Cytoskeletal regulation of the platelet glycoprotein Ib/V/IX–von Willebrand factor interaction

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3480-3489 ◽  
Author(s):  
Nayna Mistry ◽  
Susan L. Cranmer ◽  
Yuping Yuan ◽  
Pierre Mangin ◽  
Sacha M. Dopheide ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Cho Cells ◽  
Actin Polymerization ◽  
Cytochalasin D ◽  
Actin Binding ◽  
Platelet Glycoprotein ◽  
Actin Binding Protein ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Shear-induced binding of von Willebrand factor (vWf) to the platelet glycoprotein (GP) Ib/V/IX complex plays a key role in initiating platelet adhesion and aggregation at sites of vascular injury. This study demonstrated that pretreating human platelets with inhibitors of actin polymerization, cytochalasin D or latrunculin B, dramatically enhances platelet aggregation induced by vWf. The effects of these inhibitors were specific to the vWf-GPIbα interaction because they enhanced vWf-induced aggregation of Glanzmann thrombasthenic platelets and Chinese hamster ovary (CHO) cells transfected with GPIb/V/IX. Moreover, cytochalasin D enhanced the extent of platelet aggregation induced by high shear stress (5000 s−1) and also lowered the shear threshold required to induce aggregation from 3000 s−1 to as low as 500 s−1. Studies of CHO cells expressing GPIbα cytoplasmic tail truncation mutants that failed to bind actin-binding protein-280 (deletion of residues 569-610 or 535-568) demonstrated that the linkage between GPIb and actin-binding protein-280 was not required for vWf-induced actin polymerization, but was critical for the enhancing effects of cytochalasin D on vWf-induced cell aggregation. Taken together, these studies suggest a fundamentally important role for the cytoskeleton in regulating the adhesive function of GPIb/V/IX.

Cytoskeletal regulation of the platelet glycoprotein Ib/V/IX–von Willebrand factor interaction

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3480-3489 ◽  
Author(s):  
Nayna Mistry ◽  
Susan L. Cranmer ◽  
Yuping Yuan ◽  
Pierre Mangin ◽  
Sacha M. Dopheide ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Cho Cells ◽  
Actin Polymerization ◽  
Cytochalasin D ◽  
Actin Binding ◽  
Platelet Glycoprotein ◽  
Actin Binding Protein ◽  
Von Willebrand ◽  
Willebrand Factor

Shear-induced binding of von Willebrand factor (vWf) to the platelet glycoprotein (GP) Ib/V/IX complex plays a key role in initiating platelet adhesion and aggregation at sites of vascular injury. This study demonstrated that pretreating human platelets with inhibitors of actin polymerization, cytochalasin D or latrunculin B, dramatically enhances platelet aggregation induced by vWf. The effects of these inhibitors were specific to the vWf-GPIbα interaction because they enhanced vWf-induced aggregation of Glanzmann thrombasthenic platelets and Chinese hamster ovary (CHO) cells transfected with GPIb/V/IX. Moreover, cytochalasin D enhanced the extent of platelet aggregation induced by high shear stress (5000 s−1) and also lowered the shear threshold required to induce aggregation from 3000 s−1 to as low as 500 s−1. Studies of CHO cells expressing GPIbα cytoplasmic tail truncation mutants that failed to bind actin-binding protein-280 (deletion of residues 569-610 or 535-568) demonstrated that the linkage between GPIb and actin-binding protein-280 was not required for vWf-induced actin polymerization, but was critical for the enhancing effects of cytochalasin D on vWf-induced cell aggregation. Taken together, these studies suggest a fundamentally important role for the cytoskeleton in regulating the adhesive function of GPIb/V/IX.

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.

