Inhibition of Thrombin-Induced Secretion from Platelets by Chlortetracycline and Its Analogs^[*]

Summary

Chlortetracycline (CTC) (1.0 mM) blocks platelet secretion after a few seconds preincubation. The amount needed for inhibition can be reduced relative to time of preincubation. 50% inhibition is obtained with 20 ¼M CTC after 1 hr incubation. 50 ¼M CTC causes much greater inhibition, especially after short time incubation. The level of metabolic ATP is decreased about 60% with both 20 and 50 ¼M CTC. Two tetracycline analogs, anhydrotetracycline and demeclocycline (DMC), have different solubility properties in nonionic medium, but inhibit secretion at the same concentration, with little effect on the metabolic ATP level. The results suggest that CTC and its analogs do not inhibit platelet function by acting as metabolic inhibitors. While CTC causes increased leakage of cytoplasmic content at the concentrations where secretion is blocked more than 90%, DMC doses not cause leakage even at much higher concentration, so that there seems to be no connection between the induction of leakage (i.e. the membrane-active properties) and the inhibitory effect of the drugs.

Key words

Chlortetracycline - Calcium antagonists - Platelet inhibitor - Platelet metabolism - Tetracycline analogs - Platelet leakage

^* Some of these results were presented at the VII. International Congress on Thrombosis and Haemostasis, London, UK, July, 1979 (Abstract Thrombos. Haemostas. 42: 216).

• References

• 1 Caswell AH, Hutchison JD. Visualization of membrane bound cations by a fluorescent technique. Biochem Biophys Res Commun 1971; 42: 43-49
• 2 Caswell AH. The migration of divalent cations in mitochondria visualized by a fluorescent chelate probe. J Membrane Biol 1972; 7: 345-364
• 3 Schuster SM, Olson MS. Energy-dependent release of magnesium from beef heart submitochondrial particles. J Biol Chem 1973; 248: 8370-8377
• 4 Carvalho CA, Carvalho AP. Fluorimetrie monitoring of calcium binding to sarcoplasmic reticulum membranes. Biochim Biophys Acta 1977; 468: 21-30
• 5 Luthra R, Olson MS. The effects of chlorotetracyclin on calcium movements in isolated rat liver mitochondria. Arch Biochem 1978; 191: 494-502
• 6 Chandler DE, Williams JA. Intracellular divalent cation release in pancreatic acinar cells during stimulus-secretion coupling I. Use of chlorotetracycline as fluorescent probe. J Cell Biol 1978; 76: 371-385
• 7 LeBreton GC, Dinerstein RJ, Roth LJ, Feinberg H. Direct evidence for intracellular divalent cation redistribution associated with platelet shape change. Biochem Biophys Res Commun 1976; 71: 362-370
• 8 Feinstein MB. Release of intracellular membrane-bound calcium precedes the onset of stimulus-induced exocytosis in platelets. Biochem Biophys Res Commun 1980; 93: 593-600
• 9 LeBreton GC, Sandler WC, Feinberg H. Effect of D₂O and chlortetracycline on ADP-induced platelet shape change and aggregation. Thromb Res 1976; 8: 477-485
• 10 Mürer EH, Stewart GJ, Davenport K, Siojo E, Rahwan RG, Witiak DT. Effect of three calcium antagonists on platelet secretion and metabolism. Biochem Pharmac 1981; 30: 523-530
• 11 Mürer EH, Davenport K, Rausch MA, Day HJ. Metabolic aspects of the secretion of stored compounds from blood platelets V. Effect of ionophore A23187 on washed platelets. Biochim Biophys Acta 1976; 451: 1-19
• 12 Lages B, Scrutton MC, Holmsen H. Studies on gel filtered human platelets: Isolation and characterization in a medium containing no added Ca⁺⁺, Mg⁺⁺, K⁺ . J Lab Clin Med 1975; 85: 811-825
• 13 Walsh PN, Gagnatelli G. Platelet antiheparin activity: Storage sites and release mechanism. Blood 1974; 44: 157-168
• 14 Holmsen H, Robkin L, Day HJ. Effects of antimycin and 2-deoxyglucose on secretion in human platelets. Biochem J 1979; 182: 413-419
• 15 Mürer EH, Stewart GJ, Rausch MA, Day HJ. Calcium ionophore A 23187 (Eli Lilly): Effect on platelet function, structure and metabolism. Thrombos Diathes Haemorrh 1975; 34: 72-82
• 16 Mürer EH, Siojo E. Effects of 8-(N,N-diethylamino)octyl 3,4,5-trimethoxyben-zoate, HCl (TMB-8) on washed blood platelets with special reference to a specific loss of serotonin. Thromb Haemostas 1982; 47: 59-61
• 17 Dürckheimer W. Tetracyclines: Chemistry, biochemistry and structureactivity relations. Angewandte Chemie Int Ed in English 1975; 14: 721-734
• 18 Mürer EH. Release reaction and energy metabolism in blood platelets with special reference to the burst in oxygen uptake. Biochim Biophys Acta 1968; 162: 320-326
• 19 Gains N. The limitations of chlortetracycline as a fluorescent probe of divalent cations associated with membrans. Eut J Biochem 1980; 111: 199-202