scholarly journals Selective induction of a glycoprotein IIIa ligand-induced binding site by fibrinogen and von Willebrand factor

Blood ◽  
1996 ◽  
Vol 88 (10) ◽  
pp. 3824-3830 ◽  
Author(s):  
TH Mondoro ◽  
CD Wall ◽  
MM White ◽  
LK Jennings
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Binding Sites ◽  
Flow Cytometric Analysis ◽  
Clot Retraction ◽  
Recognition Sequence ◽  
Ligand Interaction ◽  
Selective Ligand ◽  
Von Willebrand ◽  
Willebrand Factor

Ligand-induced binding sites (LIBS) are neoantigenic regions of glycoprotein (GP)IIb-IIIa that are exposed upon interaction of the receptor with the ligand fibrinogen or the ligand recognition sequence (RGDS). LIBS have been suggested to contribute to postreceptor occupancy events such as full-scale platelet aggregation, adhesion to collagen, and clot retraction. This study examined the induction requirements of a GPIIIa LIBS with regard to ligand specificity. Through the use of the anti-LIBS D3, we report that this complex- activating antibody induces fibrinogen-and von Willebrand factor-binding to GPIIb-IIIa on intact platelets. Bound ligand was detected by flow cytometric analysis and platelet aggregation assays. These bound ligands increased the number of D3-binding sites and altered the affinity of D3 for GPIIb-IIIa on platelets. In contrast, activation of platelet GPIIb-IIIa by D3 did not increase the binding of another RGD- containing ligand, vitronectin. Furthermore, bound vitronectin on thrombin-stimulated platelets did not cause the expression of the D3 LIBS epitope. We conclude direct activation of GPIIb-IIIa in the absence of platelet activation results in selective ligand interaction and that D3 LIBS induction requires the binding of the multivalent ligands, fibrinogen or von Willebrand factor. Thus, the region of GPIIIa recognized by D3 may be an important regulatory domain in ligand- receptor interactions that directly mediate platelet aggregation.

Glycoprotein Ib Has a Partial Role in Platelet-von Willebrand Factor Collagen Interaction

1988 ◽  
Vol 60 (02) ◽  
pp. 182-187 ◽  
Author(s):  
Morio Aihara ◽  
Ken Tamura ◽  
Ryuko Kawarada ◽  
Keizou Okawa ◽  
Yutaka Yoshida
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Ricinus Communis ◽  
Protein S ◽  
Platelet Membrane ◽  
Membrane Glycoprotein ◽  
Normal Plasma ◽  
Ricinus Communis Agglutinin ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe adhesion of human fixed washed platelets (FWP) to collagen was decreased after treatment with Serratia marcescens protease (SP), which removed 95% of the glycocalicin from platelet membrane glycoprotein (GP) lb. However, the diminished adhesion of SP treated FWP to collagen could still be increased in the presence of purified von Willebrand factor (vWF). This ability of vWF to increase FWP adhesion to collagen is defined as collagen cofactor (CCo). The adhesion of FWP to collagen was not affected by a monoclonal antibody (MAb) to GP Ilb/IIIa (10E5), that inhibits ADP and collagen induced platelet aggregation. On the other hand, it was decreased by 50% by a MAb to GP lb (6D1), that inhibits ristocetin induced platelet aggregation. Adhesion of FWP in buffer to collagen was completely inhibited by Ricinus communis agglutinin I or concanavalin A, while Lens culinalis agglutinin and wheat germ agglutinin showed 50% inhibition. The FWP adhesion to collagen in the presence of vWF (normal plasma) was unaffected by MAbs to GP Ilb/IIIa (10E5, P2, HPL1) but was decreased to 32-38% by MAbs to GP lb (6D1, AN51, HPL11). A MAb to vWF (CLB-RAg 35), that inhibits ristocetin induced binding of vWF to platelets, decreased the CCo of normal plasma by 70%. The MAb, CLB-RAg 201, that inhibits the binding of vWF to collagen, completely inhibited the CCo of normal plasma. In conclusion, our data suggest that (1) GP lb has a partial role in FWP adhesion to collagen; (2) the binding of vWF to collagen is required for the expression of CCo; (3) CCo is partly mediated through GP lb; but (4) other platelet membrane protein(s) besides GP lb or GP Ilb/IIIa must also be involved in FWP-vWF-collagen interactions.

