IN VITRO CLOT LYSIS INDUCED BY SINGLE-CHAIN UROKINASE (scu-PA) AND ACTIVATION OF THE CONTACT PHASE SYSTEM

Scu-PA was purified from ccaiditioned medium of cell lines (HT 1080, MG 118) by immunoaffinity chrcxnatography on anti-u-PA Sepharose and passage through benzamidine-Sepharose to remove traces of two-chain u-PA (tcu-PA). The amidolytic activity determined with S-2444 was 20'000 u-PA IU/mg before and undetectable ( <700 IU/mg) after the benzamidine-Sepharose step. Pretreatment of scu-PA by plasmin yielded a specific activity of 100'000 IU/mg. We have studied, in vitro, the lysis of plasma clots containing ¹²⁵I-fibrin as tracer. To obtain 50% lysis in 90 min, 130 IU/ml of scu-PA had to be added to plasma before clotting; an equivalent rate of lysis was obtained with 45 IU/ml of tcu-PA. When, after the addition of scu-PA, the contact phase system of plasma was activated using ellagic acid and phospholipids, lysis was complete within minutes following clotting; 7 IU/ml of scu-PA induced 50% lysis in 90 min. An equivalent concentration of tcu-PA or plasmin-treated scu-PA did not bring about clot lysis after 7 h. Using plasmas deficient in prekalli-krein or in high molecular weight kininogen no lysis occured in the presence of 7 IU/ ml of scu-PA and contact phase activation; lysis reached 15% after 7 h with FXII deficient plasma and was normal with FXI deficient plasma. Clot digestion was totally abolished in the presence of a kallikrein inhibitor (SBTI 0.2 mg/ml) or antibodies directed against prekallikrein or u-PA. Hie addition of FXII inhibitor (LBTI 0.2 mg/ml) or of antibodies against FXII reduced the rate, but did not totally block the lysis. Contact dependent fibrinolysis in the presence of 7 IU/ml of scu-PA was furthermore modulated by plasminogen activator inhibitor 1 (PAI-1). A positive exponential correlation between the level of PAI-1 activity and 50% lysis time was observed (r²=0.83, n=12).

We have previously reported that kallikrein efficiently converts scu-PA into tcu-PA. The present study shows that, in normal human plasma, the fibrinolytic effect of scu-PA is twenty fold potentiated through contact activation and suggests an important physiological role for scu-PA counteracting excessive clot formation in situations associated with contact activation of the coagulation system.