The future of glycoprotein VI as an antithrombotic target


Martine Jandrot-Perrus, Inserm UMRS_698, Université Paris 7, Hôpital Xavier Bichat, 46 rue Henri Huchard, Paris F-75877, France.
Tel.: +33 1 40 25 86 00; fax: +33 1 40 25 86 02.


Summary.  The treatment of acute coronary syndromes has been considerably improved in recent years with the introduction of highly efficient antiplatelet drugs. However, there are still significant limitations: the recurrence of adverse vascular events remains a problem, and the improvement in efficacy is counterbalanced by an increased risk of bleeding, which is of particular importance in patients at risk of stroke. One of the most attractive targets for the development of new molecules with potential antithrombotic activity is platelet glycoprotein (GP)VI, because its blockade appears to ideally combine efficacy and safety. This review summarizes current knowledge on GPVI regarding its structure, its function, and its role in physiologic hemostasis and thrombosis. Strategies for inhibiting GPVI are presented, and evidence of the antithrombotic efficacy and safety of GPVI antagonists is provided.


The treatment of acute coronary syndromes has been considerably improved in recent years with the introduction of highly efficient antiplatelet drugs. The current standard treatment is based on dual antiplatelet therapy with aspirin and a P2Y12-targeting drug, either a thienopyridine compound, such as clopidogrel or prasugrel, or a direct reversible antagonist, such as ticagrelor [1]. However, this strategy still has significant limitations: the recurrence of adverse vascular events remains a problem, and the improvement in efficacy is counterbalanced by an increased risk of bleeding. This is of particular importance in patients at risk of stroke. Indeed, the therapeutic armamentarium for the treatment of thrombotic stroke is still rather limited. The most effective current treatment is based on recanalization by vascular thrombolysis, but only a few patients benefit from this, owing to the very short therapeutic window (≤ 4 h), and the rate of successful outcome does not exceed 40% [2]. The use of antiplatelet agents improves outcomes by reducing reocclusion, although it increases the risk of cerebral hemorrhage [3,4].

The search for better antiplatelet drugs that are able to efficiently prevent thrombus growth while having a minimal effect on physiologic hemostasis remains a challenge [1,5]. One of the most attractive targets for the development of new molecules with potential antithrombotic activity is platelet glycoprotein (GP)VI, because its blockade appears to ideally combine efficacy and safety [6].

It was in the 1980s that GPVI emerged as a candidate receptor for collagen, through identification of a patient with autoimmune thrombocytopenia whose platelets showed GPVI deficiency and no response to collagen [7]. In the late 1990s, GPVI was shown to be coupled to the common γ-chain of Fc receptors (FcRγ) [8,9] and to belong to the immunoglobulin-like receptor superfamily, while identification of the snake venom, convulxin, as a GPVI-specific ligand [10,11] and the synthesis of collagen-related peptide (CRP) [12] offered new possibilities for characterizing GPVI. Finally, human and mouse GPVI genes were cloned simultaneously by different approaches [13–15], and GPVI expression was shown to be restricted to platelets and megakaryocytes. Since then, numerous studies have contributed to the characterization of GPVI in physiology and pathology.

GPVI structure and signaling

GPVI is a type 1 transmembrane protein of 58 kDa, 45% of which is carbohydrate. The GPVI chain is organized in three main domains from the N-terminus to the C-terminus (Fig. 1A):

Figure 1.

 Structure of glycoprotein (GP)VI and signaling pathway. (A) Schematic representation of GPVI associated with the FcRγ homodimer: D1 and D2 represent the two immunoglobulin-like loops, and PRD is the proline-rich domain to which the Src-family kinase Lyn is constitutively bound. N-linked and O-linked glycosylated chains are also schematized. (B) Schematic representation of the signaling cascade downstream of GPVI. Protein tyrosine kinases are in red, adaptors are in green, and lipid kinase and phosphatase are in yellow. The stars indicate the ITAM tyrosines phosphorylated by the Src-family kinases Lyn and Fyn. DAG, diacylglycerol; IP3, inositol triphosphate; LAT, linker for activation of T-cells; PI3K, phosphoinositide 3-kinase; PIP3, phosphatidylinositol triphosphate; PKC, protein kinase C; PLC, phospholipase.

