The active metabolite of prasugrel inhibits ADP-stimulated thrombo-inflammatory markers of platelet activation: Influence of other blood cells, calcium, and aspirin

2007 ◽  
Vol 98 (07) ◽  
pp. 192-200 ◽  
Author(s):  
Joseph Jakubowski ◽  
You FuLi ◽  
Marc Barnard ◽  
Marsha Fox ◽  
Matthew Linden ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Whole Blood ◽  
Blood Cells ◽  
Inflammatory Markers ◽  
Active Metabolite ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Flow Cytometric Analysis ◽  
Platelet Surface

SummaryThe novel thienopyridine prodrug prasugrel, a platelet P2Y12 ADP receptor antagonist, requires in vivo metabolism for activity. Although pharmacological data have been collected on the effects of prasugrel on platelet aggregation,there are few data on the direct effects of the prasugrel’s active metabolite, R-138727, on other aspects of platelet function. Here we examined the effects of R-138727 on thrombo-inflammatory markers of platelet activation, and the possible modulatory effects of other blood cells, calcium, and aspirin. Blood (PPACK or citrate anticoagulated) from healthy donors pre- and post-aspirin was incubated with R-138727 and the response to ADP assessed in whole blood or platelet-rich plasma (PRP) by aggregometry and flow cytometric analysis of leukocyte-platelet aggregates,platelet surface P-selectin, and GPIIb-IIIa activation. Low-micromolar concentrations of R-138727 resulted in a rapid and consistent in-hibition of these ADP-stimulated thrombo-inflammatory markers.These rapid kinetics required physiological calcium levels, but were largely unaffected by aspirin. Lower IC50 values in whole blood relative to PRP suggested that other blood cells affect ADP-induced platelet activation and hence the net inhibition by R-138727. R-138727 did not inhibit P2Y12-mediated ADP-induced shape change, even at concentrations that completely inhibited platelet aggregation, confirming the specificity of R-138727 for P2Y12. In conclusion, R-138727, the active metabolite of prasugrel, results in rapid, potent, consistent, and selective inhibition of P2Y12-mediated up-regulation of thromboinflammatory markers of platelet activation.This inhibition is enhanced in the presence other blood cells and calcium,but not aspirin.

scholarly journals The Effect of Tirofiban on Fibrinogen/Agonist-Induced Platelet Shape Change and Aggregation

2008 ◽  
Vol 14 (3) ◽  
pp. 295-302 ◽  
Author(s):  
I. Anita Jagroop ◽  
Dimitri P. Mikhailidis
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Adenosine Diphosphate ◽  
Vascular Events ◽  
Spontaneous Platelet Aggregation ◽  
Induced Aggregation ◽  
Platelet Shape

There is evidence linking raised plasma fibrinogen (fib) and platelet hyperactivity with vascular events. One way to inhibit platelets is to block the platelet membrane glycoprotein (GP) IIb/IIIa receptor, which binds circulating fib or von Willebrand factor and cross-links platelets at the final common pathway to platelet aggregation. Tirofiban is a potent and specific fib receptor antagonist, used in the treatment of unstable angina. The authors assessed the effect of tirofiban on spontaneous platelet aggregation (SPA), fib-induced, serotonin (5HT)-induced, and adenosine diphosphate (ADP)-induced aggregation in whole blood by calculating the percentage free platelet count. These various agonists were used alone and in combination. The authors also measured the effect of tirofiban on agonists-induced (ADP, 5HT) platelet shape change (PSC). The effect of fib on PSC was also evaluated in platelet-rich plasma using a high-resolution (0.07 fL) channelyzer. Tirofiban significantly inhibited SPA, fib (2, 4, 8 g/L), ADP, ADP + fib combination, and 5HT-induced aggregation. Tirofiban had no effect on agonist-induced PSC. There was no apparent change in platelet volume with fib. In conclusion, tirofiban does not appear to have an effect on PSC, an early phase of platelet activation. Tirofiban seems to be a nonspecific and an effective inhibitor of platelet aggregation (a later phase of platelet activation) in whole blood. The clinical significance of these findings remains to be established.

scholarly journals Ultrastructural Studies of Platelet Aggregates From Human Subjects Receiving Clopidogrel and From a Patient With an Inherited Defect of an ADP-Dependent Pathway of Platelet Activation

