PLATELET ACTIVATION INDUCED BY A MONOCLONAL ANTIBODY AGAINST THE PLATELET GP Ilb/IIIa COMPLEX

1987 ◽  
Author(s):  
P W Modderman ◽  
J G Huisman ◽  
J A van Mourik ◽  
A E G Kr ◽  
v d Borne
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Medical Research ◽  
Platelet Activation ◽  
Dense Granules ◽  
Fibrinogen Binding ◽  
Platelet Release ◽  
Induced Aggregation ◽  
Aggregation Of Platelets

A receptor for fibrinogen on the platelet GP Ila/lIIb complex is induced by ADP, thrombin and other agonists. To study functional domains on GP Ilb/IIIa, the effects of anti-GP Ilb/IIIa monoclonal antibodies (Mab’s) on platelet function were determined. One of these Mab’s, 6C9, induced platelet aggregation. The antibody binds to the intact GP Ilb/IIIa complex only, not to free GP lib or free GP Ilia. Its epitope is different from that of C17, a Mab that inhibits ADP-induced fibrinogen binding and platelet aggregation. 6C9 induces fibrinogen-mediated aggregation rather than agglutination since 6C9-induced platelet interactions were blocked by treatments that also inhibited the effects of ADP etc., without inhibiting binding of 6C9 itself. 6C9 induces binding of 125I-fibrinogen (35.000 ± 7.300 molecules/platelet, Kd = 1.3 ± 0.4 µM) to unstirred platelets. Binding of fibrinogen was 60 to 80% inhibited by apyrase, which indicates that 6C9-induced fibrinogen binding is largely mediated via ADP released from platelets. In addition, 6C9 induced aggregation of platelets in the absence of extracellular fibrinogen. Mediation of this process by platelet fibrinogen or other a-granule proteins, released upon activation by 6C9, was implicated by the finding that aggregation of washed platelets, but not of platelets to which fibrinogen was added, could be blocked by PGI2. Platelet release was also assessed directly by measuring β-thromboglobulin (α-granules) and (14C) serotonin (dense granules) in the medium of unstirred platelets incubated with 6C9. F(ab')2 fragments of 6C9 only aggregated platelets in the presence of fibrinogen and did not release (14C) serotonin. Moreover, release induced by intact 6C9 was inhibited by anti-GP Ilb/IIIa Mab C17 but not by C17 F(ab’)2, although the latter inhibited ADP-induced platelet aggregation. These data indicate that binding of antibodies to specific sites on GP Ilb/IIIa may induce Fc-dependent platelet activation.This study was supported by the Foundation for Medical Research MEDIGON (grant no. 900-526-057.

On The Mechanism Of Heparin-Induced Potentiation Of Platelet Aggregation

1981 ◽  
Author(s):  
M Yamamoto ◽  
K Watanabe ◽  
Y Ando ◽  
H Iri ◽  
N Fujiyama ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Coagulation Factors ◽  
Marked Enhancement ◽  
Matrix Bound ◽  
Induced Aggregation ◽  
Aggregation Of Platelets ◽  
Plasma Heparin

It has been suggested that heparin caused potentiation of aggregation induced by ADP or epinephrine. The exact mechanism of heparin-induced platelet activation, however, remained unknown. In this paper, we have investigated the role of anti-thrombin III ( AT ) in heparin-induced platelet activation using purified AT and AT depleted plasma. When ADP or epinephrine was added to citrated PRP one minute after addition of heparin ( 1 u/ml, porcine intestinal mucosal heparin, Sigma Co. USA ), marked enhancement of platelet aggregation was observed, compared with the degree of aggregation in the absence of heparin. However, in platelet suspensions prepared in modified Tyrode’s solution, heparin exhibited no potentiating effect on platelet aggregation induced by epinephrine or ADP. Potentiation of epinephrine- or ADP-induced platelet aggregation by heparin was demonstrated when purified AT was added to platelet suspensions at a concentration of 20 μg/ml. AT depleted plasma, which was prepared by immunosorption using matrix-bound antibodies to AT, retained no AT, while determination of α1-antitrypsinα2- macroglobulin and fibrinogen in AT depleted plasma produced values which corresponded to those of the original plasma when dilution factor was taken into account. The activities of coagulation factors were also comparable to those of the original plasma. Heparin exhibited potentiating effect on ADP- or epinephrine-induced aggregation of platelets in original plasma, but no effect in AT depleted plasma. When purified AT was added back to AT depleted plasma at a concentration of 20 μg/ml, potentiation of platelet aggregation by heparin was clearly demonstrated.Our results suggest that effect of heparin on platelet aggregation is also mediated by anti-thrombin III.

