Biomechanical Properties of Bonds Mediated by the von Willebrand Factor A1 Domain during Platelet Adhesion under Flow Conditions.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3665-3665
Author(s):  
Marianna M. Machin ◽  
Jennifer N. Orje ◽  
Paolo Canu ◽  
Zaverio M. Ruggeri
Keyword(s):  
Residence Time ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Bond Formation ◽  
Biomechanical Properties ◽  
Hydrodynamic Forces ◽  
Flow Conditions ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The initial attachment of platelets to vascular lesions exposed to a high wall shear rate (γw) depends on the interaction between the membrane glycoprotein (GP) Ibα, a component of the GP Ib-IX-V receptor complex, and the A1 domain of surface-immobilized von Willebrand factor (VWFA1). We performed perfusion experiments under different flow conditions to measure transient platelet contacts onto immobilized recombinant VWFA1 and determine the probability of bond formation (capturing) and resistance to tensile stress (bond lifetime) of VWFA1-GP Ibα interactions. To define how molecular conformations influence the biomechanical properties of the bonds, we compared fragments exhibiting the native dimeric assembly of A1 domain with monomeric fragments obtained by selective purification of recombinant protein expressed in stable D. melanogaster cell lines. The minimum coating concentration of dimeric VWFA1 at which platelet adhesion events were statistically significantly different from nonspecific interactions on uncoated glass was 1 μg/ml. In the range of γw between 30 and 30,000 s−1, there was no threshold value for the initiation of adhesion, as seen for selectins. The number of adhering platelets first increased and then decreased with monotonically increasing γw, indicating the effect of transport phenomena as well as hydrodynamic forces on the VWFA1-GP Ibα interaction. Maximum event number was at 5,000 s−1 for dimeric and 1,500 s−1 for monomeric VWFA1. The platelet count had no statistically relevant influence on the efficiency of capturing and duration of adhesive contacts. As γw increased, a higher coating concentration of the VWF A1 domain was required to initiate platelet adhesion. The coating concentration determined the number of individual adhesion events that could occur over a defined period of time but did not affect the residence time, which is a measure of the strength of the bond between the receptor and the ligand. Hydrodynamic forces generated by blood flow shortened the duration of VWF-GP Ibα interactions. The percentage of platelets that had a residence time of less than 0.1 s increased almost linearly with increasing γw. Dimeric A1 domain was more efficient than the monomeric counterpart in promoting platelet adhesion as it displayed activity at lower coating concentrations. At permissible γw, a 10-fold higher monomer than dimer coating concentration (2 vs. 20 μg/ml) was required to obtain a similar capturing efficiency. Moreover, the upper limit of γw compatible with the initiation of adhesion was significantly higher for dimeric as compared to monomeric A1 domain. Doubling the dimer coating concentration resulted in a 5-fold increase in the γw limit for adhesion, but the same increase in the monomer coating concentration did not enhance the probability of bond formation at higher γw. In spite of the substantial difference in capturing efficiency, monomeric and dimeric VWFA1 supported platelet adhesion events of similar duration at any given γw, indicating that a different molecular conformation did not affect the lifetime of the interaction with GP Ibα. These results indicate that the dimeric assembly of A1 domains in VWF multimers may be crucial to support the initiation of platelet adhesion at high shear rates, but the duration of each adhesion event is limited by intrinsic properties of the individual VWFA1-GP Ibα bond.

scholarly journals Uremic platelets have a functional defect affecting the interaction of von Willebrand factor with glycoprotein IIb-IIIa

