The generation of thrombin in vivo induces the selective loss of high molecular weight multimers of von Willebrand factor and the reversible sequestration of platelets

1993 ◽  
Vol 85 (4) ◽  
pp. 751-760 ◽  
Author(s):  
Cheng Hock Toh ◽  
Hugh Hoogendoorn ◽  
Alan R. Giles
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Von Willebrand Factor ◽  
Selective Loss ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
High Molecular Weight Multimers

Laboratory Predictors Of Loss Of High Molecular Weight Multimers In Patients With Cardiovascular Disease-Associated Acquired Von Willebrand Syndrom

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 335-335
Author(s):  
Ewa M. Wysokinska ◽  
Dong Chen ◽  
Joseph L Blackshear
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Von Willebrand Factor ◽  
Bleeding Risk ◽  
Protein Electrophoresis ◽  
Von Willebrand Disease ◽  
Cardiovascular Disorders ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
High Molecular Weight Multimers

Abstract Background Association of acquired von Willebrand syndrome (AVWS) with various cardiovascular (CV) disorders such as cardiac valve disease and hypertrophic cardiomyopathy (HCM) is well known and documented. The mechanism is thought to be related to shear stress induced loss of high molecular weight multimers (HMWM). The gold standard test to assess for loss of HMWM is von Willebrand protein electrophoresis and then visual assessment of loss of high molecular weight bands. This is both a costly and subjective test. Ratio of von Willebrand factor activity to antigen level is useful in patients with type IIA Von Willebrand Disease caused by loss of HMW multimer, but its sensitivity to detect CV-associated AVWS is unknown. Aim Our aim was to test whether routine VWF laboratory tests could be used to predict which patients with CV conditions are going to have high molecular weight multimer loss. We also aimed to assess whether these tests could be used to predict bleeding risk in patients with CV disorders. Methods We prospectively collected laboratory data of 234 patients with cardiovascular disorders known to be associated with AVWS: aortic stenosis (66), aortic insufficiency(22), aortic and mitral valve prostheses(38), mitral valve regurgitation (51) and hypertrophic cardiomyopathy(57). All patients had Von Willebrand factor antigen (VWF:Ag), Von Willebrand factor activity by latex method (VWF:Ltx), platelet function testing via PFA-100 CADP as well as von Willebrand factor multimers tested. All patients also completed a bleeding questionnaire. We used logistic regression model to calculate the relationship between the VWF:Ltx/VWF:Ag ratio and loss of high molecular weight multimers. Same analysis was performed for PFA-100. We also tested these associations for bleeding risk. Results Mean value for VWF:Ag was 142 IU/dL, VWF:Ltx 121%, PFA-100 151 seconds and 0.86 for the VWF:Ltx/Ag ratio. Over a half of patients (56%) had VWF multimer loss noted on protein electrophoresis testing and a quarter reported bleeding on bleeding questionnaire. The ratio of VWF:Ltx to VWF:Ag had strong correlation with HMW multimer loss (p<0.001) with AUC of 0.77. Correlation with PFA-100 was even stronger with AUC of 0.83. The ratio cut off value of 0.83 had sensitivity of 60% and specificity of 83% in predicting multimer loss. With the cut off of 0.77, specificity reached 95%. With PFA 100 value of 118 seconds, specificity was 76% and sensitivity was 80%. Increasing the cut off to 198 seconds improved the specificity to 95%. The association with bleeding was present for PFA-100 (p=0.01), but did not exist for the Ltx/Ag ratio. Conclusions PFA-100 CADP as well as VWF:Ag and VWF:Ltx can be used to detect acquired Von Willebrand disease in patients with cardiovascular disorders and may decrease the need for costly and time consuming testing of multimers. PFA-100 CADP also correlates with the bleeding risk in these patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Clearance of normal and type 2A von Willebrand factor in the rat

