TMEM16F Plays a Vital Role In Platelet Microparticle Generation and Thrombosis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2294-2294 ◽  
Author(s):  
Ann-Desdemonia N Fowajuh ◽  
Debora K Mukaz ◽  
Dongjun Li ◽  
Pani A. Apostolidis ◽  
Aasma Khan ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Resolution ◽  
Thrombus Formation ◽  
Calcium Ionophore ◽  
Annexin V ◽  
Ionophore A23187 ◽  
Platelet Surface ◽  
Microparticle Formation ◽  
Dual Agonist ◽  
High Resolution Microscopy

Abstract Transmembrane (TMEM)16f is member of the TMEM16 family of ion channels, recently shown to be essential for optimal Ca2+-dependent phospholipid scrambling in platelets, platelet-dependent pro-coagulant activity and thrombosis. However, relatively little is known about the effect of TMEM16f on platelet signaling, functional activity, and microparticle formation. TMEM16f-/- mice were obtained from Andrea Vortkamp and the platelets of these mice were isolated, stimulated with either dual agonist (thrombin + convulxin) or calcium ionophore (A23187), and platelet surface exposure of phosphatidylserine (PS) and microparticle generation were assessed by both flow cytometry and high resolution immunofluorescent confocal microscopy. To measure PS exposure, annexin V binding to platelet surfaces was quantified: In response to thrombin and convulxin, only 10.65% ± 1.35% of TMEM16f-/- platelets exposed PS after dual agonist exposure, compared to 16.1% ± 2.3%PS+ WT platelets when analyzed by high resolution microscopy. When analyzed by flow cytometry, dual agonist exposure of WT platelets yielded 52.4% ± 7.0% of maximal annexin V binding achieved by 10μM A23187; in contrast, there was no significant increase in annexin V binding detected in TMEM16f-/- platelets treated with dual agonist. Responses to calcium ionophore were also reduced in TMEM16f-/- platelets relative to WT (17.3% ± 8.3% PS+ TMEM16f-/- platelets in response to 1μM A23187 compared to 40% ± 1.4% PS+ WT platelets, by high resolution microscopy). Microparticle generation from TMEM16f-/- platelets compared to WT platelets was also evaluated by high resolution immunofluorescent microscopy and flow cytometry. On average, approximately 5.7 ± 0.33 microparticles were generated by untreated WT platelets however thrombin and convulxin treated platelets yielded 10.66 ± 0.92 microparticles per platelet whereas no significant increase in microparticle generation was observed in TMEM16f-/- platelets after dual agonist treatment. These data suggest that TMEM16f is required not only for PS exposure on platelet membranes, but also for the shedding of PS+ microparticles. TMEM16f-/- mice also have a significant defect in stable occlusive thrombus formation following 10% ferric chloride injury of the carotid artery (0/6 TMEM16f-/- mice form stable thrombi compared to 5/6 WT mice forming stable thrombi under the same conditions). TMEM16f-/- platelets had a slight (15%) reduction in total fibrinogen binding stimulated by PAR4 peptide agonist compared to WT, but showed no significant differences in aggregation to PAR4 agonist peptide or ADP compared to WT control platelets, suggesting that the defect in thrombus formation is likely due to PS- or microparticle-dependent procoagulant activity. This hypothesis is supported by preliminary results demonstrating that when microparticles isolated from ionophore-stimulated WT platelets were injected into TMEM16f knockout mice, 3/3 TMEM16f-/- mice formed stable thrombi, compared to 5/6 WT mice injected with vehicle control. In conclusion, TMEM16f-/- mice are deficient in platelet PS exposure, platelet-derived microparticle formation, and injury-induced thrombus formation, and platelet-derived microparticles appear to contribute to the defect in thrombus formation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: a flow cytometry study showing a role for free sulfhydryl groups

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2554-2565 ◽  
Author(s):  
J Dachary-Prigent ◽  
JM Freyssinet ◽  
JM Pasquet ◽  
JC Carron ◽  
AT Nurden
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
Annexin V ◽  
Cytoskeletal Proteins ◽  
Procoagulant Activity ◽  
Ionophore A23187 ◽  
Calpain Activity ◽  
Platelet Surface ◽  
Reactive Agents ◽  
Platelet Secretion

