Dependence of Human Platelet Functional Responses on Divalent Cations: Aggregation and Secretion in Heparin- and Hirudin-Anticoagulated Platelet-Rich Plasma and the Effects of Chelating Agents

Summary

The dependence of ADP- and epinephrine-induced platelet aggregation and secretion on extracellular divalent cations was examined by quantitating these responses in citrate-, heparin-, and hirudin-anticoagulated platelet-rich plasma. ADP-induced ¹⁴C-5HT secretion in heparin-PRP and hirudin-PRP was generally decreased, relative to that in citrate-PRP, without corresponding reductions in aggregation, whereas in response to epinephrine, both aggregation and secretion were decreased in heparin-PRP, and abolished in hirudin-PRP. In heparin-PRP, but not in hirudin-PRP, the degree to which these responses were altered was highly variable among normal subjects, and was dependent on the anticoagulant concentration. Addition of citrate restored the extent of ADP-induced secretion and of epinephrine-induced aggregation and secretion in heparin-PRP to that observed in citrate-PRP, and increased the extent of ADP-induced secretion in hirudin-PRP. Addition of EDTA or EGTA, however, had no effect on ADP-induced secretion in heparin-PRP. These results suggest that ADP-induced aggregation and secretion, as well as responses to ADP vs. epinephrine, have different dependencies on extracellular or surface-bound divalent cations. The variable responses observed in heparin-PRP may reflect direct interactions of heparin with platelets, and this variability may account for the conflicting results of previous studies.

Keywords

Platelet aggregation - Platelet secretion - Calcium - Chelating agents

• References

• 1 Born GVR, Cross MJ. The aggregation of blood platelets. J Physiol 1963; 168: 178-195
• 2 Skoza L, Zucker MB, Jerushalmy Z, Grant RA. Kinetic studies of platelet aggregation induced by adenosine diphosphate and its inhibition by chelating agents, guanidino compounds and adenosine. Thromb Diath Haemorrh 1967; 18: 713-775
• 3 Heptinstall S. The use of a chelating ion-exchange resin to evaluate the effects of the extracellular calcium concentration on adenosine diphosphate-induced aggregation of human blood platelets. Thromb Haemostas 1976; 36: 208-220
• 4 Haslam RJ. Role of adenosine diphosphate in the aggregation of human blood platelets by thrombin and by fatty acids. Nature 1964; 202: 765-768
• 5 Holmsen H, Day HJ. The selectivity of the thrombin-induced platelet release reaction: subcellular localization of released and retained constituents. J Lab Clin Med 1970; 75: 840-855
• 6 Detwiler TC, Feinmann RD. Kinetics of the thrombin-induced release of ATP by platelets. Comparison with release of calcium. Biochemistry 1973; 12: 2462-2468
• 7 Feinman RD, Detwiler TC. Platelet secretion induced by divalent cation ionophores. Nature 1974; 249: 172-173
• 8 Mustard JF, Perry DW, Kinlough-Rathbone RL, Packham MA. Factors responsible for ADP-induced release reaction of human platelets. Am J Physiol 1975; 228: 1757-1765
• 9 Ardlie NG, Han P. Enzymatic basis for platelet aggregation and release: the significance of the platelet “atmosphere” and the relationship between platelet function and blood coagulation. Brit J Haematol 1974; 26: 331-356
• 10 MacFarlane DE, Walsh PN, Mills DCB, Holmsen H, Day HJ. The role of thrombin in ADP-induced platelet aggregation and release: a critical evaluation. Brit J Haematol 1975; 30: 457-463
• 11 Lages B, Weiss HJ. Platelet aggregation and secretion: effects of anticoagulants and divalent cation chelators. Thromb Haemostas 1977; 38: 123
• 12 Heptinstall S, Mulley GP. Adenosine diphosphate induced platelet aggregation and release reaction in heparinized platelet rich plasma and the influence of added citrate. Brit J Haematol 1977; 36: 565-571
• 13 Wessler S, Gitel S. Control of heparin therapy, in Progress in Hemostasis and Thrombosis. Spaet TH. ed. Grune and Stratton; New York: 1976. 03. 311-329
• 14 Markwardt F. Hirudin as an inhibitor of thrombin, in Methods in Enzymology. Colowick SP, and Kaplan NO. eds. Academic Press; New York: 1970. XIX. 924-932
• 15 Akkerman JWN, Holmsen H, Loughnane M. Simultaneous measurement of aggregation, secretion, oxygen uptake, proton production, and intracellular metabolites in the same platelet suspension. Anal Biochem 1979; 97: 387-393
• 16 Lages B, Scrutton MC, Holmsen H. Secretion by gel filtered human platelets: response of platelet Ca, Mg, and K to secretory agents. J Lab Clin Med 1977; 90: 873-882
• 17 Holmsen H, Setkowsky-Dangelmaier CA. Adenine nucleotide metabolism of blood platelets X. Formaldehyde stops centrifugation induced secretion after A 23187 stimulation and causes breakdown of metabolic ATP. Biochim Biophys Acta 1977; 497: 46-61
• 18 Oreskes I, Hirsch C, Douglas KS, Kupfer S. Measurement of ionized calcium in human plasma with a calcium selective electrode. Clin Chim Acta 1968; 21: 303-313
• 19 Moore EW. Ionized calcium in normal serum, ultrafiltrates, and whole blood determined by ion exchange electrodes. J Clin Invest 1970; 49: 318-334
• 20 Glusa E, Markwardt F. Adrenaline-induced reactions of human platelets in hirudin plasma. Haemostasis 1980; 9: 188-192
• 21 Thomson C, Forbes CD, Prentice CRM. The potentiation of platelet aggregation and adhesion by heparin in vitro and in vivo. Clin Sci and Mol Med 1973; 45: 485-494
• 22 Heptinstall S, Taylor PM. The effects of citrate and extracellular calcium ions on the platelet release reaction induced by adenosine diphosphate and collagen. Thromb Haemostas 1979; 42: 778-793
• 23 Scrutton MC, Egan CM. Divalent cation requirements for aggregation of human blood platelets and the role of the anticoagulant. Thromb Res 1979; 14: 713-727
• 24 Reches A, Eldor A, Salomon Y. Heparin inhibits PGE₁-sensitive adenylate cyclase and antagonizes PGE₁ antiaggregating effects in human platelets. J Lab Clin Med 1979; 93: 638-644
• 25 Lages B, Scrutton MC, Holmsen H. Studies on gel filtered human platelets: isolation and characterization in a medium containing no added Ca, Mg, or K . J Lab Clin Med 1975; 85: 811-825
• 26 Kinlough-Rathbone RL, Perry DW, Mustard JF. The role of calcium and magnesium in the reactions of ADP with platelets. Fed Proc 1974; 33: 611
• 27 Haslam RJ, Rosson GM. Aggregation of human blood platelets by vasopressin. Am J Physiol 1972; 223: 958-967