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.

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

Platelet 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.

Microfibrils (MF) Platelet Interaction: Requirement Of Von Willebrand Factor In Platelet Adhesion To A Placenta Microfibrillar Component (PMC)

1981 ◽  
Author(s):  
F Fauvel ◽  
Y J Legrand ◽  
N Gutman ◽  
J P Muh ◽  
G Tobelem ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Human Platelets ◽  
Human Artery ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Adhesion Of Platelets ◽  
Aggregation Of Platelets

It has been shown that collagenase resistant arterial microfibrils (MF) are able to interact with platelets and therefore represents, besides collagen, a second thrombogenic structure in the vessel wall. In vitro observation using a PMC purified from the villosities of human placenta by a mechanical non denaturing procedure confirm this interaction between platelets and MF. PMC was homogenous under electron microscope (feltwork of MF with a mean diameter of 120 – 130 A) and was glycoproteic in nature. PMC were able to induce an aggregation of human platelets only if the platelets were in plasma. The role of Von Willebrand factor (F VIII/WF) as a cofactor of the aggregation of platelets by MF has been postulated from the fact that twice washed platelets from normal subject resuspended in PPP obtained from a severe Von Willebrand deficient patient were not aggregated by the PMC. Furthermore, aggregation was restored after resuspension of the same platelets in the PPP of the same patient 30 and 120 minutes after perfusion of cryoprecipitate (40 units F VIII/RA per kg).F VIII/WF mediates platelet adhesion after binding to subendothelium of human artery. Our observation strongly supports the idea that MF are the subendothelial components to which F VIII/WF binds, thus promoting an adhesion of platelets.

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.

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.

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.

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

Interaction between von Willebrand factor and glycoprotein Ib activates Src kinase in human platelets: role of phosphoinositide 3–kinase

Blood ◽  
2003 ◽  
Vol 101 (9) ◽  
pp. 3469-3476 ◽  
Author(s):  
Yi Wu ◽  
Naoki Asazuma ◽  
Kaneo Satoh ◽  
Yutaka Yatomi ◽  
Toshiro Takafuta ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Kinase Inhibitor ◽  
Signal Transduction Pathway ◽  
Human Platelets ◽  
Glutathione S Transferase ◽  
Phosphoinositide 3 Kinase ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

The binding of von Willebrand factor (VWF) to glycoprotein (GP) Ib-IX-V stimulates transmembrane signaling events that lead to platelet adhesion and aggregation. Recent studies have implied that activation of Src family kinases is involved in GPIb-mediated platelet activation, although the related signal transduction pathway remains poorly defined. This study presents evidence for an important role of Src and GPIb association. In platelet lysates containing Complete, a broad-spectrum protease inhibitor mixture, Src and Lyn dynamically associated with GPIb on VWF-botrocetin stimulation. Cytochalasin D, which inhibits translocation of Src kinases to the cytoskeleton, further increased Src and GPIb association. Similar results were obtained with botrocetin and monomeric A1 domain, instead of intact VWF, with induction of both Src activation and association between GPIb and Src. These findings suggest that ligand binding of GPIb, without receptor clustering, is sufficient to activate Src. Immunoprecipitation studies demonstrated that Src, phosphoinositide 3– kinase (PI 3–kinase), and GPIb form a complex in GPIb-stimulated platelets. When the p85 subunit of PI 3–kinase was immunodepleted, association of Src with GPIb was abrogated. However, wortmannin, a specific PI 3–kinase inhibitor, failed to block complex formation between Src and GPIb. The Src-SH3 domain as a glutathione S-transferase (GST)–fusion protein coprecipitated the p85 subunit of PI 3–kinase and GPIb. These findings taken together suggest that the p85 subunit of PI 3–kinase mediates GPIb-related activation signals and activates Src independently of the enzymatic activity of PI 3– kinase.

scholarly journals The Flow Dependent Adhesion of von Willebrand Factor (VWF)-A1 Functionalized Nanoparticles in an in Vitro Coronary Stenosis Model

