Thrombin-Induced Responses In Human Platelets Differ In Their Requirement For Receptor Occupancy; Evidence For Tight Coupling Between Occupancy And Formation Of Phos- Phatidic Acid

Abstract

Thrombin causes secretion of dense granule constituents from platelets. The time course and extent of this response is unaltered if thrombin is rapidly neutralized after secretion has started, indicating that a sustained occupancy of the thrombin receptors is not required.Using a 50-fold excess of hirudin to neutralize thrombin, we show here that the shape change and aggregation responses also are independent on sustained receptor occupancy. In contrast, the secretion of acid hydrolases, liberation of [3H-]arachidonate (from phospholipids in platelets prelabeled with [3H]arachidonate) and formation of [32P]phosphatidic acid (in platelets prelabeled with 32P-orthophosphate) induced by thrombin were immedately stopped by hirudin. However, the incorporation of 32P into phosphatidylinositol and breakdown of [3H]phospha- tidyl inositol continued unaffected after thrombin removal. Thrombin-induced platelet responses can thus be subdivided into two classes according to their requirement for receptor occupancy: The first class (shape change, aggregation, dense granule secretion phosphatidylinositol breakdown and phosphatidylinositol synthesis) requires a short, initial occupancy, while the second class (acid hydrolase secretion, arachidonate liberation and phospha- tidic acid synthesis) requires an occupancy that is sustained as long as the response is executed. The increased turnover of phosphoinositides is a cyclic process, and our results show that only one step - the formation of phosphatidic acid from diacylglyceride and ATP - is tightly coupled to receptor occupancy by the agonist.

Inhibition of the Thrombin-Platelet Reaction by Hirudin

“…Pos sibly, they have not taken into account the special conditions mentioned. Holmsen et al [7] reported that, in contrast to the thrombininduced depletion of dense granules, the re lease of a-granule constituents could be im mediately stopped by hirudin. These differ ences may be due to the different velocities of the two processes with the consequence that the velocity of thrombin blockade was just sufficient for interruption of the compara tively slow a-granule secretion process [1,6], Our results are in contradiction to the concept of Martin et al [14] that the dense granule secretion is a thrombin-independent step beginning after the termination of the catalytic step.…”

“…Although plate lets were prevented from being activated [11], dense granule secretion was little or not affected by addition of hirudin after secretion was on its way [3,7,15], In this context, it has to be taken into consideration that the kinetics of thrombin-platelet reaction and its inhibition deviate in several aspects from the Michaelis-Menten kinetics valid for common enzymatic reactions. The soluble enzyme reacts with the substrate fixed on the platelet surface.…”

Phosphoinositide metabolism in resting and thrombin‐stimulated human platelets Evidence against metabolic heterogeneity

“…In resting cells [2] as well as in stimulated cells [3][4][5], evidence has been presented for multiple metabolic pools of phosphoinositides. In platelets the existence of distinct metabolic Abbreviations: PI, phosphatidylinositol; PIP, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PA, phosphatidic acid; GroPI, diacylated phosphatidylinositol pools of PA, PI and PIP2 has been suggested [6][7][8]. Recently, Vickers and Mustard studied the specific [32p]Pi and [3Hlglycerol labelling of the phosphoinositides in both resting and stimulated platelets and proposed separate pools of polyphosphoinositides [9,10].…”

Effects of DK-002, a synthesized (6aS,cis)-9,10-Dimethoxy-7,11b-dihydro-indeno[2,1-c]chromene-3,6a-diol, on platelet activity

“…The secretion of [ 14 C]serotonin from platelets was determined by centrifuging the samples at 12,000 g for 2 min and by assaying a 100 Al aliquot of the supernatant by liquid-scintillation counting. Percent [ 14 C] serotonin secretion was determined as described by Holmsen et al (Holmsen et al, 1981).…”