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 Platelet Aggregation Induced by a Monoclonal Antibody to the A1 Domain of von Willebrand Factor

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3792-3799 ◽  
Author(s):  
Hilde Depraetere ◽  
Nadine Ajzenberg ◽  
Jean-Pierre Girma ◽  
Catherine Lacombe ◽  
Dominique Meyer ◽  
...  
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Inhibitory Effect ◽  
Platelet Glycoprotein ◽  
Rotational Viscometer ◽  
Induce Platelet Aggregation ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

Shear-induced platelet aggregation (SIPA) involves von Willebrand Factor (vWF) binding to platelet glycoprotein (GP)Ib at high shear stress, followed by the activation of αIIbβ3. The purpose of this study was to determine the vWF sequences involved in SIPA by using monoclonal antibodies (MoAbs) to vWF known to interfere with its binding to GPIb and to αIIbβ3. Washed platelets were exposed to shear rates between 100 and 4,000 seconds−1 in a rotational viscometer. SIPA was quantitated by flow cytometry as the disappearance of single platelets (DSP) in the sheared sample in the presence of vWF, relative to a control in the absence of shear and vWF. At a shear rate of 4,000 seconds−1, DSP was increased from 5.9% ± 3.5% in the absence of vWF to 32.7% ± 6.3% in the presence of vWF. This increase in SIPA was not associated with an elevation of P-selectin expression. vWF-dependent SIPA was completely abolished by MoAb 6D1 to GPIb and partially inhibited by MoAb 10E5 to αIIbβ3. Three MoAbs to vWF were compared for their effect on SIPA at 4,000 seconds−1 in the presence of vWF: MoAb 328, known to block vWF binding to GPIb in the presence of ristocetin, MoAb 724 blocking vWF binding to GPIb in the presence of botrocetin, and MoAb 9, an inhibitor of vWF binding to αIIbβ3. Similar to the effect of MoAb 6D1, MoAb 328 completely inhibited the effect of vWF, whereas MoAb 9 had a partial inhibitory effect, as MoAb 10E5 did. In contrast, MoAb 724, as well as its F(ab′)2 fragments, promoted shear-dependent platelet aggregation (165% of the DSP value obtained in the absence of MoAb 724), indicating that MoAb 724 was responsible for an enhanced aggregation, which was independent of binding to the platelet Fcγ receptor. In addition, the enhancement of aggregation induced by MoAb 724 was abrogated by MoAb 6D1 or 10E5 to the level of SIPA obtained in the presence of vWF incubated with a control MoAb to vWF. Finally, the activating effect of MoAb 724 was also found under static conditions at ristocetin concentrations too low to induce platelet aggregation. Our results suggested that on binding to a botrocetin-binding site on vWF, MoAb 724 mimics the effect of botrocetin by inducing an active conformation of vWF that is more sensitive to shear stress or to low ristocetin concentration.

scholarly journals Supranormal von Willebrand factor multimers in scleroderma

Blood ◽  
1989 ◽  
Vol 73 (6) ◽  
pp. 1586-1591 ◽  
Author(s):  
PM Mannucci ◽  
R Lombardi ◽  
A Lattuada ◽  
E Perticucci ◽  
R Valsecchi ◽  
...  
Keyword(s):  
Systemic Sclerosis ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Dose Aspirin ◽  
Low Dose Aspirin ◽  
Severity Of The Disease ◽  
Partially Occluded ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Normal Controls

Abstract Platelet adhesion-aggregation reactions play an early and pivotal role in the pathogenesis of systemic sclerosis in scleroderma, but the mechanisms are incompletely understood. We determined whether or not plasma from 11 consecutive patients with scleroderma contained a subset of larger than normal (“supranormal”) multimers of von Willebrand factor (vWF) that are potent inducers of platelet aggregation and adhesion. Supranormal multimers were found in all patients on at least one of two different occasions 9 to 12 months apart, whatever the duration and severity of the disease, but in none of the normal controls. Administration of low-dose aspirin (40 mg) to five of the 11 patients for ten days to inhibit the platelet release reaction slightly reduced the amounts of supranormal multimers suggesting that they might originate in part from platelets. Supranormal multimers may contribute to the pathogenesis of systemic sclerosis by inducing platelet aggregation and enhancing adhesion to subendothelium under the conditions of elevated shear stress occurring in the partially occluded vessels of the arterial microcirculation of scleroderma.

