SR121566A

Inhibition of microsurgical thrombosis by the platelet glycoprotein IIb/IIIa antagonist SR121566A

Despite major improvements in tools and significant refinements of techniques, microsurgical anastomosis still carries a significant risk of failure due to microvascular thrombosis. The key to improving the success of microvascular surgery may lie in the pharmacologic control of thrombus formation. Central to pathologic arterial thrombosis are platelets. Glycoprotein IIb/IIIa is a highly abundant platelet surface receptor that plays a major role in platelet aggregation by binding platelets to each other through the coagulation factor fibrinogen. To explore the ability of antithrombotic agents to prevent microvascular thrombosis, a rabbit ear artery model was used in which a standardized arterial injury results in predictable thrombus formation. This model was used to examine whether SR121566A, a specific and potent glycoprotein IIb/IIIa inhibitor, can successfully prevent microsurgical thrombosis. Using a coded, double-blind experimental design, 20 rabbits (40 arteries) were assigned to four treatment groups: (1) saline injection (n = 10), (2) acetylsalicylic acid 10 mg/kg (n = 10), (3) heparin 0.5 mg/kg bolus with subsequent intermittent boluses of 0.25 mg/kg every 30 minutes (n = 10), and (4) SR121566A 2 mg/kg bolus (n = 10). After vessel damage and clamp release, arteries were assessed for patency at 5, 30, and 120 minutes by the Acland refill test. Coagulation assays, in vivo bleeding times, and ex vivo platelet aggregation studies were also conducted. Scanning electron microscopy was used to examine mural thrombus composition.A significant, fourfold increase in vessel patency following administration of SR121566A over saline control (80 percent versus 20 percent patency, respectively, at 35 minutes after reperfusion, p < 0.01) was noted. This was correlated with marked inhibition of ex vivo platelet aggregation. This antiplatelet treatment did not prolong coagulation assays (mean international normalized ratio: saline, 0.66 +/- 0.04; SR121566A, 0.64 +/- 0.03; mean thromboplastin time: saline, 19.63 +/- 0.67; SR121566A, 17.87 +/- 3.27) and bleeding times (mean bleeding time: saline, 42 +/- 4; SR121566A, 48 +/- 6). Scanning electron microscopy demonstrated extensive platelet and fibrin deposition in control vessel thrombi. In contrast, thrombi from SR121566A-treated vessels demonstrated predominance of fibrin with few platelets when examined under scanning electron microscopy.Administration of SR121566A was associated with a significant increase in vessel patency, without deleterious effects on coagulation assays or bleeding times. The increase in vessel patency was correlated with inhibition of platelet aggregation and decreased platelet deposition, as demonstrated by scanning electron microscopy. Glycoprotein IIb/IIIa antagonists represent a new class of anti-platelet agents that may be suited for inhibiting microsurgical thrombosis. This study supports further investigation into the use of these agents in microsurgery.

Differential in vitro effects of the platelet glycoprotein IIb/IIIa inhibitors abixicimab or SR121566A on platelet aggregation, fibrinogen binding and platelet secretory parameters

The aim of this study was to compare fibrinogen binding, inhibition of platelet aggregation and secretory potential of the MAb abciximab (0.5-5 microg/mL) and the peptidomimetic compound SR121566A (15-250 ng/mL) in vitro in whole blood. Fibrinogen binding was followed by flow cytometry; platelet function was evaluated by light transmittance and by impedance aggregometry. Secretory functions of platelets were evaluated using ATP as marker for early secretion by dense granulae and P-selectin (CD62) for alpha-granular secretion as well as CD63 for lysosomal degranulation. Results showed that fibrinogen binding induced by 5 microM TRAP was maximally inhibited greater than 80% at 3 microg/mL abciximab or at 250 ng/mL SR121566A. At these concentrations of antagonists, platelet aggregation induced by 5 microM ADP or 2 microg/mL collagen was inhibited completely. Expression of CD62 was reduced 34% with abciximab or 15% with SR121566A; CD63 expression was reduced 22% with both agents. With both agents, the EC50 for inhibition of CD62 and CD63 expressions was in similar magnitudes than the EC50 for fibrinogen binding inhibition. With 3 microg/mL abciximab, ATP secretion was maximally reduced to 50% of the control, whereas SR121566A at 250 ng/mL had no inhibitory effect on this parameter. A slight increase in ATP secretion was seen with 0.5 microg/mL abciximab and with SR121566A in concentrations of less than 45 ng/mL. The data suggest a discoupling between the anti-aggregatory and the antisecretory effects of IIb/IIIa antagonists. Because it is not established to what extend CD62 or CD63 expression can be reduced by any means, the reduction by 20-30% obtained by 3 microg/mL abciximab or 250 ng/mL SR121566A might already be the maximum possible inhibition by these agents.

