INTRODUCTION Platelet activation is tightly regulated by inhibitory mechanisms that limit platelet accumulation at sites of vascular injury. This chapter covers the most well-established platelet inhibitory receptors and their associated signaling pathways, including: the Gs-coupled prostacyclin (prostaglandin [PG] I2) receptor, which signals via cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA); the nitric oxide (NO) receptor soluble guanylate cyclase (sGC), which signals via cyclic GMP (cGMP) and protein kinase G (PKG); and the immunoreceptor tyrosine-based inhibition motif (ITIM)containing receptors platelet endothelial cell adhesion molecule 1 (PECAM-1) and G6b-B, which signal via the Src Homology 2 (SH2) domain-containing tyrosine phosphatases Shp1 and Shp2. A central concept underpinning how all of these receptors function is the phosphorylation and dephosphorylation of key targets modulating platelet activation. The emphasis in this chapter is on the latter two ITIM-containing receptors, classically recognized as inhibitors of immunoreceptor tyrosine-based activation motif (ITAM)containing receptors, but also implicated in regulating integrin and G protein-coupled receptor (GPCR) signaling. Congenital macrothrombocytopenia and myelofibrosis observed in G6b-B-deficient mice and humans is also discussed, illustrating the central role of G6b-B in regulating platelet homeostasis.

Platelets. https://doi.org/10.1016/B978-0-12-813456-6.00015-1 Copyright © 2019 Elsevier Inc. All rights reserved.

BACKGROUND Platelets have evolved to respond rapidly to vascular injury and prevent excessive blood loss, while at the same time, limiting thrombus formation. The degree to which platelets become activated has important implications also for the outcome of other pathophysiological processes in which they are involved, including blood-lymphatic vessel separation, infection and inflammation, maintenance of vascular integrity, and wound repair. For the purposes of this chapter, we will focus exclusively on inhibitory receptors regulating platelet activation in the context of thrombosis. Whether or not the same receptors regulate the platelet response in other physiological and disease conditions, or megakaryocyte (MK) function at sites of hematopoiesis remains to be determined. Platelet activation at sites of vascular injury is triggered by an array of agonists and their associated receptors, the latter of which can be broadly divided into tyrosine kinase-linked receptors (TKLR) and GPCRs. Engagement of extracellular matrix and plasma proteins, including VWF, collagen, fibrinogen, and laminins with their respective TKLRs, results in ligand-mediated receptor clustering and activation of tyrosine kinases that phosphorylate downstream signaling and cytoskeletal proteins, thus initiating and propagating the activation signal.1 These primary activation signals are enhanced by a multitude of positive feedback mechanisms to ensure a rapid and robust platelet response under high shear conditions. Secondary mediators of platelet activation include thromboxane A2 (TxA2) production, adenosine diphosphate (ADP) release and thrombin generation, all of which signal through GPCRs, as discussed in other chapters. These mediators also exert paracrine effects, activating other platelets, which accumulate in a growing thrombus. Yet in healthy vessels, thrombus formation is confined to the site of injury and does not result in vessel occlusion, which can have life-threatening consequences. The optimal platelet response is regulated by a combination of extrinsic factors derived from the endothelial lining and vessel wall, and intrinsic mechanisms inherent to platelets that modulate the strength and duration of activation signals. Extrinsic factors include prostaglandin I2 (PGI2) and nitric oxide (NO), released by endothelial cells, acting directly on platelets, the ADP scavenger CD39, present on the surface of endothelial cells, and the immunoreceptor tyrosine-based inhibition motif (ITIM)containing receptor platelet endothelial cell adhesion molecule 1 (PECAM-1). The latter acts as both an extrinsic and intrinsic factor, as it is present on the surface of both endothelial cells and platelets, and interacts trans-homophilically. Intrinsic factors include the platelet receptors for PGI2 and NO, namely the Gs-coupled PGI2 receptor and sGC, respectively, other ITIM-containing receptors, including G6b-B, carcinoembryonic antigen-related cell adhesion molecules 1 and 2 (CEACAM-1 and -2), and their associated signaling pathways (Fig. 15.1). Other factors such as flow rates, venous versus arterial, vessel composition, and thrombus permeability, core versus shell, also contribute to the strength and duration of activation signals (Chapter 20). Platelet inhibitory signals are transient and can be overcome by activation signals to ensure rapid response,

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Fig. 15.1 Platelet inhibitory receptors. Inhibitory receptors can be divided into G protein-coupled receptors, the intracellular soluble guanylate cyclase (sGC) and immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptors. The most well-established platelet inhibitory receptors include the prostacyclin (PGI2) receptor, sGC, PECAM-1 and G6b-B. Endothelial PGI2 binds to PGI2 receptor which activates Gs leading to adenylate cyclase (AC) activation, increased cyclic adenosine monophosphate (cAMP) synthesis, activation of protein kinase A (PKA) and platelet inhibition, whereas nitric oxide (NO) diffuses through the platelet membrane, activates sGC, leading to increased synthesis of cyclic guanosine monophosphate (cGMP), activation of protein kinase G (PKG) and platelet inhibition. PKA and PKG phosphorylate key inhibitory serine and threonine (Ser/Thr) residues in substrates. PECAM-1 signaling is evoked by trans-homophilic interactions with PECAM-1 molecules on other cells leading to phosphorylation of the ITIM and immunoreceptor tyrosine-based switch motif (ITSM) by Src family kinases (SFKs) in the cytoplasmic tail of the protein. G6b-B signaling is triggered by heparan sulfate binding to the ectodomain of the receptor resulting in phosphorylation of its ITIM and ITSM by SFKs. Phosphorylated ITIM and ITSM in PECAM-1 and G6b-B leads to recruitment and activation of the tyrosine phosphatases Shp1 and Shp2, which dephosphorylate key phospho-tyrosine residues (p-Tyr) in target proteins in activation pathways leading to platelet inhibition. (Professional illustration by Patrick Lane, ScEYEnce Studios.)

otherwise platelets would not be able to adhere to sites of injury and aggregate. Irreversible platelet activation is achieved in the core of a thrombus, where platelets are exposed to multiple, powerful agonists, over a sustained period, collectively driving platelets to a point of no return. Conversely, partially activated platelets found in the shell of a thrombus, or platelets stimulated with less potent agonists, can revert to a resting state. This is favorable, as activated platelets in the circulation can have catastrophic consequences, leading to thrombin generation and disseminated intravascular coagulation. Recent findings by Mori and co-workers demonstrated that sustained platelet activation signals, induced by deletion of key regulators of Src family kinases (SFKs), namely the inhibitory non-transmembrane tyrosine kinase C-terminal Src kinase (Csk) and the receptor-like tyrosine phosphatase CD148, results in a paradoxical reduction in platelet reactivity to vascular injury, due to the triggering of powerful negative feedback mechanisms.2 With the advent of intravital microscopy, negative feedback pathways can be visualized in action following superficial vessel injury, revealing that thrombi recede with time as the inhibitory mechanisms outcompete activation signals. What remains is a compact thrombus at the vessel wall, with limited interference on blood flow. Another key concept to bear in mind is that inhibitory mechanisms vary in different

parts of the vascular system and also within a thrombus, and that whether a thrombus grows or recedes is dependent on the net effects of activatory and inhibitory receptors. Below, we discuss in further detail two of the most wellestablished inhibitory platelet receptors and their associated mechanisms of action, namely the PGI2 receptor and sGC, and the ITIM-containing receptors and their modes of action, focusing primarily on the prototype PECAM-1 and relative newcomer, G6b-B, shown to be a critical regulator of platelet homeostasis.

PROSTACYCLIN RECEPTOR: BROAD-SPECTRUM INHIBITOR OF PLATELET ACTIVATION Prostacyclin (prostaglandin I2, PGI2) is a lipid mediator of the prostanoid family released by the healthy endothelium, which is a potent vasodilator and inhibitor of platelet activation.3 The biology of PGI2 is covered extensively in Chapter 17. Here, we discuss briefly the contribution of the PGI2 receptor (IP receptor) to platelet inhibition and highlight novel developments in this field. The relevance of PGI2 signaling to platelet inhibition in vivo is supported by human studies, including patients with PGI2 receptor mutations and with pharmacologically suppressed PGI2 production, as well as by knockout (KO) mouse studies

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utilizing various thrombosis models. Human patients with atherosclerosis who are also deficient in PGI2 signaling due to a mutation in the receptor have an increased risk of thrombosis, but patients from a low-risk cohort do not.4 These data suggest that mutations in the receptor contributes to the acceleration of cardiovascular disease, but play minimal roles in its initiation. In addition, drugs inhibiting cyclooxygenase-2 (COX-2), a central enzyme for PGI2 production, suppress PGI2 levels in patients and result in an increased risk of myocardial infarction and stroke.5 These findings are reinforced by studies using PGI2 receptor KO mice, which exhibit enhanced FeCl3-induced arterial thrombosis,6 and thrombus formation in a laser-induced thrombosis model of arterioles.7 Similarly, impaired PGI2 production in COX-2 KO mice culminates in increased thrombosis in a photochemical injury model and larger thrombi in a laser injury model.7 Interestingly, the early phase of thrombus formation was not altered in PGI2 receptor KO mice, but reduced platelet disaggregation was observed at later time stages, resulting in enhanced thrombus development.7 Of note, the PGI2 receptor KO mice do not display signs of spontaneous thrombosis and exhibit normal tail bleeding times.6 These results are in line with a model in which the PGI2 receptor restricts platelet activation and thrombus formation in settings of vascular injury or in the presence of atherosclerosis but plays no role in preventing spontaneous thrombosis in healthy, uninjured vessels. The consequence of PGI2 binding to its receptor on the platelet surface is the activation of stimulatory G-protein α subunit (Gsα), which in turn stimulates the activity of membraneassociated adenylyl cyclase (AC) resulting in cyclic adenosine monophosphate (cAMP) production. Elevated intracellular cAMP levels activate protein kinase A (PKA), a broad-spectrum serine/threonine kinase, which phosphorylates several substrate proteins involved in various aspects of platelet activation (Fig. 15.2). PKA substrates include regulators of heterotrimeric

Fig. 15.2 Broad-spectrum inhibition of platelet activation by PGI2 and NO. PGI2 and NO mediated elevation of intracellular cyclic nucleotide levels, which activate the Ser/Thr kinases PKA and PKG, respectively leading to phosphorylation of several proteins. PKA and PKG substrate proteins are involved in various aspects of platelet activation and their phosphorylation provides a generalized inhibition of platelet activation.1 AC, adenylate cyclase; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; Gs, guanine nucleotide-binding protein stimulatory; GTP, guanosine triphosphate; PKA, protein kinase A; PKG, protein kinase G; sGC, soluble guanylate cyclase. (Reproduced with permission from Coxon et al.8 Professional illustration by Patrick Lane, ScEYEnce Studios.)

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G-proteins, small G-proteins, calcium and cyclic nucleotide levels, and modulators of the actin cytoskeleton.9 Recent data from mass spectrometry-based phosphoproteomics studies show that platelet inhibition by the PGI2 receptor is more complex than previously anticipated and indicate a high number (>100) of potential PKA substrates.10 Mechanistically, PKA phosphorylation frequently alters inter-molecular interactions between substrates and their interaction partners, resulting in the modulation of several key signaling nodes, enabling them to counteract activation signals effectively.11 Intriguingly, PGI2 inhibits platelet responses to all agonists, irrespective of their mechanism of action, demonstrating that PKA substrates regulate critical nodes in platelet activation pathways. In addition to the PGI2 receptor, other Gs-coupled inhibitory receptors are also expressed in platelets including the adenosine A2a12 and A2b receptors,13 the prostanoid EP2, EP4 and DP1 receptors,14,15 and vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor 1 (VPAC1).16 These receptors transmit similar inhibitory signals as the PGI2 receptor to disrupt platelet activation, including AC stimulation, cAMP elevation, PKA activation, and potentially analogous downstream mechanisms. However, the relative share of these receptors or the role of their ligands in limiting thrombus formation are less well understood.

