Platelet Phosphatidylserine Exposure Research Articles

Ano6 regulates C2C12 myoblast proliferation and differentiation

Anocatamin 6 (Ano6) is a member of TMEM16, a conserved gene family predicted to code for eight transmembrane proteins with putative Ca2+‐activated chloride channel activity. Ano6 mutations lead to Scott syndrome (SCTS) in patients, a bleeding disorder caused by a defect in Ca2+‐activated Ano6‐mediated phosphatidylserine exposure in platelets and other blood cells. However, the function of ANO6 remains unknown in most other tissues including skeletal muscle. Here we show that Ano6 regulates muscle cell proliferation and differentiation using C2C12 cells as a model system. Ano6 is expressed in adult mouse skeletal muscle and C2C12 myocytes. To investigate the functions of Ano6 in muscle cells, we established Ano6 knockdown (KD) C2C12 cell line using lentiviral shRNA targeting Ano6. Ano6 KD significantly reduced C2C12 myoblast proliferation and promoted myoblast differentiation to form myotubes, suggesting that Ano6 plays an essential role in muscle growth. In the future, we will further define the mechanisms underlying the roles of Ano6 in muscle cells.

Both TMEM16F-dependent and TMEM16F-independent pathways contribute to phosphatidylserine exposure in platelet apoptosis and platelet activation

AbstractScott syndrome, a bleeding disorder caused by defective phospholipid scrambling, has been associated with mutations in the TMEM16F gene. The role of TMEM16F in apoptosis- or agonist-induced phosphatidylserine (PS) exposure was studied in platelets from a Scott syndrome patient and control subjects. Whereas stimulation of control platelets with the BH3-mimetic ABT737 resulted in 2 distinct fractions with moderate and high PS exposure, the high PS-exposing fraction was markedly delayed in Scott platelets. High, but not moderate, PS exposure in platelets was suppressed by chelation of intracellular Ca2+, whereas caspase inhibition completely abolished ABT737-induced PS exposure in both Scott and control platelets. On the other hand, high PS exposure induced by the Ca2+-mobilizing agonists convulxin/thrombin fully relied on mitochondrial depolarization and was virtually absent in Scott platelets. Finally, PS exposure induced by collagen/thrombin was partly affected in Scott platelets, and the residual PS positive fraction was insensitive to inhibition of caspases or mitochondrial depolarization. In conclusion, TMEM16F is not required for, but enhances, caspase-dependent PS exposure; convulxin-/thrombin-induced PS exposure is entirely dependent on TMEM16F, whereas collagen/thrombin-induced PS exposure results from 2 distinct pathways, one of which involves mitochondrial depolarization and is mediated by TMEM16F.

Binding of thrombin-activated platelets to a fibrin scaffold through α(IIb)β₃ evokes phosphatidylserine exposure on their cell surface.

Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS) and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an in-vitro experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyzed using Confocal Laser Scanning Microscopy. Almost all platelets exposed PS after treatment with tissue factor, thrombin or ionomycin. Argatroban abrogated fibrin network formation in all samples, however, platelet PS exposure was inhibited only in tissue factor- and thrombin-treated samples but not in ionomycin-treated samples. FK633, an αIIbβ3 antagonist, and cytochalasin B impaired platelet binding to the fibrin scaffold and significantly reduced PS exposure evoked by thrombin. Gly-Pro-Arg-Pro amide abrogated not only fibrin network formation, but also PS exposure on platelets without suppressing platelet binding to fibrin/fibrinogen. These results suggest that outside-in signals in platelets generated by their binding to the rigid fibrin network are essential for PS exposure after thrombin treatment.