1996 ◽  
Vol 16 (12) ◽  
pp. 1532-1543 ◽  
Author(s):  
Michel Humbert ◽  
Paquita Nurden ◽  
Claude Bihour ◽  
Jean-Max Pasquet ◽  
Joëlle Winckler ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
High Dose ◽  
Platelet Response ◽  
Platelet Surface ◽  
Ultrastructural Studies ◽  
Contact Points ◽  
Platelet Aggregates

Our study investigated the effect of the antithrombotic drug clopidogrel (75 mg/d for 7 days) on the ultrastructure of platelet aggregates induced by ADP or 2-methylthio-ADP (2-MeS-ADP) in citrated platelet-rich plasma and examined the activation state of the GP IIb/IIIa complexes. Results were compared with those obtained for patient M.L., who has a congenital disorder characterized by a reduced and reversible platelet response to ADP. When untreated normal platelets were stimulated with high-dose ADP, electron microscopy revealed large and stable aggregates often surrounded by a layer of what appeared to be degranulated platelets. The reversible aggregates of platelets from subjects receiving clopidogrel or from patient M.L. did not show this layer. Electron microscopy showed that in both situations, the aggregates were composed of loosely bound platelets with few contact points. Immunogold labeling of ultrathin sections of Lowicryl-embedded aggregates formed by ADP or 2-MeS-ADP showed a much decreased platelet surface staining by (1) a polyclonal anti-fibrinogen antibody and (2) AP-6, a murine anti–ligand-induced binding site monoclonal antibody specific for GP IIb/IIIa complexes occupied with fibrinogen. Similar findings were seen after disaggregation, when many single platelets were present that showed no signs of secretion. Flow cytometry confirmed that the number of ligand-occupied GP IIb/IIIa complexes was much lower on platelets stimulated with ADP or 2-MeS-ADP after clopidogrel treatment. As expected from previous studies, ADP-induced platelet shape change and Ca 2+ influx were unaffected by clopidogrel. These results agree with the hypothesis that platelet activation by ADP is biphasic and highlight a receptor-induced activation pathway affected by clopidogrel (or congenitally impaired in patient M.L.) that is necessary for the full activation of GP IIb/IIIa and the formation of stable macroaggregates.

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

Blood ◽  
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.

Changes in G-Actin after Platelet Activation in Platelet Rich Plasma

1992 ◽  
Vol 68 (06) ◽  
pp. 727-730 ◽  
Author(s):  
S Heptinstall ◽  
J Glenn ◽  
P Spangenberg
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Actin Polymerization ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Inhibition Assay ◽  
Activated Platelets ◽  
Lysis Buffer ◽  
Two Phases ◽  
Rapid Conversion

SummaryWe have used the DNase I inhibition assay to study changes in G-actin after platelet activation in platelet-rich plasma (PRP) induced by ADP. Because of problems associated with depolymerization of F-actin after lysis of ADP-activated platelets in the presence of plasma, G-actin was measured using a lysis buffer that contained formaldehyde to prevent any depolymerization of F-actin.Different patterns of response were seen depending on the concentration of ADP used, and these were modified by avoiding aggregation by either not stirring the sample or by adding EDTA. The results show rapid conversion of G-actin to F-actin in association with shape change, and there is a further decrease in G-actin associated with irreversible platelet aggregation. Thus evidence is presented that actin polymerization occurs in two phases after ADP stimulation.

scholarly journals Platelets as Predictors of Vascular Risk: Is There a Practical Index of Platelet Activity?

2003 ◽  
Vol 9 (3) ◽  
pp. 177-190 ◽  
Author(s):  
Stavroula Tsiara ◽  
Moses Elisaf ◽  
I. Anita Jagroop ◽  
Dimitri P. Mikhailidis
Keyword(s):  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Vascular Risk ◽  
Platelet Activity ◽  
Vascular Events ◽  
Bioactive Substances ◽  
Platelet Surface ◽  
Soluble P

