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

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.

[1]  J. Hoxie,et al.  Changes in the platelet membrane glycoprotein IIb.IIIa complex during platelet activation. , 1985, The Journal of biological chemistry.

[2]  A. Schmaier,et al.  Fibrinogen receptors in platelet adhesion to surfaces of extracorporeal circuit. , 1987, The American journal of physiology.

[3]  G. FitzGerald,et al.  11-Dehydrothromboxane B2: a quantitative index of thromboxane A2 formation in the human circulation. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[4]  S. Shattil.  Expression, regulation and detection of fibrinogen receptors on activated human platelets. , 1988, Progress in clinical and biological research.

[5]  H. Gralnick,et al.  Identification of platelet glycoprotein IIb/IIIa as the major binding site for released platelet-von Willebrand factor. , 1986, Blood.

[6]  H. T. ten Duis,et al.  Hematologic aspects of cardiotomy suction in cardiac operations. , 1980, The Journal of thoracic and cardiovascular surgery.

[7]  J. Hoxie,et al.  Detection of activated platelets in whole blood using activation- dependent monoclonal antibodies and flow cytometry , 1987 .

[8]  C. Milstein,et al.  [1] Preparation of monoclonal antibodies: Strategies and procedures , 1981 .

[9]  J. George,et al.  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. , 1986, The Journal of clinical investigation.

[10]  G. FitzGerald,et al.  Marked platelet activation in vivo after intravenous streptokinase in patients with acute myocardial infarction. , 1988, Circulation.

[11]  E. Salzman,et al.  Elevated Plasma Fibrinopeptide A ana Thromboxane B2 Levels During Cardiopulmonary Bypass , 1980, Circulation.

[12]  V. Fuster,et al.  Antithrombotic therapy: role of platelet-inhibitor drugs. I. Current concepts of thrombogenesis: role of platelets. (first of three parts). , 1981, Mayo Clinic proceedings.

[13]  G. Vilaire,et al.  The in vitro synthesis of polypeptides for the platelet membrane glycoproteins IIb and IIIa. , 1987, Blood.

[14]  B. Furie,et al.  A platelet membrane protein expressed during platelet activation and secretion. Studies using a monoclonal antibody specific for thrombin-activated platelets. , 1984, The Journal of biological chemistry.

[15]  R. McEver,et al.  A monoclonal antibody to a membrane glycoprotein binds only to activated platelets. , 1984, The Journal of biological chemistry.

[16]  C. Beurling-Harbury,et al.  Acquired decrease in platelet secretory ADP associated with increased postoperative bleeding in post-cardiopulmonary bypass patients and in patients with severe valvular heart disease. , 1978, Blood.

[17]  E. Hessel,et al.  Mechanism of abnormal bleeding in patients undergoing cardiopulmonary bypass: acquired transient platelet dysfunction associated with selective alpha-granule release. , 1980, Blood.

[18]  P. Wolf.  The Nature and Significance of Platelet Products in Human Plasma , 1967, British journal of haematology.

[19]  P. Weber,et al.  Biochemical evidence of platelet activation in patients with persistent unstable angina. , 1987, Journal of the American College of Cardiology.

[20]  L Roy,et al.  Platelet activation in unstable coronary disease. , 1986, The New England journal of medicine.

[21]  B. Lentz,et al.  Expression of coagulant activity in human platelets: release of membranous vesicles providing platelet factor 1 and platelet factor 3. , 1985, Thrombosis research.

[22]  A. Gotlieb,et al.  Review of antiplatelet drug use in preventing restenosis following percutaneous transluminal coronary angioplasty. , 1988, The Canadian journal of cardiology.

[23]  J. Malone.  Biosynthesis of thromboxane in patients with systemic sclerosis and Raynaud's phenomenon: Reilly IAG, Roy L, Fitzgerald GA. Br Med J 1986; 292:1037–1039 , 1987 .

[24]  S. Khuri,et al.  Hypothermia-induced reversible platelet dysfunction. , 1987, Annals of surgery.

[25]  A. Girolami,et al.  The release of β‐ thromboglobulin and platelet factor 4 during extracorporeal circulation for open heart surgery , 1981 .

[26]  B. V. van Oost,et al.  The effect of thrombin on the density distribution of blood platelets: detection of activated platelets in the circulation. , 1983, Blood.

[27]  V. Fuster,et al.  Coronary artery thrombosis in patients with unstable angina. , 1981, British heart journal.

