Platelet Apoptosis in Fluid-Phase and Adherent Platelets and Thrombi-Like Platelet Aggregates.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2102-2102
Author(s):  
Valery Leytin ◽  
David J. Allen ◽  
Elena Lyubimov ◽  
Anna Chavlovski ◽  
Mingyu She ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Fluid Phase ◽  
Human Platelets ◽  
Shear Stresses ◽  
Ionophore A23187 ◽  
High Shear ◽  
Platelet Surface ◽  
Platelet Suspension ◽  
Platelet Apoptosis ◽  
Platelet Aggregates

Abstract 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.

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.

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.

Thrombin Is Able To Trigger Apoptosis of Human Platelets.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1094-1094
Author(s):  
Valery Leytin ◽  
David J. Allen ◽  
Sergiy Mykhaylov ◽  
Elena Lyubimov ◽  
John Freedman
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
Caspase 3 ◽  
Coagulation Factor ◽  
Annexin V ◽  
Human Platelets ◽  
Inner Mitochondrial Membrane ◽  
Platelet Surface ◽  
Platelet Apoptosis ◽  
Platelet Storage

Abstract Although primarily known as a coagulation factor and as an inducer of platelet activation and aggregation, thrombin can modulate apoptosis in nucleated cells. Over the last decade, it has been recognized that apoptosis occurs not only in nucleated cells but also in anucleated cytoplasts and platelets. The current study investigated whether thrombin can impact apoptosis in anucleated human platelets. Using flow cytometry, we studied platelet apoptosis at the single cell level, analyzing markers of mitochondrial and cytoplasmic apoptosis (Leytin et al, Biochem Biophys Res Commun320:303, 2004; Leytin et al, Br J Haematol133:78, 2006). Western blotting was also employed, in addition to flow cytometry, for determining the expression of proapoptotic Bax and Bak proteins. We found that, in comparison to untreated platelets, human alpha-thrombin (1 U/mL) significantly induced four key manifestations of platelet apoptosis: (i) mitochondrial inner transmembrane potential depolarization (P&lt;0.01), (ii) expression of pro-apoptotic Bax (P=0.002) and Bak (P=0.04) proteins, (iii) caspase-3 activation (P=0.0009), and (iv) phosphatidylserine (PS) exposure (P&lt;0.0001). We also compared the magnitude of thrombin effects with those of A23187 and in vitro platelet storage under standard blood banking conditions. We demonstrated that the maximal level of both caspase-3 activation and PS exposure is achieved in A23187-stimulated platelets, indicating that A23187 is a useful positive control for quantifying these apoptosis events. Thrombin triggered caspase-3 activation to a level equal to that in A23187-induced platelets and significantly higher than in platelets stimulated with control buffer (P&lt;0.001) and stored for 0, 6 (P&lt;0.001) and 13 days at 22°C (P&lt;0.05). PS exposure was also markedly enhanced in thrombin-stimulated platelets resulting in increase of annexin V-positive cells from 1.2 ± 0.1% to 21.2 ± 2.5% (P=0.0002); platelet storage increased annexin V-positive cells from 1.4 ± 0.4% (Day 0) to 6.0 ± 0.6% (Day 6, P=0.006) and 47.6 ± 5.6% (Day 13 platelets, P=0.0013) and much higher PS exposure was observed with 10 μM A23187 (97.8 ± 0.4%, P&lt;0.0001). Thus, PS exposure induced by 1 U/mL thrombin is significantly higher than in platelets stored for 6 days (P&lt;0.001), but lower than in 13 day-old platelets (P&lt;0.001) and A23187-stimulated platelets (P&lt;0.0001). This study demonstrates that, aside from its ‘classical’ function as a coagulation factor and an inducer of platelet activation, thrombin can trigger platelet apoptosis. Thrombin appears to trigger platelet apoptosis by impacting on several intracellular apoptotic targets, including shifting the balance between Bcl-2 regulatory proteins in a pro-apoptotic direction, depolarizing the inner mitochondrial membrane, activating the executioner caspase-3, and stimulating aberrant exposure of phosphatidylserine on the platelet surface. Thrombin-induced platelet apoptosis may contribute to the pathophysiology of thrombocytopenia in diseases associated with enhanced thrombin generation, such as sepsis and disseminated intravascular coagulation.

