anti-GPVI Antibody Research Articles

Impact of glycoprotein VI and platelet adhesion on atherosclerosis—A possible role of fibronectin

Glycoprotein VI (GPVI) mediates binding of platelets to subendothelial collagen during acute arterial thrombosis. GPVI interactions with the activated atherosclerotic vascular endothelium during early atherosclerosis, however, are not well understood. In ApoE−/− mice, platelet adhesion to atherosclerotic arteries was increased, as measured by intravital microscopy. This platelet adhesion was significantly inhibited by IV injection of GPVI-Fc (1 mg/kg body weight). Atherosclerosis in ApoE−/− mice was attenuated both after 7 and 10 weeks of treatment with the anti-GPVI antibody JAQ1 (2 mg/kg body weight i.p. twice weekly). Binding of GPVI-Fc (1 mg/kg IV) occurred to deeper layers, but also to the luminal site of plaques in atherosclerotic rabbits, but not to the vessel wall of healthy littermates. Gene transfer of GPVI-Fc to the carotid vascular wall significantly attenuated athero-progression and endothelial dysfunction in atherosclerotic rabbits in vivo. Specific binding of the soluble GPVI receptor (GPVI-Fc) to fibronectin was found in vitro to coated ELISA plates. Platelet adhesion to fibronectin was significantly inhibited both by GPVI-Fc and by the anti-GPVI antibody 5C4 ex vivo in flow chamber experiments. GPVI plays a role in platelet adhesion to atherosclerotic endothelium in the absence of plaque rupture. Inhibition of GPVI both via GPVI-Fc and anti-GPVI-antibodies results in protection against atherosclerosis in both cholesterol-fed rabbits and ApoE−/− mice. This novel mechanism of GPVI-mediated platelet adhesion–possibly via fibronectin–could relevantly contribute to platelet-triggered atheroprogression.

Platelet-associated IgAs and impaired GPVI responses in platelets lacking WIP

AbstractThe role of the Wiskott-Aldrich syndrome protein (WASp) in platelet function is unclear because platelets that lack WASp function normally. WASp constitutively associates with WASp-interacting protein (WIP) in resting and activated platelets. The role of WIP in platelet function was investigated using mice that lack WIP or WASp. WIP knockout (KO) platelets lack WASp and thus are double deficient. WIP KO mice have a thrombocytopenia, similar to WASp KO mice, resulting in part from enhanced platelet clearance. Most WIP KO, but not WASp KO, mice evolved platelet-associated immunoglobulins (Ig) of the IgA class, which normalize their platelet survival but diminish their glycoprotein VI (GPVI) responses. Protein tyrosine phosphorylation, including that of phospholipase C-γ2, and calcium mobilization are impaired in IgA-presenting WIP KO platelets stimulated through GPVI, resulting in defects in α-granule secretion, integrin αIIbβ3 activation, and actin assembly. The anti-GPVI antibody JAQ1 induces the irreversible loss of GPVI from circulating platelets in wild-type mice, but not in WIP KO mice that bear high levels of platelet-associated IgAs. Together, the data indicate that platelet-associated IgAs negatively modulate GPVI signaling and function in WIP KO mice.

Glycoprotein (GP) VI dimer as a major collagen-binding site of native platelets: direct evidence obtained with dimeric GPVI-specific Fabs.

The platelet collagen receptor glycoprotein (GP) VI is suggested to exist as a dimer on the platelet surface, but no direct proof of the functional importance of dimer formation has been provided. To obtain direct evidence for GPVI dimers on the platelet membrane and their functional importance, Fab antibodies were developed that bind to GPVI dimer (GPVI-Fc2) but not to GPVI monomer (GPVIex) through a phage display method. Six Fabs were found: B-F, only reactive with GPVI-Fc2, and A, mainly reactive with GPVI-Fc2, with some reactivity towards GPVIex; each Fab (Fab-dHLX-MH) forms a bivalent dimer (b-Fab) by dimerizing the dHLX domains from two Fab molecules. Fab F was subcloned to a monovalent format by deleting its dHLX domain. All b-Fabs induced platelet aggregation, but the monomeric form of Fab F (m-Fab-F) specifically inhibited collagen-induced aggregation. All b-Fabs and m-Fab-F inhibited GPVI-Fc2 binding to fibrous collagen. Immunoblotting showed that b-Fab-F and m-Fab-F bound weakly to GPVI-Fc2. Adding the anti-GPVI monoclonal antibody 204-11 increased the B(max) of m-Fab-F binding to GPVI-Fc2, suggesting that 204-11 binds to GPVI-Fc2 molecules not already in the appropriate conformation to recognize the Fab, converting them to a conformation reactive to the Fab. GPVI forms a specific structure by dimerization that is necessary for the binding of this receptor to collagen fibrils. The binding of m-Fab-F to platelets directly demonstrates that GPVI is present as a functionally relevant dimer on the platelet surface.