SOLUBLE GUANYLATE CYCLASE: BROADSPECTRUM INHIBITOR OF PLATELET ACTIVATION NO is a small gaseous signaling molecule continuously generated by endothelial NO synthase (eNOS) in endothelial cells in response to shear stress. NO is of key importance to the cardiovascular system, causing smooth muscle relaxation and vasodilation,17 and is also a potent inhibitor of platelet aggregation.18 The biology of NO is detailed in Chapter 17. In this chapter, we discuss the contribution of sGC, the platelet NO receptor to inhibition of platelet activation and thrombus formation. The relevance of the NO pathway in regulating platelet activation and thrombosis has been conclusively shown by genetic alterations in key genes involved in this regulatory network.19 A loss-of-function variant of eNOS with decreased NO production is associated with higher risk of ischemic heart disease.20,21 Similarly, variants affecting sGC are associated with increased coronary artery disease22 and myocardial infarction.23 On the contrary, gain-of-function variants of eNOS and sGC with increased NO levels or increased cGMP synthesis are associated with a reduced risk of coronary heart disease and stroke.24 Although the relevance of NO to cardiovascular health and disease is well established,17 the contribution of eNOS, the main NO-producing enzyme in the vasculature, to the regulation of thrombus formation is less clear from genetically altered mouse models. Investigations of eNOS KO mice provided conflicting results, either showing normal25,26 or markedly reduced27 bleeding time. Findings from in vivo thrombosis models are also inconsistent, demonstrating either decreased25 or unaltered thrombosis in the ferric chloride model,26 or normal thrombus formation in a photochemical injury model.28 The reason for these apparent discrepancies is not known; however, both the ferric chloride- and the photochemical injuryinduced thrombosis models are driven by an excess of reactive oxygen species (ROS), which also scavenge NO. Thus, the role of NO in thrombus formation in these models is potentially masked by the generated ROS. NO diffuses through the platelet membrane and activates the intracellular sGC, also known as NO-sensitive GC (NOGC), which in turn catalyzes the synthesis of the second messenger cGMP (Fig. 15.2). In platelets, sGC is the main receptor

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for NO and loss-of-function mutations affecting the enzyme result in the loss of most NO effects.29 sGC is a heterodimer composed of α1 and β1 subunits, and deletion of either leads to a complete absence of the enzyme in platelets in mice.30,31 NO-induced inhibition of platelet aggregation is absent in mice deficient in sGC, establishing a central role for this receptor in regulating platelet activity,30–32 in agreement with data from patients with a loss-of-function mutation in this enzyme.23 Constitutive sGC KO mice and mice with a β1 subunit point mutation, rendering the enzyme insensitive to activation by NO, displayed dramatically shortened tail bleeding times.32,33 This effect, however, was not recapitulated in Pf4-Cre-generated sGC conditional KO mice.34 cGMP transmits its effects by regulating protein kinase G I (PKGI) and the phosphodiesterases PDE2A, PDE3A and PDE5A, which regulate platelet cAMP and cGMP levels and mediate the cross-talk and synergy between these cyclic nucleotides.9,35 PKGI is a broad-spectrum serine/threonine kinase encoded by the PRKG1 gene, which signals analogously to PKA by suppressing intracellular Ca2+ levels, inhibiting TXA2 synthesis, granule release, integrin activation and cytoskeletal rearrangements.9,36,37 Studies on Prkg1 KO mice established PKGI as the major downstream target of NO and cGMP, which transmits their inhibitory effects in platelets.38 These findings are in agreement with results from human patients with reduced PKGI expression.39 In vivo PKGI plays a crucial role in preventing platelet adhesion and aggregation after ischemia.38 Prkg1 KO mice exhibit prolonged tail bleeding,40 indicating that PKGI does not transmit inhibitory effects of NO in this type of injury. Numerous studies suggest that PKGI shares several substrates with PKA and regulates the same signaling nodes.9,41 Of note, certain aspects of the platelet NO signaling pathway are still under dispute, such as whether platelets generate NO and whether the NO-sGC-PKGI signaling pathway also plays a positive regulatory role in platelet activation.11,41–43

ITIM-CONTAINING RECEPTORS Platelet ITIM-containing receptors belong to the immunoglobulin superfamily and harbor either tandem consensus ITIMs in their cytosolic tail, or an ITIM and an ITIM-like sequence termed an immunoreceptor tyrosine-based switch motif (ITSM). ITIMs are defined as six amino acid sequences containing a tyrosine (Y), followed by a C-terminal hydrophobic residue at Y +3 position and preceded by a less conserved residue at Y-2 position with the consensus sequence I/V/LxYxxL/V, where x represents any amino acid.44 ITSMs are defined by the consensus sequence TxYxxV/I.45 Phosphorylation of the tyrosine residues within these motifs generate high affinity docking sites that recruit cytosolic SH2 domain-containing phosphatases, which counteract activation signals.46 Typical effector proteins include the tandem SH2 domain-containing cytosolic tyrosine phosphatases Shp1 and Shp2 and/or the single SH2 domain-containing lipid phosphatases phosphatidylinositol 5-phosphatase-1 (SHIP-1) and SHIP-2. Phosphorylated ITIMs not only localize these phosphatases to the plasma membrane, but also contribute to their activation, which in turn dephosphorylate phospho-tyrosine residues kinases and adapter proteins or the lipid second messenger phosphatidylinositol3,4,5-trisphosphate, as in the case of SHIP phosphatases. The inhibitory Fcγ receptor IIB (FcγRIIB) serves as the prototype of ITIM signaling and was originally described to inhibit B cell activation dependent on the ITAM-containing B cell receptor (BCR).47 A key concept in the mechanism underlying how ITIM-containing receptors inhibit signaling from ITAMcontaining receptors is ligand-mediated co-clustering of the two receptors. In the case of FcγRIIB and the BCR, this is

mediated by antibody-antigen complexes. The BCR binds the antigen and FcγRIIB binds the Fc portion of antibodies against the antigen, bringing the two receptors into close proximity, and allowing ITIM-associated SHIP1 to dephosphorylate PI3,4,5P3 to PI3,4P2 and attenuate BCR signaling. In addition to binding SH2 domain-containing phosphatases, ITSMs can also bind small SH2 domain-containing adaptors called SLAM-associated proteins (SAPs), which facilitate rather than inhibit cellular activation, hence conferring activatory, as well as inhibitory functions to ITSM-containing receptors. In platelets, one of the main functions of ITIM-containing receptors is to limit signal transduction by the ITAM-containing collagen receptor GPVI-FcR γ-chain complex and the hemiITAM-containing podoplanin receptor CLEC-2 (Fig. 15.3). Comparatively less work has been carried out on the role of ITIM-containing receptors in regulating FcγRIIA signaling in platelets. Importantly, the role of ITIM signaling is not confined to the regulation of (hemi-)ITAM signaling pathways, but also includes the modulation of integrin- and GPCR-mediated responses, through as yet undefined mechanisms.8 Compared to PGI2 and NO signaling, which leads to cell-wide non-responsiveness of platelets and impacts on most activation pathways, inhibition by ITIM-containing receptors results in a spatially defined signal termination only in the vicinity of the receptor, which is a key defining feature of ITIM-containing receptors. The MK lineage expresses several ITIM-containing receptors, including platelet-endothelial cell adhesion molecule-1 (PECAM-1 or CD31), G6b-B, TREM-like transcript-1 (TLT-1), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), CEACAM1 (also known as CD66a, BGP and C-CAM), CEACAM2 (only present mice), and leukocyte immunoglobulin-like receptor B2 (LILRB2), also referred to as paired immunoglobulin-like receptor B (PIR-B)8 (Fig. 15.4). As discussed in more detail below, these receptors can be grouped by their gene expression profile into receptors restricted to the MK lineage and those with broader expression patterns. These receptors control an extensive array of MK and platelet responses, exemplified best by the phenotypes of the respective KO mice, and patient mutations as in the case of G6b-B. An emerging concept in the field is that while the main effectors, Shp1 and Shp2, appear to be expressed constantly during MK differentiation,48 LAIR-1 and G6b-B display contrasting expression profiles, being expressed either from hematopoietic stem cells to immature MKs or only on mature MKs and platelets, respectively.49,50 Despite structural similarities, the phenotypes of various KO mice are distinct, suggesting nonredundant roles of each ITIM-containing receptor in platelets. The few genes encoding these inhibitory receptors underwent rapid evolutionary changes resulting in notable differences between human and mouse receptors, highlighted below. We focus, however, on PECAM-1 and G6b-B, because these receptors have been the subject of extensive research and emerge as crucial regulators of platelet function and production. We will touch on other ITIM-containing receptors implicated in regulating platelet function.

PECAM-1: Selective Inhibitor of Platelet Activation PECAM-1 was the first ITIM-containing receptor to be described in platelets and has emerged as the prototype of this class of inhibitory receptors in platelets.8 PECAM-1 is a 130 kDa type-I transmembrane receptor protein containing six extracellular Ig homology domains, a single-pass transmembrane region, an ITIM and an ITSM in its cytosolic region.51 PECAM-1 is widely expressed in vascular endothelial cells, platelets and various subpopulations of leukocytes, including monocytes, neutrophils, dendritic cells, mast cells, natural

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Fig. 15.3 Classical inhibitory function of ITIM-containing receptors. The inhibition of ITAM-containing receptor signaling through the recruitment of the Src homology 2 (SH2) domain-containing protein-tyrosine phosphatases Shp1 and Shp2, or SH2 domain-containing inositol 50 -phosphatase 1 Ship1. Btk, Bruton’s tyrosine kinase; DAG, diacylglycerol; ER, endoplasmic reticulum; IP3-R, inositol trisphosphate receptor; P, phosphate; PI3-K, phosphoinositide 3-kinase. (Reproduced with permission from Coxon et al.8 Professional illustration by Patrick Lane, ScEYEnce Studios.)

Fig. 15.4 Platelet ITIM-containing receptors. The main structural features are shown, including the extracellular IgC2-like, IgV-like and IgI2-like domains and the main intracellular signaling motifs, namely immunoreceptor tyrosine-based inhibitory motif (ITIM, consensus sequence I/V/LxYxxL/V), immunoreceptor tyrosine-based switch motif (ITSM, consensus sequence TxYxxV/I), and proline-rich region (PRR, consensus sequence PxxP) along with nonconsensus ITIM/ITSM-like tyrosine residues. All receptors have been described in platelets except for LAIR-1, which is only found in MKs. Residues are numbered according to mature mouse peptide sequences, after cleavage of the signal peptide. (Reproduced with permission from Coxon et al.8 Professional illustration by Patrick Lane, ScEYEnce Studios.)

killer cells, B cells and T cells.52 PECAM-1 was cloned in 199053–55 and has since been the focus of extensive research, establishing critical involvement in the regulation of platelet activation, thrombus formation, vascular permeability and leukocyte trafficking.56

There are estimated to be 9400 and 5566 copies of PECAM1 in human and mouse platelets, respectively, according to proteomic databases,57,58 correlating well with results from Scatchard analyses using anti-PECAM-1 antibodies.59–62 However, the expression of PECAM-1 on human platelets can vary

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between individuals, with higher surface expression correlating with reduced platelet reactivity.59,63 Human and mouse platelets display notable differences, while the former expresses mostly the full-length version of PECAM-1, the latter expresses high levels of the Δ15 PECAM-1 isoform, which possess a shorter cytosolic tail lacking serine (S)702 and S707.64 These residues are important for regulating PECAM-1 signaling,65 embedding the cytosolic tail in the plasma membrane under resting conditions and release following phosphorylation. The extracellular domain of PECAM-1 is highly glycosylated53,66 and can bind to a number of ligands, most notably to the extracellular domain of PECAM-1 on other cells. Transhomophilic interactions are mediated by the two N-terminal Ig homology domains 1 and 2 of the extracellular domain.67–70 Heterophilic binding partners of PECAM-1 include the integrin αVβ3,70–72 CD38,73 and the neutrophil-specific CD17774; however, the physiological relevance of these interactions to platelets is currently not clear.

