Abnormal Proteolytic Processing of von Willebrand Factor Arg611 Cys and Arg 611 His

1997 ◽  
Vol 77 (01) ◽  
pp. 174-182 ◽  
Author(s):  
Toshiya Nishikubo ◽  
Olivier Christophe ◽  
Jean-Maurice Lavergne ◽  
Bernadette Obert ◽  
Kyoko Nonami ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Platelet Count ◽  
Proteolytic Processing ◽  
Terminal Part ◽  
Structural And Functional Properties ◽  
N Terminus ◽  
Abnormal Pattern ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Count Change

SummaryThe structural and functional properties of plasma and platelet vWF were studied in 8 patients (5 unrelated families) with vWD demonstrating a mutation at position 611 (R611C or R611H). Following reduction, electrophoresis and immunoblotting with a polyclonal anti-reduced vWF antibody, abnormal proteolysis of vWF was demonstrated in plasma and to a lesser extent in platelets from all patients, leading to the formation of a unique 209 kDa fragment undetectable in control as well as in type 2A, 2B or 2N vWF. Immunoblotting with MoAbs to reduced vWF showed that the C-terminal end of the 209 kDa fragment was located beyond residue 1744 of the subunit and that its N-terminus was between residues 523 and 1114. Multimeric analysis of patients vWF showed an abnormal pattern in both plasma and platelets, with a moderate decrease of the HMW multimers together with a significant increase of the lowest MW forms. The specific sensitivity of vWF R611C and vWF R611H to proteolysis was further evidenced using V-8 protease. In all patient’s samples the enzyme produced a unique monomeric 80 kDa fragment, absent in V-8 digested normal vWF, which overlapped the N-terminal part of the subunit. The functional analysis of vWF showed a markedly decreased affinity of mutated plasma vWF for platelet GPIb in the presence of ristocetin. Infusion of DDAVP in two of these patients did not lead to significant platelet count change. It induced a limited increase of the HMW multimers in plasma together with a poor correction of the vWF binding to platelet GPIb. In conclusion, our data demonstrate that in addition to a normal proteolysis, vWF mutated at position 611 undergoes a specific cleavage in plasma and platelets. In contrast to the increased proteolysis observed in type 2A and 2B patients’ plasma, this additional cleavage produced a unique 209 kDa species but maintained a HMW multimer-like structure of vWF R611C and R611H.

scholarly journals N-terminal Cleavage of the Salivary MUC5B Mucin

2001 ◽  
Vol 276 (50) ◽  
pp. 47116-47121 ◽  
Author(s):  
Claes Wickström ◽  
Ingemar Carlstedt
Keyword(s):  
Von Willebrand Factor ◽  
Cleavage Site ◽  
Major Part ◽  
Structural Difference ◽  
Proteolytic Cleavage ◽  
Terminal Part ◽  
N Terminus ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Sequence Similarities

Sequence similarities between the oligomeric mucins (MUC2, MUC5AC, MUC5B) and the von Willebrand factor suggest that they may be assembled in a similar way. After oligomerization, a fragment corresponding to the D1 and D2 domains is released from the von Willebrand factor. This cleavage does not appear to occur in pig submaxillary mucin, the only mammalian mucin in which this cleavage has been examined thus far, but whether other oligomeric mucins undergo N terminus proteolysis is not known. Antibodies recognizing the D1, D2, D3, and the first Cys domains in MUC5B were established and used to investigate to what extent proteolytic cleavage occurs within the N-terminal part of salivary MUC5B. The antibodies against the D1 and D2 domains identified a polypeptide corresponding in size to a MUC5B fragment generated by cleavage within the D′ domain analogously with the von Willebrand factor propolypeptide. The antibodies did not recognize the main mucin population, suggesting that the major part of salivary MUC5B is subjected to this cleavage. An antibody recognizing the D3 domain was used to reveal a second cleavage site in the “soluble” but not in the “insoluble” MUC5B fraction: the first structural difference observed between soluble and insoluble salivary MUC5B. The identification of these cleavage events shows that the N-terminal sites for MUC5B oligomerization are present in the D3 domain and/or in domains located C-terminal to this part of the molecule.

