A novel von Willebrand disease–causing mutation (Arg273Trp) in the von Willebrand factor propeptide that results in defective multimerization and secretion

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 560-568 ◽  
Author(s):  
Simon Allen ◽  
Adel M. Abuzenadah ◽  
Joanna Hinks ◽  
Joanna L. Blagg ◽  
Turkiz Gursel ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Von Willebrand Factor ◽  
Family Members ◽  
Amino Acid Position ◽  
Von Willebrand Disease ◽  
Molecular Defect ◽  
Wild Type ◽  
Von Willebrand ◽  
Willebrand Factor

In this report we describe the molecular defect underlying partial and severe quantitative von Willebrand factor (VWF) deficiencies in 3 families previously diagnosed with types 1 and 3 Von Willebrand-disease. Analysis of the VWF gene in affected family members revealed a novel C to T transition at nucleotide 1067 of the VWF complemetary DNA (cDNA), predicting substitution of arginine by tryptophan at amino acid position 273 (R273W) of pre–pro-VWF. Two patients, homozygous for the R273W mutation, had a partial VWF deficiency (VWF:Ag levels of 0.06 IU/mL and 0.09 IU/mL) and lacked high-molecular weight VWF multimers in plasma. A third patient, also homozygous for the R273W mutation, had a severe VWF deficiency (VWF:Ag level of less than 0.01 IU/mL) and undetectable VWF multimers in plasma. Recombinant VWF having the R273W mutation was expressed in COS-7 cells. Pulse-chase experiments showed that secretion of rVWFR273W was severely impaired compared with wild-type rVWF. However, the mutation did not affect the ability of VWF to form dimers in the endoplasmic reticulum (ER). Multimer analysis showed that rVWFR273W failed to form high-molecular-weight multimers present in wild-type rVWF. We concluded that the R273W mutation is responsible for the quantitative VWF deficiencies and aberrant multimer patterns observed in the affected family members. To identify factors that may function in the intracellular retention of rVWFR273W, we investigated the interactions of VWF expressed in COS-7 cells with molecular chaperones of the ER. The R273W mutation did not affect the ability of VWF to bind to BiP, Grp94, ERp72, calnexin, and calreticulin in COS-7 cells.

A novel von Willebrand disease–causing mutation (Arg273Trp) in the von Willebrand factor propeptide that results in defective multimerization and secretion

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 560-568 ◽  
Author(s):  
Simon Allen ◽  
Adel M. Abuzenadah ◽  
Joanna Hinks ◽  
Joanna L. Blagg ◽  
Turkiz Gursel ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Von Willebrand Factor ◽  
Family Members ◽  
Amino Acid Position ◽  
Von Willebrand Disease ◽  
Molecular Defect ◽  
Wild Type ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract In this report we describe the molecular defect underlying partial and severe quantitative von Willebrand factor (VWF) deficiencies in 3 families previously diagnosed with types 1 and 3 Von Willebrand-disease. Analysis of the VWF gene in affected family members revealed a novel C to T transition at nucleotide 1067 of the VWF complemetary DNA (cDNA), predicting substitution of arginine by tryptophan at amino acid position 273 (R273W) of pre–pro-VWF. Two patients, homozygous for the R273W mutation, had a partial VWF deficiency (VWF:Ag levels of 0.06 IU/mL and 0.09 IU/mL) and lacked high-molecular weight VWF multimers in plasma. A third patient, also homozygous for the R273W mutation, had a severe VWF deficiency (VWF:Ag level of less than 0.01 IU/mL) and undetectable VWF multimers in plasma. Recombinant VWF having the R273W mutation was expressed in COS-7 cells. Pulse-chase experiments showed that secretion of rVWFR273W was severely impaired compared with wild-type rVWF. However, the mutation did not affect the ability of VWF to form dimers in the endoplasmic reticulum (ER). Multimer analysis showed that rVWFR273W failed to form high-molecular-weight multimers present in wild-type rVWF. We concluded that the R273W mutation is responsible for the quantitative VWF deficiencies and aberrant multimer patterns observed in the affected family members. To identify factors that may function in the intracellular retention of rVWFR273W, we investigated the interactions of VWF expressed in COS-7 cells with molecular chaperones of the ER. The R273W mutation did not affect the ability of VWF to bind to BiP, Grp94, ERp72, calnexin, and calreticulin in COS-7 cells.

