Measurement of platelet p-selectin expression by flow cytometry in patients with acute ischemic stroke

AbstractAims: The aim of this study was to asses the platelet activation in the acute phase of ischemic stroke and transient ischemic attack (TIA) by defining p-selectin (CD62) expression by flow cytometry in vivo – without stimulation with agonists. We also studied whether antiplatelet therapy supresses the levels of baseline p-selectin expression and verified if there is a correlation between platelet CD62 expression and the type of ischemic stroke.Methods: We determined the expression of platelet surface p-selectin using whole-blood flow cytometry within the first 48-hours after onset of cerebral symptoms in patients with atherothrombotic and lacunar ischemic stroke and in healthy volunteers. We studied the realationship between antiplatelet medication and the type of ischemic stroke to baseline p-selectin expression.Results: Patients with acute cerebral ischemia have an excess of circulating platelets that express p-selectin, compared to healthy volunteers. The difference between average p-selectin expression in the group of healthy volunteers and the group of patients with stroke was statistically significant (p-value < 0,000001). Patients with stroke without antiplatelet medication showed a higher p-selectin expression than patients with antiplatelet medication (ASA, CLP, or ASA and CLP), hovewer, the difference was not statistically significant. There is no relationship between CD62 expression and the type of stroke.Conclusions: We can conclude that p-selectin is a highly sensitive blood biomarker of increased platelet activation. Antiplatelet therapy suppresses baseline p-selectin expression only minimally, insignificantly according to our results.

Download Full-text

Effects of Flavonoid Quercetin on Thrombo-Inflammatory Processes in Patients with Sickle Cell Disease

Blood ◽

10.1182/blood-2021-148601 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2020-2020

Author(s):

Maria A. Lizarralde ◽

Brenda Merriweather ◽

Anna Conrey ◽

Ankit Saxena ◽

Arun S Shet

Keyword(s):

Flow Cytometry ◽

Sickle Cell Disease ◽

Platelet Activation ◽

P Value ◽

Cell Disease ◽

Platelet Surface ◽

Flavonoid Quercetin ◽

Pre Treatment ◽

Stimulation Of

Abstract Introduction: Altered erythrocyte physiology in Sickle Cell Disease (SCD) results in severe vascular complications, including vaso-occlusion (VOC) along with various other manifestations, such as thrombosis and end organ damage. Moreover, platelet activation endothelial dysfunction and fibrin formation, are critical processes implicated in thrombo-inflammatory vasculopathy in SCD. Since flavonoids intake is associated with lowered adverse cardiovascular disease outcomes and cancer associated thrombosis, we are evaluating the effects of the flavonoid Isoquercetin (IQ) on thrombo-inflammatory biomarkers in patients with SCD (NCT:04514510). Here we report the mechanistic effects of Quercetin (Que), an aglycone that differs from IQ by the absence of 3-linked glucoside moiety, on markers of erythrocyte and platelet activation in patients with SCD. Methods: We studied patients with SCD (Genotype SS, mean age: 39 yrs, N=8) and ethnic matched controls (Genotype AA, mean age: 42 yrs, N=8). Erythrocyte reactive oxygen species (ROS) was measured in washed red cells by flow cytometry using the fluorescent probe 2', 7'-dichlorofluorescein diacetate (DCFDA) in the presence or absence of Quercetin (Que) (100 µM). Effects of in vitro erythrocyte ROS induction by hydrogen peroxide (H 2O 2) and ROS inhibition by N-acetyl-L-cysteine (NAC) were evaluated. Baseline and agonist-induced (ADP 20 µM) platelet P-selectin expression in vitro in the presence or absence of Que (100 µM) was assessed by flow cytometry. Results: At baseline, SS erythrocytes demonstrated elevated levels of ROS reflected by greater florescent intensity with DCFDA when compared with erythrocytes from ethnic matched controls (SS mean= 1884, SD: 508.8 vs. AA mean: 1604, SD: 554.5; p value= 0.3798). Stimulation of AA erythrocytes in vitro with H 2O 2 led to accumulation of ROS that approached levels similar to those exhibited by SS erythrocytes at baseline (AA mean: 1890, SD: 748.3). Pre-treatment of SS and AA erythrocytes in vitro with the antioxidant NAC exhibited minimal effects on ROS in SS erythrocytes (SS baseline+NAC mean: 1942, SD: 526.8, p value 0.4609 and SS H 2O 2+NAC mean: 1889, SD: 437.4, p value 0.5469) but lowered ROS levels in both baseline and H 2O 2 treated AA erythrocytes (AA baseline+NAC mean: 1442, SD: 453.9, p value 0.1563 and AA H 2O 2+NAC mean: 1581, SD: 518.7, p value 0.078). Pre-treatment of SS and AA erythrocytes in vitro with Que substantially lowered erythrocyte ROS in patients and controls at the baseline (SS baseline ROS with Que mean: 1582, SD: 542.0, p value 0.0078; AA baseline ROS with Que mean: 1341, SD: 490.9, p value 0.0313; Fig. 1A), and after H 2O 2 stimulation (SS H 2O 2 induced ROS+Que mean: 1677, SD: 572.5, p value 0.0156; AA H 2O 2 induced ROS+Que mean: 1417, SD: 512, p value 0.0078). Pre-treatment of erythrocytes with a combination of Que and NAC did not reveal synergistic anti-oxidant activity (SS NAC+Que mean: 1616, SD: 464.8; AA NAC+Que mean: 1333, SD: 524.1). We simultaneously investigated the effects of Que on platelet P-selectin expression in the same subjects. As expected, baseline platelet surface P-selectin expression was higher in SS patients compared with controls (SS N=5, mean: 1.48%, Max: 4.9%, Min: 0.03%; AA N=4, mean: 0.89%, Max: 2.3%, Min: 0%; p value 0.55). Stimulation of platelet rich plasma (PRP) with ADP increased surface P-selectin expression in comparison with baseline, in both patients and controls (SS N=5, mean: 18.04%, Max: 40.4%, Min: 5.8%, p value 0.0047; AA N=4, mean: 20.60%, Max: 43.8%, Min: 5.4%; p value 0.0140). Pre-treatment of PRP with Que prior to ADP stimulation revealed a trend toward decreased platelet surface P-selectin expression in both patients and controls (SS N=5, mean: 10.49%, Max: 24.8%, Min: 3.1%; AA N=4, mean: 12.09%, Max: 21.9%, Min: 3.4%; Fig 1B) although this did not return to baseline. Conclusion: The flavonoid Quercetin appears to have a beneficial effect in lowering erythrocyte ROS and platelet surface P-selectin and warrant further evaluation as a strategy to ameliorate thrombo-inflammatory pathophysiology in sickle hemoglobinopathies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Platelet Function following Acute Cerebral Ischemia