  • The extracellular domain contains two immunoglobulin C2-like folds (D1 and D2) orientated at ∼ 90° and kept at distance from the platelet surface by a mucin-like stalk proximal to the membrane [16]. The D1D2 domain exposes the structural determinants for binding to collagen. The combined use of CRP, mutagenesis, crystallography and epitope mapping has led to a proposed model in which the apical part of the D1 loop and the hinge region contain critical structures for interaction with collagen: a cluster of basic residues (Lys41, Lys59, Arg60, and Arg166) and a second, more hydrophobic, site (Leu36, Val34, and Asn72) [17–20]. The D2 loop is also involved in binding to collagen, presumably by contributing to the formation of a back-to-back dimer [16,21,22]. Interestingly, Miura et al. [23], using either dimeric (GPVI-Fc) or monomeric (GPVI-ex) soluble recombinant GPVI, demonstrated that only GPVI dimers bind to collagen with a good affinity. The use of antibodies that are able to discriminate between monomeric and dimeric forms provided evidence that platelet GPVI exists in both forms [24]. Recent data indicate that dimerization of GPVI is an active and tightly regulated process that promotes platelet binding to collagen [25].

Near to its transmembrane domain, GPVI contains a site that is susceptible to proteolytic attack by proteases. Sustained activation of GPVI by its ligands (collagen, convulxin, and CRP) results in the production of a 55-kDa soluble ectodomain fragment and a 10-kDa platelet-associated remnant fragment [26]. Activating anti-GPVI antibodies are capable of inducing depletion of GPVI from the platelet surface by a mechanism involving metalloproteinase-mediated shedding (see below). Recently, GPVI shedding was reported to be induced by transient platelet exposure to pathophysiologic shear rates, and this mechanism was proposed to support elevated levels of soluble GPVI in the plasma of patients with significant coronary stenosis [27,28]. Coagulation-mediated GPVI shedding induced by factor Xa and, accordingly, elevated levels of soluble GPVI were detected in the plasma of patients with disseminated intravascular coagulation [29].

  • The transmembrane domain of GPVI is composed of 19 amino acids. It contains one charged residue, Arg272, that forms a salt bridge with Asp11 of FcRγ. Phosphorylation of the FcRγ ITAM motif starts the signaling cascade downstream of GPVI [8,9].

  • The cytoplasmic region of human GPVI contains 51 amino acids having no apparent homology with other receptor proteins and no tyrosine. The cytoplasmic tail of mouse GPVI lacks the 24 C-terminal residues of human GPVI [14]. Two unique functional motifs are present in the intracellular domain of human and mouse GPVI: a basic amino acid-rich region close to the transmembrane domain that allows the binding of calmodulin, which is assumed to protect GPVI from the cleavage of its ectodomain by metalloproteases [26]; and a proline-rich domain (PRD) that is a docking site for the Src homology 3 domain of the Src family tyrosine kinases Fyn and Lyn [30]. This interaction confers a ‘ready-to-go’ state to the receptor, with Lyn selectively bound in an active conformation to the PRD of GPVI [31], and tethering of the FcRγ dimer within the inner layer of the membrane is assumed to protect its ITAM from phosphorylation.