PECAM-1 Function The function of PECAM-1 has been elucidated using Pecam1 KO mice,75 and by selectively activating PECAM-1 signaling by antibody- and recombinant ectodomain-mediated crosslinking. It is clear that the trans-homophilic interactions between PECAM-1 molecules on adjacent platelets do not contribute to platelet aggregation, which is unaltered in response to ADP and thrombin in Pecam1 KO mice.75–78 Trans-homophilic interactions between platelet and endothelial cell PECAM-1 are tighter, due to higher levels of expression of the receptor on endothelial cells. Platelets from Pecam1 KO mice exhibit enhanced aggregation and dense granule secretion in response to subthreshold concentrations of collagen and CRP, demonstrating an inhibitory function of PECAM-1 on the ITAM-containing GPVI-FcR γ-chain receptor complex.76–78 Concomitant with enhanced GPVI signaling, Pecam1 KO platelets displayed enhanced adhesion and spreading on collagen and CRP surface.76,77 A similar enhancement of platelet aggregation was also reported to subthreshold concentrations of the CLEC-2 agonist rhodocytin, indicative of an inhibitory function of PECAM-1 on CLEC-2 receptor signaling.76 Based on findings from these studies, PECAM-1 has emerged primarily as an inhibitor of ITAM- and hemi-ITAM-coupled receptor signaling. Enhanced platelet aggregation responses are, however, masked at higher concentrations of (hemi-)ITAM agonists, highlighting the modulatory role of PECAM-1 on these pathways. Antibody-mediated cross-linking of PECAM-1 results in inhibition of calcium mobilization, dense granule secretion and platelet aggregation in response to a range of low dose agonists, including collagen, the GPVI-specific agonists CRP and convulxin, the CLEC-2-specific agonist rhodocytin and thrombin.76,79 The mechanism of anti-PECAM-1 antibody-mediated inhibition of aggregation involves an increase in phosphorylation of the ITIM and ITSM of PECAM-1 and a corresponding decrease in agonist-induced tyrosine phosphorylation of several proteins involved in platelet activation.79 Similarly, recombinant dimeric human PECAM-1 ectodomain mimicking trans-homophilic interactions of PECAM-1 selectively induces downstream signaling and dose-dependently inhibits platelet aggregation in response to low doses of collagen and CRP.80 PECAM-1 has also been implicated in regulating outside-in integrin αIIbβ3 signaling. An early study showed that an antiPECAM-1 antibody against the membrane proximal Ig homology domain 6 induces PECAM-1 tyrosine phosphorylation and has an enhancing effect on ADP-mediated platelet aggregation.81 Delayed clot retraction, defective cytoskeletal reorganization and decreased focal adhesion kinase tyrosine phosphorylation were subsequently reported in Pecam1 KO

platelets, suggesting defective fibrinogen engagement by the integrin αIIbβ3 and aberrant downstream signaling.78 However, these defects were not reported in a follow-up study, demonstrating no difference in clot retraction and a mild enhancement of platelet spreading on fibrinogen.76 Discrepancies between studies are most likely due to methodological differences, or possibly genetic drift in the Pecam1 KO mouse model. Further work is necessary to resolve the role of PECAM-1 in integrin-mediated responses of platelets. The physiological function of PECAM-1 in thrombus formation is well established. In a laser-induced thrombosis model of cremaster muscle arterioles, significantly larger thrombi are observed in Pecam1 KO mice, indicating a net inhibitory role for the receptor in vivo.82 Since PECAM-1 is widely expressed within the vasculature, experiments on bone marrow chimeric mice were performed to delineate the relative contributions of endothelial versus hematopoietic cell PECAM-1 to the phenotype. These results demonstrate that increased thrombosis in this model is due to the loss of platelet and leukocyte PECAM-1.82 However, the difference between wild-type and Pecam1 KO mice in the ferric chloride-induced thrombosis model of the carotid arteries were only modest.82 Interestingly, in a FITC-dextran photochemical injury-induced thrombosis model of cremaster muscle arterioles and venules, thrombus formation was found to be normal in Pecam1 KO mice.83 The nature of the injury likely accounts for the observed differences and suggest that the role of PECAM-1 in thrombus formation depends greatly on the vascular bed and type of injury. In addition, since PECAM-1 is widely expressed on different leukocyte subsets, the loss of PECAM-1 on these cells may also contribute to the observed phenotypes. Interestingly, Pecam1 KO mice exhibited prolonged bleeding, which was shown to be due to loss of PECAM-1 from endothelial cells rather than platelets through bone marrow chimera studies.84 Despite platelet counts being normal in Pecam1 KO mice under steady state conditions,84 the rate of platelet recovery following antibody-mediated platelet depletion was delayed in these mice.85 This was attributed to impaired platelet production, increased adhesion of MKs to extracellular matrix and a lack of MK polarity towards a stromal-derived factor-1α (SDF-1α) gradient due to defective compartmentalization of the SDF-1α receptor CXCR4 to the leading edge of the MK.85 Defective MK migration may also underlie the abnormal spatial distribution of MKs in the bone marrow of these mice.86

PECAM-1 Signaling PECAM-1 was initially shown to be phosphorylated in thrombin-simulated platelets,87–89 and subsequently in response to several other platelet agonists. Central to PECAM-1 signal transduction are the two tyrosine residues within the ITIM and ITSM, which undergo inducible phosphorylation in response to several stimuli, including: thrombin, thrombin receptor-activating peptide (TRAP)90–94; collagen, CRP, convulxin80,90,95; and cross-linked anti-PECAM-1 antibodies,91,94 and not surprisingly the phosphatase inhibitor pervanadate.87 The requirement of integrin αIIbβ3 signaling for PECAM-1 phosphorylation is somewhat controversial, with most studies showing enhanced phosphorylation upon platelet aggregation.80 Phosphorylation of tyrosine (Tyr)663 and Tyr686 in the cytosolic region of PECAM-1 mediates binding of Shp1 and Shp2 via their tandem SH2 domains.91–93,96,97 The binding affinities of phosphorylated ITIM and ITSM to the SH2 domains of Shp1 and Shp2 are in the nanomolar range. Binding involves highly specific interactions of phospho-Tyr663 with the N-terminal SH2 domain and phospho-Tyr686 with the C-terminal SH2 domain of Shp2. The binding of

Platelet Inhibitory Receptors

phosphorylated ITIM and ITSM to the SH2 domains not only localizes Shp1 and Shp2 to the plasma membrane, but also enhances the catalytic activity of the phosphatases, by relieving intramolecular inhibitory interactions.91,92,96 PECAM-1 contains two serine residues, Ser702 and Ser707 in its C-terminal region, which are phosphorylated in an inducible or constitutive manner, respectively. The function of S702 phosphorylation is to disrupt plasma membrane interaction of the C-terminal region, and thus to control accessibility of the ITSM.65 The ITSM and ITIM undergo sequential phosphorylation with Lyn phosphorylating the ITSM first, which provides docking site to Csk, which in turn phosphorylates the ITIM of PECAM-1.98 The lipid phosphatase SHIP-1 has also been shown to bind to PECAM-1 in vitro,99 via its SH2 domain, as well as SFKs via their SH3 domains; however, the relevance of these interactions to platelet function have not been determined.

G6b-B: Critical Regulator of Platelet Homeostasis G6b-B is a small type I transmembrane protein that is highly and exclusively expressed in MKs and platelets.49,100–104 Structurally, G6b-B consists of a single extracellular Ig variableregion-like (IgV) domain, a transmembrane domain and a cytoplasmic tail containing a juxtamembrane proline-rich region (PRR), an ITIM and an ITSM. G6b (also referred to as C6orf25 and MPIG6B) located within the major histocompatibility complex class III region of chromosome 6 was cloned from the K562 erythroleukemia cell line. Transcripts of several predicted splice variants were cloned at the same time, including G6b-A, -C, -D and -E, all of which share the same ectodomain as G6b-B101,105 (Fig. 15.5). G6b-A also shares the same transmembrane domain and proline-rich juxtamembrane region as G6b-B, with the amino acid composition of the remaining three-quarters of the G6b-A cytoplasmic tail bearing little resemblance to that of G6b-B. Indeed, the cytoplasmic tail of G6b-A lacks tyrosine residues, an ITIM and an ITSM, but is rich in serine and threonine residues. G6b-C, -D and -E lack a transmembrane domain and are presumably secreted forms. The expression of G6b-A has been confirmed in human platelets by western blotting using an isoform-specific antibody, but its function remains undefined. Expression and functional roles of G6b-C, -D, and -E are not known.

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G6b-B is highly abundant in platelets,104 with an estimated 13,700 and 29,637 copy numbers per human and mouse platelet, respectively.57,58 The extracellular IgV domain of human G6b-B contains a single N-linked glycosylation site, whereas mouse G6b-B contains two.49,101,104 As a consequence, human G6b-B migrates as a distinct doublet, migrating between 28 and 32 kDa by SDS-PAGE, whereas mouse G6b-B migrates as a smear at 45 kDa, with a tight doublet within it. The upper band in both instances represents the glycosylated form and the lower band the unglycosylated form of human and mouse G6b-B. G6b-B was first shown to bind heparin in 2005 by de Vet and co-workers.106 This was subsequently confirmed in the Senis laboratory, and G6b-B was also shown to bind tightly to the heparan sulfate side-chains of the extracellular matrix proteoglycan perlecan.107 Binding affinities of recombinant dimeric G6b-B to heparin and heparan sulfate are in the low nanomolar range by surface plasmon resonance, whereas the binding affinity of recombinant monomeric G6b-B is low millimolar. Binding was not detected to other proteoglycans found in the vasculature, including syndecan and agrin, or extracellular matrix proteins, including collagen, laminin, fibronectin, and fibrinogen.107 Binding of G6b-B to perlecan was lost following heparanase treatment of perlecan to remove heparan sulfate side-chains, demonstrating that it does not bind to the core protein. These ligands induce specific G6b-B phosphorylation, Shp1 and Shp2 engagement, strongly suggesting that G6b-B is a heparan sulfate/heparin receptor. MKs may encounter heparan sulfate and heparin in the vessel wall, whereas platelets will only come into contact with these ligands when the vessel wall is breached or during heparin therapy. Further work is required to elucidate the biological and physiological significance of these interactions to MKs and platelets.

G6b-B Function G6b-B has first proposed to be an inhibitory receptor based on an ITIM present in its cytoplasmic tail and its interaction with Shp1 and Shp2 in transiently transfected cells treated with the phosphatase inhibitor pervanadate.101 G6b-B phosphorylation and binding of Shp1 and Shp2 was subsequently confirmed in resting and activated human and mouse platelets.49,103,104,108 The first evidence for an inhibitory function of G6b-B came

G6b A

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Fig. 15.5 G6b-B splice isoforms. Established and putative isoforms of human G6b. The main structural features are shown, including the IgV domain, ITIM, ITSM, and PRR. G6b-A and -B contain transmembrane regions and are therefore represented as surface receptors. G6b-C, -D, and -E, identified in transcriptome analysis but not as expressed protein, are predicted to be secreted because they lack the transmembrane domain. Residues are numbered according to mature human peptide sequences, after cleavage of the signal peptide. (Reproduced with permission from Coxon et al.8 Professional illustration by Patrick Lane, ScEYEnce Studios.)