Aspirin Induces Platelet Apoptosis

Abstract 5144Aspirin is widely used in the treatment of a number of clinical conditions, such as fever, pain, strokes, and heart attacks. Although aspirin is being thought of a relatively “safety” medicine, it also has some side effects, particularly the risk of bleeding which may be severe and lead to death. The mechanisms, however, are not totally understood. The effectiveness of aspirin has been attributed to its ability to inhibit prostaglandin production by inhibiting the cyclooxygenase (COX) enzyme. Interestingly, it has been reported recently that aspirin induces apoptosis in many cell types. Platelet apoptosis induced by either physical or chemical compounds or platelet storage occurs widely. We have reported that the glycoprotein Ibα-von Willebrand factor interaction, hyperthermia, and calmodulin antagonists induce platelet apoptosis, suggesting that platelet apoptosis might play important roles in controlling the number and function of circulated platelet under physiological conditions, or in the development of platelet-related hemorrhagic diseases. Thus, the aim of the current study is to explore whether aspirin induces platelet apoptosis. Washed platelets (3×108/ml) were incubated with various concentrations of aspirin (2. 5 mM, 5 mM, 10 mM, 20 mM) at 37 °C for 2 hours. The effect of aspirin on platelet mitochondrial membrane potential (ΔΨm) depolarization and phosphatidylserine (PS) exposure were analyzed by cell-permeable lipophilic cationic dye tetramethylrhodamine ethyl ester (TMRE) and annexin V binding, respectively. The data show that ΔΨms depolarization and PS exposures were dose-dependently induced by aspirin in platelets. In order to further confirm that aspirin incurs platelet apoptosis, caspase-3 activation was measured in platelets incubated with aspirin by detecting 32 kD pro-caspase-3 and 17 kD cleaved caspase-3 fragment, and the result shows that aspirin induced caspase-3 activation. Furthermore, platelet shrinkage appeared in platelets incubated with aspirin (20 mM). Caspase inhibitor z-VAD-fmk inhibited apoptotic platelet shrinkage, and blocked ΔΨm depolarization, but had no effect on PS exposure, suggesting that caspase-3 acts upstream of ΔΨm depolarization, and plays a key role in aspirin-induced platelet apoptosis. Whereas, protein kinase C (PKC), protein kinase B (PKB), p38 mitogen-activated protein kinases (MAPK), protein 53 (p53), reactive oxygen species (ROS), and mitogen-activated protein kinase kinase (MAPKK) were not involved in aspirin-induced platelet apoptosis. In addition, platelets incubated with another COX inhibitor indomethacin did not incur ΔΨm depolarazation and PS exposure in platelets. Taken together, the data indicate that aspirin induces platelet apoptosis via caspase-3 activation, and independent of the COX signaling. Disclosures:No relevant conflicts of interest to declare.

Shear Stress Initiates Cyclophilin D-Dependent Phosphatidylserine Exposure and Integrin αIIbβ3 Cleavage in Platelets

Abstract 3301When platelets are simultaneously stimulated by multiple agonists, such as thrombin and collagen, a subpopulation of procoagulant platelets is formed that is characterized by phosphatidylserine (PS) exposure, integrin αIIbβ3 inactivation, and a rounded morphology in a process that is dependent on the mitochondrial permeability transition pore (mPTP) regulatory protein cyclophilin D (CypD). Recently, we have found that, in the absence of platelet CypD, platelet accumulation is markedly accentuated both in vitro and in vivo indicating that CypD-dependent procoagulant platelet formation may limit thrombus growth. Interestingly, the importance of CypD in limiting platelet accumulation was most evident in shear stress conditions consistent with arterial flow. In low (300 s−1) and high (1500 s−1) shear conditions, CypD null platelet accumulation was increased 1.5- and 4-fold compared to wild type platelets, respectively.Platelet activation and procoagulant platelet formation were examined in various shear conditions. In platelets subjected to increasing shear, PS exposure, but not P-selectin expression or integrin αIIbβ3 activation, was observed in a platelet subpopulation. When subjected to high shear in vitro as many as 70 % of platelets expressed high levels of PS on their surface within 5 min, and shear-dependent PS exposure was observed in as little as 30 seconds in high shear stress conditions. Previously we demonstrated that agonist-initiated PS exposure is closely associated with integrin αIIbβ3 cleavage and inactivation. In shear-treated, as in strongly-stimulated platelets, integrin αIIbβ3 cleavage was closely associated with PS exposure. PS exposure and integrin αIIbβ3 cleavage were not observed in the absence of VWF. In contrast to agonist-initiated PS exposure, shear stress-induced PS exposure was observed even in the absence of extracellular calcium.In a previous study, platelet PS exposure and apoptosis induced by ristocetin were found to be closely associated with an increase in the expression of the BH3-proteins, Bax and Bak. This finding, along with the observation that shear-stress dependent PS exposure occurred even in the absence of extracellular calcium led us to examine the role of Bax and Bak. However, shear stress-induced PS exposure was unaffected in Bax/Bak null platelets. In contrast, both shear stress-induced PS exposure and integrin αIIbβ3 cleavage were markedly blunted in CypD null platelets. Furthermore, shear stress induced-PS exposure was closely associated with loss of mitochondrial transmembrane potential, together indicating the importance of mPTP formation in the regulation of PS exposure in high shear stress conditions. These data identify a novel VWF- and CypD-dependent pathway that results in rapid PS exposure and integrin αIIbβ3 cleavage in high-shear stress conditions. This shear stress-initiated pathway of PS exposure, distinct from thrombin-collagen initiated PS exposure, does not require extracellular calcium and is not associated with P-selectin expression. Disclosures:Jobe:Baxter: Membership on an entity's Board of Directors or advisory committees.