VON WILLEBRAND FACTOR (vWF) PRO-POLYPEPTIDE IS REQUIRED FOR vWF MULTIMER FORMATION

1987 ◽  
Author(s):  
C L Verweij ◽  
M Hart ◽  
H Pannekoek
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Vascular Endothelial Cells ◽  
Expression System ◽  
Proteolytic Processing ◽  
Information Encoding ◽  
Interchain Interactions ◽  
Von Willebrand ◽  
Willebrand Factor

The von Willebrand factor (vWF) is a multimeric plasma glycoprotein synthesized in vascular endothelial cells as a pre-pro-polypeptide with a highly repetitive domain structure, symbolized by the formula:(H)-D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-(0H).A heterologous expression system, consisting of a monkey kidney cell line (C0S-1), transfected with full-length vWF cDNA, is shown to mimic the constitutively, secretory pathway of vWF in endothelial cells. The assembly of pro-vWF into multimers and the proteolytic processing of these structures is found to oro-ceed along the following, consecutive steps. Pro-vWF subunits associate to form dimers, a process that does not involve the pro-polypeptide of pro-vWF. This observation is derived from transfection of C0S-1 cells with vWF cDNA, lacking the genetic information encoding the pro-polypeptide, composed of the domains D1 and D2. Pro-vWF dimers are linked intracellularly to form a regular series of multimeric structures that are secreted and cannot be distinguished from those released constitutively by endothelial cells. The presence of the pro-polypeptide, embedded in pro-vWF, is obligatory for multimerization since the deletion mutant lacking the D1 and D2 domains fails to assemble beyond the dimer stage. It is argued that the D domains are involved in interchain interactions.

scholarly journals Influence of Hepatocellular Carcinoma on Platelet Aggregation in Cirrhosis

Cancers ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 1150
Author(s):  
Alberto Zanetto ◽  
Marco Senzolo ◽  
Elena Campello ◽  
Cristiana Bulato ◽  
Sabrina Gavasso ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Platelet Count ◽  
Platelet Aggregation ◽  
Platelet Function ◽  
Adenosine Diphosphate ◽  
Compensated Cirrhosis ◽  
Impedance Aggregometry ◽  
Child Class ◽  
Von Willebrand ◽  
Willebrand Factor

Hyper-functional platelets are being proposed as a potential therapeutic target in multiple cancers. Whether this can be considered in patients with cirrhosis and hepatocellular carcinoma (HCC) is unknown as their platelet function has not yet been investigated. We evaluated platelet function in cirrhosis patients with HCC. Patients with cirrhosis with and without HCC were prospectively recruited. Platelet aggregation, a marker of platelet function, was assessed by impedance aggregometry with adenosine diphosphate (ADP), arachidonic acid (ASPI), and thrombin (TRAP) stimulation. Plasmatic levels of Von Willebrand factor antigen (VWF) were also determined. One-hundred patients were recruited (50 cirrhotics with and 50 without HCC). Cirrhosis severity by Child class and platelet count were comparable between cirrhotics with and without HCC. Cirrhotics with HCC had higher ADP- (45 vs. 28; p < 0.001), ASPI- (47 vs. 28; p < 0.001), and TRAP- (85 vs. 75; p = 0.01) induced platelet aggregation than cirrhotics without HCC, all indicative of platelet hyper-function. The relatively increased platelet aggregation in patients with HCC was confirmed after adjusting the analysis for platelet count/severity of thrombocytopenia. Levels of VWF were higher in patients with vs. without HCC (348 vs. 267; p = 0.006), particularly in compensated cirrhosis. In patients with cirrhosis, HCC is associated with increased platelet aggregation and higher VWF. The clinical implications of these findings deserve further investigation.