EFFECTS OF SOME ORGANIC SOLVENTS ON GP lb AND ACTIN-BINDING PROTEIN IN BLOOD PLATELETS

1987 ◽  
Author(s):  
A M Aakhus ◽  
N O Solum ◽  
I Hagen
Keyword(s):  
Organic Solvents ◽  
Von Willebrand Factor ◽  
Early Stage ◽  
Binding Protein ◽  
Blood Platelets ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Von Willebrand ◽  
Willebrand Factor

Effects on filamentous proteins appear to be a central phenomenon in the neuronal toxic effects of organic solvents. We have therefore compared the effects of some organic solvents (particularly isopropylalcohol, IPA) to the previously observed effects of dibucaine (DBC) on platelet cytoskeletal proteins. Incubation of platelets with 6% IPA at 37° C, like DBC, initiates a degradation of actin-binding protein (ABP) as substrate for a calcium activated protease (CAP), shown by SDS-PAGE. IPA leads to an increase followed by a decrease in bovine von Willebrand factor-induced agglutination. The decrease is accompanied by a release of glycocali-cin from the GP lb α-chain. The process was also studied using CIE of Triton X-100 extracts of platelets against antiserum to glycocalicin. Incubation of platelets with IPA before extraction in the presence of 4.2 mM leupeptin leads to a time-dependent transformation of GP Ib-related immunoprecipitates from that of the slow-migrating peak III complex (probably between ABP and GP lb) to the faster migrating GP Ib-precipitate. Our working hypothesis is that IPA induces an activation of the CAP by mobilizing calcium. This leads to degradation of ABP and liberation of GP lb from the cytoskeleton accompanied by an increased tendency for agglutination. The following decrease is explained by degradation of the glycocalicin part of the GP lb enchain which contains the binding-site for von Willebrand factor. We conclude that IPA has a similar effect on GP lb and ABP as DBC. Preliminary studies with 1% DMSO and 0,005% toluene at 37° C revealed that these organic solvents have some similar effects on platelets as described for IPA. Possibly the described effects are characteristic of certain cells at an early stage in a process ultimately leading to cell lysis.

Isolation from commercial aurintricarboxylic acid of the most effective polymeric inhibitors of von Willebrand factor interaction with platelet glycoprotein Ib. Comparison with other polyanionic and polyaromatic polymers

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2291-2298 ◽  
Author(s):  
M Weinstein ◽  
E Vosburgh ◽  
M Phillips ◽  
N Turner ◽  
L Chute-Rose ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Glycoprotein ◽  
Glycoprotein Ib ◽  
Aurintricarboxylic Acid ◽  
Coronary Artery Thrombosis ◽  
Exclusion Chromatography ◽  
Inhibitory Components ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Solutions of commercial aurintricarboxylic acid (ATA) inhibit ristocetin- or shear stress-induced, von Willebrand factor (vWF)- mediated platelet aggregation by interacting with vWF and blocking its attachment to platelet membrane glycoprotein Ib. ATA has also been shown to prevent cyclic platelet clumping in a dog model of coronary artery thrombosis. Because these ATA solutions are actually a heterogeneous mixture of polyanionic, polycarboxylic polyaromatic polymers of molecular weight (Mr) 200 to greater than 6,000, we separated the most effective inhibitory components of commercial ATA using exclusion chromatography. ATA polymers larger than Mr 700 inhibited ristocetin-induced, vWF-mediated platelet aggregation more effectively than smaller ATA polymers, whereas shear-induced, vWF- mediated platelet aggregation was optimally inhibited by ATA polymers of Mr greater than or equal to 2,500. Platelet aggregation mediated by vWF was not inhibited by a nonphenolic, polyanionic polymer (polyglutamic acid) or by a polyphenolic ATA-like polymer (aurin) devoid of carboxyl groups. Polyanionic, polysulfonated aromatic polymers (polystyrene sulfonate) of Mr 35, 17.4, 8, and 4.6 x 10(3) inhibited ristocetin- and shear-induced, vWF-mediated aggregation with less potency on a mass/volume basis than large polymers of ATA. We conclude that a polyanionic, polycarboxylated, polyphenolic ATA polymer of Mr 2,500 is optimally potent as an inhibitor of shear- and ristocetin-induced, vWF-mediated platelet aggregation and is likely to be more effective than solutions of commercial ATA as an anti-arterial thrombotic agent.