Non-Invasive Molecular Imaging of Fibrosis Using a Collagen-Targeted Peptidomimetic of the Platelet Collagen Receptor Glycoprotein VI

BackgroundFibrosis, which is characterized by the pathological accumulation of collagen, is recognized as an important feature of many chronic diseases, and as such, constitutes an enormous health burden. We need non-invasive specific methods for the early diagnosis and follow-up of fibrosis in various disorders. Collagen targeting molecules are therefore of interest for potential in vivo imaging of fibrosis. In this study, we developed a collagen-specific probe using a new approach that takes advantage of the inherent specificity of Glycoprotein VI (GPVI), the main platelet receptor for collagens I and III.Methodology/Principal FindingsAn anti-GPVI antibody that neutralizes collagen-binding was used to screen a bacterial random peptide library. A cyclic motif was identified, and the corresponding peptide (designated collagelin) was synthesized. Solid-phase binding assays and histochemical analysis showed that collagelin specifically bound to collagen (Kd 10−7 M) in vitro, and labelled collagen fibers ex vivo on sections of rat aorta and rat tail. Collagelin is therefore a new specific probe for collagen. The suitability of collagelin as an in vivo probe was tested in a rat model of healed myocardial infarctions (MI). Injecting Tc-99m-labelled collagelin and scintigraphic imaging showed that uptake of the probe occurred in the cardiac area of rats with MI, but not in controls. Post mortem autoradiography and histological analysis of heart sections showed that the labeled areas coincided with fibrosis. Scintigraphic molecular imaging with collagelin provides high resolution, and good contrast between the fibrotic scars and healthy tissues. The capacity of collagelin to image fibrosis in vivo was confirmed in a mouse model of lung fibrosis.Conclusion/SignificanceCollagelin is a new collagen-targeting agent which may be useful for non-invasive detection of fibrosis in a broad spectrum of diseases.

Measuring soluble platelet glycoprotein VI in human plasma by ELISA

Recent experimental evidence demonstrates that the platelet-specific collagen receptor, glycoprotein (GP)VI is essentially all uncleaved on normal circulating platelets, but is shed from the platelet surface in a metalloproteinase-dependent manner in response to GPVI ligands (including collagen), anti-GPVI antibodies or activation at the platelet Fc receptor, FcγRIIa. This raises the question of whether shed ectodomain fragment in plasma could be a useful biomarker of thrombotic risk and/or autoimmune thrombocytopenia. In this study, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring soluble GPVI in human plasma, using rabbit anti-GPVI polyclonal antibody in the solid-phase, murine anti-GPVI monoclonal antibody (1A12) in the fluid-phase and horseradish peroxidase (HRP)-coupled anti-mouse antibody and enhanced chemiluminescence (ECL) for detection. The ELISA was optimized for sensitivity, reproducibility, inter- and intra-assay precision, addition and recovery and detected GPVI in plasma with a lower detection limit of ∼1 ng/mL. Effects of different anti-coagulants (trisodium citrate, acid-citrate-dextrose or EDTA) were negligible. In ten healthy donors, soluble plasma GPVI levels were 18.9 ± 4.1 ng/mL. Treating normal platelet-rich plasma with a GPVI ligand (collagen-related peptide, CRP), calmodulin inhibitor W7 (that induces GPVI shedding without platelet activation) or N-ethylmaleimide (that directly activates platelet sheddases), under conditions previously shown to induce GPVI shedding, also increased plasma GPVI levels by up to ∼7-fold, compared to previously reported autoimmune (anti-GPVI) patient plasma where soluble GPVI was ∼10-fold higher than normal. Characterization of this sensitive ELISA should facilitate analysis of functional/diagnostic role(s) for soluble GPVI in human plasma associated with thrombotic/immune dysfunction.

Regulation of An Acquired Inhibitor to the GPVI Platelet Collagen Receptor in a Patient with An Autoimmune Syndrome and Kidney Disease.

