title: Analysis of the role of von Willebrand factor, platelet glycoprotein VI-, and α2β1-mediated collagen binding in thrombus formation.
abstract: 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 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.
authors: Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, Canada;
authors: Yasuaki
authors: Y
authors: Shida
authors: Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, Canada;
authors: Natalia
authors: N
authors: Rydz
authors: University Hospital and Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany; and.
authors: David
authors: D
authors: Stegner
authors: Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, Canada;
authors: Christine
authors: C
authors: Brown
authors: Division of Cancer Biology and Genetics, Queen's University, Kingston, ON, Canada.
authors: Jeffrey
authors: J
authors: Mewburn
authors: Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, Canada;
authors: Kate
authors: K
authors: Sponagle
authors: Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, Canada;
authors: Ozge
authors: O
authors: Danisment
authors: Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, Canada;
authors: Bredon
authors: B
authors: Crawford
authors: Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, Canada;
authors: Barbara
authors: B
authors: Vidal
authors: Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, Canada;
authors: Carol A
authors: CA
authors: Hegadorn
authors: Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, Canada;
authors: Cynthia M
authors: CM
authors: Pruss
authors: University Hospital and Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany; and.
authors: Bernhard
authors: B
authors: Nieswandt
authors: Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, Canada;
authors: David
authors: D
authors: Lillicrap
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002712:Chlorides / Q000009:adverse effects / Q000494:pharmacology; D003094:Collagen / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D005290:Ferric Compounds / Q000009:adverse effects / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D038982:Integrin alpha2beta1 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D020125:Mutation, Missense; D009676:Noxae / Q000009:adverse effects / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013927:Thrombosis / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism*
chemical_list: D002712:Chlorides; D005290:Ferric Compounds; D038982:Integrin alpha2beta1; D009676:Noxae; D010980:Platelet Membrane Glycoproteins; C098341:platelet membrane glycoprotein VI; D014841:von Willebrand Factor; D003094:Collagen; C024555:ferric chloride