Studies on the Binding of 3H-SR121566, an Inhibitor of Gp IIb-IIIa Activation

Abstract

SummarySR121566 is a new synthetic agent which inhibits the binding of fibrinogen to activated platelets, and platelet aggregation. 3H-SR121566 bound with nanomolar affinity (KD ranging from 45 to 72 nM) to Gp IIb-IIIa expressing cells only. On activated human platelets, this ligand allowed the detection of a maximal number of 100-140,000 binding sites. The binding of SR121566 to platelets, was displaced by several agents including RGD-containing peptides and synthetic RGD mimetics, but not by ReoPro®, a humanised monoclonal antibody which inhibits the binding of fibrinogen to the Gp IIb-IIIa complex. Neither the fibrinogen dodecapeptide nor fibrinogen itself were able to compete with SR121566 whether platelets were activated or not.Flow cytometry studies indicated that SR121566 which did not activate Gp IIb-IIIa by itself, dose-dependently prevented the detection of activation-induced binding sites on TRAP-stimulated platelets in the presence or absence of exogenous fibrinogen, indicating a direct effect on the activation state of the Gp IIb-IIIa complex. Moreover, SR121566 was able to reverse the activation of Gp IIb-IIIa and to displace the binding of fibrinogen when added up to 5 min after TRAP stimulation of platelets. When added at later times (15 to 30 min), SR121566 failed to displace fibrinogen binding, even if SR121566 binding sites were still accessible and the Gp IIb-IIIa complex not activated.In conclusion, our study is in accordance with the finding that fibrinogen is recognised by the activated Gp IIb-IIIa complex through the dodecapeptide sequence present on its gamma chain, and that this interaction is inhibited by SR121566 by preventing and reversing the activated conformation of Gp IIb-IIIa and not by direct competition with fibrinogen.

Integrin-dependent interaction of lipid rafts with the actin cytoskeleton in activated human platelets

“…A similar result was obtained with the compound SR-121566 (Fig. 3B), another competitive antagonist for fibrinogen binding to αIIbβ3 that prevents platelet aggregation (Savi et al, 2001).…”

“…5B), we observed a rapid dissociation of lipid rafts from the actin cytoskeleton. The SR-121566 compound is known to displace the fibrinogen bound to αIIbβ3 and to induce the reversion of aggregation (Savi et al, 2001). When SR-121566 was added to the platelet suspension 1.5 minutes after TRAP stimulation, the platelet disaggregation (Fig.…”

“…The extent of cytoskeleton-raft interaction paralleled the platelet aggregation intensity and was stable during an irreversible aggregation. Under conditions where platelet aggregation was reversed by addition of either SR-121566, a potent fibrinogen binding antagonist (Savi et al, 2001), or PI 3-kinase inhibitors (Trumel et al, 1999) after TRAP stimulation, the raft-cytoskeleton association became reversible. Thus, both the initiation and the stability of this interaction require sustained αIIbβ3 engagement and a stable aggregation.…”

Characterisation of Rac activation in thrombin‐ and collagen‐stimulated human blood platelets

“…2, right panel). Since RGD peptides may be partial integrin agonists [16], we also used another α IIb β 3 inhibitor, the synthetic agent SR121566 [17]. Although this compound strongly inhibited platelet aggregation, it did not affect Rac activation upon thrombin or collagen stimulation (Fig.…”

Comparative effects of two synthetic oligosaccharides on platelet activation induced by plasma from HIT patients

“…PAC1 is a monoclonal antibody that recognizes the active conformation of human GpIIb–IIIa [15] present at the platelet surface. It has been used for evaluating the activity of several drugs, including anti‐GpIIb–IIIa [16] and ADP receptor (anti‐P2Y12) antagonists such as thienopyridines [17]. The activation of GpIIb–IIIa is clearly correlated with the ability of the complex to bind soluble fibrinogen and result in platelet aggregation.…”