Comprehensive analysis of the role of von Willebrand factor and platelet glycoprotein VI- and α2β1-mediated collagen binding in thrombus formation

Rare missense mutations in the von Willebrand factor (VWF) A3 domain that disrupt collagen binding, have been found in patients with a mild bleeding phenotype. However, the analysis of these aberrant VWF-collagen interactions has been limited. Here, we have developed mouse models of collagen binding mutants and analyzed the function of the A3 domain using comprehensive in vitro and in vivo approaches. 5 loss-of-function (p.S1731T, p.W1745C, p.S1783A, p.H1786D, A3 deletion) and 1 gain-of-function (p.L1757A) variant were generated in the mouse VWF cDNA. The results of these various assays were consistent, although the magnitude of the effects were different: the gain-of-function (p.L1757A) variant showed consistent enhanced collagen binding while the loss-of-function mutants showed variable degrees of functional deficit. We further analyzed the impact of direct platelet-collagen binding by blocking glycoprotein VI (GPVI) and integrin α2β1 in our ferric chloride murine thrombosis model. The inhibition of GPVI demonstrated a comparable functional defect in thrombosis formation to the VWF mice whereas α2β1 inhibition demonstrated a milder bleeding phenotype. Furthermore, a delayed and markedly reduced thrombogenic response was still evident in VWF, GPVI and α2β1 blocked animals, suggesting that alternative primary hemostatic mechanisms can partially rescue the bleeding phenotype associated with these defects. For personal use only. on September 14, 2017. by guest www.bloodjournal.org From