Glycoprotein V (GPV) is a subunit of the platelet GPIb-V-IX receptor for von Willebrand factor and thrombin. GPV is cleaved from the platelet surface during activation by thrombin, but its role in hemostasis is still unknown. It is reported that GPV knockout mice had a decreased tendency to form arterial occluding thrombi in an intravital thrombosis model and abnormal platelet interaction with the subendothelium. In vitro, GPV-deficient platelets exhibited defective adhesion to a collagen type I-coated surface under flow or static conditions. Aggregation studies demonstrated a decreased response of the GPV-deficient platelets to collagen, reflected by an increased lag phase and reduced amplitude of aggregation. Responses to adenosine diphosphate, arachidonic acid, and the thromboxane analog U46619 were normal but were enhanced to low thrombin concentrations. The defect of GPV null platelets made them more sensitive to inhibition by the anti-GPVI monoclonal antibody (mAb) JAQ1, and this was also the case in aspirin-or apyrase-treated platelets. Moreover, an mAb (V.3) against the extracellular domain of human GPV selectively inhibited collagen-induced aggregation in human or rat platelets. V.3 injected in rats as a bolus decreased the ex vivo collagen aggregation response without affecting the platelet count. Finally, surface plasmon resonance studies demonstrated binding of recombinant soluble GPV on a collagen-coupled matrix. In conclusion, GPV binds to collagen and appears to be required for normal platelet responses to this agonist. IntroductionPlatelets play a central role in hemostasis through their ability to adhere to a damaged vessel wall and to aggregate in response to agonists such as thrombin, collagen, or adenosine diphosphate (ADP), 1 and these properties are known to be mediated by cell surface glycoproteins. 2,3 However, the exact functions of numerous platelet glycoproteins that have been characterized biochemically remain unknown. Glycoprotein V (GPV, Mr 82 kd) is one of the most abundant glycoproteins at the surface of blood platelets and has long been identified, 4-6 but its functional role is still subject to speculation. GPV is noncovalently linked to the GPIb-IX von Willebrand factor (vWF) receptor on the platelet surface. 7,8 This type I transmembrane protein has a large extracellular domain comprising 15 Leu-rich motifs followed by a thrombin cleavage site. 9,10 The specific release of a soluble 69-kd extracellular domain fragment (GPVf1) by thrombin has led to its proposal as a thrombin receptor, 6 whereas cloning of the gene in rat and mouse revealed a well-conserved thrombin cleavage site. 11 Studies in transfected cells have shown that GPV is required for efficient thrombin binding, possibly through a direct interaction with GPIb␣. 12,13 The release of a soluble fragment by thrombin has been used to develop a specific enzyme-linked immunosorbent assay for GPV that is being tested as a means of monitoring platelet activation under conditions of clinical thrombosis. 14 The role of GPV a...

SummaryGPIb is disulfide-linked to GPIbα to form GPIb, a platelet receptor for von Willebrand factor (vWF). GPIb is in turn non covalently linked to GPIX and GPV to form the GPIb/V/IX complex. Apart from its contribution to controlling surface expression of the complex, the exact function of GPIbβ is not well established due to a lack of suitable ligands or antibodies. The present report describes a monoclonal antibody (RAM.1) that labeled the 26 kDa GPIbβ subunit on western blots and coprecipitated the three subunits of the GPIb/IX complex from lysates of platelets and transfected CHO and K562 cells. RAM.1 bound to GPIbβ deleted of its intracellular domain whereas Gi27, directed against intracellular GPIbβ, did not. Using synthetic peptides, the RAM.1 epitope was mapped to a putative cysteine loop within the COOH-terminal leucine-rich flanking region. In functional assays, RAM.1 had no effect on platelet aggregation induced by ADP, collagen or thrombin, but inhibited ristocetin induced platelet agglutination and botrocetin induced vWF binding. RAM.1 inhibited adhesion of GPIb/V/IX transfected K562 cells to a vWF matrix under flow, increased their rolling velocity and decreased the resistance of cells to detachment at high shear. This study suggests a role of GPIbβ in modulating the adhesive properties of GPIb/V/IX and describes a useful tool to analyze the exact functions of GPIbβ.

Glycoprotein (GP)Ib