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

Abstract

Glycoprotein 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 longterm antithrombotic protection in mice. An alternative agent to interfere with GPVIcollagen 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. IntroductionRupture of the atherosclerotic plaque leads to the exposure of subendothelial fibrillar collagens to the flowing blood, which triggers the adhesion, aggregation, and procoagulant activity of platelets. [1][2][3][4] Platelet-collagen interactions are complex and involve a number of different receptors and signaling pathways. Besides glycoprotein Ib-V-IX (GPIb-V-IX), which indirectly interacts with collagen through von Willebrand factor, 5 the direct collagen receptor GPVI has been identified as playing a central role in this process because it mediates the activation of integrins, thereby enabling firm platelet adhesion and thrombus growth. 6 GPVI is a 62-kDa type 1 transmembrane receptor that belongs to the immunoglobulin superfamily 7,8 and is noncovalently associated with the signal-transducing FcR␥ chain. 9,10 A few patients with GPVI deficiency have been described who have mild bleeding diatheses, but their platelets show severely impaired responses to collagen. [11][12][13] Similarly, platelets from GPVI-14 or FcR␥/GPVIdeficient 15,16 mice are unresponsive to collagen, but no major bleeding has been observed in those animals, suggesting that GPVI might be an interesting target for safe antithrombotic therapy. Experimental evidence in support of this hypothesis came from studies using anti-GPVI monoclonal antibodies (mAbs) in mice. Injection of such mAbs results in down-regulation of the receptor and abolished collagen responses in circulating platelets. 17,18 Such GPVI-depleted mice are for at least 2 weeks protected against collagen-dependent thromboembolism and arterial thrombus formation but only show slightly increased bleeding times. 17,19 Similarly, FcR␥ chain-deficient mice, which lack GPVI, 16 are protected against arterial throm...

Revacept: Collagen GP VI receptor inhibitor, a new & a formidable target for antiplatelet action

“…1 Various studies done on animals had proved the superiority of revacept, as regards to the effect and safety profile, when compared to the other anti GPVI antibodies or the Fc GPVI proteins. 1,[5][6][7] The drug is currently under phase 2 trials in humans. 8 …”

“…This was not seen with revacept. 5 Hence revacept, an inhibitor of GPVI holds promise as an antiplatelet agent that potently inhibits collagen induced aggregation of human platelets but not thrombin-or ADP-induced aggregation, thereby having minimal bleeding risk.…”

“…1 Hence, revacept (PR-15) is technically a dimeric glycoprotein [GP] VI-Fc and is almost equally effective as the other known antiplatelet drugs, without any additional complication of bleeding as seen in various animal studies. 5 Revacept acts against collagen which gets exposed in endothelial lesions, and hence inhibits the activation of the platelet receptors which bind to collagen and start the platelet aggregation process. Therefore, revacept acts at a very early stage in acute vascular syndromes.…”

Direct evidence of a native GPVI dimer at the platelet surface

“…The very large discrepancy between the apparent affinities of monomeric and dimeric GPVI for fibrillar collagen is greater than would be expected from a simple avidity effect, suggesting the dimer may recognize a specific conformational epitope in collagen. The GPVI-Fc fusion protein has been used in physiologically relevant settings to demonstrate effective inhibition of thrombus formation, consistent with the ability of the dimeric receptor to bind fibrillar collagen with high specificity and reasonably high affinity [6][7][8][9].…”

“…The very large discrepancy between the apparent affinities of monomeric and dimeric GPVI for fibrillar collagen is greater than would be expected from a simple avidity effect, suggesting the dimer may recognize a specific conformational epitope in collagen. The GPVI-Fc fusion protein has been used in physiologically relevant settings to demonstrate effective inhibition of thrombus formation, consistent with the ability of the dimeric receptor to bind fibrillar collagen with high specificity and reasonably high affinity [6][7][8][9].The crystal structure of the collagen-binding domain of GPVI was solved in 2006, revealing two receptor chains arranged back-to-back in a dimeric conformation [10]. Computational docking identified a shallow groove on the top of the N-terminal Ig-like domain as the most likely binding site for a collagen triple helix, which agreed with prior mutational results [10][11][12].…”