scholarly journals Effects of OKM5, a monoclonal antibody to glycoprotein IV, on platelet aggregation and thrombospondin surface expression [see comments]

Blood ◽  
1990 ◽  
Vol 76 (12) ◽  
pp. 2501-2509 ◽  
Author(s):  
ML Aiken ◽  
MH Ginsberg ◽  
V Byers-Ward ◽  
EF Plow
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Membrane Protein ◽  
Adenosine Diphosphate ◽  
Surface Expression ◽  
Inhibitory Effect ◽  
Target Antigen ◽  
Functional Responses ◽  
Fibrinogen Binding ◽  
Intact Antibody

The monoclonal antibody, OKM5, recognizes an 88-Kd monocyte membrane protein and also binds to the platelet membrane protein, GPIV (GPIIIb, CD36). In this study, we have found that the OKM5 target epitope is present at approximately 12,000 copies per platelet and that interaction with the antibody has both stimulatory and inhibitory effects on platelet function. In the absence of other stimuli, OKM5 induced platelet aggregation, secretion, and expression of fibrinogen receptors. These stimulatory responses required intact antibody as F(ab')2 fragments were not active but blocked the stimulatory activity of the intact antibody. In contrast, exposure of platelets to OKM5 followed by another strong stimulus such as thrombin resulted in a marked suppression of fibrinogen, fibronectin, and von Willebrand factor binding to the cells. This effect was not noted when a weak stimulus, adenosine diphosphate, was the second agonist. At OKM5 concentrations that interfered with fibrinogen binding to thrombin- stimulated platelets by 80% to 90%, platelet binding of exogenous thrombospondin, or surface expression of endogenous thrombospondin was not affected. The inhibitory effect of OKM5 on fibrinogen binding to thrombin-stimulated platelets was related to the formation of massive platelet aggregates in the samples. These results show that interaction of OKM5 with its target antigen on platelets can elicit diverse functional responses from the cells.

scholarly journals Effects of OKM5, a monoclonal antibody to glycoprotein IV, on platelet aggregation and thrombospondin surface expression [see comments]

Blood ◽  
1990 ◽  
Vol 76 (12) ◽  
pp. 2501-2509 ◽  
Author(s):  
ML Aiken ◽  
MH Ginsberg ◽  
V Byers-Ward ◽  
EF Plow
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Membrane Protein ◽  
Adenosine Diphosphate ◽  
Surface Expression ◽  
Inhibitory Effect ◽  
Target Antigen ◽  
Functional Responses ◽  
Fibrinogen Binding ◽  
Intact Antibody

Abstract The monoclonal antibody, OKM5, recognizes an 88-Kd monocyte membrane protein and also binds to the platelet membrane protein, GPIV (GPIIIb, CD36). In this study, we have found that the OKM5 target epitope is present at approximately 12,000 copies per platelet and that interaction with the antibody has both stimulatory and inhibitory effects on platelet function. In the absence of other stimuli, OKM5 induced platelet aggregation, secretion, and expression of fibrinogen receptors. These stimulatory responses required intact antibody as F(ab')2 fragments were not active but blocked the stimulatory activity of the intact antibody. In contrast, exposure of platelets to OKM5 followed by another strong stimulus such as thrombin resulted in a marked suppression of fibrinogen, fibronectin, and von Willebrand factor binding to the cells. This effect was not noted when a weak stimulus, adenosine diphosphate, was the second agonist. At OKM5 concentrations that interfered with fibrinogen binding to thrombin- stimulated platelets by 80% to 90%, platelet binding of exogenous thrombospondin, or surface expression of endogenous thrombospondin was not affected. The inhibitory effect of OKM5 on fibrinogen binding to thrombin-stimulated platelets was related to the formation of massive platelet aggregates in the samples. These results show that interaction of OKM5 with its target antigen on platelets can elicit diverse functional responses from the cells.