Combined effects of plasma von Willebrand factor and platelet measures on the risk of incident venous thromboembolism

Blood ◽  
2021 ◽  
Author(s):  
Magnus Sandvik Edvardsen ◽  
Ellen-Sofie Hansen ◽  
Kristian Hindberg ◽  
Vânia Maris Morelli ◽  
Thor Ueland ◽  
...  
Keyword(s):  
Platelet Count ◽  
Venous Thromboembolism ◽  
Von Willebrand Factor ◽  
Platelet Reactivity ◽  
High Plasma ◽  
Combined Effects ◽  
Exposure Group ◽  
Increased Risk ◽  
Von Willebrand ◽  
Willebrand Factor

Plasma von Willebrand factor (VWF) and platelet reactivity are both risk factors for venous thromboembolism (VTE), and VWF can promote hemostasis by interaction with platelets. In this study, we explored the combined effects of plasma VWF and platelet measures on the risk of incident VTE. A population-based nested case-control study with 403 cases and 816 controls was derived from the Tromsø Study. VWF, platelet count and mean platelet volume (MPV) were measured in blood samples drawn at baseline. Odds ratios (ORs) with 95% confidence intervals (CIs) for VTE were estimated across VWF tertiles, within predefined MPV (&lt;8.5, 8.5-9.5, ≥9.5 fL) and platelet count (&lt;230, 230-299, ≥300·109 L-1) strata. Here, participants with VWF levels in the highest tertile and MPV ≥9.5 fL had an OR of 1.98 (95% CI 1.17-3.36) for VTE compared with those in the lowest VWF tertile and with MPV &lt;8.5 fL in the age- and sex-adjusted model. In the joint exposure group, 48% (95% CI 15% to 96%) of VTEs were attributable to the biological interaction between VWF and MPV. Similarly, individuals with VWF in the highest tertile and platelet count ≥300·109 L-1 had an OR of 2.91 (95% CI 1.49-5.67) compared with those with VWF in the lowest tertile and platelet count &lt;230, and 39% (95% CI -2% to 97%) of VTEs in the joint exposure group were explained by the interaction. Our results suggest that both platelet reactivity and platelet count interact biologically with high plasma VWF, resulting in an increased risk of incident VTE.

BLEEDING TIME IN NORMAL SUBJECTS: ITS RELATIONSHIP WITH SEX, AGE, BLOOD GROUP, HEMATOCRIT, PLATELET COUNT AND PLASMA VON WILLEBRAND FACTOR LEVEL

1987 ◽  
Author(s):  
F Rodeghiero ◽  
G Castaman ◽  
E Di Bona ◽  
M Ruggeri
Keyword(s):  
Platelet Count ◽  
Blood Group ◽  
Von Willebrand Factor ◽  
Bleeding Time ◽  
Physiological Parameters ◽  
Factor Level ◽  
Significant Difference ◽  
Von Willebrand ◽  
Willebrand Factor

Bleeding time (BT) is the most important test for in "vivo" Vevaluation of primary hemostasis. Several physiological parameters namely sex, age, blood group, hematocrit, platelet count and von Willebrand factor level could exert a significant influenceIn this study, the relationships between BT (Simplate II,General Diagnostics)and these physiological parameters havebeen examined in 58 subjects aging 17-71 (32 males and 26 females; 26 of 0 and 32 of non-0 group). In all the subjects bleedingdiathesis was excluded by interview. They were not taking anvmedicament for at least 10 days and showed normal platelet aggregation by ADP and ArachidonateMean BT value (seconds) was 318± 65 (range 195-A95)Statistical analysis failed to show any significant difference realated to sex and blood group. There was no significant relationship with hematocrit (0.01), platelet count (−0.2A), age (−0.28) and von Willebrand factor level, mesured as ristocetin cofactor (−0.2A). In particular, our data indicate that higher von Willebrand factor levels found in non-0 group in comparison with O-group (113.3 vs 83.5, P < 0:001) do not exert any apparent influence on bleeding time

Binding of ADAMTS13 to Von Willebrand Factor Under Static Conditions and Under Fluid Shear Stress