The signaling pathway coupled to the GPVI–FcRγ complex is globally similar to that of other immunoreceptors (for a review, see [32]), and consists of a cascade of tyrosine phosphorylation (Fig. 1B). The bridging of several GPVI–FcRγ complexes by multivalent ligands causes unmasking of the FcRγ cytoplasmic domain, allowing phophorylation of the ITAM motif by GPVI-bound Lyn. This leads to the engagement of the tyrosine kinase spleen tyrosine kinase (Syk) via its two Src homology 2 (SH2) domains and its subsequent activation. Syk then orchestrates the assembly of a large signaling complex built on the following adaptor proteins: linker for activation of T-cells (LAT), Gads, and SH2-domain containing leukocyte-specific phosphoprotein of 76 kDa (SLP76). The sequential activation of effector enzymes, including phosphatidylinositol 3-kinases α and β, and tyrosine kinases of the Bruton family (Btk and Tec), ends in the activation of phospholipase Cγ2, which generates diacylglycerol and inositol triphosphate, leading to protein kinase C activation and the mobilization of intracellular calcium stores, respectively. Activation of additional pathways, including the small G-protein Rac1, the GTPase Rap1, and the GTP exchange factors Vav1 and Vav3, potentiates platelet responses [33,34].

GPVI function

GPVI acts as a major receptor for platelet activation by collagen in vitro. It is activated by fibrillar collagens of types I and III, but not by soluble collagen. The smallest GPVI-binding motif at the surface of collagen fibers contains two glycine–proline–hydroxyproline triplets [35]. In healthy vessels, fibrillar collagens of types I and III are predominant, play an important mechanical role, provide an anchorage for cells, and modulate cell function through receptor-mediated contacts. In atherosclerotic plaques, collagen type I is the major component of the fibrous cap, the rupture of which leads to formation of the thrombus.

Heterologous expression of GPVI at the surface of transfected cells induces cell attachment to collagen under static conditions [14]. GPVI is also required for stable platelet adhesion and thrombus growth on immobilized collagen under flow conditions [36–38]. However, synergism between collagen receptors supports optimal firm thrombus formation in flowing blood [17,39,40]. An elegant study using receptor-specific synthetic peptides confirmed that the initial association of platelets with collagen occurs via the transient interaction of von Willebrand factor (VWF) with the GPIb–V–IX complex, and that engagement of integrin α2β1 and GPVI mediates firm adhesion and platelet accumulation [41]. GPIb–V–IX becomes increasingly important at shear rates > 1000 s−1, whereas GPVI plays an activating role at all shear rates. Interestingly, the observation that shear-induced interaction of GPIb–V–IX with VWF initiates GPVI dimerization, and therefore its binding to collagen, further supports this model [25] (Fig. 2).

Figure 2.

 Glycoprotein (GP)VI fate: this diagram depicts the different steps of GPVI processing during platelet activation. In the healthy vessel, GPVI is maintained in a monomeric form by prostacyclin (prostaglandin I2 [PGI2]) and NO. At the level of the arterial lesion, the decreased levels of inhibitory compounds, and GPIb interaction with von Willebrand factor, promote GPVI dimerization and binding to collagen, leading to strong activation signals reinforced by the secretion of soluble agonists. Adhesion is stabilized by the interaction of collagen with activated α2β1, and binding of fibrinogen to activated αIIbβ3 allows aggregation and thrombus growth. Strong and prolonged GPVI activation results in the shedding of its extracellular domain by activated metalloproteases, although it is not known whether this has consequences for the stability of the thrombi. TXA2, thromboxane A2.

One important characteristic of GPVI-mediated platelet activation is related to its capacity to trigger the release of soluble secondary agonists, mainly ADP and thromboxane A2 (TXA2), that are able to activate and recruit circulating platelets. It is well known that collagen-induced platelet aggregation is impaired in the absence of ADP and TXA2, and evidence has been provided for crosstalk between GPVI and Gi-coupled receptors [42]. Such amplification loops ensure the high efficacy of thrombus formation on collagen surfaces.

Finally, collagen has long been recognized as a potent physiologic agonist inducing platelet procoagulant activity [43]. Importantly, GPVI plays a central role in collagen-induced exposure of procoagulant phospholipids at the platelet surface, allowing efficient thrombin generation [44].