Two novel type 2N von Willebrand disease–causing mutations that result in defective factor VIII binding, multimerization, and secretion of von Willebrand factor

Blood ◽  
2000 ◽  
Vol 95 (6) ◽  
pp. 2000-2007 ◽  
Author(s):  
Simon Allen ◽  
Adel M. Abuzenadah ◽  
Joanna L. Blagg ◽  
Joanna Hinks ◽  
I. Mandy Nesbitt ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
High Molecular Weight ◽  
Von Willebrand Factor ◽  
Amino Acid Position ◽  
Von Willebrand Disease ◽  
Mild Reduction ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
High Molecular Weight Multimers

Abstract Two novel mutations, a T-to-C transition at nucleotide 2612 and a T-to-G transversion at nucleotide 3923 of the von Willebrand factor (vWF) complementary DNA, were detected by analysis of the vWF gene in DNA from members of 2 families with atypical von Willebrand disease. The T2612C transition predicts substitution of cysteine by arginine at amino acid position 788 (C788R), and the T3923G transversion predicts substitution of cysteine by glycine at position 1225 (C1225G) of pre-pro-vWF. The patients homozygous for the C788R and C1225G mutations both had a partial vWF deficiency (0.18 IU/mL and 0.07 IU/mL vWF antigen, respectively); vWF in plasma from patients homozygous for either the C788R or the C1225G mutation failed to bind factor VIII and lacked high molecular weight multimers. Recombinant (r) vWF molecules having the C788R or C1225G mutation were expressed in COS-7 cells. Both rvWF C788R and rvWF C1225G exhibited significantly impaired secretion and failed to bind factor VIII. Recombinant vWF C788R in COS-7 culture medium showed a severe reduction in high molecular weight multimers, whereas rvWF C1225G showed a very mild reduction in high molecular weight multimers when compared with wild-type rvWF.

Effect of Type IIB von Willebrand Disease Mutation Arg(545)Cys on Platelet Glycoprotein Ib Binding – Studies with Recombinant von Willebrand Factor

1993 ◽  
Vol 70 (06) ◽  
pp. 1058-1062 ◽  
Author(s):  
Aida Inbal ◽  
Nurit Kornbrot ◽  
Paul Harrison ◽  
Anna M Randi ◽  
J Evan Sadler
Keyword(s):  
Molecular Weight ◽  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Wild Type ◽  
Binding Studies ◽  
Function Type ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryType IIB von Willebrand disease (vWD) is characterized by a selective loss of high molecular weight von Willebrand factor (vWF) multimers in plasma due to their abnormally enhanced reactivity with platelets. Several missense mutations in the platelet glycoprotein lb (GPIb) binding domain of vWF were recently characterized that cause type IIB vWD. The effect of type IIB mutation Arg(545)Cys on vWF binding to platelet GPIb was studied using recombinant wild type (rvWFWT) and mutant rvWFR545C expressed in COS-7 cells. In the absence of ristocetin, 50% of rvWFR545C bound spontaneously to platelet GPIb and the binding increased to 70% in the presence of 0.2 mg/ml ristocetin; rvWFWT did not bind significantly under either condition. Botrocetin-induced binding of rvWFR545C was only slightly increased compared to rvWFWT. These data demonstrate that the Arg(545)Cys mutation increases the affinity of vWF for GPIb, resulting in the characteristic gain-of-function type IIB vWD phenotype.

Recombinant Human Von Willebrand Factor (rhVWF): First-In-Human Study Evaluating Pharmacokinetics, Demonstrating Safety and Tolerability In Type 3 Von Willebrand Disease