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from antibody-mediated cross-linking studies, demonstrating that a polyclonal antibody directed against its ectodomain induces phosphorylation, increased binding of Shp1 and attenuation of CRP- and ADP-induced platelet aggregation.103 G6b-B was subsequently shown to inhibit GPVI and CLEC-2 signaling in transiently transfected DT40 chicken B cell line. This effect was lost if consensus tyrosine residues in the ITIM and ITSM of G6b-B were mutated to phenylalanine, abrogating binding of Shp1 and Shp2. However, an inhibitory effect was also observed in DT40 cells lacking Shp1, Shp2, and SHIP1 expression, raising questions about the model and mechanism of action. Much of what is currently known about the physiological function of G6b-B comes from studies of mouse models. Targeted deletion of G6b in mice results in severe macrothrombocytopenia, aberrant platelet function, mild-to-moderate bleeding, megakaryocytosis at sites of hematopoiesis and focal myelofibrosis around MK clusters. Platelet count in G6b KO mice is reduced by 80% of normal and platelet volume is increased by 35%.49 The primary cause of this effect is reduced ability of G6b KO MKs to produce platelets. Increased platelet clearance is also a factor in this phenotype, presumably due to platelets being in a pre-activated state and cleared more rapidly from the circulation. Platelets from these mice also had increased levels of IgM and IgG on their surface, suggesting that auto-antibodies were being generated against platelet surface receptors. Serum thrombopoietin (Tpo) levels were increased in these mice due to the reduction in platelet counts, leading to increased megakaryopoiesis, myelofibrosis, and bone marrow destruction. Intriguingly, Tpo signaling was intact in primary bone marrow-derived G6b KO MKs, which developed normally ex vivo.49 However, MKs lacking G6b-B failed to spread normally on fibrinogen-, fibronectin- and collagencoated surfaces or to form proplatelets to the same extent as control MKs on fibrinogen- or fibronectin-coated surfaces, suggesting a central role for G6b-B in regulating integrin-mediated functions of MKs and proplatelet formation. This was supported by a reduction in integrin signaling in fibrinogenadhered G6b KO MKs. The cause of reduced proplatelet formation in these MKs remains undefined. Impairment of platelet reactivity to collagen and paradoxical bleeding in G6b KO mice were surprising aspects of this phenotype. The reason for the loss of response to collagen was a significant down-regulation of GPVI-FcR γ-chain expression, presumably as a means of attenuating tonic GPVI signaling in the absence of G6b-B. In contrast, CLEC-2 expression was only marginally reduced in G6b KO platelets, hence platelets hyper-responded to antibody-mediated receptor activation. Other platelet defects of note were attenuated aggregation in response to thrombin, reduced spreading of thrombin-treated platelets on fibrinogen and reduced GPIbα surface expression. Collectively, the reduction in platelet count and accompanying reduction in platelet reactivity to various agonists resulted in reduced thrombus formation and enhanced bleeding in these mice, highlighting the critical role played by negative feedback pathways in preventing thrombosis in the absence of G6b-B.49 A strikingly similar phenotype was recently reported in harboring loss-of-function mutants in G6b-B. G6b-B diY/phenylalanine (F) mice expressing a mutant form of G6b-B in which tyrosine residues within the ITIM and ITSM were mutated to phenylalanine, uncoupling G6b-B from Shp1 and Shp2.109 These findings demonstrate that G6b-B expression alone is insufficient to fulfill its biological functions and it must be able to signal via Shp1 and Shp2. This mouse model also demonstrates that other isoforms of G6b have little or no biological functions on their own.109 An equally important role of G6b-B in regulating human platelet homeostasis has emerged from patient and humanized

mouse studies. Children from five unrelated families have recently been identified with autosomal recessive congenital macrothrombocytopenia with focal myelofibrosis, due to loss-of-function mutations in G6b.110,111 In total, four unique germline mutations have been identified in G6b to date by linkage analysis and whole exome sequencing, including p. Cys108*, p.20fs, p.49fs and p.Gly157Arg. The pathologies exhibited by these patients mimic those found in G6b KO and loss-of-function mice, including mild-to-moderate bleeding, macrothrombocytopenia, atypical MKs associated with a distinctive, focal, perimegakaryocytic pattern of bone marrow fibrosis.110 Interestingly, patients exhibit variable leukocytosis and anemia, occasionally also observed in mouse models described above. It is presently unclear whether effects on other hematopoietic lineages are a direct consequence of the absence of functional G6b-B or a secondary effect arising from low platelet counts, bone marrow destruction or skewed hematopoiesis with high MK output. None of the patients had somatic mutations in JAK2, MPL, or CALR, typically associated with primary myelofibrosis in BCR-ABL-negative myeloproliferative neoplasms in adults, making G6b-B the first inhibitory ITIMcontaining receptor associated with congenital primary myelofibrosis in humans. Further evidence of similar functions of human and mouse G6b-B comes from a humanized mouse study, demonstrating that replacement of mouse G6b with human G6b by homologous recombination rescues the G6b KO phenotype.110 Mild defects in platelet count and function seen in humanized G6b-B mice arise primarily because of a 75% reduction in human G6b-B expression in mouse platelets, possibly due to differential glycosylation. Collectively, these findings establish G6b-B as a critical regulator of MK and platelet function in humans and mice, absence or loss-of-function of which culminates in macrothrombocytopenia, MK clusters at sites of hematopoiesis and myelofibrosis.

G6b-B Signaling Unlike other platelet ITIM-containing receptors, G6b-B is phosphorylated to a high stoichiometry and associated with Shp1 and Shp2 in resting platelets, suggesting it constitutively attenuates platelet reactivity, preventing them from becoming preactivated. Conserved tyrosine residues within the ITIM and ITSM of G6b-B are phosphorylated by SFKs and mediate the association of the tandem SH2 domains of Shp1 and Shp2. Recently, tonic high SFK activity was demonstrated to lead to the downregulation of the ITAM-containing GPVI-FcR γ-chain complex from the platelet surface and a concomitant increase in G6b-B levels, enhanced G6b-B phosphorylation and association with Shp1 and Shp2, further demonstrating that SFKs trigger both ITAM and ITIM signaling in parallel (Fig. 15.6).2 Currently, it is not known how the phosphorylation of ITAM-containing activatory and ITIM-containing inhibitory receptors is balanced upon SFK activation, warranting further investigation. Regarding other interaction partners of G6b-B, there is currently no evidence that it binds the lipid phosphatase SHIP1 or SAP adaptor proteins via its ITSM. There is, however, in vitro evidence suggesting it can bind Fyn, Src, and Syk, possibly facilitating phosphorylation and association of Shp1 and Shp2.108 However, these interactions have not been confirmed in platelets. There is also no evidence that the proximal proline-rich regions in G6b-B mediate binding of SFKs or any other SH3 domain-containing signaling proteins, or whether numerous serine and threonine residues in the cytoplasmic tail of G6b-A are phosphorylated and mediate signaling in activated platelets. Engagement of Shp1 and Shp2 is essential for G6b-B to mediate its biological effects, as evidenced in the G6b-B diY/F mouse model.109 Phosphorylated G6b-B can bind both Shp1

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Fig. 15.6 Initiation of G6b-B signaling. SFKs phosphorylate activating and inhibitory pathways in parallel. Once a threshold level of SFK activity is reached, ITAM, hemi-ITAM, integrin, and ITIM signaling are triggered. The kinetics and order of activation depend upon the agonist and level of SFK activity. (Reproduced with permission from Mori et al.2 Professional illustration by Patrick Lane, ScEYEnce Studios.)

and Shp2; however, all of the evidence suggests it preferentially binds and signals via Shp2. Shp2 has a 100-fold higher binding affinity for phosphopeptides corresponding to the ITIM and ITSM of G6b-B. In addition, mouse platelets contain six-fold higher levels of Shp2 than Shp1, which can outcompete for binding to G6b-B, and the phenotype of Shp2 conditional KO mice more closely resembles that of G6b-B KO mice. Presumably, the same holds true in human platelets, despite expressing 2.5-fold higher levels of Shp1 than Shp2. Downstream consequences of binding Shp1 or Shp2 are probably negligible, as the two phosphatases are likely to be interchangeable as they target many of the same substrates. Specificity derives more from distinct patterns of expression and compartmentalization rather than differences in substrate recognition. The primary downstream target of Shp2 identified to date is Syk, which is hyper-phosphorylated in G6b and Shp2 KO mouse platelets48,49; however, the exact tyrosine residues dephosphorylated by Shp2 remain undefined. Undoubtedly, Shp1 and Shp2 dephosphorylate other key substrates in platelets, but their identities also remain elusive. The fact that human G6b-B could rescue the phenotype of G6b KO mice, demonstrates that the two receptors perform orthologous functions in regulating platelet production and function, despite differences in the relative proportions of Shp1 and Shp2 expression in human versus mouse platelets.110 Residual platelet defects reported in humanized G6b-B mice were most likely due to lower human G6b-B expression in mouse platelets, rather than species-specific differences in how G6b-B signals or engages with its ligands. Nuclear magnetic resonance analysis using recombinant SH2 domains of Shp2 and phosphopeptides of the ITIM and ITSM of G6b-B reveals that the N-SH2 of Shp2 preferentially binds to the phosphorylated ITIM and C-SH2 to the phosphorylated Cterminal ITSM.109 This orientation provides the maximal extended conformation of associated Shp2, allowing it to dephosphorylate substrates in its immediate vicinity. Phosphorylation of the ITIM and ITSM of G6b-B and association of Shp1 and Shp2 increases significantly in response to collagen and thrombin stimulation, which may seem counter-intuitive, but presumably is a way of dampening GPVI, PAR1, and PAR4 signaling, and preventing sustained or uncontrolled signaling.109

Engagement of heparan sulfate and heparin also induces phosphorylation of the ITIM and ITSM of G6b-B, and association of Shp1 and Shp2, suggesting this would increase the inhibitory effect of G6b-B. However, this is greatly dependent on the concentration and complexity of the ligand.107 Low and intermediate concentrations of both ligands in fact enhance platelet aggregation to low concentrations of collagen and ADP, which is not observed at high concentrations of both ligands. In contrast, anti-platelet anti-coagulant (APAC), a higher order structure in which single-chains of heparin are covalently linked to a bovine serum albumin core, yields robust phosphorylation of G6b-B, and subsequent Shp1 and Shp2 binding, and has a significant inhibitory effect on collagen-mediated platelet aggregation, and an enhancing effect on ADP mediated platelet aggregation. Interestingly, perlecan has little effect on collagen- or ADP-mediated platelet aggregation, presumably due to the opposing effects of the N-terminal heparan sulfate side-chains acting on G6b-B,107 and endorepellin, the C-terminal domain of perlecan, binding to the integrin α2β1.112 It is worth noting that perlecan is a very large proteoglycan that binds to many other extracellular proteins,113 including collagen and laminin, which are also likely altering the platelet response. An inhibitory effect of perlecan on platelets is seen on platelet adhesion when platelets are seeded on a perlecan-coated surface. This effect is lost if perlecan is treated with heparanase, removing the heparan sulfate side-chains.107 The working model is that heparan sulfate and heparin can either cluster or repel G6b-B with itself or other receptors, depending on the concentration and complexity of the ligand.

OTHER PLATELET ITIM-CONTAINING RECEPTORS Below follows a summary of our current understanding of the functional roles of other ITIM-containing receptors (Fig. 15.4) in regulating platelet and MK function. It should be noted that some of these receptors are only expressed during megakaryopoiesis and are either not present or expressed at very low levels in human and mouse platelets. As such, they play minimal roles in regulating the hemostatic and prothrombotic activity of platelets. However, this is not the case for TLT-1, which is

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the most abundant ITIM-containing receptor in human and mouse platelets and has been implicated in regulating the platelet response to inflammatory challenges.

