Lactadherin: An unappreciated haemostasis regulator and potential therapeutic agent

Vascular Pharmacology 101 (2018) 21–28

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Review

Lactadherin: An unappreciated haemostasis regulator and potential therapeutic agent Agnieszka Kamińskaa, Francisco J. Enguitab, Ewa Ł. Stępieńa,

T

⁎

a

Department of Medical Physics, Marian Smoluchowski Institute of Physics, Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian University, Prof. Stanisława Łojasiewicza 11 Street, Kraków 30-348, Poland b Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, Lisboa 1649-028, Portugal

A R T I C L E I N F O

A B S T R A C T

Keywords: Coagulation MFG-E8 Microvesicles Phosphatidylserine RGD

Lactadherin is a small (53–66 kDa) multifunctional glycoprotein belonging to the secreted extracellular matrix protein family. It has a multi-domain structure and is involved in many biological and physiological processes, including phagocytosis, angiogenesis, atherosclerosis, tissue remodeling, and haemostasis regulation. Lactadherin binds phosphatidylserine (PS)-enriched cell surfaces in a receptor-independent manner. Interaction between lactadherin and PS is crucial for regulation of blood coagulation processes. This review summarizes recent knowledge on the possible role of lactadherin in haemostasis control, emphasizing the great significance of the interaction between lactadherin and PS expressed on activated platelets and extracellular vesicles. The possible role of lactadherin as a therapeutic target and biomarker is also discussed.

1. Introduction 1.1. Haemostasis principle Haemostasis is a complex and tightly controlled physiological process which maintains blood in the fluid state under normal circumstances, or stops bleeding after an injury. The main components involved in haemostasis regulation are: platelets, the vessel wall, plasma coagulation factors, and a fibrinolytic system [1,2]. To preserve haemostasis, maintenance of an appropriate balance between procoagulant and anticoagulant plasma activity is very important. The traditional model of haemostasis divides this process into two stages - primary and secondary haemostasis. Primary haemostasis refers to vasoconstriction, platelet aggregation and adhesion in the site of injury, while secondary haemostasis refers to the formation of a stable fibrin clot generated by the coagulation cascade [3]. Nevertheless, the key element of a proper functioning blood coagulation system is the interaction of platelet extracellular matrix (ECM) adhesion receptors with ECM proteins originating from different cells lining central or peripheral organs such as the liver, vessel walls, smooth muscles, or even platelets [4]. The main components within the ECM that interact directly with platelets are

laminin, fibronectin, vitronectin, and collagen. Among these proteins, one specific glycoprotein released upon macrophage activation – lactadherin – seems to play a regulatory role in haemostasis via the initiation and propagation of coagulation processes. This review summarizes recent knowledge on the role of lactadherin in haemostasis regulation, emphasizing a great significance for the interaction between lactadherin and phospholipids (PLs), especially phosphatidylserine (PS), involved in this process. 1.2. Decisive role of platelets in haemostasis Platelets are very lipid-rich cell-derived blood elements [5]. As a result of their small size (1–2 μm), their plasma membrane surface is relatively large in relation to their volume and they are enriched in various haemostatic elements including surface glycoproteins such as CD36, CD41, CD42a, and integrins (α2β1, α5β1, α6β1, αIIbβ3 (IIbIIIa CD49b/CD61), αvβ3), which helps in intercellular trafficking and adhesion [6,7]. Platelet adhesion, activation, and forthcoming aggregation after injury are crucial stages in primary haemostasis. Platelets play a decisive role in the maintenance of haemostasis by adhesion to damaged endothelium in order to participate in clot formation [8].

