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.

Endothelial von Willebrand factor recruits platelets to atherosclerosis-prone sites in response to hypercholesterolemia

Blood ◽  
2002 ◽  
Vol 99 (12) ◽  
pp. 4486-4493 ◽  
Author(s):  
Gregor Theilmeier ◽  
Carine Michiels ◽  
Erik Spaepen ◽  
Ingrid Vreys ◽  
Désiré Collen ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Perfusion Studies ◽  
Von Willebrand ◽  
Willebrand Factor

Platelets are thought to play a causal role during atherogenesis. Platelet-endothelial interactions in vivo and their molecular mechanisms under shear are, however, incompletely characterized. Here, an in vivo platelet homing assay was used in hypercholesterolemic rabbits to track platelet adhesion to plaque predilection sites. The role of platelet versus aortic endothelial cell (EC) activation was studied in an ex vivo flow chamber. Pathways of human platelet immobilization were detailed during in vitro perfusion studies. In rabbits, a 0.125% cholesterol diet induced no lesions within 3 months, but fatty streaks were found after 12 months. ECs at segmental arteries of 3- month rabbits expressed more von Willebrand factor (VWF) and recruited 5-fold more platelets than controls (P < .05, n = 5 and 4, respectively). The 3-month ostia had an increased likelihood to recruit platelets compared to control ostia (56% versus 18%, P < .0001, n = 89 and 63, respectively). Ex vivo, the adhesion of 3-month platelets to 3-month aortas was 8.4-fold increased compared to control studies (P < .01, n = 7 and 5, respectively). In vitro, endothelial VWF–platelet glycoprotein (GP) Ib and platelet P-selectin– endothelial P-selectin glycoprotein ligand 1 interactions accounted in combination for 83% of translocation and 90% of adhesion (P < .01, n = 4) of activated human platelets to activated human ECs. Platelet tethering was mainly mediated by platelet GPIbα, whereas platelet GPIIb/IIIa contributed 20% to arrest (P < .05). In conclusion, hypercholesterolemia primes platelets for recruitment via VWF, GPIbα, and P-selectin to lesion-prone sites, before lesions are detectable.

scholarly journals The binding domain of von Willebrand factor to sulfatides is distinct from those interacting with glycoprotein Ib, heparin, and collagen and resides between amino acid residues Leu 512 and Lys 673

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2310-2317 ◽  
Author(s):  
O Christophe ◽  
B Obert ◽  
D Meyer ◽  
JP Girma
Keyword(s):  
Von Willebrand Factor ◽  
Competitive Inhibition ◽  
Binding Domain ◽  
Functional Site ◽  
Platelet Glycoprotein ◽  
Functional Domain ◽  
Amino Acid Residues ◽  
Glycoprotein Ib ◽  
Von Willebrand ◽  
Willebrand Factor

A series of proteolytic fragments of human von Willebrand Factor (vWF) was purified to characterize the functional site that supports its interaction with sulfatides. SpIII, an N-terminal homodimer generated by V-8 protease (amino acids [AA] 1 to 1365), bound to sulfatides in a dose-dependent and saturable way. SpIII also totally inhibited the binding of vWF to sulfatides and SpIII binding was completely abolished by vWF. In contrast, SpII, the complementary C-terminal homodimer (AA 1366 to 2050), did not exhibit any binding affinity for sulfatides. Four purified fragments overlapping the sequence of SpIII were also tested for their ability to interact with sulfatides. An N-terminal monomeric 34-Kd fragment (P34, AA 1 to 272) generated by plasmin, a central monomer (SpI, AA 911 to 1365) produced by digestion with V-8 protease, and a tetrameric fragment III-T2 (comprising a pair of the two sequences AA 273 to 511 and AA 674 to 728) produced by secondary digestion of SpIII with trypsin did not interact with sulfatides. In contrast, a monomeric 39/34-Kd fragment produced by dispase (AA 480 to 718) bound specifically and with a high affinity to sulfatides and totally displaced vWF or SpIII binding. Conversely, binding of the 39/34-Kd species was totally abolished by vWF or SpIII. Thus, a functional site responsible for sulfatide binding was localized between AA 480 and 718 and comparison of the binding properties of the 39/34-Kd and III-T2 fragments indicated that the sequence 512 to 673 is necessary for the binding to sulfatides. Further mapping of this new functional domain of vWF, based on experiments of competitive inhibition of binding by either heparin or monoclonal antibodies directed toward vWF, showed that the site interacting with sulfatides is distinct from those involved in binding to platelet glycoprotein Ib, collagen, or heparin. This finding was confirmed by experiments using synthetic peptides which also indicated that the sequence comprising AA 569 to 584 is part of the sulfatide-binding domain or influences its activity.

