The mechanism of plasminogen activation and fibrin dissolution by single chain urokinase-type plasminogen activator in a plasma milieu in vitro.

The relative contribution of several mechanisms to plasminogen activation and fibrin dissolution by urokinase-type plasminogen activator (u-PA) in vitro was quantitated. The activation of plasminogen by recombinant single chain u-PA (rscu-PA), by its two chain derivative (rtcu-PA) and by a plasmin-resistant mutant, rscu-PA-Glu158, obeys Michaelis-Menten kinetics with catalytic efficiencies of 0.00064, 0.046, and 0.00005 L/mumol.s for native plasminogen (Glu-plasminogen) and of 0.0061, 1.21, and 0.0004 L/mumol.s for partially degraded plasminogen (Lys-plasminogen). In a purified system consisting of a fibrin clot submerged in a plasminogen solution, the equi-effective doses (50% lysis in one hour) for rscu-PA, rtcu-PA, and rscu-PA-Glu158 were 16, 6.5, and 32,000 ng/mL for Glu-plasminogen and two- to fourfold lower for Lys-plasminogen. In a plasma milieu, 50% lysis in two hours was obtained for a plasma clot with 2.1 micrograms/mL rscu-PA, 0.5 micrograms/mL rtcu-PA, and greater than 200 micrograms/mL rscu-PA-Glu158 and for a purified fibrin clot with 1.3 micrograms/mL rscu-PA and 0.27 microgram/mL rtcu-PA. After predigestion of a purified fibrin clot with plasmin, the apparent potency of rscu-PA and rtcu-PA increased by 40% and 20%, respectively. In conclusion, rscu-PA has an intrinsic plasminogen activating potential that is only about 1% of that of rtcu-PA and that is 13 times higher than that of rscu-PA-Glu158. Conformational transition of Glu-plasminogen to Lys-plasminogen enhances its sensitivity to activation by all u-PA moieties ten- to 20-fold. Predigestion of fibrin clots with associated increased binding of plasminogen results in a minor apparent increase of the fibrinolytic potency of rscu-PA and rtcu-PA. The relative fibrinolytic potency of rtcu-PA is two to three orders of magnitude higher than that of rscu-PA-Glu158 but only two- to five-fold higher than that of rscu-PA, both in purified systems and in a plasma milieu. These results indicate that conversion of rscu-PA to rtcu-PA constitutes the primary mechanism of fibrin dissolution.

Plasmin activity and complement activation during storage of citrated platelet concentrates.

Platelet concentrates were studied for evidence of plasmin activity and complement activation during a 7-to-10-day storage period. When measured by an amidolytic activity assay, plasmin reached a level of 845 +/- 540 nkats/L on day 7 (n = 9). Fibrin(ogen) degradation product (FDP) levels became markedly elevated on the tenth day of storage, rising to 45 +/- 22 micrograms/ml (n = 5). Antiplasmin levels decreased in platelet concentrates by 18% +/- 6% (n = 5) over 7 days, but there was no significant decrease in stored platelet-poor plasma (-1.7%, n = 5, p = 0.5). The amount of plasminogen in platelet concentrate converted to plasmin was estimated to be less than 3% by assay of total plasminogen. Supernatant plasma from stored platelet concentrates was examined for the presence of the complement activation peptides C3a and C5a. From day 0 to day 10 of storage, mean C3a levels rose from 327 ng/ml to 6690 ng/ml. An equivalent increase in C3a levels, from 336 ng/ml at day 0 to 6866 ng/ml at day 10, was also observed in stored platelet-poor plasma. C5a was not detected (less than 10 ng/ml) at any point during the storage period; however, we noted a small decrease of borderline significance (p = 0.04) in total C5 from day 0 (117 micrograms/ml) to day 10 (108 micrograms/ml). Only trace amounts of C3 fragments were found on stored platelets, and there was no evidence of the membrane attack complex. These findings indicate the presence of plasmin activity and conversion of C3 during storage of platelet concentrates.

High-dose, brief-duration intravenous infusion of streptokinase in acute myocardial infarction: description of effects in the circulation.

The effects in the circulating blood of a 1-hour intravenous infusion of 1.5 X 10(6) units of streptokinase (SK) were measured during the subsequent 24-hour period in 7 patients with acute myocardial infarction. At the end of the infusion, the activator activity, expressed in SK units, averaged 65 U/ml, all of the plasminogen had disappeared, only a small amount of free plasmin was still present and functionally active alpha 2 antiplasmin had been reduced to 21% of the preinfusion level. All of the native fibrinogen had been degraded and the thrombin-coagulable protein was composed entirely of fragment X species, but the circulating plasma also contained significant amounts of the more extensively degraded fragments Y, D and E. The biologic half-life of the SK-induced activator activity was 23 minutes and that of the fibrinogen degradation products was 6.3 hours. The lytic effects persisted for 4 hours before any signs of recovery from the hemostatic defect were evident; considerable recovery was present at 25 hours.