Annexin V, a protein with a high affinity and a strict specificity for aminophospholipids at physiologic calcium concentrations, was used to probe platelet activation and the development of procoagulant activity. Platelet secretion was studied in parallel using VH10, a murine monoclonal antibody specific for GMP-140, an alpha-granule membrane glycoprotein. Both proteins were labeled with fluorescein isothiocyanate and platelet activation was assessed by flow cytometry. Microparticles, which are shed from the platelet surface and also support procoagulant activity, were distinguished from platelets according to their associated light scattering signal. The relative ability of different inducers to trigger exposure of the procoagulant surface and microparticle formation was: ionophore A23187 = thrombin plus collagen = collagen = thrombin. The density of aminophospholipid on microparticles was higher than on remnant platelets. Platelet activation by these agonists was accompanied by GMP-140 exposure, both on platelets and microparticles. Here, thrombin was the most efficient agonist. The mechanisms responsible for the above processes were investigated using E-64-d, a specific membrane-permeable inhibitor of Ca(2+)-activated protease (calpain); tetracaine, an activator of calpain; and N-ethylmaleimide and diamide, two sulfhydryl-reactive agents. These agents were added to platelets alone or before stimulation by agonists. Calpain activity was assessed by the hydrolysis of cytoskeletal proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results showed that calpain activity is not essential for aminophospholipid translocation or for secretion. In contrast, although sulfhydryl-reactive agents alone can trigger procoagulant activity, they inhibit microvesicle formation and platelet secretion induced by the above agonists, suggesting that different mechanisms account for these phenomena. The use of annexin V in flow cytometry is a rapid method to assess procoagulant activity in platelets and the loss of phospholipid asymmetry in cell membranes.

scholarly journals Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: a flow cytometry study showing a role for free sulfhydryl groups

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2554-2565 ◽  
Author(s):  
J Dachary-Prigent ◽  
JM Freyssinet ◽  
JM Pasquet ◽  
JC Carron ◽  
AT Nurden
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
Annexin V ◽  
Cytoskeletal Proteins ◽  
Procoagulant Activity ◽  
Ionophore A23187 ◽  
Calpain Activity ◽  
Platelet Surface ◽  
Reactive Agents ◽  
Platelet Secretion

Abstract Annexin V, a protein with a high affinity and a strict specificity for aminophospholipids at physiologic calcium concentrations, was used to probe platelet activation and the development of procoagulant activity. Platelet secretion was studied in parallel using VH10, a murine monoclonal antibody specific for GMP-140, an alpha-granule membrane glycoprotein. Both proteins were labeled with fluorescein isothiocyanate and platelet activation was assessed by flow cytometry. Microparticles, which are shed from the platelet surface and also support procoagulant activity, were distinguished from platelets according to their associated light scattering signal. The relative ability of different inducers to trigger exposure of the procoagulant surface and microparticle formation was: ionophore A23187 = thrombin plus collagen = collagen = thrombin. The density of aminophospholipid on microparticles was higher than on remnant platelets. Platelet activation by these agonists was accompanied by GMP-140 exposure, both on platelets and microparticles. Here, thrombin was the most efficient agonist. The mechanisms responsible for the above processes were investigated using E-64-d, a specific membrane-permeable inhibitor of Ca(2+)-activated protease (calpain); tetracaine, an activator of calpain; and N-ethylmaleimide and diamide, two sulfhydryl-reactive agents. These agents were added to platelets alone or before stimulation by agonists. Calpain activity was assessed by the hydrolysis of cytoskeletal proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results showed that calpain activity is not essential for aminophospholipid translocation or for secretion. In contrast, although sulfhydryl-reactive agents alone can trigger procoagulant activity, they inhibit microvesicle formation and platelet secretion induced by the above agonists, suggesting that different mechanisms account for these phenomena. The use of annexin V in flow cytometry is a rapid method to assess procoagulant activity in platelets and the loss of phospholipid asymmetry in cell membranes.

scholarly journals Mitochondrial Inner Membrane Depolarization as a Marker of Platelet Apoptosis

2016 ◽  
Vol 23 (2) ◽  
pp. 139-147 ◽  
Author(s):  
Armen V. Gyulkhandanyan ◽  
David J. Allen ◽  
Sergiy Mykhaylov ◽  
Elena Lyubimov ◽  
Heyu Ni ◽  
...  
Keyword(s):  
Mitochondrial Permeability Transition ◽  
Calcium Ionophore ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Calcium Ionophore A23187 ◽  
Clinical Settings ◽  
Ionophore A23187 ◽  
Platelet Apoptosis ◽  
Mitochondrial Inner Membrane ◽  
Microparticle Formation