The specificity of GPVI is not restricted to collagen. Laminin, present in the basement membrane, supports weak platelet activation under arterial shear flow via interactions with α6β1 and GPVI [45]. Fibronectin was also reported to interact with GPVI, and anti-GPVI antibodies reduced platelet adhesion to fibronectin [46].

GPVI in hemostasis

As GPVI is an important receptor for platelet activation, one would expect that GPVI deficiency would be of critical importance for normal hemostasis. GPVI deficiency has been identified in a few patients [47] and, surprisingly, is most often associated with only a mild bleeding tendency. This suggests that the lack of GPVI is not intrinsically a bleeding risk, but that its association with other genetic factors affecting platelet and/or endothelium hemostatic functions or environmental factors could occasionally lead to more severe hemorrhagic manifestations. The most frequent cause of GPVI deficiency is acquired immune thrombocytopenia. Usually, patients present with mild to severe thrombocytopenia and a profound defect in collagen-induced platelet activation resulting from antibody-induced GPVI depletion as described below [7,48–50]. Thrombocytopenia does not always correlate with the bleeding tendency, and GPVI deficiency does not always regress upon platelet count normalization. Patients with suspected congenital GPVI deficiency [51–54] have a normal platelet count, and a molecular defect has been identified in two of them and in both cases involves compound heterozygous GPVI mutations [53,54].

In genetically engineered mice, the lack of GPVI is obtained by invalidation of the genes coding for GPVI [37,55] or its coreceptor, FcRγ, which is required for surface expression of the complex [56]. An acquired GPVI deficiency is also induced by injection of the anti-GPVI mAb JAQ1. This results in transient thrombocytopenia and long-lasting GPVI depletion [57]. The absence of GPVI has no serious hemorrhagic impact in mice, and the longer bleeding time that is sporadically observed may be related to modifier genes [58].

Together, these observations indicate that GPVI is not an absolute requirement for normal hemostasis, the initial adhesion phase facilitated by vasoconstriction being ensured by the VWF–GPIb axis [59].

GPVI and experimental thrombosis in healthy vessels

Despite some controversies, GPVI appears to be an important contributor to thrombus formation in experimental models. The first evidence was the protective effect of JAQ1-induced GPVI deficiency on lethal thromboembolism induced by the injection of a mixture of collagen and epinephrine [57]. GPVI-deficient mice are also protected from platelet adhesion, thrombus growth, leukocyte recruitment and neointimal hyperplasia after mechanical arterial injury [60–62]. The role of GPVI was found to be important in the FeCl3-induced vascular injury model in some studies [62] but not in others [59,63], and to depend on the degree of injury in the laser-induced model [63,64]. These differences in the effect of GPVI deficiency on thrombus formation in healthy vessels can be explained by differences in the cause of the GPVI deficiency (GPVI-depleted, FcRγ−/−, or GPVI−/−), the extent and quality of collagen exposed at the site of the lesion, or by the extent of thrombin generation [65]. Nevertheless, taken together, these data tell us that, in the healthy vessel, GPVI-dependent platelet activation and tissue factor-triggered thrombin generation strongly cooperate to optimize the rate of thrombus growth and stabilization.

GPVI in atherothrombosis

It is assumed that abundant collagen fibers exposed by eroded atherosclerotic plaques tether and activate platelets, leading to acute thrombus formation. Exposure of tissue factor concomitantly triggers activation of the coagulation cascade, and, together with collagen-activated platelets, supports thrombin generation. Indeed, thrombi were observed to develop sequentially on homogenates of human atherosclerotic plaques obtained from aorta or carotid endarterectomy in arterial flow conditions, with collagen-induced platelet accumulation being followed by tissue factor-mediated fibrin formation. In two independent studies, blockade of GPVI by antibodies, 9O12 Fab and human scFv 10B12, respectively, drastically inhibited plaque-induced thrombus formation, demonstrating the key role of collagen–GPVI interaction in inducing atherothrombosis [66,67]. The importance of GPVI for thrombus formation in pathologic arteries has been recently validated in models of in vivo thrombosis induced by acute plaque rupture in ApoE−/− mice. Kuijpers et al. [68] showed that GPVI deficiency significantly impaired thrombus formation caused by ultrasound-induced plaque rupture. Hechler and Gachet [69] confirmed this observation, and also showed that GPVI is critical in a model in which the plaque is injured by a needle.

