Dissociation of aggregated platelet by small molecular substance obtained from blood

Abstract

Heat stable small molecular weight substance having platelet aggregation inhibiting activity was obtained from blood by Funahara & Miki (Blood & Vessel 10; 431-435, 1979). The following studies were made to learn a feature of platelet aggregation inhibiting activity of the inhibitor. The inhibitor was isolated from serum using Sephadex G25 column (Fig. 1). Six milligram of the inhibitor was obtained from 1ml of serum. The effects of 12mg/ml (final concentration) of the inhibitor on platelet aggregation induced by 0.5μM, 5μM and 50μM (final concentration) of ADP were 68, 33 and 22%, respectively. Platelet aggregation induced by 0.5μM of ADP was completely inhibited by 15.5mg/ml of the inhibitor. The effect of the inhibitor on thrombin—or epinephrine—induced aggregation was about 30% stronger than that on ADP-induced aggregation.Addition of 100μl of ADP (final concentration 0.5μM) to 900μl of platelet suspending medium (700μl of Ca++-free Tyrode solution plus 200μl of fresh human PRP) induced decrease of free platelet number (Fig. 2-a —*—), but addition of 16mg/ml of the inhibitor at 3min after ADP addition induced increase of free platelet number (Fig. 2-a —_??_—). Preincubation of the inhibitor with 20mM CaCl2 did not inhibit the activity to increase free platelet number (Fig. 2-a —_??_—). The activity was also observed when thrombin or epinephrine was used as inducer of platelet aggregation (Fig. 2-b, c). Incubation of the inhibitor in 1N HCl or NaOH solution at 100°C for 30min did not inhibit the activity. The inhibitor did not have ADP-removing activity.From the results obtained, it was concluded that blood has small molecular weight stable substance having the activity to dissociate aggregated platelet.