Blood ◽  
1990 ◽  
Vol 76 (7) ◽  
pp. 1336-1340 ◽  
Author(s):  
G Escolar ◽  
A Cases ◽  
E Bastida ◽  
M Garrido ◽  
J Lopez ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Indirect Evidence ◽  
Flow Conditions ◽  
Experimental Conditions ◽  
Shear Rates ◽  
Functional Defect ◽  
Uremic Patients ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Uremic patients have an impaired platelet function that has been related to membrane glycoprotein (GP) abnormalities. Using a perfusion system, we have studied the interaction of normal and uremic platelets with vessel subendothelium (SE) under flow conditions. Reconstituted blood containing washed platelets, purified von Willebrand factor (vWF) (1 U/mL), and normal washed red blood cells was exposed to de- endothelialized rabbit segments for 10 minutes at two different shear rates (800 and 1,600 seconds-1). In some experiments a monoclonal antibody to the GPIIb-IIIa complex (EDU3) was added to the perfusates. With normal platelets, the percentage of the vessel covered by platelets (%CS) was 23.1% +/- 3.7% at 800 seconds-1 and 30% +/- 4.3% at 1,600 seconds-1. Platelets were observed in contact or forming monolayers on vessel SE. EDU3 inhibited the spreading of normal platelets. The %CS (11.1% +/- 3.3%) was statistically decreased (P less than .01) and most of the platelets were observed in contact with the vessel surface. These data indicate that, under flow conditions, the interaction of vWF with GPIIb-IIIa can support the spreading of normal platelets in the absence of exogenous fibrinogen. Under the same experimental conditions, the interaction of uremic platelets with SE was markedly impaired at both shear rates studied (P less than .01 v normal platelets). The presence of EDU3 did not modify the interaction of uremic platelets. These results confirm the impairment of the platelet adhesion observed in uremic patients. Furthermore, they indicate the presence of a functional defect in the interaction of vWF with GPIIb-IIIa. The fact that perfusions with normal and uremic platelets in the presence of an antibody to the GPIIb-IIIa complex did not show any differences gives indirect evidence on a functionally normal interaction vWF/GPIb in uremic patients.

Cytosolic Calcium Changes in a Process of Platelet Adhesion and Cohesion on a von Willebrand Factor-Coated Surface Under Flow Conditions

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1149-1155 ◽  
Author(s):  
Mitsuhiro Kuwahara ◽  
Mitsuhiko Sugimoto ◽  
Shizuko Tsuji ◽  
Shigeki Miyata ◽  
Akira Yoshioka
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Cytosolic Calcium ◽  
Flow Chamber ◽  
Flow Conditions ◽  
Color Changes ◽  
Coated Surface ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Calcium Green

Recent flow studies indicated that platelets are transiently captured onto and then translocated along the surface through interaction of glycoprotein (GP) Ib with surface-immobilized von Willebrand factor (vWF). During translocation, platelets are assumed to be activated, thereafter becoming firmly adhered and cohered on the surface. In exploring the mechanisms by which platelets become activated during this process, we observed changes in platelet cytosolic calcium concentrations ([Ca2+]i) concomitantly with the real-time platelet adhesive and cohesive process on a vWF-coated surface under flow conditions. Reconstituted blood containing platelets loaded with the Ca2+ indicators Fura Red and Calcium Green-1 was perfused over a vWF-coated glass surface in a flow chamber, and changes in [Ca2+]i were evaluated by fluorescence microscopy based on platelet color changes from red (low [Ca2+]i) to green (high [Ca2+]i) during the platelet adhesive and cohesive process. Under flow conditions with a shear rate of 1,500 s−1, no change in [Ca2+]i was observed during translocation of platelets, but [Ca2+]i became elevated apparently after platelets firmly adhered to the surface. Platelets preincubated with anti-GP IIb-IIIa antibody c7E3 showed no firm adhesion and no [Ca2+]i elevation. The intracellular Ca2+chelator dimethyl BAPTA did not inhibit firm platelet adhesion but completely abolished platelet cohesion. Although both firm adhesion and cohesion of platelets have been thought to require activation of GP IIb-IIIa, our results indicate that [Ca2+]i elevation is a downstream phenomenon and not a prerequisite for firm platelet adhesion to a vWF-coated surface. After platelets firmly adhere to the surface, [Ca2+]i elevation might occur through the outside-in signaling from GP IIb-IIIa occupied by an adhesive ligand, thereby leading to platelet cohesion on the surface.

scholarly journals Role of the glycoprotein Ib-binding A1 repeat and the RGD sequence in platelet adhesion to human recombinant von Willebrand factor