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 258-258
Author(s):  
Hendrik B Feys ◽  
Patricia J Anderson ◽  
J. Evan Sadler
Keyword(s):  
Shear Stress ◽  
Von Willebrand Factor ◽  
Time Course ◽  
Fluid Shear Stress ◽  
Proteolytic Processing ◽  
Full Length ◽  
Fluid Shear ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract ADAMTS13 is a plasma metalloprotease that is essential for the normal proteolytic processing of von Willebrand factor (VWF). Dysfunctional ADAMTS13 may lead to thrombotic thrombocytopenic purpura, as uncleaved and unusually large VWF multimers accumulate in the blood and cause intravascular platelet aggregation. Many studies indicate that proteolysis of multimeric VWF involves conformational changes in the VWF A2 domain that expose the Y1605-M1606 scissile bond and also allow substrate binding to multiple exosites on ADAMTS13. For example, VWF is resistant to proteolysis by ADAMTS13 unless the VWF is subjected to fluid shear stress, mild denaturation with guanidine or urea, or adsorption onto a surface. However, the functional interactions between shear stress, various ADAMTS13 binding sites and VWF cleavage are not understood. Therefore, we investigated the effect of fluid shear stress and ADAMTS13 structure on ADAMTS13-VWF binding and VWF cleavage. Upon mixing recombinant VWF (rVWF) and ADAMTS13 in a physiological buffer (50 mM HEPES, 5 mM CaCl2, 1 μM ZnCl2, 150 mM NaCl, pH 7.4), we found that immunoprecipitation with anti-VWF also pulled down substantial amounts of ADAMTS13. Although less striking, a similar result was obtained with purified plasma VWF. Therefore, ADAMTS13 can bind VWF without gaining access to the cleavage site in VWF domain A2. When fluid shear stress was applied for 2 min with a bench-top vortexer, ADAMTS13 binding increased 3-fold and VWF was also cleaved. Lowering the ionic strength markedly increased the rate of VWF cleavage but did not affect ADAMTS13 binding, which suggests that cleavage and binding depend on distinct VWF-ADAMTS13 interactions. Shear-induced binding was reversible slowly upon removal of unbound ADAMTS13 or rapidly by addition of SDS. ADAMTS13-VWF binding was stable for at least 24 h after cessation of shear stress, indicating that the structural change in VWF that promotes binding was not readily reversible. Using a catalytically inactive ADAMTS13 variant to simplify the analysis of binding assays, 30 nM ADAMTS13(E231Q) bound to 30 μg/ml rVWF (120 nM subunits) with a stoichiometry of 0.012 ± 0.004 under static conditions and 0.098 ± 0.023 after shearing (mean ± SD, n = 3, P = 0.019). With 120 nM ADAMTS13(E231Q) the stoichiometry increased to 0.086 ± 0.036 under static conditions and 0.469 ± 0.033 after shearing for 2 min. Recombinant ADAMTS13 truncated after TSP-1 repeat 8 (lacking the C-terminal CUB domains, delCUB), or truncated after the Spacer domain (consisting of domains MDTCS), did not bind rVWF under static conditions, implicating the CUB domains in binding to VWF. In contrast, full-length ADAMTS13, delCUB and MDTCS bound similarly to rVWF after shearing. In a previous study, delCUB and MDTCS did not cleave VWF subjected to fluid shear stress (Zhang et al, Blood2007; 110: 1887–1894). However, under the conditions employed in these experiments, MDTCS and delCUB displayed significant proteolytic activity, cleaving VWF at a rate comparable to that of full length ADAMTS13 when shear stress was applied over a time course of 0–160 sec. We conclude that ADAMTS13 CUB domains contribute to binding a few sites on multimeric VWF under static conditions, whereas ADAMTS13 MDTCS domains are sufficient to bind many sites in an altered conformation of VWF that is induced by fluid shear stress. Binding of ADAMTS13 to unsheared VWF multimers may facilitate the cleavage of VWF within a growing thrombus.

Association of Quantitative and Qualitative Abnormalities of Von Willebrand Factor and Risk of Death In Hemodialysis Patients