Activated platelets play a role in the pathogenesis of coronary heart disease (CHD). Following activation, platelets change shape, aggregate, and release several bioactive substances. The aim of this review is to identify if there is a simple and cost-effective method that indicates platelet activation and predicts the risk of CHD and vascular events. The rationale for identifying high-risk patients is to reduce their risk of vascular events by administering appropriate and effective antiplatelet treatment, like aspirin, clopidogrel, or combination regimens. Many laboratory tests estimating platelet activity have been described. Some are relatively simple, such as spontaneous or agonist-induced platelet aggregation. Other tests include measuring the mean platelet volume (MPV) or plasma soluble P-selectin levels. Some more complex tests include flow cytometry to determine platelet GP Ilb/Illa receptors, platelet surface P-selectin, plateletmonocyte aggregates, and microparticles. Only few prospective studies assessed the predictive value of platelet activation in healthy individuals. Although the MPV seems an 'easy method, there are insufficient data supporting its ability to predict the risk of a vascular event in healthy adults. Platelet aggregation, in whole blood or in platelet-rich plasma was not consistently predictive of vascular risk. Soluble P-selectin measurement is a promising method but it needs further evaluation. Flow cytometry methods are costly, time-consuming, and need specialized equipment. Thus, they are unlikely to be useful in estimating the risk in large numbers of patients. There is as yet no ideal test for the detection of platelet activation. Each currently available test has merits and disadvantages. Simple methods such as the MPV and the determination of platelet release products need further evaluation.

The Two Phases of Heparin-Induced Thrombocytopenia (HIT): Early Monocyte/Tissue Factor (TF) Phase and Late Platelet Phase

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 270-270
Author(s):  
Lubica Rauova ◽  
Valerie Tutwiler ◽  
Hyun Sook Ahn ◽  
Vincent M. Hayes ◽  
Richard H. Aster ◽  
...  
Keyword(s):  
Cell Surface ◽  
Tissue Factor ◽  
Platelet Activation ◽  
Whole Blood ◽  
Blood Cells ◽  
Flow Cytometric Analysis ◽  
Passive Immunization ◽  
Prothrombotic State ◽  
Monocyte Activation ◽  
Platelet Factor

Abstract Abstract 270 HIT is an immune thrombocytopenic disorder associated with a high risk of thrombosis. Mechanistic studies have focused on circulating platelet factor 4 (PF4)/heparin complexes and on the subsequent activation of platelets. We and others have shown that binding of PF4 to cell surface glycosaminoglycans (GAGs) not only makes platelets critical targets in the pathogenesis of HIT, but monocytes and neutrophils as well. Our previous observations that monocyte depletion using clodronate-laden liposomes prior to induction of HIT in a passive immunization model mitigated the prothrombotic state, but paradoxically exacerbated initial thrombocytopenia, suggest that our understanding of the relationship between monocyte activation, platelet activation, thrombocytopenia and thrombosis is unsettled. Chondroitin sulfate is the predominant GAG expressed by platelets; therefore, they bind PF4 less avidly than monocytes, which have a cell surface rich in heparan and dermatan sulfates. Based on this cell-type difference in surface affinity for PF4, we hypothesized that: 1) monocytes and perhaps other vascular cells bind PF4 and form surface PF4/HIT antigenic complexes preferentially when compared with platelets, and 2) depletion of this high-affinity (monocyte) “sink” shifts PF4 binding to platelets making them more targeted. Flow cytometric analysis of whole mouse and human blood support these hypotheses as monocytes bind ∼100-fold more FITC-labeled human (h) PF4 than platelets or red blood cells and ∼10-fold more than neutrophils or lymphocytes. When isolated platelets and white blood cells are admixed, the amount of exogenously added hPF4 bound to platelets is inversely related to the leukocyte:platelet ratio. In vitro, exposure of whole blood to the HIT-like monoclonal antibody KKO plus recombinant hPF4 generated an intense platelet activation characterized by binding of annexin V and factor Xa, consistent with formation of coated platelets. Importantly, formation of coated platelets was attenuated by monocyte depletion from whole blood samples or by inhibition of thrombin by PPACK. We then infused KKO into transgenic mice expressing hPF4 and hFcgRIIA to induce HIT and followed the temporal profile of antibody binding to various cell types. Binding of KKO to monocytes was detected within 30 min of injection. These monocytes remained the predominant target over the first 4 hrs, after which binding decreased as the circulating monocytes were depleted. The mechanism and implications of this relatively late monocytopenia during HIT is under study. However, these data provide an explanation of how clodronate-laden monocyte depletion prior to inducing HIT exacerbated thrombocytopenia in the murine model of HIT, while decreasing the prothrombotic state: Early in the disease when PF4 is limited, monocytes selectively bind the PF4 and are targeted by HIT antibodies, which induces tissue factor and generates thrombin, but limits initial thrombocytopenia. Later, after induction of large amounts of TF, activated monocytes are cleared, shifting the target of antigen-antibody interactions to the surface of platelets, enhancing their response to available thrombin, thereby establishing a feed-forward prothrombotic cycle and more platelet clearance. In conclusion, we propose that HIT evolves from a monocyte-focused to a platelet-focused disease and that early intervention to prevent monocyte activation provides a new important potential therapeutic target for intervention at the earliest stages of disease recognition that may become less effective as time passes. Disclosures: Cines: Amgen Inc.: Consultancy; GlaxoSmithKline: Consultancy; Eisai: Consultancy.

