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.

CALPAIN-MEDIATED CYTOSKELETAL DEGRADATION CORRELATES WITH STIMULATION OF PLATELET PROCOAGULANT ACTIVITY

1987 ◽  
Author(s):  
P F J verhallen ◽  
E M Bevers ◽  
P Comfurius ◽  
W M A Linkskens ◽  
R F A Zwaal
Keyword(s):  
Outer Surface ◽  
Time Course ◽  
Degradation Products ◽  
Cytoskeletal Proteins ◽  
Procoagulant Activity ◽  
Ionophore A23187 ◽  
Calpain Activity ◽  
Similar Time ◽  
Coagulant Activity ◽  
Relationship Of

We have shown earlier that the negatively charged phospholipid phosphatidylserine (PS), which becomes translocated from the inner surface to the outer surface of the plasma membrane upon platelet activation, is responsible for platelet procoagulant activity. Studies with erythrocytes have suggested a role for cytoskeletal proteins in the regulation of transmembrane asymmetry of PS. The possibility that platelet cytoskeletal proteins are involved in the loss of transmembrane asymmetry of PS, was explored by correlative investigations of both platelet prooagulant activity and activity of calpain, an endogenous Ca 2+ -dependent thiol-protease, known to hydrolyze major cytoskeletal proteins (e.g.: filamin, talin, myosin). Platelet procoagulant activity was assayed by determination of the prothrombinase activity under conditions at which the catalytic PS-surface was rate-limiting. Calpain-activity was monitored by the appearance of known degradation products of major cytoskeletal proteins. The following results were obtained: (1) The ability of thrombin, collagen, collagen & thrombin, or the Ca -ionophore A23187 to stimulate platelet procoagulant activity closely correlated with their ability to stimulate platelet calpain-activity (2). Generation of platelet procoagulant activity upon platelet stimulation by collagen & thrombin or by A23187 exhibited a time course identical to the development of calpain-activity. In addition, the local anesthetics dibucaine and tetracaine, shown to gradually stimulate calpain activity, were able to generate platelet procoagulant activity with a similar time course. (3) Using a Ca2+ buffering system and A23187 to equilibrate intracellular- and extracellular free Ca2+ , it was found that the Ca2+ -response relationship of both platelet calpain- and pro-coagulant-activity was identical. From these findings we conclude that the degradation of cytoskeletal proteins destroys their putative interactions with PS, enabling this lipid to participate in transbilayer movement, leading to the formation of a procoagulant outer surface of the platelet.

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.

High-dose epinephrine enhances platelet aggregation at the expense of procoagulant activity

2021 ◽  
Author(s):  
Alessandro Aliotta ◽  
Debora Bertaggia Calderara ◽  
Maxime G Zermatten ◽  
Lorenzo Alberio
Keyword(s):  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Light Transmission ◽  
Shape Change ◽  
Annexin V ◽  
Cytosolic Calcium ◽  
Calcium Mobilization ◽  
Procoagulant Activity ◽  
High Dose

Platelet activation is characterized by shape change, granule secretion, activation of fibrinogen receptor (glycoprotein [GP] IIb/IIIa) sustaining platelet aggregation, and externalization of negatively charged aminophospholipids contributing to platelet procoagulant activity. Epinephrine alone is a weak platelet activator. However, it is able to potentiate platelet activation initiated by other agonists. In this work, we investigated the role of epinephrine in the generation of procoagulant platelets. Human platelets were activated with convulxin (CVX), thrombin (THR) or protease-activated receptor (PARs) agonists, epinephrine (EPI), and combination thereof. Platelet aggregation was assessed by light transmission aggregometry or with PAC-1 binding by flow cytometry. Procoagulant collagen-and-thrombin (COAT) platelets, induced by combined activation with CVX-and-THR, were visualized by flow cytometry as Annexin-V-positive and PAC-1-negative platelets. Cytosolic calcium fluxes were monitored by flow cytometry using Fluo-3 indicator. EPI increased platelet aggregation induced by all agonist combinations tested. On the other hand, EPI dose-dependently reduced the formation of procoagulant COAT platelets generated by combined CVX-and-THR activation. We observed a decreased Annexin-V positivity and increased binding of PAC-1 with the triple activation (CVX+THR+EPI) com-pared with CVX+THR. Calcium mobilization with triple activation was decreased with the higher EPI dose (1000 µM) compared with CVX+THR calcium kinetics. In conclusion, when platelets are activated with CVX-and-THR, the addition of increasing concentrations of EPI (triple stimulation) modulates platelet response reducing cytosolic calcium mobilization, decreasing procoagulant activity and en-hancing platelet aggregation.

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<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<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<0.001) and stored for 0, 6 (P<0.001) and 13 days at 22°C (P<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<0.0001). Thus, PS exposure induced by 1 U/mL thrombin is significantly higher than in platelets stored for 6 days (P<0.001), but lower than in 13 day-old platelets (P<0.001) and A23187-stimulated platelets (P<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.

scholarly journals von Willebrand factor bound to glycoprotein Ib is cleared from the platelet surface after platelet activation by thrombin