Availability of universal marker for the diagnosis of platelet apoptosis is an important but currently unresolved goal of platelet physiology investigations. Mitochondrial inner transmembrane potential (▵Ψm) depolarization is frequently used as a marker of apoptosis in nucleated cells and anucleate platelets. Since ▵Ψm depolarization in platelets is also frequently associated with concurrent induction of other apoptotic responses, it may appear that ▵Ψm depolarization is a good universal marker of platelet apoptosis. However, data presented in the current study indicate that this is incorrect. We report here fundamental differences in the effects of potassium ionophore valinomycin and calcium ionophore A23187 on human platelet apoptosis. Although both A23187-triggered and valinomycin-triggered ▵Ψm depolarization are strongly induced, the former is dependent on the opening of mitochondrial permeability transition pore (MPTP) and the latter is MPTP-independent. Furthermore, effects of calcium and potassium ionophores on other apoptotic events are also basically different. A23187 induces caspase-3 activation, proapoptotic Bax and Bak protein expression, phosphatidylserine exposure, and microparticle formation, whereas valinomycin does not induce these apoptotic manifestations. Discovery of targeted ▵Ψm depolarization not associated with apoptosis in valinomycin-treated platelets indicates that this marker should not be used as a single universal marker of platelet apoptosis in unknown experimental and clinical settings as it may lead to a false-positive apoptosis diagnosis.

DETECTION OF ACTIVATED PLATELETS IN WHOLE BLOOD BY FLOW CYTOMETRY

1987 ◽  
Author(s):  
S J Shattil ◽  
J A Hoxie ◽  
M Cunningham ◽  
C S Abrahms ◽  
J O’Brien ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
Platelet Activation ◽  
Whole Blood ◽  
Thrombus Formation ◽  
Membrane Surface ◽  
White Blood Cells ◽  
Color Flow ◽  
Platelet Surface ◽  
Activated Platelets

Platelets may become activated in a number of clinical disorders and participate in thrombus formation. We have developed a direct test for activated platelets in whole blood that utilizes dual-color flow cytometry and requires no washing steps. Platelets were distinguished from erythrocytes and white blood cells in the flow cytometer by labeling the platelets with biotin-AP1, an antibody specific for membrane glycoprotein lb, and analyzing the cells for phycoerythrin-streptavidin fluorescence. Membrane surface changes resulting from platelet activation were detected with three different FITC-labeled monoclonal antibodies: 1) PAC1, an antibody specific for the fibrinogen receptor on activated platelets; 2) 9F9, which binds to the D-domain of fibrinogen and detects platelet-bound fibrinogen; and 3) S12, which binds to an alpha-granule membrane protein that associates with the platelet surface during secretion. Unstimulated platelets demonstrated no PAC1, 9F9, or S12-specific fluorescence, indicating that they did not bind these antibodies. Upon stimulation with agonists, however, the platelets demonstrated a dose-dependent increase in FITC-fluorescence. The binding of 9F9 to activated platelets required fibrinogen. Low concentrations of ADP and epinephrine, which induce fibrinogen receptors but little secretion, stimulated near-maximal PAC1 or 9F9 binding but little S12 binding. On the other hand, a concentration of phorbol myristate acetate that evokes full platelet aggregation and secretion induced maximal binding of all three antibodies. When blood samples containing activated and non-activated platelets were mixed, as few as 0.8% activated platelets could be detected by this technique. There was a direct correlation between ADP-induced FITC-PAC1 binding and binding determined in a conventional 125I-PAC1 binding assay (r = 0.99; p < 0.001). These studies demonstrate that activated platelets can be reliably detected in whole blood using activation-dependent monoclonal antibodies and flow cytometry. This method may be useful to assess the degree of platelet activation and the efficacy platelet inhibitor therapy in thrombotic disorders.

Cocaine’s Direct Effect on Either Sickle or Normal Erythrocytes Does Not Increase Phosphatidylserine Exposure, Calcium Pumping, or Scramblase Activity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3692-3692
Author(s):  
Kitty DeJong ◽  
Robert Hagar
Keyword(s):  
Flow Cytometry ◽  
Pulmonary Hypertension ◽  
Calcium Influx ◽  
Plasma Concentrations ◽  
Calcium Ionophore ◽  
Annexin V ◽  
Cell Types ◽  
Red Cell ◽  
Sickle Cells ◽  
Red Cell Membranes

