RESUMO
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. Five loss-of-function (p.S1731T, p.W1745C, p.S1783A, p.H1786D, A3 deletion) and 1 gain-of-function (p.L1757A) variants were generated in the mouse VWF complementary DNA. 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 whereas 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 α2ß1 in our ferric chloride murine thrombosis model. The inhibition of GPVI demonstrated a comparable functional defect in thrombosis formation to the VWF(-/-) mice whereas α2ß1 inhibition demonstrated a milder bleeding phenotype. Furthermore, a delayed and markedly reduced thrombogenic response was still evident in VWF(-/-), GPVI, and α2ß1 blocked animals, suggesting that alternative primary hemostatic mechanisms can partially rescue the bleeding phenotype associated with these defects.
Assuntos
Colágeno/metabolismo , Integrina alfa2beta1/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Trombose/metabolismo , Fator de von Willebrand/metabolismo , Substituição de Aminoácidos , Animais , Cloretos/efeitos adversos , Cloretos/farmacologia , Colágeno/genética , Modelos Animais de Doenças , Compostos Férricos/efeitos adversos , Compostos Férricos/farmacologia , Células HEK293 , Humanos , Integrina alfa2beta1/genética , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Noxas/efeitos adversos , Noxas/farmacologia , Glicoproteínas da Membrana de Plaquetas/genética , Estrutura Terciária de Proteína , Trombose/induzido quimicamente , Trombose/genética , Trombose/patologia , Fator de von Willebrand/genéticaRESUMO
BACKGROUND: The multimeric glycoprotein von Willebrand factor (VWF) mediates platelet adhesion and aggregation at the site of vessel injury. The adhesive activity of VWF is influenced by its multimer length which is regulated by the metalloprotease ADAMTS13. The ability of ADAMTS13 to regulate platelet thrombus growth in a shear-dependent manner has been described, however, the mechanistic basis of this action has not been well characterized. METHODS: We developed an mCherry-tagged murine ADAMTS13 protein and utilized an ex vivo flow chamber system to visualize the localization of ADAMTS13 within the platelet thrombus under different conditions of shear. Using this system, we also assessed the influence of platelet-mediated tensile force on ADAMTS13 localization within the thrombus using gain-of-function GPIb binding and loss-of-function GPIIbIIIa binding mutants in VWF/ADAMTS13 DKO mice. RESULTS: ADAMTS13 was visualized on the growing platelet thrombus under very high shear using ADAMTS13-mcherry. ADAMTS13-mCherry localized particularly at the top portion of the thrombus and reduced thrombus size as it grew to occlusion. At the pathological high shear of 7500 s-1, platelet-mediated tensile force, involving GPIb but not GPIIbIIIa receptors, influenced localization of ADAMTS13 to the thrombus under conditions of shear. CONCLUSIONS: Tensile force applied on VWF produced by shear stress and platelet GPIb binding has a crucial role in ADAMTS13 activity at the site of thrombus formation. These results suggest that ADAMTS13 activity at the site of platelet thrombus formation is regulated by a shear stress and platelet-dependent feedback mechanism to prevent vessel occlusion and pathological thrombosis.