LAIR-1 Human LAIR-1 is a type I transmembrane glycoprotein containing a single Ig constant-region-type-2-like domain, an ITIM and an ITSM in its cytoplasmic tail. Mouse LAIR-1 exhibits an overall sequence homology of 50% to the human protein and contains an ITIM and a non-consensus ITIM-like motif. The physiological ligand of LAIR-1 is collagen.114 LAIR-1 is expressed in a variety of leukocytes, including NK cells, T cells, B cells, monocytes, dendritic cells, eosinophils, basophils, mast cells.115 It has also been identified in hematopoietic progenitors and immature MKs, but not in mature MKs or platelets.50,116 Platelets from Lair1 KO mice display increased aggregation in response to low doses of collagen and CRP similarly to other platelet ITIM-containing receptors. Interestingly, these mice exhibit mild thrombocythemia and increased proplatelet formation in vitro demonstrating an inhibitory role for LAIR-1 in platelet formation.50 This inhibitory effect is likely mediated by the tyrosine kinase Csk, which was demonstrated to bind phosphorylated LAIR-1.117

TLT-1 TLT-1 is a type I transmembrane receptor with a single IgV-like domain, an ITIM and a non-consensus ITIM-like motif in its cytoplasmic tail,118 which is highly and specifically expressed in human and mouse MKs and platelets.119 There are estimated to be 14,200 and 154,769 copies of TLT-1 per human and mouse platelet, respectively, using proteomics-based approaches,57,58 making it the most abundant platelet ITIMcontaining receptor. It is tyrosine phosphorylated, and binds Shp1 and Shp2 in transiently transfected cell lines and platelets. However, unlike other ITIM-containing platelet receptors that negatively regulate platelet activation, TLT-1 acts as a positive regulator that facilitates platelet activation,120–122 making it a non-conventional ITIM-containing receptor. In addition, it is present in the membranes of α-granules, also containing a soluble splice-isoform of TLT-1 that translocates to the platelet surface and is secreted from activated platelets, respectively. The ectodomain of TLT-1 is shed from activated platelets, contributing to total soluble TLT-1 in the plasma. Interestingly, soluble TLT-1 levels are significantly elevated in septic patients suffering from chronic inflammatory conditions.122 However, the physiological function and clinical significance of elevated levels of soluble TLT-1 are not known. TLT-1 binds fibrinogen, but whether ligand engagement induces downstream signaling has not been shown. Mice lacking TLT-1 are mildly thrombocytopenic, exhibit marginally prolonged bleeding following excision of the tail tip and reduced platelet aggregation to thrombin. Most interestingly, lipopolysaccharide-treated TLT1-deficient mice developed higher levels of plasma tissue necrosis factor and D-dimers than control mice, and were more likely to succumb to the challenge.122 TLT-1 KO mice were also predisposed to hemorrhage associated with localized inflammatory lesions. Collectively, these findings suggest that TLT-1 plays a protective role in regulating the inflammatory response to injury.122

CEACAM1 and CEACAM2 CEACAM1 and CEACAM2 are structurally related members of the immunoglobulin superfamily. CEACAM1 is expressed in a variety of epithelial, endothelial, and hematopoietic cell types.123,124 CEACAM1 undergoes extensive alternative

splicing with 11 isoforms present in humans and four isoforms in mice.125 The human receptor consists of an N-terminal IgV-like domain, followed by up to three Ig constant-regiontype-2-like domains, a transmembrane domain and a variable cytoplasmic tail, with the longest isoform containing two ITIMs. Mouse CEACAM1 contains an ITIM and an ITSM. Similar to PECAM-1, the extracellular domain of CEACAM1 is heavily glycosylated and can mediate homophilic interactions.125 Low expression of CEACAM1 has been detected in both human and mouse platelets by flow cytometry,126 with mouse platelets estimated to contain 868 copies per platelet by proteomics,58 which is supported by gene expression data in mouse MKs and platelets.100,127 In contrast, CEACAM1 is not detected in human platelets using various proteomicsbased approaches.57,104,128 Similarly, gene expression studies also do not support its presence on human platelets.127 The reason for this apparent discrepancy is not clear, but is most likely the consequence of low amounts of CEACAM1 in human platelets. The identities of splice isoforms of CEACAM1 expressed in human and mouse platelets have not yet been determined. CEACAM1 has, however, been shown to be upregulated on the surface of thrombin-stimulated mouse platelets, demonstrating an intracellular pool of the receptor.126 Platelets from Ceacam1 KO mice exhibit enhanced aggregation and granule secretion in response to subthreshold concentrations of collagen, CRP and rhodocytin, and increased adhesion on a collagen-coated surface,126,129 suggesting that CEACAM1 attenuates (hemi-)ITAM receptor-mediated responses, presumably through the compartmentalization and activation of Shp1 and Shp2. Increased thrombus size and stability was observed following ferric chloride-induced injury of mesenteric arterioles in Ceacam1 KO mice.126 In addition, CEACAM1 has been implicated in positively regulating integrin αIIbβ3 function.130 CEACAM2 contains an ITIM and an ITSM in its cytoplasmic tail, and has also been implicated in regulating platelet activation. Ceacam2 is present in the mouse genome; however, the orthologous gene is not present in the human genome.131 It displays a more limited gene expression pattern compared to CEACAM1, being expressed in kidney, uterus, testis, brain and select intestinal epithelial cells.132–134 The expression of CEACAM2 on mouse platelets is likely to be low129 as the protein was not detected by mass spectrometry,58 and gene expression data indicates only low levels of Ceacam2 transcripts in platelets.127 However, mouse MKs were shown to express Ceacam2.100 Intriguingly, Ceacam2 KO mice exhibit a similar platelet phenotype to that reported in Ceacam1 KO mice, including: increased aggregation and secretion to subthreshold concentrations of agonists; increased thrombus formation; increased (hemi-)ITAM signaling; and positive regulation of integrin signaling.126,129,135 Key questions that remain to be addressed are whether CEACAM1 and CEACAM2 are able to partially compensate for one-another, and the platelet-specific contributions to the phenotypes observed in the KO mouse models.

PIR-B The human leukocyte immunoglobulin-like receptor (LILR) family contains 11 genes, five of which encode inhibitory receptors (LILRB1–5). LILRB2 contains an extracellular domain composed of four Ig-like domains, an ITIM, and two nonconsensus ITIM-like motifs in its cytosolic tail. In contrast, in mice there is a single inhibitory receptor in this family, namely paired Ig-like receptor B (PIR-B) with six Ig-like domains, an ITIM and three non-consensus ITIM-like motifs, which is the proposed mouse ortholog of LILRB2.136 LILRB2 and PIR-B

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are expressed in hematopoietic stem cells, myeloid cells, dendritic cells, mast cells, B cells, and platelets.137–139 It is noteworthy, that the protein is not detected in either human or mouse platelets by proteomics-based approaches, suggesting low abundance.57,58 Platelets from transgenic mice lacking the cytosolic tail of PIR-B are hyper-responsive to low doses of CRP demonstrating negative regulation of ITAM signaling, similar to that reported for PECAM-1 and CEACAM1/2.137 These mice exhibit mild thrombocythemia with a higher number of MKs, suggesting a role for PIR-B in MK development or function.137 This is supported by the presence of Pirb transcript in mouse MKs,100 and PIR-B protein in a proportion of MK progenitors.140

CONCLUSION Platelets have evolved a diverse repertoire of inhibitory receptors and signaling mechanisms to prevent and modulate platelet activation, without which pathological thrombosis would occur. All of these mechanisms are transient and reversible; otherwise, platelets could not respond to their surroundings. Broad-spectrum inhibitors of platelet activation, including PGI2 and NO, help to maintain platelets in a resting state by increasing intracellular cAMP and cGMP levels and activating PKA and PKG, respectively resulting in Ser/Thr phosphorylation of key receptors, signaling proteins and adaptors. ITIMcontaining receptors provide a more subtle and selective means of inhibiting platelet activation, involving receptor-ligand engagement in the lumen or vessel wall, mediating compartmentalization and activation of lipid and tyrosine phosphatases that dephosphorylate and inactivate key signaling components downstream of activation receptors. PECAM-1 plays an important role in limiting platelet activation and thrombus size at sites of vascular injury, whereas G6b-B plays a broader role in regulating not only the reactivity, but also the number of platelets in the circulation. Several other ITIMcontaining receptors, including CEACAM1 and CEACAM2 provide additional levels of regulation in parallel with PECAM-1, whereas LAIR-1 and PIR-B contribute to the maintenance of platelet homeostasis with G6b-B, acting at the level of MKs. The complexity and complementarity of these systems is not surprising, as they must cooperate in order to control the number and response of platelets to any pathological condition they encounter. Further work is needed to understand better how these diverse systems integrate in order to prevent lifethreatening thrombosis from occurring. Such physiological mechanisms of platelet inhibition could be exploited as a means of preventing or treating thrombosis. REFERENCES 1. Senis YA, Mazharian A, Mori J. Src family kinases: at the forefront of platelet activation. Blood 2014;124:2013–24. 2. Mori J, Nagy Z, Di Nunzio G, Smith CW, Geer MJ, Al Ghaithi R, van Geffen JP, Heising S, Boothman L, Tullemans BME, Correia JN, Tee L, Kuijpers MJE, Harrison P, Heemskerk JWM, Jarvis GE, Tarakhovsky A, Weiss A, Mazharian A, Senis YA. Maintenance of murine platelet homeostasis by the kinase Csk and phosphatase CD148. Blood 2018;131:1122–44. 3. Moncada S, Gryglewski R, Bunting S, Vane JR. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature 1976;263:663–5. 4. Arehart E, Stitham J, Asselbergs FW, Douville K, Mackenzie T, Fetalvero KM, Gleim S, Kasza Z, Rao Y, Martel L, Segel S, Robb J, Kaplan A, Simons M, Powell RJ, Moore JH, Rimm EB, Martin KA, Hwa J. Acceleration of cardiovascular disease by a dysfunctional prostacyclin receptor mutation: potential implications for cyclooxygenase-2 inhibition. Circ Res 2008;102: 986–93.

