Thrombin-Induced Increase in Intracellular Cyclic 3′,5′-Adenosine Monophosphate in Human Platelets

Abstract

A B S T R A C T The present data disagree with earlier suggestions that thrombin's effect on platelets is to cause a decrease in intracellular cyclic 3',5'-adenosine monophosphate. Washed humani platelets or plateletrich plasma were incubated at 37°C with human thrombin. After centrifugation, the supernates were assayed for nucleotides and calcium released. The platelet pellets, anid in some experiments the supernates as well, were assayed by radioiimmunoassay for intracellular cyclic AMP. In the washed platelet systenm, increasing doses of thrombin to 0.5 U/cc induced increasing release of nucleotides and calcium. This was accompanied by an average twofold increase in intracellular cyclic AMP levels. Prostaglandin E1, which inhibited 30-50% of release, induced a four-to fivefold increase in cyclic AMP levels that was additive to the cyclic AMP-stimulatory effect of thrombin. Theophylline. which inhibited only 20-40%4 of nucleotide release, was synergistic with thrombin in the intracellular accumulation of cyclic AMP. The timiie-course of cyclic AMP accumulation in response to thrombinl was slower thani thrombin-induced nucleotide release. Similar findings were made in the platelet-rich plasma system where thrombin stimulation of nucleotide release also resulted in a marked accumulation of intracellular cyclic AMP. Thrombin did not appear to stimiiulate the release of intracellular cyclic AMP.The mechanism underlying these observations was not apparent. The thrombin had no measurable inhibitory effect on platelet phosphodiesterase activity in either intact washed cells or the platelet homogenate supernates. Furthermore, thrombin inhibited, rather than stimulated, platelet adenyl cyclase activity in both

Enhancement of platelet function by superoxide anion.

“…Thrombin, and other aggregating agents, clearly inhibit adenyl cyclase activity (25)(26)(27). The fact that thrombin also synergizes with O2 to produce aggregation and release suggests that the two stimuli may act on similar or closely related membrane sites.…”

Modulation of adenylate cyclase of human platelets by phorbol ester

“…In membrane preparations, such a receptor-induced, Ni-mediated adenylate cyclase inhibition has been described for epinephrine and norepinephrine (acting via a2 adrenoceptors), for ADP, vasopressin (via VI receptors), platelet-activating factor and, very recently, also for thrombin epinephrine inhibits adenylate cyclase in membranes and reduces intracellular cyclic AMP levels in intact platelets, whereas thrombin causing an even more pronounced adenylate cyclase inhibition in membranes than epinephrine [6] does not generally reduce total platelet cyclic AMP levels. This platelet-activating agent has even been reported to increase basal platelet cyclic AMP levels [7]. The discrepancy between the effects of some platelet agonists observed in membrane preparations and intact cells suggested that there must be intracellular mechanism(s) counteracting the effects of these agonists on the membrane-bound adenylate cyclase.…”

Thrombin‐Induced Platelet Prostaglandin and Cyclic AMP Production and a Possible Intrinsic Modulation of Platelet Function*

“…These data conflicted with earlier descriptions of a decrease in intracellular cyclic AMP in response to various aggregating agents (Robinson et a1 1969, Salzman & Neri 1969, Marquis et a1 1970. A discussion of differences in methodology between the various studies failed to account satisfactorally for the apparent descrepancy of observations (Droller & Wolfe 1972). This led us to seek a plausible mechanism to explain the thrombin-induced increments in cyclic AMP that we observed.…”

“…The PRP was directly used in some of the experiments. For other studies, washed platelets were prepared as previously described (Droller & Wolfe 1972) and resuspended in 25 mM Tris-HC1 buffer, pH 7.4, containing 140 mM NaCl and 0.3 mM EDTA. Final platelet counts were 200-400 x lo9/].…”

“…This led us to seek a plausible mechanism to explain the thrombin-induced increments in cyclic AMP that we observed. On the basis both of time course studies in which the accumulation of cyclic AMP lagged the Occurrence of release by several seconds (Droller & Wolfe 1972), and of work by Smith & Willis (1971) in which thrombin was found to stimulate the production of PGE, and PGE,,, we suggested that the increments of cyclic AMP may have been caused by the stimulation of adenyl cyclase activity by the prostaglandins that the platelets had themselves produced (Droller & Wolfe 1972, Droller 1973. This implied the possible existence of a prostaglandin-cyclic AMP regulatory or modulatory mechanism of platelet function.…”