The info obtained in the current study suggest that GPIIbIIIa may be also involved in positive and negative modulation of platelet apoptosis triggered by thrombin- and calcium ionophore-mediated pathways, when apoptosis is potentiated by sending pro-apoptotic signals through active GPIIbIIIa, and inhibited by binding of the fibrinogen-mimetic therapeutics, Integrilin and Aggrastat. In conclusion, we have shown a novel platelet-directed activity of two clinically used GPIIbIIIa antagonist drugs, eptifibatide (Integrilin) and tirofiban (Aggrastat), with ability to inhibit apoptosis executioner caspase-3 induced by potent platelet agonists, thrombin and A23187, and the absence of adverse pro-apoptotic effects on resting platelets. also in anucleated platelets stimulated with thrombin, calcium ionophores, very high shear stresses and platelet storage (see references in68). It has been further reported that platelet LY450108 activation and apoptosis may be induced by different mechanisms and/or require different levels of triggering stumuli.9,10Recently, we have shown that injection of anti-GPIIb antibody induced caspase-3 activation in mouse platelets,11suggesting that direct GPIIbIIIa-mediated pro-apoptotic signaling is able to trigger caspase-3 activation within platelets. The current study aimed to examine, for the first time, the effect of Integrilin and Aggrastat on caspase-3 activation in human platelets. We studied the effects of Integrilin and Aggrastat on caspase-3 activation in resting platelets which express GPIIbIIIa receptors in their non-active (closed) conformation, and in platelets stimulated with thrombin and calcium ionophore A23187, which induce transition of GPIIbIIIa receptors into active (open) conformation. Table 1Ashows that treatment of resting platelets with Integrilin and Aggrastat did not affect caspase-3 activation (p>0.05). In contrast, a 2.32.7-fold increase in caspase-3 activation was observed in platelets after thrombin and A23187 stimulation (Table 1B and C,p<0.01). However, when platelets were preincubated with Integrilin and Aggrastat before agonist treatment, these drugs significantly inhibited agonist-induced caspase-3 activation (Figure 1andTable 1B and C,p<0.05). == Table 1. == Effect of GPIIbIIIa antagonists, eptifibatide (Integrilin) and tirofiban (Aggrastat), on caspase-3 activation in resting human platelets and platelets stimulated with thrombin and calcium ionophore A23187. == Figure 1. DICER1 == Flow cytometric histograms illustrating inhibitory effect of eptifibatide (Integrilin) on thrombin-induced caspase-3 activation in human platelets. Platelets were treated with control buffer and 1 U/mL thrombin or preincubated with 0.48 M Integrilin before treatment with thrombin and caspase-3 activation was determined by the cell-penetrating carboxyfluorescein-labeled probe FAM-DEVD-FMK, which covalently binds to active caspase-3 (Online Supplementary Appendix). Note that treatment of platelets with thrombin induces caspase-3 activation in comparison with platelets treated with control buffer, whereas preincubation with Integrilin markedly attenuates thrombin-induced cas-pase-3 activation. The fact that Integrilin and Aggrastat do not promote caspase-3 activation in unstimulated platelets (Table 1A) suggests that these GPIIbIIIa antagonists do not induce transmission of pro-apoptotic transmembrane signals inside platelets through inactive GPIIbIIIa integrin. They also do not activate caspase-3 directly by interacting with Arg-Gly-Asp (RGD)-binding motif of pro-caspase-3, as was reported for cell-penetrating GPIIbIIIa antagonists, orbofiban and xemilofiban, in rat cardiomyocytes,5indicating that the chemically different Integrilin and Aggrastat do not penetrate human platelets. The inhibitory effect of Integrilin and Aggrastat on thrombin- and A23187-induced caspase-3 activation (Table 1 LY450108 B and C) suggests a role for GPIIbIIIa integrin in caspase-3 activation induced by these platelet agonists. In the absence of Integrilin and Aggrastat, caspase-3 activation induced by thrombin and A23187 is primarily triggered by pro-apoptotic signal transduction through pro-teinase-activated receptors (PAR)7,12and by calcium mobilization/overloading,8respectively; GPIIbIIIa integrins are then secondarily activated by inside-out signaling, bind plasma- and/or platelet-derived fibrinogen and/or von Willebrand factor (VWF)1,2and transmit pro-apoptotic outside-in signaling further promoting thrombin- or A23187-induced caspase-3 activation. In the presence of Integrilin or Aggrastat, on the other hand, the GPIIbIIIa antagonist inhibits caspase-3 activation induced by thrombin or A23187, attenuating transmission of pro-apoptotic outside-in signaling through active GPIIbIIIa by inhibiting fibrinogen/VWF binding and/or preventing conversion of GPIIbIIIa to a fully active conformation. We have previously shown that injection of anti-GPIIb antibody is able to trigger caspase-3 activation in mouse plateletsin vivo,11indicating that GPIIbIIIa integrin may be directly involved in pro-apoptotic signal transduction inside platelets. The data obtained in the current study suggest that GPIIbIIIa may be also involved in positive and negative modulation of platelet apoptosis triggered by thrombin- and calcium ionophore-mediated pathways, when apoptosis is potentiated by sending pro-apoptotic signals through active GPIIbIIIa, and inhibited by binding of the fibrinogen-mimetic therapeutics, Integrilin and Aggrastat. In conclusion, we have shown a novel platelet-directed LY450108 activity of two clinically used GPIIbIIIa antagonist drugs, eptifibatide (Integrilin) and tirofiban (Aggrastat), with ability to inhibit apoptosis executioner caspase-3 induced by potent platelet.