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5. Grosser T, Yu Y, Fitzgerald GA. Emotion recollected in tranquility: lessons learned from the COX-2 saga. Annu Rev Med 2010;61: 17–33. 6. Murata T, Ushikubi F, Matsuoka T, Hirata M, Yamasaki A, Sugimoto Y, Ichikawa A, Aze Y, Tanaka T, Yoshida N, Ueno A, Oh-Ishi S, Narumiya S. Altered pain perception and inflammatory response in mice lacking prostacyclin receptor. Nature 1997;388: 678–82. 7. Yu Y, Ricciotti E, Scalia R, Tang SY, Grant G, Yu Z, Landesberg G, Crichton I, Wu W, Pure E, Funk CD, Fitzgerald GA. Vascular COX2 modulates blood pressure and thrombosis in mice. Sci Transl Med 2012;4:132ra54. 8. Coxon CH, Geer MJ, Senis YA. ITIM receptors: more than just inhibitors of platelet activation. Blood 2017;129:3407–18. 9. Smolenski A. Novel roles of cAMP/cGMP-dependent signaling in platelets. J Thromb Haemost 2012;10:167–76. 10. Beck F, Geiger J, Gambaryan S, Veit J, Vaudel M, Nollau P, Kohlbacher O, Martens L, Walter U, Sickmann A, Zahedi RP. Time-resolved characterization of cAMP/PKA-dependent signaling reveals that platelet inhibition is a concerted process involving multiple signaling pathways. Blood 2014;123:e1–e10. 11. Nagy Z, Smolenski A. Cyclic nucleotide-dependent inhibitory signaling interweaves with activating pathways to determine platelet responses. Res Pract Thromb Haemost 2018;2(3):558–71. 12. Ledent C, Vaugeois JM, Schiffmann SN, Pedrazzini T, El Yacoubi M, Vanderhaeghen JJ, Costentin J, Heath JK, Vassart G, Parmentier M. Aggressiveness, hypoalgesia and high blood pressure in mice lacking the adenosine A2a receptor. Nature 1997;388:674–8. 13. Yang D, Chen H, Koupenova M, Carroll SH, Eliades A, Freedman JE, Toselli P, Ravid K. A new role for the A2b adenosine receptor in regulating platelet function. J Thromb Haemost 2010;8:817–27. 14. Hubertus K, Mischnik M, Timmer J, Herterich S, Mark R, Moulard M, Walter U, Geiger J. Reciprocal regulation of human platelet function by endogenous prostanoids and through multiple prostanoid receptors. Eur J Pharmacol 2014;740:15–27. 15. Kuriyama S, Kashiwagi H, Yuhki K, Kojima F, Yamada T, Fujino T, Hara A, Takayama K, Maruyama T, Yoshida A, Narumiya S, Ushikubi F. Selective activation of the prostaglandin E2 receptor subtype EP2 or EP4 leads to inhibition of platelet aggregation. Thromb Haemost 2010;104:796–803. 16. Freson K, Hashimoto H, Thys C, Wittevrongel C, Danloy S, Morita Y, Shintani N, Tomiyama Y, Vermylen J, Hoylaerts MF, Baba A, van Geet C. The pituitary adenylate cyclase-activating polypeptide is a physiological inhibitor of platelet activation. J Clin Invest 2004;113:905–12. 17. Murad F. Shattuck lecture. Nitric oxide and cyclic GMP in cell signaling and drug development. N Engl J Med 2006;355:2003–11. 18. Mellion BT, Ignarro LJ, Ohlstein EH, Pontecorvo EG, Hyman AL, Kadowitz PJ. Evidence for the inhibitory role of guanosine 30 ,50 monophosphate in ADP-induced human platelet aggregation in the presence of nitric oxide and related vasodilators. Blood 1981;57:946–55. 19. Wobst J, Schunkert H, Kessler T. Genetic alterations in the NOcGMP pathway and cardiovascular risk. Nitric Oxide 2018;76:105–12. 20. Casas JP, Bautista LE, Humphries SE, Hingorani AD. Endothelial nitric oxide synthase genotype and ischemic heart disease: metaanalysis of 26 studies involving 23028 subjects. Circulation 2004;109:1359–65. 21. Loscalzo J. Nitric oxide signaling and atherothrombosis redux: evidence from experiments of nature and implications for therapy. Circulation 2018;137:233–6. 22. Kessler T, Wobst J, Wolf B, Eckhold J, Vilne B, Hollstein R, von Ameln S, Dang TA, Sager HB, Moritz Rumpf P, Aherrahrou R, Kastrati A, Bjorkegren JLM, Erdmann J, Lusis AJ, Civelek M, Kaiser FJ, Schunkert H. Functional characterization of the GUCY1A3 coronary artery disease risk locus. Circulation 2017;136:476–89. 23. Erdmann J, Stark K, Esslinger UB, Rumpf PM, Koesling D, de Wit C, Kaiser FJ, Braunholz D, Medack A, Fischer M, Zimmermann ME, Tennstedt S, Graf E, Eck S, Aherrahrou Z, Nahrstaedt J, Willenborg C, Bruse P, Braenne I, Nothen MM, Hofmann P, Braund PS, Mergia E, Reinhard W, Burgdorf C, Schreiber S,

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39.

40. 41.

PART I Platelet Biology Balmforth AJ, Hall AS, Bertram L, Steinhagen-Thiessen E, Li SC, Marz W, Reilly M, Kathiresan S, Mcpherson R, Walter U, Cardiogram Ott J, Samani NJ, Strom TM, Meitinger T, Hengstenberg C, Schunkert H. Dysfunctional nitric oxide signalling increases risk of myocardial infarction. Nature 2013;504:432–6. Emdin CA, Khera AV, Klarin D, Natarajan P, Zekavat SM, Nomura A, Haas M, Aragam K, Ardissino D, Wilson JG, Schunkert H, Mcpherson R, Watkins H, Elosua R, Bown MJ, Samani NJ, Baber U, Erdmann J, Gormley P, Palotie A, Stitziel NO, Gupta N, Danesh J, Saleheen D, Gabriel S, Kathiresan S. Phenotypic consequences of a genetic predisposition to enhanced nitric oxide signaling. Circulation 2018;137:222–32. Marjanovic JA, Li Z, Stojanovic A, Du X. Stimulatory roles of nitricoxide synthase 3 and guanylyl cyclase in platelet activation. J Biol Chem 2005;280:37430–8. Ozuyaman B, Godecke A, Kusters S, Kirchhoff E, Scharf RE, Schrader J. Endothelial nitric oxide synthase plays a minor role in inhibition of arterial thrombus formation. Thromb Haemost 2005;93:1161–7. Freedman JE, Sauter R, Battinelli EM, Ault K, Knowles C, Huang PL, Loscalzo J. Deficient platelet-derived nitric oxide and enhanced hemostasis in mice lacking the NOSIII gene. Circ Res 1999;84:1416–21. Dayal S, Wilson KM, Leo L, Arning E, Bottiglieri T, Lentz SR. Enhanced susceptibility to arterial thrombosis in a murine model of hyperhomocysteinemia. Blood 2006;108:2237–43. Friebe A, Koesling D. The function of NO-sensitive guanylyl cyclase: what we can learn from genetic mouse models. Nitric Oxide 2009;21:149–56. Friebe A, Mergia E, Dangel O, Lange A, Koesling D. Fatal gastrointestinal obstruction and hypertension in mice lacking nitric oxide-sensitive guanylyl cyclase. Proc Natl Acad Sci U S A 2007;104:7699–704. Mergia E, Friebe A, Dangel O, Russwurm M, Koesling D. Spare guanylyl cyclase NO receptors ensure high NO sensitivity in the vascular system. J Clin Invest 2006;116:1731–7. Dangel O, Mergia E, Karlisch K, Groneberg D, Koesling D, Friebe A. Nitric oxide-sensitive guanylyl cyclase is the only nitric oxide receptor mediating platelet inhibition. J Thromb Haemost 2010;8:1343–52. Thoonen R, Cauwels A, Decaluwe K, Geschka S, Tainsh RE, Delanghe J, Hochepied T, de Cauwer L, Rogge E, Voet S, Sips P, Karas RH, Bloch KD, Vuylsteke M, Stasch JP, van de Voorde J, Buys ES, Brouckaert P. Cardiovascular and pharmacological implications of haem-deficient NO-unresponsive soluble guanylate cyclase knock-in mice. Nat Commun 2015;6:8482. Zhang G, Xiang B, Dong A, Skoda RC, Daugherty A, Smyth SS, Du X, Li Z. Biphasic roles for soluble guanylyl cyclase (sGC) in platelet activation. Blood 2011;118:3670–9. Walter U, Gambaryan S. cGMP and cGMP-dependent protein kinase in platelets and blood cells. Handb Exp Pharmacol 2009;533–48. Aburima A, Walladbegi K, Wake JD, Naseem KM. cGMP signaling inhibits platelet shape change through regulation of the RhoA-rho kinase-MLC phosphatase signaling pathway. J Thromb Haemost 2017;15:1668–78. Antl M, von Bruhl ML, Eiglsperger C, Werner M, Konrad I, Kocher T, Wilm M, Hofmann F, Massberg S, Schlossmann J. IRAG mediates NO/cGMP-dependent inhibition of platelet aggregation and thrombus formation. Blood 2007;109:552–9. Massberg S, Sausbier M, Klatt P, Bauer M, Pfeifer A, Siess W, Fassler R, Ruth P, Krombach F, Hofmann F. Increased adhesion and aggregation of platelets lacking cyclic guanosine 30 ,50 monophosphate kinase I. J Exp Med 1999;189:1255–64. Eigenthaler M, Ullrich H, Geiger J, Horstrup K, Honig-Liedl P, Wiebecke D, Walter U. Defective nitrovasodilator-stimulated protein phosphorylation and calcium regulation in cGMP-dependent protein kinase-deficient human platelets of chronic myelocytic leukemia. J Biol Chem 1993;268:13526–31. Li Z, Xi X, Gu M, Feil R, Ye RD, Eigenthaler M, Hofmann F, Du X. A stimulatory role for cGMP-dependent protein kinase in platelet activation. Cell 2003;112:77–86. Makhoul S, Walter E, Pagel O, Walter U, Sickmann A, Gambaryan S, Smolenski A, Zahedi RP, Jurk K. Effects of the

42. 43. 44. 45. 46. 47. 48.

49.

50.

51.

52. 53.

54. 55.

56. 57.

58. 59.

60. 61. 62.

NO/soluble guanylate cyclase/cGMP system on the functions of human platelets. Nitric Oxide 2018;76:71–80. Estevez B, Du X. New concepts and mechanisms of platelet activation signaling. Physiology (Bethesda) 2017;32:162–77. Gambaryan S, Tsikas D. A review and discussion of platelet nitric oxide and nitric oxide synthase: do blood platelets produce nitric oxide from L-arginine or nitrite? Amino Acids 2015;47:1779–93. Daeron M, Jaeger S, du Pasquier L, Vivier E. Immunoreceptor tyrosine-based inhibition motifs: a quest in the past and future. Immunol Rev 2008;224:11–43. Cannons JL, Tangye SG, Schwartzberg PL. SLAM family receptors and SAP adaptors in immunity. Annu Rev Immunol 2011;29:665–705. Newman DK. The Y’s that bind: negative regulators of Src family kinase activity in platelets. J Thromb Haemost 2009;7(Suppl. 1): 195–9. Smith KG, Clatworthy MR. FcgammaRIIB in autoimmunity and infection: evolutionary and therapeutic implications. Nat Rev Immunol 2010;10:328–43. Mazharian A, Mori J, Wang YJ, Heising S, Neel BG, Watson SP, Senis YA. Megakaryocyte-specific deletion of the protein-tyrosine phosphatases Shp1 and Shp2 causes abnormal megakaryocyte development, platelet production, and function. Blood 2013;121:4205–20. Mazharian A, Wang YJ, Mori J, Bem D, Finney B, Heising S, Gissen P, White JG, Berndt MC, Gardiner EE, Nieswandt B, Douglas MR, Campbell RD, Watson SP, Senis YA. Mice lacking the ITIM-containing receptor G6b-B exhibit macrothrombocytopenia and aberrant platelet function. Sci Signal 2012;5:ra78. Smith CW, Thomas SG, Raslan Z, Patel P, Byrne M, Lordkipanidze M, Bem D, Meyaard L, Senis YA, Watson SP, Mazharian A. Mice lacking the inhibitory collagen receptor LAIR-1 exhibit a mild thrombocytosis and hyperactive platelets. Arterioscler Thromb Vasc Biol 2017;37:823–35. Newman PJ, Newman DK. Signal transduction pathways mediated by PECAM-1: new roles for an old molecule in platelet and vascular cell biology. Arterioscler Thromb Vasc Biol 2003;23:953–64. Woodfin A, Voisin MB, Nourshargh S. PECAM-1: a multifunctional molecule in inflammation and vascular biology. Arterioscler Thromb Vasc Biol 2007;27:2514–23. Newman PJ, Berndt MC, Gorski J, White GC, Lyman S, Paddock C, Muller WA. Pecam-1 (Cd31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily. Science 1990;247:1219–22. Simmons DL, Walker C, Power C, Pigott R. Molecular cloning of CD31, a putative intercellular adhesion molecule closely related to carcinoembryonic antigen. J Exp Med 1990;171:2147–52. Stockinger H, Gadd SJ, Eher R, Majdic O, Schreiber W, Kasinrerk W, Strass B, Schnabl E, Knapp W. Molecular characterization and functional analysis of the leukocyte surface protein CD31. J Immunol 1990;145:3889–97. Privratsky JR, Newman PJ. PECAM-1: regulator of endothelial junctional integrity. Cell Tissue Res 2014;355:607–19. Burkhart JM, Vaudel M, Gambaryan S, Radau S, Walter U, Martens L, Geiger J, Sickmann A, Zahedi RP. The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways. Blood 2012;120:e73–82. Zeiler M, Moser M, Mann M. Copy number analysis of the murine platelet proteome spanning the complete abundance range. Mol Cell Proteomics 2014;13:3435–45. Mazurov AV, Vinogradov DV, Kabaeva NV, Antonova GN, Romanov YA, Vlasik TN, Antonov AS, Smirnov VN. A monoclonal antibody, VM64, reacts with a 130 kDa glycoprotein common to platelets and endothelial cells: heterogeneity in antibody binding to human aortic endothelial cells. Thromb Haemost 1991;66:494–9. Metzelaar MJ, Korteweg J, Sixma JJ, Nieuwenhuis HK. Biochemical characterization of PECAM-1 (CD31 antigen) on human platelets. Thromb Haemost 1991;66:700–7. Newman PJ. The role of PECAM-1 in vascular cell biology. Ann N Y Acad Sci 1994;714:165–74. Wu XW, Lian EC. Binding properties and inhibition of platelet aggregation by a monoclonal antibody to CD31 (PECAM-1). Arterioscler Thromb Vasc Biol 1997;17:3154–8.

