Platelet-VWF complexes get the chop

Blood ◽  
2008 ◽  
Vol 111 (2) ◽  
pp. 475-475
Author(s):  
Michael C. Berndt ◽  
Robert K. Andrews
Keyword(s):  
Ligand Binding ◽  
Von Willebrand Factor ◽  
Tensile Force ◽  
Inhibitory Effect ◽  
Platelet Glycoprotein ◽  
A1 Domain ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor ◽  
Binding Subunit

In this issue of Blood, Shim and colleagues define a dual role for platelet glycoprotein (GP)Ibα (the major ligand-binding subunit of the GPIb-IX-V complex) in regulating ADAMTS13-mediated cleavage of von Willebrand factor (VWF) under shear: it alleviates an inhibitory effect of the VWF A1 domain on cleavage of the A2 domain,1 and it allows tensile force to be exerted on the A2 domain through at least 2 platelets binding per VWF multimer via the A1 domain (see figure).

scholarly journals Humanized GPIbα–von Willebrand factor interaction in the mouse

2018 ◽  
Vol 2 (19) ◽  
pp. 2522-2532 ◽  
Author(s):  
Sachiko Kanaji ◽  
Jennifer N. Orje ◽  
Taisuke Kanaji ◽  
Yuichi Kamikubo ◽  
Yosuke Morodomi ◽  
...  
Keyword(s):  
Carotid Artery ◽  
Von Willebrand Factor ◽  
Mouse Strain ◽  
Platelet Rich Plasma ◽  
Platelet Glycoprotein ◽  
Transgenic Mouse Strain ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Knockin Mouse

Abstract The interaction of platelet glycoprotein Ibα (GPIbα) with von Willebrand factor (VWF) initiates hemostasis after vascular injury and also contributes to pathological thrombosis. GPIbα binding to the VWF A1 domain (VWFA1) is a target for antithrombotic intervention, but attempts to develop pharmacologic inhibitors have been hindered by the lack of animal models because of the species specificity of the interaction. To address this problem, we generated a knockin mouse with Vwf exon 28–encoding domains A1 and A2 replaced by the human homolog (VWFh28). VWFh28 mice (M1HA) were crossbred with a transgenic mouse strain expressing human GPIbα on platelets (mGPIbαnull;hGPIbαTg; H1MA) to generate a new strain (H1HA) with humanized GPIbα-VWFA1 binding. Plasma VWF levels in the latter 3 strains were similar to those of wild-type mice (M1MA). Compared with the strains that had homospecific GPIbα-VWF pairing (M1MA and H1HA), M1HA mice of those with heterospecific pairing had a markedly greater prolongation of tail bleeding time and attenuation of thrombogenesis after injury to the carotid artery than H1MA mice. Measurements of GPIbα-VWFA1 binding affinity by surface plasmon resonance agreed with the extent of observed functional defects. Ristocetin-induced platelet aggregation was similar in H1HA mouse and human platelet-rich plasma, and it was comparably inhibited by monoclonal antibody NMC-4, which is known to block human GPIbα-VWFA1 binding, which also inhibited FeCl3-induced mouse carotid artery thrombosis. Thus, the H1HA mouse strain is a fully humanized model of platelet GPIbα-VWFA1 binding that provides mechanistic and pharmacologic information relevant to human hemostatic and thrombotic disorders.

scholarly journals Platelet Aggregation Induced by a Monoclonal Antibody to the A1 Domain of von Willebrand Factor

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3792-3799 ◽  
Author(s):  
Hilde Depraetere ◽  
Nadine Ajzenberg ◽  
Jean-Pierre Girma ◽  
Catherine Lacombe ◽  
Dominique Meyer ◽  
...  
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Inhibitory Effect ◽  
Platelet Glycoprotein ◽  
Rotational Viscometer ◽  
Induce Platelet Aggregation ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