The preferred interaction of GPVI with the atherosclerotic plaque was illustrated in vivo by gamma imaging after injection of 125I-labeled recombinant soluble GPVI (GPVI-Fc) into ApoE−/− mice [70]. The same group also reported that inhibition of GPVI reduced neointima formation after wire-induced injury of the carotid artery in ApoE−/− mice, and resulted in marked attenuation of carotid atheroprogression in cholesterol-fed rabbits and ApoE−/− mice [46,70], suggesting a GPVI-mediated platelet contribution to atheroprogression.

Together, these studies provide strong arguments for considering GPVI as a therapeutic target of great interest. The predicted strengths of a GPVI clinical antagonist include:

  • 1 Good antithrombotic efficacy resulting from:

    • Early inhibition of platelet activation focused at the site of arterial lesions;

    • Reduced thrombin generation that would further limit thrombus growth;

  • 2 Preserved physiologic hemostasis and hence reduced bleeding risk.

  • 3 High specificity because of the platelet-restricted expression of GPVI.

GPVI blockade

Several strategies may be employed to inhibit GPVI-triggered platelet activation. GPVI–collagen interaction can be disrupted by collagen-binding molecules (GPVI mimics), by GPVI-binding compounds (aptamers, small molecules, and antibodies), or by GPVI depletion (Fig. 3).

Figure 3.

 How to target glycoprotein (GP)VI: different strategies for blocking GPVI activation are depicted. MMP, matrix metalloprotease.

GPVI mimics

The soluble immunoadhesin GPVI-Fc is composed of two extracellular domains of GPVI fused to the constant domain (Fc) of human IgG1. This dimeric soluble form of GPVI has a high affinity for collagen ([4 ± 0.2] × 10−9 m) and competes with platelet GPVI for collagen binding, hence limiting collagen-induced platelet aggregation [14]. Massberg et al. [71] observed that intravenous injection of GPVI-Fc reduced platelet recruitment following endothelial denudation caused by carotid artery ligation, but Gruner et al. [72] did not confirm this observation. Nevertheless, reduced thrombus formation after balloon-induced carotid injury in rabbits and protection from injury-induced remodeling and atheroprogression were observed in GPVI-Fc-treated animals [70,73]. Very recently, a phase I study demonstrated that GPVI-Fc (Revacept) efficiently inhibited collagen-induced platelet aggregation ex vivo with no alteration of primary hemostasis in 30 healthy human volunteers [74]. Potential limitations to the use of GPVI-Fc are evaluation of the amount of collagen exposed on arterial lesions, and hence the amount of product to inject, and the risk of immunization resulting from neoepitopes exposed by the fusion protein.

Another approach consists of developing low molecular weight GPVI mimics. Our team has obtained one peptidomimetic of GPVI that binds to collagen but has no inhibitory properties, owing to its lower affinity for collagen than that of dimeric GPVI. This compound is under evaluation as a promising tracer for in vivo imaging of fibrosis [75].

