Effect of recombinant von Willebrand factor reproducing type 2B or type 2M mutations on shear-induced platelet aggregation

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3796-3803 ◽  
Author(s):  
Nadine Ajzenberg ◽  
Anne-Sophie Ribba ◽  
Ghassem Rastegar-Lari ◽  
Dominique Meyer ◽  
Dominique Baruch
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
High Shear ◽  
Shear Rates ◽  
High Shear Rates ◽  
Consistent Increase ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The aim was to better understand the function of von Willebrand factor (vWF) A1 domain in shear-induced platelet aggregation (SIPA), at low (200) and high shear rate (4000 seconds-1) generated by a Couette viscometer. We report on 9 fully multimerized recombinant vWFs (rvWFs) expressing type 2M or type 2B von Willebrand disease (vWD) mutations, characterized respectively by a decreased or increased binding of vWF to GPIb in the presence of ristocetin. We expressed 4 type 2M (-G561A, -E596K, -R611H, and -I662F) and 5 type 2B (rvWF-M540MM, -V551F, -V553M, -R578Q, and -L697V). SIPA was strongly impaired in all type 2M rvWFs at 200 and 4000 seconds-1. Decreased aggregation was correlated with ristocetin binding to platelets. In contrast, a distinct effect of botrocetin was observed, since type 2M rvWFs (-G561A, -E596K, and -I662F) were able to bind to platelets to the same extent as wild type rvWF (rvWF-WT). Interestingly, SIPA at 200 and 4000 seconds-1 confirmed the gain-of-function phenotype of the 5 type 2B rvWFs. Our data indicated a consistent increase of SIPA at both low and high shear rates, reaching 95% of total platelets, whereas SIPA did not exceed 40% in the presence of rvWF-WT. Aggregation was completely inhibited by monoclonal antibody 6D1 directed to GPIb, underlining the importance of vWF-GPIb interaction in type 2B rvWF. Impaired SIPA of type 2M rvWF could account for the hemorrhagic syndrome observed in type 2M vWD. Increased SIPA of type 2B rvWF could be responsible for unstable aggregates and explain the fluctuant thrombocytopenia of type 2B vWD.

Effect of recombinant von Willebrand factor reproducing type 2B or type 2M mutations on shear-induced platelet aggregation

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3796-3803 ◽  
Author(s):  
Nadine Ajzenberg ◽  
Anne-Sophie Ribba ◽  
Ghassem Rastegar-Lari ◽  
Dominique Meyer ◽  
Dominique Baruch
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
High Shear ◽  
Shear Rates ◽  
High Shear Rates ◽  
Consistent Increase ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

The aim was to better understand the function of von Willebrand factor (vWF) A1 domain in shear-induced platelet aggregation (SIPA), at low (200) and high shear rate (4000 seconds-1) generated by a Couette viscometer. We report on 9 fully multimerized recombinant vWFs (rvWFs) expressing type 2M or type 2B von Willebrand disease (vWD) mutations, characterized respectively by a decreased or increased binding of vWF to GPIb in the presence of ristocetin. We expressed 4 type 2M (-G561A, -E596K, -R611H, and -I662F) and 5 type 2B (rvWF-M540MM, -V551F, -V553M, -R578Q, and -L697V). SIPA was strongly impaired in all type 2M rvWFs at 200 and 4000 seconds-1. Decreased aggregation was correlated with ristocetin binding to platelets. In contrast, a distinct effect of botrocetin was observed, since type 2M rvWFs (-G561A, -E596K, and -I662F) were able to bind to platelets to the same extent as wild type rvWF (rvWF-WT). Interestingly, SIPA at 200 and 4000 seconds-1 confirmed the gain-of-function phenotype of the 5 type 2B rvWFs. Our data indicated a consistent increase of SIPA at both low and high shear rates, reaching 95% of total platelets, whereas SIPA did not exceed 40% in the presence of rvWF-WT. Aggregation was completely inhibited by monoclonal antibody 6D1 directed to GPIb, underlining the importance of vWF-GPIb interaction in type 2B rvWF. Impaired SIPA of type 2M rvWF could account for the hemorrhagic syndrome observed in type 2M vWD. Increased SIPA of type 2B rvWF could be responsible for unstable aggregates and explain the fluctuant thrombocytopenia of type 2B vWD.

