ALBERT

Description: Abstract 2998 Poster Board II-976 Introduction: The platelet surface receptor glycoprotein (GP) IIbIIIa (integrin αaIIbβ3) mediates platelet aggregation and plays a key role in hemostasis and thrombosis. Numerous GPIIbIIIa antagonists have been designed and tested as inhibitors of platelet aggregation. Two of these antagonists, eptifibatide (Integrilin) and tirofiban (Aggrastat) have been approved by the U.S. Food and Drug Administration (FDA) and widely used for preventing and treating thrombotic complications in patients undergoing percutaneous coronary intervention and in patients with acute coronary syndromes. It has been reported, however, that some GPIIbIIIa antagonists, such as orbofiban and xemilofiban, promote apoptosis in cardiomyocytes by activation of the apoptosis executioner caspase-3, raising the possibility that platelets also may be susceptible to pro-apoptotic effects of eptifibatide and tirofiban. Over the past decade it has been well-documented that apoptosis occurs not only in nucleated cells but also in anucleated platelets stimulated with thrombin, calcium ionophores, very high shear stresses and platelet storage (Leytin et al, J Thromb Haemost 4: 2656, 2006; Mason et al, Cell 128: 1173, 2007). It has been further reported that platelet activation and apoptosis may be induced by different mechanisms and/or require different levels of triggering stumuli (Leytin et al, Br J Haematol 136: 762, 2007; Br J Haematol 142: 494, 2008). Recently, we have shown that injection of anti-GPIIb antibody induced caspase-3 activation in mouse platelets in vivo (Leytin et al, Br J Haematol 133: 78, 2006), suggesting that direct GPIIbIIIa-mediated pro-apoptotic signaling is able to trigger caspase-3 activation within platelets. Study Design and Methods: The current study aimed to examine, for the first time, the effect of eptifibatide and tirofiban on caspase-3 activation in human platelets. We studied the effects of eptifibatide and tirofiban on caspase-3 activation in resting platelets, which express GPIIbIIIa receptors in their non-active (“closed”) conformation, and in platelets stimulated with thrombin or calcium ionophore A23187, which induce transition of GPIIbIIIa receptors into active (“open”) conformation. Resting platelets were treated with control buffer, 0.48 μM eptifibatide or 0.48 μM tirofiban, and stimulated platelets were treated with 1 U/mL thrombin or 10 μM A23187, or preincubated with eptifibatide or tirofiban before treatment with thrombin or A23187. Caspase-3 activation was determined by flow cytometry using the cell-penetrating FAM-DEVD-FMK probe, which covalently binds to active caspase-3. Results and Discussion: We found that treatment of resting platelets with eptifibatide and tirofiban did not affect caspase-3 activation (P〉0.05, n=7). In contrast, a 2.3-2.7-fold increase of caspase-3 activation was observed in platelets after thrombin or A23187 stimulation (P

Description: Platelet storage lesion is a serious problem limiting clinical use of platelet concentrates (PC) after extended and long-term storage. Platelet activation is a well-known manifestation of platelet storage lesion. However, over the last decade, platelet apoptosis has been also recognized in stored PCs and in platelets following exposure to thrombin, calcium ionophores, anti-platelet antibodies and very high shear stresses. The aim of this study was to elucidate the contribution of platelet activation and apoptosis to the platelet storage lesion during conventional (Days 2–5), extended (Days 6–8) and long-term (Days 11–16) PC storage. We prepared seven prestorage-leukoreduced PC by the platelet-rich plasma (PRP) method, stored PC for 2–16 days at 22°C, and used flow cytometry for determining platelet activation as P-selectin (CD62) exposure and platelet apoptosis as depolarization of mitochondrial inner membrane potential (ΔΨm), activation of executioner caspase-3, exposure of phosphatidylserine (PS) and release of apoptotic platelet fragments microparticles (MP). Platelet activation and apoptotic responses were also determined in fresh (Day 0) PRP using thrombin titration. We found a significant increase of platelet activation under conventional PC storage for 2–5 days (38.6 ± 3.1% CD62 positive cells, P 〈 0.0001). With extended (Days 6–8) storage, platelet activation was increased to 66.5 ± 5.3% and reached the maximal level of 92.0 ± 1.1% after 11–12 storage days (P 〈 0.0001). In contrast, ΔΨm depolarization and caspase-3 activation did not increase in comparison with Day 0 platelets, even after PC storage for 12 days (P 〉 0.05) and storage for 13–16 days was required for significant triggering these apoptotic events (P 〈 0.05-0.0001). Similarly, although we observed a slight increase of PS exposure (5–10%) and MP release (9–11%) during PC storage for 2–12 days, incubation for 13–16 days was required for a stronger (30–60%) stimulation of these apoptotic manifestations (P 〈 0.001-0.0001). Paired comparison between the effects of PC storage on CD62 exposure and apoptotic events clearly demonstrated for all storage times a significantly higher level of platelet activation than levels of ΔΨm depolarization, caspase-3 activation, PS exposure and MP release (P 〈 0.01-0.0001). Furthermore, we found that when fresh (Day 0) PRP was treated with different thrombin doses, ranging from 0.05 to 10 U/ml, a much higher maximal level of platelet activation (~90%) was reached, in comparison to the level of apoptosis (30–40%), and 100- to 200-fold lower dose of thrombin were required for maximal induction of activation (0.05–0.1 U/ml) than for stimulation of apoptosis (10 U/ml). Taken together, these data indicate that (i) during PC storage, platelet activation is triggered much earlier than platelet apoptosis, (ii) platelet activation rather than apoptosis contributes most to the platelet storage lesion during conventional (Days 2–5) and extended (Days 6–8) PC storage whereas during long-term (Days 13–16) storage both responses are involved in platelet deterioration, and (iii) platelet activation and apoptosis are different phenomena; they may be induced by different mechanisms and/or require quite different levels of triggering stimuli.