The stimulation by thrombin of glucose oxidation in human platelets.

Embden-Meyerhof pathway.The biochemical mechanism of the stimulation was investigated. MethodsMaterials.Purified bovine thrombin,' the activity of which was assayed by determining its ability to convert fibrinogen to fibrin (18), was stored in lyophilized form and used within 1 week of rehydration.Substrates 2 labeled with 14C were tested for radiochemical purity by paper chromatography: at least 98% of the radioactivity traveled with the authentic compound, and no contaminants were found.Solutions of puromycin dihydrochloride,3 adenosine diphosphate (ADP),4 and ethylenediaminetetra- acetic acid (EDTA) 5 were prepared fresh for each experiment.Human platelets were isolated daily from 500 ml of blood drawn from fasting donors and mixed with 50 ml of 1.5% EDTA.All preparatory steps were carried out with siliconized pipettes at 5° C in cellulose nitrate vessels.Erythrocytes were removed by centrifugation at 1,400 X g for 4 minutes.The supernatant plasma was centrifuged at 120 X g for 10 minutes to remove leukocytes.The platelets were harvested from the plasma by centrifugation at 1,400 X g for 15 minutes, washed once in 0.85% saline, and suspended in 0.85% saline at a final concentra- tion of 1 to 2 X 10' platelets per ml.The final platelet suspension contained no aggregates.Platelet counts were performed by phase microscopy.Average erythrocyte and leukocyte contamination was less than 1 cell per 3,000 platelets and was found to contribute no more than 0.1% of the observed radiochemical yield in the experiments to be described.Experiments were begun within 3 hours after the blood had been obtained.Experimental procedure.Incubations for determining substrate oxidation were performed in 10-ml Erlenmeyer flasks containing the appropriate labeled substrate; 1.5 ml of fresh Krebs-Ringer bicarbonate buffer (19) (modified to contain one-half of the recommended calcium concen- tration) containing glucose or other unlabeled substrates; and 0.5 ml of 0.85% saline in which were dissolved throm- 1 Generously provided by Dr.