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 1035-1042 ◽  
Author(s):  
H Lankhof ◽  
YP Wu ◽  
T Vink ◽  
ME Schiphorst ◽  
HG Zerwes ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Static Condition ◽  
Site Directed Mutagenesis ◽  
Flow Conditions ◽  
Rgd Sequence ◽  
Absolute Requirement ◽  
Platelet Spreading ◽  
Von Willebrand ◽  
Willebrand Factor

To assess the relative importance of the glycoprotein (GP) Ib binding domain and the RGDS binding site in platelet adhesion to isolated von Willebrand factor (vWF) and to collagen preincubated with vWF, we deleted the A1 domain yielding delta A1-vWF and introduced an aspartate- to-glycine substitution in the RGDS sequence by site-directed mutagenesis (RGGS-vWF). Recombinant delta A1-vWF and RGGS-vWF, purified from transfected baby hamster kidney cells, were compared with recombinant wild-type vWF (WT-vWF) in platelet adhesion under static and flow conditions. Purified mutants were coated on glass or on a collagen type III surface and exposed to circulating blood in a perfusion system. Platelet adhesion under static condition, under flow conditions, and in vWF-dependent adhesion to collagen has an absolute requirement for GPIb-vWF interaction. The GPIIb/IIIa-vWF interaction is required for adhesion to coated vWF under flow conditions. Under static condition and vWF-dependent adhesion to collagen, platelet adhesion to RGGS-vWF is similar as to WT-vWF, but platelet spreading and aggregation are abolished.

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.

Vimentin Exposed On Platelets Serves As an Adhesive Receptor for Von Willebrand Factor

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 259-259
Author(s):  
Miguel A. Cruz ◽  
K. Vinod Vijayan
Keyword(s):  
Binding Site ◽  
Platelet Adhesion ◽  
Flow Conditions ◽  
Platelet Receptor ◽  
Coated Surface ◽  
Initial Interaction ◽  
A Domains ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 259 Platelet adhesion, activation, and aggregation in the vasculature are necessary events in both life-saving hemostasis and pathological thrombosis. Thrombosis may occur in patients presenting with several clinical conditions including atherosclerosis, cardiovascular disease, and inflammation. Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that plays a critical role in mediating platelet adhesion, activation, and aggregation on the exposed subendothelium in order to maintain hemostasis under arterial flow conditions. On the other hand, VWF permits the stabilization of platelets adherent to components of ruptured atherosclerotic plaques, leading to artery-occluding thrombus formation. The initial interaction of activated or hyperadhesive VWF with platelets occurs via the interaction between the A1 domain of VWF and the platelet receptor glycoprotein (GP)Ibα. This engagement is responsible for reducing the velocity of rapidly flowing platelets, allowing the rolling platelets to interact with the second binding site on VWF for the platelet receptor GPIIb/IIIa; a binding site that is located within the C domains of VWF. Therefore, the hyperadhesive property of VWF apparently relies on the synchronized interaction of the two platelet surface receptors, GPIbα and GPIIb/IIIa. Despite this concept, we and others have speculated that other binding site in VWF synergistically works with the A1 domain to quickly capture the extremely fast flowing platelets. We have obtained interesting results from studies using a monomeric A1A2A3 domain protein that lacks the binding site for GPIIb/IIIa. For example, the rolling velocity of platelets over an A1A2A3-coated surface was markedly lower than that seen with use of the single A1 domain. This observation suggests the possibility of an additional binding site in the A domains for platelets. Given the similar hyperadhesive features of the A1A2A3 protein and plasma VWF, we proposed to look for a potential receptor on platelets with a recognition site within the A domains of VWF. We suggested examining vimentin because, it was identified as a binding protein for the isolated A2 domain of VWF in our laboratory, and vimentin has been found on the surface of platelets. First, both full length VWF and recombinant A1A2A3 proteins efficiently bound to human vimentin only in the presence of the modulator ristocetin, indicating that vimentin preferably interact with the active conformation of VWF. In fact, a constitutively active A1A2A3 protein (containing a gain-of-function mutation in A1 domain) had a binding activity for vimentin higher than that of wild type (WT) A1A2A3 in the absence of ristocetin. Second, anti-vimentin monoclonal antibody blocked the interaction of that mutant A1A2A3 to activated washed platelets using flow cytometry. Third, we then examined the effect of anti-vimentin antibody on flow-dependent platelet adhesion to A1A2A3-coated surface at high shear stress. In comparison to whole blood incubated with irrelevant IgG molecule as a negative control, the anti-vimentin antibody blocked 75% platelet adhesion to the triple-A domain protein. Finally, whole blood from vimentin-deficient or WT mice was perfused over a surface coated with murine VWF at high shear rate. In comparison to platelets from WT mice, vimentin-deficient platelets had a significant reduced platelet adhesion to VWF (25% of WT). Similarly, vimentin-deficient platelets had a reduced platelet adhesion to collagen (20% of WT) under high flow conditions. This platelet-collagen interaction is initially mediated by VWF. These interesting results indicate that vimentin on platelets serves as a receptor for VWF, and this binding may participate in the initial interaction of circulating platelets with VWF under flow conditions. Disclosures: No relevant conflicts of interest to declare.

