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.

A new method has been developed for the quantitative assessment of energy consuming processes in platelets. Under carefully controlled metabolic conditions ATP resynthesis is abruptly blocked by a cocktail of metabolic inhibitors. This leads to a fall in metabolic ATP, which is linear with time between 0 and 30 sec after addition of the inhibitors. Evidence is presented that this fall reflects the velocity by which the platelets consume metabolic energy prior to addition of the inhibitors. Resting platelets consume 4 μmol ATP equivalents/min/1011 cells at 37° and 0.5 μmol (same units) at 15°C. When thrombin (5 U/ml) is included in the inhibitor-mixture, aggregation and secretion of dense granules (3H-serotonin), α-granules (β-thromboglobulin) and lysosomal granules (N acetyl β glucosaminidase) follow despite the arrest in ATP resynthesis. The fall in metabolic ATP is now much steeper, reflecting an increase in energy consumption during these functions. Using changes in temperature as a means to affect secretion and energy metabolism, secretion velocity (measured between 0 and 10 sec after thrombin addition) can be compared with simultaneous energy consumption (measured between 0 and 30 sec after thrombin addition). At a consumption of 12 ymol ATP/min/1011 cells secretion velocity of dense-, α- and lysosomal granules is 100, 95 and 50% of uninhibited suspensions, respectively. At 6 μmol (same units) these percentages are 70, 35 and 25%.If thrombin is added after addition of the inhibitors thereby initiating secretion at lowered metabolic ATP levels, secretion is slower as metabolic ATP is lower. Again lysosomal granule secretion is more inhibited than α-granule secretioiy. which is slower than dense granule secretion. These data reflect an increasing need for metabolic energy in the order: dense-, α- and lysosomal granule secretion.

ADP-induced aggregation was determined at various times of pre-incubation with ADP in unstirred platelet rich plasma to which adenosine deaminase was added. In the early stages of incubation the shape change response was absent, the aggregation response was poor and not reversible. As incubation proceeded, these three parameters returned towards normal while there was still ADP in the system. Log dose response (rate of aggregation) curves for the platelets incubated with 1 μM of ADP had shifted to higher concentrations of ADP and there was a small decrease in the maximal height of the curves. Gelfiltered platelets in calcium-free Tyrode solution were incubated with 1 μM of ADP at 37° C. Log dose response curves obtained at different times of incubation showed a greater shift to higher ADP concentrations than those obtained with platelets in plasma. There was also a pronounced decrease in the maximal height of the curves. These two observations became more apparent as incubation proceeded. Addition of apyrase (0.1 mg/ml) at various times of incubation prevented the progessive impairment of the aggregation response and this even slowly increased towards that of the control. The time of incubation with ADP before addition of apyrase did not have any influence on the ability of the platelets to recover their aggregability towards ADP.

Reduction of [ATP] and accumulation of hypoxanthine occurs in platelets·when consumption exceeds resynthesis of ATP. The intermediate IMP accumulates transiently ([IMP]↑) to a very varying degree. Several findings indicate that [IMP] is controlled by cytoplasmic [Ca++]: (1) The transient nature of [IMP]↑ would follow the release and subsequent uptake of cytoplasmic Ca++, the latter resulting in a return to resting state. (2) Under certain conditions antimycin does not cause [IMP]↑, but in combination with fluoride, which itself may cause release of intracellular Ca++, [IMP]↑ is prolonged, suggesting that Ca++ uptake is energy-requiring. (3) Concomitant addition of antimycin, deoxyglucose and gluconolactone causes irreversible [IMP]↑, suggesting that a rapid fall in [ATP] causes leakage of Ca++-from intracellular sites. Irreversible (IMP]↑ is also seen with high [A23l87] when cytoplasmic [Ca++] is irreversibly enhanced. (4) Secretion induced by 2-propyl-3-dimethylamino-5,6-methylenedioxyindene is accompanied by [IMP]↑. Chlortetracycline inhibits secretion and abolishes [IMP]↑. (5) The conversion of IMP to inosine + hypoxanthine in platelet lysates is enhanced by EGTA + Mg++ and inhibited by Ca++. This indicates that Ca++ inhibits Mg++-dependents 5′-nucleotidase responsible for IMP↑ inosine, which may explain the proposed regulatory effect of cytoplasmic Ca++ on [IMP]↑ in platelets. Since no reliable direct method is known for monitoring Ca++-movements in platelets, [IMP]↑ may be a useful indicator for [Ca++] in platelet cytoplasm.

The effect of H2O2 on platelet metabolism, aggregation and release was studied by the in vitro exposure of 3H-adenine labelled platelet rich plasma (PRP) to H2O2. On incubation of PRP with H2O2 (100 to 500 μM final cone.) over a 30 minute period, there was a mean drop of 38% in the baseline steady state levels of radioactive metabolic ATP, the fall occurring in the first 3 minutes of incubation, with a corresponding increase in the levels of radioactive inosinemonophosphate and hypoxanthine. This was not a nonspecific lytic effect on the platelet since no extracellular leakage of platelet nucleotides occurred during the incubation. Further, mean decreases of 8 to 38% in steady state levels of platelet metabolic ATP were observed during incubations of PRP with 5 to 500 μM H2O2 respectively. Finally, the action of H2O2 on adenosine diphosphate (ADP) induced biphasic aggregation and release was studied in PRP preincubated for 3 minutes with H2O2 (100 to 500 μM). Partial inhibition of the primary wave, and complete inhibition of the second wave of ADP induced aggregation was observed in the H2O2 pretreated platelets, con-commitant with inhibition of release of platelet non-metabolic ATP and ADP, when compared to the control saline preincubated platelets. Since H2O2 is generated in vivo by bacteria and leucocytes during phagocytosis, the in vivo effects of the described inhibition of H2O2 and its possible role in platelet-leucocyte-bacterial interaction requires further elucidation.