[28]  J. Dawes,et al.  Platelet alpha granule depletion: findings in patients with prosthetic heart valves and following cardiopulmonary bypass surgery. , 1983, Thrombosis research.

[29]  E. Blackstone,et al.  Complement activation during cardiopulmonary bypass: evidence for generation of C3a and C5a anaphylatoxins. , 1981, The New England journal of medicine.

[30]  E. Engvall,et al.  Enzyme immunoassay ELISA and EMIT. , 1980, Methods in enzymology.

[31]  B. Osterud,et al.  Platelet storage lesion: formation of platelet fragments with platelet factor 3 activity. , 1987, Thrombosis research.

[32]  P. Newman,et al.  Human platelet fibrinogen: purification and hemostatic properties. , 1985, Blood.

[33]  Edward W. Merrill,et al.  Does the conformation of adsorbed fibrinogen dictate platelet interactions with artificial surfaces , 1986 .

[34]  J. V. van Oosterhout,et al.  Studies with a monoclonal antibody against activated platelets: evidence that a secreted 53,000-molecular weight lysosome-like granule protein is exposed on the surface of activated platelets in the circulation. , 1987, Blood.

[35]  V. Fuster,et al.  10. Antithrombotic therapy: role of platelet-inhibitor drugs. II. Pharmacologic effects of platelet-inhibitor drugs (second of three parts). , 1981, Mayo Clinic proceedings.

[36]  L. Brass,et al.  Induction of the fibrinogen receptor on human platelets by intracellular mediators. , 1987, The Journal of biological chemistry.

[37]  C. Milstein,et al.  Preparation of monoclonal antibodies: strategies and procedures. , 1981, Methods in enzymology.

[38]  S. Karpatkin,et al.  Red-cell and platelet fragmentation in idiopathic autoimmune thrombocytopenic purpura. , 1977, The New England journal of medicine.

[39]  J. Moake,et al.  Shear-induced platelet aggregation requires von Willebrand factor and platelet membrane glycoproteins Ib and IIb-IIIa. , 1987, Blood.

[40]  J. Westwick,et al.  Investigation of the Interaction of Blood Platelets with the Coagulation System at the Site of Plug Formation In Vivo in Man - Effect of Low-Dose Aspirin , 1987, Thrombosis and Haemostasis.

[41]  G. FitzGerald,et al.  Increased thromboxane biosynthesis in a human preparation of platelet activation: biochemical and functional consequences of selective inhibition of thromboxane synthase. , 1986, Circulation.

[42]  J. Hoxie,et al.  Inhibition of fibrinogen binding to stimulated human platelets by a monoclonal antibody. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[43]  S. P. Levine.  10 – Secreted Platelet Proteins as Markers for Pathological Disorders* , 1986 .

[44]  P. Insel,et al.  Expression of fibrinogen receptors during activation and subsequent desensitization of human platelets by epinephrine. , 1986, Blood.

[45]  G. Köhler,et al.  A better cell line for making hybridomas secreting specific antibodies , 1978, Nature.

[46]  R. Montgomery,et al.  Diagnosis of Bernard-Soulier syndrome and Glanzmann's thrombasthenia with a monoclonal assay on whole blood. , 1983, The Journal of clinical investigation.

[47]  G. Vilaire,et al.  Exposure of platelet fibrinogen receptors by ADP and epinephrine. , 1979, The Journal of clinical investigation.

[48]  H. Nieuwenhuis,et al.  Studies with a monoclonal antibody against activated platelets: evidence that a secreted 53,000-molecular weight lysosome-like granule protein is exposed on the surface of activated platelets in the circulation , 1987 .

[49]  E. Merrill,et al.  Does the conformation of adsorbed fibrinogen dictate platelet interactions with artificial surfaces? , 1986, Blood.

[50]  P. Sims,et al.  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. , 1988, The Journal of biological chemistry.

[51]  S. Karpatkin,et al.  Microthrombocytosis and Platelet Fragmentation Associated with Idiopathic/Autoimmune Thrombocytopenic Purpura , 1975, British journal of haematology.

[52]  L. Hillis,et al.  Release of prostaglandins and thromboxane into the coronary circulation in patients with ischemic heart disease. , 1981, The New England journal of medicine.

[53]  J. George,et al.  Platelet membrane microparticles in blood bank fresh frozen plasma and cryoprecipitate. , 1986, Blood.

[54]  G. FitzGerald,et al.  Biosynthesis of thromboxane in patients with systemic sclerosis and Raynaud's phenomenon. , 1986, British medical journal.