Angiology ◽

10.1177/0003319709332959 ◽

2009 ◽

Vol 60 (3) ◽

pp. 362-369 ◽

Cited By ~ 13

Author(s):

Jonathan Smout ◽

Alexander Dyker ◽

Marcus Cleanthis ◽

Gary Ford ◽

Patrick Kesteven ◽

...

Keyword(s):

Ischemic Stroke ◽

Platelet Aggregation ◽

Acute Ischemic Stroke ◽

Platelet Activation ◽

Initial Level ◽

Antiplatelet Agents ◽

Platelet Surface ◽

Acute Cerebral Ischemia ◽

Platelet Aggregates ◽

Sensitive Marker

Background Studies have previously identified increased levels of platelet activation following acute ischemic stroke. In order to evaluate new antiplatelet agents and their combinations, there is a need for accurate measures of platelet activation. Methods Blood was taken from 17 patients within 24 hours of an acute ischemic stroke, and then at 3, 7, 14 and 42 days. For comparison, a group of 18 stable arteriopaths had identical tests performed. Platelet aggregation was measured using a free platelet counting technique, and platelet surface P-selectin and monocyte platelet aggregates (MPAs) were measured using flow cytometry. Soluble P-selectin and D-dimers were measured by an enzyme linked immune assay. Results The initial level of MPAs was significantly raised in the stroke patients compared with the stable patients ( p = 0.04, 14.2% vs. 9.3%); however, this difference was not significantly higher than later study points (14.2%, 10.1%, 9.3%, 11.9%, 11.3%; days 1, 3, 7, 14 and 42 respectively. Day 1 vs. day 7 p = 0.07 ANOVA). No changes in P-selectin or platelet aggregation were identified. D-dimer levels were significantly higher on day 7 than day 42 ( p < 0.01), and fibrinogen levels were elevated on both days 3 and 14 compared with day 42. Fibrinogen levels were not elevated compared with stable patients. Conclusions MPA levels are elevated following an acute ischemic stroke compared to stable patients, but no significant change was seen with other platelet markers. This study suggests MPAs are a more sensitive marker of platelet activation than either P-selectin or aggregation.

Download Full-text

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

Blood ◽

10.1182/blood.v79.8.2011.2011 ◽

1992 ◽

Vol 79 (8) ◽

pp. 2011-2021 ◽

Cited By ~ 30

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.