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2011-2021 ◽  
Author(s):  
P Hourdille ◽  
HR Gralnick ◽  
E Heilmann ◽  
A Derlon ◽  
AM Ferrer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Surface Expression ◽  
Von Willebrand Disease ◽  
Platelet Surface ◽  
Immunogold Staining ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We recently reported that after activation of human platelets by thrombin, glycoprotein (GP) Ib-IX complexes are translocated to the surface-connected canalicular system (SCCS) (Blood 76:1503, 1990). As GPIb is a major receptor for von Willebrand factor (vWF) in platelet adhesion, we have now examined the consequences of thrombin activation on the organization of vWF bound to GPIb on the platelet surface. Studies were performed using monoclonal or polyclonal antibodies in either immunogold staining and electron microscopy (Au-EM) or in flow cytometry. When unstirred platelet-rich plasma was incubated with ristocetin, bound vWF was located by Au-EM as discrete masses regularly distributed over the cell surface. Platelets from a patient with Glanzmann's thrombasthenia, lacking GPIIb-IIIa complexes, gave a similar pattern, confirming that this represented binding to GPIb. That ristocetin was not precipitating vWF before their binding to the platelets was shown by the detection of similar masses on the surface of platelets of a patient with type IIB von Willebrand disease. Experiments were continued using washed normal platelets incubated in Tyrode-EDTA, the purpose of the EDTA being to limit the surface expression of endogenous vWF after platelet stimulation. Under these conditions, platelets were treated with ristocetin for 5 minutes at 37 degrees C in the presence of increasing amounts of purified vWF. This was followed by incubation with thrombin (0.5 U/mL) for periods of up to 10 minutes. Flow cytometry showed a time-dependent loss in the surface expression of vWF bound to GPIb and these changes were confirmed by Au-EM. In particular, immunogold staining performed on ultrathin sections showed that the bulk of the vWF was being cleared to internal membrane systems. Surface clearance of vWF during thrombin- induced platelet activation is a potential mechanism for regulating platelet adhesivity.

scholarly journals Factor XIIIa binding to activated platelets is mediated through activation of glycoprotein IIb-IIIa

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 1006-1016 ◽  
Author(s):  
AD Cox ◽  
DV Devine
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Activation ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
A Chain ◽  
Functional Receptor

Abstract Stabilization of a clot is dependent on fibrin cross-linking mediated by the transglutaminase, factor XIIIa (FXIIIa). In addition to fibrin stabilization, FXIIIa acts on a number of platelet-reactive proteins, including fibronectin and vitronectin, as well as the platelet proteins, glycoprotein (GP) IIb-IIIa, myosin, and actin. However, conditions inducing the platelet-activation dependent binding of FXIIIa have not been characterized nor have the sites mediating FXIIIa binding been identified. The generation of FXIIIa and consequent detection of FXIIIa on the platelet surface were compared with other thrombin- induced activation events; the rate at which FXIIIa bound to activated platelets was much slower than platelet degranulation or fibrin(ogen) binding. Whereas platelets could be rapidly induced to express a functional receptor for FXIIIa, the rate of FXIIIa binding to platelets is limited by the rate of conversion of FXIII to FXIIIa. Immunoprecipitation of radiolabeled platelets using polyclonal anti- FXIII A-chain antibody identified two proteins corresponding to GPIIb and GPIIIa. Preincubation of intact platelets with 7E3, a monoclonal antibody that blocks the fibrinogen binding site, or GRGDSP peptide inhibited FXIIIa binding by about 95% when measured by flow cytometry; FXIIIa binding to purified GPIIb-IIIa was also inhibited by 7E3. The binding of FXIIIa to purified GPIIb-IIIa was enhanced by the addition of fibrinogen, but not by that of fibronectin or thrombospondin, suggesting that FXIIIa also binds to fibrinogen associated with the complex. These observations suggest that activated platelets bearing FXIIIa may enhance stabilization of platelet-rich thrombi through surface-localized cross-linking events.

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.

Flow Cytometry Detection of Platelet Procoagulant Activity and Microparticles in Patients with Unstable Angina Treated by Percutaneous Coronary Angioplasty and Stent Implantation

2001 ◽  
Vol 86 (09) ◽  
pp. 784-790 ◽  
Author(s):  
Catherine Vidal ◽  
Christian Spaulding ◽  
Françoise Picard ◽  
Frédéric Schaison ◽  
Josiane Melle ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
Stent Implantation ◽  
Whole Blood ◽  
Coronary Angioplasty ◽  
Annexin V ◽  
Percutaneous Coronary Angioplasty ◽  
Blood Samples ◽  
Percutaneous Coronary ◽  
Whole Blood Samples

SummaryPlatelet activation is known to participate to the pathogenesis of acute coronary syndromes. Aminophospholipid exposure and micro-particles shedding are hallmarks of full platelet activation and may account for the dissemination of prothrombotic seats. Using flow cytometry analysis of annexin V binding to externalized aminophospholipids, we followed platelet procoagulant activity (PPA) and platelet microparticles (PMP) shedding in venous and coronary whole blood samples from 30 patients with unstable angina before and after percutaneous coronary angioplasty (PTCA) and stent implantation. Baseline values of PPA and PMP were significantly more elevated in patients than in control subjects (p <0.005). PMP percentage was significantly higher in coronary than in venous blood, and in coronary blood of patients with proximal instead of mid/distal lesions of coronary arteries. No enhancement of platelet reactivity to TRAP and collagen was induced by procedure. Whereas activated GpIIb-IIIa and P-selectin expression decreased 24 h and 48 h after procedure, PPA and PMP remained as elevated as before. Thus, flow cytometry is a reliable method for detection of fully activated platelets in whole blood samples. Annexin V binding analysis demonstrates the persistance of in vivo platelet activation, despite the use of antiaggregating agents.

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.

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