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 237-237 ◽  
Author(s):  
Tobias Suiter ◽  
Michael Laffan ◽  
Pier M. Mannucci ◽  
Christine L. Kempton ◽  
Edward H Romond ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Von Willebrand Factor ◽  
Phase 1 ◽  
Von Willebrand Disease ◽  
Fviii Activity ◽  
Post Infusion ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 237 Von Willebrand Disease (VWD) is an inherited rare bleeding disorder caused by a deficiency of von Willebrand factor (VWF). VWF is the largest soluble multimeric plasma glycoprotein, which facilitates platelet aggregation and stabilizes FVIII in the circulation. Patients with type 3 disease display severe hemorrhagic symptoms, mainly in mucosal tissues, muscle and joints. Replacement of VWF stabilizes endogenous FVIII to hemostatic levels within hours. Commercially available VWF/FVIII concentrates are plasma-derived (pd) and subject to limitations such as donor dependency, risk of blood-borne pathogen transmission, lack of high molecular weight VWF multimers, and variation in multimer composition. A novel recombinant human VWF (rhVWF) has been developed using a plasma-free method, which represents the largest protein ever produced using recombinant technology. Safety, tolerability and pharmacokinetics of the rhVWF combined at a fixed ratio with rFVIII were investigated in a Phase 1 multicenter, international clinical study in 31 patients with type 3 VWD and severe type 1 VWD. Four concentrations of rhVWF (2, 7.5, 20 and 50 IU VWF:RCo/kg) were administered in a dose-escalating manner in separate cohorts. rhVWF was well tolerated, and no thrombotic events, VWF inhibitors or other serious adverse reactions were observed. Pharmacokinetics of rhVWF/rhFVIII (50 IU VWF:RCo/kg and 38.5 IU FVIII/kg) compared with pdVWF/pdFVIII (50 IU VWF:RCo/kg and 21 IU/kg FVIII/kg) were evaluated in a sub-group of 8/31 patients using a randomized, crossover design (8-day minimum washout period). Interim data in 8 subjects show a higher degree of secondary FVIII activity with rhVWF/rhFVIII compared to pdVWF/pdFVIII (see Figure 1) that is not solely due the difference in the rhVVF:FVIII infusion ratio (1.3:1 rhVWF/rhFVIII vs. approximately 2:1 pdVWF/pdFVIII). The pharmacokinetics of the rhVWF:RCo and pdVWF:RCo were comparable and were also reflected in the VWF:Ag and collagen binding activity. Evidence is also provided for the in vivo cleavage of the ultra-high molecular weight multimers of rhVWF by endogenous ADAMTS13. In summary, interim data from the ongoing Phase 1 study, demonstrate that rhVWF is safe and well tolerated, has VWF:RCo pharmacokinetics that are comparable to pdVWF and enhances stabilization of endogenous FVIII. Multiple doses of rhVWF/rhFVIII would be expected to have beneficial effects in major surgery and severe mucosal bleeding events. These data would also support the treatment concept to administer rhVWF alone once a therapeutic baseline level of endogenous FVIII is achieved (after 1–2 doses).Figure 1:Preliminary PK data from 8 subjects post-infusion of either rhVWF/rhFVIII or pdVWF/pdFVIII. Endogenous FVIII activity reached a plateau after 6 hours and remained stable for at least 30 hours. FVIII was still elevated well above baseline at 96 hoursFigure 1:. Preliminary PK data from 8 subjects post-infusion of either rhVWF/rhFVIII or pdVWF/pdFVIII. Endogenous FVIII activity reached a plateau after 6 hours and remained stable for at least 30 hours. FVIII was still elevated well above baseline at 96 hours Disclosures: Suiter: Baxter BioScience: Employment. Laffan:Baxter BioScience: Consultancy. Mannucci:Baxter BioScience: Consultancy. Kempton:Baxter BioScience: Consultancy. Romond:Baxter BioScience: Consultancy. Shapiro: Baxter BioSci- ence: Consultancy. Birschmann:Baxter BioScience: Consultancy. Gill:Baxter BioScience: Consultancy. Ragni:Baxter BioScience: Consultancy. Turecek:Baxter BioScience: Employment. Ewenstein:Baxter Bioscience: Employment. Baxter BioScience:Baxter BioScience: Employment.

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 Analysis of Arg834Gln and Val902Glu type 2A von Willebrand disease mutations: studies with recombinant von Willebrand factor and correlation with patient characteristics

Blood ◽  
1996 ◽  
Vol 87 (7) ◽  
pp. 2788-2794 ◽  
Author(s):  
T Englender ◽  
A Lattuada ◽  
PM Mannucci ◽  
JE Sadler ◽  
A Inbal
Keyword(s):  
Molecular Weight ◽  
Von Willebrand Factor ◽  
Patient Characteristics ◽  
Von Willebrand Disease ◽  
Wild Type ◽  
Disease Mutations ◽  
Normal Transport ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Intracellular Proteolysis

Type 2A von Willebrand disease (vWD), the most common qualitative form of vWD, is characterized by a relative decrease in circulating intermediate and high molecular weight (HMW) multimers. We studied the biosynthesis of recombinant von Willebrand factor (vWF) containing each of two type 2A vWD mutations previously reported by us, Arg834Gln and Val902Glu. The structure of recombinant Arg834Gln vWF within transfected COS-7 cells and the secretion of HMW multimers were similar to wild type vWF. The normal transport and secretion of Arg834Gln vWF, categorizes it as a group II type 2A mutation. In contrast, the Val90- 2Glu mutation resulted in intracellular proteolysis of vWF with the generation of a 176-kD fragment and retention of vWF between the endoplasmic reticulum and the Golgi complex. Moreover, the 176-kD fragment was also increased in plasma from patients with the Val902Glu mutation. Significantly impaired secretion and intracellular proteolysis of Val902Glu vWF categorizes a new sub-group of type 2A mutations. The intracellular proteolysis of vWF Val902Glu explains the lack of response to 1-deamino 8-D-arginine vasopressin (DDAVP) in patients who carry the mutation.