Small GPVI antagonists

Few small GPVI-binding molecules have so far been identified. A preliminary report described aptamers able to block GPVI interaction with collagen [76]. Losartan, a non-peptide AT1 receptor antagonist, was reported to inhibit collagen-induced platelet adhesion and activation, and structural analysis showed a hydrophobic interaction between losartan and the GPVI D1 domain [77,78]. However, the therapeutic effect of losartan is mainly to reduce blood pressure. Compounds preventing GPVI dimerization or oligomerization have been proposed as having potential therapeutic interest (for a review, see [79]). Agents increasing cAMP levels are expected to limit GPVI dimerization and binding to collagen, but their effect is non-specific, as cAMP has a global inhibitory effect on platelets. The efficacy of extracellular agents blocking homotypic interactions between the D2 domains is uncertain, as indicated by the partial inhibition of GPVI binding to CRP by scFv 1C3 [22]. Disruption of the intrareceptor GPVI–FcRγ transmembrane interactions or the homotypic interactions between the FcRγ cytoplasmic domains by small membrane-permeable molecules is expected to disconnect the receptor from downstream signaling [79]. A potential drawback of this approach is a lack of specificity, as FcRγ is the signaling subunit of several other immune receptors.

GPVI antibodies

Antibodies directed against GPVI are of interest, because some have the ability to block the interaction of GPVI with collagen [18,38], and others induce platelet GPVI depletion [80] (Table 1). The strengths of antibodies are (i) their specificity, and (ii) their capacity to easily saturate their target, owing to their high affinity and the relatively low number of GPVI copies at the platelet surface (4000 ± 1000).

Table 1.   Properties of anti-glycoprotein (GP)VI antibodies
NameTypePlatelet activationShedding of GPVIBlockade GPVI binding to collagenInhibition of GPVI-induced platelet activationReference
  1. ND, not determined.

JAQ-1Rat anti-mouse GPVIYesYes By GPVI depletion[57]
Mouse mAb against human GPVI
Chimeric mAb
NoNo, but induce GPVI internalization By GPVI depletion[80]
mF1201Mouse mAb against human GPVIYesYes By GPVI depletion[80]
9O12Mouse mAb against human GPVIYes (IgG)Yes (IgG)Yes (IgG, Fab)Yes (Fab)[38]
204-11Mouse mAb against human GPVIYesNDYes [88]
5C4Rat mAb against human GPVINDNDYesYes (Fab)[46,71]
1G5Mouse mAb against human GPVIYesYes Yes (Fab)[85]
OM2Mouse mAb against human GPVIYesNo Yes (Fab)[89]
OM4Mouse mAb against human GPVI cross-reacts with rat GPVI ND Yes (Fab)[91]
10B12hscFv against human GPVINoNDYesYes[18]
BLO8-1dAb against human GPVINoNDYes (CRP)Yes[87]

GPVI-depleting antibodies  As mentioned above, the first report of a GPVI-depleting antibody was made by Sugiyama et al. [7] in a GPVI-deficient patient. Interestingly, the antibody-induced GPVI deficiency observed in vivo could not be reproduced in vitro. Similarly, the GPVI downregulation induced by JAQ1 antibodies in mice occurs exclusively in vivo [57]. Several cases of antibody-induced GPVI depletion have since been reported, with heterogeneous behavior: some induce GPVI depletion in vivo [81] and others in vivo and in vitro [50], and in some cases, but not all, the shedding of GPVI is shown by the presence of soluble GPVI in plasma [50,82]. The mechanism of GPVI downregulation appears to be dependent on activating signals and to involve internalization of the antigen–antibody complexes and/or shedding of the GPVI extracellular domain by metalloproteases [83]. Recently, Takayama et al. [80] obtained recombinant antibodies mimicking the patient’s antibody. When injected into monkeys, the mouse mAb mF1232 and the chimeric antibody cF1232 induced loss of platelet surface GPVI by endocytosis without thrombocytopenia, and, interestingly, GPVI internalization was reproduced in vitro in the presence of cAMP-elevating agents. Antibody-induced GPVI deficiency is proposed as a novel therapeutic approach, with the potential disadvantage that the depletion is long-lasting.

Inhibitory antibodies  A small number of inhibitory antibodies have been identified. It is important to consider that inhibitory antibodies should preferably be monovalent, to avoid clustering of GPVI or interaction with FcγRIIA. This is exemplified by the mAbs 9O12 and 1G5, which activate platelets in an FcγRIIA-dependent and FcγRIIA-independent manner, and which both induce activation-dependent shedding of GPVI in vitro [84,85].