Abbreviations: ADP, adenosine diphosphate; ATP, adenosine triphosphate; APL, acute promyelocytic leukemia; Del-1, developmental endothelial locus-1; ECM, extracellular matrix; EGF, endothelial growth factor; EVs, extracellular vesicles; FA, fatty acid; LDL, low density lipoprotein; MFG-E8, milk fat globule-epidermal growth factor 8; MPs, microparticles; P2Y1/ P2Y12, G protein-coupled purinergic signaling receptors; PAR1/4, protease-activated receptors 1/4; PE, phosphatidylethanolamine; PL, phospholipid; PMVs, platelet-derived microvesicles; PS, phosphatidylserine; RBC, red blood cell; RGD, arginine-glycine-aspartic acid; SED1, secreted protein containing EGF-like repeats and discoidin/F5/8 complement domains; SLE, systemic lupus erythematosus; SP, sphingolipid; ST, steroid; TF, tissue factor; vWF, von Willebrand Factor ⁎ Corresponding author at: Zakład Fizyki Medycznej, Instytut Fizyki im. Mariana Smoluchowskiego, Uniwersytet Jagielloński, ul. Prof. S. Łojasiewicza 11, Kraków 30-348, Poland. E-mail addresses: [email protected] (A. Kamińska), [email protected] (F.J. Enguita), [email protected] (E.Ł. Stępień). https://doi.org/10.1016/j.vph.2017.11.006 Received 31 July 2017; Received in revised form 19 September 2017; Accepted 18 November 2017 Available online 21 November 2017 1537-1891/ © 2017 Elsevier Inc. All rights reserved.

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stimulates the assembly of prothrombinase and tenase complexes - crucial activators of the coagulation cascade which accelerate fibrin formation [19,20].

According to our canonical knowledge about the circulation system, primary haemostasis is initiated by injured endothelium leading to exposure of the above mentioned procoagulant ECM proteins (laminin, collagens, fibronectin, and vitronectin). Additionally, endothelial cells secrete von Willebrand Factor (vWF) which facilitates platelet aggregation. Platelet adhesion is the result of direct interaction between platelet receptors α2β1 or GPVI and collagen, this process is preceded by the formation of the GP1b-IX-V/vWF complex which then interacts with collagen. The interaction between platelet surface receptors and endothelium matrix proteins leads to the next stage, platelet activation, which induces a change in their shape to a more amorphous form with long protrusions. Finally, activated platelets release their content in the degranulation process. It has been proposed that, platelets release four types of granules through their canalicular system: (1) α-granules with adhesive proteins (platelet factor 4, vWF, fibrinogen, fibronectin, factor V, factor XI, protein S, and PAI-1); (2) dense δ-granules (nucleotides, bioactive amines – serotonin, and Ca2 +); and the lesser known (3) λgranules and (4) γ-granules similar to lysosomes with hydrolytic enzymes [9,10]. The platelet changes during the activation process are induced by numerous agonists such as thrombin, ADP, collagen, or thromboxane A2, which interact with specific receptors on the platelet plasma membrane (Table 1, Fig. 1).