GPVI is a major platelet collagen signaling receptor associated with the ITAM-containing FcRγ in the platelet membrane. Autoantibodies to GPVI result in ADAM10-mediated cleavage and loss of receptor function. We now report a young woman with a history of autoimmune and kidney disease associated with systemic lupus erythematosus, and a strong circulating antibody to GPVI. The patient has been followed over a 3-year period. Particularly studied were (i) the platelet aggregation response to collagen (and convulxin (Cvx)), (ii) platelet GPVI levels (flow cytometry and Western blot (WB)), (iii) plasma soluble GPVI levels (GPVIs, dual antibody ELISA with capture and biotinylated detction MoAbs), and (iv) circulating anti-GPVI antibody titres (ELISA using recombinant GPVIs). When first studied in 2006, the patient had a normal platelet count but a virtual absence of platelet aggregation to collagen and Cvx. Nevertheless, her platelets responded to other agonists including cross-linking FcRγIIA by way of the monoclonal antibody, IV.3, showing that the PLCγ2-dependent signaling pathway shared with GPVI-FcRγ was retained. Western blotting with a series of MoAbs to GPVI (extracellular and intracellular domains) and other platelet receptors showed a major and specific deficit of GPVI with appearance of GPVI dimer and a 47 kDa degradation product. Other platelet receptors known to be sensitive to metalloprotease cleavage, GPIbα, P-selectin, TLT-1 were not reduced. However, FcRγ was moderately decreased in WB. Interestingly, the patient's plasma was able to activate normal platelets, a phenomenon blocked by recombinant GPVI-Fc protein but not by IV.3 showing that it was independent of FcRγIIA and probably related to GPVI dimerization. Her plasma also specifically blocked the response of control platelets to Cvx in a time-dependent manner. Studies continued in 2007 when the patient additionally experienced neurological and vascular problems. Platelet expression of GPVI continued to be severely reduced by WB, and ELISA confirmed strong anti-GPVI plasma antibody titers. Plasma GPVIs levels were undetectable compared to control donors. Transfusion of donor platelets prior to a renal biopsy was unsuccessful; they were rapidly cleared and failed to correct the platelet defect. The patient was now subjected to increasingly strict immunosuppressive medication including corticoids (prednisolone), imurel (azathioprine) then endoxan (cyclophosphamide). A large reduction in plasma anti-GPVI levels, the appearance of GPVIs in the patient's plasma (the ELISA results were confirmed by WB of plasma samples), the partial restoration of both platelet GPVI levels and the platelet collagen response accompanied these treatments which was paralleled by an improvement in her nephrotic syndrome. Our results suggest that this patient developed a potent anti-GPVI antibody responsible for the loss of platelet GPVI, with the possible clearance of GPVIs by immune complex formation. The activity of the antibody was much reduced by immunotherapy; further studies are required to know whether this is related to the improvement in her clinical state.

Mice Lacking Wasp-Interacting Protein Evolve Anti-Platelet IgAs That Impair Platelet Responses Mediated by the Collagen Receptor GPVI.

Wiskott-Aldrich Syndrome (WAS) is a hematopoietic disorder characterized by immune deficiency, eczema and severe thrombocytopenia with small platelets. However, the role of the WAS protein (WASp), mutated or absent in WAS, is unclear in platelets because platelets that lack WASp function normally. WASp constitutively associates with WIP in resting and activated platelets. We investigated the role of the platelet WIP-WASp complex by using mice that lack either WIP or WASp. WIP knockout (KO) platelets lack WASp and WIP expression is reduced in WASp KO platelets, indicating a requirement for both proteins for each other's integrity. WIP KO, but not WASp KO mice evolve platelet-associated antibodies of the IgA class, as evidenced by immunoblot analysis and flow cytometry. We present evidence that platelet-associated IgAs specifically target the collagen receptor GPVI in WIP KO mice:protein tyrosine phosphorylation, including that of phospholipase C-γ2, and calcium mobilization are impaired in IgA-presenting WIP KO platelets stimulated through GPVI, resulting in α-granule secretion, integrin αIIbβ3 activation and actin assembly defects;binding of the anti-GPVI antibody JAQ1 to normally expressed GPVI is reduced in IgA-presenting WIP KO platelets;JAQ1 induces the irreversible loss of GPVI from circulating platelets in WT mice, but not in WIP KO mice that bear anti-platelet IgAs.Together, the data indicate that platelet-associated IgAs negatively modulate GPVI signaling in WIP KO mice.