Human single-chain antibodies have been obtained by phage display [18,22,86]. The 10B12 antibody binds to the D1 domain, and inhibits platelet interactions with CRP and collagen and thrombus formation. The 1C3 antibody maps a region of the D2 domain involved in the clustering of the receptor, and partly inhibits GPVI binding to its ligands. BLO8-1 is a human domain antibody that recognizes an epitope within the collagen-binding domain of GPVI, and inhibits CRP-induced platelet activation [87]. The limit of natural antibodies is their relatively low affinity.

Several anti-human mAbs also have inhibitory properties. The 9O12, 204-11 and 5C4 antibodies were reported to limit the binding of GPVI to collagen [38,71,88]. The Fab fragments of 5C4, 1G5, OM2, OM4 and 9O12 have inhibitory effects on GPVI-mediated platelet activation in vitro and ex vivo or in vivo [38,46,85,89–93]. OM2 and OM4 were obtained by immunizing GPVI-deficient mice with human recombinant GPVI, and have good affinity for GPVI. The injection of OM2 Fab into cynomolgous monkeys resulted in inhibition of ex vivo collagen-induced platelet aggregation [90]. In a model of cyclic flow reduction in the rat carotid artery, the number of complete occlusions was significantly reduced by intravenous administration of OM4 Fab, which cross-reacts with rat GPVI without prolonging the bleeding time [91].

Numerous studies have been conducted with 9O12, and their results support the assumption that targeting GPVI has a good antithrombotic efficacy with no bleeding side effects. The 9O12 antibody is a mAb with high affinity for human GPVI (10−9 M). In addition to blocking platelet adhesion and aggregation under static and arterial flow conditions, 9O12 Fab reduced platelet procoagulant activity by preventing collagen-induced phosphatidylserine exposure [38,94]; 9O12 Fab also impaired platelet adhesion and aggregation, and reduced phosphatidylserine exposure when blood was perfused at arterial shear rates on extracts of atherosclerotic plaques [66]. The specificity of 9O12 is restricted to human and non-human primate GPVI. After administration of an intravenous bolus dose of 9O12 Fab to cynomolgous monkeys, neither the platelet count nor GPVI expression at the platelet surface was modified [92]. Binding of 9O12 Fab to the platelet surface was rapid (peak at 30–150 min postinjection) and reversible, with a 75% decrease at day 1. There was no sign of bleeding. In contrast, ex vivo collagen-induced platelet aggregation was totally inhibited, platelet adhesion to collagen and thrombus growth at arterial shear rates were reduced as well as thrombin generation.

These data were promising, but there was still a need to obtain more evidence of efficacy and safety in arterial thrombosis models. Mice are obviously not entirely satisfactory for preclinical studies, as most of the blocking antibodies directed against human GPVI do not bind to mouse GPVI, and also because sequence variability in the intracellular and extracellular domains of GPVI could influence collagen-induced responses [14,18]. We thus constructed a transgenic mouse expressing human GPVI, to be subjected to various models of arterial thrombosis [93]. These mice, obtained by knock-in, were viable and fertile, and platelets exhibited normal responses to all of the agonists tested. Surface-expressed human GPVI had a similar density to that observed on human platelets, and bound 9O12 Fab. After an intravenous bolus injection, platelet-bound 9O12 Fab peaked at 30 min. No modifications in the platelet count or GPVI expression were observed, and tail bleeding time and blood loss were not increased as compared with controls, in agreement with the data obtained in monkeys. However, ex vivo collagen-induced platelet aggregation and thrombus formation under flow conditions were profoundly impaired. The 9O12 Fab injection protected human GPVI-expressing mice against lethal thromboembolism induced by injection of a collagen/adrenaline mixture. In addition, 9O12 Fab provided protection against thrombosis after superficial laser injury of mesenteric arterioles, after deep laser injury under conditions where thrombin was blocked, and after mechanical injury to the aorta. The human GPVI-expressing mouse also allows study of the effects of GPVI antagonists on atherothrombosis by grafting of their bone marrow into irradiated ApoE−/− mice. The administration of 9O12 Fab proved to be highly effective in preventing arterial thrombosis triggered by the injury of carotid artery atherosclerotic plaques in these mice (Hechler B, Mangin P, Loyau S, Jandrot-Perrus M, Gachet C, manuscript in preparation).