Modulation of von Willebrand Factor A1 Domain-Mediated Platelet Aggregation/Agglutination by α-Thrombin.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 628-628
Author(s):  
Grazia Loredana Mendolicchio ◽  
Reha Celikel ◽  
Kottayil I. Varughese ◽  
Brian Savage ◽  
Zaverio M. Ruggeri
Keyword(s):  
Platelet Aggregation ◽  
Shear Rate ◽  
Von Willebrand Factor ◽  
Platelet Glycoprotein ◽  
Shear Rates ◽  
Amino Terminal ◽  
Low Responder ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Evaluation of the crystal structures of the amino terminal domain of platelet glycoprotein (GP) Ibα bound to the von Willebrand factor A1 domain (VWFA1) or to α-thrombin indicate the absence of significant steric hindrance in a putative triple complex of the two ligands interacting with the same receptor molecule. Superposition of the models reveals that intermolecular contacts may be established between VWFA1 and α-thrombin concurrently bound to GP Ibα, and suggests that these additional interactions could stabilize the intrinsically low affinity binding of the VWF A1 domain. To verify the predictions of the model, we used gel electrophoresis under native conditions and purified components in solution to demonstrate directly the formation of a triple complex. We then sought to evaluate whether α-thrombin could influence the functional effects of the VWF-GP Ibα interaction. For this purpose, we established a model of platelet agglutination/aggregation dependent on the interaction between recombinant dimeric VWFA1 domain, purified from the culture medium of stably transfected D. melanogaster cell lines, and GP Ibα. In this assay, platelet rich plasma prepared from individual donor blood collected with the thrombin inhibitor D-phenyl alanyl-L-prolyl-L-arginine chloromethyl ketone dihydrochloride (PPACK) as an anticoagulant (80 μM) was mixed with varying concentrations of dimeric VWFA1 (0.5-10 μg/ml) and exposed to variable shear rate levels in a cone-and-plate viscometer. Platelet aggregation was observed at shear rates between 6 and 108 dyn/cm2. The response in different normal controls was reproducible but variable in extent, and individuals could be assigned to one of two categories, low responder and high responder. An agglutination response was observed after platelets were treated with 10 μM prostaglandin E1 to block activation, and the distinction between low and high responders remained true under these conditions. For simplicity, agglutinated platelets were still defined as “aggregates”. With activation blocked platelets, aggregates were stable up to a shear rate of 30 dyn/cm2, but began to dissipate at higher levels. The addition of α-thrombin with the active site irreversibly blocked by PPACK at concentrations between 5 and 10 μg/ml substantially increased the extent of the platelet response. This was demonstrated by a faster rate of platelet agglutination/aggregation, a greater stability of aggregates at higher shear rates, and an overall increase in the size of aggregates formed. To demonstrate the latter, samples were exposed to shear stress under selected conditions and immediately fixed with 1% glutaraldehyde for quantitative image analysis. Maximum aggregate size was increased several fold in the presence of α-thrombin, and the difference was particularly evident in low responder individuals in whom dimeric VWFA1 alone caused the formation of small and unstable aggregates. PPACK-blocked thrombin by itself had no effect on platelet aggregate formation at any shear rate tested. Our findings delineate a mechanism through which α-thrombin may stabilize platelet-platelet contacts by mediating a tighter association between VWF A1 domain and GP Ibα receptor. Such a function, independent of proteolytic activity, may enhance platelet deposition at sites of vascular injury.