1.4. Platelet-derived microvesicles and their role in the coagulation processes Platelet activation leads to their degranulation and release of nanoand micro-sized extracellular vesicles (EVs) [21]. These EVs, usually called platelet-derived microvesicles (PMVs), present PS on their surface to allow them to contribute to procoagulant activity [22]. The PMVs are shed from the platelet surface membrane, in a process that involves cytoskeleton reorganization, membrane budding, and finally changes in lipid membrane asymmetry, resulting in external exposure of PS [23–25]. Increased PMVs formation is very often associated with pathology and is postulated to be a recognized mechanism of a hypercoagulation state [23,26,27]. In their study, Van der Zee et al. demonstrated that subpopulations of CD63- and P-selectin-positive PMVs reflect platelet activation and their concentrations are higher in patients with myocardial infarction and peripheral arterial disease [28]. Zhao et al. showed increased exposure of PS on blood cells and microparticles (MPs) which may contribute to enhanced procoagulant activity in patients with internal carotid artery stenosis who have undergone carotid artery stenting [29]. In cancer patients, levels of PS-positive platelets and PMVs were increased significantly in stage III/IV colon cancer patients, leading to the conclusion that PS-positive platelets and MPs contribute to hypercoagulability and represent a potential therapeutic target to prevent thrombosis [30]. Additionally, PMVs have a high procoagulant potential related to the PS presence on their surface and they promote the competence of tissue factor (TF) which accelerates the formation of coagulation complexes [28,31–33]. Owing to their specific properties including small size, molecular composition, and electrostatic charge, PMVs create an additional catalytic surface not only for blood coagulation factors but also for other proteins involved in coagulation and fibrinolysis processes. It is known that PMVs display surface receptors involved in haemostasis maintenance and control, including integrins (GPIIb-IIIa – αIIbβ3; GPIa-IIa – α2β1), glycoproteins (GPIb-V-IX; CD36), selectins (P-selectin), receptors for coagulation factors (VIII), and anticoagulant plasma proteins (protein S) (Table 1) [34–37]. Selectins, which are present in the α-granules of platelets and the Weibel-Palade bodies of endothelial cells can be transferred via MPs even to distant locations, and they have a crucial role in thrombus formation. It has been demonstrated that platelet origin selectin (P-selectin) induces the expression of TF on monocytes, mediates the binding of platelets to monocytes and neutrophils, and it is involved in inflammation, wound healing, and immune response [38–42].

1.3. Platelet lipids as a component of the coagulation system Platelet activation is related to changes in lipid membrane asymmetry. The lipid fraction of platelets contains common PLs, sphingolipids (SPs), steroids (STs), prenol lipids, and minor structural derivatives (positional isomers and hydrocarbon saturates of fatty acids (FA)), among which, phosphatidylethanolamine (PE) and phosphatidylserine (PS) are most the abundant [11,12]. PS is a negatively-charged aminophospholipid belonging to the family of glycerophospholipids. It represents between 2 and 10% of all lipids in nucleated mammalian cells [13]. Under normal conditions, PS is located preferentially in the cytosolic inner membrane leaflet. After platelet activation, PS is distributed from the inner space and exposed on the platelet membrane surface to facilitate platelet procoagulant activity (Fig. 1) [14]. Platelet PS exposure is necessary, but not sufficient for coagulation promotion and amplification [13,15]. Exposure of surface PS during the platelet aggregation process promotes activity of the coagulation pathway by forming electrostatic and hydrophobic interactions involved in the binding site formation for a number of vitamin K-dependent coagulation factors including activated enzymatic factors IX (IXa), X (Xa), nonenzymatic activated cofactors V (Va), VIII (VIIIa), and prothrombin [16,17]. This PS-binding has consequential biochemical and clinical implications including local increase in coagulation factors, restriction of the 2-D mobility of clotting proteins and, more essentially, induction of conformational changes upon binding [18]. What is more, PS

2. Lactadherin – structure and biological function Lactadherin also known as MFG-E8 (Milk Fat Globule-Epidermal Growth Factor 8) or SED1 (secreted protein containing EGF-like repeats and discoidin/F5/8 complement domains) belongs to the family of secreted ECM glycoproteins. In most species, lactadherin occurs as two splice variants: ~53 kDa and ~66 kDa that includes an O-glycosylated proline/threonine rich sequence [44]. The smaller variant is present in the mammary glands of adolescent female animals as well as other tissues and organs (sweat glands, bile ducts) and body fluids (serum, urine, cerebrospinal fluid). It can be expressed and released by activated macrophages, epithelial cells, immature dendritic cells, pancreatocytes, and keratinocytes [45–47]. The larger variant of the lactadherin globule is secreted into milk by mammary epithelial cells of humans, cows, or mice and it is most abundant in the fraction of milkfat-globule membranes [44,48]. The protein sequence of bovine lactadherin contains 427 amino acids and occurs in two glycosylation variants: PAS-6 (52 kDa) and PAS-7