Protocatechuic Acid Protects Platelets from Apoptosis via Inhibiting Oxidative Stress-Mediated PI3K/Akt/GSK3β Signaling

2021 ◽  
Author(s):  
Fuli Ya ◽  
Kongyao Li ◽  
Hong Chen ◽  
Zezhong Tian ◽  
Die Fan ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Caspase 3 ◽  
Protocatechuic Acid ◽  
Human Platelets ◽  
Ros Generation ◽  
Inhibitory Effects ◽  
Platelet Apoptosis ◽  
Phosphatidylserine Exposure ◽  
Underlying Mechanisms

AbstractOxidative stress plays crucial roles in initiating platelet apoptosis that facilitates the progression of cardiovascular diseases (CVDs). Protocatechuic acid (PCA), a major metabolite of anthocyanin cyanidin-3-O-β-glucoside (Cy-3-g), exerts cardioprotective effects. However, underlying mechanisms responsible for such effects remain unclear. Here, we investigate the effect of PCA on platelet apoptosis and the underlying mechanisms in vitro. Isolated human platelets were treated with hydrogen peroxide (H2O2) to induce apoptosis with or without pretreatment with PCA. We found that PCA dose-dependently inhibited H2O2-induced platelet apoptosis by decreasing the dissipation of mitochondrial membrane potential, activation of caspase-9 and caspase-3, and decreasing phosphatidylserine exposure. Additionally, the distributions of Bax, Bcl-xL, and cytochrome c mediated by H2O2 in the mitochondria and the cytosol were also modulated by PCA treatment. Moreover, the inhibitory effects of PCA on platelet caspase-3 cleavage and phosphatidylserine exposure were mainly mediated by downregulating PI3K/Akt/GSK3β signaling. Furthermore, PCA dose-dependently decreased reactive oxygen species (ROS) generation and the intracellular Ca2+ concentration in platelets in response to H2O2. N-Acetyl cysteine (NAC), a ROS scavenger, markedly abolished H2O2-stimulated PI3K/Akt/GSK3β signaling, caspase-3 activation, and phosphatidylserine exposure. The combination of NAC and PCA did not show significant additive inhibitory effects on PI3K/Akt/GSK3β signaling and platelet apoptosis. Thus, our results suggest that PCA protects platelets from oxidative stress-induced apoptosis through downregulating ROS-mediated PI3K/Akt/GSK3β signaling, which may be responsible for cardioprotective roles of PCA in CVDs.

scholarly journals Modulation of thrombin-mediated activation of factor VIII:C by calcium ions, phospholipid, and platelets

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 53-58
Author(s):  
MB Hultin
Keyword(s):  
Calcium Ions ◽  
Calcium Concentration ◽  
Human Platelets ◽  
Ionophore A23187 ◽  
Factor X ◽  
Platelet Activity ◽  
Platelet Surface ◽  
New Finding ◽  
Factor X Activation ◽  
External Calcium

The activation of factor VIII:C by thrombin appears to be an important prerequisite for the function of factor VIII:C as a cofactor in factor X activation in coagulation. The possible modulation of factor VIII:C activation by potential cofactors such as calcium ions, phospholipid, and platelets was studied systematically. Factor VIII:C activation could not be studied in the complete absence of Ca2+, since factor VIII:C activity decayed rapidly in calcium-free buffers, EDTA, or ethylene glycol tetra-acetic acid (EGTA), with only partial or no recovery of activity after readdition of Ca2+, Mn2+, or Mg2+. Added calcium chloride at 1.25, 2.5, 4, 10, 50, and 200 mmol/L produced progressive inhibition of factor VIII:C activation, with complete inhibition achieved by 50 mmol/L. Crude phospholipid preparations gave varying results, while purified phospholipids either had no effect or inhibited activation. This paper reports the new finding that fresh washed human platelets markedly potentiated factor VIII:C activation by a low concentration of thrombin (0.02 U/mL), even with prostaglandin E1 (PGE1) or dibutyryl cyclic AMP (cAMP) added to the washed platelets. However, the activity of platelets in factor VIII:C activation was inhibited by inclusion of PGE1 or dibutyryl cAMP during platelet washing, and ionophore A23187 increased this platelet activity; these data suggest that platelet stimulation is involved in the development of this activity. When platelets were maximally stimulated by thrombin (0.5 U/mL), the external calcium concentration increased 55 to 160 mumol/L, as measured with murexide, supporting the possible modulation of factor VIII:C activation by a transient increase in Ca2+ at the platelet surface.