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1692-1699 ◽  
Author(s):  
JH Jr Stoddart ◽  
J Andersen ◽  
DC Lynch
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Von Willebrand Factor ◽  
Clearance Rate ◽  
Similar Rate ◽  
Metabolic Clearance ◽  
Wild Type ◽  
Von Willebrand ◽  
Willebrand Factor

A model for the in vivo clearance of normal and mutant forms of human von Willebrand factor (vWF) has been established using catheterized rats. vWF clearance rates in rat plasma were determined by quantitation of reduced vWF subunits on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and multimeric vWF was analyzed using nondenaturing SDS-agarose gels. Normal vWF derived from human umbilical vein endothelial cells displayed a biphasic pattern of clearance, with half times of 35 minutes (T 1/2 a; SD 15. min.) and 245 minutes (T 1/2 b; SD 76. min.); metabolic clearance rate = 0.65%/minute. High molecular weight multimers of vWF were cleared more rapidly than dimeric vWF. vWF containing the S1613P mutation found in some type 2A von Willebrand disease (vWD) patients was observed to undergo proteolysis in vivo resulting in a reduction of high molecular weight vWF and concomitant appearance of rapidly-migrating satellite species, although the overall clearance rate of vWF antigen was similar to wild type vWF. These results provide direct in vivo evidence that the S1613P mutation causes the characteristic type 2A vWD phenotype. Full-length recombinant vWF produced from transfected Chinese hamster ovary cells was cleared at a similar rate to endothelial cell-derived vWF, and recombinant vWF devoid of O-linked carbohydrates was cleared significantly faster. vWF devoid of sulfate was cleared at a similar rate as wild type vWF, indicating the sulfate moiety of vWF does not regulate in vivo clearance. This animal model should prove useful in subsequent in vivo analysis of additional forms of vWD and in the development of protease inhibitor therapy for 2A vWD.

Impact of mutations in the von Willebrand factor A2 domain on ADAMTS13-dependent proteolysis

Blood ◽  
2006 ◽  
Vol 107 (6) ◽  
pp. 2339-2345 ◽  
Author(s):  
Wolf Achim Hassenpflug ◽  
Ulrich Budde ◽  
Tobias Obser ◽  
Dorothea Angerhaus ◽  
Elke Drewke ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Von Willebrand ◽  
A2 Domain ◽  
The Impact ◽  
Willebrand Factor ◽  
High Molecular Weight Multimers

Abstract Classical von Willebrand disease (VWD) type 2A, the most common qualitative defect of VWD, is caused by loss of high-molecular-weight multimers (HMWMs) of von Willebrand factor (VWF). Underlying mutations cluster in the A2 domain of VWF around its cleavage site for ADAMTS13. We investigated the impact of mutations commonly found in patients with VWD type 2A on ADAMTS13-dependent proteolysis of VWF. We used recombinant human ADAMTS13 (rhuADAMTS13) to digest recombinant full-length VWF and a VWF fragment spanning the VWF A1 through A3 domains, harboring 13 different VWD type 2A mutations (C1272S, G1505E, G1505R, S1506L, M1528V, R1569del, R1597W, V1607D, G1609R, I1628T, G1629E, G1631D, and E1638K). With the exception of G1505E and I1628T, all mutations in the VWF A2 domain increased specific proteolysis of VWF independent of the expression level. Proteolytic susceptibility of mutant VWF in vitro closely correlated with the in vivo phenotype in patients. The results imply that increased VWF susceptibility for ADAMTS13 is a constitutive property of classical VWD type 2A, thus explaining the pronounced proteolytic fragments and loss of HMWM seen in multimer analysis in patients.

scholarly journals Multimer size dependence of von Willebrand factor binding to crosslinked or noncrosslinked fibrin

