Shielding the front-strand β3 of the von Willebrand factor A1 domain inhibits its binding to platelet glycoprotein Ibα

Blood ◽  
2003 ◽  
Vol 101 (4) ◽  
pp. 1375-1383 ◽  
Author(s):  
Arnaud Bonnefoy ◽  
Hiroshi Yamamoto ◽  
Chantal Thys ◽  
Morikazu Kito ◽  
Jos Vermylen ◽  
...  
Keyword(s):  
Binding Site ◽  
Von Willebrand Factor ◽  
Amino Acid Sequences ◽  
Platelet Glycoprotein ◽  
Surface Distribution ◽  
Perfusion Studies ◽  
Mechanistic Basis ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Platelet adhesion to damaged vessel wall and shear-induced platelet aggregation necessitate binding of the von Willebrand factor (VWF) A1 domain to platelet GPIbα. Blocking this interaction represents a promising approach to the treatment of arterial thrombosis. Comparison of amino acid sequences of the VWF A1 domain in several species, expressing VWF recognized by the blocking monoclonal antibody AJvW-2, suggested 9 residues (His563, Ile566, Asp570, Ala581, Val584, Ala587, Arg616, Ala618, and Met622) to contribute to the epitope for AJvW-2 or to be part of the GPIbα-binding site. Glutathione-S-transferase (GST)–human VWF A1 fusion proteins, in which these amino acids were mutated to their murine counterparts, were tested for their capacity to bind AJvW-2 or heparin, to interfere with botrocetin- or ristocetin-mediated VWF binding to GPIb, or to induce flow-dependent platelet tethering in a perfusion chamber. Thus, mutations His563Arg, Ile566Leu, Asp570Ala, and Ala587Thr, clustered on the outer surface of the A1 domain, dramatically impaired binding of AJvW-2 to A1. The His563Arg, Ile566Leu, and Asp570Ala mutations also impaired the binding of heparin, which competes with AJvW-2 for binding to A1. Perfusion studies revealed that His563, Ile566, Asp570, Arg616, and Ala618 take part in GPIbα binding, their mutation-impairing platelet recruitment. In agreement with the surface distribution of VWF type 2M mutations, this study demonstrates overlapping of the epitope for AJvW-2 and the GPIbα-binding site, located around the front pocket of the A1 domain and defined by strands β3, β4, and helix α3, and it provides a mechanistic basis for VWF neutralization by this antibody.

Conformational Changes in the A3 Domain of von Willebrand Factor Modulate the Interaction of the A1 Domain With Platelet Glycoprotein Ib

Blood ◽  
1999 ◽  
Vol 93 (6) ◽  
pp. 1959-1968 ◽  
Author(s):  
Bernadette Obert ◽  
Anne Houllier ◽  
Dominique Meyer ◽  
Jean-Pierre Girma
Keyword(s):  
Monoclonal Antibody ◽  
Binding Site ◽  
Von Willebrand Factor ◽  
Conformational Changes ◽  
Platelet Glycoprotein ◽  
Terminal Part ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Dose Dependent