Table 1 Major platelet receptors and their ligands. Receptor

Ligand

Receptor type

GPIb-V-IX (CD42) PAR1, PAR4 P2Y12, P2Y1 GPIIb-IIIa (αIIbβ3) GPVI GPIa-IIa (α2β1) GPIV (CD36) TPα, TPβ

vWF Thrombin ADP Fibrinogen, Laminin, vWF Collagen Collagen/laminins Thrombospondin 1, LDL Thromboxane 2

Glycoprotein G protein-coupled receptor G protein-coupled receptor Integrin Glycoprotein Integrin Glycoprotein G protein-coupled receptor

Abbreviations: ADP – adenine triphosphate; CD36 – platelet glycoprotein 4 (GPIV); GPIbV-IX – platelet glycoprotein receptor; GPVI – glycoprotein VI; LDL – low density lipoprotein; PAR1/4 – protease-activated receptors 1/4; P2Y1, P2Y12 – purinergic signaling receptors; TPα – thromboxane receptor α, TPβ – thromboxane receptor β; vWF – von Willebrand Factor (according to Heemskerk et al. [43]).

22

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Fig. 1. Platelet membrane with its receptors upon activation. Platelet activation occurs via platelet agonists and ligands (ECM proteins: laminin, fibronectin, vitronectin, lactadherin, collagen, fibrinogen), most of them contribute to phosphatidylserine (PS) exposure upon activation (Table 1). Abbreviations: ADP – adenosine diphosphate; ATP – adenosine triphosphate; CD36 – platelet glycoprotein 4 (GPIV); GPIb-V-IX – platelet glycoprotein receptor; GPVI – glycoprotein VI; LDL – low density lipoprotein; PAR1/4 – protease-activated receptors 1/4; P2Y1/P2Y12 – G protein-coupled purinergic signaling receptors; PS – phosphatidylserine.

in PL binding is described below in this review (2.2). The lactadherin EGF2 domain contains a very conservative RGD celladhesion motif which is recognized by integrin heterodimers: αVβ3 and αVβ5 [51]. The ~66 kDa splice variant includes an O-glycosylated proline/threonine rich sequence which is inserted between the EGF2 domain and C1 discoidin domain and which contains galactose, Nacetylgalactosamine, and fucose [44,52]. Nevertheless, the role of Oglycosylation is not well known, and it may increase the efficiency of secretion (in vitro, the long form has been observed in the culture supernatant whereas the short one has been shown to accumulate in cells) or it may have a substantial function in protein stability and affinity [53].

(47 kDa) with a common polypeptide core [49]. Bovine lactadherin is composed of four functional domains starting from the N-terminus: EGF1-EGF2-C1-C2 where the EGF2-like domain (homology with epidermal growth factor) contains an arginine-glycine-aspartic acid (RGD) motif and the C2 discoidin domain has homology to coagulation factors VIII and V [50]. Human lactadherin is shorter that bovine, it is composed of three domains (EGF-C1-C2), and shares 64% amino acid sequence identity with bovine lactadherin (Fig. 2). Given the homology of the C2 domain to coagulation factors VIII and V and the competition between them for PL membrane-binding sites, lactadherin could function as an anticoagulant and thereby regulate haemostasis. The role of the C2 domain 23

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Fig. 2. Structure of bovine lactadherin domains and scheme of interactions between selected domains (EGF and C2) and their receptors in target cells (integrin αVβ3, PS) during the opsonization process. Model for EGF-like domains 1 and 2 was generated by homology modelling with Phyre2 [96] and using the crystal structure of the RGD finger from the human developmental endothelial cell locus-1 (Del-1) glycoprotein as a template (PDB code: 4D90). Atomic model for C1 domain was generated by homology modelling using the same protocol and the crystal structure of the prothrombinase complex from Pseudonaja textilis as a structural template (PDB code: 4BXS). The atomic coordinates for the C2 domain correspond to the crystal structure of the bovine lactadherin C2 domain determined at 1.67 Å resolution (PDB code: 3BN6). All the structural representations were generated with PyMOL Viewer [97].

