Exterior and Interior Events in Early Stage of Thrombin-induced Platelet Aggregation

Abstract

SummaryTreatment of washed rabbit platelets with 1 u/ml of thrombin at 37° C resulted in a disappearance from platelets of a protein with 250,000 dalton molecular weight which was shown to be originated from plasma membrane. Parallel loss of adenyl cyclase was noted, and both reactions were complete within 30 sec. From the patterns of disc electrophoretograms, the importance of quick suppression of thrombin action in demonstrating the primary event was stressed.Thrombin induced an apparent activation of membrane bound phosphodiesterase. This reaction was also complete within 30 sec. The cellular component which contained the enzyme activity was distinct from plasma membrane. Soluble phosphodiesterase was not influenced by thrombin at all.These reactions required intact platelet cells to react with thrombin, and no reaction was detected when subcellular preparations were treated with thrombin.Possibility of collaboration of changes in externally located synthetic enzyme with those in internally located degrading enzyme in the early phase of thrombin action on platelets was suggested.

The Effect of Mitomycin C on Platelet Aggregation and Adenosine 3′, 5′-Monophosphate Metabolism

“…Evidence has been presented in recent years suggesting that platelet functions are controlled by variations in intracellular adenosine 3', 5'-rnonophosphate (cyclic AMP) level (Salzman andWeisenberger 1972, Haslam 1975). It has previously been shown in this laboratory that thrombin, a potent inducer of platelet aggregation and release reaction, not only decreases platelet adenyl cyclase activity but also stimulates structurally bound phosphodiesterase (Hashimoto et al 1977). This paper reports that Mitomycin C inhibits rabbit platelet aggregation induced by adenosine diphosphate (ADP) or thrombin, possibly by stabilizing the membranes, with a simultaneous increase in the level of cyclic AMP.…”

“…Concentrated PRP was prepared from PRP with acid-citrate-dextrose (ACD) as described in the previous paper (Hashimoto et al 1975). Washed platelets prepared from PRP with ACD (Hashimoto et al 1977) were finally suspended in an isotonic saline medium (139 mM NaCI and 15 mM Tris-HCI buffer, pH 7.4, with 1 mg per ml glucose). For determination of sulfhydryl groups, washed platelet suspension was prepared according to the method of Ardlie et al (1970).…”

“…Washed platelets that had been preincubated with or without Mitomycin C were incubated at 37°C in isotonic saline with or without thrombin (1 U per ml) plus 2.5 mM CaCl 2 as described previously (Hashimoto et al 1977). Incubation was terminated by freezing on acetone-solid carbon dioxide.…”

“…Membrane solubilization and polyacrylamide gel electrophoresis with sodium lauryl sulfate were carried out as described previously using 80 ug protein from each membrane sample per 5 x 60 mm gel (Hashimoto et al 1977). The gels were stained with Coomassie Brilliant Blue and destained electrophoretically.…”

“…Phosphodiesterase activity in the soluble platelet fraction (100,000 G supernatant fraction of the dialyzed sonicates) or 50,000 G sediment was assayed at 37°C for 10 min in 4.6 mM MgClz 4 , 1 mM U_ 1 4C cyclic AMP (0.16-0.25 mCi per mmole) and 38.5 mM Tris-HCI, pH 8.0 (Hashimoto et al 1977).…”