Taken together, these data tell us that the human GPVI-expressing mouse is suitable for the preclinical in vivo evaluation of GPVI antagonists in terms of safety and efficacy.

Because the immunogenicity of murine antibody fragments is a major obstacle to their clinical development, it was necessary to carry out a humanization procedure. A humanized 9O12 scFv was obtained by grafting the complementarity-determining regions onto closely related human antibody variable domains and carrying out structural refinements [95]. The humanized single-chain antibody (hscFv 9O12) retained the affinity, specificity and functionality of 9O12 Fab, including inhibition of collagen-induced platelet activation. Therefore, hscFv 9O12 represents the building block for the production of a monovalent humanized Fab with pharmacokinetic properties that are more suitable for therapeutic purposes than those of scFvs.

Disruption of GPVI signaling

Blockade of GPVI signaling might also be achieved by the use of kinase inhibitors such as those developed for cancer therapy. As an example, inhibitors of Syk may be useful, and fostamatinib is a clinically available Syk inhibitor that has been investigated for the treatment of chronic lymphocytic leukemia, rheumatoid arthritis, or asthma. The fact that no bleeding side effects have so far been reported is in favor of the safety of targeting GPVI. The major drawback of this approach is its lack of specificity, for at least two reasons: Syk is a crucial player in diverse GPVI-independent biological functions, and the effects of Syk inhibitors are probably mediated by inhibition of several Syk-dependent and Syk-independent signaling pathways.

Conclusions and future directions

The development of new pharmaceutical compounds with high efficacy for thrombosis but that respect physiologic hemostasis represents a challenge. Drugs targeting GPVI appear to be promising, as they can interrupt the initial phase of platelet activation specifically in diseased vessels, and thus the subsequent cascade of events ending in thrombosis, with no identified secondary bleeding effects.

Antagonists of GPVI have great potential for the treatment of acute atherothrombotic stroke, which constitutes a therapeutic emergency. Current treatment is based on thrombolysis, with two major limits: a short therapeutic window, and limited efficacy [2]. Administration of a GPVI antagonist at the time of thrombolysis is expected to increase the rate of recanalization by limiting the contribution of platelets to the reconstruction of the clot. Interestingly, both an increase in GPVI expression at the platelet surface and an increase in plasma soluble GPVI levels were reported in patients with acute ischemic stroke, suggesting a contribution of GPVI to the pathology [96,97]. For the majority of patients (85%), who do not benefit from recanalization, there is currently no well-defined protocol [98]. Antiplatelet therapy is usually recommended to reduce the risk of recurrent stroke but, in the absence of standardized large-scale clinical studies, the current guidelines for ischemic stroke/transient ischemic attack (TIA) do not favor one antiplatelet agent over another. Dual antiplatelet therapy with aspirin and clopidogrel did not demonstrate any clear benefit as compared with aspirin alone, but did show a significant increase in bleeding [3,4]. The safety of targeting GPVI with regard to the bleeding risk offers a new possibility for the medical prevention of recurrent ischemic events.

Indeed, GPVI antagonists could also be of interest in the treatment of coronary artery disease. If the proof of concept of their efficacy and safety is obtained in stroke, the place of such new molecules in the treatment of acute coronary syndrome should be reconsidered. It is, in particular, expected that targeting of GPVI during angioplasty will be of interest to prevent thrombosis and restenosis.

Disclosure of conflict of interests

The authors state that they have no conflict of interest.