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2217-2217
Author(s):  
Rachel Holden ◽  
Angie Tuttle ◽  
Francis MacLeod ◽  
Toni Burbidge ◽  
Carol Hegadorn ◽  
...  
Keyword(s):  
Chronic Kidney Disease ◽  
Kidney Disease ◽  
Vascular Disease ◽  
Von Willebrand Factor ◽  
Functional Activity ◽  
Conflicts Of Interest ◽  
Proteolytic Processing ◽  
Risk Of Death ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 2217 In order to evaluate the possible role of abnormalities of von Willebrand factor in the hemostatic defects seen in indivdiuals with chronic kidney disease (CKD), a cohort study was performed evaluating pre- and post-dialysis levels of von Willebrand factor (VWF), VWF multimer profiles and levels of its cleaving protease, ADAMTS-13. There were 57 subjects (31 males, 26 females) enrolled with CKD with a mean age of 75 years (range 60 – 90). Subjects with known vascular disease were recruited; 49 (86%) had documented ischemic heart disease, 16 (29%) had cerebrovascular disease and 17 (31%) had peripheral vascular disease. A little over half had diabetes mellitus (30 subjects or 54%), 37 (67%) were on antiplatelet therapy and 7 (13%) were chronically anticoagulated with warfarin. Blood samples were drawn immediately pre- and again post-dialysis and all results were compared with a group of age-matched normal controls (Table 1). As has been previously reported, VWF antigen levels (VWF:Ag) and VWF functional activity as measured by the ristocetin cofactor assay (VWF:RCo) were higher in the pre-dialysis samples compared with controls, and both levels were increased even further following dialysis. Additionally, the percentage of high molecular weight VWF multimers (% HMWM) were significantly increased in the pre-dialysis samples compared with controls. This is a novel finding, and the level of % HMWM seen in the subjects is similar to what has been reported in individuals with Thrombotic Thrombocytopenic Purpura (TTP). This difference decreased following dialysis, potentially due to the effect of shear stress on VWF and the resultant proteolytic processing, however still remained significantly higher when compared with controls. ADAMTS-13 functional activity was lower in the subjects compared with controls, providing a possibly explanation for the increase in % HMWM. IL-6 levels are higher in subjects compared with controls. IL-6, which is an inflammatory cytokine known to be increased in patients with CKD, has been previously reported as a marker of inactivation of ADAMTS-13. Two years after enrollment, follow up of the subjects revealed that 22 had died, 17 from documented cardiovascular events. Higher VWF levels at the time of enrollment significantly correlated with risk of death (p=0.041) during the study period. The increase in % HMWM suggests that a “TTP-like phenotype” may also be playing a role. Taken together, these data suggest that both quantitative and qualitative abnormalities of VWF contribute to the risk of thrombotic death in chronic kidney disease. Disclosures: No relevant conflicts of interest to declare.

Altered von Willebrand factor subunit proteolysis and multimer processing associated with the Cys2362Phe mutation in the B2 domain

2007 ◽  
Vol 97 (04) ◽  
pp. 527-533 ◽  
Author(s):  
Luigi Marco ◽  
Lisa Gallinaro ◽  
Maryta Sztukowska ◽  
Mario Mazzuccato ◽  
Monica Battiston ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Proteolytic Processing ◽  
Triplet Structure ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor ◽  
Ristocetin Cofactor ◽  
Ristocetin Cofactor Activity ◽  
Marked Drop

SummaryThe normal von Willebrand factor (vWF) multimer pattern results from the ADAMTS-13 cleavage of the Tyr1605-Met1606 bond in the A2 domain of vWF. We identified a patient with severe von Willebrand disease (vWD) homozygously carrying a Cys to Phe mutation in position 2362 of vWF with markedly altered vWF multimers and an abnormal proteolytic pattern. The proband’s phenotype was characterized by a marked drop in plasma vWF antigen and ristocetin cofactor activity, and a less pronounced decrease in FVIII. The vWF multimers lacked any triplet structure, replaced by single bands with an atypical mobility, surrounded by a smear, and abnormally large vWF multimers. Analysis of the plasma vWF subunit's composition revealed the 225 kDa mature form and a single 205 kDa fragment, but not the 176 kDa and 140 kDa fragments resulting from cleavage by ADAMTS-13.The 205 kDa fragment was distinctly visible, along with the normal vWF cleavage products, in the patient's parents who were heterozygous for the Cys2362Phe mutation. Their vWF levels were mildly decreased and vWF multimers were organized in triplets, but also demonstrated abnormally large forms and smearing. Our findings indicate that a proper conformation of the B2 domain, which depends on critical Cys residues, may be required for the normal proteolytic processing of vWF multimers.

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