Proteolytic Inhibitors, Contact and Other Variables in the Release Reaction of Human Platelets

Summary

Platelet-rich plasma (PRP) containing platelets labelled with ¹⁴C-serotonin was used to characterize the release reaction. Heparin promotes release, and the reduced release response to epinephrine observed in heparinized compared to citrated PRP is probably due to the earlier reaction. The release reaction can occur in citrated PRP at pH 7.0, is marked between pH 7.2 and 7.6, and decreases above pH 8.0. It is retained better when PRP is stored below pH 7.7. Adenosine, prostaglandin E₁, and theophylline inhibit both aggregation and release, probably because they elevate cyclic AMP. D- and L-tosyl-phenylalanine chloromethylketone (TPCK) also inhibit both aggregation and release at a concentration of 0.1 mM whereas 0.1 mM D- and l-L-chloro-2-tosylamido-7-amino-2-heptanone (TLCK) and 5 mM diisopropylfluoro-phosphate (DFP), like aspirin, inhibit only release. Colchicine (0.1 mM) alters platelet shape but does not affect release or aggregation. Centrifugation of PRP at 37° C causes release which responds to inhibitors and pH changes in a manner similar to release induced by aggregation with ADP or epinephrine. Release occurs in thromb-asthenic platelets centrifuged at 37° C and is increased by epinephrine. Release of ATP and ADP correlates significantly with release of ¹⁴C-serotonin. Thrombin plus CaCl₂ causes release of β-glucuronidase but not of zinc.