Shear-induced platelet aggregation (SIPA) involves von Willebrand Factor (vWF) binding to platelet glycoprotein (GP)Ib at high shear stress, followed by the activation of αIIbβ3. The purpose of this study was to determine the vWF sequences involved in SIPA by using monoclonal antibodies (MoAbs) to vWF known to interfere with its binding to GPIb and to αIIbβ3. Washed platelets were exposed to shear rates between 100 and 4,000 seconds−1 in a rotational viscometer. SIPA was quantitated by flow cytometry as the disappearance of single platelets (DSP) in the sheared sample in the presence of vWF, relative to a control in the absence of shear and vWF. At a shear rate of 4,000 seconds−1, DSP was increased from 5.9% ± 3.5% in the absence of vWF to 32.7% ± 6.3% in the presence of vWF. This increase in SIPA was not associated with an elevation of P-selectin expression. vWF-dependent SIPA was completely abolished by MoAb 6D1 to GPIb and partially inhibited by MoAb 10E5 to αIIbβ3. Three MoAbs to vWF were compared for their effect on SIPA at 4,000 seconds−1 in the presence of vWF: MoAb 328, known to block vWF binding to GPIb in the presence of ristocetin, MoAb 724 blocking vWF binding to GPIb in the presence of botrocetin, and MoAb 9, an inhibitor of vWF binding to αIIbβ3. Similar to the effect of MoAb 6D1, MoAb 328 completely inhibited the effect of vWF, whereas MoAb 9 had a partial inhibitory effect, as MoAb 10E5 did. In contrast, MoAb 724, as well as its F(ab′)2 fragments, promoted shear-dependent platelet aggregation (165% of the DSP value obtained in the absence of MoAb 724), indicating that MoAb 724 was responsible for an enhanced aggregation, which was independent of binding to the platelet Fcγ receptor. In addition, the enhancement of aggregation induced by MoAb 724 was abrogated by MoAb 6D1 or 10E5 to the level of SIPA obtained in the presence of vWF incubated with a control MoAb to vWF. Finally, the activating effect of MoAb 724 was also found under static conditions at ristocetin concentrations too low to induce platelet aggregation. Our results suggested that on binding to a botrocetin-binding site on vWF, MoAb 724 mimics the effect of botrocetin by inducing an active conformation of vWF that is more sensitive to shear stress or to low ristocetin concentration.

scholarly journals von Willebrand factor self-association is regulated by the shear-dependent unfolding of the A2 domain

2019 ◽  
Vol 3 (7) ◽  
pp. 957-968 ◽  
Author(s):  
Changjie Zhang ◽  
Anju Kelkar ◽  
Sriram Neelamegham
Keyword(s):  
Von Willebrand Factor ◽  
Ex Vivo ◽  
Association Studies ◽  
Apolipoprotein A1 ◽  
Platelet Glycoprotein ◽  
Functional Domain ◽  
Self Association ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

Abstract von Willebrand factor (VWF) self-association results in the homotypic binding of VWF upon exposure to fluid shear. The molecular mechanism of this process is not established. In this study, we demonstrate that the shear-dependent unfolding of the VWF A2 domain in the multimeric protein is a major regulator of protein self-association. This mechanism controls self-association on the platelet glycoprotein Ibα receptor, on collagen substrates, and during thrombus growth ex vivo. In support of this, A2-domain mutations that prevent domain unfolding due to disulfide bridging of N- and C-terminal residues (“Lock-VWF”) reduce self-association and platelet activation under various experimental conditions. In contrast, reducing assay calcium concentrations, and 2 mutations that destabilize VWF-A2 conformation by preventing coordination with calcium (D1498A and R1597W VWD type 2A mutation), enhance self-association. Studies using a panel of recombinant proteins that lack the A1 domain (“ΔA1 proteins”) suggest that besides pure homotypic A2 interactions, VWF-A2 may also engage other protein domains to control self-association. Addition of purified high-density lipoprotein and apolipoprotein-A1 partially blocked VWF self-association. Overall, similar conditions facilitate VWF self-association and ADAMTS13-mediated proteolysis, with low calcium and A2 disease mutations enhancing both processes, and locking-A2 blocking them simultaneously. Thus, VWF appears to have evolved 2 balancing molecular functions in a single A2 functional domain to dynamically regulate protein size in circulation: ADAMTS13-mediated proteolysis and VWF self-association. Modulating self-association rates by targeting VWF-A2 may provide novel methods to regulate the rates of thrombosis and hemostasis.