Mitochondrial Calcium and Reactive Oxygen Species Regulate Agonist-Initiated Platelet Phosphatidylserine Exposure

To study the interactions of cytoplasmic calcium elevation, mitochondrial permeability transition pore (mPTP) formation, and reactive oxygen species formation in the regulation of phosphatidylserine (PS) exposure in platelets. mPTP formation, but not the degree of cytoplasmic calcium elevation, was associated with PS exposure in wild-type, cyclophilin D-null, ionomycin-treated, and reactive oxygen species-treated platelets. In the absence of the mPTP regulator cyclophilin D, agonist-initiated mPTP formation and high-level PS exposure were markedly blunted, but cytoplasmic calcium transients were unchanged. Mitochondrial calcium (Ca(2+)(mit)) transients and reactive oxygen species, key regulators of mPTP formation, were examined in strongly stimulated platelets. Increased reactive oxygen species production occurred in strongly stimulated platelets and was dependent on extracellular calcium entry, but not the presence of cyclophilin D. Ca(2+)(mit) increased significantly in strongly stimulated platelets. Abrogation of Ca(2+)(mit) entry, either by inhibition of the Ca(2+)(mit) uniporter or mitochondrial depolarization, prevented mPTP formation and exposure but not platelet aggregation or granule release. Sustained cytoplasmic calcium levels are necessary, but not sufficient, for high-level PS exposure in response to agonists. Increased Ca(2+)(mit) levels are a key signal initiating mPTP formation and PS exposure. Blockade of Ca(2+)(mit) entry allows the specific inhibition of platelet procoagulant activity.

Chrysophanol-8-O-glucoside, an Anthraquinone Derivative in Rhubarb, Has Antiplatelet and Anticoagulant Activities

Rhubarb is a widely used traditional medicine and has been reported to elicit a number of biological effects including anti-inflammatory and antiplatelet effects. In the present study, we investigated the effects of anthraquinone derivatives isolated from rhubarb on platelet activity. Of four anthraquinone derivatives isolated from rhubarb examined, chrysophanol-8-O-glucoside (CP-8-O-glc) was found to have the most potent inhibitory effect on collagen- and thrombin-induced platelet aggregation. CP-8-O-glc–treated mice showed significantly prolonged bleeding times. Furthermore, CP-8-O-glc was found to have a significant inhibitory effect on rat platelet aggregation ex vivo and on thromboxane A2 formation in vitro. In coagulation tests, CP-8-O-glc did not alter prothrombin time, and it prolonged the activated partial thromboplastin time. However, CP-8-O-glc only inhibited platelet phosphatidylserine exposure, but not exert direct inhibition on intrinsic factors. This study demonstrates the antiplatelet and anticoagulant effects of CP-8-O-glc and suggests that this compound might be of therapeutic benefit for the prevention of platelet-related cardiovascular diseases.

Doxorubicin-Induced Platelet Procoagulant Activities: An Important Clue for Chemotherapy-Associated Thrombosis

Thrombotic risk associated with chemotherapy including doxorubicin (DOX) has been frequently reported; yet, the exact mechanism is not fully understood. Here, we report that DOX can induce procoagulant activity in platelets, an important contributor to thrombus formation. In human platelets, DOX increased phosphatidylserine (PS) exposure and PS-bearing microparticle (MP) generation. Consistently, DOX-treated platelets and generated MPs induced thrombin generation, a representative marker for procoagulant activity. DOX-induced PS exposure appeared to be from intracellular Ca²⁺ increase and ATP depletion, which resulted in the activation of scramblase and inhibition of flippase. Along with this, apoptosis was induced by DOX as determined by the dissipation of mitochondrial membrane potential (Δψ), cytochrome c release, Bax translocation, and caspase-3 activation. A Ca²⁺ chelator ethylene glycol tetraacetic acid, caspase inhibitor Q-VD-OPh, and antioxidants (vitamin C and trolox) can attenuate DOX-induced PS exposure and procoagulant activity significantly, suggesting that Ca²⁺, apoptosis, and reactive oxygen species (ROS) were involved in DOX-enhanced procoagulant activity. Importantly, rat in vivo thrombosis model demonstrated that DOX could manifest prothrombotic effects through the mediation of platelet procoagulant activity, which was accompanied by increased PS exposure and Δψ dissipation in platelets.

