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: Apoptosis, or programmed cell death, is the physiologic mechanism that serves for controlled deletion of unwanted cells. Apoptosis was initially attributed exclusively to nucleated cells but over the past decade it has been recognized that apoptosis also occurs in anucleated cytoplasts and platelets. In this study, using flow cytometry we analyzed in human platelets three critical manifestations of mitochondrial, cytoplasmic and plasma membrane apoptosis, mitochondrial inner transmembrane potential (Δψm) depolarization, caspase-3 activation and phosphatidylserine (PS) externalization, respectively. We found that these hallmarks of apoptosis can be induced in human platelet suspension by diverse stimuli, including human α-thrombin (1, 10, 100 nM), calcium ionophore A23187 (3, 5, 10 μM), high shear stresses generated by cone-and-plate viscometer (120, 200, 390 dyn/cm2) and prolonged storage of platelet concentrates in blood banking conditions at 22°C for 6 and 13 days. We also demonstrated that these apoptotic markers can be induced in mouse platelets in vivo in a murine model of immune thrombocytopenia caused by injection of anti-glycoprotein (GP) IIb (rat anti-mouse GPIIb, MWReg30) antibody. Other manifestations of apoptosis were detected in human platelets, including expression of proapoptotic members of Bcl-2 family proteins (Bax and Bak) induced by thrombin, and platelet shrinkage and shedding of microparticles induced by high shear stresses. In addition to apoptosis in fluid-phase platelets, apoptosis was also revealed by confocal fluorescent microscopy in adherent human platelets and thrombi-like platelet aggregates deposited on thrombogenic immobilized human vascular collagen types I and III, as detected by PS exposure and shedding of PS-exposed microparticles. Taken together, these data suggest that platelet apoptosis is a phenomenon that can be triggered by a wide diversity of chemical and physical stimuli using different mechanisms mediated by thrombin-, collagen- and integrin GPIIbIIIa-receptors, mechanoreceptors and Ca2+-overloading. These stimuli trigger platelet apoptosis by impacting on several intracellular apoptotic targets, including shifting the balance between Bcl-2 regulatory proteins in a proapoptotic direction, depolarizing the inner mitochondrial membrane, activating the executioner caspase-3, stimulating aberrant PS exposure on the platelet surface and, eventually, resulting in ‘terminal’ stages of platelet apoptosis, such as platelet shrinkage and shedding of PS-exposed microparticles resembling apoptotic bodies. Platelet apoptosis can be induced both in fluid-phase and adherent platelets and thrombi-like platelet aggregates. These data also indicate that natural PL agonists thrombin and subendothelial vascular collagens and hemodynamic shear forces, can be involved not only in the processes of hemostasis, thrombosis and blood coagulation but also can trigger platelet death via apoptosis. Platelet apoptosis may contribute to the pathophysiology of thrombocytopenia in diseases associated with enhanced thrombin generation, such as sepsis and disseminated intravascular coagulation, as well as in autoimmune and alloimmune thrombocytopenias.

Description: The kinetics of the intracellular iron were studied in an invitro system using reticulocyte-rich rabbit red cells incubated with Fe59-labeled plasma. The incubation was stopped at intervals of a few seconds to 70 minutes, and the cells were fractionated by differential centrifugation and by chemical means. In being selectively taken up by reticulocytes, Fe59 is first associated with particulate fractions and then gradually released to the soluble cytoplasm, when the iron is incorporated into hemoglobin and into a transient nonhemoglobin protein phase. The entire process from the initial binding of iron by cell surfaces receptors until the incorporation of iron into hemoglobin requires only 6 to 8 minutes at 37 C. It is probable that in suitable concentrations, lead allows Fe59 to accumulate in stroma but blocks its entry into the nonhemoglobin protein iron phase and into hemoglobin.

Description: 1. The concentrations of certain amino acids were measured by ion exchange chromatography in mature red cells, in red cells with a high percentage of reticulocytes, and in the plasma in which these red cells had been suspended. The concentration ratio as a measure of net accumulation of the red cells for amino acids was derived from these data. 2. Adult red cells can accumulate glycine and alanine, but not methionine, valine, isoleucine or leucine. 3. Reticulocytes can accumulate glycine best, then alanine, then methionine, isoleucine, and leucine to approximately the same extent. They were unable to maintain a concentration gradient for valine. 4. In two patients with thalassemia, no abnormality was found in the plasma or red cell levels of citrulline, proline, glycine, alanine, valine, isoleucine, tyrosine, or phenylalanine. In one of the thalassemic patients no abnormality of histidine was found. The amino acid accumulation by thalassemic, or fetal red cells was the same as for normal red cells of comparable reticulocyte percentage.