Regulation of Von Willebrand Factor A1 Domain by Mechanical Force and Neighboring Domains

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2117-2117
Author(s):  
Wendy E Thomas ◽  
Rebecca A Penkala ◽  
Elaine Hillenmeyer ◽  
Matthew Whitfield ◽  
An-yue Tu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Shear Stress ◽  
Von Willebrand Factor ◽  
Mechanical Force ◽  
Platelet Glycoprotein ◽  
Bond Rupture ◽  
C Terminus ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 2117 Regulation of the bond between platelet glycoprotein (GP) Ibα of the GPIb-IX-V complex, and the von Willebrand Factor (VWF) A1 domain is critical to the balance between hemostasis and thrombosis, particularly in high shear conditions. The GPIbα-A1 interaction is known to be activated by shear stress and inhibited by neighboring domains in VWF, but the role of neighboring domains in the shear-dependence remained unknown. Here it is shown that platelet aggregation required shear stress in the presence of VWF proteins that contain the neighboring D′D3 domain (Plus D′D3 or plasma VWF) but that platelets aggregate spontaneously with a protein that lacks this region (Delta D′D3). Moreover, platelets and microspheres coated with the N-terminal 300 amino acids of GPIbα (GC300) bind to immobilized VWF in a shear-enhanced manner for Plus D′D3 but not for Delta D′D3. In single-molecule force spectroscopy experiments, the D′D3 domain decreased the number of GPIbα-A1 bonds that formed, but did not alter bond rupture force, consistent with the hypothesis that D′D3 shields the A1 domain. By expressing recombinant VWF fragments that contain the A1 domain and various lengths of the N-terminal region, we determined that most of the inhibition by the D′D3 domain was conferred by 23 amino acids in the linker between the A1 domain and the D′D3 domain. By anchoring the fragments to the surface in an oriented manner, we demonstrated that binding was much stronger when force was applied between GPIbα and the A1 C-terminus, than when force was applied between GPIbα and the A1 N-terminus, similar to what has been observed for integrins. Based on these results, we propose the following model for regulation of VWF by mechanical force. When multimeric VWF is stretched in flow, the D′D3 domains are pulled away from the A1 domains, exposing the latter to bind platelets. When force is applied between GPIbα and the C-terminus of A1, it induces an activating conformational change that could be analogous to that seen in integrins. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Platelet adhesion and aggregate formation controlled by immobilised and soluble VWF

2020 ◽  
Author(s):  
Matthias F. Schneider ◽  
Mohammad A. Fallah ◽  
Christian Mess ◽  
Tobias Obser ◽  
Reinhard Schneppenheim ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Platelet Glycoprotein ◽  
Aggregate Formation ◽  
Cooperative Phenomena ◽  
Interference Contrast Microscopy ◽  
A Domains ◽  
A1 Domain ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

Abstract BackgroundIt has been demonstrated that von Willebrand factor (VWF) mediated platelet-endothelium and platelet-platelet interactions are shear dependent. The VWF's mobility under dynamic conditions (e.g. flow) is pivotal to platelet adhesion and VWF-mediated aggregate formation in the cascade of VWF-platelet interactions in haemostasis.ResultsCombining microfluidic tools with fluorescence and reflection interference contrast microscopy (RICM), here we show, that specific deletions in the A-domains of the biopolymer VWF affect both, adhesion and aggregation properties independently. Intuitively, the deletion of the A1-domain led to a significant decrease in both adhesion and aggregate formation of platelets. Nevertheless, the deletion of the A2-domain revealed a completely different picture, with a significant increase in formation of rolling aggregates (gain of function). We predict that the A2-domain effectively ‘masks’ the potential between the platelet glycoprotein (GP) Ib and the VWF A1-domain. Furthermore, the deletion of the A3-domain led to no significant variation in either of the two functional characteristics.ConclusionsThese data demonstrate that the macroscopic functional properties i.e. adhesion and aggregate formation cannot simply be assigned to the properties of one particular domain, but have to be explained by cooperative phenomena. The absence or presence of molecular entities likewise affects the properties (thermodynamic phenomenology) of its neighbours, therefore altering the macromolecular function.