Ticlopidine Selectively Inhibits Human Platelet Responses to Adenosine Diphosphate

1991 ◽  
Vol 66 (06) ◽  
pp. 694-699 ◽  
Author(s):  
Marco Cattaneo ◽  
Benjaporn Akkawat ◽  
Anna Lecchi ◽  
Claudio Cimminiello ◽  
Anna M Capitanio ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Inhibitory Effect ◽  
Clot Retraction ◽  
Fibrinogen Binding ◽  
Low Concentrations ◽  
Binding Inhibition ◽  
High Concentrations ◽  
Formalin Fixed

SummaryPlatelet aggregation and fibrinogen binding were studied in 15 individuals before and 7 days after the oral administration of ticlopidine (250 mg b.i.d.). Ticlopidine significantly inhibited platelet aggregation induced by adenosine diphosphate (ADP), the endoperoxide analogue U46619, collagen or low concentrations of thrombin, but did not inhibit platelet aggregation induced by epinephrine or high concentrations of thrombin. Ticlopidine inhibited 125I-fibrinogen binding induced by ADP, U46619 or thrombin (1 U/ml). The ADP scavengers apyrase or CP/CPK, added in vitro to platelet suspensions obtained before ticlopidine, caused the same pattern of aggregation and 125I-fibrihogen binding inhibition as did ticlopidine. Ticlopidine did not inhibit further platelet aggregation and 125I-fibrinogen binding induced in the presence of ADP scavengers. After ticlopidine administration, thrombin or U46619, but not ADP, increased the binding rate of the anti-GPIIb/IIIa monoclonal antibody 7E3 to platelets. Ticlopidine inhibited clot retraction induced by reptilase plus ADP, but not that induced by thrombin or by reptilase plus epinephrine, and prevented the inhibitory effect of ADP, but not that of epinephrine, on the PGE1-induced increase in platelet cyclic AMP. The number of high- and low-affinity binding sites for 3H-ADP on formalin-fixed platelets and their K d were not modified by ticlopidine. These findings indicate that ticlopidine selectively inhibits platelet responses to ADP.

Inhibition of Integrin-Mediated Platelet Aggregation, Fibrinogen-Binding, and Interactions with Extracellular Matrix by Nonpeptidic Mimetics of Arg-Gly-Asp

1993 ◽  
Vol 70 (06) ◽  
pp. 1030-1036 ◽  
Author(s):  
David Varon ◽  
Ofer Lider ◽  
Rima Dardik ◽  
Boris Shenkman ◽  
Ronen Alon ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Platelet Aggregation ◽  
Specific Interaction ◽  
Adenosine Diphosphate ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Rgd Sequence ◽  
Fibrinogen Binding ◽  
Inhibitory Potential ◽  
Dose Dependent

SummaryThe interaction of the activated platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa) with fibrinogen and von-Wille-brand factor (vWF) is essential for platelet aggregation. The minimal structure required for this integrin’s binding to fibrinogen is the Arg-Gly-Asp (RGD) sequence. Inasmuch as normal level of GPIIb-IIIa-RGD interactions are required for maintaining hemostasis, elevated platelet aggregation can cause adverse pathological effects. We have previously reported that nonpeptidic mimetics of RGD, consisting of carboxylate and guanidinium groups of Asp and Arg divided by a linear 11-atom spacer, acquired a significant affinity for the GPIIb-IIIa integrin and inhibited platelet aggregation. The structural requirements for the interactions of the RGD sequence with GPIIb-IIIa and the inhibitory potential of a newly designed series of mimetics on platelet aggregation and interactions with extracellular matrix (ECM) were assayed herein. Adenosine-diphosphate (ADP)-induced platelet aggregation was inhibited in a dose-dependent manner by various RGD mimetics, with a maximal inhibition of 80-100% with an IC50 of 3 μM for the most potent inhibitor, NS-11 which a six-membered ring was introduced into the spacer chain, which exceeded the IC50 attained with the original RGDS peptide. The inhibitory effect of the RGD mimetics was attributed to their specific interaction with the GPIIb-IIIa integrin, since these mimetics inhibited the binding of the PAC-1 mAb to GPIIb-IIIA. Furthermore, the binding of 125I-labeled fibrinogen to platelets was inhibited by the RGD surrogates in a dose-dependent and saturable manner. The RGD-mimetics also inhibited up to 70% the adhesion, aggregation, and deposition of platelets onto ECM. Thus, we suggest that the novel nonpeptidic mimetics of RGD described herein, which were shown to be resistant to proteolytic digestion, would be valuable in novel therapeutic approaches to treat in RGD-dependent pathological disorders involving platelet-ECM interactions.