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2679 ◽  
Author(s):  
Yathreb Asaad ◽  
Mark Epshtein ◽  
Andrew Yee ◽  
Netanel Korin
Keyword(s):  
Von Willebrand Factor ◽  
Coronary Stenosis ◽  
Nano Particles ◽  
Adhesive Properties ◽  
Polystyrene Nanoparticles ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

In arterial thrombosis, von Willebrand factor (VWF) bridges platelets to sites of vascular injury. The adhesive properties of VWF are controlled by its different domains, which may be engineered into ligands for targeting nanoparticles to vascular injuries. Here, we functionalized 200 nm polystyrene nanoparticles with the VWF-A1 domain and studied their spatial adhesion to collagen or collagen-VWF coated, real-sized coronary stenosis models under physiological flow. When VWF-A1 nano-particles (A1-NPs) were perfused through a 75% stenosis model coated with collagen-VWF, the particles preferentially adhered at the post stenotic region relative to the pre-stenosis region while much less adhesion was detected at the stenosis neck (~ 65-fold less). When infused through collagen-coated models or when the A1 coating density of nanoparticles was reduced by 100-fold, the enhanced adhesion at the post-stenotic site was abolished. In a 60% stenosis model, the adhesion of A1-NPs to collagen-VWF-coated models depended on the location examined within the stenosis. Altogether, our results indicate that VWF-A1 NPs exhibit a flow-structure dependent adhesion to VWF and illustrate the important role of studying cardiovascular nano-medicines in settings that closely model the size, geometry, and hemodynamics of pathological environments.

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 von Willebrand factor binds to native collagen VI primarily via its A1 domain

1997 ◽  
Vol 324 (1) ◽  
pp. 185-191 ◽  
Author(s):  
Marc F. HOYLAERTS ◽  
Hiroshi YAMAMOTO ◽  
Katarine NUYTS ◽  
Ingrid VREYS ◽  
Hans DECKMYN ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Von Willebrand Factor ◽  
Fibroblast Cell ◽  
Human Platelets ◽  
Collagen Vi ◽  
Human Lung Fibroblast ◽  
Ic50 Values ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Collagen VI is abundant in the arterial subendothelium. To investigate its mechanism of interaction with von Willebrand factor (vWF), collagen VI was isolated from human placenta and from the extracellular matrix of the human lung fibroblast cell line MRC-5. Purified vWF bound to non-digested collagen VI with moderately high affinity (EC50 ≈ 5 nM) and could be inhibited by the Hirudo medicinalis collagen inhibitor calin. The anti-(human vWF A1 domain) monoclonal antibody (AJvW-2), as well as aurin tricarboxylic acid (ATA), at concentrations that saturate the vWF A1 domain, also inhibited this binding. In contrast, the monoclonal anti-(human vWF A3 domain) antibody (82D6A3) inhibited vWF binding to collagens I, III and IV, but had no effect on vWF binding to collagen VI. Likewise, vWF binding to collagen VI was not inhibited by the recombinant vWF domain D4. Polyclonal anti-(collagen VI) antibodies, specifically neutralizing the binding of vWF to collagen VI, confirmed that in the intact endothelial cell extracellular matrix, collagen VI was accessible for interaction with vWF. This binding was only marginally affected by 82D6A3 but was dose-dependently inhibited by AJvW-2, ATA and the A1 domain analogue VCL (recombinant A1 domain of vWF), with IC50 values comparable to those found for the inhibition of vWF binding to isolated collagen VI. The weak interaction of isolated human platelets with collagen VI was mediated via the platelet collagen receptor (GPIa/IIa) and was competitively inhibited by vWF but not by VCL, suggesting that vWF and GPIa/IIa bind to neighbouring but distinct sites on collagen VI. We conclude that vWF binds to collagen VI primarily via its A1 domain, which distinguishes it from the vWF A3 domain-mediated binding to fibrillar collagens.

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