2.1. The role of lactadherin in phagocytosis

The biological function of lactadherin cannot be overestimated. It contributes to a variety of cellular interactions, and plays an important role in many physiological and pathological processes such as phagocytosis, the pathogenesis of atherosclerosis, angiogenesis, virus protection, as well as many others [52,54–56]. It may facilitate neoangiogenesis and modulate function as a cell adhesion protein in the connection of smooth muscles to fibers in arteries [57]. Furthermore, lactadherin can inhibit enzymes engaged in coagulation processes such as prothrombinase, factor X, and VIIa [58,59]. The structural model of the bovine lactadherin molecule explains its principal functional properties (Fig. 3).

The role of lactadherin in the initialization of phagocytosis has been shown in a number of studies. Lactadherin acts as an opsonin that targets apoptotic cells. After their secretion by macrophages, lactadherin molecules form a kind of a bridge between PS exposed on apoptotic cells and αVβ3 integrin on phagocytes for uptake by macrophages [52,60,61]. Dasgupta and Thiagarajan studied the effect of lactadherin on the phagocytosis of sickle red blood cells (RBCs). They showed that αVβ3 integrin present on macrophages works as a mediator of lactadherin-induced phagocytosis. In the presence of lactadherin, a higher amount of abnormal RBCs were bound to macrophages [62]. The shortened RBCs survival in sickle cell anemia is due to extravascular destruction mediated by the macrophages. The presence of PS in the 24

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Fig. 3. Structural model of bovine lactadherin showing its domains and principal functional features. The model was obtained by combining the homology models of the individual domains presented in Fig. 2 and subsequent threading with the I-TASSER suite [98]. Secondary structure and surfaces were calculated by PyMOL Viewer [97]. The position of the integrin-interacting cell attachment motif (RGD) and the surface spikes in the C2 domain, which are involved in the interaction with membrane PC, are highlighted as sticks in the model. An interactive online model prepared by the Autodesk Molviewer platform is available at the following address: https://molviewer.com/?session=c651bbf0-93d2-11e7-859b277f1a8fcbd8&tV=presentation.

motifs are both non-specific electrostatic residues and serine-headgroup specific coordinates stabilized with Ca2 +[69]. Two structural biology groups determined the crystal structure of the C2 lactadherin domain: B. Furie's group and G. E. Gilbert's group [71,72]. Lin et al. determined the crystal structure of the bovine lactadherin C2 domain at a resolution of 2.4 Å (PDB code: 2PQS) and showed it has a discoidin-like fold containing two β-sheets of five and three anti-parallel β-strands packed against one another. One β-turn and two loops containing solvent exposed hydrophobic residues extend from the C2 domain β-sandwich core [71]. Phosphatidyl-L-serine interactions with the C2 lactadherin domain have been revealed by Shao et al. and have been shown to display a more efficient PL binding of lactadherin as compared to that of factors VIII and V, despite their significant homology [72]. Interestingly, the structure by Shao et al. has been obtained at higher resolution (1.7 Å) and showed a few minor conformational changes in the spike loops (PDB code: 3BN6). Mutagenesis studies confirmed the contribution of the membrane-interactive spikes in greater flexibility and membrane conformability than the corresponding loops of factor VIII-C2 and factor V-C2 [72]. It has been previously reported that lactadherin binds PS in a stereospecific and calcium (Ca2 +)-independent manner [52]. Otzen et al. showed the detailed binding kinetics of lactadherin to PS-containing membranes (see Fig. 2). The authors suggested that lactadherin binds PS in a two-step mechanism which is sensitive to vesicles size and composition [69]. In previous work, it was also demonstrated that lactadherin prefers binding to membranes with higher curvature. The use of fluorescein-stained lactadherin demonstrated that small unilamellar vesicles have 4-time more PS-binding sites then larger ones [73]. This suggests that lactadherin can serve as a microvesicle (exosome) scavenger. In another study, it has been proven that positively-charged residues in the C2 domain are engaged in the binding to PS-exposed cells and that hydrophobic aromatic residues (Trp26, Phe81) stabilize the docking of PS with the C2 domain [74]. Several studies have compared the dynamics of PS binding to different PS-specific proteins including annexin V and lactadherin. Hu et al. demonstrated greater sensitivity of lactadherin for the detection of PS in comparison with annexin V [69]. Moreover, the lactadherin binding to PS on activated platelets or RBCs is more efficient in comparison with that of annexin V [75]. As a result of the high and Ca2 +-independent affinity of lactadherin to PS exposed on the cell surface, this protein can detect apoptotic cells earlier than annexin V. Additionally, their