Platelet Inhibitory Receptors 63. Jones CI, Garner SF, Moraes LA, Kaiser WJ, Rankin A, Bloodomics C, Ouwehand WH, Goodall AH, Gibbins JM. PECAM-1 expression and activity negatively regulate multiple platelet signaling pathways. FEBS Lett 2009;583:3618–24. 64. Bergom C, Paddock C, Gao C, Holyst T, Newman DK, Newman PJ. An alternatively spliced isoform of PECAM-1 is expressed at high levels in human and murine tissues, and suggests a novel role for the C-terminus of PECAM-1 in cytoprotective signaling. J Cell Sci 2008;121:1235–42. 65. Paddock C, Lytle BL, Peterson FC, Holyst T, Newman PJ, Volkman BF, Newman DK. Residues within a lipid-associated segment of the PECAM-1 cytoplasmic domain are susceptible to inducible, sequential phosphorylation. Blood 2011;117:6012–23. 66. Newton JP, Hunter AP, Simmons DL, Buckley CD, Harvey DJ. CD31 (PECAM-1) exists as a dimer and is heavily N-glycosylated. Biochem Biophys Res Commun 1999;261:283–91. 67. Newton JP, Buckley CD, Jones EY, Simmons DL. Residues on both faces of the first immunoglobulin fold contribute to homophilic binding sites of PECAM-1/CD31. J Biol Chem 1997;272: 20555–63. 68. Paddock C, Zhou D, Lertkiatmongkol P, Newman PJ, Zhu J. Structural basis for PECAM-1 homophilic binding. Blood 2016;127:1052–61. 69. Sun J, Williams J, Yan HC, Amin KM, Albelda SM, Delisser HM. Platelet endothelial cell adhesion molecule-1 (PECAM-1) homophilic adhesion is mediated by immunoglobulin-like domains 1 and 2 and depends on the cytoplasmic domain and the level of surface expression. J Biol Chem 1996;271:18561–70. 70. Sun QH, Delisser HM, Zukowski MM, Paddock C, Albelda SM, Newman PJ. Individually distinct Ig homology domains in PECAM-1 regulate homophilic binding and modulate receptor affinity. J Biol Chem 1996;271:11090–8. 71. Buckley CD, Doyonnas R, Newton JP, Blystone SD, Brown EJ, Watt SM, Simmons DL. Identification of alpha v beta 3 as a heterotypic ligand for CD31/PECAM-1. J Cell Sci 1996;109 (Pt 2):437–45. 72. Piali L, Hammel P, Uherek C, Bachmann F, Gisler RH, Dunon D, Imhof BA. CD31/PECAM-1 is a ligand for alpha v beta 3 integrin involved in adhesion of leukocytes to endothelium. J Cell Biol 1995;130:451–60. 73. Deaglio S, Morra M, Mallone R, Ausiello CM, Prager E, Garbarino G, Dianzani U, Stockinger H, Malavasi F. Human CD38 (ADP-ribosyl cyclase) is a counter-receptor of CD31, an Ig superfamily member. J Immunol 1998;160:395–402. 74. Sachs UJ, Andrei-Selmer CL, Maniar A, Weiss T, Paddock C, Orlova VV, Choi EY, Newman PJ, Preissner KT, Chavakis T, Santoso S. The neutrophil-specific antigen CD177 is a counterreceptor for platelet endothelial cell adhesion molecule-1 (CD31). J Biol Chem 2007;282:23603–12. 75. Duncan GS, Andrew DP, Takimoto H, Kaufman SA, Yoshida H, Spellberg J, de la Pompa JL, Elia A, Wakeham A, Karan-Tamir B, Muller WA, Senaldi G, Zukowski MM, Mak TW. Genetic evidence for functional redundancy of platelet/endothelial cell adhesion molecule-1 (PECAM-1): CD31-deficient mice reveal PECAM-1dependent and PECAM-1-independent functions. J Immunol 1999;162:3022–30. 76. Dhanjal TS, Ross EA, Auger JM, Mccarty OJ, Hughes CE, Senis YA, Buckley CD, Watson SP. Minimal regulation of platelet activity by PECAM-1. Platelets 2007;18:56–67. 77. Patil S, Newman DK, Newman PJ. Platelet endothelial cell adhesion molecule-1 serves as an inhibitory receptor that modulates platelet responses to collagen. Blood 2001;97:1727–32. 78. Wee JL, Jackson DE. The Ig-ITIM superfamily member PECAM-1 regulates the “outside-in” signaling properties of integrin alpha (IIb)beta3 in platelets. Blood 2005;106:3816–23. 79. Cicmil M, Thomas JM, Leduc M, Bon C, Gibbins JM. Platelet endothelial cell adhesion molecule-1 signaling inhibits the activation of human platelets. Blood 2002;99:137–44. 80. Jones KL, Hughan SC, Dopheide SM, Farndale RW, Jackson SP, Jackson DE. Platelet endothelial cell adhesion molecule-1 is a negative regulator of platelet-collagen interactions. Blood 2001;98:1456–63. 81. Varon D, Jackson DE, Shenkman B, Dardik R, Tamarin I, Savion N, Newman PJ. Platelet/endothelial cell adhesion molecule-1 serves as a costimulatory agonist receptor that

82.

83. 84. 85.

86. 87.

88.

89.

90.

91.

92.

93.

94.

95.

96.

97. 98.

291

modulates integrin-dependent adhesion and aggregation of human platelets. Blood 1998;91:500–7. Falati S, Patil S, Gross PL, Stapleton M, Merrill-Skoloff G, Barrett NE, Pixton KL, Weiler H, Cooley B, Newman DK, Newman PJ, Furie BC, Furie B, Gibbins JM. Platelet PECAM-1 inhibits thrombus formation in vivo. Blood 2006;107:535–41. Vollmar B, Schmits R, Kunz D, Menger MD. Lack of in vivo function of CD31 in vascular thrombosis. Thromb Haemost 2001;85:160–4. Mahooti S, Graesser D, Patil S, Newman P, Duncan G, Mak T, Madri JA. PECAM-1 (CD31) expression modulates bleeding time in vivo. Am J Pathol 2000;157:75–81. Dhanjal TS, Pendaries C, Ross EA, Larson MK, Protty MB, Buckley CD, Watson SP. A novel role for PECAM-1 in megakaryocytokinesis and recovery of platelet counts in thrombocytopenic mice. Blood 2007;109:4237–44. Wu Y, Welte T, Michaud M, Madri JA. PECAM-1: a multifaceted regulator of megakaryocytopoiesis. Blood 2007;110:851–9. Modderman PW, von dem Borne AE, Sonnenberg A. Tyrosine phosphorylation of P-selectin in intact platelets and in a disulphide-linked complex with immunoprecipitated pp60c-src. Biochem J 1994;299:613–21. Pt 3. Newman PJ, Hillery CA, Albrecht R, Parise LV, Berndt MC, Mazurov AV, Dunlop LC, Zhang J, Rittenhouse SE. Activation-dependent changes in human platelet PECAM-1: phosphorylation, cytoskeletal association, and surface membrane redistribution. J Cell Biol 1992;119:239–46. Zehnder JL, Hirai K, Shatsky M, Mcgregor JL, Levitt LJ, Leung LL. The cell adhesion molecule CD31 is phosphorylated after cell activation. Down-regulation of CD31 in activated T lymphocytes. J Biol Chem 1992;267:5243–9. Cicmil M, Thomas JM, Sage T, Barry FA, Leduc M, Bon C, Gibbins JM. Collagen, convulxin, and thrombin stimulate aggregation-independent tyrosine phosphorylation of CD31 in platelets—evidence for the involvement of Src family kinases. J Biol Chem 2000;275:27339–47. Edmead CE, Crosby DA, Southcott M, Poole AW. Thrombininduced association of SHP-2 with multiple tyrosinephosphorylated proteins in human platelets. FEBS Lett 1999;459:27–32. Jackson DE, Kupcho KR, Newman PJ. Characterization of phosphotyrosine binding motifs in the cytoplasmic domain of platelet endothelial cell adhesion molecule-1 (PECAM-1) that are required for the cellular association and activation of the protein-tyrosine phosphatase, SHP-2. J Biol Chem 1997;272: 24868–75. Jackson DE, Ward CM, Wang RG, Newman PJ. The proteintyrosine phosphatase SHP-2 binds platelet/endothelial cell adhesion molecule-1 (PECAM-1) and forms a distinct signaling complex during platelet aggregation—evidence for a mechanistic link between PECAM-1- and integrin-mediated cellular signaling. J Biol Chem 1997;272:6986–93. Relou IAM, Gorter G, Ferreira IA, van Rijn HJM, Akkerman JWN. Platelet endothelial cell adhesion molecule-1 (PECAM-1) inhibits low density lipoprotein-induced signaling in platelets. J Biol Chem 2003;278:32638–44. Lee FA, van Lier M, Relou IAM, Foley L, Akkerman JWN, Heijnen HFG, Farndale RW. Lipid rafts facilitate the interaction of PECAM-1 with the glycoprotein VI-FcR gamma-chain complex in human platelets. J Biol Chem 2006;281:39330–8. Hua CT, Gamble JR, Vadas MA, Jackson DE. Recruitment and activation of SHP-1 protein-tyrosine phosphatase by human platelet endothelial cell adhesion molecule-1 (PECAM-1)— Identification of immunoreceptor tyrosine-based inhibitory motif-like binding motifs and substrates. J Biol Chem 1998;273: 28332–40. Lu TT, Yan LG, Madri JA. Integrin engagement mediates tyrosine dephosphorylation on platelet-endothelial cell adhesion molecule 1. Proc Natl Acad Sci U S A 1996;93:11808–13. Tourdot BE, Brenner MK, Keough KC, Holyst T, Newman PJ, Newman DK. Immunoreceptor tyrosine-based inhibitory motif (ITIM)-mediated inhibitory signaling is regulated by sequential phosphorylation mediated by distinct nonreceptor tyrosine kinases: a case study involving PECAM-1. Biochemistry 2013;52: 2597–608.