Description: Apoptosis, or programmed cell death, is appreciated as the main physiologic mechanism that regulates cell life-span and serves for controlled deletion of unwanted cells. Since its discovery in 1972, apoptosis was long attributed exclusively to nucleate cells. It took more than 20 years to recognize apoptosis in enucleated cells cytoplasts and anucleate platelets. During the following years, apoptosis has been demonstrated in platelets treated with natural and artificial agonists, in platelet concentrates aged during storage under standard blood banking conditions, and in animal models of suppressed thrombopoiesis and thrombocytopenia. Other studies documented that mechanical forces (shear stresses) stimulate platelet activation and signaling in the absence of exogenous chemical stimuli. We analysed whether shear stresses can trigger platelet apoptosis, a question that has not yet been studied. Using a cone-and-plate viscometer (CAP-2000, Brookfield Engineering Labs, Inc., Middleboro, MA), we exposed human platelet-rich plasma to different shear stresses, ranging from physiologic arterial and arterioles levels (10–44 dynes/cm2) to pathologic high levels (117–388 dynes/cm2) occurring in stenosed coronary, peripheral or cerebral arteries. We found that pathologic shear stresses induce not only platelet activation (P-selectin upregulation and GPIb-alpha downregulation) but also trigger apoptosis events, including mitochondrial transmembrane potential depolarization, caspase 3 activation, phosphatidylserine exposure, and platelet shrinkage and fragmentation into microparticles, whereas physiologic shear stresses are not effective. Platelets subjected to pathologic shear stresses are characterized by impaired platelet function as shown by the absence of ADP-induced platelet aggregation. Apoptosis changes were also induced by the treatment of platelets with calcium ionophore A23187 (10 μM) and thrombin (1 U/mL). Thus, in the present work, we have demonstrated that platelet apoptosis can be induced by chemical stimuli and by mechanical rheological forces (pathologic high shear stresses). Most of shear-induced activation and apoptosis events occur inside of the platelet, including translocation of CD62 from alpha-granules to the platelet surface, depolarization of mitochondrial inner membrane potential, activation of cytosolic enzyme caspase 3, and translocation of phosphatidylserine from the inner to the outer plasma membrane leaflet. These data suggest that the effects of shear stress on platelet activation and apoptosis are mediated by mechanoreceptor(s) that transmit activation and apoptosis signals to the cell interior. The platelet paradigm of apoptosis induced by chemical agonists and shear stresses suggests that apoptotic cytoplasmic machinery may function without nuclear participation.

Description: A single nucleotide polymorphism (SNP) at position 196 in the β3 integrin causes a Leu33Pro substitution in the mature protein. Alloimmunization against the β3Leu33 form (human platelet antigen [HPA]-1a, PlA1, Zwa) in patients who are β3Pro33 homozygous (HPA-1b1b, PlA2A2, Zwbb) causes neonatal alloimmune thrombocytopenia, posttransfusion purpura, or refractoriness to platelet transfusion. Studies with recombinant proteins have demonstrated that amino acids 1 to 66 and 288 to 490 of the β3 integrin contribute to HPA-1a epitope formation. In determining the HPA-1a status of more than 6000 donors, we identified a donor with an HPA-1aweak phenotype and an HPA-1a1b genotype. The platelets from this donor had normal levels of surface αIIbβ3 but reacted only weakly with monoclonal and polyclonal anti–HPA-1a by whole blood enzyme-linked immunosorbent assay (ELISA), flow cytometry, and sandwich ELISA. We reasoned that an alteration in the primary nucleotide sequence of the β3Leu33 allele of this donor was disrupting the HPA-1a epitope. In agreement with this hypothesis, sequencing platelet RNA-derived αIIb and β3 cDNA identified a novel G/A SNP at position 376 of the β3 integrin that encodes for an Arg93Gln replacement in the β3Leu33 allele. Coexpression of the β3Leu33Gln93 encoding cDNA in Chinese hamster ovary cells with human αIIb cDNA showed that the surface-expressed αIIbβ3 reacted normally with β3 integrin–specific monoclonal antibodies but only weakly with monoclonal anti-HPA-1a. Our results show that an Arg93Gln mutation in the β3Leu33 encoding allele disrupts the HPA-1a epitope, suggesting that Arg93 contributes to the formation of the HPA-1a B-cell epitope.

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.