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.

scholarly journals A murine monoclonal antibody that blocks fibrinogen binding to normal platelets also inhibits fibrinogen interactions with chymotrypsin- treated platelets

Blood ◽  
1984 ◽  
Vol 64 (1) ◽  
pp. 59-63 ◽  
Author(s):  
EI Peerschke ◽  
BS Coller
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Molecular Weights ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
Unstimulated Control ◽  
Lines Of Evidence

We recently described a monoclonal antibody, 10E5 , that completely blocks adenosine diphosphate (ADP) induced fibrinogen binding to platelets and aggregation induced by ADP, epinephrine, and thrombin. Multiple lines of evidence indicate that 10E5 binds to platelet membrane glycoproteins IIb and/or IIIa. Because it has been reported that platelets treated with chymotrypsin aggregate when fibrinogen is added, we tested the effect of 10E5 antibody on chymotrypsin-induced fibrinogen binding and platelet aggregation. Aspirin-treated human platelets were washed in modified Tyrode's buffer (pH 7.5), incubated for 5 minutes at 22 degrees C with 300 micrograms/mL chymotrypsin, and washed again. The amount of 10E5 antibody bound to these platelets (37,232 +/- 2,928 molecules/platelet; mean +/- SEM, N=9) was similar to that bound to unstimulated control platelets (36,910 +/- 2,669) and did not differ significantly from the amount of antibody bound to ADP- treated platelets (P less than .01, N = 5). The amount of 10E5 bound to chymotrypsin-treated platelets correlated directly with the amount of fibrinogen bound to separate aliquots of the same platelet samples (r = .876, P less than .001). The 10E5 antibody caused virtually complete inhibition of both the binding of fibrinogen to chymotrypsin-treated platelets and the aggregation induced by exogenous fibrinogen. Immunoprecipitation studies of 125I-labeled chymotrypsin-treated platelets revealed that the 10E5 antibody bound proteins with molecular weights characteristic of glycoproteins IIb and IIIa. These data suggest that the fibrinogen receptor on chymotrypsin-treated platelets is identical to that on ADP-treated platelets and that this receptor is either near to, or on, the glycoprotein IIb/IIIa complex.

scholarly journals A murine monoclonal antibody that blocks fibrinogen binding to normal platelets also inhibits fibrinogen interactions with chymotrypsin- treated platelets

Blood ◽  
1984 ◽  
Vol 64 (1) ◽  
pp. 59-63 ◽  
Author(s):  
EI Peerschke ◽  
BS Coller
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Molecular Weights ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
Unstimulated Control ◽  
Lines Of Evidence