scholarly journals Platelet Apoptosis in Adult Immune Thrombocytopenia. Relationship with Auto-Antibodies, Platelet Function and Treatment

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2792-2792 ◽  
Author(s):  
Geraldine Contrufo ◽  
Ana C Glembotsky ◽  
Nora P Goette ◽  
Paola R Lev ◽  
Matías Grodzielski ◽  
...  
Keyword(s):  
Platelet Count ◽  
Platelet Activation ◽  
Immune Thrombocytopenia ◽  
Calcium Ionophore ◽  
Activation Parameters ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Platelet Surface ◽  
Platelet Apoptosis ◽  
Cell Penetrating

Abstract Similarly to nucleated cells, platelet life span is also controlled by an intrinsic apoptotic program that triggers collapse of the mitochondrial inner membrane potential, activation of caspases-3, -8 and -9, phosphatidylserine (PS) externalization and microparticle shedding. The aim of the present study was to investigate platelet apoptosis in adult patients with immune thrombocytopenia (ITP) under different treatment conditions and to search for its relationship with the type of auto-antibody and the platelet-activation status. Twenty-four patients with chronic ITP, age 42 (21-80) years (median and range) diagnosed according to current criteria (Rodeghiero et al, 2009) were included after written informed consent in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee from Instituto de Investigaciones Medicas Alfredo Lanari. Platelet count was 38x109/L (6-85). Platelet apoptosis was evaluated by phosphatidylserine (PS) exposure on the platelet surface using FITC-conjugated Anexin-V, mitochondrial electrochemical potential changes (ΔΨm) using the cell penetrating lipophilic cationic fluorochrome JC-1, and activated caspase-3 (a-casp3) measured by the cell-penetrating carboxyfluorescein-labelled fluoromethyl ketone tetrapeptide (FAM-DEVD-FMK). These parameters were studied in resting platelets and after stimulation with calcium ionophore (A23187). Platelet activation was evaluated by FITC-PAC-1 binding to activated GPIIb-IIIa and GPIb-IX internalization using PE-CD42b, in resting conditions and after stimulation either with ADP or TRAP. Apoptosis and activation parameters were evaluated by flow cytometry. In resting conditions, platelets from ITP patients showed increased PS expression and a- casp3 and abnormal ΔΨm (table 1). TablePSΔΨma-casp3Patients19.6 (1.9-82.0)31.2 (5.8-92.4)11.3 (1.8-40.9)Controls4.7 (1.9-10.8)10.3 (2.2-27.5)4.3 (1.9-8.2)p (Mann-Whitney)0.002<0.00010.012n212312 After stimulation with A23187, ITP platelets had similar levels of PS expression (p=0.305, n=20) and ΔmΨ (p=0.383, n=25) compared to normal controls. However, an increased sensitivity to the apoptotic stimulus was evidenced by elevated levels of a-casp3 at low and high A23187 concentrations (1-3 mM, p=0.097; 6-10 mM, p=0.002). Platelet apoptosis was not related to platelet activation, as PAC-1 binding was not increased in ITP platelets (basal p=0.847, ADP-induced p=0.059, TRAP-induced p=0.103, n=16). Besides, internalization of GPIb-IX after ADP and TRAP stimulation was also normal (p=NS, n=9 for both agonists). Platelets from ITP patients bearing two of the most frequently found auto-antibodies (5 with anti-GPIIb-IIIa and 1 with anti-GPIb-IX) had similar levels of PS expression and ΔmΨ at resting conditions than those who were negative for these auto-antibodies (n=14) (p=0.265 and 0.148, respectively). There were no differences in apoptosis markers either in resting platelets or after stimulation when comparing untreated patients (n=9) vs patients under any kind of treatment (n=15) (resting conditions, PS p=0.737; ΔmΨ p=0.270; stimulated, PS p=0.966; ΔmΨ p=0.987), although platelet count was similar in both groups. Normal platelets incubated during 1 hour with plasma from ITP had higher a-casp3 than those incubated with normal plasma (n=12 and 9, respectively, p=0.027), suggesting a plasmatic component could be responsible for the apoptotic stimulus. In conclusion, increased platelet apoptosis in ITP patients could be induced by a plasmatic factor, contributing to thrombocytopenia in this entity. Disclosures Riveros: Roche: Speakers Bureau.