PKC inhibition markedly enhances Ca2+ signaling and phosphatidylserine exposure downstream of protease‐activated receptor‐1 but not protease‐activated receptor‐4 in human platelets

Cytosolic calcium concentration is a critical regulator of platelet activation, and so platelet Ca(2+) signaling must be tightly controlled. Thrombin-induced Ca(2+) signaling is enhanced by inhibitors of protein kinase C (PKC), suggesting that PKC negatively regulates the Ca(2+) signal, although the mechanisms by which this occurs and its physiological relevance are still unclear. To investigate the mechanisms by which PKC inhibitors enhance thrombin-induced Ca(2+) signaling, and to determine the importance of this pathway in platelet activation. Cytosolic Ca(2+) signaling was monitored in fura-2-loaded human platelets. Phosphatidylserine (PS) exposure, a marker of platelet procoagulant activity, was measured by annexin V binding and flow cytometry. PKC inhibition by bisindolylmaleimide-I (BIM-I) enhanced α-thrombin-induced Ca(2+) signaling in a concentration-dependent manner. PAR1 signaling, activated by SFLLRN, was enhanced much more strongly than PAR4, activated by AYPGKF or γ-thrombin, which is a potent PAR4 agonist but a poor activator of PAR1. BIM-I had little effect on α-thrombin-induced signaling following treatment with the PAR1 antagonist, SCH-79797. BIM-I enhanced Ca(2+) release from intracellular stores and Ca(2+) entry, as assessed by Mn(2+) quench. However, the plasma membrane Ca(2+) ATPase inhibitor, 5(6)-carboxyeosin, did not prevent the effect of BIM-I. PKC inhibition strongly enhanced α-thrombin-induced PS exposure, which was reversed by blockade of PAR1. Together, these data show that when PAR1 is stimulated, PKC negatively regulates Ca(2+) release and Ca(2+) entry, which leads to reduced platelet PS exposure.

Store-operated calcium entry and non-capacitative calcium entry have distinct roles in thrombin-induced calcium signalling in human platelets

Phosphatidylserine (PS)-exposing platelets accelerate coagulation at sites of vascular injury. PS exposure requires sustained Ca2+ signalling. Two distinct Ca2+ entry pathways amplify and sustain platelet Ca2+ signalling, but their relative importance in human platelets is not known. Here we examined the relative roles of store-operated Ca2+ entry (SOCE) and non-capacitative Ca2+ entry (NCCE) in thrombin-induced Ca2+ signalling and PS exposure by using two Ca2+ channel blockers. BTP-2 showed marked selectivity for SOCE over NCCE. LOE-908 specifically blocked NCCE under our conditions. Using these agents we found that SOCE is important at low thrombin concentrations whereas NCCE became increasingly important as thrombin concentration was increased. PS exposure was reduced by LOE-908, and only activated at thrombin concentrations that also activate NCCE. In contrast, BTP-2 had no effect on PS exposure. We suggest that SOCE amplifies and sustains Ca2+ signalling in response to low concentrations of thrombin whereas both NCCE and SOCE are important contributors to Ca2+ signalling at higher thrombin concentrations. However, despite being involved in Ca2+ signalling at high thrombin concentrations, SOCE is not important for thrombin-induced PS exposure in human platelets. This suggests that the route of Ca2+ entry is an important regulator of thrombin-induced PS exposure in platelets.

Critical Role for CD38-mediated Ca2+ Signaling in Thrombin-induced Procoagulant Activity of Mouse Platelets and Hemostasis

CD38, a multifunctional enzyme that catalyzes the synthesis of intracellular Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), is known to be expressed on platelets. However, the role of CD38 in platelets remains unclear. Our present results show that treatment of platelets with thrombin results in a rapid and sustained Ca(2+) signal, resulting from a coordinated interplay of Ca(2+)-mobilizing messengers, inositol 1,4,5-trisphosphate, cADPR, and NAADP. By dissecting the signaling pathway using various agents, we delineated that cADPR and NAADP are sequentially produced through CD38 internalization by protein kinase C via myosin heavy chain IIA following phospholipase C activation in thrombin-induced platelets. An inositol 1,4,5-trisphosphate receptor antagonist blocked the thrombin-induced formation of cADPR and NAADP as well as Ca(2+) signals. An indispensable response of platelets relying on cytosolic calcium is the surface exposure of phosphatidylserine (PS), which implicates platelet procoagulant activity. Scrutinizing this parameter reveals that CD38(+/+) platelets fully express PS on the surface when stimulated with thrombin, whereas this response was decreased on CD38(-/-) platelets. Similarly, PS exposure and Ca(2+) signals were attenuated when platelets were incubated with 8-bromo-cADPR, bafilomycin A1, and a PKC inhibitor. Furthermore, in vivo, CD38-deficient mice exhibited longer bleeding times and unstable formation of thrombus than wild type mice. These results demonstrate that CD38 plays an essential role in thrombin-induced procoagulant activity of platelets and hemostasis via Ca(2+) signaling mediated by its products, cADPR and NAADP.