Abstract We recently described a monoclonal antibody, 10E5 , that completely blocks adenosine diphosphate (ADP) induced fibrinogen binding to platelets and aggregation induced by ADP, epinephrine, and thrombin. Multiple lines of evidence indicate that 10E5 binds to platelet membrane glycoproteins IIb and/or IIIa. Because it has been reported that platelets treated with chymotrypsin aggregate when fibrinogen is added, we tested the effect of 10E5 antibody on chymotrypsin-induced fibrinogen binding and platelet aggregation. Aspirin-treated human platelets were washed in modified Tyrode's buffer (pH 7.5), incubated for 5 minutes at 22 degrees C with 300 micrograms/mL chymotrypsin, and washed again. The amount of 10E5 antibody bound to these platelets (37,232 +/- 2,928 molecules/platelet; mean +/- SEM, N=9) was similar to that bound to unstimulated control platelets (36,910 +/- 2,669) and did not differ significantly from the amount of antibody bound to ADP- treated platelets (P less than .01, N = 5). The amount of 10E5 bound to chymotrypsin-treated platelets correlated directly with the amount of fibrinogen bound to separate aliquots of the same platelet samples (r = .876, P less than .001). The 10E5 antibody caused virtually complete inhibition of both the binding of fibrinogen to chymotrypsin-treated platelets and the aggregation induced by exogenous fibrinogen. Immunoprecipitation studies of 125I-labeled chymotrypsin-treated platelets revealed that the 10E5 antibody bound proteins with molecular weights characteristic of glycoproteins IIb and IIIa. These data suggest that the fibrinogen receptor on chymotrypsin-treated platelets is identical to that on ADP-treated platelets and that this receptor is either near to, or on, the glycoprotein IIb/IIIa complex.

The Inhibition of Platelet Aggregation by an Aorta Intima Extract

1974 ◽  
Vol 32 (02/03) ◽  
pp. 417-431 ◽  
Author(s):  
A. du P Heyns ◽  
D. J van den Berg ◽  
G. M Potgieter ◽  
F. P Retief
Keyword(s):  
Platelet Aggregation ◽  
Degradation Products ◽  
Adenosine Diphosphate ◽  
Inhibitory Effect ◽  
Protective Role ◽  
Vascular Trauma ◽  
Wear And Tear ◽  
Sequential Addition ◽  
Aggregation Activity

SummaryThe platelet aggregating activity of extracts of different layers of the arterial wall was compared to that of Achilles tendon. Arterial media and tendon extracts, adjusted to equivalent protein content as an index of concentration, aggregated platelets to the same extent but an arterial intima extract did not aggregate platelets. Platelet aggregation induced by collagen could be inhibited by mixing with intima extract, but only to a maximum of about 80%. Pre-mixing adenosine diphosphate (ADP) with intima extracts diminished the platelet aggregation activity of the ADP. Depending on the relationship between ADP and intima extract concentrations aggregating activity could either be completely inhibited or inhibition abolished. Incubation of ADP with intima extract and subsequent separation of degradation products by paper chromatography, demonstrated a time-dependent breakdown of ADP with AMP, adenosine, inosine and hypoxanthine as metabolic products; ADP removal was complete. Collagen, thrombin and adrenaline aggregate platelets mainly by endogenous ADP of the release reaction. Results of experiments comparing inhibition of aggregation caused by premixing aggregating agent with intima extract, before exposure to platelets, and the sequential addition of first the intima extract and then aggregating agent to platelets, suggest that the inhibitory effect of intima extract results from ADP breakdown. It is suggested that this ADP degradation by intima extract may play a protective role in vivo by limiting the size of platelet aggregates forming at the site of minimal “wear and tear” vascular trauma.

Effects of a Monoclonal Antibody to Glycoprotein IIb/IIIa (P256) and of Enzymically Derived Fragments of P256 on Human Platelets

1991 ◽  
Vol 65 (04) ◽  
pp. 432-437 ◽  
Author(s):  
A W J Stuttle ◽  
M J Powling ◽  
J M Ritter ◽  
R M Hardisty
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Calcium Ions ◽  
Human Platelet ◽  
Human Platelets ◽  
In Vivo Detection ◽  
Fibrinogen Binding ◽  
Specific Antagonist