Bitiscetin has recently been shown to induce von Willebrand factor (vWF)-dependent aggregation of fixed platelets (Hamako J, et al,Biochem Biophys Res Commun 226:273, 1996). We have purified bitiscetin from Bitis arietans venom and investigated the mechanism whereby it promotes a form of vWF that is reactive with platelets. In the presence of bitiscetin, vWF binds to platelets in a dose-dependent and saturable manner. The binding of vWF to platelets involves glycoprotein (GP) Ib because it was totally blocked by monoclonal antibody (MoAb) 6D1 directed towards the vWF-binding site of GPIb. The binding also involves the GPIb-binding site of vWF located on the A1 domain because it was inhibited by MoAb to vWF whose epitopes are within this domain and that block binding of vWF to platelets induced by ristocetin or botrocetin. However, in contrast to ristocetin or botrocetin, the binding site of bitiscetin does not reside within the A1 domain but within the A3 domain of vWF. Thus, among a series of vWF fragments, 125I-bitiscetin only binds to those that overlap the A3 domain, ie, SpIII (amino acid [aa] 1-1365), SpI (aa 911-1365), and rvWF-A3 domain (aa 920-1111). It does not bind to SpII corresponding to the C-terminal part of vWF subunit (aa 1366-2050) nor to the 39/34/kD dispase species (aa 480-718) or T116 (aa 449-728) overlapping the A1 domain. In addition, bitiscetin that does not bind to DeltaA3-rvWF (deleted between aa 910-1113) has no binding site ouside the A3 domain. The localization of the binding site of bitiscetin within the A3 domain was further supported by showing that MoAb to vWF, which are specific for this domain and block the interaction between vWF and collagen, are potent inhibitors of the binding of bitiscetin to vWF and consequently of the bitiscetin-induced binding of vWF to platelets. Thus, our data support the hypothesis that an interaction between the A1 and A3 domains exists that may play a role in the function of vWF by regulating the ability of the A1 domain to bind to platelet GPIb.

Characterization of the Binding Between the Polymeric Platelet Adhesive Proteins, Multimerin 1 and Von Willebrand Factor

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1144-1144
Author(s):  
D'Andra Parker ◽  
Subia Tasneem ◽  
Nola Fuller ◽  
J. Evan Sadler ◽  
Philip G de Groot ◽  
...  
Keyword(s):  
Binding Site ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Platelet Glycoprotein ◽  
High Shear ◽  
A Domains ◽  
Adhesive Proteins ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 1144 Introduction: Multimerin 1 (MMRN1) is a massive variably-sized homopolymeric protein that is stored in platelet and endothelial cell secretion granules, for release with vascular injury. Recently, MMRN1 was identified to support platelet adhesion in vitro and in vivo. At high shear, MMRN1 supports platelet adhesion by a von Willebrand factor (VWF)-dependent, but integrin-independent mechanism, involving platelet glycoprotein (GP) Ibα. Direct binding of MMRN1 to GP Ibα has not been demonstrated. These data led us to postulate that VWF binds MMRN1 at site(s) distinct from the GP Ibα binding site, and test the roles of VWF A domains in MMRN1 binding. Methods: Modified enzyme linked immunosorbent assays (ELISA) and surface plasmon resonance (SPR) were used to assess binding interactions between wildtype (WT) MMRN1 and WT or domain deleted VWF constructs, and VWF polypeptides. Protein constructs tested included: multimeric VWF deletion constructs ΔA1A2A3-VWF, ΔA1A3-VWF, and ΔA1-VWF, and monomeric VWF polypeptides A1A2A3, A1A2, A1 and A3. Bovine serum albumin (BSA) coated surfaces were used as the negative control. Results: Unlike WT-VWF, VWF lacking the A domains (ΔA1A2A3-VWF) or the combination of the A1 and A3 domains (ΔA1A3-VWF) did not detectably bind to MMRN1 (p < 0.001). VWF lacking the A1 domain (ΔA1-VWF) showed MMRN1 binding comparable to WT-VWF (p = 0.39), excluding the possibility that MMRN1 binding site is located in VWF A1 domain (the region that binds GP Ibα). VWF polypeptides A1A2A3, A1A2 and A3 bound to MMRN1 (p < 0.001), unlike the VWF polypeptide A1 (p = 0.137), although the A1A2 polypeptide showed reduced binding compared to A1A2A3 (p < 0.001). SPR analyses confirmed that MMRN1 binding was supported by VWF peptides containing the A3 and/or A2 domains. Conclusions: The regions of VWF that support MMRN1 includes the A3, and possibly A2 domains, which respectively contain binding sites for collagen and ADAMTS-13. Our data suggest that the mechanism by which GP Ibα and VWF support platelet adhesion to MMRN1 at high shear include: VWF binding to GP Ibα via the A1 domain, and to MMRN1 via the A3 and possibly A2 domains. These findings have implications for the molecular mechanisms that support platelet adhesion at sites of vessel injury. Disclosures: No relevant conflicts of interest to declare.