Platelet GPVI binds to collagenous structures in the core region of human atheromatous plaque and is critical for atheroprogression in vivo

Platelet adhesion to the atherosclerotic vascular wall induces thrombosis and boosters vascular inflammation and atheroprogression. In the present study we studied the binding of the platelet collagen receptor glycoprotein (GP) VI to human atherosclerotic plaques (AP) and the role of GPVI-mediated platelet adhesion for atheroprogression. Soluble GPVI-Fc fusion protein bound to immobilized collagen type I, collagen type III, and predominantly to the core region of human carotid atheromatous plaques. The pattern of GPVI-Fc binding was similar to the immunostaining pattern of collagen type III and differed from the immunostaining of collagen type I, which was more intense in the cap than in the core. Plaque-induced platelet aggregation in stirred blood and platelet adhesion/aggregate formation under flow were inhibited by the anti-GPVI monoclonal antibody 5C4 or by pretreatment of plaques with anti-collagen type I and anti-collagen type III antibody, or GPVI-Fc. However, there was no correlation between GPVI-Fc binding and platelet aggregating activity of individual plaques. GPVI bound also to atherosclerotic arteries of ApoE-deficient mice in vivo as assessed by small animal positron emission tomography (PET). Prolonged administration of soluble GPVI attenuated atheroprogression in ApoE-deficient mice. In humans, GPVI binding to collagenous type I and type III structures of the plaque core region mediates plaque-induced platelet adhesion and aggregation, but GPVI binding is not the sole platelet-activating determinant of plaques. In mice, GPVI-mediated platelet adhesion to the atherosclerotic vascular wall is involved in atheroprogression in vivo. Taken together, our data suggests that GPVI is a relevant target to prevent atherothrombotic events and atheroprogression.

Ex vivo inhibition of thrombus formation by an anti-glycoprotein VI Fab fragment in non-human primates without modification of glycoprotein VI expression.

Glycoprotein (GP)VI is an attractive target for the development of new antithrombotic drugs. Its deficiency protects animals in several models of thrombosis, arterial stenosis and ischemia--reperfusion while inducing no major bleeding tendency. The Fab fragment of one anti-GPVI monoclonal antibody (9O12.2) inhibits all GPVI functions in vitro. The aim of this study was to determine the ex vivo effects of 9O12.2 Fab on hemostasis, coagulation and thrombosis in non-human primates. Blood samples were collected from cynomolgus monkeys before and after (30, 90 and 150 min, 1 and 7 days) a bolus injection of 9O12.2 Fab (4 mg kg(-1)) or vehicle. Platelet counts and coagulation tests (prothrombin time, activated partial thromboplastin time) were not modified following Fab injection. The PFA-100 closure time increased during the first hours and returned to initial values on day + 1. Platelet-bound Fab was detected from 30 min to 24 h after Fab injection without GPVI depletion at any time. Collagen-induced platelet aggregation was selectively and fully inhibited at 30 min. Thrombus formation on collagen in flowing whole blood (1500 s(-1)) was delayed and decreased, and collagen-induced or tissue factor-induced thrombin generation in platelet-rich plasma was profoundly inhibited. The anti-GPVI 9O12.2 Fab inhibits thrombus formation ex vivo in non-human primates with a composite effect on platelet activation and thrombin generation in the absence of GPVI depletion.

Abstract 806: Protein Disulfide Isomerase (PDI) signals Tissue Factor (TF)-dependent Fibrin formation in vivo

Background: Pathological fibrin formation is a predominant cause for arterial thrombosis, triggering myocardial infarction and stroke. Tissue factor (TF), which is expressed by vascular endothelium and cell-derived microparticles, is a central trigger of intravascular fibrin generation. In intact blood vessels, TF is only minimally active, and the coagulation system is down-regulated. In contrast, TF is rapidly stimulated following endothelial disruption. However, the molecular mechanisms that trigger fibrin formation via regulation of TF activation are largely undefined. Methods and Results: Here, we have identified a novel pathway that leads to TF activation in vitro and contributes to intravascular coagulation in vivo. We show that the protein disulfide isomerase (PDI) induces disulfide formation of the Cys186/Cys209 pair located in the extracellular domain of TF. In vitro, PDI-mediated disulfide formation led to profound activation of TF. In vivo, we found that PDI is not present in the intact vessel wall, but becomes abundantly expressed following vessel damage. We show that injured smooth muscle cells and adherent/activated platelets are the major cellular sources of PDI at sites of vascular injury. We then assessed fibrin formation following injury of the common carotid artery using intravital microscopy. We observed that PDI-blockade strongly reduced fibrin formation by approximately 45– 60%. Likewise, intravenous infusion of PDI significantly enhanced TF-dependent fibrin formation. Together, this implies that PDI initiates coagulation in vitro and also during arterial thrombosis in vivo. Notably, PDI did not act on platelets to trigger intravascular coagulation. Correspondingly, PDI blockade also inhibited fibrin formation, when platelet adhesion was prevented using a function-blocking anti-GPVI antibody. Conclusion: These findings reveal for the first time in vivo that the thiol isomerase PDI is a major molecular trigger of blood coagulation, which is exposed after vessel injury and converts TF into its functionally active form. Hence, PDI represents a promising novel target for the treatment of pathologic fibrin formation in patients with thrombotic disorders.

