L.C. Best

Effects of epoxymethano analogues of prostaglandin endoperoxides on aggregation, on release of 5-hydroxytryptamine and on the metabolism of 3′,5′-cyclic AMP and cyclic GMP in human platelets

Abstract The effects on human platelets of two synthetic analogues of prostaglandin endoperoxides were examined in order to explore the relationship between aggregation and prostaglandin and cyclic nucleotide metabolism, and to help elucidate the role of the natural endoperoxide intermediates in regulating platelet function. Both analogues (Compound I, (15 S )-hydroxy-9 α ,11 α -(epoxymethano)-prosta-(5 Z ,13 E )-dienoic acid, and Compound II, (15 S )-hydroxy-11 α ,9 α -(epoxymethano)-prosta-(5 Z ,13 E )-dienoic acid) caused platelets to aggregate, an effect which could be inhibited by prostaglandin E 1 but not by indomethacin. Compound II produced primary, reversible aggregation at concentrations which did not induce release of 5-hydroxytryptamine. Production of thromboxane B 2 and malonyldialdehyde was monitored as an index of endogenous production of prostaglandin endoperoxides and thromboxane A 2 and were increased after incubation of human platelets with thrombin, collagen or arachidonic acid. However, neither malonydialdehyde nor thromboxane B 2 levels were significantly influenced by the endoperoxide analogues. Both analogues produced a small elevation of adenylate cyclase activity in platelet membranes and of cyclic AMP content in intact platelets, but neither had any modifying effect on the much greater stimulation of adenylate cyclase and cyclic AMP levels by prostaglandin E 1 . Of all the aggregating agents tested, only arachidonic acid produced any significant increase in platelet cyclic GMP levels. These results suggest that the epoxymethano analogues of prostaglandin endoperoxides induce platelet aggregation independently of thromboxane biosynthesis and without inhibiting adenylate cyclase or lowerin platelet cyclic AMP levels. They therefore differ from better known aggregating agents such as ADP, epinephrine and collagen, which increase thromboxane A 2 production and reduce cyclic AMP levels, at least in platelets previously exposed to prostaglandin E 1 .

Strontium ions induce production of thromboxane B2 and secretion of 5-hydroxytryptamine in washed human platelets

Abstract The addition of strontium chloride to suspensions of washed human platelets induced biosynthesis of thromboxane B 2 (TxB 2 ) and secretion of [ 14 C]-5-hydroxytryptamine (5HT) in a dose dependent manner. Both responses occurred maximally within four minutes and were inhibited competitively by the presence of calcium ions. Magnesium and lanthanum ions also inhibited both the production of thromboxane B 2 and secretion of 5HT in response to strontium ions. Inhibition of platelet thromboxane generation by aspirin resulted in an almost total inhibition of the secretion response after adding strontium ions. In addition, both the secretion of 5HT and thromboxane B 2 production in the presence of strontium ions were inhibited by dibutyryl cyclic AMP and by prostaglandin E 1 . The results suggest that strontium ions induce platelet secretion largely via the liberation of arachidonate from membrane phospholipid with the subsequent biosynthesis of thromboxane A 2 .

Strontium ions stimulate phosphoinositide metabolism in human blood platelets

Phospholipid metabolism is thought to play a key role in the activation of a wide variety of cell types [1,2]. In platelets, arachidonate is released from membrane phospholipids [3] and subsequently converted to thromboxane A2, a potent inducer of platelet aggregation and secretion [4]. In addition, one or possibly more thromboxane-independent mechanisms of platelet activation exist which also appear to involve phospholipid metabolism [5,6]. In response to thrombin [7,8] and to collagen [7], platelet phosphatidylinositol is degraded by phopholipase C with the formation of diglyceride [9] and subsequently phosphatidic acid [10]. The mechanism by which these events are coupled to secretion and aggregation is not clear, although it has been postulated that the phosphatidylinositol effect may be associated in some way with a rise in cytosol calcium concentration [1 ]. We have shown that secretion of 5-hydroxytryptamine in platelets can be triggered by the addition of strontium ions [ 11 ], possibly by mimicking a rise in intracellular calcium. We report here that strontium ions cause a specific turnover of the platelet phosphatidylinositol pool accompanied by an apparent stimulation of polyphosphoinositide metabolism. The latter class of phospholipids is also thought to be involved in the regulation of cell activation during stimulation and may thus play a role in the control of granule secretion and other processes which occur during aggregation.

Lanthanum stimulates the accumulation of cyclic AMP and inhibits secretion and thromboxane B2 formation in human platelets.

La3+ was found to inhibit the secretion of 5-hydroxytryptamine and the production of thromboxane B2 by washed platelets exposed to collagen or thrombin. In addition, La3+ inhibited secretion in response to sodium arachidonate, although the conversion of arachidonate to thromboxane B2 was not affected. La3+ was also found to enhance the accumulation of cyclic AMP under basal conditions and in response to prostaglandin E1, in washed platelets. The inhibition of cyclic AMP accumulation by ADP was prevented by La3+, suggesting that the effect of ADP on cyclic AMP metabolism was dependent upon the presence or flux of calcium at the platelet membrane. La3+ inhibited the activity of adenylate cyclase in platelet lysates both in response to prostaglandin E1 and to F-, indicating a possible effect at the catalytic subunit of the enzyme. None of the observed effects of La3+ could be reversed by the addition of Ca2+ up to 10 mM. The stimulation of cyclic AMP production by La3+ may largely explain the inhibitory effect of La3+ upon platelet secretion and thromboxane B2 production. These results also suggest that Ca2+ localised at the platelet plasma membrane may be important in the regulation of cyclic AMP metabolism.

Phosphoinositide metabolism in human blood platelets: Effects of two types of divalent cation chelators

Phospholipid metabolism was investigated in platelets in relation to secretion of 5-hydroxytryptamine. Both thrombin and the ionophore, A23187, induced secretion of 5-hydroxytryptamine and platelet aggregation accompanied by the specific breakdown of phosphatidyl inositol. The products detected corresponded to phosphatidic acid and a substance which co-chromatographed with polyphosphoinositides and lysophosphatidyl inositol. Thrombin-induced aggregation, secretion and phosphatidyl inositol metabolism were all prevented by EDTA, but only the secretory response was inhibited by the intracellular calcium chelator chlortetracycline (CTC). In contrast, A23187 induced turnover of phosphatidyl inositol in the presence of EDTA even though aggregation and secretion were prevented. All three responses to A23187 were inhibited by chlortetracycline. Thus, platelet aggregation, secretion and phospholipid metabolism can be dissociated by the use of two types of divalent cation chelators: EDTA and CTC. Furthermore, these findings suggest that thrombin and A23187 induce phosphoinositide metabolism by two distinct mechanisms.