Download Full-text

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

Blood ◽

10.1182/blood.v83.4.1006.1006 ◽

1994 ◽

Vol 83 (4) ◽

pp. 1006-1016 ◽

Cited By ~ 29

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.

Download Full-text

DETECTION OF ACTIVATED PLATELETS IN WHOLE BLOOD BY FLOW CYTOMETRY

10.1055/s-0038-1643830 ◽

1987 ◽

Cited By ~ 4

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.

Download Full-text

Fucosyltransferase VI Induces Platelet Activation: A Novel Property of a Plasma Glycosyltransferase.

Blood ◽

10.1182/blood.v114.22.4016.4016 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4016-4016

Author(s):

José-Tomás Navarro ◽

Shwan Tawfiq ◽

Roland Wohlgemuth ◽

Karin M. Hoffmeister ◽

Robert Sackstein

Keyword(s):

Flow Cytometry ◽

Platelet Aggregation ◽

Platelet Activation ◽

Light Transmission ◽

Conflicts Of Interest ◽

Platelet Rich Plasma ◽

Surface Protein ◽

Surface Flow ◽

Platelet Surface ◽

Biochemical Studies

Abstract Abstract 4016 Poster Board III-952 A number of glycosyltransferases are present in human plasma with the α(1→3) fucosyltransferase, Fucosyltransferase VI (FTVI), having the highest plasma concentration. Notably, elevated plasma levels of FTVI are associated with a variety of cancers and correlate with tumor load/progression. The well-known association of neoplasia with thromboembolic complications prompted us to examine whether FTVI has direct effect(s) on platelet function. We obtained human platelets from blood of healthy donors and separated from platelet-rich plasma by differential centrifugation. Freshly isolated platelets (x108/ml) were stirred and exposed at 37°C to varying concentrations (20, 40, 60 and 80 mU/mL) of glycosyltransferases FTVI, β-1-4-galactosyltransferase-I (βGalT-I), or α,2-3-N-sialyltransferase (α2,3-N-ST), or to 1 U/mL thrombin. Platelet aggregation and activation was assessed by aggregometry (light transmission) or by flow cytometry of FSC/SSC characteristics and of surface expression of P-Selectin, respectively. FT-VI reproducibly induced platelet aggregation and activation, whereas other glycosyltransferases (β4GalT-I and α2,3-N-ST) had no effect on platelets. FTVI activation of platelets was concentration-dependent, and the aggregation curve for FTVI was one wave, similar to that for thrombin. FTVI-induced platelet activation was independent of catalytic conversion of surface glycans, but was inhibited by FTVI denaturation, indicating that FTVI-induced platelet activation is a lectin-mediated process. To determine the membrane target(s) mediating FTVI-induced platelet activation, biochemical studies were performed after catalytic exofucosylation of the platelet surface. Flow cytometry after platelet exofucosylation showed formation of the carbohydrate structure sLex, detected by the mAb Heca452, but no formation of Lex (CD15). Western blot showed that enforced fucosylation induced sLex on a single platelet surface protein, and further biochemical studies revealed that this protein is GPIbα. These findings unveil a previously unrecognized property of FTVI as an activator of platelets, mediated via a specific lectin/carbohydrate interaction on GP1ba, and offer novel perspectives on the pathobiology of tumor-associated thrombogenesis. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Lack of uniform platelet activation in patients after ischemic stroke and choice of antiplatelet therapy

Thrombosis Research ◽

10.1016/j.thromres.2004.03.002 ◽

2004 ◽

Vol 113 (3-4) ◽

pp. 197-204 ◽

Cited By ~ 25

Author(s):

Victor L Serebruany ◽

Alex I Malinin ◽

Benjamin R Oshrine ◽

David C Sane ◽

Aviv Takserman ◽

...

Keyword(s):

Ischemic Stroke ◽

Antiplatelet Therapy ◽

Platelet Activation

Download Full-text

Clopidogrel responder status is uninfluenced by CYP2C19*2 in Danish patients with stroke

PLoS ONE ◽

10.1371/journal.pone.0236260 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0236260

Author(s):

Charlotte Lützhøft Rath ◽

Niklas Rye Jørgensen ◽

Troels Wienecke

Keyword(s):