Ligand Binding Rapidly Induces Disulfide-dependent Dimerization of Glycoprotein VI on the Platelet Plasma Membrane

Thrombus formation in hemostasis or thrombotic disease is initiated by adhesion of circulating platelets to damaged blood vessel walls. Exposed subendothelial collagen interacting with platelet glycoprotein (GP) VI leads to platelet activation and integrin alpha(IIb)beta(3)-mediated aggregation. We previously showed that ligand binding to GPVI also induces metalloproteinase-dependent shedding, generating an approximately 55-kDa soluble ectodomain fragment and an approximately 10-kDa membrane-associated remnant. Here, treatment of platelets with collagen or the GPVI-targeting rattlesnake toxin convulxin also induces rapid (10-30 s) formation of a high molecular weight GPVI complex (GPVIc) under nonreducing conditions, as detected by immunoblotting with anti-GPVI antibodies. The appearance of an approximately 20-kDa remnant detectable using a polyclonal antibody against the GPVI cytoplasmic tail under nonreducing, but not reducing, conditions after ectodomain shedding and nonreduced/reduced two-dimensional SDS-polyacrylamide gel analysis of biotinylated platelets confirmed that that GPVIc was a homodimer. Formation of disulfide-linked GPVIc was prolonged in the presence of metalloproteinase inhibitor GM6001 and was independent of GPVI signaling because it was unaffected by inhibitors of Src kinases, Syk, or phosphoinositide 3-kinase. To identify the thiol involved in disulfide bond formation, wild-type or mutant GPVI, where two available sulfhydryls (Cys-274 and Cys-338) were individually mutated to serine, was expressed in rat basophilic leukemia cells. Dimerization of wild-type and C274S GPVI, but not the C338S mutant, was observed after treating cells with convulxin. We conclude that (i) a subpopulation of GPVI forms a constitutive dimer on the platelet surface, facilitating rapid disulfide cross-linking, (ii) convulxin or other GPVI agonists induce disulfide-linked GPVI dimerization independent of GPVI signaling, and (iii) the penultimate residue of the GPVI cytoplasmic tail, Cys-338, mediates disulfide-dependent dimer formation.

Diverging signaling events control the pathway of GPVI down-regulation in vivo

AbstractCoronary artery thrombosis is often initiated by platelet activation on collagen-rich subendothelial layers in the disrupted atherosclerotic plaque. The activating platelet collagen receptor glycoprotein VI (GPVI) noncovalently associates with the Fc receptor γ-chain (FcRγ), which signals through its immunoreceptor-tyrosine–based activation motif (ITAM) via the adaptor LAT leading to the activation of phospholipase Cγ2 (PLCγ2). GPVI is a promising antithrombotic target as anti-GPVI antibodies induce the irreversible loss of the receptor from circulating platelets by yet undefined mechanisms in humans and mice and long-term antithrombotic protection in the latter. However, the treatment is associated with transient but severe thrombocytopenia and reduced platelet reactivity to thrombin questioning its clinical usefulness. Here we show that GPVI down-regulation occurs through 2 distinct pathways, namely ectodomain shedding or internalization/intracellular clearing, and that both processes are abrogated in mice carrying a point mutation in the FcRγ-associated ITAM. In mice lacking LAT or PLCγ2, GPVI shedding is abolished, but the receptor is irreversibly down-regulated through internalization/intracellular clearing. This route of GPVI loss is not associated with thrombocytopenia or altered thrombin responses. These results reveal the existence of 2 distinct signaling pathways downstream of the FcRγ-ITAM and show that it is possible to uncouple GPVI down-regulation from undesired side effects with obvious therapeutic implications.