The A/T1381 polymorphism in the A1-domain of von Willebrand factor influences the affinity of von Willebrand factor for platelet glycoprotein Ibα

2007 ◽  
Vol 98 (07) ◽  
pp. 178-185 ◽  
Author(s):  
Tímea Szántó ◽  
Ágota Schlammadinger ◽  
Stephanie Staelens ◽  
Simon De Meyer ◽  
Kathleen Freson ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Association Studies ◽  
Platelet Glycoprotein ◽  
Platelet Receptor ◽  
Increased Risk ◽  
Static Conditions ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryMany polymorphisms in vonWillebrand factor (VWF) have been reported and their association with VWF plasma levels or cardiovascular diseases has been investigated. The aim of this study was to examine whether the amino acid polymorphis mA/T1381 in the VWF A1-domain would affect VWF binding to platelet GPIbα. Sixty-one normal individuals were genotyped at the A/T1381 locus. Twenty-one A/A1381 homozygotes, 30 A/T1381 heterozygotes and 10 T/T1381 homozygotes were identified. Remarkably, when compared to VWF of A/T1381 and A/A1381 individuals, VWF of individuals carrying the T/T1381 variant showed an increased affinity for its platelet receptor GPIbα under static conditions, as reflected by an increased sensitivity to low concentrations of ristocetin or botrocetin. In addition, also the rVWF-T1381 demonstrated a higher affinity for GPIbα than rVWF-A1381. Interestingly, this enhanced affinity of the T/T variant over the A/T and A/A variant was, however, too subtle to affect platelet adhesion under physiological flow conditions, which fully corroborates the normal haemostatic phenotype of all individuals. We demonstrate that the VWF A/T1381 polymorphism plays an important role in inter-individual variability of the affinity of VWF for GPIbα, with T/T variants having a higher affinity than A/A and A/T variants, at least under static conditions in vitro. Further genetic linkage and association studies are necessary to establish whether the A/T1381 polymorphism could correlate with an increased risk of thrombotic events.

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.

scholarly journals Platelet Aggregation Induced by a Monoclonal Antibody to the A1 Domain of von Willebrand Factor

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3792-3799 ◽  
Author(s):  
Hilde Depraetere ◽  
Nadine Ajzenberg ◽  
Jean-Pierre Girma ◽  
Catherine Lacombe ◽  
Dominique Meyer ◽  
...  
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Inhibitory Effect ◽  
Platelet Glycoprotein ◽  
Rotational Viscometer ◽  
Induce Platelet Aggregation ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractShear-induced platelet aggregation (SIPA) involves von Willebrand Factor (vWF) binding to platelet glycoprotein (GP)Ib at high shear stress, followed by the activation of αIIbβ3. The purpose of this study was to determine the vWF sequences involved in SIPA by using monoclonal antibodies (MoAbs) to vWF known to interfere with its binding to GPIb and to αIIbβ3. Washed platelets were exposed to shear rates between 100 and 4,000 seconds−1 in a rotational viscometer. SIPA was quantitated by flow cytometry as the disappearance of single platelets (DSP) in the sheared sample in the presence of vWF, relative to a control in the absence of shear and vWF. At a shear rate of 4,000 seconds−1, DSP was increased from 5.9% ± 3.5% in the absence of vWF to 32.7% ± 6.3% in the presence of vWF. This increase in SIPA was not associated with an elevation of P-selectin expression. vWF-dependent SIPA was completely abolished by MoAb 6D1 to GPIb and partially inhibited by MoAb 10E5 to αIIbβ3. Three MoAbs to vWF were compared for their effect on SIPA at 4,000 seconds−1 in the presence of vWF: MoAb 328, known to block vWF binding to GPIb in the presence of ristocetin, MoAb 724 blocking vWF binding to GPIb in the presence of botrocetin, and MoAb 9, an inhibitor of vWF binding to αIIbβ3. Similar to the effect of MoAb 6D1, MoAb 328 completely inhibited the effect of vWF, whereas MoAb 9 had a partial inhibitory effect, as MoAb 10E5 did. In contrast, MoAb 724, as well as its F(ab′)2 fragments, promoted shear-dependent platelet aggregation (165% of the DSP value obtained in the absence of MoAb 724), indicating that MoAb 724 was responsible for an enhanced aggregation, which was independent of binding to the platelet Fcγ receptor. In addition, the enhancement of aggregation induced by MoAb 724 was abrogated by MoAb 6D1 or 10E5 to the level of SIPA obtained in the presence of vWF incubated with a control MoAb to vWF. Finally, the activating effect of MoAb 724 was also found under static conditions at ristocetin concentrations too low to induce platelet aggregation. Our results suggested that on binding to a botrocetin-binding site on vWF, MoAb 724 mimics the effect of botrocetin by inducing an active conformation of vWF that is more sensitive to shear stress or to low ristocetin concentration.

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