Requirement Of Fibrinogen And Calcium For Inter-Platelet Bridges In Platelet Aggregation: A Hypothesis

1981 ◽  
Author(s):  
Elizabeth Kornecki ◽  
Stefan Niewiarowski
Keyword(s):  
Platelet Aggregation ◽  
Binding Sites ◽  
Proteolytic Enzymes ◽  
Divalent Cations ◽  
Platelet Surface ◽  
Fibrinogen Binding ◽  
Platelet Aggregates ◽  
High Concentrations ◽  
Induced Aggregation ◽  
Aggregation Of Platelets

Fibrinogen and calcium are required for the aggregation of platelets stimulated by ADP or pre-treated with proteolytic enzymes. Specific platelet surface fibrinogen binding sites (receptors) are exposed after platelets are stimulated by ADP or pre-treated with Chymotrypsin or pronase. It has previously been shown in our laboratory that an intact, symmetrical fibrinogen molecule is essential for fibrinogen binding and fibrinogen-induced aggregation of both ADP-stimulated and proteolytically-treated platelets. Here we propose that the mechanism by which fibrinogen and calcium aggregate platelets is by forming inter-platelet bridges linking the fibrinogen receptors of adjacent platelets together. In support of this proposition are the following new lines of evidence: 1) The fibrinogen-induced aggregations of ADP-stfiliulated or proteolytically-treated platelets are inhibited by high concentrations of fibrinogen (Ki=2.6 and 8.5 × 10 5M, respectively). The fibrinogen binding sites on adjacent platelets, at these concentrations, would be saturated by fibrinogen and therefore no inter-platelet fibrinogen bridges could be formed to hold the platelets together. 2) ADP-stimulated or chymotrypsin-treated platelets aggregated by fibrinogen are deaggregated by Chymotrypsin or pronase and this deaggregation coincides with the loss of 125I-fibrinogen from the platelet surface. 3) Preincubation of platelets with EDTA results in inhibition of both platelet aggregation and 125I-fibrinogen binding. Following the aggregations of ADP-stimulated or of chymotrypsin-treated platelets by fibrinogen, the addition of EDTA to the platelet aggregates results in both their deaggregation and their loss of bound 125I-fibrinogen. Thus it appears that divalent cations, especially calcium, are essential for the formation of fibrinogen-linked platelet aggregates.

Aggregation to Thrombin and Collagen of Platelets from a Glanzmann Thrombasthenic Patient Lacking Glycoproteins IIb and IIIa

1989 ◽  
Vol 62 (03) ◽  
pp. 962-967 ◽  
Author(s):  
Lilian McGregor ◽  
Michel Hanss ◽  
Amal Sayegh ◽  
Juan J Calvette ◽  
Marie-Christine Trzeciak ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Membrane Glycoproteins ◽  
Albumin Solution ◽  
Western Blots ◽  
Glycoprotein Complex ◽  
Antigen Present ◽  
High Concentrations ◽  
Induced Aggregation

