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

Catherine Vidal; Christian Spaulding; Françoise Picard; Frédéric Schaison; Josiane Melle; Simon Weber; Michaëla Fontenay-Roupie

doi:10.1055/s-0037-1616132

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2001; 86(03): 784-790
DOI: 10.1055/s-0037-1616132

Review Articles

Schattauer GmbH

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

Catherine Vidal

¹Service de Cardiologie

,

Christian Spaulding

¹Service de Cardiologie

,

Françoise Picard

²Laboratoire central d’Hématologie, Hôpital Cochin, Paris, France

,

Frédéric Schaison

¹Service de Cardiologie

,

Josiane Melle

²Laboratoire central d’Hématologie, Hôpital Cochin, Paris, France

,

Simon Weber

¹Service de Cardiologie

,

Michaëla Fontenay-Roupie

²Laboratoire central d’Hématologie, Hôpital Cochin, Paris, France

› Author Affiliations

Abstract

Summary

Platelet 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.

Keywords

Human blood platelets - flow cytometry - annexin V - microvesiculation - unstable angina

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References

References
1 Gawaz M, Neumann FJ, Ott I, May A, Schöming A. Platelet activation and coronary stent implantation. Effect of thrombolytic therapy. Circulation 1996; 94: 279-85.
2 Gawaz M, Neumann FJ, Ott I, May A, Rüdiger S, Schömig A. Role of activation-dependent platelet membrane glycoproteins in development of subacute occlusive coronary stent thrombosis. Coron Art Dis 1997; 8: 121-8.
3 Schömig A, Neumann FJ, Kastrati A, Schihlen H, Blasini R, Hadamitzki M, Walter H, Zitzmann-Roth EM, Richard G, Alt E, Schmitt C, Ulm K. A randomized comparison of antiplatelet and anticoagulant therapy after the placement of coronary stents. N Engl J Med 1996; 334: 1084-9.
4 The EPIC investigators.. Use of a monoclonal antibody directed against the platelet glycoprotein IIb/IIIa receptor in high-risk coronary angioplasty. The EPIC investigation. N Engl J Med 1994; 330: 956-61.
5 Nurden P. Bidirectional trafficking of membrane glycoproteins following platelet activation in suspension. Thromb Haemost 1997; 78: 1305-15.
6 Zwaal RFA, Bevers EM, Comfurius P, Rosing J, Tilly RHJ, Verhallen PFJ. Loss of membrane phospholipid asymmetry during activation of blood platelets and sickled red cells; mechanisms and physiological significance. Mol Cell Biochem 1981; 91: 23-31.
7 Sims PJ, Wiedmer T, Esmon CT, Weiss HJ, Shattil SJ. Assembly of the platelet prothrombinase complex is linked to vesiculation of the platelet plasma membrane. J Biol Chem 1989; 264: 17049-53.
8 Sims PJ, Faioni EM, Wiedmer T, Shattil SJ. Complement proteins C5b-9 cause release of membrane vesicles from the platelet surface that are enriched in the membrane receptor for coagulation factor Va and express prothrombinase activity. J Biol Chem 1988; 263: 18205-9.
9 Miyazaki Y, Nomura S, Miyake T, Kagawa H, Kitada C, Taniguchi H, Komiyama Y, Fujimura Y, Ikeda Y, Fukuhara S. High shear stress can initiate both platelet aggregation and shedding of procoagulant containing microparticles. Blood 1996; 88: 3456-64.
10 Tans G, Rosing J, Thomassen CLGD, Heeb MJ, Zwaal RFA, Griffin JH. Comparison of anticoagulant and procoagulant activities of stimulated platelets and platelet-derived microparticles. Blood 1991; 77: 2641-8.
11 Stuart MC, Bevers EM, Comfurius P, Zwaal RF, Reutelingsperger CP, Frederik PM. Ultrastructural detection of surface exposed phosphatidylserine on activated blood platelets. Thromb Haemost 1995; 74: 1145-56.
12 Owens MR, Holme S, Cardinali S. Platelet microvesicles adhere to subendothelium and promote adhesion of platelets. Thromb Res 1992; 66: 247-56.
13 Siljander P, Carpen O, Lassila R. Platelet-derived microparticles associate with fibrin during thrombosis. Blood 1996; 87: 4651-63.
14 Mallat Z, Hugel B, Ohan J, Lesèche G, Freyssinet J-M, Tedgui A. Shed membrane microparticles with procoagulant potential in human athero-sclerotic plaques. Circulation 1999; 99: 348-51.
15 Gawaz M, Neumann FJ, Ott I, Schiessler A, Schöming A. Platelet function in acute myocardial infarction treated with direct angioplasty. Circulation 1996; 93: 229-37.
16 George JN, Pickett EB, Saucerman S, McEver RP, Kunicki TJ, Kieffer N, Newman PJ. Platelet surface glycoproteins: Studies on resting and activated platelets and platelet membrane microparticles in normal subjects, and observations in patients during adult respiratory distress syndrome and cardiac surgery. J Clin Invest 1986; 78: 340-50.
