The platelet glycoproteins (GPs) Ib, integrin ␣ 2  1 , and GPVI are considered central to thrombus formation. Recently, their relative importance has been re-evaluated based on data from murine knockout models. To examine their relationship during human thrombus formation on collagen type I fibers at high shear (1000 s ؊1 ), we tested a novel antibody against GPVI, an immunoglobulin single-chain variable fragment, 10B12, together with specific antagonists for GPIb␣ (12G1 Fab 2 ) and ␣ 2  1 (6F1 mAb or GFOGER-GPP peptide).GPVI was found to be crucial for aggregate formation, Ca 2؉ signaling, and phosphatidylserine (PS) exposure, but not for primary adhesion, even with more than 97% receptor blockade. Inhibiting ␣ 2  1 revealed its involvement in regulating Ca 2؉ signaling, PS exposure, and aggregate size. Both GPIb␣ and ␣ 2  1 contributed to primary adhesion, showing overlapping function. The coinhibition of receptors revealed synergism in thrombus formation: the coinhibition of adenosine diphosphate (ADP) receptors with collagen receptors further decreased adhesion and aggregation, and, crucially, the complete eradication of thrombus formation required the coinhibition of GPVI with either GPIb␣ or ␣ 2  1 . In summary, human platelet deposition on collagen depends on the concerted interplay of several receptors: GPIb in synergy with ␣ 2  1 mediating primary adhesion, reinforced by activation through GPVI, which further regulates the thrombus formation.
IntroductionThe platelet response to exposed subendothelial matrix is fundamental to thrombosis and hemostasis. Uniquely, collagen, the most abundant vessel wall protein, mediates platelet adhesion and activation, localizing and regulating the hemostatic response at sites of injury. Discovering the molecular mechanisms that control platelet-collagen interaction is crucial for understanding the pathogenesis of arteriothrombotic diseases such as stroke and myocardial infarction. Under high shear rate conditions, the glycoprotein (GP) Ib/V/IX complex allows initial platelet rolling over von Willebrand factor (VWF) bound to subendothelial collagen fibers, and subsequently collagen receptors come into contact with their specific binding sequences in the collagen. For the next step, platelet arrest and activation, firm evidence exists of a role for only 2 receptors, integrin ␣ 2  1 and immunoglobulin superfamily member GPVI, despite the apparent redundancy in collagen receptors (for a review, see Siljander PR-M and Farndale RW 1 ).According to the 2-site, 2-step model, high-affinity interaction through ␣ 2  1 stops the platelet, allowing low-affinity binding of GPVI, which generates signaling required for the subsequent thrombus formation. Platelet deposition under flow was found to be dependent on GPIb/V/IX and ␣ 2  1 , 2-4 whereas no platelet deposition occurred on the GPVI-specific substrate collagen-related peptide (CRP), even under low shear rates. 5 The limited number of studies with human platelets deficient in either GPVI or ␣ 2  1 support the 2-site, ...