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.

scholarly journals Conformational Changes in the A1 Domain of von Willebrand Factor Modulating the Interaction with Platelet Glycoprotein Ib

1996 ◽  
Vol 271 (15) ◽  
pp. 9046-9053 ◽  
Author(s):  
Shigeki Miyata ◽  
Shinya Goto ◽  
Augusto B. Federici ◽  
Jerry Ware ◽  
Zaverio M. Ruggeri
Keyword(s):  
Von Willebrand Factor ◽  
Conformational Changes ◽  
Platelet Glycoprotein ◽  
Glycoprotein Ib ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

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.

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.

Porcine von Willebrand Factor Binding to Human Platelet GPIb Induces Transmembrane Calcium Influx

1996 ◽  
Vol 75 (04) ◽  
pp. 655-660 ◽  
Author(s):  
Mario Mazzucato ◽  
Luigi De Marco ◽  
Paola Pradella ◽  
Adriana Masotti ◽  
Francesco I Pareti
Keyword(s):  
Monoclonal Antibody ◽  
Shear Stress ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Human Platelet ◽  
Platelet Glycoprotein ◽  
Dependent Manner ◽  
Transmembrane Flux ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryPorcine von Willebrand factor (P-vWF) binds to human platelet glycoprotein (GP) lb and, upon stirring (1500 rpm/min) at 37° C, induces, in a dose-dependent manner, a transmembrane flux of Ca2+ ions and platelet aggregation with an increase in their intracellular concentration. The inhibition of P-vWF binding to GP lb, obtained with anti GP lb monoclonal antibody (LJ-Ib1), inhibits the increase of intracellular Ca2+ concentration ([Ca2+]i) and platelet aggregation. This effect is not observed with LJ-Ib10, an anti GP lb monoclonal antibody which does not inhibit the vWF binding to GP lb. An anti GP Ilb-IIIa monoclonal antibody (LJ-CP8) shown to inhibit the binding of both vWF and fibrinogen to the GP IIb-IIIa complex, had only a slight effect on the [Ca2+]i rise elicited by the addition of P-vWF. No inhibition was also observed with a different anti GP IIb-IIIa monoclonal antibody (LJ-P5), shown to block the binding of vWF and not that of fibrinogen to the GP IIb-IIIa complex. PGE1, apyrase and indomethacin show a minimal effect on [Ca2+]i rise, while EGTA completely blocks it. The GP lb occupancy by recombinant vWF fragment rvWF445-733 completely inhibits the increase of [Ca2+]i and large aggregates formation. Our results suggest that, in analogy to what is seen with human vWF under high shear stress, the binding of P-vWF to platelet GP lb, at low shear stress and through the formation of aggregates of an appropriate size, induces a transmembrane flux of Ca2+, independently from platelet cyclooxy-genase metabolism, perhaps through a receptor dependent calcium channel. The increase in [Ca2+]i may act as an intracellular message and cause the activation of the GP IIb-IIIa complex.

scholarly journals Epitope mapping by cDNA expression of a monoclonal antibody which inhibits the binding of von Willebrand factor to platelet glycoprotein IIb/IIIa

1992 ◽  
Vol 284 (3) ◽  
pp. 711-715 ◽  
Author(s):  
G Piétu ◽  
A S Ribba ◽  
G Chérel ◽  
D Meyer
Keyword(s):  
Monoclonal Antibody ◽  
Von Willebrand Factor ◽  
Fusion Proteins ◽  
Solid Phase ◽  
Platelet Glycoprotein ◽  
Rgd Sequence ◽  
Adhesive Proteins ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Beta Galactosidase

In order to study the structure-function relationship of von Willebrand Factor (vWF), we have located the epitope of a well-characterized monoclonal antibody (MAb) to vWF (MAb 9). This MAb reacts with the C-terminal portion of the vWF subunit, SPII fragment [amino acids (aa) 1366-2050], which includes an Arg-Gly-Asp (RGD) sequence at positions 1744-1746, and totally inhibits vWF and SPII binding to platelet membrane glycoprotein IIb/IIIa (GPIIb/IIIa). A recombinant DNA library was constructed by cloning small (250-500 nucleotides) vWF cDNA fragments into the lambda gt11 vector and these inserts were expressed as fusion proteins with beta-galactosidase. Immunological screening of the library with 125I-MAb 9 identified three immunoreactive clones. vWF inserts were amplified by the PCR and their sequences demonstrated overlapping nucleotides from positions 7630 to 7855 of vWF cDNA, coding for aa residues 1698-1773 of the mature subunit, indicating that this is the epitope of MAb 9. vWF-beta-galactosidase fusion protein reacted with 125I-MAb 9 by Western blotting. In a solid-phase radioimmunoassay, the purified fusion proteins decreased the binding of vWF to 125I-MAb 9 by 50%, and this inhibition was dose-dependent between 3.5 and 120 nM. Therefore the epitope of MAb 9 is located within aa 1698-1773 of the vWF subunit, which includes the RGD sequence implicated in the binding of adhesive proteins of GPIIb/IIIa.

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 Activation of Human Platelets by the Membrane-Expressed A1 Domain of von Willebrand Factor

Blood ◽  
1997 ◽  
Vol 90 (11) ◽  
pp. 4425-4437 ◽  
Author(s):  
Jan Schulte am Esch ◽  
Miguel A. Cruz ◽  
Jonathan B. Siegel ◽  
Josef Anrather ◽  
Simon C. Robson
Keyword(s):  
Von Willebrand Factor ◽  
Conformational Changes ◽  
Human Platelets ◽  
Pathological Process ◽  
Xenograft Rejection ◽  
Modifying Factors ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractPlatelet activation and microthrombus formation are invariable features of xenograft rejection and the vascular injury observed when porcine organs are transplanted into primates. This pathological process could be mediated, at least in part, by aberrant interactions of von Willebrand Factor (vWF) associated with the donor vasculature with host platelets. Unlike human vWF, native porcine vWF (pvWF) interacts with human GPIb independently of shear stress or nonphysiological stimuli, eg, ristocetin. We therefore contrasted the potential of isolated human and porcine vWF–A1-domains to interact with human platelets in vitro. Both human and porcine vWF–A1-domains expressed as glycosyl phosphatidylinositol–linked FLAG fusion proteins on COS-7 cells induced GPIb-dependent aggregation and intracellular Ca++ uptake of platelets, independent of both the remainder of the vWF protein and additional modifying factors. Porcine A1-domains were more potent than human homologues, and in addition ristocetin could boost platelet aggregation only with the human A1-domain. Putative conformational changes in the porcine A1-domain could result in the heightened, ristocetin-independent interactions observed with human platelets and may be of importance for xenograft survival.

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.

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