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.

Human von Willebrand Disease Mutations in Mouse von Willebrand Factor Alter Its Cleavage by ADAMTS13.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2708-2708
Author(s):  
Cynthia M. Pruss ◽  
Carol A. Hegadorn ◽  
Colleen R.P. Notley ◽  
Rouzbeh Chegeni ◽  
Aly S. Dhala ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Von Willebrand Disease ◽  
Full Length ◽  
P Value ◽  
Wild Type ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

Abstract Von Willebrand Factor (VWF) is a large multimeric glycoprotein that mediates platelet adhesion to the damaged blood vessel wall and subsequent platelet aggregation at the site of vascular injury. The size of VWF multimers in plasma is regulated by the specific VWF cleaving protease, ADAMTS13, that cleaves VWF at the Y1605-M1606 bond in the VWF A2 domain. The adhesive properties of VWF is directly related to the multimer size, with loss of high molecular weight VWF leading to the bleeding phenotype in Type 2A von Willebrand disease (VWD) and ultra high molecular weight VWF multimers observed in the prothrombotic state of thrombotic thrombocytopenic purpura (TTP). VWF mutations leading to multimer changes have not been examined in an animal model. Although the human and mouse VWF genes are highly conserved, mutations in the two genes have not been compared experimentally. To that end, we have made mutations in the mouse VWF (mVWF) cDNA to compare the ADAMTS13 cleavage patterns seen with human VWF mutations in two in vitro assays. Recombinant multimerized full-length mVWF was digested with different concentrations of recombinant mouse ADAMTS13 (mADAMTS13) and 1.5M urea, and analyzed via multimer migration distance. The GST and histidine-tagged G1554-T1668 A2 domain region of mVWF (mVWF115) was assayed via ELISA as follows: the mVWF115 was bound to anti-GST coated plates, digested with mADAMTS13, and intact mVWF115 detected via HRP-labeled anti-histidine tag antibody. We examined R1597W (VWD Type 2A), R1306W, (VWD Type 2B), Y1584C (VWD Type 1), as well as two previously described changes that drastically lower VWF cleavage, Y1605A/M1606A, and D1614A/E1615A/K1617A. Y1584C showed a statistically insignificant 41% decrease in cleavage in the full-length assay, but a 20% increase in the mVWF115 assay. Conversely, R1597W had a 64% decrease in the full-length assay, but a 28% increase in the mVWF115 assay. Y1605A/M1606A showed a dramatic loss of cleavage, with no observable loss of high molecular weight multimers at 32 units/ml of mADAMTS13, and a 126-fold higher mADAMTS13 concentration than wild type in the mVWF115 assay. This contrasted with the 8-fold increase observed with D1614A/E1615A/K1617A. The R1306W A1 domain mutation showed a significant increase in cleavage, with 63% less ADAMTS13 necessary in the full-length assay. The triple mutant R1306W/Y1605A/M1606A did not cleave at the highest concentration, 32 U/ml, similar to that of Y1605A/M1606A, showing that the R1306W mutation was not able to increase cleavage of the Y1605A/M1606A change. These results show a strong correlation between the mouse mutations described here and previously reported human VWF mutations in their susceptibility to ADAMTS13 cleavage. These results provide a rationale for the development of mouse models of type 1, 2A, and 2B VWD, as well as mVWF mutations that might lead to a prothrombotic state similar to TTP in humans. mADAMTS13 Concentration for 50% mVWF Cleavage ADAMTS13 Concentration (Units/ml) Relative mADAMTS13 Concentration (to Wild Type) mVWF Multimer (n=1) mVWF115 (n=4) mVWF Multimer (n=1) mVWF115 (n=4) *: P value <0.05, **: P Value < 0.001 in 2 tailed T test. Wild Type 0.301 0.197 1 1 Y1584C 0.423 0.157* 1.41 0.80* R1597W 0.109** 0.253* 0.36** 1.28* Y1605A/M1606A >32** 24.8** >100** 126** D1614A/E1615A/K1617A (−) 1.58** (−) 8.0** R1306W 0.112** (−) 0.37** (−) R1306W/Y1605A/M1606A >32** (−) >100** (−)

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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