Reversible Inhibition of Human Platelet Activation by Hypothermia In Vivo and In Vitro

1994 ◽  
Vol 71 (05) ◽  
pp. 633-640 ◽  
Author(s):  
Alan D Michelson ◽  
Hollace MacGregor ◽  
Marc R Barnard ◽  
Anita S Kestin ◽  
Michael J Rohrer ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Whole Blood ◽  
Peripheral Blood ◽  
Bleeding Time ◽  
Human Platelet ◽  
Platelet Surface ◽  
Shed Blood

SummaryA hypothermia-induced hemorrhagic diathesis is associated with cardiopulmonary bypass, major surgery, and multiple trauma, but its pathophysiological basis is not well understood. We examined the hypothesis that hypothermia reversibly inhibits human platelet activation in vitro and in vivo. Platelet activation was studied in normal volunteers by whole blood flow cytometric analysis of modulation of platelet surface GMP-140 and the glycoprotein (GP) Ib-IX complex in: a) shed blood emerging from a standardized in vivo bleeding time wound; b) peripheral blood activated in vitro with either thrombin (in the presence of gly-pro-arg-pro, an inhibitor of fibrin polymerization) or the stable thromboxane (TX) A2 analogue U46619. Platelets in peripheral whole blood were activated at temperatures between 22° C and 37° C. the forearm skin temperature was maintained at temperatures between 22° C and 37° C prior to and during the bleeding time incision. Platelet aggregation was studied in shed blood by flow cytometry and in peripheral blood by aggregometry. Generation of TXB 2 (the stable metabolite of TXA 2) was determined by radioimmunoassay. In vitro, hypothermia inhibited both thrombin- and U46619-induced upregulation of GMP-140, downregulation of the GPIb-IX complex, platelet aggregation, and TXB2 generation. These inhibitory effects of hypothermia were all completely reversed by rewarming the blood to 37° C. In vivo, platelet activation was inhibited by hypothermia as shown by 5 independent assays of shed blood: upregulation of GMP-140, downregulation of the GPIb-IX complex, platelet aggregate formation, TXB 2 ggeneration, and the bleeding time. In summary, by a combination of immunologic, biochemical, and functional assays, we demonstrate that hypothermia inhibits human platelet activation in whole blood in vitro and in vivo. Rewarming hypothermic blood completely reverses the activation defect. These results suggest that maintaining normothermia or rewarming a hypothermic bleeding patient may reduce the need for platelet transfusions.

Potentiation by red blood cells of shear-induced platelet aggregation: relative importance of chemical and physical mechanisms

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1200-1206 ◽  
Author(s):  
RC Reimers ◽  
SP Sutera ◽  
JH Joist
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Platelet Rich Plasma ◽  
Adenine Nucleotides ◽  
Laminar Shear Stress ◽  
Platelet Surface ◽  
Surface Transport ◽  
Laminar Shear