The Fab Fragment of a Novel Anti-GPVI Monoclonal Antibody, OM4, Reduces In Vivo Thrombosis Without Bleeding Risk in Rats

Inhibition of GPVI has been proposed as a useful antithrombotic strategy; however, in vivo proof-of-concept animal studies targeting GPVI are lacking. We evaluated a novel anti-human GPVI monoclonal antibody OM4 Fab in rats. OM4 Fab specifically inhibited collagen-induced aggregation of rat platelets in vitro with an IC50 of 20 to 30 microg/mL but not ADP and AA-induced platelet aggregation. After intravenous administration of OM4 Fab, a rapid inhibition of ex vivo platelet aggregation was observed with a gradual recovery within 60 to 90 minutes which corresponded to the decline in OM4 Fab plasma concentration and time-dependent decrease in platelet-bound OM4 Fab. In contrast to previous reports in mice, intravenous OM4 Fab did not deplete platelet GPVI. Injection of OM4 IgG caused acute thrombocytopenia. In a modified Folts model of cyclic flow reduction in rat carotid artery, the number of complete occlusions was significantly reduced by intravenous administration of OM4 Fab (20 mg/kg) before or after mechanical injury to the vessel, without prolongation of bleeding time. Fab fragment of the monoclonal antibody OM4 effectively inhibits collagen induced platelet aggregation in vitro and ex vivo, and in vivo thrombosis in rats without prolonging bleeding time. Antibodies against GPVI may have therapeutic potential, inhibiting thrombosis without prolonging bleeding time.

Identification of human platelet glycoprotein VI-specific IgG autoantibody and its fragments

Glycoprotein VI (GPVI), as a major receptor for collagen on the platelet surface, mediates the initial platelet contact with collagen, and causes platelet aggregation and thrombosis. Agents of anti-GPVI can inhibit the adhesion, activation and aggregation function of platelets, which can be used for preventing thrombotic diseases. To make humanized monoclonal antibodies by phage surface display technology, we studied plasmas from 44 patients with chronic idiopathic thrombocytopenic purpura using modified monoclonal antibody immobilization of platelet antigen assays. GPVI-specific antibodies were found in six (13.6%) patients. Among these, only one showed significant inhibition of platelet aggregation induced by collagen. The IgG antibody and F(ab')2 fragments of this patient's plasma were further purified and their immunoreactivities and effects on platelet aggregation were reanalyzed. It was found that purified IgG and its F(ab')2 fragments from the patient not only retained the ability to bind to platelet GPVI, but also inhibited collagen-induced platelet aggregation. In this study, therefore, the specific anti-platelet GPVI autoantibody that inhibits collagen-induced platelet aggregation has been successfully screened out, which can be used to develop completely humanized anti-GPVI phage antibody.

Establishment of a flow cytometric assay for determination of human platelet glycoprotein VI based on a mouse polyclonal antibody

Glycoprotein VI (GPVI) is the major signaling receptor for collagen on platelets. Recent studies suggest that the surface density of GPVI is related to the activation of platelets by collagen. To measure the level of GPVI on platelets, a mouse polyclonal antibody BJ010 was prepared using an amplified fragment of extracellular domain in GPVI. The specific reactivity of BJ010 was identified by anti-GPVI specific monoclonal antibody 11a12 using immunoprecipitation, sandwich enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The antibody BJ010 recognized both native and denatured human GPVI so that it was used to set up a flow cytometric assay to detect the level of GPVI in normal subjects. The relative level of GPVI on platelets was detected in 101 healthydonors. The median geometric mean fluorescence intensity (GMFI) of platelet GPVI level was 57.7. The variation between minimum and maximum values of platelet GPVI and integrin alpha2beta1 were found to be 3.5- and 4.1-fold, respectively, in the normal subjects. There was a week correlation between the amount of GPVI and integrin alpha2beta1 on platelet surfaces. For the method, the intraassay and interassay coefficient of variation was 6.3% and 8.8%, respectively. The flow cytometric assay described here provides a simple, reliable, reproducible, and readily available means of quantitation of collagen receptor GPVI density on the platelet surface in a larger number of blood samples.