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.

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).

The von Willebrand factor antagonist (GPG-290) prevents coronary thrombosis without prolongation of bleeding time

2007 ◽  
Vol 98 (08) ◽  
pp. 397-405 ◽  
Author(s):  
Michael Wadanoli ◽  
Dianne Sako ◽  
Gray Shaw ◽  
Robert Schaub ◽  
Qin Wang ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Coronary Thrombosis ◽  
Platelet Glycoprotein ◽  
Closure Time ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe interaction between von Willebrand factor (VWF) and platelet glycoprotein Ibα (GPIbα) is a critical step that allows platelet adhesion, activation and subsequent thrombus formation to the injured vessel wall under high-shear conditions. In this study, we sought to investigate 1) whether GPG-290, a recombinant human GPIbα chimeric protein, would prevent thrombosis in a canine model of coronary thrombosis by blocking VWFGPIbα interaction; and 2) whether desmopressin (DDAVP), a VWF release stimulant, could reduce the prolonged bleeding time caused by a 10x efficacious dose of GPG-290. The antithrombotic efficacy of GPG-290 was evaluated by the in-vivo ability to prevent cyclic flow reductions (CFRs) and ex-vivo inhibition of platelet adhesion/aggregation reflected by prolongation of Platelet Function Analyzer (PFA-100®) collagen /ADP closure time. The anti-hemostatic effect was assessed by template bleeding time. GPG-290 at doses of 25, 50 and 100 μg/kg abolished CFRs in 67%,100% and 100% of the treated dogs without bleeding time prolongation, respectively; GPG-290 dose-dependently prolonged the ex-vivo collagen/ADP-closure time, while it had no effects on plasma VWF antigen level (VWF:Ag) and VWF-collagen binding activity (VWF:CB); the prolonged template bleeding time caused by 500 μg/kg of GPG-290 was prevented by intravenous infusion of DDAVP (0.3 μg/kg). In conclusion, GPG-290 appears to be an effective agent for treating arterial thrombosis without bleeding time prolongation.

scholarly journals The binding domain of von Willebrand factor to sulfatides is distinct from those interacting with glycoprotein Ib, heparin, and collagen and resides between amino acid residues Leu 512 and Lys 673

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2310-2317 ◽  
Author(s):  
O Christophe ◽  
B Obert ◽  
D Meyer ◽  
JP Girma
Keyword(s):  
Von Willebrand Factor ◽  
Competitive Inhibition ◽  
Binding Domain ◽  
Functional Site ◽  
Platelet Glycoprotein ◽  
Functional Domain ◽  
Amino Acid Residues ◽  
Glycoprotein Ib ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract A series of proteolytic fragments of human von Willebrand Factor (vWF) was purified to characterize the functional site that supports its interaction with sulfatides. SpIII, an N-terminal homodimer generated by V-8 protease (amino acids [AA] 1 to 1365), bound to sulfatides in a dose-dependent and saturable way. SpIII also totally inhibited the binding of vWF to sulfatides and SpIII binding was completely abolished by vWF. In contrast, SpII, the complementary C-terminal homodimer (AA 1366 to 2050), did not exhibit any binding affinity for sulfatides. Four purified fragments overlapping the sequence of SpIII were also tested for their ability to interact with sulfatides. An N-terminal monomeric 34-Kd fragment (P34, AA 1 to 272) generated by plasmin, a central monomer (SpI, AA 911 to 1365) produced by digestion with V-8 protease, and a tetrameric fragment III-T2 (comprising a pair of the two sequences AA 273 to 511 and AA 674 to 728) produced by secondary digestion of SpIII with trypsin did not interact with sulfatides. In contrast, a monomeric 39/34-Kd fragment produced by dispase (AA 480 to 718) bound specifically and with a high affinity to sulfatides and totally displaced vWF or SpIII binding. Conversely, binding of the 39/34-Kd species was totally abolished by vWF or SpIII. Thus, a functional site responsible for sulfatide binding was localized between AA 480 and 718 and comparison of the binding properties of the 39/34-Kd and III-T2 fragments indicated that the sequence 512 to 673 is necessary for the binding to sulfatides. Further mapping of this new functional domain of vWF, based on experiments of competitive inhibition of binding by either heparin or monoclonal antibodies directed toward vWF, showed that the site interacting with sulfatides is distinct from those involved in binding to platelet glycoprotein Ib, collagen, or heparin. This finding was confirmed by experiments using synthetic peptides which also indicated that the sequence comprising AA 569 to 584 is part of the sulfatide-binding domain or influences its activity.