Abstract Evidence has been reported to indicate that red blood cells (RBCs) may potentiate platelet adherence and platelet aggregation (PAG) in different flow systems in vitro as well as hemostatic platelet plug formation in response to vascular injury. In this study, we demonstrate that RBCs enhance PAG induced by well-defined, low-intensity, uniform, laminar shear stress. Potentiation by RBCs of shear-induced PAG was associated with appreciable loss of adenine nucleotides from 14C- adenine-labeled RBCs, the extent of which increased with increasing RBC concentration. The concentrations of RBC-derived ADP measured in the medium after shear, as determined by both high pressure liquid chromatography and the luciferin/luciferase system, were within the range of concentrations of ADP which may trigger PAG or potentiate PAG induced by low concentrations of other platelet agonists in the aggregometer. To assess the relative contribution of chemical (ADP) and physical (platelet surface transport) mechanisms in the RBC-mediated potentiation of shear-induced PAG, aliquots of citrated platelet-rich plasma (C-PRP) were exposed to shear stress in the presence of untreated RBCs or RBCs exposed to an antihemolytic concentration (5 mumol/L) of the membrane stabilizing agent, chlorpromazine (CPZ). Potentiation of shear-induced PAG in the RBC-CPZ system was significantly less than that in the untreated RBC system. However, CPZ- induced reduction of PAG potentiation was associated with an increase rather than a decrease in loss of adenine nucleotides from RBC. Furthermore, shear-induced PAG in C-PRP as well as ADP- and collagen- induced PAG in C-PRP in the aggregometer was significantly inhibited by 5 mumol/L CPZ, indicating that the observed reduced potentiation of shear-induced PAG by RBCs in the presence of CPZ was due to a direct inhibitory effect of the drug on platelets rather than a reduction of shear-induced liberation of ADP from RBCs. When aliquots of C-PRP were exposed to shear stress in the presence of RBCs completely depleted of ADP by fixation in 1% glutaraldehyde, potentiation of PAG was approximately half of that observed with intact RBCs. These findings indicate that both RBC-derived ADP and RBC-mediated platelet surface transport are involved in the potentiation by RBCs of PAG induced by laminar shear stress.

scholarly journals Potentiation by red blood cells of shear-induced platelet aggregation: relative importance of chemical and physical mechanisms

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1200-1206 ◽  
Author(s):  
RC Reimers ◽  
SP Sutera ◽  
JH Joist
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Platelet Rich Plasma ◽  
Adenine Nucleotides ◽  
Laminar Shear Stress ◽  
Platelet Surface ◽  
Surface Transport ◽  
Laminar Shear

Evidence has been reported to indicate that red blood cells (RBCs) may potentiate platelet adherence and platelet aggregation (PAG) in different flow systems in vitro as well as hemostatic platelet plug formation in response to vascular injury. In this study, we demonstrate that RBCs enhance PAG induced by well-defined, low-intensity, uniform, laminar shear stress. Potentiation by RBCs of shear-induced PAG was associated with appreciable loss of adenine nucleotides from 14C- adenine-labeled RBCs, the extent of which increased with increasing RBC concentration. The concentrations of RBC-derived ADP measured in the medium after shear, as determined by both high pressure liquid chromatography and the luciferin/luciferase system, were within the range of concentrations of ADP which may trigger PAG or potentiate PAG induced by low concentrations of other platelet agonists in the aggregometer. To assess the relative contribution of chemical (ADP) and physical (platelet surface transport) mechanisms in the RBC-mediated potentiation of shear-induced PAG, aliquots of citrated platelet-rich plasma (C-PRP) were exposed to shear stress in the presence of untreated RBCs or RBCs exposed to an antihemolytic concentration (5 mumol/L) of the membrane stabilizing agent, chlorpromazine (CPZ). Potentiation of shear-induced PAG in the RBC-CPZ system was significantly less than that in the untreated RBC system. However, CPZ- induced reduction of PAG potentiation was associated with an increase rather than a decrease in loss of adenine nucleotides from RBC. Furthermore, shear-induced PAG in C-PRP as well as ADP- and collagen- induced PAG in C-PRP in the aggregometer was significantly inhibited by 5 mumol/L CPZ, indicating that the observed reduced potentiation of shear-induced PAG by RBCs in the presence of CPZ was due to a direct inhibitory effect of the drug on platelets rather than a reduction of shear-induced liberation of ADP from RBCs. When aliquots of C-PRP were exposed to shear stress in the presence of RBCs completely depleted of ADP by fixation in 1% glutaraldehyde, potentiation of PAG was approximately half of that observed with intact RBCs. These findings indicate that both RBC-derived ADP and RBC-mediated platelet surface transport are involved in the potentiation by RBCs of PAG induced by laminar shear stress.

Comparison of platelet aggregation parameters and expression of platelet granule markers

2019 ◽  
Author(s):  
В.В. Малышева ◽  
О.А. Шустова ◽  
С.Г. Хаспекова ◽  
Я.А. Наймушин ◽  
А.В. Мазуров
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Light Transmission ◽  
Platelet Rich Plasma ◽  
Thrombin Receptor ◽  
Platelet Surface ◽  
Dense Granules ◽  
Turbidimetric Method ◽  
Healthy Donors ◽  
Aggregation Parameters

