Cloning, expression and functional characterization of the full‐length murine ADAMTS13

Bruno, Katharina; Völkel, Dirk; Plaimauer, Barbara; Antoine, Gerhard; Pable, Sabrina; Motto, David G.; Lemmerhirt, Heidi L.; Dorner, Friedrich; Zimmermann, Klaus F.; Scheiflinger, F.

doi:10.1111/j.1538-7836.2005.01246.x

Cited by 11 publications

(11 citation statements)

References 43 publications

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“…The mouse VWF protein amino acid sequence has 83% identity and 91% homology to that of human VWF, with similar variation in the ADAMTS13 sequences. 29,33 Mouse ADAMTS13 has poor activity on the human VWF substrate in both full-length and A2 domain VWF73 assays. 34 This is further exacerbated by a retrotransposon insertion in C57Bl/6 mice that leads to the premature truncation of ADAMTS13.…”

Section: Discussionmentioning

confidence: 99%

Section: In Vitro Adamts13 Digests Of Recombinant Vwfmentioning

confidence: 99%

“…EDTA at a final concentration of 50mM was added to stop the reaction. 29 Samples were frozen at Ϫ80°C until multimer analysis. Analysis of the relative multimer migration for full-length digests was analyzed as previously described, using a 4-parameter curve fit to determine EC 50 , or ADAMTS13 concentration to cause 50% loss of relative multimer distance.…”

mentioning

confidence: 99%

“…Analysis of the relative multimer migration for full-length digests was analyzed as previously described, using a 4-parameter curve fit to determine EC 50 , or ADAMTS13 concentration to cause 50% loss of relative multimer distance. 29 Curve fit F tests for logEC 50 were performed to determine statistical significance.The hVWF115 and mVWF115 ADAMTS13 digestion ELISA assays were modified from the ADAMTS13 activity ELISA. 27 Reacti-Bind Anti-GST plates (Pierce) were coated with 1.25 mg/mL VWF115 in PBS, pH 7.2 for 1 hour, and washed with PBS/0.05% Tween-20 (PBS/Tween).…”

mentioning

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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Section: Discussionmentioning

confidence: 99%

Section: In Vitro Adamts13 Digests Of Recombinant Vwfmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

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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“…The reactions were stopped through the addition of ethylenediaminetetraacetic acid at a final concentration of 50mM. 15 Samples were frozen at Ϫ80°C until multimer analysis. Analysis of the relative multimer migration was performed as previously described.…”

Section: Adamts13 Digestionmentioning

confidence: 99%

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

Golder¹,

Pruss²,

Hegadorn³

et al. 2010

Blood

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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...

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ADAMTS13 endopeptidase 3.4.24.87

Schomburg¹,

Schomburg²

2013

Class 3.4–6 Hydrolases, Lyases, Isomerases, Ligases

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Cloning, expression and functional characterization of the full‐length murine ADAMTS13

Cited by 11 publications

References 43 publications

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

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

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

ADAMTS13 endopeptidase 3.4.24.87

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