SummaryThe anti-platelet monoclonal antibody P256 is currently undergoing development for in vivo detection of thrombus. We have examined the actions of P256 and two fragments on human platelet function. P256, and its divalent fragment, caused aggregation at concentrations of 10−9−3 × 10−8 M. A monovalent fragment of P256 did not cause aggregation at concentrations up to 10−7 M. P256–induced platelet aggregation was dependent upon extracellular calcium ions as assessed by quin2 fluorescence. Indomethacin partially inhibited platelet aggregation and completely inhibited intracellular calcium mobilisation. Apyrase caused partial inhibition of aggregation. Aggregation induced by the divalent fragment was dependent upon fibrinogen and was inhibited by prostacyclin. Aggregation induced by the whole antibody was only partially dependent upon fibrinogen, but was also inhibited by prostacyclin. P256 whole antibody was shown, by flow cytometry, to induce fibrinogen binding to indomethacin treated platelets. Monovalent P256 was shown to be a specific antagonist for aggregation induced by the divalent forms. In–111–labelled monovalent fragment bound to gel-filtered platelets in a saturable and displaceable manner. Monovalent P256 represents a safer form for in vivo applications

Partial Inhibition of Platelet Aggregation and Fibrinogen Binding by a Murine Monoclonal Antibody to GPIIIa: Requirement for Antibody Bivalency

1994 ◽  
Vol 72 (06) ◽  
pp. 964-972 ◽  
Author(s):  
Jeffery L Kutok ◽  
Barry S Coller
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Murine Monoclonal Antibody ◽  
Platelet Glycoprotein ◽  
Igg Antibody ◽  
Surface Receptors ◽  
Partial Inhibition ◽  
Fibrinogen Binding ◽  
Igg Binding ◽  
Agonist Concentration

SummaryWe produced a murine monoclonal antibody, 7H2, and localized its epitope to one or more small regions on platelet glycoprotein (GP) Ilia. 7H2-IgG and 7H2-F(ab’)2 completely inhibit platelet aggregation and fibrinogen binding at low agonist concentrations, but only partially inhibit aggregation and fibrinogen binding at high agonist concentrations; 7H2-Fab has no effect on aggregation or fibrinogen binding at any agonist concentration. 7H2-IgG binds to the entire platelet population as judged by flow cytometry. At near saturating concentrations, ∼40,000 7H2-IgG antibody molecules bind per platelet. In contrast, ∼80,000 7H2 Fab molecules bind per platelet, suggesting that 7H2-IgG binding is bivalent. 7H2 was unable to inhibit fibrinogen binding to purified, immobilized GPIIb/IIIa. These data indicate that the bivalent binding of 7H2 to GPIIIa is required for its partial inhibition of fibrinogen binding to platelets, perhaps through dimerization of GPIIb/IIIa surface receptors (or more complex GPIIb/IIIa redistribution triggered by 7H2 binding) resulting in limited accessibility of fibrinogen to its binding site(s).

Inhibition of Platelet Function by Antiarrhythmic Drugs, Verapamil and Disopyramide

1982 ◽  
Vol 47 (02) ◽  
pp. 150-153 ◽  
Author(s):  
P Han ◽  
C Boatwright ◽  
N G Ardlie
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Antiarrhythmic Drugs ◽  
Adenosine Diphosphate ◽  
Inhibitory Effect ◽  
Second Phase ◽  
Ionophore A23187 ◽  
High Concentrations ◽  
Artery Disease

SummaryVarious cardiovascular drugs such as nitrates and propranolol, used in the treatment of coronary artery disease have been shown to have an antiplatelet effect. We have studied the in vitro effects of two antiarrhythmic drugs, verapamil and disopyramide, and have shown their inhibitory effect on platelet function. Verapamil, a calcium channel blocker, inhibited the second phase of platelet aggregation induced by adenosine diphosphate (ADP) and inhibited aggregation induced by collagen. Disopyramide similarly inhibited the second phase of platelet aggregation caused by ADP and aggregation induced by collagen. Either drug in synergism with propranolol inhibited ADP or collagen-induced platelet aggregation. Disopyramide at high concentrations inhibited arachidonic add whereas verapamil was without effect. Verapamil, but not disopyramide, inhibited aggregation induced by the ionophore A23187.

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