Ex vivo evaluation of anti-GPVI antibody in Cynomolgus monkeys: Dissociation between anti-platelet aggregatory effect and bleeding time

Recent progress in the understanding of thrombus formation has suggested an important role of glycoprotein (GP)VI. In contrast to its pivotal role in collagen-induced platelet activation, it has been suggested that its blockade does not induce massive bleeding tendency. To demonstrate the dissociation between inhibitory effect on platelet aggregation and bleeding by GPVI blockade, we examined the effects of Fab fragment of OM2, an anti-human GPVI monoclonal antibody on ex vivo collagen-induced platelet aggregation and skin bleeding time after intravenous injection in cynomolgus monkeys. In a dose-escalation study, OM2 potently (> 80%) inhibited collagen-induced platelet aggregation at the cumulative dose of 0.2 mg/kg with a slight prolongation of bleeding time (1.3 times baseline value). Furthermore, at 18.8 mg/kg, the highest dose tested, prolongation of bleeding time was still mild (1.9 times). In contrast, abciximab, Fab fragment of anti-GPIIb/IIIa antibody prolonged bleeding time by 5.0 times at 0.35 mg/kg, the lowest effective dose on platelet aggregation. In a pharmacodynamic study, a bolus injection of OM2 at 0.4 mg/kg produced potent inhibition of collagen-induced aggregation up to six hours after injection, showing longer half-life than that of abciximab. The injection of OM2 Fab did not induce thrombocytopenia and GPVI depletion in monkeys. These results suggest that blockade of GPVI by antibody can exert a potent inhibitory effect on collagen-induced platelet aggregation with a milder prolongation of bleeding time than blockade of GPIIb/IIIa. This study indicates that OM2 has the potential to be developed as a new class of therapeutic tool.

Characterization of Anti-Human GPVI Autoantibodies Mediating Persistent Target Immnunodepletion over a 15-Year Period.

The first reported patient with platelet GPVI deficiency (Blood 1987; 69:1712) had anti-GPVI antibodies (YA-Abs) in the patient's serum, which contributed to later progress in identification of GPVI as a novel collagen receptor. Although there is now a concept that anti-GPVI Ab induces immunodepletion of GPVI in vivo and may become a promising therapeutic antithrombotic agent, precise nature of anti-human GPVI Ab mediating immunodepletion of GPVI is unknown. Since the patient's platelets are still lacking GPVI and unresponsive to collagen stimulation, we purified YA-Abs from the patient's serum kept in 1988 (YA-Abs-88) and in 2003 (YA-Abs-03) using sequential affinity chromatography on protein A-Sepharose and recombinant human GPVI fused to Fc (rhGPVI-Fc)-Sepharose. The amount of YA-Abs-03 in the serum was estimated to be about 3 μg/mL and twenty times less than that of YA-Abs-88. We also generated 75 mouse monoclonal antibodies (mAbs) against the extracellular part of human GPVI. Among those, several mAbs cross-reacted with YA-Abs for the binding to rhGPVI-Fc, indicating oligoclonal nature of YA-Abs. Two representative mAbs with no cross-reactivity, F1201 and F1232, cross-reacted with YA-Abs-88 and with both YA-Abs-88 and -03, respectively. Previous studies identified two distinct collagen binding sites of human GPVI; the one is a sequence including Lys59 of human GPVI which is recognized by 10B12 Ab, (Blood 2004; 103:903) and the other is a sequence including Gly30, Val34 and Leu36 which involves the binding to 9O12 Ab (J.Biol. Chem. 2004; 279:52293). Point mutated rhGPVI-Fc at Lys59 (K59E) was recognized by YA-Abs-88, -03, and F1232 but not by F1201. Another mutated rhGPVI-Fc in which human GPVI sequence from Pro31 to Leu36 was substituted for mouse GPVI counter sequence was recognized by any of those Abs. YA-Abs-88 and F1201but not F1232 evoked surface P-selectin expression or aggregation of human platelets, while F1232 inhibited collagen-induced aggregation. Since both F1201 and F1232 were found to react with nonhuman primate platelets, either mAb was intravenously injected into monkeys at a dose of 0.3 mg/kg that reached its maximum plasma concentration of 10 μg/mL. Within 1 to 2 days after injection, their platelets showed the lack of both GPVI and collagen-induced aggregation. Thus, GPVI-deficiency of the patient's platelets is now maintained by unexpectedly much decreased amount of specific Abs which contain F1232-like Ab but not F1201-like Ab included in YA-Abs-88. Immunodepletion of GPVI can be safely induced by specific Ab like F1232 recognizing different epitope from previously reported collagen binding sites where their cross-linking causes platelet activation.