SummaryThe aim of this study was to investigate the platelets of a Glanzmann thrombasthenic patient, which in citrated PRP failed to respond to various agonists, but aggregated and secreted to high concentrations of thrombin (0.36, 0.72 and 1 U/ml) and collagen (4, 10 and 20 μg/ml) when washed and resuspended in a Tyrode-albumin solution (containing 2 mM Ca2+). Aggregation of the patient platelets was not affected by anti-IIb/IIIa monoclonal antibody (P18) which strongly inhibits thrombin or collagen induced aggregation of normal platelets. Washed platelets of this patient did not aggregate to ADP (10-100 μM) in the presence of added fibrinogen (2 mg/ml) nor bind 125I-labelled fibrinogen (40 to 320 μg/ml) when thrombin-stimulated. Different anti-IIb/IIIa monoclonal antibodies (P2, P18) when used in binding or crossed immunoelectrophoretic studies showed a complete absence of the IIb-IIIa glycoprotein complex on the patient platelets. Moreover, glycoproteins IIb or IIIa were absent on silver-stained twodimensional (non-reduced/reduced) polyacrylamide gel separations of the patient platelets and were not detected by Western blots used in combination with anti-PLA1 (antigen present on Ilia), anti-Leka (antigen present on IIb). This study shows that platelets lacking glycoproteins IIb or IIIa can aggregate in response to high concentrations of collagen or thrombin when resuspended in the presence of physiological concentrations of calcium. Results obtained in this study could indicate the existence of other mechanisms (other than the IIb-IIIa glycoprotein complex) involving glycolipids, heparans, proteoglycans, and/or unknown membrane glycoproteins to mediate platelet aggregation of stimulated thrombasthenic platelets.

Epinephrine-Induced Aggregation of Rabbit Platelets Refractory to ADP

1985 ◽  
Vol 53 (03) ◽  
pp. 366-371 ◽  
Author(s):  
C Lalau Keraly ◽  
R L Kinlough-Rathbone ◽  
J F Mustard
Keyword(s):  
Platelet Aggregation ◽  
Adrenergic Receptor ◽  
Fibrinogen Binding ◽  
Rabbit Platelets ◽  
Induced Aggregation ◽  
Aggregation Of Platelets

SummaryThe mechanisms involved in platelet aggregation induced by epinephrine are unclear. Although epinephrine does not aggregate washed rabbit platelets, platelets made refractory to ADP will aggregate in response to epinephrine in the presence of ADP. We have examined whether the mechanism(s) by which epinephrine induces aggregation of refractory platelets involves fibrinogen binding and Ca2+ association. With normal platelets, ADP causes aggregation, fibrinogen binding and Ca2+ association in a medium containing 0.2 mM 45Ca2+. After 3 min of incubation with ADP, fibrinogen dissociates from platelets, but 45Ca2+ does not. Epinephrine alone does not cause aggregation, fibrinogen binding or 45Ca2+ association. Platelets that are refractory to ADP do not aggregate and bind fibrinogen upon addition of ADP, but aggregate and bind fibrinogen in response to epinephrine, provided ADP is still present. These effects of epinephrine are mediated by the α-adrenergic receptor since they are blocked by phentolamine or verapamil and potentiated by propranolol. However, epinephrine-induced aggregation of platelets refractory to ADP does not involve further detectable increase in the amount of 45Ca2+ associated with the platelets.

STIMULUS-DEPENDENT CHANGES IN THE SURFACE EXPRESSION OF GPIIb/IIIa AND FIBRINOGEN RECEPTORS. RELATIONSHIP TO PLATELET AGGREGATION

1987 ◽  
Author(s):  
K Niiya ◽  
E Hodson ◽  
R Bader ◽  
V Byers-Ward ◽  
E F Plow ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Divalent Cation ◽  
Surface Expression ◽  
The Other ◽  
Fab Fragment ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Dissociation Constant Kd

Platelet stimulation altered the binding of three monoclonal antibodies (monovalent Fab’ fragment) directed against the glycoprotein (GP)IIb/IIIa complex. We found that 47,600-60,300 molecules of antibody bound per platelet before stimulation, as compared to 89,200-146,500 molecules per platelet after thrombin stimulation. These changes were observed in parallel with a small but significant increase in the dissociation constant (Kd) of two antibodies. In contrast, no statistically significant changes were observed with ADP-stimulated platelets. The increased binding of LJ-CP3, but not of the other two antibodies, to activated platelets decreased by 3040% in the presence of EDTA at 22-25°C, suggesting the occurrence of divalent-cation mediated, activation-dependent changes in the corresponding GPIIb/IIIa epitope. Platelets stimulated by thrombin bound more fibrinogen than those stimulated by ADP, and significant differences in the extent but not in the affinity of fibrinogen binding were observed with different platelet agonists. When the pool of GPIIb/IIIa molecules exposed on the surface of unstimulated platelets was reacted with monoclonal antibody LJ-CP3 to block ADP-induced fibrinogen binding and platelet aggregation, thrombin stimulation still induced substantial binding and aggregation. This effect of thrombin required exposure of platelets to the active agonist and was not mediated by molecules released by thrombin into the medium. Therefore, platelets activated with “strong” agonists exhibit increased number of surface-oriented epitopes associated with GPIIb/IIIa. The GPIIb/IIIa molecules bearing these newly exposed epitopes are functional in that they bind fibrinogen and mediate platelet aggregation.