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.

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 &lt; .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 &lt; .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 &lt; .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 &lt; .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 &lt; .05). In conclusion, hypercholesterolemia primes platelets for recruitment via VWF, GPIbα, and P-selectin to lesion-prone sites, before lesions are detectable.

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.

Von Willebrand factor C1C2 domain is involved in platelet adhesion to polymerized fibrin at high shear rate

Blood ◽  
2004 ◽  
Vol 103 (5) ◽  
pp. 1741-1746 ◽  
Author(s):  
Jeffrey F. W. Keuren ◽  
Dominique Baruch ◽  
Paulette Legendre ◽  
Cécile V. Denis ◽  
Peter J. Lenting ◽  
...  
Keyword(s):  
Shear Rate ◽  
Binding Site ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
High Shear Rate ◽  
High Shear ◽  
Perfusion Studies ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractFibrin is actively involved in platelet reactions essential for thrombus growth, in which von Willebrand factor (VWF) might be an important mediator. The aim of this study was to localize VWF domains that bind to fibrin and to determine their relevance in platelet adhesion. VWF binds specifically to fibrin with an apparent Kd of 2.2 μg/mL. Competition in the presence of 2 complementary fragments, SpIII (residues 1-1365) and SpII (residues 1366-2050), indicated that the high affinity binding site for fibrin is located in the C-terminal part, thus distinct from the A domains. Comparison of 2 deleted rVWF (ΔD4B-rVWF, ΔC1C2-rVWF) suggested that the C1C2 domains contained a fibrin binding site. This site is distinct from RGD, as shown by binding of D1746G-rVWF to fibrin. Perfusion studies at high shear rate demonstrated that C1C2 domains were required for optimal platelet adhesion to fibrin. With the use of a VWF-deficient mouse model, it was found that plasma VWF is critical for platelet tethering and adhesion to fibrin. These results suggest a dual role of fibrin-bound VWF in thrombus formation: first, fibrin-bound VWF is critical in the recruitment of platelets by way of glycoprotein (GP) Ib, and, second, it contributes to stationary platelet adhesion by way of binding to activated αIIbβ3.

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.

scholarly journals Identification of platelet glycoprotein IIb/IIIa as the major binding site for released platelet-von Willebrand factor [published erratum appears in Blood 1987 Jun;69(6):1789]

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 732-736 ◽  
Author(s):  
RI Parker ◽  
HR Gralnick
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Von Willebrand Factor ◽  
Plasma Fibrinogen ◽  
Platelet Glycoprotein ◽  
Surface Binding ◽  
Human Fibrinogen ◽  
Platelet Surface ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We studied the effects(s) of two monoclonal antibodies, 6D1 and 10E5 (directed against platelet glycoprotein Ib [GPIb] and the GPIIb/IIIa complex, respectively), and purified human plasma fibrinogen on the binding of released platelet-von Willebrand factor (vWf) to the platelet surface. Neither of the monoclonal antibodies nor fibrinogen had any effect on the amount of platelet-vWf expressed on unstimulated platelets or on the amount expressed on platelets stimulated in the absence of extracellular Ca++. However, the antibody directed against GPIIb/IIIa inhibited 72% of the thrombin-induced increase in the platelet-vWf bound to the platelet surface when platelets were stimulated in the presence of 5 mmol/L Ca++. The antibody against GPIb did not inhibit the surface expression of platelet-vWf on stimulated platelets in the presence of Ca++. Purified normal human fibrinogen inhibited the surface binding of platelet-vWf to thrombin-stimulated platelets to a degree similar to that observed with the monoclonal antibody directed against the GPIIb/IIIa complex. These data indicate that platelet-vWf released from platelets binds primarily to the GPIIb/IIIa complex at or near the plasma fibrinogen binding site.

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