Vitamin E inhibition on platelet procoagulant activity: Involvement of aminophospholipid translocase activity

Background Activated platelets provide an important procoagulant surface, because exposed negatively charged phosphatidylserine (PS) is an important cofactor of the coagulation cascade. Aminophospholipid translocase (APLT) can transport PS from the outer to the inner membrane leaflet. Although vitamin E has been investigated for its anti-aggregating effect on platelets, its effect on platelet procoagulant activity has not been reported. Methods Phorbol 12-myristate 13-acetate (PMA), a well-known PKC activator, and thrombin were used to induce PS exposure on platelet surface. The expression of PS was measured by annexin A5 binding with flow cytometry. Platelet procoagulant activity was measured by a prothrombinase assay. APLT activity was measured by flow cytometry by determining the percent of 1-palmitoyl-2-[6-[(7-nitro-2–1,3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphatidylserine (NBD-PS) translocated from the outer to the inner membrane leaflet. Inhibition effects of vitamin E on platelet aggregation were simultaneously measured by a Multiplate aggregometer, a Chrono-log aggregometer, and a PFA-100 system. Results Vitamin E significantly attenuated PMA-induced conformational change of glycoprotein IIb/IIIa and P-selectin expression. Vitamin E significantly inhibited PMA and thrombin-induced PS externalization and reduced prothrombinase activity on platelet surfaces both in vitro and ex vivo. APLT activity was increased by vitamin E in a dose-dependent manner, indicating that reduced procoagulant activity may be attributed, at least in part, to this increased APLT activity. Vitamin E inhibited platelet aggregation induced by combined chemokine SDF-1 and low-dose ADP as well as by usual doses of ADP or collagen when measured by the Multiplate and Chrono-log aggregometers but not when measured by PFA-100. Conclusions These in vitro and ex vivo results showed that vitamin E inhibited platelet PS exposure and procoagulant activity partly by increasing APLT activity. These actions of vitamin E on platelet function provide new insights into the anticoagulation properties of vitamin E.

βγ-CAT, a non-lens betagamma-crystallin and trefoil factor complex, induces calcium-dependent platelet apoptosis

In recent years, it has been reported that apoptosis may occur in platelets and play a role in the clearance of effete platelets. βγ-CAT, a newly identified non-lens βγ-crystallin and trefoil factor complex from frog Bombina maxima skin secretions, caused several in vivo toxic effects on mammals. Through determined haematological parameters of rabbits, it has been found that βγ-CAT significantly reduced the number of platelets in a time-dependent manner. Here, in order to explore the effect of βγ-CAT on platelets, washed platelets were incubated with various concentrations of βγ-CAT for 30 minutes. We found that βγ-CAT induced several apoptosis events in human platelets, including caspase-3 activation, phosphatidylserine (PS) exposure, depolarisation of mitochondrial inner transmembrane potential (ΔΨm), cytochrome c release and strong expression of pro-apoptotic Bax and Bak proteins. However, βγ-CAT did not significantly induce platelet activation as detected by P-selectin surface expression, GPIIb/IIIa activation and platelet aggregation. In addition, we observed that βγ-CAT-induced PS exposure and ΔΨm depolarisation in platelets are Ca2+-dependent. Taken together, βγ-CAT can induce Ca2+-dependent platelet apoptosis but does not cause platelet activation.

