The activation of human plasminogen (P) by two-chain tissue plasminogen activator (A) was studied in the presence of fibrin films (F) of increasing size and surface density. Initial rates of plasminogen activation (v) were determined as a function both of the plasminogen and fibrin concentration. The activation rate was strongly dependent on the presence of fibrin and plots of 1/v versus 1/ [p] or 1 /[F] yielded straight lines. The kinetic data were in agreement with the following reaction scheme.According to this model tissue plasminogen activator would bind to fibrin with a dissociation constant (KF of 0.2 µM and this complex fixes plasminogen with a Michaelis constant (Kp’) of 0.15 µM (Glu-plasminogen) or 0.02 µM (Lys-plasminogen) to form a ternary complex, converted to plasmin with a catalytic rate constant kcat = 0.05 s-1. This mechanism implies that both plasminogen and tissue plasminogen activator are concentrated on the fibrin surface through formation of a fibrin bridge. Activation of plasminogen in the absence of fibrin occurs with Km = 65 µM (Glu-plasminogen) or Km= 19 µM (Lys-plasminogen) and kcat = 0.05 s-1. Our data suggest that fibrin enhances the activation rate of plasminogen by tissue plasminogen activator by increasing the affinity of plasminogen for fibrin-bound tissue plasminogen activator and not by influencing the catalytic efficiency of the enzµMe. These data also support the hypothesis that fibrinolysis is both triggered by and directed towards fibrin.Generated plasmin was quantitated by measuring the rate of solubilization of 125I-labeled fibrin.

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During thrombolytic therapy of myocardial infarction (MI) with urokinase or streptokinase (SK), levels of fibrin(ogen) degradation products in serum are often dramatically elevated as a result of a combination of systemic fibrinogenolysis and local thrombolysis. Others have measured increased levels of D-dimer in serum of MI patients after SK therapy and postulated that thrombolysis could be monitored during SK therapy by measuring D-dimer levels. In the present study rt-PA was infused into healthy volunteers to analyse if elevated FbDP levels in MI patients really reflect coronary thrombolysis or could be due to a systemic effect. Over a period of 60 min., three groups (n = 6 each) were given i.v. 0.23 mg rt-PA/'kg (group I), 0.50 mg rt-PA/kg (group II) and a placebo infusion (group III), respectively. Two blood samples were taken from an anticubital vein in the arm contralateral to the site of infusion (one on citrate/ aprotinin, the other on citrate alone) at different time points. Using a new enzyme immunoassay (EIA), based on monoclonals and development by us, we measured FbDP in plasma (not serum). Before infusion all volunteers had FbDP levels ≪ 0.5µg/ml. Upon infusion FbDP levels in groups I and II increased to average values of 1.0 ± 0.4/µg/ml and 0.8 ± 0.2/µg/ml, respectively, for the samples taken in citrate/aprotinin. The values in citrate alone did not differ significantly, and were 1.1 ± 0.5/µg/ml and 0.8 ± 0.3/µg/ml for groups I and II, respectively. FbDP levels in group III remained ≪ 0.5/µg/ml. The results show that FbDP levels increase upon rt-PA infusion, even in healthy volunteers. This suggests lysis of systemic fibrin. We conclude that lysis of systemic fibrin limits the value of FbDP levels as a measure for coronary thrombolysis.

It is known that plasminogen activation in blood samples taken during thrombolytic therapy with tissue-type plasminogen activator (t-PA) may continue during plasma handling, leading to artificially low fibrinogen (Fbg) and α2-antiplasmin (AP) values. Addition of D-Phe-Pro-Arg-CH2Cl or quenching antibodies against t-PA prevents this phenomenon, but these additions do not allow measurement of t-PA activity. The question of this study is, why the in vitro effects occur, even during freezing of the samples. Normal plasma was supplemented with various amounts of two-chain melanoma or recombinant t-PA and stored at -20°C, with and without a prior snap-freeze procedure. AP consumption (chromo-genic substrate assay) and Fbg degradation (Clauss method), measured in thawed samples, were most pronounced in the non snap-frozen samples. As it took a relatively long time before these samples were really frozen, the time course of the effects was studied at different temperatures. Plasma samples containing 1000 IU t-PA per ml were incubated at 37, 25,10, 0 and -8°C between 0 and 120 min. AP reduction was most rapid at 37°C (50% after 13 min), was less at 25°C (50% after 30 min), but did not further decrease at lower temperatures. The AP reduction at temperatures between 25 and -8°C corresponded to the effect of 40% t-PA activity at 37°C. The Fbg values gave a similar picture: the most rapid reduction occurred at 37°C, a slower reduction at 25°C, but no further reduction (even a small increase) was found from 25 to -8°C. The experiments were repeated in a purified system, consisting of t-PA, plasminogen, Fbg and AP. In contrast to the plasma system, AP reduction gradually decreased from 37 to 0°C. The apparent t-PA activity at 0°C was 6% of the activity at 37 °C.It is concluded that the in vitro effects in plasma samples containing t-PA can be, at least partially, explained by an abnormally strong plasminogen activation around 0°C. A normal temperature dependency in the purified system strongly suggests that unknown plasma factors enhance plasminogen activation at low temperatures.

In order to localize the binding site(s) for tissue-type plasminogen activator (t-PA) in the fibrin(ogen) molecule, two-chain t-PA was immobilized onto microtitration plates and incubated with fibrinogen and various fibrinogen fragments. The extent of binding was quantified with an enzyme immunoassay. Hardly any binding to t-PA was observed with fibrinogen, fragments X, Y and E. A moderate binding was observed with fragments D(cate) and D(EGTA) and a strong binding with the cyanogen bromide fragment FCB-2 (Kd apparent = 65 nM). Results of control experiments, in which the binding of fibrinogen and its fragments to immobilized Lys-plasminogen was measured, using the same assay, were in line with literature data: hardly any binding was found with fibrinogen, fragments X and Y. A moderate binding was observed with fragments D and E and a strong binding with FCB-2 (Kd apparent = 100 nM). The stimulatory capacity of the various fragments on the Lys-plasminogen activation by t-PA, as studied in a spectrophotometric assay, was found to be absent for fragment E, low for fibrinogen, fragments X, Y and D, and high for FCB-2. It is concluded that the t-PA binding site in the fibrin (ogen) molecule resides in the distal domains from which fragments D and FCB-2 are derived. The site is apparently hidden in fibrinogen and early fibrinogen degradation products. Binding of both plasminogen and t-PA is required for stimulation of the plasminogen activation, as illustrated by fragment E which binds plasminogen and no t-PA, and has no stimulatory capacity.