• References

• 1 Austen K. F. 1970. Histamine and other mediators of allergic reactions. In: Samter M. (Ed.) Immunological Diseases. Little, Brown and Co.; Boston, Mass.: Vol. 1 332.
• 2 Born G. V. R, and Bricknell J. 1959; The uptake of 5-hydroxytryptamine by blood platelets in the cold. Journal of Physiology 147: 153.
• 3 Day H. J, and Holmsen H. 1971; Concepts of the blood platelet release reaction. Series Haematologica 4 (01) 3.
• 4 Foley B, Johnson S. A, Hackley B, Smith Jr. J. C., and Halstead J. A. 1968; Zinc content of human platelets. Proceedings of the Society for Experimental Biology and Medicine 128: 265.
• 5 Friedberg N. M, and Zucker M. B. 1972; ADP as the cause of reversible inhibition of platelet retention in glass-bead columns. Journal of Laboratory and Clinical Medicine 80: 603.
• 6 Goldstein A. 1964. Biostatistics, an Introductory Text. The Macmillan Company; New York:
• 7 Hampton J. R, and Mitchell J. R. A. 1966; Effect of glass contact on the electrophoretic mobility of human blood platelets. Nature 209: 470.
• 8 Harada K, and Zucker M. B. 1971; Simultaneous development of platelet factor 4 activity and release of ¹⁴C-serotonin. Thrombosis et Diathesis Haemorrhagica 25: 41.
• 9 Holmsen H, and Day H. J. 1971; Adenine nucleotides and platelet function. Series Haematologica 4 (01) 28.
• 10 Holmsen H, Holmsen I, and Bernhardsen A. 1966; Microdetermination of adenosine diphosphate and adenosine triphosphate in plasma with the firefly luciferase system. Analytical Biochemistry 17: 456.
• 11 Hovig T. 1963; Release of a platelet aggregating substance (adenosine diphosphate) from rabbit blood platelets induced by saline “extract” of tendons. Thrombosis et Diathesis Haemorrhagica 9: 264.
• 12 Jerushalmy Z, and Zucker M. B. 1966; Some effects of fibrinogen degradation products (FDP) on blood platelets. Thrombosis et Diathesis Haemorrhagica 15: 413.
• 13 Jobin F, and Sirois M. 1971; Réactions plaquettaires et processus immunitaires. V. Inhibition par des substrats et inhibiteurs de la chymotrypsine de la libération d’ADP par les plaquettes. Thrombosis et Diathesis Haemorrhagica 25: 81.
• 14 Macmillan D. C. 1966; Secondary dumping effect in human citrated platelet-rich plasma produced by adenosine diphosphate and adrenaline. Nature 211: 140.
• 15 Massini P, and Lüsches E. F. 1971; The induction of the release reaction in human blood platelets by close cell contact. Thrombosis et Diathesis Haemorrhagica 25: 13.
• 16 Mills D. C. B, Robb I. A, and Roberts G. C. K. 1968; The release of nucleotides, 5-hydroxy-tryptamine and enzymes from human blood platelets during aggregation. Journal of Physiology 195: 715.
• 17 Mustard J. F, Packham M. A, and Senyi A. 1967; The effect of DFP on platelet function in vitro and in vivo. Journal of Laboratory and Clinical Medicine 70: 1003 (Abstract).
• 18 Niewiarowski S, and Thomas D. P. 1969; Platelet factor 4 and adenosine diphosphate release during human platelet aggregation. Nature 222: 1269.
• 19 O’Brien J. R. 1966; Changes in platelet membranes possibly associated with platelet stickiness. Nature 212: 1057.
• 20 O’Brien J. R, Shoobrldge S. M, and Finch W. J. 1969; Comparison of the effect of heparin and citrate on platelet aggregation. Journal of Clinical Pathology 22: 28.
• 21 O’Brien J. R, and Woodhouse M. A. 1968; Platelets: their size, shape and stickiness in vitro: degranulation and propinquity. Experimental Biology and Medicine 3: 90.
• 22 Reinhold J. G, Pascoe E, and Kfoury G. A. 1968; Capillary diameter and rate of aspiration as factors affecting the accuracy of zinc analysis in serum by atomic absorption spectrophotometry. Analytical Biochemistry 25: 557.
• 23 Rogers A. B. 1972; Effect of pH on human platelet aggregation induced by epinephrine and ADP. Proceedings of the Society for Experimental Biology and Medicine 139: 1100.
• 24 Salzman E. W, and Levine L. 1971; Cyclic 3’,5’-adenosine monophosphate in human blood platelets. Journal of Clinical Investigation 50: 131.
• 25 Shaw E, and Glover G. 1970; Further observations on substrate-derived chloromethyl ketones that inactivate trypsin. Archives of Biochemistry and Biophysics 139: 298.
• 26 Sixma J. J, and Nijessen J. G. 1970; Characteristics of platelet factor 3 release during ADP-induced aggregation. Thrombosis et Diathesis Haemorrhagica 24: 206.
• 27 Skoza L, Zucker M. B, Jerushalmy Z, and Grant R. 1967; Kinetic studies of platelet aggregation induced by adenosine diphosphate and its inhibition by chelating agents, guanidino compounds, and adenosine. Thrombosis et Diathesis Haemorrhagica 18: 713.
• 28 Spaet T. H, and Zucker M. B. 1964; Mechanism of platelet plug formation and role of adenosine diphosphate. American Journal of Physiology 206: 1267.
• 29 Stormorken H. 1969 The release reaction of secretion. Scandinavian Journal of Haematology. suppl. 9
• 30 Talalay P, Fishman W. H, and Huggins C. 1946; Chromogenic substrates. IL Phenolphthalein glucuronic acid as substrate for the assay of glucuronidase activity. Journal of Biological Chemistry 166: 457.
• 31 Thomas D. P. 1967; Effect of catecholamines on platelet-aggregation caused by thrombin. Nature 215: 298.
• 32 Valdorf-Hansen J. F, and Zucker M. B. 1971; Effect of temperature and inhibitors on serotonin-¹⁴ release from human platelets. American Journal of Physiology 220: 105
• 33 Weiss H. J. 1967; Platelet aggregation, adhesion and adenosine diphosphate release in thrombo-pathia (platelet factor 3 deficiency); a comparison with Glanzmann’s thrombasthenia and von Willebrand’s disease. American Journal of Medicine 43: 570.
• 34 White J. G. 1969; Effects of colchicine and vinca alkaloids on human platelets. III. Influence on primary internal contraction and secondary aggregation. American Journal of Pathology 54: 467.
• 35 Zucker M. B, and Jerushalmy Z. 1967. Studies on platelet shape and aggregation; the effect of inhibitors on these and other platelet characteristics. In: Johnson S. A, and Seegers W. H. (eds.) Physiology of Hemostasis and Thrombosis. Charles C. Thomas; Springfield, Illinois: 249.
• 36 Zucker M. B, Pert J. H, and Hilgartner M. W. 1966; Platelet function in a patient with thrombasthenia. Blood 28: 524.
• 37 Zucker M. B, and Peterson J. 1968; Inhibition of adenosine diphosphate-induced secondary aggregation and other platelet functions by acetylsalicylic acid ingestion. Proceedings of the Society for Experimental Biology and Medicine 127: 547.
• 38 Zucker M. B, and Peterson J. 1970; Effect of acetylsalicylic acid, other nonsteroidal antiinflammatory agents, and dipyridamole on human blood platelets. Journal of Laboratory and Clinical Medicine 76: 66.
• 39 Zucker-Franklin D. 1969; Microfibrils of blood platelets : their relationship to microtubules and the contractile protein. Journal of Clinical Investigation 48: 165.