Hypothesis: Cocaine directly affects the exposure of phosphatidylserine, calcium pumping, and scramblase activity in sickle cells. Background: Cocaine is highly associated with pulmonary hypertension in both normal and sickle cell disease patients. Given the growing interest in pulmonary hypertension as a major morbidity factor, the causes and determinants of pulmonary hypertension need to be elucidated to allow rational study designs. Phosphatidylserine (PS) exposure is known to occur in sickle cells and appears to be relevant for vascular damage. Although the mechanism underlying PS exposure is poorly understood, signal transduction processes appear to play a role. We wondered if cocaine could activate these processes and cause PS exposure, or interfere with the proteins involved in the transbilayer movement of PS. Methods: Sickle and normal erythrocytes were exposed to cocaine HCl (from a 10% topical solution, Roxane Labs) at concentrations between 100 and 1000 ng/ml, which is the range of determined plasma concentrations after cocaine use. PS exposure after cocaine treatment was measured by labeling with fluorescently conjugated annexin V (AV) and analysis by flow cytometry. Alterations in scramblase activity were determined by assessing the percentage of PS-exposing cells following loading of the cells with 0.1 mM calcium using calcium ionophore. Calcium influx and Ca-ATPase-mediated calcium efflux were monitored using the fluorescent probe Fluo4, and flippase activity was assessed using NBD-PS followed by analysis with flow cytometry. Results: Sickle cells differ from normal cells with respect to most of the measured parameters, such as having more PS-exposing cells, lower flippase activity, and alterations in calcium kinetics. However, cocaine did not have any effect on PS exposure, calcium-induced scrambling, calcium influx, calcium pump activity, or flippase activity. The cell scatter patterns did not show any gross changes in red cell shape or density after cocaine treatment. Discussion: The lack of effect implies that cocaine is working through other mechanisms than effects on red cell membranes. Therefore, future studies should focus on other vascular cell types and membrane pathways. Alternatively, cocaine metabolites should be evaluated for their activities on red cells.

Chemical Agonists and High Shear Stress Induce Apoptosis in Human Platelets.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3875-3875
Author(s):  
Valery Leytin ◽  
Sergiy Mykhaylov ◽  
David J. Allen ◽  
Lukasz Miz ◽  
Elena V. Lyubimov ◽  
...  
Keyword(s):  
Shear Stress ◽  
Platelet Activation ◽  
Caspase 3 ◽  
Calcium Ionophore ◽  
Shear Stresses ◽  
Ionophore A23187 ◽  
High Shear ◽  
Platelet Surface ◽  
Platelet Apoptosis ◽  
Chemical Stimuli

Abstract 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.

scholarly journals Collagen But Not Fibrinogen Surfaces Induce Bleb Formation, Exposure of Phosphatidylserine, and Procoagulant Activity of Adherent Platelets: Evidence for Regulation by Protein Tyrosine Kinase-Dependent Ca2+ Responses

Blood ◽  
1997 ◽  
Vol 90 (7) ◽  
pp. 2615-2625 ◽  
Author(s):  
Johan W.M. Heemskerk ◽  
Wim M.J. Vuist ◽  
Marion A.H. Feijge ◽  
Chris P.M. Reutelingsperger ◽  
Theo Lindhout
Keyword(s):  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Imaging System ◽  
Morphological Changes ◽  
Annexin V ◽  
Ionophore A23187 ◽  
Platelet Surface ◽  
Protein Tyrosine ◽  
The Matrix ◽  
Bleb Formation

With a combined phase-contrast and fluorescence video imaging system, changes in morphology and cytosolic [Ca2+]i were investigated of fura-2–loaded platelets during adhesion to fibrinogen or collagen matrices. The Ca2+ signals were, on the level of single platelets, compared to the secretion and procoagulant responses, using fluorescent-labeled AK-6 antibody against P-selectin and labeled annexin V for detection of surface-exposed phosphatidylserine (PS), respectively. Platelets in contact with fibrinogen developed filapods and spread over the matrix, in most of the cells without detectable Ca2+ signal. Thrombin induced repetitive spiking in [Ca2+]i , followed by the expression of P-selectin but not of PS on the platelet surface. Platelet interaction with collagen resulted in spreading and transformation of the cells into blebbing, “balloon”-like structures (diameter about 5 μm). The latter morphological changes were accompanied by high and prolonged increases in [Ca2+]i , by the exposure of both P-selectin and PS, and by the ability of the platelets to convert prothrombin into thrombin. Thrombin addition accelerated the onset of the Ca2+ signals and the appearance of surface-exposed PS. Collagen-induced PS exposure was slightly reduced by treatment of the platelets with aspirin, and strongly inhibited by suppression of the Ca2+ responses with prostaglandin E1 or the Ca2+ chelator, dimethyl-BAPTA. Inhibition of protein tyrosine phosphorylation with genistein, U73343, or wortmannin resulted in spiking Ca2+ responses in many of the platelets and in almost complete reduction of bleb formation and PS exposure. In contrast, genistein did not suppress bleb formation and PS exposure of platelets stimulated with the Ca2+ ionophore A23187. We conclude that a collagen but not fibrinogen matrix acts as a potent activator of the procoagulant response through activation of tyrosine kinases and subsequent generation of sustained intracellular Ca2+ signals.