Relative antithrombotic effect of soluble GPVI dimer compared with anti-GPVI antibodies in mice

AbstractGlycoprotein VI (GPVI) is an essential platelet collagen receptor; therefore, the inhibition of GPVI-collagen interactions may be an attractive antithrombotic strategy. We have previously shown that targeting of GPVI with antibodies leads to the depletion of the receptor and to long-term antithrombotic protection in mice. An alternative agent to interfere with GPVI-collagen interactions might be soluble GPVI acting as a competitive inhibitor, thereby averting undesired effects on platelets. To test this, we expressed soluble dimeric human GPVI, comprising the extracellular domain of the receptor fused to the human immunoglobulin Fc domain (GPVI-Fc), and compared its antithrombotic potential with that of anti-GPVI antibodies in mice. In contrast to a recent report, we found by intravital fluorescence microscopy and ultrasonic flow measurements that GPVI-Fc had no effect on platelet adhesion and thrombus formation at the injured arterial wall, whereas anti-GPVI antibodies profoundly inhibited these processes. Similar results were obtained with a fusion protein comprising the extracellular domain of mouse GPVI and human IgG-Fc. This indicates that direct targeting of GPVI provides significantly stronger protection against arterial thrombosis than soluble GPVI dimer.

Anti–Glycoprotein VI Treatment Severely Compromises Hemostasis in Mice With Reduced α 2 β 1 Levels or Concomitant Aspirin Therapy

Platelet inhibition is a major strategy to prevent arterial thrombosis, but it is frequently associated with increased bleeding because of impaired primary hemostasis. The activating platelet collagen receptor, glycoprotein VI (GP VI), may serve as a powerful antithrombotic target because its inhibition or absence results in profound protection against arterial thrombosis but no major bleeding in mice. Mice lacking (-/-) or expressing half-levels (+/-) of the other major platelet collagen receptor, integrin alpha2beta1, were injected with the anti-GP VI antibody JAQ1 and analyzed on day 5. Anti-GP VI treatment resulted in a marked hemostatic defect in alpha2-/- or alpha2+/- mice, as shown by dramatically prolonged tail bleeding times. Platelet adhesion to collagen was studied in an ex vivo whole-blood perfusion system under high shear conditions. Weak integrin activation by thromboxane A2 (TxA2) receptor stimulation restored defective adhesion of anti-GP VI-treated wild-type but not alpha2-/- or alpha2+/- platelets to collagen. This process required the simultaneous activation of the G(q) and G13 signaling pathways, as demonstrated by use of the respective knockout strains. Conversely, inhibition of TxA2 production by aspirin severely compromised hemostasis in anti-GP VI-treated or GP VI/Fc receptor gamma-chain-deficient but not control mice. Anti-GP VI therapy may result in defective hemostasis in patients with reduced alpha2beta1 levels or concomitant aspirin therapy. These observations may have important implications for a potential use of anti-GP VI-based therapeutics in the prevention of cardiovascular disease.

Surface expression of collagen receptor Fc receptor-gamma/glycoprotein VI is enhanced on platelets in type 2 diabetes and mediates release of CD40 ligand and activation of endothelial cells.

Diabetes is associated with an enhanced collagen-mediated platelet activation that contributes significantly to thromboischemic complications. In this study, the platelet collagen receptor glycoprotein VI (GPVI) was studied in 385 patients with type 2 diabetes. Surface expression of the platelet Fc receptor that forms a functional complex with GPVI was significantly increased in patients with diabetes compared with those without diabetes (P = 0.02). Fc receptor expression correlated with GPVI expression and was found to be independently associated with diabetes (r = 0.529, P < 0.001). Stimulation of GPVI through a specific anti-GPVI monoclonal antibody significantly enhanced surface expression of CD40L (P = 0.006). Because CD40L is a potent platelet-derived cytokine that is involved in thrombosis and atherosclerosis, we evaluated the effect of GPVI-mediated release of CD40L on activation of endothelial cells. Coincubation of GPVI-stimulated platelets resulted in substantial enhanced endothelial surface expression of CD62P, alphavbeta3, and intercellular adhesion molecule 1 (P < 0.05) and secretion of monocyte chemoattractant protein 1 of cultured human umbilical vein endothelial cells (P < 0.01). These results suggest that the function of collagen receptor GPVI is altered in type 2 diabetes and may play an important role in atherothrombotic complications. Inhibition of GPVI may be a promising pharmacological target in the treatment of high-risk diabetic patients.