Thrombin Interaction with Human Platelets Potentiation of Thrombin-induced Aggregation and Release by Inactivated Thrombin

David R. Phillips

doi:10.1055/s-0038-1647686

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1974; 32(01): 207-215
DOI: 10.1055/s-0038-1647686

Original Article

Schattauer GmbH

Thrombin Interaction with Human Platelets Potentiation of Thrombin-induced Aggregation and Release by Inactivated Thrombin

Authors

David R. Phillips With technical assistance of Patricia Poh Agin

¹Laboratories of Biochemistry, St. Jude Children’s Research Hospital Memphis, Tennessee 38101, U.S.A

Abstract

Summary

The possibility that thrombin acts on platelets by a mechanism other than proteolysis was investigated. The proteolytic site of thrombin was modified with phenylmethylsulfonyl fluoride (PMSF). This modified enzyme did not induce platelet aggregation or the platelet release reaction. Platelets were then incubated with the inactivated enzyme (PMS-thrombin) and later with active thrombin. In this sequence of incubation, PMS-thrombin enhanced not only platelet aggregation induced by active thrombin but also the thrombin-induced release reaction. Preincubation with PMS-thrombin was essential for this enhancement as the inhibited enzyme did not affect aggregation if added after active thrombin. The effect of PMS-thrombin was limited to thrombin-induced reactions of the platelet. The inhibited enzyme had no effect on aggregation induced by adenosine diphosphate or collagen, or on thrombininduced coagulation of fibrinogen. These results suggest (1) that both proteolytic and binding sites for thrombin are present on the human platelet plasma membrane ; and (2) that interaction of thrombin with the binding site potentiates the activity of the proteolytic site.

Full Text

References

References
1 Baeziger N. L, Brodie G. N, Majerus P. W. 1971; A Thrombin-Sensitive Protein of Human Platelet Membranes. Proceedings of the National Academy of Sciences, U.S.A 68: 240.
2 Born G. V. R, Cross M. J. 1963; The Aggregation of Blood Platelets. Journal of Physiology 168: 178.
3 Cohen I, Bohak I, DeVries A, Katchalski E. 1969; Purification and Interaction with Thrombin. European Journal of Biochemistry 10: 388.
4 Davey M. G, Lüscher E. F. 1967; Actions of Thrombin and Other Coagulant and Proteolytic Enzymes on Blood Platelets. Nature 216: 857.
5 Detwiler T. C, Feinman R. D. 1973; Kinetics of the Thrombin-Induced Release of Ca (11) by Platelets. Biochemistry 12: 282.
6 Ganguly P. 1971; Isolation and Properties of a Thrombin-Sensitive Protein from Human Blood Platelets. Journal of Biological Chemistry 246: 4286.
7 Glover G, Shaw E. 1971; The Purification of Thrombin and Isolation of a Peptide Containing the Active Center of Histidine. Journal of Biological Chemistry 246: 4598.
8 Grette K. 1962; Studies on the Mechanism of Thrombin-Catalyzed Hemostatic Reactions in Blood Platelets. Acta Physiologica Scandinavica 56: 5.
9 Haslam R. J. 1964; Role of Adenosine Diphosphate in the Aggregation of Human Blood Platelets by Thrombin and by Fatty Acids. Nature 202: 765.
10 Kiesselbach T. H, Wagner R. H. 1966; Fibrin-Stabilizing Factor: A ThrombinLabile Platelet Protein. American Journal of Physiology 211: 1472.
11 Lowry O. H, Rosebrotjgh N. J, Farr A. L, Randall R. J. 1951; Journal of Biological Chemistry. 193: 265.
12 Lundblad R. L. 1971; A Rapid Method for the Purification of Bovine Thrombin and the Inhibition of the Purified Enzyme with Phenylmethylsulfonyl fluoride. Biochemistry 10: 2501.
13 Lüscher E. F. 1956; Viscous Metamorphosis of Blood Platelets and Clot Retraction. Vox Sanguinis 01: 133.
14 Majertjs P. W, Brodie G. N. 1972; The Binding of Phytohemagglutinins to Human Platelet Plasma Membranes. Journal of Biological Chemistry 247: 4253.
15 Massini P, Lüscher E. F. 1971; The Induction of the Release Reaction in Human Blood Platelets by Close Cell Contact. Thrombosis Diathesis et Haemorrhagica 25: 13.
16 Nachman R. L. 1965; Immunologic Studies of Platelet Protein. Blood 25: 703.
17 O’Brien J. R. 1962; Platelet Aggregation II. Some Results from a New Method of Study. Journal of Clinical Pathology 15: 452.
18 Phillips D. R. 1972; Effect of Trypsin on the Exposed Polypeptides and Glycoproteins in the Human Platelet Membrane. Biochemistry 11: 4582.
19 Phillips D. R, Agin P. P. 1973; Thrombin-Induced Alterations in the Surface Structure of the Human Platelet Plasma Membrane. Series Haematologica VI: 292.
20 Zucker M. B, Borrelli J. 1959; Viscous Metamorphosis, Clot Retraction and Other Morphologic Alterations in Blood Platelets. Journal of Applied Physiology 14: 575.
21 Zucker M. B. 1972; Proteolytic Inhibitors, Contact and Other Variables in the Release Reaction of Human Platelets. Thrombosis Diathesis et Haemorrhagica 28: 383.