17 Bihour C, Durrieu-Jaïs, Besse P, Nurden P, Nurden AT. Flow cytometry reveals activated GpIIb-IIIa complexes on platelets from patients undergoing thrombolytic therapy after acute myocardial infarction. Blood Coag Fib 1995; 6: 395-410.
18 Thiagarajan P, Tait JF. Binding of annexin V/placental anticoagulant protein 1 to platelets: evidence for phosphatidylserine exposure in the procoagulant response of activated platelets. J Biol Chem 1990; 265: 17420-3.
19 Dachary-Prigent J, Freyssinet J-M, Pasquet J-M, Carron J-C, Nurden AT. Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: a flow cytometry study showing a role for free sulfydryl groups. Blood 1993; 81: 2554-65.
20 Dachary-Prigent J, Pasquet J-M, Freyssinet J-M, Nurden AT. Calcium involvement in aminophospholipid exposure and microparticle formation during platelet activation: a study using Ca2+-ATPase inhibitors. Biochemistry 1995; 34: 11625-34.
21 Shattil SJ, Cunningham M, Hoxie JA. Detection of activated platelets using activation-dependent monoclonal antibodies and flow cytometry. Blood 1987; 70: 307-12.
22 Michelson AD. Flow cytometry: a clinical test of platelet function. Blood 1996; 87: 4925-36.
23 Schmitz G, Rothe G, Ruf A, Barlage S, Tschöpe D, Clemetson KJ, Goodall AH, Michelson AD, Nurden AT. Vincent Shankey T for the European Working group on clinical cell analysis.. European Working group on clinical cell analysis: Consensus protocol for the flow cytometric characterisation of platelet function. Thromb Haemost 1998; 79: 885-96.
24 Toti F, Satta N, Fressinaud E, Meyer D, Freyssinet J-M.. Scott syndrome, characterized by impaired transmembrane migration of procoagulant phosphatidylserine and hemorrhagic complications, is an inherited disorder. Blood 1996; 87: 1409-15.
25 Abrams CS, Ellison N, Budzynski AZ, Shattil SJ. Direct detection of activated platelets and platelet-derived microparticles in humans. Blood 1990; 75: 128-38.
26 Yano Y, Kambayashi JI, Kawasaki T, Sakon M. Quantitative determination of circulating platelet microparticles by flow cytometry. Int J Cardiol 1994; 47 Suppl S13-9.
27 Aupeix K, Hugel B, Martin T, Bischoff P, Lill H, Pasquali JL, Freyssinet JM. The significance of shed membrane particles during programmed cell death in vitro, and in vivo, in HIV-1 infection. J Clin Invest 1997; 99: 1546-50.
28 Hugel B, Socié G, Vu T, Toti F, Gluckman E, Freyssinet JM, Scribohaci ML. Elevated levels of circulating procoagulant microparticles in patients with paroxysmal noctural hemoglobinuria and aplastic anemia. Blood 1999; 93: 3451-6.
29 Singh PJ, Gemmell CH, Daly PA, Yeo EL. Elevated platelet-derived micro-particle levels during unstable angina. Can J Cardiol 1995; 11: 1015-21.
30 Tschoepe D, Schultheiss HP, Kolarov P, Schwippert B, Dannehl K, Volsw D, Nieuwenhuis HK, Kehrel B, Strauer B, Gries FA. Platelet membrane activation markers are predictive for increased risk of acute ischemic events after PTCA. Circulation 1993; 88: 37-42.
31 Furman MI, Benoit SE, Barnard MR, Valeri CR, Borbone ML, Becker RC, Hechtman HB, Michelson AD. Increased platelet rectivity and circulating monocyte-platelet aggregates in patients with stable coronary artery disease. J Am Coll Cardiol 1998; 31: 352-8.
32 Michelson AD, Barnard MR, Hechtman HB, MacGregor H, Connolly RJ, Loscalzo J, Valeri CR. In vivo tracking of platelets: Circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function. Proc Natl Acad Sci USA 1996; 93: 11877-82.
33 Ruf A, Patscheke H. Flow cytometry detection of activated platelets: comparison of determining shape change, fibrinogen binding and P-selectin expression. Semin Thromb Hemostasis 1995; 21: 146-51.
34 Gawaz M, Ruf A, Neumann F-J, Pogatsa-Murray G, Dickfeld T, Zohlnhöfer D, Schöming A. Effect of gycoprotein IIb-IIIa receptor antagonism on platelet membrane glycoproteins after coronary stent placement. Thromb Haemost 1998; 80: 994-1001.
35 Neumann FJ, Gawaz M, Ott I, May A, Mössmer G, Schömig A. Prospective evaluation of hemostatic predictors of subacute stent thrombosis after coronary Palmaz-Schatz stenting. J Am Coll Cardiol 1996; 27: 15-21.
36 Klein LW, Weintraub WS, Agarwal JB, Schneider RM, Seelans PA, Katz RI, Helfant RH. Prognostic significance of severe narrowing of the proximal portion of the left anterior descending coronary artery. Am J Cardiol 1986; 58: 42-6.
37 Hannan EL, Racz MJ, McCallister BD, Ryan TJ, Arani DT, Isom OW, Jones RH. A comparison of three-year survival following coronary artery bypass graft surgery and percutaneous transluminal coronary angioplasty. J Am Coll Cardiol 1999; 33: 63-72.