Changes In Mitochondrial Permeability Initiate Platelet Phosphatidylserine Exposure In Response to Elevated Cytoplasmic Calcium Levels

AbstractAbstract 2024Platelet phosphatidylserine (PS) exposure and procoagulant activity occur in a subset of activated platelets following stimulation with multiple or high levels of agonists. Key among the upstream regulators of agonist-initiated PS exposure is elevated cytoplasmic calcium levels. Additionally, the importance of mitochondrial permeability transition pore (mPTP) formation in regulating PS exposure has been highlighted by recent studies using cyclophilin D null (CypD -/-) platelets. CypD is a critical regulatory protein in the initiation of mPTP formation, and in the absence of CypD agonist-initiated PS exposure is markedly impaired. An alternative, slower pathway of PS exposure, initiated by the BH3 mimetic ABT-737, is regulated by members of the Bcl-2 protein pathway.It is currently unclear how mitochondrial and calcium-signaling pathways interact to regulate PS exposure in platelets. mPTP formation has been reported as both a consequence and a cause of elevated intracellular calcium levels in other cells. To clarify the relative roles of these two upstream signals, we examined the sequence of events that occur upon platelet activation using calcium and mitochondrial transmembrane potential (ΔΨm) sensitive fluorescent probes. In isolated platelets studied by flow cytometry and among adherent platelets evaluated by confocal microscopy, elevated intracellular calcium levels were observed prior to mPTP formation (as determined by loss of ΔΨm). A rise in mitochondrial calcium levels as measured by rhod-2 occurred coincidentally with elevations in cytoplasmic calcium levels. Similar increases in intracellular calcium levels were observed in CypD -/- and CypD +/+ platelets, despite the almost complete absence of mPTP formation and PS exposure in the CypD -/- platelets. These results are consistent with calcium acting as an upstream signal regulating mPTP formation.Ionomycin (3 μM) stimulation can overcome the defect in PS exposure in CypD -/- platelets. This result has been interpreted to indicate that calcium activates effector pathways downstream of the mPTP. However, an alternative explanation is that ionomycin raises cytoplasmic calcium to an elevated level that is sufficient to initiate mPTP formation even in CypD's absence. Dose response studies indicated that the calcium sensitivity of the mPTP was decreased in CypD -/- platelets, but ionomycin (3uM) caused mPTP opening and PS externalization in both CypD +/+ and CypD -/- platelets. Furthermore, in both CypD +/+ and CypD -/- platelets, mPTP formation and PS exposure were closely correlated at all doses of ionomycin utilized. These results indicate that high doses of ionomycin overcome the CypD -/- defect in PS exposure by initiating non-CypD-dependent mPTP formation.The roles of the mitochondrial and calcium pathways in regulating ABT-737-mediated PS exposure were examined. Extracellular calcium was not required for PS exposure initiated by ABT-737, distinct from its importance in agonist-initiated PS exposure. In contrast to previously reported results (Schoenwaelder et al, Blood 2009) we observed that ABT-737 stimulation causes a loss of ΔΨm coincident with PS exposure. Since disruptions in oxidative metabolism could also cause the observed effect on ΔΨm, ABT-737's effects on mitochondrial permeability were confirmed using a calcein-cobalt quenching assay. Unlike agonist-initiated PS exposure, both increased mitochondrial permeability and PS exposure were not dependent on CypD.Together these results indicate a central role for alterations in mitochondrial membrane permeability in mediating PS exposure. In agonist-stimulated platelets increases in cytoplasmic calcium result in mPTP formation and mitochondrial permeabilization, triggering downstream events that result in PS exposure. In ABT-737 stimulated platelets non-CypD-dependent mitochondrial permeability changes mediated by Bcl-2 proteins may also act as an important initiator of the events that result in PS exposure. Disclosures:No relevant conflicts of interest to declare.