scholarly journals New Method for Assessment of Plasma Von Willebrand Factor

1977 ◽  
Author(s):  
T. Sano ◽  
T. Motomiya ◽  
N. Mashimo ◽  
H. Yamazaki
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Normal Subjects ◽  
Induce Platelet Aggregation ◽  
Platelet Suspension ◽  
Maximal Plasma ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Diabetes Melitus

As much interests have been focused on von Willebrand factor (vWF) in diabetes melitus and atherosclerosis, request to determine vWF has been increasing recently. Two methods for assessment of plasma vWF level, without platelet aggregometer, were devised. 1) Platelet-rich plasma (PRP) sensitivity to ristocetin-induced platelet aggregation (RIPA): PRP was separated without centrifugation from citrated blood. Serially two-fold diluted restocetin (16 to 16x2-10 mg/ml) was prepared in a Cooke Microtiter tray and PRP (25 μl each) was added to each concentration of ristocetin. Then the ristocetin-PRP mixture was agitated for 15 seconds using a Kowa Kizai Micromixer and the minimum effective final concentration of ristocetin to give platelet aggregation was obtained microscopically and this was defined as PRP sensitivity to RIPA. This method is convenient for screening test. 2) vWF assay:Serially two-fold diluted plasma (2 to 1024 times, in Tris-salin pH 7.2 containing 12 mg/ml bovine serum albumin), fixed and washed platelet suspension (6x105 /μl, Macfarlane et al.1975) and 3 mg/ml ristocetin were mixed (25 μl each) in a microtiter tray and agitated for 15 seconds. The maximal plasma dilution to induce platelet aggregation was obtained microscopically and defined as the titer of plasma vWF. In normal subjects, minimum effective ristocetin concentration (PRP sensitivity to RIPA) was around 1 to 0.5 mg/ml and maximal plasma dilution to give platelet aggregation (vWF titer) was around 16 to 32 times. The present methods have a good reproducibility and are performed easily without aggregometer and thought to be useful clinically.

The Importance of the Carbohydrate Moiety of the Factor VIII/Von Willebrand Factor Protein in Binding to and Causing Agglutination of Human Platelets

1979 ◽  
Author(s):  
H.R. Gralnick ◽  
D.K. Morisato
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Binding Sites ◽  
Human Platelets ◽  
Galactose Oxidase ◽  
Scatchard Analysis ◽  
Human Fibrinogen ◽  
Number Of Binding Sites ◽  
Von Willebrand ◽  
Willebrand Factor

We have investigated the binding of radiolabelled factor VIII/von Willebrand factor (f. VIII/vWf) protein to human platelets (P) in the presence of ristocetin (R). In these atudies we have delineated the importance of the carbohydrate (CHO) moiety(s) in both the binding to the P and in cauaing agglutination of P. Binding of the f.VIII/vWf protein to human P was time and temperature dependent and dependent on the concentration of R. Binding was specific in that it could not be blocked by human fibrinogen but was inhibited by unlabelled f.VIII/vWf protein. In studies utilizing varying amounts of the f.VIII/vWf protein or by varying the number of P in the assay, the number of binding sites for the f. VIII/vWf protein were estimated at 9,500-9,800 per platelet. Scatchard analysis revealed 11,000 binding sites with 3,600 of high affinity and 7,400 of low affinity. Removal of the sialic acid of the f.VIII/vWf protein resulted in no significa nt change in its ability to bind to the P surface or cause agglutination in the presence, IR. Removal of the galactose by 6-galactosijase resulted in a 75% reduction of binding of the f.VIII/vWf protein and a 91% decrease in the agglutination of human P. Similar studies with galactose oxidase showed that oxidation of the penultimate galactose residue s results in a decrease in agglutination comparable to that seen with 6-galactosidase treatment. These studies indicate that the CHO moiety of the f.VIII/vWf protein is important in both binding to the P surface as well as causing agglutination of human P.

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