The Effect of Calcium and Magnesium on Rabbit Blood Platelet Aggregation in Vitro^[*]

Summary

The effect of calcium and magnesium on the aggregation of rabbit blood platelets in vitro was studied, with the following results:

1. Platelet aggregation induced by ADP or collagen could be prevented by EGTA or EDTA. The aggregating effect was restored by recalcification. The effect was also restored by addition of magnesium in EDTA-PRP, but not in EGTA-PRP unless a surplus of calcium was present.

2. Calcium remained in concentrations of the order of 0.15–0.25 mM after dialysis or cation exchange of plasma. Aggregation of washed platelets resuspended in such plasma could not be produced with ADP or collagen, unless the calcium concentration was increased or that magnesium was added.

3. The adhesiveness of blood platelets to collagen was reduced in EGTA-PRP and EDTA-PRP. Release of ADP from platelets influenced by collagen could not be demonstrated either in EGTA-PRP (presence of magnesium) or in EDTA-PRP.

4. It is concluded that calcium is a necessary factor both for the reaction leading to release of ADP and for the the aggregation produced by ADP.

5. Thrombin induced aggregation of washed platelets suspended in tris-buffered saline in the presence of calcium. No effect of magnesium could be observed unless small quantities of calcium were present.

• References

• 1 Berwick L, Coman D. R. Some chemical factors in cellular adhesion and stickiness. Cancer Res. 22: 982 1962;
• 2 Bounemeaux Y. Sur le mécanisme de la retraction du caillot et de la métamorphose visqueuse des plaquettes. Rev. Hémat. 12: 16 1957;
• 3 Bounameaux Y. Action de la thrombine et de la reptilase sur la métamorphose visqueuse des plaquettes de diverses espèces animales. Rev. franc. Etud. clin. biol. 4: 54 1959;
• 4 Brinkhoits K. M, LeRoy E. C, Cornell W. P, Brown R. C, Hazlehurst J. L, Vennart G. P. Macroscopic studies of platelet agglutination ; nature of thrombocyte agglutinating activity of plasma. Proc. Soc. exp. Biol. (N.Y.) 98: 379 1958;
• 5 Flaschka H. EDTA titrations. London: Pergamon Press; 122 1959
• 6 Fruton J. S, Simmonds S. General biochemistry. 2. ed.. New York: John Wiley & Sons; 1077 1959
• 7 Godai H. G. The effect of EDTA on human fibrinogen and its significance for the coagulation of fibrinogen with thrombin. Scand. J. clin. Lab. Invest. 12 (Suppl. 53) 20 1960;
• 8 Grette K. Studies on the mechanism of thrombin-catalyzed hemostatic reactions in blood platelets. Actaphysiol. scand. 56 (Suppl. 195) 83 1962;
• 9 Halse K. Unpublished data.
• 10 Hellem A. J. The adhesiveness of human blood platelets in vitro. Scand. J. clin. Lab. Invest. 12 (Suppl. 51) 117 1960;
• 11 Hovig T. The ultrastructure of rabbit blood platelet aggregates. Thrombos. Diathes. haemorrh. (Stuttg.) 8: 455 1962;
• 12 Hovig T. Aggregation of rabbit blood platelets produced in vitro by saline “extract” of tendons. Thrombos. Diathes. haemorrh. (Stuttg.) 9: 248 1963;
• 13 Hovig T. Release of a platelet-aggregating substance (adenosine diphosphate) from rabbit blood platelets induced by saline “extract” of tendons. Thrombos. Diathes. haemorrh. (Stuttg.) 9: 264 1963;
• 14 Karnovshy N. J. Simple methods for “staining with lead” at high pH in electron microscopy. J. biophys. biochem. Cytol. 11: 729 1961;
• 15 Käser-Glanzmann R, Lüscher E. F. The mechanism of platelet aggregation in relation to hemostasis. Thrombos. Diathes. haemorrh. (Stuttg.) 7: 480 1962;
• 16 Lundevall J. Serological studies of human blood platelets. Scand. J. clin. Lab. Invest. 10 (Suppl. 34) 122 1958;
• 17 Lüscher E. F. Viscous metamorphosis of blood platelets and clot retraction. Vox Sang. 1: 133 1956;
• 18 Maltaner F. Enchancing effect of Mg-f + on clotting activity of Ca++. Proc. Soc. exp. Biol. (N.Y.) 67: 301 1948;
• 19 Oellgaard E. Macroscopic studies of platelet aggregation. Nature of an aggregating factor in red blood cells and platelets. Thrombos. Diathes. haemorrh. (Stuttg.) 6: 86 1961;
• 20 Palade C. E. A study of fixation for electron microscopy. J. exp. Med. 95: 285 1952;
• 21 Ringbom A, Pensar E. Wänninen A compleximetric titration method for determining calcium in the presence of magnesium. Anal. Chem. 19: 525 1958;
• 22 Ryter A, Kellenberger E. L’inclusion au polyester pour l’ultramicrotomie. J. Ultrastruct. Res. 2: 200 1958;
• 23 Schmid H. J, Jackson D. P, Gonley D. L. Mechanism of action of thrombin on platelets. J. clin. Invest. 41: 543 1962;
• 24 Shermer R, Mason W.R. G, Wagner R. H, Brinkhous K. M. Studies on thrombin-induced platelet agglutination. J. exp. Med. 114: 905 1961;
• 25 Skâlhegg B. A, Hellem A. J, Oedegaard A. E. Investigations on adenosine diphosphate (ADP) induced platelet adhesiveness in vitro. Thrombos. Diathes. haemorrh. (Stuttg.). 11: 305 317 1964;
• 26 Weiss L. Studies on cellular adhesion in tissue culture. III. Some effects of calcium. Exp. Cell Res. 21: 71 1960;
• 27 Weiss L. The adhesion of cells. Int. Rev. Cytol. 9: 187 1960;
• 28 Zucker M. B, Borrelli J. Viscous metamorphosis of blood platelets produced by thrombin. Fed. Proc. 14: 168 1955;
• 29 Zucker M. B, Borrelli J. Viscous metamorphosis, clot retraction and other morphologic alterations of blood platelets. J. appi. Physiol. 14: 575 1959;
• 30 Zucker M. B, Borrelli J. Platelet clumping produced by connective tissue suspensions and collagen. Proc. Soc. exp. Biol. (N.Y.) 109: 779 1962;