Mutation-specific hemostatic variability in mice expressing common type 2B von Willebrand disease substitutions

Abstract

Type 2B von Willebrand disease (2B VWD) results from von Willebrand factor (VWF) A1 mutations that enhance VWF-GPIb␣ binding. These "gain of function" mutations lead to an increased affinity of the mutant VWF for platelets and the binding of mutant highmolecular-weight VWF multimers to platelets in vivo, resulting in an increase in clearance of both platelets and VWF. Three common 2B VWD mutations (R1306W, V1316M, and R1341Q) were independently introduced into the mouse Vwf cDNA sequence and the expression vectors delivered to 8-to 10-week-old C57Bl6 VWF ؊/؊ mice, using hydrodynamic injection. The resultant phenotype was examined, and a ferric chloride-induced injury model was used to examine the thrombogenic effect of the 2B VWD variants in mice. Reconstitution of only the plasma component of VWF resulted in the generation of the 2B VWD phenotype in mice. Variable thrombocytopenia was observed in mice expressing 2B VWF, mimicking the severity seen in 2B VWD patients: mice expressing the V1316M mutation showed the most severe thrombocytopenia. Ferric chlorideinduced injury to cremaster arterioles showed a marked reduction in thrombus development and platelet adhesion in the presence of circulating 2B VWF. These defects were only partially rescued by normal platelet transfusions, thus emphasizing the key role of the abnormal plasma VWF environment in 2B VWD. IntroductionType 2B von Willebrand disease (2B VWD) is a qualitative variant of VWD, in which there is an increased affinity of the mutant von Willebrand factor (VWF) for platelet glycoprotein Ib␣ (GPIb␣). 1 Inherited in an autosomal-dominant manner, it arises as a result of missense mutations clustered within exon 28 of the VWF gene, the region that encodes the VWF A1 protein domain involved in the binding of VWF to GPIb␣. 1,2 The gain-of-function phenotype appears to arise through the destabilization of the A1 domain, mimicking the structural changes seen when immobilized VWF is activated through shear stress and allowing the binding of VWF to GPIb␣ in the absence of vascular injury. 3,4 The bleeding phenotype seen in 2B VWD patients probably arises through a multifactorial mechanism: (1) a decrease in plasma high-molecular-weight (HMW) multimers, (2) the occurrence of thrombocytopenia, and (3) the inability of platelets to interact with immobilized VWF at the site of vascular damage. 3 The thrombocytopenia and decrease in HMW multimers arise as a result of increased clearance of both platelets and VWF. 3 In addition, 2B VWF is more susceptible to ADAMTS13-mediated cleavage. 5 The VWF mutation database lists more than 50 reports of 24 different mutations leading to 2B VWD. 6 Of these, the mutations R1306W, V1316M, and R1341Q are the most common, having been reported 10, 9, and 7 times, respectively. 6 The recent study of Federici et al 7 of a cohort of 67 2B VWD patients showed a heterogeneous clinical presentation, dependent on the VWF A1 domain mutation. Of the 11 mutations present in this cohort, the V1316M mutation resulted in the most significant thro...

von Willebrand factor mutation promotes thrombocytopathy by inhibiting integrin αIIbβ3

“…Three different experimental models were compared: (a) a murine model for vWD-type 2B, in which the mvWF/p.V1316M mutation is expressed via hydrodynamic injection (11,18); (b) analysis of platelets isolated from a patient with vWD-type 2B heterozygous for the p.V1316M mutation; and finally, (c) a model in which isolated normal human platelets are activated after preincubation with recombinant hvWF/p.V1316M.…”

Pathologic mechanisms of type 1 VWD mutations R1205H and Y1584C through in vitro and in vivo mouse models

“…21,31,32 Male mice were anesthetized via intraperitoneal injection of ketamine/xylazine/ atropine. The jugular vein was cannulated for injection of rhodamine 6G (40 ng; Sigma-Aldrich) to fluorescently label platelets in vivo and the cremaster exteriorized.…”

“…This experimental strategy has already recapitulated the human disease phenotypes for defective binding to collagen and GPIIbIIIa, 19 and type 2B VWD. 20,21 However, until now, quantitative VWD phenotypes have not been explored using this methodology.…”

“…This experimental strategy has already recapitulated the human disease phenotypes for defective binding to collagen and GPIIbIIIa, 19 and type 2B VWD. 20,21 However, until now, quantitative VWD phenotypes have not been explored using this methodology.Both R1205H and Y1584C have been well described in type 1 VWD patient populations. However, neither mutation has been examined for longer-term expression in an animal model system to evaluate the associated pathogenic mechanisms in a controlled setting.…”

von Willebrand disease: advances in pathogenetic understanding, diagnosis, and therapy

“…The phenotype is transmitted as a highly penetrant dominant trait, and clinical and mouse model evidence indicates that the bleeding severity and accompanying thrombocytopenia is related predominantly to the specific type 2B mutation inherited. [12][13][14] In addition, acquired influences such as acute stress and pregnancy, in which the levels of mutant protein are increased, will also result in worsening of the in vivo platelet clumping and thrombocytopenia, thus aggravating the risk of bleeding. 12 This disorder has a genocopy in platelet-type VWD in which gain-of-function mutations in the GPIb␣ gene result in enhanced binding to VWF.…”