Aggregation of Platelets and Inert Particles Induced by Thrombin

Frangis C. Chao; James L. Tullis; Gail S. Conneely; John W. Lawler

doi:10.1055/s-0038-1647970

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1976; 35(03): 717-736
DOI: 10.1055/s-0038-1647970

Original Article

Schattauer GmbH

Aggregation of Platelets and Inert Particles Induced by Thrombin

Authors

Frangis C. Chao

¹Center for Blood Research; Department of Medicine, New England Deaconess Hospital and Harvard Medical School, and Department of Physics, Boston College, Boston, Massachusetts, U.S.A.
James L. Tullis

¹Center for Blood Research; Department of Medicine, New England Deaconess Hospital and Harvard Medical School, and Department of Physics, Boston College, Boston, Massachusetts, U.S.A.
Gail S. Conneely

¹Center for Blood Research; Department of Medicine, New England Deaconess Hospital and Harvard Medical School, and Department of Physics, Boston College, Boston, Massachusetts, U.S.A.
John W. Lawler

¹Center for Blood Research; Department of Medicine, New England Deaconess Hospital and Harvard Medical School, and Department of Physics, Boston College, Boston, Massachusetts, U.S.A.

Abstract

Summary

Thrombin-induced platelet aggregation and release were investigated in washed platelet suspensions and in suspensions of inert particles in order to evaluate the role of fibrinogen-fi-brin transformation in aggregometer tracings. Thrombin (0.25–2.0 U/ml) produced two waves of light transmission increase (LTI) in both platelet and inert particle suspensions containing fibrinogen, and concomittantly aggregates were observed under phase microscopy. Without fibrinogen, thrombin induced rapid release of platelet ADP but failed to cause second wave of LTI. The kinetics of LTI in platelet and inert particle systems were related to both thrombin and fibrinogen concentrations. A rapid second wave of LTI could be produced by direct interaction of thrombin-treated platelets or inert particles with polymerizing fibrin, and was inhibited by sodium sulfite and low pH of 5.1 which prevent fibrin monomer polymerization. No fibrin strands were noted in platelet aggregates fixed at the completion of the second wave of LTI. Apyrase and PGE₁ inhibited the rate of first but not that of second wave LTI. The results suggest that the release of platelet ADP induced by thrombin primarily affects the first phase aggregation, and the second phase may result from interaction of thrombin-exposed platelets and polymerizing fibrin. Thus, the blood coagulation mechanism may be directly involved in platelet aggregation.