Platelet Apoptosis and Agonist Mediated Activation In Myelodysplastic Syndromes

AbstractAbstract 1866Myelodysplastic syndromes (MDS) comprise distinct disorders characterized by dysplastic and ineffective hematopoiesis that seems to be related to an increased apoptosis of bone marrow cells (Nimer, Blood 111: 4841, 2008). Clinical manifestations in MDS range from a diverse degree of anemias, leuko- or thrombocytopenias to severe transfusion-dependent peripheral pancytopenias. Thrombocytopenia and platelet dysfunction contribute to hemorrhagic complications observed in MDS. Many of the features of apoptosis such as membrane fragmentation, microvesiculation and phosphatidylserine (PS) exposure are observed during platelet activation to a procoagulant state, raising the possibility that apoptosis may regulate platelet function.The aim of this work was to determine whether a correlation exists between apoptosis and activation processes in platelets from MDS patients.Twenty six patients diagnosed MDS and classified according to WHO-2008 were included: 6 with refractory anemia (RA), 7 with RA with ringed sideroblasts (RARS), 6 with refractory RA with excess blasts-1 (RAEB-1) and 7 with cytopenia with multilineage dysplasia (RCMD) associated with isolated 5q deletion. Twenty six healthy donors were included as control group.Apoptosis was determined by flow cytometry analysis through FITC-annexin V binding to platelet membrane PS exposed under basal conditions and after stimulation with a PAR-1 receptor agonist (TRAP, SFLLRN, thrombin receptor-activating peptide 6). Levels of pro- apoptotic Bax and anti-apoptotic Bcl-2 proteins were determined by densitometric analysis of western blots performed with platelet lysates.Platelet activation was determined through FITC-fibrinogen, FITC-PAC-1 (a mAb that recognizes activated conformation of fibrinogen receptor) and FITC-P-selectin mAb binding to quiescent and 100 mM TRAP activated platelets by flow cytometry. Surface expression of fibrinogen receptor (aIIb and b3 subunits) was determined by flow cytometry with specific mAbs.Platelets from RA, RARS and RCMD patients expressed more PS than control ones under basal conditions (p<0.05) as well as after 100 mM TRAP stimulation (p<0.05). Moreover, platelets from these MDS patients expressed more Bax protein than control group (p<0.05).On the other hand, PS exposure and Bax content in platelets from RAEB-1 patients were similar to controls, but they expressed a higher amount of Bcl-2 (p<0.05). No correlation was observed between PS exposure or Bax expression and platelet number.Platelets from all MDS patients showed an impaired activation by TRAP, even when PS exposure was higher than in control group. This diminished response to TRAP was not due to a reduction in surface expression of fibrinogen receptor in platelets from MDS patients.Our results suggest that platelets from RA, RARS and RCMD patients are more apoptotic than control ones and that a correlation between platelet surface PS and activation does not seem to exist. Moreover, dissimilarity in expression pattern of apoptotic proteins among MDS types indicates differences in the intracellular mechanisms underlying these pathologies. Disclosures:No relevant conflicts of interest to declare.

Roles of Platelet STIM1 and Orai1 in Glycoprotein VI- and Thrombin-dependent Procoagulant Activity and Thrombus Formation

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca(2+) entry (SOCE) with Orai1 as principal Ca(2+) entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca(2+) entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1(-/-) and Orai1(-/-) platelets had greatly impaired glycoprotein (GP) VI-dependent Ca(2+) signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2(-/-) platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca(2+) signals of Stim1(-/-) and Orai1(-/-) platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1(-/-) and Orai1(-/-) platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca(2+) entry, inhibited Ca(2+) and procoagulant responses even in Stim1(-/-) and Orai1(-/-) platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca(2+) entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca(2+) entry and PS exposure, only one relying on STIM1-Orai1 interaction.

Platelet Senescence and Phosphatidylserine Exposure.

Abstract 2117Poster Board II-94The transbilayer movement of phosphatidylserine from the inner to the outer leaflet of membrane bilayer occurs during platelet activation and is essential for platelet procoagulant activity. Expression of phosphatidylserine also occurs in platelets stored more than 5 days in the blood bank. We have examined the role of mitochondrial pathways in activation-induced platelet procoagulant activity and during in vitro senescence. Collagen and thrombin-induced exposure of phosphatidylserine is associated with a decrease in mitochondrial membrane potential (MMP). Cyclosporin A, a known inhibitor of cyclophilin D, inhibited phosphatidylserine exposure and the loss of MMP. Consistent with this, platelets from CypD deficient mice had decreased loss of MMP and impaired phosphatidylserine exposure when treated with a combination of thrombin and collagen. However, there is no release of cytochrome c into the cytosol. Also there is no activation of caspase-3 or Rho associated kinase I (ROCK1). In sharp contrast to this, in platelets stored for more than 5 days, phosphatidylserine exposure is found to be associated with caspase-3 and ROCK1 activation. To delineate the mechanism we used ABT737, a BH3 mimetic that induces mitochondrial pathway of apoptosis via Bcl-2 family of proteins. ABT737-induced phosphatidylserine exposure was also associated with cytochrome c release, caspase-3 and ROCKI activation and it is unaffected in platelets isolated from CypD knock out mice or in platelets treated with cyclosporine A prior to activation. Caspase-3 inhibitor (z-DVED-fmk and ROCK1 inhibitor Y-27632 reduced ABT-737 induced phosphatidylserine expression.Our results also show that activation-induced phosphatidylserine exposure is dependent on mitochondrial membrane depolarization and CypD but is independent of cytochrome c release or caspase-3 or ROCK1 activation. In contrast, the phosphatidylserine exposure in stored platelets occurs via mitochondrial cytochrome c release, caspase-3 activation and ROCKI activation and does not depend on mitochondrial membrane depolarization or CypD. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Two distinct pathways regulate platelet phosphatidylserine exposure and procoagulant function