scholarly journals Modulation of thrombin-mediated activation of factor VIII:C by calcium ions, phospholipid, and platelets

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 53-58 ◽  
Author(s):  
MB Hultin
Keyword(s):  
Calcium Ions ◽  
Calcium Concentration ◽  
Human Platelets ◽  
Ionophore A23187 ◽  
Factor X ◽  
Platelet Activity ◽  
Platelet Surface ◽  
New Finding ◽  
Factor X Activation ◽  
External Calcium

Abstract The activation of factor VIII:C by thrombin appears to be an important prerequisite for the function of factor VIII:C as a cofactor in factor X activation in coagulation. The possible modulation of factor VIII:C activation by potential cofactors such as calcium ions, phospholipid, and platelets was studied systematically. Factor VIII:C activation could not be studied in the complete absence of Ca2+, since factor VIII:C activity decayed rapidly in calcium-free buffers, EDTA, or ethylene glycol tetra-acetic acid (EGTA), with only partial or no recovery of activity after readdition of Ca2+, Mn2+, or Mg2+. Added calcium chloride at 1.25, 2.5, 4, 10, 50, and 200 mmol/L produced progressive inhibition of factor VIII:C activation, with complete inhibition achieved by 50 mmol/L. Crude phospholipid preparations gave varying results, while purified phospholipids either had no effect or inhibited activation. This paper reports the new finding that fresh washed human platelets markedly potentiated factor VIII:C activation by a low concentration of thrombin (0.02 U/mL), even with prostaglandin E1 (PGE1) or dibutyryl cyclic AMP (cAMP) added to the washed platelets. However, the activity of platelets in factor VIII:C activation was inhibited by inclusion of PGE1 or dibutyryl cAMP during platelet washing, and ionophore A23187 increased this platelet activity; these data suggest that platelet stimulation is involved in the development of this activity. When platelets were maximally stimulated by thrombin (0.5 U/mL), the external calcium concentration increased 55 to 160 mumol/L, as measured with murexide, supporting the possible modulation of factor VIII:C activation by a transient increase in Ca2+ at the platelet surface.

PLATELET DISAGGREGATION IN PLASMA--A NOVEL EFFECT OF TISSUE PLASMINOGEN ACTIVATOR

1987 ◽  
Author(s):  
D E Vaughan ◽  
J Loscalzo
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Vascular Events ◽  
Platelet Surface ◽  
Platelet Disaggregation ◽  
Tissue Plasminogen ◽  
Circulating Platelet Aggregates ◽  
Platelet Aggregates

Platelet aggregates are thought to play a significant role in many clinically important ischemic vascular events. Recently it has been shown that the platelet surface binds plasminogen and, in so doing, enhances its conversion to plasmin by tissue plasminogen activator (tPA). Since fibrinogen, an alternative substrate for plasmin, serves as the cohesive link among platelets induced to aggregate by a variety of agonists, we hypothesized that the local production of plasmin at the platelet surface may be important in promoting the disaggregation of aggregated platelets. When added to a suspension of human platelets induced to aggregate in plasma with adenosine 5'-diphosphate, tPA promoted disaggregation over several minutes. The rate of disaggregation and its extent were dependent on the concentration of tPA as well as on its time of addition. Preincubation of platelet-rich plasma with excess alpha-2-antiplasmin inhibited disaggregation by tPA. While platelet surface fibrinogen receptors did not appear to be proteolyzed by plasmin in this plasma system, platelet-bound cohesive fibrinogen was selectively proteolyzed compared with proteolysis of ambient fibrinogen. The rate of disaggregation correlated best with the rate of loss of platelet-bound fibrinogen and not with the rate of ambient fi-brinogenolysis. These data demonstrate that tPA facilitates platelet disaggregation through the plasmin-mediated proteolysis of cohesive fibrinogen. This phenomenon may be important in the dispersal of circulating platelet aggregates and may be operative in the thrombolysis of platelet-rich clots.

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