SummaryA group of patients with clinical hemorrhagic disease who exhibited a prolonged bleeding time and a short prothrombin consumption time classified as Thrombocytopathy A were studied utilizing procedures involving isolated platelets and purified prothrombin. These frozen and thawed platelet extracts had poor platelet factor 3 activity which was normal after the extracts had been treated with ultrasonic oscillations. The electron microscope studies of the morphology of these platelets were abnormal. It is concluded that these platelets contain adequate amounts of platelet factor 3 but are resistant to disintegration and the activity is only liberated with difficulty.The authors wish to thank Miss Jeanne M. Riddle, M.S. medical technologist in Hematology at Henry Ford Hospital for technical assistance in providing the platelet counts for this investigation.

SummaryThe procoagulant or P fraction obtained from human urine was analyzed to determine how it functions in the activation of purified prothrombin. Prothrombin converts to thrombin in the presence of P fraction, Ac-globulin, calcium chloride and either whole platelets or platelet factor 3. The yield of thrombin can be made to depend upon the concentration of P fraction. This conversion of prothrombin to thrombin can be partially or completely inhibited by adding various concentrations of soybean trypsin inhibitor introduced into the activation mixture.When purified prothrombin was activated with platelet factor 3 and calcium chloride, the prothrombin activity, as measured by the two-stage assay, decreased. When it was at the lowest possible level, P fraction was added. Quite quickly prothrombin activity began to reappear, and soon 100% of the original prothrombin activity was accounted for as either prothrombin-R or thrombin.

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SummaryNumerous chemical enzyme inhibitors were employed to determine the active polar groups in the prothrombin, thrombin and autoprothrombin C molecules. Prothrombin and autoprothrombin C activities were decreased by the reduction of disulfide bonds, but thrombin activity was affected only at higher concentrations of reducing agent. Amino groups were not essential for the activity of autoprothrombin C; however, the blocking of such groups decreased the activities of prothrombin and thrombin. The blocking of carbonyl groups decreased the activity of both prothrombin and autoprothrombin C, but thrombin was not inactivated by hydroxylamine. The activities of none of the three were decreased by sulfhydry] blocking agents. Nonspecific oxidizing agents reduced the activities of all three molecules ; however, oxidizing agents which specifically oxidize sulfhydryl groups, were incapable of decreasing the activities of prothrombin, thrombin or autoprothrombin C. The sulfhydryl groups are not essential for the activities of prothrombin, thrombin or autoprothrombin C.

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SummaryThrombocytopenic serum supports a short prothrombin consumption time, however, the same serum contains as little prothrombin as normal serum. The prothrombin consumption test of thrombocytopenic serum can be prolonged by adsorption on BaCO3 or reduced again by the addition of the sodium citrate eluate. The antihemophilic activity of thrombocytopenic blood disappears when the blood clots as in normal blood and autoprothrombin I is present in much smaller amounts than in normal serum. The authors suggest that a new factor, a possible derivative of prothrombin, is responsible for the short prothrombin consumption value in thrombocytopenic blood.

SummaryThe presence of hirudin at the time adsorbed plasma and serum were combined in TGT inhibited the formation of the prothrombin converting activity. Hirudin, a potent antithrombin, probably exerted its inhibitory effect by neutralizing any thrombin which might form. If adsorbed plasma and serum were incubated together for a period as short as 1 to 3 min before the addition of hirudin, calcium and lipid, hirudin no longer exerted the same inhibitory effect, but, in order to exert the same inhibitory effect, had to be present in higher concentration. Preincubation of factor VIII deficient plasma with serum and calcium was not effective in overcoming hirudin inhibition, but preincubation with factor IX deficient serum with adsorbed plasma and calcium was able to overcome inhibition due to hirudin equally as well as normal serum.Once again the importance of traces of thrombin in the formation of activity capable of rapidly clotting substrate plasma in TGT has been shown by using a specific thrombin inhibitor. A thrombin dependent interaction appears to take place between adsorbed plasma and serum and also between product I and phospholipid.