Ex vivo--in vitro interaction between aspirin, clopidogrel, and the glycoprotein IIb/IIIa inhibitors abciximab and SR121566A

Objectives: To assess the interaction between aspirin and clopidogrel in healthy male volunteers and the interaction of the glycoprotein IIb/IIIa (GPIIb/IIIa) inhibitors abciximab and SR121566A with blood from those pretreated subjects (ex vivo-in vitro).
Methods: Aspirin (300 mg/day), clopidogrel (75 mg/day), or the combination of both drugs were administered orally for 8 days. Group 1 (n = 5) started with aspirin and group 2 (n = 5) with clopidogrel. From day 4 to day 8, subjects of both groups received the combined treatment. Blood from these subjects was spiked with abciximab (0.5 and 1.5 microg x mL(-1)) and SR121566A (31 and 62 ng x mL(-1)).
Results: In vivo, average bleeding times were 6.8 minutes at baseline, 20.3 minutes for clopidogrel alone (P < .01), 10.9 minutes for aspirin alone (difference not significant), and 24.0 minutes (P < .01) for the combined treatment. Fibrinogen binding to the platelet GPIIb/IIIa receptor was reduced for aspirin to 69% (difference not significant), to 63% for clopidogrel (difference not significant), and to 63% for the clopidogrel plus aspirin combination (P < .01). CD62 expression as a marker of platelet granular secretion was reduced to 66% by clopidogrel (P < .01) and to 41% by the combination of clopidogrel and aspirin; aspirin alone had no effect. In vitro, with pretreatment with aspirin and clopidogrel, inhibitory effects of the GPIIb/IIIa inhibitors on fibrinogen binding were additive to changes observed with aspirin or clopidogrel alone. No effect on CD62 expression was observed with either GPIIb/IIIa inhibitor. Aspirin and clopidogrel reinforced effects of the GPIIb/IIIa inhibitors on adenosine diphosphate (5 micromol/L)-induced aggregation in an additive manner, a supra-additive effect was observed with collagen (2 microg x mL(-1))-induced aggregation.
Conclusion: The augmentation of the antiaggregatory effects of GPIIb/IIIa inhibitors by aspirin and clopidogrel and the lack of antisecretory effects of GPIIb/IIIa inhibitors may favor their combination with clopidogrel.

Effect of SR121566A, a potent GP IIb-IIIa antagonist on platelet-mediated thrombin generation in vitro and in vivo

The effect of SR121566A, a new non-peptide GP IIb-IIIa antagonist was studied in vitro with regard to thrombin generation in platelet rich plasma and in vivo on stasis-induced venous thrombosis in the rabbit. SR121566A inhibited ADP-, arachidonic acid- and collagen-induced human platelet aggregation with IC50 values of 46 +/- 7.5, 56 +/- 6 and 42 +/- 3 nM, respectively. In the same experimental conditions, SR121566A strongly inhibited thrombin generation triggered by low concentrations of tissue factor. SR121566A reduced in a dose-dependent manner both the area under the curve and the thrombin peak concentration but did not affect the lag phase (defined as the time until 10 nM thrombin was generated). Aspirin (100 microg/ml) did not affect thrombin generation. One hour after intravenous administration to rabbits, SR121566A exhibited a potent ex vivo inhibitory effect against ADP-, arachidonic acid- and collagen-induced platelet aggregation. The ID50 were 0.6 +/- 0.25, 0.7 +/- 0.08 and 0.13 +/- 0.08 mg/kg, respectively. The ability of aspirin and SR121566A to affect venous stasis was determined in a stasis-induced venous thrombosis model in rabbits under high and low thrombogenic challenges. While aspirin was ineffective in both conditions, SR121566A significantly inhibited thrombus formation under low thrombogenic challenge demonstrating for the first time that a potent non-peptide platelet GP IIb-IIIa antagonist inhibits thrombin generation in vivo and exhibits a strong antithrombotic effect with regard to stasis-induced venous thrombosis. These results therefore confirm the existence of a close relationship between platelet activation and thrombin generation leading to blood coagulation but also emphasise the key role of platelets in the development of venous thrombosis, most likely through activation of the GP IIb-IIIa complex.

Effect of SR121566A, a potent GP IIb-IIIa antagonist, on the HIT serum/heparin-induced platelet mediated activation of human endothelial cells

Heparin-induced thrombocytopenia (HIT) is a common adverse effect of heparin therapy that carries a risk of serious thrombotic events. This condition is caused by platelet aggregation, which is mediated by anti-heparin/platelet factor 4 antibodies. Sera from patients with HIT in the presence of platelets, induced the expression of E-selectin, VCAM, ICAM-1 and tissue factor and the release of IL1beta, IL6, TNFalpha and PAI-1 by human umbilical vein endothelial cells (HUVECs) in vitro and initiated platelet adhesion to activated HUVECs. These effects which occurred in a time-dependent manner were significant in the first 1-2 h of incubation and reached a maximum after 6 to 9 h. The GP IIb-1IIa receptor antagonist SR121566A which has been shown to block platelet aggregation induced by a wide variety of agonists including HIT serum/heparin, reduced in a dose-dependent manner the HIT serum/heparin-induced, platelet mediated expression and release of the above mentioned proteins. The IC50 for inhibition of HIT serum/ heparin-induced platelet dependent HUVEC activation by SR121566A was approximately 10-20 nM. ADP, but not serotonin release, also appeared to be involved as apyrase and ATPgammaS blocked platelet-dependent, HIT serum/heparin-induced cell surface protein expression and cytokine release by HUVECs. Increased platelet adherence to HIT serum/heparin-activated HUVECs was inhibited by SR121566A and, to a lesser extent, by apyrase and ATPgammaS, showing that platelet activation and release was at the origin of the HIT serum/heparin-induced expression of these proteins by HUVECs. Thus, sera from patients with HIT induced the expression of adhesive and coagulation proteins and the release of cytokines by HUVECs through the activation of platelets which occurred in a GP IIb-IIIa-dependent manner, a process that could be selectively blocked by SR121566A.