Ischemic Stroke ◽

Platelet Reactivity ◽

Adenosine Diphosphate ◽

Platelet Inhibition ◽

Nucleotide Polymorphisms ◽

Transient Ischemic Attacks ◽

Platelet Surface ◽

Antiplatelet Medication ◽

Long Term Treatment ◽

Adp Receptor

Background Antiplatelet therapy is a cornerstone of secondary stroke prevention, but the responsiveness to antiplatelet medication varies among patients. Clopidogrel is a pro-drug that requires hepatic transformation to reach its active metabolite. Single nucleotide polymorphisms (SNPs) in key enzymes or the target adenosine diphosphate (ADP) receptor on the platelet surface are believed to be involved in clopidogrel-mediated platelet inhibition and decreased antiplatelet effect with high-on-treatment platelet reactivity (HTPR). Objective This study investigated whether specific SNPs in key hepatic enzymes (CYP2C19*2, *3, *17, CYP3A4*1G, and NR1I2) or the ADP receptor (PR2Y12) are associated with HTPR to clopidogrel. Patients & methods This observational study included patients with ischemic stroke (IS) and transient ischemic attacks (TIAs) receiving clopidogrel at a dose of 75 mg/day. Patients were genotyped for eight different SNPs in the genes encoding CYP2C19, CYP3A4, NR1I2, and the P2Y12 receptor. Results Of the 103 patients that were included, 30.7% carried the CYP2C19*2 allele and had higher platelet reaction unit (PRU) values than non-carriers, but no patients showed HTPR. Carriers of the *17 allele had higher platelet inhibition but showed no difference in PRU values compared with non-carriers. The remaining SNPs were neither associated with PRU nor with platelet inhibition. Conclusions Patients with IS and TIAs treated with 75 mg clopidogrel/day do not have HTPR. A genetic analysis of CYP2C19*2, *3, *17, CYP3A4*1G, and NR1I2 revealed no associations with clopidogrel HTPR. CYP2C19*2 carriers and patients with HTPR in the acute phase after ischemic stroke or transient ischemic attacks exhibit higher PRU values, but not long-term treatment HTPR.

Download Full-text

Direct detection of activated platelets and platelet-derived microparticles in humans

Blood ◽

10.1182/blood.v75.1.128.bloodjournal751128 ◽

1990 ◽

Vol 75 (1) ◽

pp. 128-138 ◽

Cited By ~ 3

Author(s):

CS Abrams ◽

N Ellison ◽

AZ Budzynski ◽

SJ Shattil

Keyword(s):

Flow Cytometry ◽

Cardiopulmonary Bypass ◽

Platelet Activation ◽

Bleeding Time ◽

Relative Proportion ◽

Open Heart Surgery ◽

Normal Range ◽

In Vivo Models ◽

Platelet Surface ◽

Activated Platelets

Flow cytometry was used to determine whether activated platelets and platelet-derived microparticles can be detected directly in whole blood after a hemostatic insult. Two different in vivo models of platelet activation were examined: (1) a standardized bleeding time, and (2) cardiopulmonary bypass. Platelets and microplatelets were identified with a biotinylated anti-glycoprotein (GP)lb antibody and a fluorophore, phycoerythrin-streptavidin. Microparticles were distinguished from platelets by light scatter. Activated platelets were detected with three fluorescein-labeled monoclonal antibodies (MoAbs): (1) PAC1, which binds to the activated form of GPIIb-IIIa; (2) 9F9, a newly developed antibody that is specific for fibrinogen bound to the surface of activated platelets; and (3) S12, which binds to an alpha- granule membrane protein expressed on the platelet surface after granule secretion. In nine normal subjects, bleeding times ranged from 4.5 to 7.5 minutes. Over this time, there was a progressive increase in the amount of PAC1, 9F9, and S12 bound to platelets in blood emerging from the bleeding time wound. With all three antibodies, platelet activation was apparent as early as 30 seconds after the incision (P less than .03). Activation was accompanied by a progressive decrease in the concentration of platelets in blood from the wound, while the concentration of microparticles increased slightly. In nine patients undergoing open heart surgery, 1 hour of cardiopulmonary bypass caused a 2.2-fold increase in the relative proportion of microparticles in circulating blood (P less than .001). Moreover, bypass caused platelet activation as evidenced by a mean two- to threefold increase in PAC1 binding to platelets. Although this increase was significant (P less than .02), PAC1 binding exceeded the normal range for unstimulated control platelets in only 5 of 9 patients, and 9F9 and S12 binding exceeded the normal range in only two patients. Taken together, these studies demonstrate that it is now feasible using flow cytometry to evaluate the extent of platelet activation and the presence of platelet- derived microparticles in the circulation of humans.

Download Full-text

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

Blood ◽

10.1182/blood.v81.10.2554.bloodjournal81102554 ◽

1993 ◽

Vol 81 (10) ◽

pp. 2554-2565 ◽

Cited By ~ 5

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.

Download Full-text