Blood ◽  
1990 ◽  
Vol 75 (7) ◽  
pp. 1460-1465
Author(s):  
JA Ribes ◽  
CW Francis
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Von Willebrand Factor ◽  
Factor Xiii ◽  
Primary Cultures ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
High Molecular Weight Multimers

von Willebrand factor (vWF) is synthesized in endothelial cells (EC) and may be either secreted constitutively or stored in Weibel-Palade bodies (WPB) for regulated release. Because fibrin stimulates rapid vWF release from EC, we examined the binding of EC synthesized vWF to fibrin. Culture medium containing constitutively secreted vWF was removed from metabolically labeled primary cultures of human umbilical vein EC, and vWF released from WPB was obtained after stimulation by A23187. vWF-deficient fibrinogen with or without factor XIII was added to releasate or media and clotted with thrombin to form crosslinked or noncrosslinked fibrin. vWF was immunopurified from releasate or media before and after clotting, and the amount and multimeric pattern of vWF bound was determined after sodium dodecyl sulfate agarose gel electrophoresis. High molecular weight multimers of vWF, whether secreted constitutively or released from WPB, bound preferentially to fibrin. Multimers of greater than 20 subunits represented 60% +/- 4% (SEM) of A23187 released vWF and 11% +/- 5% of media vWF, but binding to fibrin was similar, 96% +/- 1% and 94% +/- 2%, respectively. A progressively smaller proportion of vWF bound as multimer size decreased, and dimeric vWF binding was least, with 34% +/- 5% binding from A23187 releasate and 51% +/- 4% from media. The amount of vWF binding to crosslinked or noncrosslinked fibrin was similar, and preferential binding of high molecular weight multimers occurred with both. As measured by enzyme-linked immunosorbent assay, 45% +/- 2% of constitutively secreted vWF bound to crosslinked fibrin and 50% +/- 2% to noncrosslinked fibrin. The propolypeptide of vWF did not bind to fibrin. These findings indicate that binding of EC secreted vWF binding to fibrin depends on multimeric size but not on factor XIII crosslinking. This suggests that vWF released from EC in the presence of fibrin will bind locally, thereby facilitating platelet adhesion to the hemostatic plug or thrombus.

scholarly journals Clearance of normal and type 2A von Willebrand factor in the rat

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1692-1699 ◽  
Author(s):  
JH Jr Stoddart ◽  
J Andersen ◽  
DC Lynch
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Von Willebrand Factor ◽  
Clearance Rate ◽  
Similar Rate ◽  
Metabolic Clearance ◽  
Wild Type ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract A model for the in vivo clearance of normal and mutant forms of human von Willebrand factor (vWF) has been established using catheterized rats. vWF clearance rates in rat plasma were determined by quantitation of reduced vWF subunits on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and multimeric vWF was analyzed using nondenaturing SDS-agarose gels. Normal vWF derived from human umbilical vein endothelial cells displayed a biphasic pattern of clearance, with half times of 35 minutes (T 1/2 a; SD 15. min.) and 245 minutes (T 1/2 b; SD 76. min.); metabolic clearance rate = 0.65%/minute. High molecular weight multimers of vWF were cleared more rapidly than dimeric vWF. vWF containing the S1613P mutation found in some type 2A von Willebrand disease (vWD) patients was observed to undergo proteolysis in vivo resulting in a reduction of high molecular weight vWF and concomitant appearance of rapidly-migrating satellite species, although the overall clearance rate of vWF antigen was similar to wild type vWF. These results provide direct in vivo evidence that the S1613P mutation causes the characteristic type 2A vWD phenotype. Full-length recombinant vWF produced from transfected Chinese hamster ovary cells was cleared at a similar rate to endothelial cell-derived vWF, and recombinant vWF devoid of O-linked carbohydrates was cleared significantly faster. vWF devoid of sulfate was cleared at a similar rate as wild type vWF, indicating the sulfate moiety of vWF does not regulate in vivo clearance. This animal model should prove useful in subsequent in vivo analysis of additional forms of vWD and in the development of protease inhibitor therapy for 2A vWD.

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