Requirements for Aggregation of Washed Human Platelets Suspended in Buffered Salt Solutions

Christina B Harbury; J. Edward Hershgold; Stanley L Schrier

doi:10.1055/s-0038-1649036

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1972; 28(01): 002-013
DOI: 10.1055/s-0038-1649036

Original Article

Schattauer GmbH

Requirements for Aggregation of Washed Human Platelets Suspended in Buffered Salt Solutions

Authors

Christina B Harbury

¹Division of Hematology, Department of Medicine, Stanford University School of Medicine, Stanford, California, and Veterans Administration Hospital, Palo Alto, California
J. Edward Hershgold

¹Division of Hematology, Department of Medicine, Stanford University School of Medicine, Stanford, California, and Veterans Administration Hospital, Palo Alto, California
Stanley L Schrier

¹Division of Hematology, Department of Medicine, Stanford University School of Medicine, Stanford, California, and Veterans Administration Hospital, Palo Alto, California

Abstract

Summary

With the use of washed human platelets suspended in a buffered salt solution, we have studied the factors capable of acting directly at the platelet plasma membrane to produce aggregation as opposed to those which produce the platelet release reaction and secondary aggregation. Platelet aggregation was measured in an aggregometer and the release reaction followed by assays of the release products.

Ca and fibrinogen will not independently induce aggregation in washed human platelets suspended in a buffered salt solution. However, in their absence ADP-, 5HT-and epinephrine-mediated aggregation will not proceed. Of particular interest was the finding that in the presence of Ca, threshold doses of thrombin will produce aggregation earlier if μg amounts of fibrinogen are added. Ca and fibrinogen therefore appear to be absolutely required for aggregation, whether the aggregation is produced by the addition or by the release of required components.

If either ADP, 5HT or epinephrine is added singly to buffer-suspended platelets in the presence of Ca and fibrinogen, 10–20% aggregation is observed. The combination of either ADP-5HT, ADP-epinephrine or 5HT-epinephrine produces 60–70% aggregation. The addition of ADP, 5HT and epinephrine together produces 80–90% aggregation.

No release reaction is produced by the addition of ADP or 5HT alone or in combination. A new finding in this study therefore is that 5HT and ADP can act directly at the platelet plasma membrane to produce extensive aggregation in the absence of any release reaction.

Epinephrine does produce a small release reaction, and may therefore produce its enhancement of aggregation either by acting directly at the platelet plasma membrane or by the release of something other than Ca, fibrinogen, ADP, 5HT, or epinephrine.

In this study using washed human platelets suspended in a buffered salt solution, no evidence was found for the requirement of a plasma protein other than fibrinogen in platelet aggregation.

Full Text

References

References
1 Aster R. H, Jandl J. H. 1964; Platelet sequestration in man. I. Methods in rat platelets. The Journal of Clinical Investigation 43: 843.
2 Baumgartner H. R, and Born G. V. R. 1968; Effects of 5-hydroxytryptamine on platelet aggregation. Nature 218: 137.
3 Born G. V. R. 1962; Quantitative investigations into the aggregation of blood platelets. The Journal of Physiology 162: 67P.
4 Born G. V. R, and Cross M. J. 1963; The aggregation of blood platelets. The Journal of Physiology 168: 178.
5 Born G. V. R, and Cross M. J. 1964; Effects of inorganic ions and plasma proteins on the aggregation of blood platelets by adenosine diphosphate. The Journal of Physiology 170: 397.
6 Born G. V. R, and Smith J. B. 1970; Uptake, metabolism and release of ³H-adrenaline. British Journal of Pharmacology 39: 765.
7 Cronberg S, Kubisz P, and Caen J. P. 1970; Demonstration of a plasmatic cofactor different from fibrinogen necessary for platelet release by ADP and adrenaline. Thrombosis et Diathesis Haemorrhagica 24: 409.
8 Cross M. J. 1964; Effects of fibrinogen on the aggregation of platelets by adenosine diphosphate. Thrombosis et Diathesis Haemorrhagica 12: 524.
9 Davey M. G, and Luscher E. F. 1968; Release reactions of human platelets induced by thrombin and other agents. Biochimica et Biophysica Acta 165: 490.
10 Day J. H, Holmsen H, and Hovig T. 1969 Subcellular particles of human platelets. Scandinavian Journal of Haematology, Supplement 7.
11 Day J. H, and Holmsen H. 1971; Concepts of the blood platelet release reaction. Series Haematologica 4: 3.
12 Deykin D, Pritzker C. R, and Scolnick E. M. 1965; Plasma cofactors in adenosine diphos-phase induced aggregation of human platelets. Nature 208: 296.
13 Gaarder A, Jonsen J, Laland S, Hellem A, and Owren P. A. 1961; Adenosine diphosphate in red cells as a factor in the adhesiveness of human blood platelets. Nature 192: 531.
14 Goodman L. S, and Gilman A. (eds.) 1966. The pharmacological basis of therapeutics. Third Edition.. The MacMillan Company; New York:
15 Haslam R. J. 1964; Role of adenosine diphosphate in the aggregation of human blood platelets by thrombin and by fatty acids. Nature 202: 765.
16 Holmsen H, Day J. H, and Stormorken H. 1969 The blood platelet release reaction. Scandinavian Journal of Haematology, Supplement #8.
17 Holmsen H, and Day J. H. 1970; The selectivity of the thrombin-induced platelet release reaction: Subcellular localization of released and retained constituents. The Journal of Laboratory and Clinical Medicine 75: 840.
18 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.
19 Lowry O. H, Rosebrough N. J, Fare A. L, and Randall R. J. 1951; Protein measurement with the folin phenol, reagent. The Journal of Biological Chemistry 193: 265.
20 Macmillan D. C. 1966; Secondary clumping effect in human citrated platelet-rich plasma produced by adenosine diphosphate and adrenaline. Nature 211: 140.
21 Massini P, and Luscher E. F. 1971; The induction of the release reaction in human blood platelets by close cell contact. Thrombosis et Diathesis Haemorrhagica 25: 13.
22 Mills D. C. B, and Roberts G. C. K. 1967; Effects of adrenalin on human blood platelets. The Journal of Physiology 193: 443.
23 Mills D. C. B, Robb I. A, and Roberts C. K. 1968; The release of nucleotides, 5-hydroxy-tryptamine and enzymes from human blood platelets during aggregation. The Journal of Physiology 195: 715.
24 Mosseson M. W, and Finlayson J. S. 1963; Subfraction of human fibrinogen. The Journal of Laboratory and Clinical Medicine 62: 663.
25 Murer E. H. 1968; Release reaction and energy metabolism in blood platelets with special reference to burst in oxygen uptake. Biochimica et Biophysica Acta 162: 32.
26 Mürer E. H. 1969; A comparative study of the action of release inducers upon platelet release and phosphorus metabolism. Biochimica et Biophysica Acta 192: 138.
27 Mustard J. F, and Packham M. A. 1970; Factors influencing platelet function. Pharmacological Reviews 22: 97.
28 Rossi E. C. 1967; Some effects on artificial media upon glycolysis in rat platelets. Blood 30: 758.
29 Solum N. O. 1970; ADP-Induced aggregation of washed platelets. Scandinavian Journal of Haematology 7: 236.
30 Thomas D. P, Niewiarowski S, and Ream V. J. 1970; Release of adenine nucleotide and platelet Factor IV from platelets of man and four other species. The Journal of Laboratory and Clinical Medicine 75: 607.