Expression and Function of the Mouse Collagen Receptor Glycoprotein VI Is Strictly Dependent on Its Association with the FcRγ Chain

Abstract

Platelet glycoprotein (GP) VI has been proposed as the major collagen receptor for activation of human platelets. Human GPVI belongs to the immunoglobulin superfamily and is noncovalently associated with the FcR␥ chain that is involved in signaling through the receptor. In mice, similar mechanisms seem to exist as platelets from FcR␥ chain-deficient mice do not aggregate in response to collagen. However, the activating collagen receptor on mouse platelets has not been definitively identified. In the current study we examined the function and in vivo expression of GPVI in control and FcR␥ chain-deficient mice with the first monoclonal antibody against GPVI (JAQ1). On wild type platelets, JAQ1 inhibited platelet aggregation induced by collagen but not PMA or thrombin. Cross-linking of bound JAQ1, on the other hand, induced aggregation of wild type but not FcR␥ chain-deficient platelets. JAQ1 stained platelets and megakaryocytes from wild type but not FcR␥ chaindeficient mice. Furthermore, JAQ1 recognized GPVI (approximately 60 kDa) in immunoprecipitation and Western blot experiments with wild type but not FcR␥ chain-deficient platelets. These results strongly suggest that GPVI is the collagen receptor responsible for platelet activation in mice and demonstrate that the association with the FcR␥ chain is critical for its expression and function.Collagen is one of the major components of the vessel wall responsible for platelet adhesion and activation at sites of vascular injury (1). A variety of collagens have been identified, seven of which are found in the subendothelial layer. The interaction between platelets and collagen can either occur indirectly via intermediary proteins like von Willebrand factor, which complexes to collagen(s) in the vessel wall and concomitantly binds to the platelet receptors glycoprotein (GP) 1 Ib-

Tetraspanin Tspan9 regulates platelet collagen receptor GPVI lateral diffusion and activation

“…Fab fragments from anti-GPVI monoclonal antibody JAQ1 [42] and anti-CD9 monoclonal antibody Nyn.H3 were coupled to Atto 647N or 565 as described previously [43]; 500 µL of washed mouse platelets at 2 × 10 7 /mL was allowed to spread directly on cleaned glass coverslips for 35 minutes at 37°C. Non-adhered platelets were removed by gentle washing with modified Tyrode’s buffer [33].…”

CEACAM1 negatively regulates platelet-collagen interactions and thrombus growth in vitro and in vivo

“…18 Antirabbit fluorescein isothiocyanate (FITC) was purchased from Dako (Botany, Australia). Anti-mouse GPVI, 19 anti-mouse GPIb␣/IX/V complex, 20 anti-mouse integrin ␣2␤1, 21 anti-mouse CD9, 22 and anti-mouse GPVI (JAQ1) 23 were purchased or obtained from Emfret Analytics (Wü rzburg, Germany). Anti-mouse integrin ␣IIb␤3, 24 anti-mouse CD44 25 and HRP-conjugated anti-phosphotyrosine RC20 antibody were obtained from BD Biosciences Pharmingen (Franklin Lakes, NJ).…”

Variation in Human Platelet Glycoprotein VI Content Modulates Glycoprotein VI–Specific Prothrombinase Activity

“…18 In addition, in genetically engineered mice that do not express FcR ␥-chain, there is a reproducible impairment of collagen-induced platelet activation. 19,20 CVX is a protein from the venom of C durissus terrificus that binds uniquely to GPVI. Because it is polymeric, CVX readily induces platelet activation.…”