scholarly journals von Willebrand factor is present on the surface of platelets stimulated in plasma by ADP

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1362-1366
Author(s):  
B Adelman ◽  
P Carlson ◽  
P Powers
Keyword(s):  
Binding Site ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Surface Binding ◽  
Platelet Surface ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand factor (vWf) can bind to glycoprotein (GP) IIb/IIIa on activated platelets. The significance of this interaction is unclear, however, because it has not been possible to detect vWf binding to GPIIb/IIIa on platelets stimulated in plasma. We have developed an indirect, flow cytometry assay that uses fluorescein-labeled antibodies to detect vWf and fibrinogen on platelets. Using this assay, we found vWf on the surface of platelets stimulated in plasma by ADP. The number of platelets that bound vWf increased in proportion to ADP concentration and incubation time. Washed platelets in a protein-free buffer activated by 1 mumol/L calcium ionophore A23187 or 10 mumol/L ADP also bound vWf, suggesting that we were detecting surface binding of alpha-granule-derived vWf. Monoclonal antibodies against the vWf binding site on GPIb (6D1) and the vWf and fibrinogen binding sites on GPIIb/IIIa (LJP5 and LJ-CP8, respectively) were used to characterize the mechanism of vWf binding to stimulated platelets. Ristocetin- induced binding of vWf was inhibited by 6D1, and ADP-induced binding of fibrinogen was inhibited by LJ-CP8. None of these antibodies inhibited ADP-induced vWf binding. Aspirin and prostaglandin E1 also inhibited ADP-induced binding of vWf in platelet-rich plasma. During platelet activation in plasma, vWf derived from alpha-granules becomes bound to the platelet surface possibly being transferred already associated with a binding site.

Platelet Apoptosis Events Can Be Triggered by Shear Stresses and Chemical Agonists.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3940-3940
Author(s):  
Valery Leytin ◽  
Sergiy Mykhaylov ◽  
David J. Allen ◽  
John J. Freedman
Keyword(s):  
Platelet Activation ◽  
Caspase 3 ◽  
Platelet Rich Plasma ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Shear Stresses ◽  
Ionophore A23187 ◽  
Platelet Surface ◽  
Platelet Apoptosis ◽  
Chemical Stimuli

Abstract 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 GPIbα 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 α-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.

The GPIIbIIIa Antagonist Drugs Eptifibatide and Tirofiban Inhibit Caspase-3 Activation in Thrombin- and Calcium Ionophore-Stimulated Human Platelets.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2998-2998
Author(s):  
Valery Leytin ◽  
Asuman Mutlu ◽  
Sergiy Mykhaylov ◽  
David J. Allen ◽  
Armen V. Gyulkhandanyan ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Caspase 3 ◽  
Conflicts Of Interest ◽  
Calcium Ionophore ◽  
Human Platelets ◽  
Shear Stresses ◽  
Ionophore A23187 ◽  
Platelet Surface ◽  
Apoptotic Effects

Abstract 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<0.01, n=7). However, when platelets were preincubated with eptifibatide and tirofiban before agonist treatment, these drugs significantly inhibited agonist-induced caspase-3 activation by an average of 44-50% (P<0.05, n=7). The fact that eptifibatide and tirofiban do not promote caspase-3 activation in unstimulated platelets suggests that these GPIIbIIIa antagonists do not induce transmission of pro-apoptotic transmembrane signals inside platelets through inactive GPIIbIIIa integrin. The inhibitory effect of eptifibatide and tirofiban on thrombin- and A23187-induced caspase-3 activation suggests a role of GPIIbIIIa integrin in caspase-3 activation induced by these platelet agonists. Conclusions: We have demonstrated 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. Taken together with earlier reported data (Leytin et al, Br J Haematol 133: 78, 2006), the current study indicates that, aside from their well-known participation in platelet activation and aggregation, GPIIbIIIa receptors are involved in the modulation of platelet apoptosis. This GPIIbIIIa-mediated mechanism of apoptosis modulation may be very efficient given the extremely large number of GPIIbIIIa copies (≈80,000) on the platelet surface. Disclosures: No relevant conflicts of interest to declare.

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