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

Golder, Mia; Pruss, Cynthia M.; Hegadorn, Carol; Mewburn, Jeffrey; Laverty, Kimberly; Sponagle, Kate; Lillicrap, David

doi:10.1182/blood-2009-11-253120

Cited by 37 publications

(39 citation statements)

References 26 publications

(38 reference statements)

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“…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.…”

Section: Figurementioning

confidence: 99%

von Willebrand factor mutation promotes thrombocytopathy by inhibiting integrin αIIbβ3

Casari¹,

Berrou²,

Lebret³

et al. 2013

J. Clin. Invest.

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von Willebrand disease type 2B (vWD-type 2B) is characterized by gain-of-function mutations in von Willebrand factor (vWF) that enhance its binding to the glycoprotein Ib-IX-V complex on platelets. Patients with vWD-type 2B have a bleeding tendency that is linked to loss of vWF multimers and/or thrombocytopenia. In this study, we uncovered evidence that platelet dysfunction is a third possible mechanism for bleeding tendency. We found that platelet aggregation, secretion, and spreading were diminished due to inhibition of integrin αIIbβ3 in platelets from mice expressing a vWD-type 2B-associated vWF (vWF/p.V1316M), platelets from a patient with the same mutation, and control platelets pretreated with recombinant vWF/p.V1316M. Impaired platelet function coincided with reduced thrombus growth. Further, αIIbβ3 activation and activation of the small GTPase Rap1 were impaired by vWF/p.V1316M following exposure to platelet agonists (thrombin, ADP, or convulxin). Conversely, thrombin-or ADP-induced Ca 2+ store release, which is required for αIIbβ3 activation, was normal, indicating that vWF/p.V1316M acts downstream of Ca 2+ release and upstream of Rap1. We found normal Syk phosphorylation and PLCγ2 activation following collagen receptor signaling, further implying that vWF/p.V1316M acts directly on or downstream of Ca 2+ release. These data indicate that the vWD-type 2B mutation p.V1316M is associated with severe thrombocytopathy, which likely contributes to the bleeding tendency in vWD-type 2B.

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Section: Figurementioning

confidence: 99%

von Willebrand factor mutation promotes thrombocytopathy by inhibiting integrin αIIbβ3

Casari¹,

Berrou²,

Lebret³

et al. 2013

J. Clin. Invest.

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“…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.…”

Section: Intravital Microscopy For the Ferric Chloride Injury Model Omentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

Pruss

Golder

Bryant

et al. 2011

Blood

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Type 1 VWD is the mild to moderate reduction of VWF levels. This study examined the mechanisms underlying 2 common type 1 VWD mutations, the severe R1205H and more moderate Y1584C. In vitro biosynthesis was reduced for both mutations in human and mouse VWF, with the effect being more severe in R1205H. VWF knockout mice received hydrodynamic injections of mouse Vwf cDNA. Lower VWF antigen levels were demonstrated in both homozygous and heterozygous forms for both type 1 mutations from days 14-42. Recombinant protein infusions and hydrodynamicexpressed VWF propeptide to antigen ratios demonstrate that R1205H mouse VWF has an increased clearance rate, while Y1584C is normal. Recombinant AD-AMTS13 digestions of Y1584C demonstrated enhanced cleavage of both human and mouse VWF115 substrates. Hydrodynamic-expressed VWF shows a loss of high molecular weight multimers for Y1584C compared with wild-type and R1205H. At normal physiologic levels of VWF, Y1584C showed reduced thrombus formation in a ferric chloride injury model while R1205H demonstrated similar thrombogenic activity to wild-type VWF. This study has elucidated several novel mechanisms for these mutations and highlights that the type 1 VWD phenotype can be recapitulated in the VWF knockout hydrodynamic injection model. IntroductionThe large multimeric glycoprotein VWF is critical to normal hemostasis through mediating platelet-subendothelial interactions as well as binding to platelets to support their aggregation at the site of endothelial damage. The disease phenotype of type 1 VWD is a mild to moderate quantitative reduction of supposedly functionally normal VWF, with plasma VWF levels between 5% and 50% of normal. 1 This disease can be caused by a wide array of defects including defective RNA or protein synthesis, premature protein degradation before cellular release, ineffective secretion, rapid plasma clearance, or a mutation that results in a null allele. 2 R1205H, the Vicenza mutation, has a relatively severe type 1 phenotype that involves accelerated VWF clearance. Often occurring with a second VWF variation, M740I, the Vicenza mutation shows a significant reduction in VWF antigen (VWF:Ag) to ϳ 0.15 U/mL, VWF Ristocetin Cofactor Activity (VWF:RCo) ϳ 0.20 U/mL, and Factor VIII levels Ͻ 0.30 U/mL, but maintains normal platelet VWF levels and function. [3][4][5][6] Patient bleeding scores, a marker of VWD severity, range between 2-17 (n ϭ 18), with a mean of 8 (bleeding score Ն 4 is positive). 1,7-9 Accelerated clearance of the mutant protein has been demonstrated via desmopressin (DDAVP) studies 3 and human recombinant protein infusion in the VWF knockout mouse, 10 as well as through high VWFpp/ VWF:Ag ratios, with observed ratios of 10 or greater for this indirect measurement of VWF clearance from the plasma. 4 R1205H VWF also often displays an increase in high molecular weight multimers along with occasional alteration in the typical multimer triplet band pattern, [3][4][5]11 and has been attributed to the rapid clearance of the protein and thus red...

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“…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.…”

Section: Type 2b Vwdmentioning

confidence: 99%

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

Lillicrap

2013

Hematology

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von Willebrand disease (VWD) is the most common autosomally inherited bleeding disorder. The disease represents a range of quantitative and qualitative pathologies of the adhesive glycoprotein von Willebrand factor (VWF). The pathogenic mechanisms responsible for the type 2 qualitative variants of VWF are now well characterized, with most mutations representing missense substitutions influencing VWF multimer structure and interactions with platelet GPIb␣ and collagen and with factor VIII. The molecular pathology of type 3 VWD has been similarly well characterized, with an array of different mutation types producing either a null phenotype or the production of VWF that is not secreted. In contrast, the pathogenetic mechanisms responsible for type 1 VWD remain only partially resolved. In the hemostasis laboratory, the measurement of VWF:Ag and VWF:RCo are key components in the diagnostic algorithm for VWD, although the introduction of direct GPIb␣-binding assays may become the functional assay of choice. Molecular genetic testing can provide additional benefit, but its utility is currently limited to type 2 and 3 VWD. The treatment of bleeding in VWD involves the use of desmopressin and plasma-derived VWF concentrates and a variety of adjunctive agents. Finally, a new recombinant VWF concentrate has just completed clinical trial evaluation and has demonstrated excellent hemostatic efficacy and safety.

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

Cited by 37 publications

References 26 publications

von Willebrand factor mutation promotes thrombocytopathy by inhibiting integrin αIIbβ3

von Willebrand factor mutation promotes thrombocytopathy by inhibiting integrin αIIbβ3

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

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

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