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.

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.

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 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.

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

Abstract 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 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

Abstract 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.

Baboon Fibrinogen Adsorption and Platelet Adhesion to Polymeric Materials

1991 ◽  
Vol 65 (05) ◽  
pp. 608-617 ◽  
Author(s):  
Joseph A Chinn ◽  
Thomas A Horbett ◽  
Buddy D Ratner
Keyword(s):  
Platelet Adhesion ◽  
Coagulation Factor ◽  
Polymeric Materials ◽  
Platelet Suspension ◽  
Perfusion Chamber ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Fibrinogen Adsorption

SummaryThe role of fibrinogen in mediating platelet adhesion to polymers exposed to blood plasma was studied by comparison of the effect of plasma dilution on fibrinogen adsorption and platelet adhesion, and by the use of coagulation factor deficient plasmas. Polyetherurethane substrates were first preadsorbed with dilute plasma, then contacted with washed platelets suspended in a modified, apyrase containing Tyrode’s buffer. Platelet adhesion was studied under static conditions in Multiwell dishes, and also under shearing conditions using a parallel plate perfusion chamber. Fibrinogen adsorption and platelet adhesion were measured using 125I radiolabeled baboon fibrinogen and min radiolabeled baboon platelets, respectively. Surfaces were characterized by electron spectroscopy for chemical analysis (ESCA).When fibrinogen adsorption to Biomer was measured after 2 h contact with a series of dilute plasma solutions under static conditions, a peak in adsorption was observed from 0.26% plasma, i.e., adsorption was greater from 0.26% plasma than from either more or less dilute plasma. A peak in subsequent platelet adhesion to the plasma preadsorbed surfaces, measured after 2 h static incubation with washed platelets, was also observed but occurred on Biomer preadsorbed with 1.0% plasma.When fibrinogen adsorption was measured after 5 min contact under shearing conditions, the fibrinogen adsorption peak occurred on surfaces that had been exposed to 1.0% plasma. A peak in platelet adhesion to these preadsorbed surfaces, measured after 5 min contact with the platelet suspensions under shearing conditions, was observed on Biomer preadsorbed with 0.1% plasma. Shifts between the positions of the peaks in protein adsorption and platelet adhesion occurred on other polymers tested as well.Platelet adhesion was almost completely inhibited when baboon and human plasmas lacking fibrinogen (i. e., serum, heat defibrinogenated plasma, and congenitally afibrinogénémie plasma) were used. Platelet adhesion was restored to near normal when exogenous fibrinogen was added to fibrinogen deficient plasmas. Adhesion was also inhibited completely when a monoclonal antibody directed against the glycoprotein IIb/IIIa complex was added to the platelet suspension. Platelet adhesion to surfaces preadsorbed to von Willebrand factor deficient plasma was the same as to surfaces preadsorbed with normal plasma.While it appears that surface bound fibrinogen does mediate the initial attachment of platelets to Biomer, the observation that the fibrinogen adsorption and platelet adhesion maxima do not coincide exactly also suggests that the degree of subsequent platelet adhesion is dictated not only by the amount of surface bound fibrinogen but also by its conformation.

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.

scholarly journals The functions of the A1A2A3 domains in von Willebrand factor include multimerin 1 binding

2016 ◽  
Vol 116 (07) ◽  
pp. 87-95 ◽  
Author(s):  
D'Andra Parker ◽  
Subia Tasneem ◽  
Richard Farndale ◽  
Dominique Bihan ◽  
J. Sadler ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Structural Features ◽  
High Shear ◽  
The Individual ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryMultimerin 1 (MMRN1) is a massive, homopolymeric protein that is stored in platelets and endothelial cells for activation-induced release. In vitro, MMRN1 binds to the outer surfaces of activated platelets and endothelial cells, the extracellular matrix (including collagen) and von Willebrand factor (VWF) to support platelet adhesive functions. VWF associates with MMRN1 at high shear, not static conditions, suggesting that shear exposes cryptic sites within VWF that support MMRN1 binding. Modified ELISA and surface plasmon resonance were used to study the structural features of VWF that support MMRN1 binding, and determine the affinities for VWF-MMRN1 binding. High shear microfluidic platelet adhesion assays determined the functional consequences for VWF-MMRN1 binding. VWF binding to MMRN1 was enhanced by shear exposure and ristocetin, and required VWF A1A2A3 region, specifically the A1 and A3 domains. VWF A1A2A3 bound to MMRN1 with a physiologically relevant binding affinity (KD: 2.0 ± 0.4 nM), whereas the individual VWF A1 (KD: 39.3 ± 7.7 nM) and A3 domains (KD: 229 ± 114 nM) bound to MMRN1 with lower affinities. VWF A1A2A3 was also sufficient to support the adhesion of resting platelets to MMRN1 at high shear, by a mechanism dependent on VWF-GPIbD binding. Our study provides new information on the molecular basis of MMRN1 binding to VWF, and its role in supporting platelet adhesion at high shear. We propose that at sites of vessel injury, MMRN1 that is released following activation of platelets and endothelial cells, binds to VWF A1A2A3 region to support platelet adhesion at arterial shear rates.

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.

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