Adhesive Domains in the Collagen III Fragment α1(III)CB4 that Support α2β1- and von Willebrand Factor-mediated Platelet Adhesion under Flow Conditions

1999 ◽  
Vol 82 (09) ◽  
pp. 1137-1144 ◽  
Author(s):  
Martin IJsseldijk ◽  
Glenda Heijnen-Snyder ◽  
Eric Huizinga ◽  
Laurence Morton ◽  
C. Graham Knight ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Binding Sites ◽  
Platelet Adhesion ◽  
Recognition Site ◽  
Flow Conditions ◽  
Binding Studies ◽  
Collagen Iii ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

SummarySeven overlapping peptides derived from the bovine α1(III)CB4 fragment of collagen III support static platelet adhesion, and an integrin α2β1-recognition site has been assigned within this fragment to residues 522-528 of the collagen α1(III) chain; (25). In this study we found that two of the peptides, CB4(III)-6 and -7, were able to support platelet adhesion under flow conditions, whereas the other peptides showed either very little (CB4(III)-1 and -4) or no platelet adhesion at all (CB4(III)-2, -3 and -5). Using the recombinant leech anti-platelet protein (rLAPP), known to prevent both α2β1 integrin- and von Willebrand factor (vWF)-binding to collagen, we observed almost complete inhibition of platelet adhesion to peptides CB4(III)-6 and -7. In solidphase binding assays rLAPP bound to CB4(III)-6 and -7 and to CB4(III)-6/7, containing the peptide 6/7 overlap sequence, and not to any other peptide. Our results suggest that the overlap sequence GPP*-GPRGGAGPP*GPEGGK (single-letter amino acid code, P* = hydroxyproline), corresponding to residues 523-540 of the α1(III) collagen chain, contains a binding site for rLAPP. Monoclonal antibodies (MoAbs) directed against the α2 subunit of integrin α2β1 inhibited platelet adhesion to both CB4(III)-6 and -7 by about 50%, showing that the α2β1-recognition site in this locality in α1(III)CB4 detected under static conditions is of sufficient affinity to withstand shear forces. Solid-phase binding studies indicated that vWF binds to CB4(III)-7 and to a lesser extent to CB4(III)-4. Furthermore, rLAPP competed with vWF in binding to CB4(III)-7. Our results indicate that residues 541-558 of the α1(III)-chain may contain one of the critical vWF-binding sites involved in the initial phase of platelet adhesion to collagen III. MoAbs against vWF (A1 and A3 domain) and glycoprotein (GP)Ib confirmed that vWF is involved in adhesion to CB4(III)-7 and showed that vWF is also involved in adhesion to CB4(III)-6 despite the absence of direct binding of vWF to the peptide. The existence of α2β1-, vWF- and rLAPP-binding sites all in close proximity in α1(III)CB4 testifies to the importance of this locus in collagen III for its platelet reactivity.