Введение. Активация тромбоцитов стимулирует их агрегацию и ассоциированный с секрецией экзоцитоз внутриклеточных гранул. Агрегацию чаще всего изучают турбидиметрическим методом, а экзоцитоз гранул методом проточной цитофлуориметрии, определяя экспрессию их маркеров на поверхности тромбоцитов. Цель исследования: сравнение чувствительности двух методов оценки активации тромбоцитов, исследования их агрегации и экспрессии маркеров внутриклеточных гранул. Материалы и методы. Тромбоциты здоровых доноров активировали АДФ и пептидом, активирующим рецептор тромбина (thrombin receptor activating peptide, TRAP). Агрегацию тромбоцитов изучали турбидиметрическим методом в обогащенной тромбоцитами плазме, регистрируя максимальный уровень светопропускания (Т макс). Экспрессию маркеров гранул изучали в цельной крови с помощью проточной цитофлуориметрии, регистрируя процент тромбоцитов, окрашенных антителами против маркеров альфагранул (CD62P) и плотных гранул (CD63). Результаты. У всех доноров 20 мкМ АДФ и 10 мкМ TRAP стимулировали мощную, необратимую агрегацию тромбоцитов (62,1 10,3 и 65,1 5,1 T макс, соответственно). Средние уровни агрегации были ниже при ее стимуляции 2,5 мкМ АДФ (30,9 23,2 T макс) и 1 мкМ TRAP (24,4 29,1 T макс). Экспрессия маркеров гранул была максимальной при активации тромбоцитов 10 мкМ TRAP (77,6 13,7 CD62P и 73,9 13,3 CD63), ниже при активации 1 мкМ TRAP (46,3 25,1 CD62P и 38,3 23,8 CD63), еще ниже при активации 20 мкМ АДФ (19,8 8,5 CD62P и 13,6 5,2 CD63) и минимальной при активации 2,5 мкМ АДФ (8,0 4,2 CD62P и 8,3 3,3 CD63). Адреналин (20 мкМ) не стимулировал экспрессию маркеров гранул, но при совместном добавлении с 20 мкМ АДФ повышал ее в 1,9 раза для обоих маркеров CD62P и CD63. Заключение. При активации тромбоцитов АДФ исследование агрегации является более чувствительным методом, чем определение экспрессии маркеров гранул. При активации тромбоцитов TRAP чувствительность обоих методов приблизительно одинакова. Экспрессия маркеров гранул увеличивается при совместном добавлении к тромбоцитам АДФ и адреналина, хотя адреналин сам по себе не стимулирует их экспрессию. Introduction. Platelet activation stimulates their aggregation and secretion associated exocytosis of intracellular granules. Aggregation is usually studied by turbidimetric method and granule exocytosis by detecting expression of their markers on platelet surface using flow cytofluorimetry. Aim: сomparison of the sensitivity of two methods for evaluation of platelet activation, studies of their aggregation and expression of intracellular granule markers. Materials and methods. Healthy donors platelets were activated by ADP and thrombin receptor activating peptide (TRAP). Platelet aggregation was investigated in platelet rich plasma by turbidimetric method, assessed by the maximal level of light transmission (T max). Expression of granule markers was detected in whole blood by fl ow cytofl uorimetry registering the percent of platelets stained with antibodies against the markers of alphagranules (CD62P) and dense granules (CD63). Results. In all donors 20 M ADP and 10 M TRAP stimulated strong irreversible platelet aggregation (62.1 10.3 and 65.1 5.1 T max). Mean aggregation levels were lower when it was stimulated by 2.5 M ADP (30.9 23.2 T max) and 1 M TRAP (24.4 29.1 T max). Expression of granule markers was maximal at platelet activation by 10 M TRAP (77.6 13.7 CD62P and 73.9 13.3 CD63), lower at activation by 1 M TRAP (46.3 25.1 CD62P and 38.3 23.8 CD63), more lower at activation by 20 M ADP (19.8 8.5 CD62P and 13.6 5.4 CD63), and minimal at activation by 2.5 M ADP (8.0 4.2 CD62P and 8.3 3.3 CD63). Epinephrine (20 M) did not stimulate expression of granule markers but at combined addition with 20 M ADP increased its level by 1.9 for both markers, CD62P and CD63. Conclusion. Platelet aggregation is more sensitive method than detection of expression of granule markers when platelets are activated by ADP. Both methods demonstrate about the same sensitivity when platelets are activated by TRAP. Expression of granule markers is increased at combined addition of ADP and epinephrine although epinephrine by itself fails to stimulate their expression.

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