scholarly journals Platelet von Willebrand factor: evidence for its involvement in platelet adhesion to collagen

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1214-1217
Author(s):  
E Fressinaud ◽  
D Baruch ◽  
C Rothschild ◽  
HR Baumgartner ◽  
D Meyer
Keyword(s):  
Shear Rate ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Von Willebrand Disease ◽  
High Shear ◽  
Shear Rates ◽  
High Shear Rates ◽  
Von Willebrand ◽  
Willebrand Factor

Although it is well established that plasma von Willebrand Factor (vWF) is essential to platelet adhesion to subendothelium at high shear rates, the role of platelet vWF is less clear. We studied the respective role of both plasma and platelet vWF in mediating platelet adhesion to fibrillar collagen in a parallel-plate perfusion chamber. Reconstituted blood containing RBCs, various mixtures of labeled washed platelets and plasma from controls or five patients with severe von Willebrand disease (vWD), was perfused through the chamber for five minutes at a shear rate of 1,600 s-1. Platelet-collagen interactions were estimated by counting the radioactivity in deposited platelets and by quantitative morphometry. When the perfusate consisted of normal platelets suspended in normal plasma, platelet deposition on the collagen was 24.7 +/- 3.6 X 10(6)/cm2 (mean +/- SEM, n = 6). Significantly less deposition (16 +/- 2.3) was observed when vWD platelets were substituted for normal platelets. In mixtures containing vWD plasma, significantly greater deposition (9 +/- 2.2) was obtained with normal than with vWD platelets (1 +/- 0.4) demonstrating a role for platelet vWF in mediating the deposition of platelets on collagen. Morphometric analysis confirmed these data. Our findings indicate that platelet, as well as plasma, vWF mediates platelet-collagen interactions at a high shear rate.

Platelet von Willebrand factor: evidence for its involvement in platelet adhesion to collagen

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1214-1217 ◽  
Author(s):  
E Fressinaud ◽  
D Baruch ◽  
C Rothschild ◽  
HR Baumgartner ◽  
D Meyer
Keyword(s):  
Shear Rate ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Von Willebrand Disease ◽  
High Shear ◽  
Shear Rates ◽  
High Shear Rates ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Although it is well established that plasma von Willebrand Factor (vWF) is essential to platelet adhesion to subendothelium at high shear rates, the role of platelet vWF is less clear. We studied the respective role of both plasma and platelet vWF in mediating platelet adhesion to fibrillar collagen in a parallel-plate perfusion chamber. Reconstituted blood containing RBCs, various mixtures of labeled washed platelets and plasma from controls or five patients with severe von Willebrand disease (vWD), was perfused through the chamber for five minutes at a shear rate of 1,600 s-1. Platelet-collagen interactions were estimated by counting the radioactivity in deposited platelets and by quantitative morphometry. When the perfusate consisted of normal platelets suspended in normal plasma, platelet deposition on the collagen was 24.7 +/- 3.6 X 10(6)/cm2 (mean +/- SEM, n = 6). Significantly less deposition (16 +/- 2.3) was observed when vWD platelets were substituted for normal platelets. In mixtures containing vWD plasma, significantly greater deposition (9 +/- 2.2) was obtained with normal than with vWD platelets (1 +/- 0.4) demonstrating a role for platelet vWF in mediating the deposition of platelets on collagen. Morphometric analysis confirmed these data. Our findings indicate that platelet, as well as plasma, vWF mediates platelet-collagen interactions at a high shear rate.