AbstractProcoagulant platelets exhibit hallmark features of apoptotic cells, including membrane blebbing, microvesiculation, and phosphatidylserine (PS) exposure. Although platelets possess many well-known apoptotic regulators, their role in regulating the procoagulant function of platelets is unclear. To clarify this, we investigated the consequence of removing the essential mediators of apoptosis, Bak and Bax, or directly inducing apoptosis with the BH3 mimetic compound ABT-737. Treatment of platelets with ABT-737 triggered PS exposure and a marked increase in thrombin generation in vitro. This increase in procoagulant function was Bak/Bax- and caspase-dependent, but it was unaffected by inhibitors of platelet activation or by chelating extracellular calcium. In contrast, agonist-induced platelet procoagulant function was unchanged in Bak−/−Bax−/− or caspase inhibitor–treated platelets, but it was completely eliminated by extracellular calcium chelators or inhibitors of platelet activation. These studies show the existence of 2 distinct pathways regulating the procoagulant function of platelets.

Intradialytic and postdialytic platelet activation, increased platelet phosphatidylserine exposure and ultrastructural changes in platelets in children with chronic uremia

The present research evaluated the intradialytic and postdialytic changes in platelet factor-4 and beta-thromboglobulin plasma levels by enzyme-linked immunoadsorbent assay method and platelet aggregation by ADP as well as flow cytometric percentage of annexin-V-positive platelets as a measure of phosphatidylserine externalization and ultrastructural examination of platelets in 37 uremic patients on regular hemodialysis and 25 age-matched and sex-matched controls. Platelet factor-4 plasma levels increased, remain consistently high during hemodialysis session (20.24 +/- 3.05 IU/ml after 30 min, P < 0.001 and 23.67 +/- 3.68 IU/ml after 240 min, P < 0.001) and returned to control values (6.10 +/- 1.54 IU/ml) only after 24 h following the end of the session. beta-Thromboglobulin showed a trend similar to that of platelet factor-4. Platelet aggregation by ADP showed reduced function in comparison with controls (69.32 +/- 12.37 versus 91.95 +/- 1.59%, P < 0.001). Flow cytometric percentage of annexin-V-positive platelet was significantly elevated (P < 0.001) in uremic patients when compared with normal controls. Ultrastructural studies of platelets 30 min after starting of dialysis showed degranulation of its granules and at 240 min showed complete degranulation, whereas in the postdialytic phase (12 h after the end of dialysis) refilled alpha-granules started to appear. Positive correlations were found between platelet concentration and platelet factor-4 and beta-thromboglobulin plasma levels during and after dialysis (P < 0.001) and with annexin-V-positive platelets percentage (P < 0.001). In conclusion, activated platelets were found in chronic hemodialysis patients, a finding that may explain why uremic patients often suffer from thrombotic accidents. The platelet activation is associated with exposure of phosphatidylserine on the platelet exterior. Platelet factor-4 and beta-thromboglobulin are released from platelets as a result of a defect in their granules membrane as shown by the electron microscopy, mainly as a consequence of the blood-membrane contact during dialysis, and they return only slowly to control values.

Role of cAMP-dependent protein kinase in the regulation of platelet procoagulant activity

The membrane microparticle (MP) formation and phosphatidylserine (PS) exposure evoked by platelet activation provide catalytic surfaces for thrombin generation. Several reports have indicated the effects of cAMP-elevating agents on agonist-induced MP formation and PS exposure; however, the mechanism still remains unclear. Here we show that inhibition of basal cyclic AMP-dependent protein kinase (PKA) activity incurred platelet MP formation and PS exposure. Pretreatment of platelets with cAMP-elevating agent, forskolin, abolished thrombin plus collagen-induced MP formation and PS exposure, and obviously decreased calcium ionophore-evoked MP shedding. Moreover, the inhibitory effects of forskolin on agonists-induced MP formation and PS exposure were reversed by the PKA inhibitor H89. PKA inhibitor-induced MP formation was dose-dependently inhibited by calpain inhibitor MDL28170, and forskolin abrogated thrombin plus collagen-induced calpain activation. In conclusion, PKA plays key roles in the regulation of platelet MP formation and PS exposure. PKA-mediated MP shedding is dependent on calpain activation.