outer erythrocyte membrane can be a signal for sickle RBCs recognition by macrophages and phagocytosis [63,64]. The devoting role of lactadherin in mediated phagocytosis and procoagulant/fibrinolytic activity has been demonstrated on acute promyelocytic leukemia (APL) blasts. APL blasts pretreated with lactadherin showed a more efficient engulfment by monocyte-derived macrophages and endothelial cells. This resulted in APL cell phagocytosis and the significant reduction of both thrombin and plasmin formation [65]. If phagocytosis is not efficient, apoptotic cells are not properly removed and undergo secondary necrosis resulting in the release of toxic or immunogenic intracellular materials. Such a pathomechanism has been observed in systemic lupus erythematosus (SLE), where patients with higher MFG-E8 levels have a stronger immune response with higher inflammatory biomarkers levels. This study suggests that phagocytosis is increased in a dose dependent manner at low concentrations of MFG-E8, but above the optimal level, the effect shifts toward the inhibition of phagocytosis. Elevated serum levels of MFG-E8 are associated with the presence of high-intensity cerebral lesions in SLE patients what may be associated with impaired apoptosis [64]. Furthermore, lactadherin absence leads to reduced interleukin-10 synthesis, increased interferon-γ expression, and apoptotic cell accumulation which contributes to atherosclerosis progression in MFGE8-deficient mice [52]. It was also demonstrated that lactadherin is a negative regulator of tissue fibrosis by facilitating the clearance of apoptotic cells [66].

2.2. PS and lactadherin interaction PS is a negatively charged cell abundant PL with a well described role and localization in cell membranes. PS contributes 5 to 10% of total cellular lipid content and its biological function as a signaling molecule is related to the exposure of PS on the outer side of the bilayer [67]. The list of potential PS-binding proteins is very long and extends rapidly for different categories of enzymatic and non-enzymatic proteins including annexins, coagulation factors, and SED1/MFG-E8/Del-1 proteins [55,61,68,69]. Some PS effectors (PS-binding proteins) have relatively simple structural motifs that interact with PS, like the specific discoidintype C2 domains present in coagulation factors V and VIII and lactadherin[70]. Other coagulation factors, VII, IX, X, and prothrombin have Gla domains which are characterized by coordinates specific to serineheadgroups and a Ca2 +-binding center [18,69,71]. Annexin PS-binding 25

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smooth muscle cells proliferation and promotes their invasiveness [94]. In diabetic patients, serum concentrations of MFG-E8 were negatively associated with the risk of microvascular complications [95]. These data suggest that impaired lactadherin signaling contributes to aging processes leading to atherosclerosis and neurodegeneration.

properties to bind small PS-positive vesicles can be considered as possible regulators of microvesicle docking processes. Lactadherin binds to PS more efficiently than annexin V and requires a lesser amount of PS – below 2.5%, which is the threshold for annexin V [76]. The same feature of lactadherin was presented by Poulsen et al. who investigated, in pigs, the kinetics of the PS marker technetium99 m-labelled lactadherin in comparison with PS tracer technetium99 m-labelled annexin V. Also, in this case, marker labelled lactadherin had higher affinity for PS than that of annexin V [77]. Additionally, Waehrens et al. proved that lactadherin cell attachment is mediated by PS binding without interaction with other plasma membrane components [78]. Dasgupta et al. showed that lactadherin-deficient mice had increased concentration of PMVs and therefore lactadherin plays a significant role in their clearance [79].