Soluble Monomeric Von Willebrand Factor A1A2A3 (R1450E) Mutant Fragment Enhances Shear-Induced Platelet Aggregation and Platelet Adhesion to Fibrin(ogen) Via αIIbβ3 Under High Shear Stress.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4025-4025
Author(s):  
Miguel A. Cruz ◽  
Katie E. Sowa ◽  
Scott M. Smith
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Surface Coverage ◽  
High Shear ◽  
Shear Rates ◽  
High Shear Rates ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 4025 Poster Board III-961 Recently, we described that the gain of function mutation R1450E in the A1 domain of von Willebrand factor (VWF) eliminates the formation of catch bond with glycoprotein (GP)Ibα, prolonging the bond lifetimes at low forces. Because those studies were performed with the mutant immobilized on a plastic surface, we further characterize the effect of this mutant on platelet function in solution and under shear stress. Both wild type (WT) and mutant A1A2A3 proteins were expressed in HEK293 cells and purified to homogeneity. The monomeric state of A1A2A3 proteins were assessed by gel filtration chromatography and neither of the proteins had formed dimers or any higher order aggregates. The recombinant A1A2A3 mutant bound spontaneously to GPIbα without the modulator ristocetin with a half-maximal binding observed at 65 ± 8 nM. This apparent dissociation constant was comparable to that of WT (50 ± 10 nM) in the presence of ristocetin. The mutant failed to induce spontaneous platelet aggregation under stirring conditions, and blocked 100% ristocetin-induced platelet agglutination (RIPA) at concentration of 250 nM. At the same concentration, the mutant increased shear-induced platelet aggregation (SIPA) at 500s-1 and 5000s-1 shear rates, reaching 42% and 66%, respectively, while SIPA did not exceed 18% in the presence of WT. The anti-αIIbβ3 antibody 7E3 blocked the effect of the mutant on SIPA. Blood was then incubated with the mutant (250 nM) and perfused over a surface coated with fibrin(ogen) at different shear rates. Blood containing WT resulted in <10% surface coverage by platelets after 1.5 minutes while platelets from blood containing the mutant rapidly bound covering 100% of the fibrin(ogen) surface area at 1500s-1. At shear rate of 2500s-1, surface coverage was 20% for the mutant and 0% for WT fragment. EDTA and antibodies 6D1 (GPIbα) and 10E5 (αIIbβ3) effectively blocked mutant-mediated platelet adhesion and thrombus formation under high shear rates. The addition of ristocetin (0.5 mg/ml) to whole blood prior perfusion reproduced the effect of the mutant. Here, we describe an A1A2A3 mutant that bound spontaneously to GPIbα but affected differently RIPA and SIPA. These results suggest that hydrodynamic forces directly act on the GPIbα-mutant A1A2A3 complex, regulating signaling. In addition, platelet activation induced by the binding of soluble mutant A1A2A3 or plasma VWF results in αIIbβ3-mediated platelet adhesion to fibrin(ogen) under high shear rates. Disclosures: No relevant conflicts of interest to declare.

