Kinetic Studies of Platelet Aggregation Induced by Adenosine Diphosphate and Its Inhibition by Chelating Agents, Guanidino Compounds, and Adenosine^[*]

Summary

The kinetics, activation, pH dependence and inhibition of platelet aggregation induced by ADP were studied by recording the OD of stirred PRP from blood anticoagulated with acid-citrate-dextrose solution. There was a lag period between the addition of ADP and the initiation of aggregation. Addition of ADP in the absence of stirring or ionized calcium did not cause aggregation. When aggregation was subsequently initiated by stirring or the addition of CaCl₂ there was no lag period. The steepest slope of the OD curve was used as the reaction velocity (V). It was affected by the concentration of calcium ions and was maximum at pH 8.0. When the ADP concentration was varied in the presence of different concentrations of ionized calcium, the overall kinetics revealed a series of rectangular hyperbola segments which did not pass through the origin. These observations led to the conclusion that the overall reaction can be depicted as a chain reaction: ADP interacts reversibly with platelets; when calcium is present these platelets become “sticky” and, when stirred, they aggregate. Kinetic studies of inhibition indicated that adenosine is a competitive inhibitor of ADP. The guanidino compounds tested were noncompetitive with respect to ADP but their inhibitory activity was counteracted by calcium.

• References

• 1 Sharp A. A. Present status of platelet aggregation. New Engl. J. Med. 272: 89 1965;
• 2 Marcus A. J, Zucker M. B. The physiology of blood platelets; recent biochemical, morphologic and clinical research. Grune & Stratton; New York: 1965
• 3 Zucker M. B, Jerushalmy Z. Studies on platelet shape and aggregation; the effect of inhibitors on these and other platelet characteristics. In: Physiology of hemostasis and thrombosis. Johnson S. A, Seegers W. H. Eds. Charles C Thomas; Springfield: 111 1967
• 4 Johnson S. A, Van Horn D. L, Pederson H. J, Marr J. The function of platelets; a review. Transfusion (Philad.) 06: 3 1966;
• 5 Johnson S. A. Red blood cells, fibrin and platelets in hemostasis. In: Physiology of hemostasis and thrombosis. Johnson S. A, Seegers W. H. Eds. Charles C Thomas; Springfield: 111 1967
• 6 Born G. V. R, Gross M. J. Effects of inorganic ions and of plasma proteins on the aggregation of blood platelets by adenosine diphosphate. J. Physiol. (Lond.) 170: 397 1964;
• 7 Mitchell J. R. A, Sharp A. A. Platelet clumping in vitro. Brit. J. Haemat. 10: 78 1964;
• 8 Hovig T. The effect of calcium and magnesium on rabbit blood platelet aggregation in vitro. Thrombos. Diathes. haemorrh. (Stuttg.) 12: 179 1964;
• 9 McLean J. R, Maxwell R. E, Hertler D. Fibrinogen and adenosine phosphate-induced aggregation of platelets. Nature (Lond.) 202: 605 1964;
• 10 Deykin D, Pritzker C. R, Scolnick E. M. Plasma co-factors in adenosine diphosphate- induced aggregation of human platelets. Nature (Lond.) 208: 296 1965;
• 11 O’Brien J. R. Platelet aggregation. Part I. Some effects of the adenosine phosphates, thrombin, and cocaine upon platelet adhesiveness. Part II. Some results from a new method of study. J. clin. Path. 15: 446 1962;
• 12 Born G. V. R, Gross M. J. The aggregation of blood platelets. J. Physiol. (Lond.) 168: 178 1963;
• 13 Salzman E. W, Chambers D. A. Inhibition of ADP-inducedplatelet aggregation by substituted amino acids. Nature (Lond.) 204: 698 1964;
• 14 Brinkhous K. M, Bead M. S, Mason R. G. Plasma thrombocyte-agglutinating activity and fibrinogen; synergism with adenosine diphosphate. Lab. Invest. 14: 335 1965;
• 15 Jerushalmy Z, Skoza L, Zucker M. B, Grant R. Inhibition of ADP-induced platelet aggregation by guanidino compounds. Biochem. Pharmacol. 15: 1791 1966;
• 16 Zucker M. B, Borrelli J. Reversible alterations in platelet morphology produced by anticoagulants and by cold. Blood. 09: 602 1954;
• 17 Flatow F. A, Levin B. H, Freireich E. J. Preparation of platelet concentrates for transfusion employing adenosine diphosphate. Transfusion (Philad.) 06: 205 1966;
• 18 Flatow Jr F. A, Freireich E. J. The increased effectiveness of platelet concentrates prepared in acidified plasma. Blood. 27: 449 1966;
• 19 Zucker M. B, Zaccardi J. B. Platelet shape change induced by adenosine diphosphate and prevented by adenosine monophosphate. Fed. Proc. (No. 2, Part I.) 23: 299 1964;
• 20 Dixon M, Webb E. C. Enzymes. Academic Press; New York: 1964
• 21 Macmillan D. C, Oliver M. F. The initial changes in platelet morphology following the addition of adenosine diphosphate. J. Atheroscler. Res. 05: 440 1965;
• 22 Bull B. S, Zucker M. B. Changes in platelet volume produced by temperature, metabolic inhibitors, and aggregating agents. Proc. Soc. exp. Biol. (N. Y.) 120: 296 1965;
• 23 Zucker M. B, Pert J. H, Hilgartner M. W. Platelet function in a patient with thrombasthenia. Blood. 28: 524 1966;
• 24 O’Brien J. R. Effects of adenosine diphosphate and adrenaline on mean platelet shape. Nature (Lond.) 207: 300 1965;
• 25 Cohn M. Magnetic resonance studies of metal activation of enzymic reactions of nucleotides and other phosphate substrates. Biochemistry. 02: 623 1963;
• 26 Martell A. E, Schwarzenbach G. Adenosinphosphat und Triphosphat als Komplexbildner für Calcium und Magnesium. Helv. chim. Acta. 39: 653 1956;
• 27 Walaas E. Stability constants of metal complexes with mononucleotides. Acta chem. scand. 12: 528 1958;
• 28 Taqui Khan M. M, Martell A. E. Metal chelates of adenosinediphosphoric and adenosine- monophosphoric acids. J. Amer. chem. Soc. 84: 3037 1962;
• 29 Macmillan D. C. Secondary clumping effect in human citrated platelet-rich plasma produced by adenosine diphosphate and adrenaline. Nature (Lond.) 211: 140 1966;
• 30 Mills D. G. B, Boberts G. C. K. Membrane active drugs and the aggregation of human blood platelets. Nature (Lond.) 213: 35 1967;
• 31 Pressman B. C, Park J. K. Competition between Mg⁺⁺ and guanidine for mitochondrial binding sites. Biochem. biophys. Res. Commun. 11: 182 1963;