The In Vitro Production of Thromboxane B₂ by Platelets of Diabetic Patients Is Normal at Physiological Concentrations of lonized Calcium

Summary

Platelets of patients with diabetes and no evidence of macroangiopathy produce normal amounts of thromboxane (Tx) B₂ in vivo, whereas they usually show increased production in vitro. Since in vitro studies have been usually performed in citrated PRP, we tested the hypothesis that the discrepancy between in vivo and in vitro studies is due to the low concentration of plasma ionized calcium ([Ca ²⁺ ]_o) that is present in citrated PRP. In fact, low [Ca ²⁺ ]_o artifactually potentiates the platelet TxB₂ production in vitro. Forty patients with diabetes mellitus and 37 matched Controls were studied. Blood was anticoagulated with citrate, the thrombin inhibitor D-phenylalanyl-l-prolyl-l-chloromethylketone (PPACK) or both anticoagulants. Platelet aggregation, release of ¹⁴C-serotonin and TxB₂ production were induced in platelet rieh plasma (PRP) by several agonists. The following results were obtained: i) Citrated PRP: Arachidonic acid induced aggregation (p <0.01) and TxB₂ production (p <0.02) were significantly greater in patients than in Controls. No statistically significant differences were found with other agonists. ii) PPACK PRP: No statistically significant difference was found between diabetic platelets and Controls, iii) PPACK plus citrate PRP: The results were not different from those obtained with citrate alone. Therefore, our results show that diabetic platelets produce normal amounts of TxB₂ in vitro when the [Ca²⁺]_o is physiological.

• References

• 1 Sagel J, Colwell JA, Crook L. Increased platelet aggregation in early diabetes mellitus. Ann Intern Med 1975; 82: 733-738
• 2 O'Malley BC, Timperley WR, Ward JD, Porter NR, Preston FE. Platelet abnormalities in diabetic peripheral neuropathy. Lancet 1975; II: 1274-1276
• 3 Butkus A, Skrinska VA, Schumacher P. Thromboxane production and platelet aggregation in diabetic subjects with clinical complications. Thromb Res 1980; 19: 211-223
• 4 Halushka PV, Curtis RogersR, Boyd LoadholtC, Colwell JA. Increased platelet thromboxane synthesis in diabetes mellitus. J Lab Clin Med 1981; 97: 87-96
• 5 Davi G, Rini GB, Averna M, Novo S, Di FedeG, Pinto A, Notarbartolo A, Strano A. Thromboxane B₂ formation and platelet sensitivity to prostacyclin in insulin-dependent and insulin-independent diabetics. Thromb Res 1982; 26: 359-370
• 6 McDonald JWD, Dupre J, Rodger NW, Champion MC, Webb CD, Ali M. Comparison of platelet thromboxane synthesis in diabetic patients on conventional insulin therapy and continuous insulin infusions. Thromb Res 1982; 28: 705-712
• 7 Fritschi J, Christe M, Lammle B, Marbet GA, Berger W, Duckert F. Platelet aggregation, β-thromboglobulin and platelet factor 4 in diabetes mellitus and in patients with vasculopathy. Thromb Haemostas 1984; 52: 236-237
• 8 Di MinnoG, Silver MJ, Cerbone AM, Riccardi G, Rivellese A, Mancini M, Thiagarajan P. Increased binding of fibrinogen to platelets in diabetes; the role of prostaglandins and thromboxane. Blood 1985; 65: 156-162
• 9 Jones DB, Davis TM, Bown E, Carter RD, Mann JI, Prescott RJ. Determinants of ADP-induced platelet aggregation in diabetes mellitus. Diabetologia 1986; 29: 291-294
• 10 Kobbah AM, Ewald U, Tuvemo T. Platelet aggregability during the first two years of Type I (insulin-dependent) diabetes mellitus in children. Diabetologia 1989; 32: 729-735
• 11 Nievelstein PFEM, Sixma JJ, Ottenhof-Rovers M, Wynne HJ, De GrootPG, Banga JD. Platelet adhesion and aggregate formation in Type I diabetes under flow conditions. Diabetes 1991; 40: 1410-1417
• 12 Winocour PD. The role of platelets in the pathogenesis of diabetic vascular disease. In: Complications of Diabetes Mellitus. Molecular and Cellular Biology of Diabetes Mellitus, Vol III Draznin B, Melmed S, LeRoith D. (eds) Alan R. Liss, Inc.; New York: 1989. pp 37-47
• 13 Alessandrini P, McRae J, Feman S, Fitzgerald GA. Thromboxane biosynthesis and platelet function in type I diabetes mellitus. N Engl J Med 1988; 319: 208-212
• 14 Fitzgerald GA, Brash AR, Blair IA, Lawson J. Analysis of urinary metabolites of thromboxane and prostacyclin by negative ion-chemical ionization gas chromatography-mass spectrometry. In: Advances in Prostaglandin Thromboxane and Leukotriene Research. Vol 15 Hayaishi O, Yamamoto S. (eds) Raven Press; New York: 1985. pp 87-90
• 15 Davi G, Catalano I, Averna M, Notarbartolo A, Strano A, Ciabattoni G, Patrono C. Thromboxane biosynthesis and platelet function in type II diabetes mellitus. N Engl J Med 1990; 322: 1769-1774
• 16 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
• 17 Heptinstall S, Mulley GP. Adenosine diphosphate induced platelet aggregation and release reaction in heparinized platelet rieh plasma and the influence of added citrate. Br J Haematol 1977; 36: 565-571
• 18 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
• 19 Lages B, Weiss HJ. 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. Thromb Haemostas 1981; 45: 173-179
• 20 Packham MA, Kinlough-Rathbone RL, Mustard JF. Thromboxane A₂ causes feedback amplification involving extensive thromboxane A₂ formation on close contact of human platelets in media with a low concentration of ionized calcium. Blood 1987; 70: 647-651
• 21 Packham MA, Bryant NL, Guccione MA, Kinlough-Rathbone RL, Mustard JF. Effect of the concentration of Ca²⁺ in the suspending medium on the responses of human and rabbit platelets to aggregating agents. Thromb Haemostas 1989; 62: 968-976
• 22 Packham MA, Rand ML, Ruben DH, Kinlough-Rathbone RL. Effect of calcium concentration and inhibitors on the responses of platelets stimulated with collagen: contrast between human and rabbit platelets. Comp Biochem Physiol 1991; 99A: 551-557
• 23 Cattaneo M, Mustard JF, Canciani MT, Richardson M, Federici AB, Mannucci PM. Conditions influencing the interaction of asialo von Willebrand factor with human platelets, the effects of external ionized calcium concentration and the role of arachidonate pathway. Thromb Haemostas 1988; 60: 280-288
• 24 Tomaselli L, Cerletti C, de Gaetano G, Notarbartolo S, Davi G, Pupillo M. Normal platelet function, but increased platelet activation in vivo in diabetic patients. Thromb Haemostas 1990; 64: 604