Modulation of von Willebrand Factor A1 Domain-Mediated Platelet Aggregation/Agglutination by α-Thrombin.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 628-628
Author(s):  
Grazia Loredana Mendolicchio ◽  
Reha Celikel ◽  
Kottayil I. Varughese ◽  
Brian Savage ◽  
Zaverio M. Ruggeri
Keyword(s):  
Platelet Aggregation ◽  
Shear Rate ◽  
Von Willebrand Factor ◽  
Platelet Glycoprotein ◽  
Shear Rates ◽  
Amino Terminal ◽  
Low Responder ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Evaluation of the crystal structures of the amino terminal domain of platelet glycoprotein (GP) Ibα bound to the von Willebrand factor A1 domain (VWFA1) or to α-thrombin indicate the absence of significant steric hindrance in a putative triple complex of the two ligands interacting with the same receptor molecule. Superposition of the models reveals that intermolecular contacts may be established between VWFA1 and α-thrombin concurrently bound to GP Ibα, and suggests that these additional interactions could stabilize the intrinsically low affinity binding of the VWF A1 domain. To verify the predictions of the model, we used gel electrophoresis under native conditions and purified components in solution to demonstrate directly the formation of a triple complex. We then sought to evaluate whether α-thrombin could influence the functional effects of the VWF-GP Ibα interaction. For this purpose, we established a model of platelet agglutination/aggregation dependent on the interaction between recombinant dimeric VWFA1 domain, purified from the culture medium of stably transfected D. melanogaster cell lines, and GP Ibα. In this assay, platelet rich plasma prepared from individual donor blood collected with the thrombin inhibitor D-phenyl alanyl-L-prolyl-L-arginine chloromethyl ketone dihydrochloride (PPACK) as an anticoagulant (80 μM) was mixed with varying concentrations of dimeric VWFA1 (0.5-10 μg/ml) and exposed to variable shear rate levels in a cone-and-plate viscometer. Platelet aggregation was observed at shear rates between 6 and 108 dyn/cm2. The response in different normal controls was reproducible but variable in extent, and individuals could be assigned to one of two categories, low responder and high responder. An agglutination response was observed after platelets were treated with 10 μM prostaglandin E1 to block activation, and the distinction between low and high responders remained true under these conditions. For simplicity, agglutinated platelets were still defined as “aggregates”. With activation blocked platelets, aggregates were stable up to a shear rate of 30 dyn/cm2, but began to dissipate at higher levels. The addition of α-thrombin with the active site irreversibly blocked by PPACK at concentrations between 5 and 10 μg/ml substantially increased the extent of the platelet response. This was demonstrated by a faster rate of platelet agglutination/aggregation, a greater stability of aggregates at higher shear rates, and an overall increase in the size of aggregates formed. To demonstrate the latter, samples were exposed to shear stress under selected conditions and immediately fixed with 1% glutaraldehyde for quantitative image analysis. Maximum aggregate size was increased several fold in the presence of α-thrombin, and the difference was particularly evident in low responder individuals in whom dimeric VWFA1 alone caused the formation of small and unstable aggregates. PPACK-blocked thrombin by itself had no effect on platelet aggregate formation at any shear rate tested. Our findings delineate a mechanism through which α-thrombin may stabilize platelet-platelet contacts by mediating a tighter association between VWF A1 domain and GP Ibα receptor. Such a function, independent of proteolytic activity, may enhance platelet deposition at sites of vascular injury.

Distinct Functional Conformations of von Willebrand Factor A1 Domain in Support of Platelet-Surface or Platelet-Platelet Interactions.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 5182-5182
Author(s):  
Gianmarco Podda ◽  
James R. Roberts ◽  
Richard A. McClintock ◽  
Zaverio M. Ruggeri
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Conformational Changes ◽  
Platelet Adhesion ◽  
Shear Rates ◽  
Amino Terminal ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Positively Charged

Abstract The adhesive protein, von Willebrand factor (VWF), is generally considered a key substrate for platelet adhesion to the vessel wall, yet its role in platelet cohesion (aggregation) may be equally important for normal thrombus formation. In either case, the function of VWF is mediated by the primary interaction of the VWF A1 domain (VWF-A1) with glycoprotein (GP) Ibα, a component of the GPIb-IX-V receptor complex on the platelet membrane. Because normal plasma VWF in solution and GPIb coexist in circulating blood without any appreciable interaction, it has been postulated that conformational changes occur when VWF becomes immobilized and/or under the effect of pathologically elevated shear stress, such that binding to the receptor becomes possible and resultis in platelet tethering to a surface and shear-induced aggregation. Changes of the molecular shape of VWF, from coiled to extended, have been shown under the effect of hemodynamic forces, but evidence for conformational changes within VWF-A1 has remained elusive. The crystal structure of VWF-A1 in complex with a GPIbα amino terminal fragment has revealed that the VWF-A1 residues involved in the interaction are comprised between positions 544–614 and, in particular, do not include several positively charged Arg and Lys residues located in helices α4 and 5 (residues 627–668). The latter appear as likely candidates to interact with negatively charged residues in GPIbα as a consequence of potential conformational changes induced by tensile stress on the bond following an initial ligand-receptor contact. We tested this hypothesis by evaluating the ability of selected VWF-A1 mutants to support platelet adhesion or aggregation, respectively, under controlled flow conditions. Methods. We expressed in insect cells and purified a series of VWF-A1 fragments comprising residues 445–733. One fragment had native sequence and 8 had single or multiple substitutions of positively charged amino acid residues in helices α4 and/or α5. None of the substituted residues contribute to contacts with GP Ibα in the known crystal structures of the corresponding complex, and all except one were between 8 and 20 angstroms away from the closest GPIbα residue. All the fragments were dimeric (d) owing to the presence of interchain disulfide bond(s). Results: Native dVWF-A1 in solution supported platelet aggregation in a laminar flow field. Of the 8 mutants, 5 had variably decreased function (up to 95% less aggregation) and 2 had increased function (up to 200% increase in aggregation). The same results were observed with platelet-rich plasma in suspension or by measuring platelet aggregate formation with blood cells perfused over immobilized VWF-A1 at wall shear rates as high as 10,000 1/s. In contrast, as judged by the number of tethered platelets and their rolling velocities, all mutants supported adhesion as well as or better that the native VWFA-1 at all shear rates tested (500–25,000 1/s). Conclusions: These results provide structural evidence for the existence of different VWF-A1 conformers that can modulate adhesive properties with distinct effects on platelet adhesion to a surface or platelet aggregation.