Effect of Three Japanese Kampo Medicines on Platelet Activation by Monoclonal Anti-platelet Membrane Glycoprotein Antibodies

1994 ◽  
Vol 22 (01) ◽  
pp. 71-76 ◽  
Author(s):  
Toshihiro Kawakatsu ◽  
Shosaku Nomura ◽  
Hirofumi Kido ◽  
Kazuyuki Yamaguchi ◽  
Tsutomu Fukuroi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Antibody Binding ◽  
Platelet Glycoprotein ◽  
Membrane Glycoprotein ◽  
Fc Gamma Receptor ◽  
Gamma Receptor ◽  
Induced Aggregation

We studied the effect of three Japanese kampo medicines on platelet activation by an anti-CD9 monoclonal antibody (NNKY1-19) and an anti-human Fc gamma receptor II monoclonal antibody (NNKY3-2). Sho-saiko-to (TJ-9) and Sairei-to (TJ-114) partially suppressed platelet aggregation induced by NNKYl-19, while Juzen-taiho-to (TJ-48) suppressed aggregation induced by NNKY3-2. TJ-9 and TJ-114 also suppressed collagen-induced aggregation, but TJ-48 did not. Flow cytometry showed that the three medicines did not affect antibody binding to the platelets. Thus, all three kampo medicines suppressed platelet activation by anti-platelet glycoprotein antibodies without inhibiting antibody binding.

scholarly journals Platelet activation by a synthetic hydrophobic polymer, polymethylmethacrylate

Blood ◽  
1991 ◽  
Vol 78 (7) ◽  
pp. 1713-1721
Author(s):  
JA Ware ◽  
J Kang ◽  
MT DeCenzo ◽  
M Smith ◽  
SC Watkins ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Gamma Chain ◽  
Fibrinogen Receptor ◽  
Hydrophobic Polymer ◽  
Fibrinogen Binding ◽  
Initial Stage ◽  
Artificial Surfaces ◽  
Induced Aggregation

Platelets adhere to artificial surfaces in the initial stage of thrombus formation, but the subsequent steps in signal transduction that lead to platelet activation by artificial surfaces are not understood. When 0.325-micron diameter beads composed of a hydrophobic polymer, polymethylmethacrylate (PMMA), were added to gel-filtered aequorin-loaded platelets suspended in media containing Ca2+, the platelets aggregated; addition of fibrinogen was not required. Platelet aggregation was preceded by an increase in cytoplasmic Ca2+ and was accompanied by phosphorylation of the 47-Kd substrate of protein kinase C (PKC), 5-hydroxytryptamine (5-HT) release, and accumulation of phosphatidic acid. All these effects were partially inhibited by apyrase and aspirin. Monoclonal antibodies (MoAbs) 7E3 and M148 and the synthetic peptides RGDS and fibrinogen gamma chain fragment 400–411, all of which bind to the platelet fibrinogen receptor glycoprotein IIb- IIIa (GPIIb-IIIa) and inhibit fibrinogen binding, prevented PMMA- induced aggregation but did not inhibit the Ca2+ increase. Chymotrypsin- treated platelets aggregated after addition of fibrinogen, but not PMMA. We conclude that platelets interact initially with PMMA at membrane sites other than those required for fibrinogen binding, leading to activation of membrane phospholipases and PKC, an increase in cytoplasmic Ca2+, release of 5-HT, ADP, and fibrinogen from storage granules, and to platelet aggregation.