Full Text

References

References
1 Ardlie N. G, Han P. 1974; Enzymatic basis for platelet aggregation and release: The significance of the ‘platelet atmosphere’ and the relationship between platelet function and blood coagulation. British Journal of Hematology 26: 331.
2 Ashcroft G, Crawford B, Binns J. K, MacDougall E. J. 1964; Estimation of 5-hydroxytryp-tamine in human blood. Clinica chimica acta 9: 364.
3 Baenziger N. L, Brodie G. N, Majerus P. W. 1971; A thrombinsensitive protein of human platelet membrane. Proceedings of the National Academy of Sciences 68: 240.
4 Boyer M. H, Shainoff J. R, Ratnoff O. D. 1972; Acceleration of fibrin polymerization by calcium ions. Blood 39: 382.
5 Chao F. C, Tullis J. L. 1973; The in vitro effects of mithramycin on the aggregation and the calcium uptake of human platelets. Thrombosis et Diathesis Haemorrhagica 29: 712.
6 Chao F. C, Tullis J. L, Kenney D. M, Conneely G. S, Doyle J. R. 1974 Concentration effects of platelets, fibrinogen and thrombin on platelet aggregation and fibrin clotting. Thrombosis et Diathesis Haemorrhagica. 32
7 Doolittle R. F. 1973. Structural aspects of the fibrinogen to fibrin conversion. In: Anfinsen C. B, Edsall J. T, Richards F. M. (eds.) Advances in Protein Chemistry.. Academic Press; New York: 1.
8 Elias T. P, Iyers G. Y. N. 1967; Some inhibitors of the fibrinogen-fibrin conversion. Thrombosis et Diathesis Haemorrhagica 18: 499.
9 Fang P, Chao F. C, Tullis J. L. 1974; Kinetic analysis of thrombin-induced aggregation of human platelets. I. Thrombin dependency. Microvascular Research 8: 70.
10 Ganguly P. 1974; Binding of thrombin to human platelets. Nature 247: 306.
11 Glover G, Shaw E. 1971; The purification of thrombin and isolation of a peptide containing the active center histidine. The Journal of Biological Chemistry 246: 4594.
12 Haslam R. J. 1967. Mechanism of blood platelet aggregation. In: Johnson S. A, Seegers W. H. (eds.) Physiology of Hemostasis and Thrombosis.. Charles C. Thomas; Springfield, Illinois: 88.
13 Holmsen H, Holmsen I, Bernhardsen A. 1966; Micro determination of adenosine dephosphate and adenosine triphosphate in plasma with the firefly luciferase system. Analytical Biochemistry 17: 456.
14 Horowitz H. I, Papayoanou M. F. 1968; Activation of platelet factor 3 by adenosine 5’ diphosphate. Thrombosis et Diathesis Haemorrhagica 19: 18.
15 Hovig T. 1962; The ultrastructure of rabbit blood platelet aggregates. Thrombosis et Diathesis Haemorrhagica 8: 455.
16 Hugues J. 1959; Agglutination precoce des plaquettes au cours de la formation du clou hemostatique. Thrombosis et Diathesis Haemorrhagica 3: 177.
17 Johnson S. A. 1971. Platelets in hemostasis and thrombosis. In: Johnson S. A. (ed.) The Circulating Platelet.. Academic Press; New York: 355.
18 Klein P. D, Seegers W. E. 1950; The nature of plasma anti-thrombin activity. Blood 5: 742.
19 Larrieu M. J, Rigollot C, Kubisz P. 1970; Platelet aggregation induced by soluble fibrin monomer complexes. Life Science 9: 1111.
20 Luft J. H. 1961; Improvements in epoxy resin embedding methods. Journal Biophysics Biochemistry Cytology 9: 409.
21 Macfarlane R. G. 1972. Hemostasis. In: Biggs R. (ed.) Human Blood Coagulation, Hemostasis and Thrombosis.. Blackwell; Oxford: 543.
22 Mommaerts W. F. M. 1945; On the nature of forces operating in blood clotting. II. The clotting of fibrinogen as a two-step reaction. Journal of General Physiology 29: 113.
23 Müller-Berghaus G, Heinrich D. 1972; Fibrin monomer and platelet aggregation in vitro and in vivo . British Journal of Haematology 23: 177.
24 Mustard J. F, Packham M. A. 1970; Factors influencing platelet function: adhesion, release and aggregation. Pharmacological Review 22: 97.
25 Niewiarowski S, Regoeczi E, Stewart G. J, Senyi A. F, Mustard J. F. 1973; Platelet interaction with polymerizing fibrin. Journal of Clinical Investigation 51: 685.
26 Packham M. A, Guccione M. A, Chang P. L, Mustard J. F. 1973; Platelet aggregation and release: effects of low concentrations of thrombin or collagen. American Journal of Physiology 225: 38.
27 Phillips D. R, Agin P. P. 1974; Thrombin substrates and the proteolytic site of thrombin action on human-platelet plasma membrane. Biochimica et Biophysica Acta 352: 218.
28 Reynolds E. S. 1963; The use of lead citrate at high pH as an electron opaque stain in electron microscopy. Journal of Cell Biology 17: 208.
29 Solum N. O. 1966; Platelet aggregation during fibrin polymerization. Scandinavian Journal of Clinical and Laboratory Investigation 18: 577.
30 Solum N. O, Stormorken H. 1965; Influence of fibrinogen on the aggregation of washed human blood platelets induced by adenosine diphosphate, thrombin, collagen and adrenaline. Scandinavian Journal of Clinical and Laboratory Investigation 17 (suppl 84) 170.
31 Steiner M. 1973; Effect of thrombin on the platelet membrane. Biochimica et Biophysica Acta 323: 653.
32 Stemerman M. B. 1974. Platelet interaction with intimal connective tissue. In: Baldini M. G, Ebbe S. (eds.) Platelets: Production, Function and Storage.. Grune S. Stratton; New York: 157.
33 Tangen O, Berman H. J, Marfey P. 1971; Gel filtration. A new technique for separation of blood platelets from plasma. Thrombosis et Diathesis Haemorrhagica 25: 268.
34 Thomas D. P. 1967; Effect of catecholamines on platelet aggregation caused by thrombin. Nature 215: 298.
35 Tollefsen D. M, Feagler J. R, Majerus P. W. 1974; The binding of thrombin to the surface of human platelets. The Journal of Biological Chemistry 249: 2646.
36 Tullis J. L, Tinch R. J, Baudanza P, Gibson J. G, Diforte S, Conneely G, Murthy K. 1971; Plateletpheresis in a disposable system. Transfusion 11: 368.
37 Walsh P. N. 1972; The role of platelets in the contact phase of blood coagulation. British Journal of Haematology 22: 237.
38 Zucker M. B. 1972. Platelet function. In: Williams W. J, Beutler E, Erslev A. J, Rundles R. W. (eds.) Hematology.. McGraw-Hill; New York: 1014.