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99. Pumphrey NJ, Taylor V, Freeman S, Douglas MR, Bradfield PF, Young SP, Lord JM, Wakelam MJO, Bird IN, Salmon M, Buckley CD. Differential association of cytoplasmic signalling molecules SHP-1, SHP-2, SHIP and phospholipase C-gamma l with PECAM-1/CD31. FEBS Lett 1999;450:77–83. 100. de Graaf CA, Choi J, Baldwin TM, Bolden JE, Fairfax KA, Robinson AJ, Biben C, Morgan C, Ramsay K, Ng AP, Kauppi M, Kruse EA, Sargeant TJ, Seidenman N, D’Amico A, D’Ombrain MC, Lucas EC, Koernig S, Baz Morelli A, Wilson MJ, Dower SK, Williams B, Heazlewood SY, Hu Y, Nilsson SK, Wu L, Smyth GK, Alexander WS, Hilton DJ. Haemopedia: an expression atlas of murine hematopoietic cells. Stem Cell Rep 2016;7:571–82. 101. de Vet EC, Aguado B, Campbell RD. G6b, a novel immunoglobulin superfamily member encoded in the human major histocompatibility complex, interacts with SHP-1 and SHP-2. J Biol Chem 2001;276:42070–6. 102. Macaulay IC, Tijssen MR, Thijssen-Timmer DC, Gusnanto A, Steward M, Burns P, Langford CF, Ellis PD, Dudbridge F, Zwaginga JJ, Watkins NA, van der Schoot CE, Ouwehand WH. Comparative gene expression profiling of in vitro differentiated megakaryocytes and erythroblasts identifies novel activatory and inhibitory platelet membrane proteins. Blood 2007;109:3260–9. 103. Newland SA, Macaulay IC, Floto AR, De Vet EC, Ouwehand WH, Watkins NA, Lyons PA, Campbell DR. The novel inhibitory receptor G6B is expressed on the surface of platelets and attenuates platelet function in vitro. Blood 2007;109:4806–9. 104. Senis YA, Tomlinson MG, Garcia A, Dumon S, Heath VL, Herbert J, Cobbold SP, Spalton JC, Ayman S, Antrobus R, Zitzmann N, Bicknell R, Frampton J, Authi KS, Martin A, Wakelam MJ, Watson SP. A comprehensive proteomics and genomics analysis reveals novel transmembrane proteins in human platelets and mouse megakaryocytes including G6b-B, a novel immunoreceptor tyrosine-based inhibitory motif protein. Mol Cell Proteomics 2007;6:548–64. 105. Ribas G, Neville M, Wixon JL, Cheng J, Campbell RD. Genes encoding three new members of the leukocyte antigen 6 superfamily and a novel member of Ig superfamily, together with genes encoding the regulatory nuclear chloride ion channel protein (hRNCC) and an N omega-N omega-dimethylarginine dimethylaminohydrolase homologue, are found in a 30-kb segment of the MHC class III region. J Immunol 1999;163:278–87. 106. de Vet EC, Newland SA, Lyons PA, Aguado B, Campbell RD. The cell surface receptor G6b, a member of the immunoglobulin superfamily, binds heparin. FEBS Lett 2005;579:2355–8. 107. V€ ogtle T, Rajesh S, Douglas MR, Senis YA. OC 66.4 Perlecan: a physiological ligand for the inhibitory platelet receptor G6b-B. Res Pract Thromb Haemost 2017;1:251. 108. Coxon CH, Sadler AJ, Huo J, Campbell RD. An investigation of hierarchical protein recruitment to the inhibitory platelet receptor, G6B-b. PLoS One 2012;7:e49543. 109. Geer MJ, van Geffen JP, Gopalasingam P, Vogtle T, Smith CW, Heising S, Kuijpers MJE, Tullemans BME, Jarvis GE, Eble JA, Jeeves M, Overduin M, Heemskerk JWM, Mazharian A, Senis YA. Uncoupling of the ITIM receptor G6b-B from the tyrosine phosphatases Shp1 and Shp2 disrupts platelet homeostasis in mice. Blood 2018;132(13):1413–25. 110. Hofmann I, Geer MJ, Vogtle T, Crispin A, Campagna DR, Barr A, Calicchio ML, Heising S, van Geffen JP, Kuijpers MJE, Heemskerk JWM, Eble JA, Schmitz-Abe K, Obeng EA, Douglas M, Freson K, Pondarre C, Favier R, Jarvis GE, Markianos K, Turro E, Ouwehand WH, Mazharian A, Fleming MD, Senis YA. Congenital macrothrombocytopenia with focal myelofibrosis due to mutations in human G6b-B is rescued in humanized mice. Blood 2018;132(13):1399–412. 111. Melhem M, Abu-Farha M, Antony D, Madhoun AA, Bacchelli C, Alkayal F, Alkhairi I, John S, Alomari M, Beales PL, Alsmadi O. Novel G6B gene variant causes familial autosomal recessive thrombocytopenia and anemia. Eur J Haematol 2017;98:218–27. 112. Bix G, Iozzo RA, Woodall B, Burrows M, Mcquillan A, Campbell S, Fields GB, Iozzo RV. Endorepellin, the C-terminal angiostatic module of perlecan, enhances collagen-platelet responses via the alpha2beta1-integrin receptor. Blood 2007;109:3745–8.

113. Gubbiotti MA, Neill T, Iozzo RV. A current view of perlecan in physiology and pathology: a mosaic of functions. Matrix Biol 2017;57–58:285–98. 114. Lebbink RJ, de Ruiter T, Adelmeijer J, Brenkman AB, van Helvoort JM, Koch M, Farndale RW, Lisman T, Sonnenberg A, Lenting PJ, Meyaard L. Collagens are functional, high affinity ligands for the inhibitory immune receptor LAIR-1. J Exp Med 2006;203:1419–25. 115. Meyaard L. The inhibitory collagen receptor LAIR-1 (CD305). J Leukoc Biol 2008;83:799–803. 116. Steevels TA, Westerlaken GH, Tijssen MR, Coffer PJ, Lenting PJ, Akkerman JW, Meyaard L. Co-expression of the collagen receptors leukocyte-associated immunoglobulin-like receptor-1 and glycoprotein VI on a subset of megakaryoblasts. Haematologica 2010;95:2005–12. 117. Verbrugge A, Rijkers ES, de Ruiter T, Meyaard L. Leukocyteassociated Ig-like receptor-1 has SH2 domain-containing phosphatase-independent function and recruits C-terminal Src kinase. Eur J Immunol 2006;36:190–8. 118. Washington AV, Quigley L, Mcvicar DW. Initial characterization of TREM-like transcript (TLT)-1: a putative inhibitory receptor within the TREM cluster. Blood 2002;100:3822–4. 119. Washington AV, Schubert RL, Quigley L, Disipio T, Feltz R, Cho EH, Mcvicar DW. A TREM family member, TLT-1, is found exclusively in the α-granules of megakaryocytes and platelets. Blood 2004;104:1042–7. 120. Barrow AD, Astoul E, Floto A, Brooke G, Relou IA, Jennings NS, Smith KG, Ouwehand W, Farndale RW, Alexander DR, Trowsdale J. Cutting edge: TREM-like transcript-1, a platelet immunoreceptor tyrosine-based inhibition motif encoding costimulatory immunoreceptor that enhances, rather than inhibits, calcium signaling via SHP-2. J Immunol 2004;172:5838–42. 121. Giomarelli B, Washington VA, Chisholm MM, Quigley L, Mcmahon JB, Mori T, Mcvicar DW. Inhibition of thrombininduced platelet aggregation using human single-chain Fv antibodies specific for TREM-like transcript-1. Thromb Haemost 2007;97:955–63. 122. Washington AV, Gibot S, Acevedo I, Gattis J, Quigley L, Feltz R, de la Mota A, Schubert RL, Gomez-Rodriguez J, Cheng J, Dutra A, Pak E, Chertov O, Rivera L, Morales J, Lubkowski J, Hunter R, Schwartzberg PL, Mcvicar DW. TREM-like transcript-1 protects against inflammation-associated hemorrhage by facilitating platelet aggregation in mice and humans. J Clin Invest 2009;119:1489–501. 123. Kammerer R, Hahn S, Singer BB, Luo JS, von Kleist S. Biliary glycoprotein (CD66a), a cell adhesion molecule of the immunoglobulin superfamily, on human lymphocytes: structure, expression and involvement in T cell activation. Eur J Immunol 1998;28:3664–74. 124. Prall F, Nollau P, Neumaier M, Haubeck HD, Drzeniek Z, Helmchen U, Loning T, Wagener C. CD66a (BGP), an adhesion molecule of the carcinoembryonic antigen family, is expressed in epithelium, endothelium, and myeloid cells in a wide range of normal human tissues. J Histochem Cytochem 1996;44: 35–41. 125. Gray-Owen SD, Blumberg RS. CEACAM1: contact-dependent control of immunity. Nat Rev Immunol 2006;6:433–46. 126. Wong C, Liu Y, Yip J, Chand R, Wee JL, Oates L, Nieswandt B, Reheman A, Ni H, Beauchemin N, Jackson DE. CEACAM1 negatively regulates platelet-collagen interactions and thrombus growth in vitro and in vivo. Blood 2009;113:1818–28. 127. Rowley JW, Oler AJ, Tolley ND, Hunter BN, Low EN, Nix DA, Yost CC, Zimmerman GA, Weyrich AS. Genome-wide RNA-seq analysis of human and mouse platelet transcriptomes. Blood 2011;118:e101–11. 128. Lewandrowski U, Wortelkamp S, Lohrig K, Zahedi RP, Wolters DA, Walter U, Sickmann A. Platelet membrane proteomics: a novel repository for functional research. Blood 2009;114: e10–9. 129. Alshahrani MM, Yang E, Yip J, Ghanem SS, Abdallah SL, Deangelis AM, O’Malley CJ, Moheimani F, Najjar SM, Jackson DE. CEACAM2 negatively regulates hemi (ITAM-bearing) GPVI and CLEC-2 pathways and thrombus growth in vitro and in vivo. Blood 2014;124:2431–41.

Platelet Inhibitory Receptors 130. Yip J, Alshahrani M, Beauchemin N, Jackson DE. CEACAM1 regulates integrin alphaIIbbeta3-mediated functions in platelets. Platelets 2016;27:168–77. 131. Zebhauser R, Kammerer R, Eisenried A, Mclellan A, Moore T, Zimmermann W. Identification of a novel group of evolutionarily conserved members within the rapidly diverging murine Cea family. Genomics 2005;86:566–80. 132. Han E, Phan D, Lo P, Poy MN, Behringer R, Najjar SM, Lin SH. Differences in tissue-specific and embryonic expression of mouse Ceacam1 and Ceacam2 genes. Biochem J 2001;355: 417–23. 133. Heinrich G, Ghosh S, Deangelis AM, Schroeder-Gloeckler JM, Patel PR, Castaneda TR, Jeffers S, Lee AD, Jung DY, Zhang Z, Opland DM, Myers Jr MG, Kim JK, Najjar SM. Carcinoembryonic antigen-related cell adhesion molecule 2 controls energy balance and peripheral insulin action in mice. Gastroenterology 2010;139:644–52. 652 e1. 134. Robitaille J, Izzi L, Daniels E, Zelus B, Holmes KV, Beauchemin N. Comparison of expression patterns and cell adhesion properties of the mouse biliary glycoproteins Bbgp1 and Bbgp2. Eur J Biochem 1999;264:534–44. 135. Alshahrani MM, Kyriacou RP, O’Malley CJ, Heinrich G, Najjar SM, Jackson DE. CEACAM2 positively regulates integrin alphaIIbbeta3mediated platelet functions. Platelets 2016;27:743–50.

293

136. van der Touw W, Chen HM, Pan PY, Chen SH. LILRB receptormediated regulation of myeloid cell maturation and function. Cancer Immunol Immunother 2017;66:1079–87. 137. Fan X, Shi P, Dai J, Lu Y, Chen X, Liu X, Zhang K, Wu X, Sun Y, Wang K, Zhu L, Zhang CC, Zhang J, Chen GQ, Zheng J, Liu J. Paired immunoglobulin-like receptor B regulates platelet activation. Blood 2014;124:2421–30. 138. Kubagawa H, Cooper MD, Chen CC, Ho LH, Alley TL, Hurez V, Tun T, Uehara T, Shimada T, Burrows PD. Paired immunoglobulin-like receptors of activating and inhibitory types. Curr Top Microbiol Immunol 1999;244:137–49. 139. Zheng J, Umikawa M, Cui C, Li J, Chen X, Zhang C, Huynh H, Kang X, Silvany R, Wan X, Ye J, Canto AP, Chen SH, Wang HY, Ward ES, Zhang CC. Inhibitory receptors bind ANGPTLs and support blood stem cells and leukaemia development. Nature 2012;485:656–60. 140. Schumacher A, Denecke B, Braunschweig T, Stahlschmidt J, Ziegler S, Brandenburg LO, Stope MB, Martincuks A, Vogt M, Gortz D, Camporeale A, Poli V, Muller-Newen G, Brummendorf TH, Ziegler P. Angptl4 is upregulated under inflammatory conditions in the bone marrow of mice, expands myeloid progenitors, and accelerates reconstitution of platelets after myelosuppressive therapy. J Hematol Oncol 2015;8:64.

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