Modulation of Platelet Function by Recombinant Thrombomodulin Hematologic Implications.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3898-3898
Author(s):  
Cafer Adiguzel ◽  
Omer Iqbal ◽  
Daniel Fareed ◽  
Debra Hoppensteadt ◽  
Walter Jeske ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Whole Blood ◽  
Platelet Function Tests ◽  
Flow Cytometric ◽  
Platelet Release ◽  
Recombinant Thrombomodulin ◽  
Microparticle Formation ◽  
Induced Aggregation

Abstract Several recombinant thrombomodulin (r-TM) preparations have been developed for different clinical indications. While the in vitro effects of r-TM on blood coagulation parameters are extensively studied, the effect of this agent on platelet function tests such as the adhesion, aggregation, activation and secretion are not fully explored. The purpose of this study was to investigate the effect of a recombinant version of thrombomodulin (ART 123, Asahi, Pharmaceutical, Japan) on various platelet function tests. Platelet aggregation, platelet release and platelet activation by tissue factor (TF) utilizing flow cytometry studies were carried out. In the platelet aggregation studies, citrated whole blood was supplemented with graded amounts of r-TM, in a concentration range of 0–10 ug/ml in the blood of normal healthy volunteers (n=25). Platelet rich plasma (PRP) was prepared by controlled centrifugation (800g) for 15 minutes. Platelet count in the PRP was adjusted to 250,000/ul and aggregation studies were carried out using ADP (5 and 2.5 uM), and alpha thrombin (0.5 U/ml). Platelet activation studies were carried out using flow cytometric method utilizing citrated whole blood and recombinant TF and ADP as activators. In this procedure whole blood was supplemented with TM in a concentration range of 0–10 ug/ml and incubated. TF was then added and further incubated for an additional 2 minutes. Platelets were fixed and incubated with CD 61 and CD 62 antibodies and analyzed using the flow cytometer. Platelet release assays included the 14C serotonin release (SRA) from the washed platelets. In the platelet aggregation assay r-TM did not produce any significant inhibition of agonist induced aggregation with ADP and epinephrine, however, r-TM produced a strong concentration inhibition of thrombin induced aggregation with an IC 50 of 0.42 ug/ml. In the flow studies, r-TM produced an initial augmentation of the generation of microparticles at concentrations up to 0.31 ug/ml (ranges; 5–20%). However at concentrations > 0.31 ug/ml r-TM produced a concentration dependent inhibition of the microparticle formation with an IC 50 of 2.5 ug/ml. At concentrations of >5.0 ug/ml a complete inhibition of TF mediated microparticle formation was noted. Interestingly, r-TM did not produce any inhibition of the p-selectin expression at all concentrations studied. In the SRA, r-TM did not produce any release at concentrations up to 10 ug/ml. However, r-TM produced a strong inhibition of the alpha thrombin induced SRA release. These studies demonstrate that although in the agonist induced platelet aggregation studies r-TM does not produce any modulation of platelet aggregation responses with the exception of thrombin, in the flow cytometric studies it produces a biophasic response. In a concentration range of 0 - .31 ug/ml it produced a slight augmentation of the TF mediated platelet activation. However, at higher concentrations it produced an inhibition of the platelet microparticle formation. Interestingly, there was no effect of r-TM on p-selectin activation. These studies suggest that although r-TM does not produce any inhibition of the agonist induced aggregation of platelets, it can inhibit the TF mediated microparticle formation. Moreover, since r-TM at concentrations of up to 10 ug/ml does not produce any effect on p-selectin expression. It is unlikely to produce any primary hemostatic compromise in a therapeutic range of 2–6 ug/ml.

Differential Inhibition of Arachidonic Acid Mediated Aggregation of Platelets by Branded and Generic Argatroban Preparations

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5131-5131
Author(s):  
Christopher Aranda ◽  
Debra Hoppensteadt ◽  
Omer Iqbal ◽  
Bruce E Lewis ◽  
Jawed Fareed
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Serotonin Release ◽  
Final Concentration ◽  
Release Mechanism ◽  
Clinical Effects ◽  
Induced Aggregation ◽  
Aggregation Of Platelets ◽  
Inhibition Of Thrombin