3. Conclusions According to the literature, lactadherin is a multifunctional protein with roles that include phagocytosis, angiogenesis, pathogenesis of arteriosclerosis, wound healing or tissue remodeling, and blood coagulation. The data presented here show that lactadherin offers a possible target for haemostasis and thrombosis regulation. The interaction between PS present on platelets or platelet-derived microvesicles and the lactadherin C2 domain plays a major role in this regulation. It has been demonstrated that the blockade of PS by lactadherin leads to inhibition of coagulation enzyme complexes; therefore, this protein may change coagulant enzyme activity and act as an effective anticoagulant. Furthermore, because of its promotional role in angiogenesis and effective clearance of apoptotic cells, lactadherin can be also seen as a therapeutic target. In conclusion, lactadherin can have two distinct potential uses in disease treatment: it can serve as (1) a therapeutic agent in regulation of the coagulation process, or (2) a therapeutic target as an active phagocytosis or a plaque formation regulator. The biological activity of lactadherin can be inhibited by blocking it with a specific antibody. This approach creates a new solution for anticancer therapy. In contrast, delivery of lactadherin as a medical agent could be used for atherosclerosis treatment by preventing plaque formation. It has been also demonstrated, that lactadherin is overexpressed in some diseases, including diabetes and bladder tumor development, making it a potential marker of disease progression.

2.3. Anticoagulant activity of lactadherin A number of studies have shown that increased exposure of PS as the effect of disease leads to increased procoagulant activity [26,27,63,80]. PS has an impact on haemostasis by binding to Xa factor which activates the transformation of prothrombin to thrombin in the blood coagulation process. What is more, PS-containing membranes could downregulate Xa factor at higher Ca2 + concentrations and upregulate it at lower concentrations of Ca2 +[81]. Lactadherin can act as an anticoagulant factor through its ability to bind PS, inositol, and glycerol on cell membranes. Shi and Gilbert indicated that lactadherin can function as a potential PL-blocking anticoagulant and the amount of lactadherin necessary to inhibit enzymatic complexes (prothrombinase, the factors X, and VIIa) is proportional to the PL concentration [14,55]. Another author showed that lactadherin can extend coagulation time 2–4 fold and is a more effective anticoagulant compared with annexin V for platelet storage [82]. Gao et al. showed that lactadherin reduces around 80% of procoagulant activity on peripheral blood cells, endothelial cells, and microparticles by blocking PS [83]. What is more, blocking of PS on the surface of platelets by lactadherin inhibits tenase and prothrombinase activity 98%. These two complexes facilitate thrombin generation.

Conflict of interest None.

2.4. Lactadherin as a regulator of vascularization

Acknowledgments

It has been shown that lactadherin is expressed in vascular tissues (aorta, endothelial and smooth muscle cells of arterioles and capillaries, and around tumor blood vessels) [56,84]. Silvestre et al. demonstrated the pro-angiogenic role of lactadherin. The interaction of this protein with αVβ3 integrin is a crucial step in VEGF-dependent (vascular endothelial growth factor) neovascularization. Lactadherin can be a potential regulator of angiogenesis because of the homology of its C2 domain with the protein Del-1 (developmental endothelial locus-1) which promotes vessel remodeling [85]. During the last decade, a part of research has focused on the role of lactadherin in cancer development [53,86]. It was demonstrated that lactadherin localizes primarily around tumor blood vessels in melanoma knockout mice, therefore it may be involved in tumor vessel formation [87]. On the other hand, lactadherin is involved in skin wound healing and improves cardiac remodeling after myocardial infarction [88,89].

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