SummaryHuman extrinsic (tissue-type) plasminogen activator (EPA) was highly purified from the culture fluid of a human melanoma cell line, both as a one-chain or as a two-chain molecule. Its specific fibrinolytic effect on human whole blood clots or plasma clots with different degrees of fibrin crosslinking was evaluated in an in vitro system, composed of a 125I-fibrin labeled clot, hanging in circulating human plasma. After infusion of EPA (30 IU per ml over 3 hrs), non-crosslinked clots lysed more extensively (75-100 percent in 5 hrs) than totally-crosslinked clots (50-65 percent), and no difference was found between one-chain or two-chain EPA. The extent of lysis of totally-crosslinked human or animal plasma clots hanging in autologous plasma induced by EPA varied markedly from one species to the other. When 90 IU of EPA were infused over 3 hrs, crosslinked human plasma clots dissolved for over 95 percent within 5 hrs. Under comparable conditions, the degree of lysis was 80 percent in primate plasma (cynomolgus fascicularis), 60 percent in cat and rabbit plasma, 30 percent in dog plasma and only 10 percent in rat plasma. Systemic activation of the fibrinolytic system in the circulating plasmas was minor and dose-dependent in all species, but complete fibrinogen breakdown was not observed in any species following infusion of up to 90 IU EPA per ml plasma.It is concluded that the human system is more susceptible to EPA induced fibrinolysis than the other animal systems which were investigated, and that even totally-crosslinked clots can be lysed after infusion of EPA.

SummaryThe turnover of highly purified human extrinsic plasminogen activator (EPA) (one- and two-chain form) was studied in rabbits. Following intravenous injection, EPA-activity declined rapidly. The disappearance rate of EPA from the plasma could adequately be described by a single exponential term with a t ½ of approximately 2 min for both the one-chain and two-chain forms of EPA.The clearance and organ distribution of EPA was studied by using 125I-labeled preparations. Following intravenous injection of 125I-1abeled EPA the radioactivity disappeared rapidly from the plasma also with a t ½ of approximately 2 min down to a level of 15 to 20 percent, followed by a small rise of blood radioactivity. Gel filtration of serial samples revealed that the secondary increase of the radioactivity was due to the reappearance of radioactive breakdown products in the blood. Measurement of the organ distribution of 125I at different time intervals revealed that EPA was rapidly accumulated in the liver, followed by a release of degradation products in the blood.Experimental hepatectomy markedly prolonged the half-life of EPA in the blood. Blocking the active site histidine of EPA had no effect on the half-life of EPA in blood nor on the gel filtration patterns of 125I in serial plasma samples.It is concluded that human EPA is rapidly removed from the blood of rabbits by clearance and degradation in the liver. Recognition by the liver does not require a functional active site in the enzyme. Neutralization in plasma by protease inhibitors does not represent a significant pathway of EPA inactivation in vivo.

Recent data published on the prevalence of inhibitors to factor VIII in hemophiliacs on treatment show great variations, with prevalence rates ranging from 3.6 to 14.2%. We have studied the cumulative risk of inhibitor development in a cohort of 62 patients with hemophilia A. All patients were born after 1960, were natives of the Vienna area, had a factor VIII activity of less than 5%, and were treated at least once. Using the method of Cutler and Ederer, the cumulative risk of inhibitor development was found to be 24% at the age of 25 years. Most inhibitors developed between the ages of 3 and 7 years. The current prevalence of F VIII inhibitors in the group of patients studied is 17.5%. It is concluded that prevalence data underestimate the true risk of inhibitor development.

SummaryThe incidence of arterial embolism (AE) and pulmonary embolism (PE) during treatment with oral anticoagulants (OA) or without OA therapy was studied in 38 patients with dilated cardiomyopathy (DCMP). AE/PE occurred in 17 patients (44.7%) before initiation of OA treatment. The severity of DCMP was a risk factor for AE/PE, but not the presence of atrial fibrillation or intracardial thrombi. No AE/PE episodes occurred during the period of OA therapy. No major bleeding complications were seen, probably due to the moderate intensity of OA therapy (therapeutic range 5-15% Thrombotest® [TT], 2.1-4.8 International Normalized Ratio [INR], median TT value 11%, median INR 2.6). Recurrence of AE was observed in 4 of 5 patients in whom treatment with OA had been discontinued.

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SummaryThe fibrinolytic capacity of 121 patients with a history of venous thrombosis and/or pulmonary embolism was studied by venous occlusion technique, at earliest 3 months after the last thromboembolic episode. After discontinuation of oral anticoagulation treatment the clinical course of the patients was followed and new thromboembolic episodes were noted. During the observation period of 56 ± 18.8 months 45 of 121 patients experienced recurrence of thrombosis. The recurrence-rate was significantly lower in patients with a post-occlusion ELT shorter than 60 min (4.8%/year) than in patients with an ELT longer than 60 min (10.3%/year). It is concluded that the fibrinolytic capacity is a useful parameter for determining the risk of recurrence in patients with venous thrombosis.

SummaryHuman extrinsic plasminogen activator (EPA), highly purified from a melanoma cell culture fluid is inactivated in human plasma with a half-life (t ½) of 90–105 min. Gel filtration on Ultrogel AcA 34 of mixtures of 125I-labeled EPA and human plasma, incubated at 37°C, revealed the progressive formation of two radioactive components, one with an apparent Mr of 150,000 and one eluting at the void volume. The component with an Mr of 150,000 was identified as consisting at least in part of EPA-α2-antiplasmin complex since: 1) it reacted with antibodies against α2-antiplasmin, but not with antibodies against the other known plasma protease inhibitors, and 2) formation of this component was strongly reduced in plasma specifically depleted in α2-antiplasmin or when the active site of EPA was blocked. The component eluting at the void volume was identified as consisting at least in part of EPA-α2-macroglobulin complex since: 1) it only reacted with antibodies against these two proteins and 2) was not formed in plasma depleted in α2-macroglobulin or when the active site of EPA was blocked.In purified systems α2-antiplasmin inhibited one-chain EPA with a rate constant of 60 M-1s-1 and two-chain EPA with a rate constant of 130 M-1s-1, which corresponds to a t ½ in plasma of 180 min or 90 min, respectively. α2-Macroglobulin inhibited one-chain EPA with a rate constant of 15 M-1s-1 and two-chain EPA with a rate constant of 30 M-1s-1, which corresponds to a t ½ plasma of 4 or 2 hrs.All these findings taken together indicate that EPA is slowly neutralized in human plasma primarily by α2-antiplasmin and to a lesser extent by α2-macroglobulin. There appears to be no specific inhibitor in human plasma, which would inactivate EPA either rapidly or to a significant extent.