Abstract Abstract 5131 Argatroban represents a parentral antithrombin agent which is used in the management of anticoagulation in heparin compromised patients. Its main mechanism of action is mediated via inhibition of thrombin and its generation. While its effect on platelet activation inhibition by thrombin have been reported, very little information on the effects of this drug on thromboxane formation and Arachidonic Acid mediated activation of platelets is available. Argatroban and its generic versions namely Slovastan, Argaron and Gartban, may modulate Arachidonic Acid mediated platelet aggregation and release processes. To test this hypothesis a branded version of Argatroban (Mitsubishi – Tanade, Tokyo, Japan) and three generic versions of Argatroban namely Slovastan, Gartban, and Slovastan were compared for their effects on Arachidonic Acid mediated aggregation of platelets in normal healthy male and female volunteers (and n = 4). Other agonists such as Epinephrine, Collagen, and ADP were also used. The effect of Arachidonic Acid on serotonin release was also measured using an Elisa technique for the measurement of serotonin. All of the generic and branded versions of Argatroban produce varying levels of the inhibition of the Arachidonic Acid mediated aggregation of platelets, ranging from the 24 to 36 percent in comparison to the control at a concentration of 1 mg/ mL (p value < 0.05). Interestingly all of the Argatrobans produced a relatively weaker inhibition of the Arachidonic Acid mediated aggregation of platelets 24–26 percent inhibition versus 36 percent at the final concentration of 1 mg/mL. No differences were noted in the aggregation profile of ADP, Collagen, and Epinephrine between the control and the Argatrboban at a final concentration of 1 mg /mL. No differences were noted between the generic and branded Argatroban on all of the other agonists induced aggregation. In the serotonin release assays, all of the generic and branded Argatroban produced a concentration dependent inhibition of serotonin release which was stronger with the branded version of Argatroban. These results indicate that besides the inhibition of thrombin Argatroban is capable of inhibiting platelet activation via other mechanisms. Moreover, the generic versions of Argatroban exhibit a weaker inhibition of Arachidonic Acid mediated platelet aggregation and release. These studies suggest that beside thrombin mediated aggregation Argatroban and its generic versions can modulate platelet activation and release reactions. Furthermore a difference is observed between the generic and branded product which may impact the safety inefficacy profile in these agents. Argatroban is used commonly in the management of patients with heparin induced thrombocytopenia where multiple mechanisms of platelet activation are contributory to the pathogenesis of this syndrome. Modulation of thrombanxane formation and platelet release mechanism by Argatroban may represent an additional mechanism of the clinical effects of this parentral antithrombin agent. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Platelet activation by a synthetic hydrophobic polymer, polymethylmethacrylate

Blood ◽  
1991 ◽  
Vol 78 (7) ◽  
pp. 1713-1721 ◽  
Author(s):  
JA Ware ◽  
J Kang ◽  
MT DeCenzo ◽  
M Smith ◽  
SC Watkins ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Gamma Chain ◽  
Fibrinogen Receptor ◽  
Hydrophobic Polymer ◽  
Fibrinogen Binding ◽  
Initial Stage ◽  
Artificial Surfaces ◽  
Induced Aggregation

Abstract Platelets adhere to artificial surfaces in the initial stage of thrombus formation, but the subsequent steps in signal transduction that lead to platelet activation by artificial surfaces are not understood. When 0.325-micron diameter beads composed of a hydrophobic polymer, polymethylmethacrylate (PMMA), were added to gel-filtered aequorin-loaded platelets suspended in media containing Ca2+, the platelets aggregated; addition of fibrinogen was not required. Platelet aggregation was preceded by an increase in cytoplasmic Ca2+ and was accompanied by phosphorylation of the 47-Kd substrate of protein kinase C (PKC), 5-hydroxytryptamine (5-HT) release, and accumulation of phosphatidic acid. All these effects were partially inhibited by apyrase and aspirin. Monoclonal antibodies (MoAbs) 7E3 and M148 and the synthetic peptides RGDS and fibrinogen gamma chain fragment 400–411, all of which bind to the platelet fibrinogen receptor glycoprotein IIb- IIIa (GPIIb-IIIa) and inhibit fibrinogen binding, prevented PMMA- induced aggregation but did not inhibit the Ca2+ increase. Chymotrypsin- treated platelets aggregated after addition of fibrinogen, but not PMMA. We conclude that platelets interact initially with PMMA at membrane sites other than those required for fibrinogen binding, leading to activation of membrane phospholipases and PKC, an increase in cytoplasmic Ca2+, release of 5-HT, ADP, and fibrinogen from storage granules, and to platelet aggregation.

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