Platelet adhesion at high shear rates: The roles of von Willebrand factor/GPIb and the β1 integrin α2β1

1996 ◽  
Vol 81 (1) ◽  
pp. 113-119 ◽  
Author(s):  
Harvey R. Gralnicks ◽  
Wendy S. Kramer ◽  
Laurie P. McKeown ◽  
Leonard Garfinkel ◽  
Amos Pinot ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Β1 Integrin ◽  
High Shear ◽  
Shear Rates ◽  
High Shear Rates ◽  
Von Willebrand ◽  
Willebrand Factor

Activation of pp125FAK by type 2B recombinant von Willebrand factor binding to platelet GPIb at a high shear rate occurs independently of αIIbβ3 engagement

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4363-4371 ◽  
Author(s):  
Médina Mekrache ◽  
Christilla Bachelot-Loza ◽  
Nadine Ajzenberg ◽  
Abdelhafid Saci ◽  
Paulette Legendre ◽  
...  
Keyword(s):  
Shear Rate ◽  
Von Willebrand Factor ◽  
Thrombus Formation ◽  
High Shear Rate ◽  
Von Willebrand Disease ◽  
High Shear ◽  
High Level ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Shear-induced platelet aggregation (SIPA) involves the sequential interaction of von Willebrand factor (VWF) with both glycoprotein Ib (GPIb) and αIIbβ3 receptors. Type 2B recombinant VWF (2B-rVWF), characterized by an increased affinity for GPIb, induces strong SIPA at a high shear rate (4000 s–1). Despite the increased affinity of 2B-rVWF for GPIb, patients with type 2B von Willebrand disease have a paradoxical bleeding disorder, which is not well understood. The purpose of this study was to determine if SIPA induced by 2B-rVWF was associated with αIIbβ3-dependent platelet activation. To this end, we have addressed the influence of 2B-rVWF (Val553Met substitution) on SIPA-dependent variations of tyrosine protein phosphorylation (P-Tyr) and the effect of αIIbβ3 blockers. At a high shear rate, 2B-rVWF induced a strong SIPA, as shown by a 92.7% ± 0.4% disappearance of single platelets (DSP) after 4.5 minutes. In these conditions, increased P-Tyr of proteins migrating at positions 64 kd, 72 kd, and 125 kd were observed. The band at 125 kd was identified as pp125FAK using anti–phospho-FAK antibody. This effect, which required a high level of SIPA (&gt; 70% DSP), was observed at 4000 s–1 but not at 200 s–1. Monoclonal antibodies (MoAbs) 6D1 (anti-GPIb) and 328 (anti-VWF A1 domain), completely abolished SIPA and p125FAK phosphorylation mediated by 2B-rVWF. In contrast, neither RGDS peptide nor MoAb 7E3, both known to block αIIbβ3 engagement, had any effect on SIPA and pp125FAK. The size of aggregates formed at a high shear rate in the presence of 2B-rVWF was decreased by genistein, demonstrating the biologic relevance of pp125FAK. These findings provide a unique mechanism whereby the enhanced interaction of 2B-rVWF with GPIb, without engagement of αIIbβ3, is sufficient to induce SIPA but does not lead to stable thrombus formation.

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