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

AbstractShear-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 Assembly and GPIIIa content of cytoskeletal core in platelets agglutinated with bovine von Willebrand factor

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1572-1579 ◽  
Author(s):  
EG Puszkin ◽  
EA Mauss ◽  
MB Zucker
Keyword(s):  
Von Willebrand Factor ◽  
Inhibitory Action ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cytochalasin D ◽  
Actin Binding ◽  
Insoluble Residue ◽  
Sds Page ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The association between occupancy of the von Willebrand factor (vWf) receptor glycoprotein (GP) Ib, agglutination, and the assembly and composition of the cytoskeletal core was studied in 125I-surface- labeled aspirin-treated washed platelets. Binding of ligands to GPIIb- IIIa and platelet aggregation were abolished by addition of EDTA. Platelet agglutination induced by bovine vWf generated a complete cytoskeletal core (Triton-insoluble residue), shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to be composed of actin-binding protein (ABP) (260 Kd), 235-Kd protein, myosin heavy chain (200 Kd), alpha-actinin (100 Kd), and actin (43 Kd). In addition, autoradiography of the gels showed a 125I 105-Kd GP, identified by immunoblot as GPIIIa, as well as GPIb, GPIIb, and another band at 87 Kd, probably GPIV. Neither cytoskeletal assembly nor GPIIa incorporation was altered if calpain was inhibited with leupeptin. Platelet suspensions exposed to bovine vWf without stirring (ie, nonagglutinated) or platelets in which agglutination was inhibited with ADP showed smaller cytoskeletons with little ABP, 235 Kd protein, and alpha-actinin. Autoradiographs showed mainly GPIb. Cytochalasin D (CD) and monobromobimane (MB) enhanced agglutination and prevented the inhibitory action of ADP on bovine vWf-induced platelet agglutination. CD markedly inhibited the assembly of the cytoskeletal core as well as GPIIIa retention, whereas MB resulted in a large Triton-insoluble residue which contained GPIIIa. Thus, development of a platelet cytoskeletal core is apparently not required for agglutination, but when a cytoskeletal core is assembled in agglutinated platelets, GPIIIa is retained.

Disruption of the long-range GPIIIa Cys5-Cys435 disulfide bond results in the production of constitutively active GPIIb-IIIa (αIIbβ3) integrin complexes

Blood ◽  
2002 ◽  
Vol 100 (6) ◽  
pp. 2094-2101 ◽  
Author(s):  
Qi-Hong Sun ◽  
Chao-Yan Liu ◽  
Ronggang Wang ◽  
Cathy Paddock ◽  
Peter J. Newman
Keyword(s):  
Platelet Aggregation ◽  
Long Range ◽  
Disulfide Bond ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Receptor Activation ◽  
Platelet Glycoprotein ◽  
Adhesive Properties ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The major platelet integrin αIIbβ3, also known as the platelet glycoprotein (GP) IIb-IIIa complex, mediates platelet aggregation by serving as the receptor for fibrinogen and von Willebrand factor. In addition to its physiologic role, GPIIb-IIIa also bears a number of clinically important alloantigenic determinants. Previous studies have shown that disruption of the long-range Cys5-Cys435 disulfide bond of the β3 subunit results in the production of isoforms that bind some, but not all, anti-PlA1 alloantibodies, suggesting that mutations in this so-called long-range disulfide bond can alter the conformation of GPIIIa. The purpose of this study was to examine the effects of either the Cys5Ala or Cys435Ala substitution of GPIIIa on the adhesive properties of the GPIIb-IIIa complex. We found that both Ala5GPIIIa and Ala435GPIIIa were capable of associating with GPIIb and were expressed normally on the cell surface when cotransfected into Chinese hamster ovary (CHO) cells. CHO cells expressing GPIIb-Ala5GPIIIa or GPIIb-Ala435IIIa bound well-characterized, conformationally sensitive ligand-induced binding site (LIBS) antibodies, and were capable of constitutively binding the fibrinogen-mimetic monoclonal antibodies Pl-55 and PAC-1, as well as soluble fibrinogen. Both GPIIb-Ala5IIIa– and GPIIb-Ala435IIIa–transfected CHO cells also bound more avidly to immobilized fibrinogen and were capable of mediating the tyrosine phosphorylation of pp125FAK on cell adhesion. These data are consistent with the notion that these regions of GPIIIa participate in the conformational change associated with receptor activation. Additionally, these studies may provide a molecular explanation for the previously reported ability of mild reducing agents to activate the GPIIb-IIIa complex and promote platelet aggregation.

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