An Anti-von Willebrand Factor Aptamer Reduces Platelet Adhesion Among Patients Receiving Aspirin and Clopidogrel in an Ex Vivo Shear-Induced Arterial Thrombosis

2010 ◽  
Vol 17 (6) ◽  
pp. E70-E78 ◽  
Author(s):  
Dabit Arzamendi ◽  
Firas Dandachli ◽  
Jean-François Théorêt ◽  
Gregory Ducrocq ◽  
Mark Chan ◽  
...  
Keyword(s):  
Antiplatelet Therapy ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Ex Vivo ◽  
Platelet Glycoprotein ◽  
Artery Disease ◽  
A Site ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

The von Willebrand factor (vWF) aptamer, ARC1779 that blocks the binding of vWF A1-domain to platelet glycoprotein 1b (GPIb) at high shear, may deliver a site-specific antithrombotic effect. We investigated the efficiency of ARC1779 on platelet function in patients with coronary artery disease (CAD) on double antiplatelet therapy. Blood from patients taking aspirin and clopidogrel and from normal volunteers was treated ex vivo with ARC1779 or abciximab, either prior to perfusion (pretherapy) or 10 minutes following the initiation of perfusion (posttherapy) on damaged arteries. Under pre- but not posttherapy, platelet adhesion was significantly reduced by ARC1779 at 83 and 250 nmol/L and by abciximab (100 nmol/L) versus placebo (4.8, 3.8, and 2.9 vs 7.3 platelets × 106/cm2, P < .05). In contrast to abciximab, ARC1779 did not significantly affect platelet aggregation, P-selectin expression, and platelet−leukocyte binding. These proof-of-concept data may constitute the framework for randomized clinical investigations of this novel antiplatelet therapy among patients with CAD.

scholarly journals von Willebrand factor self-association on platelet GpIbα under hydrodynamic shear: effect on shear-induced platelet activation

Blood ◽  
2010 ◽  
Vol 116 (19) ◽  
pp. 3990-3998 ◽  
Author(s):  
Kannayakanahalli M. Dayananda ◽  
Indrajeet Singh ◽  
Nandini Mondal ◽  
Sriram Neelamegham
Keyword(s):  
Shear Stress ◽  
Von Willebrand Factor ◽  
Cell Activation ◽  
Cell Surface Receptor ◽  
Platelet Glycoprotein ◽  
Fluid Shear ◽  
Hydrodynamic Shear ◽  
Self Association ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The function of the mechanosensitive, multimeric blood protein von Willebrand factor (VWF) is dependent on its size. We tested the hypothesis that VWF may self-associate on the platelet glycoprotein Ibα (GpIbα) receptor under hydrodynamic shear. Consistent with this proposition, whereas Alexa-488–conjugated VWF (VWF-488) bound platelets at modest levels, addition of unlabeled VWF enhanced the extent of VWF-488 binding. Recombinant VWF lacking the A1-domain was conjugated with Alexa-488 to produce ΔA1-488. Although ΔA1-488 alone did not bind platelets under shear, this protein bound GpIbα on addition of either purified plasma VWF or recombinant full-length VWF. The extent of self-association increased with applied shear stress more than ∼ 60 to 70 dyne/cm2. ΔA1-488 bound platelets in the milieu of plasma. On application of fluid shear to whole blood, half of the activated platelets had ΔA1-488 bound, suggesting that VWF self-association may be necessary for cell activation. Shearing platelets with 6-μm beads bearing either immobilized VWF or anti-GpIbα mAb resulted in cell activation at shear stress down to 2 to 5 dyne/cm2. Taken together, the data suggest that fluid shear in circulation can increase the effective size of VWF bound to platelet GpIbα via protein self-association. This can trigger mechanotransduction and cell activation by enhancing the drag force applied on the cell-surface receptor.

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