KINETIC BEHAVIOUR OF VON WILLEBRAND FACTOR AND FIBRONECTIN EXPLAINS DEPENDENCE OF PLATELET ADHESION ON PHYSICAL PARAMETERS

1987 ◽  
Author(s):  
Hans H F I van Breugel ◽  
Philip G de Groot ◽  
Jan J Sixma
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Physical Parameters ◽  
Flow Conditions ◽  
Binding Studies ◽  
Platelet Suspension ◽  
Coated Surface ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

To study the kinetics of the contribution of von Willebrand Factor (vWF) and fibronectin (FN) in platelet adhesion we developed a method with which we can perform binding studies of platelets to these purified proteins under static and flow conditions. Glass coverslips were incubated for one hour with vWF (50 (jg/ml) or FN (300 pg/ml) in saline and were perfused with washed platelets (resuspended in human albumin solution) in the flat perfusion chamber as developed by Sakariassen (J.Lab.Clin.Med. 102, 522-535, 1983). Static conditions were achieved by incubating the coated coverslips with the platelet suspension.In this system, adhesion of platelets to FN coated coverslips strongly decreased at shear rates above 300 /s. The adhesion to this surface could be inhibited with antibodies against platelet glycoprotein Ilbllla and against lb, under static and under flow conditions.Adhesion to vWF coated surfaces increased with increasing shear rate and ultimately reached a plateau at about 800 /s. Adhesion to a vWF coated surface could be totally inhibited by anti GP-Ib and only partially by GP-IIbllla.When after perfusion of a FN coated surface with platelets, the same surface was perfused with a platelet free perfusate, the coverage of platelets on this surface decreased. No decrease in platelet coverage was found when this experiment was performed with a vWF coated coverslip.From these results we conclude that platelets bind to FN at a high rate and with a low affinity, while they bind slowly but with a high affinity to vWF, probablyvia similar platelet receptors.

scholarly journals Analysis of the Involvement of the von Willebrand Factor–Glycoprotein Ib Interaction in Platelet Adhesion to a Collagen-Coated Surface Under Flow Conditions

Blood ◽  
1997 ◽  
Vol 90 (11) ◽  
pp. 4413-4424 ◽  
Author(s):  
Masaaki Moroi ◽  
Stephanie M. Jung ◽  
Shosaku Nomura ◽  
Sadayoshi Sekiguchi ◽  
Antonio Ordinas ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Initial Reaction ◽  
Adhesion Assay ◽  
Flow Conditions ◽  
Coated Surface ◽  
Von Willebrand ◽  
Willebrand Factor

The requisite initial reaction for in vivo thrombus formation in flowing blood is platelet adhesion to the exposed surface of the extracellular matrix. The contribution of von Willebrand factor (vWF ) in plasma and glycoprotein (GP) Ib on the platelet membrane to platelet adhesion has been well-documented. We have recently developed a procedure (the “flow adhesion assay”) for measuring platelet adhesion under flow conditions that allowed us to characterize platelet adhesion to a collagen-coated surface. Here, we apply our method to analyze platelet adhesion to a vWF-coated surface to determine how this might differ from adhesion to a collagen-coated surface. Platelet adhesion to the vWF-coated surface was monitored as the linear increase in the area occupied by adherent platelets. The fluorescence image showed that platelets adhering to the vWF surface were mainly single platelets, and if any were present, the platelet aggregates were small, this being the primary difference from the adhesion to a collagen surface, where adherent platelets were mostly in aggregates. The flow adhesion assay detected the movement of platelets on the vWF surface, suggesting the reversible binding of vWF with platelets. The velocity of the platelets increased at higher shear rates or at lower vWF densities on the surface. Treatment of the vWF-coated surface with the aggregating agent botrocetin before initiation of blood flow increased platelet adhesion while dramatically decreasing the velocity of platelet movement. The present observations on the adhesion of platelets to the vWF-pretreated collagen surface and measurements of the velocity of platelets moving on the collagen surface suggest that the first interaction on the collagen-coated surface is the binding of vWF molecules to the collagen surface. This small number of vWF molecules would serve to attract and slow platelets flowing near the surface. This would facilitate the actual adhesion to the collagen surface that is mainly generated by the interaction between platelet collagen receptors, including GP Ia/IIa and GP VI, with collagen.

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