Postoperative changes in the plasmatic levels of tissue-type plasminogen activator and its fast-acting inhibitor--relationship to deep vein thrombosis and influence of prophylaxis.

Fibrinogen, euglobulin lysis time (ELT), tissue-type plasminogen activator (t-PA) antigen, plasminogen activator inhibitor activity (PA-inhibitor) and alpha 2-antiplasmin (alpha 2-AP) were measured pre- and postoperatively in 60 patients undergoing total hip replacement. Reduced fibrinolytic activity as assessed by the prolongation of euglobulin lysis time, decrease of t-PA and increase of PA-inhibitor and alpha 2-AP could be demonstrated. These changes did not correlate with the postoperative deep vein thrombosis (DVT) diagnosed with the 125I-fibrinogen test. However, preoperative PA-inhibitor activity was significantly higher in patients with postoperative DVT (p less than 0.01). The prophylactic treatment with aspirin (20 patients) and with heparin plus dihydroergotamine (20 patients) induced significant changes in some of those parameters. This study shows that the decrease of t-PA and the increase of PA-inhibitor may contribute to the reduced postoperative fibrinolytic activity after total hip replacement. PA-inhibitor level might be a useful marker in evaluating the risk of developing DVT in patients undergoing total hip replacement.

Turnover of tissue plasminogen activator in normal and hepatectomized rabbits.

The distribution, and clearance of the tissue plasminogen activator (tPA) was studied in rabbits, rats and mice. Following an intravenous injection of I-labelled tPA a large portion of the radioactive dose was rapidly accumulated in the liver. After one hour the radioactivity in the liver was less than 5 per cent of the injected dose. The highest activity was then found in the intestine, stomach and in the blood. Gel filtration of plasma taken one hour after the injection revealed that the major part (60%) of the radioactivity was present as low molecular weight metabolites or free iodine. The remaining activity was present as high molecular weight inhibitor complexes (26%) or as free tPA (15%). In order to study in vivo reactions between tPA and plasma inhibitors without hepatic interference, plasma turnover was also studied in hepatectomized rabbits. High amounts of radioactivity remained in the plasma after one hour. Gel filtration of this plasma revealed that 54 per cent of the radioactivity was bound to inhibitors, 34 per cent circulated as free tPA while a minor portion (12%) was found as free iodine or metabolites. The half-life of fibrinolytically active tPA in hepatectomized rabbits was 40 minutes compared to 2 minutes in intact rabbits. The increase in the half-life of tPA in hepatectomized rabbits also resulted in an improved thrombolytic effect after treatment with 0.5 mg tPA.

Fibrinogenolysis and fibrinolysis in normal volunteers and patients with thrombosis after infusion of urokinase.

We have studied the effects of urokinase (UK) on concentration changes of alpha 2 antiplasmin (alpha 2 AP) and on fibrino(geno)lysis. Medium dose (480,000 u) or large dose (960,000 u) of UK was given to each of seven normal volunteers by intravenous drip infusion within six hours, and then blood and urine analyses were carried out. Total alpha 2 AP, which includes free alpha 2 AP and alpha 2 AP-plasmin complex, decreased to about 50% of the original value with large dose of UK. alpha 2 AP-plasmin complex appeared in the plasma one hr after UK infusion and increased up to 50% of total alpha 2 AP at the end of UK infusion. B beta peptides, which are liberated from fibrin(ogen) at the very early stage of fibrino(geno)lysis, increased significantly with UK infusion, and was 65 times as much as the normal range at the end of UK infusion. Urinary B beta peptides increased as well as plasma B beta peptides. On the other hand, fibrin(ogen) degradation products (FDP) measured with enzyme immunoassay (EIA) increased only slightly, and moreover, urinary FDP was not detectable at any time. Plasma fibrinogen levels did not decrease and changed within the normal range in both groups. We then gave 960,000 u of UK to four patients with deep vein thrombosis and blood analyses were carried out as with normal volunteers. The most significant observation different from that of normal volunteers was shown in FDP levels. Serum FDP levels of four patients increased significantly in comparison with normal volunteers. Urinary FDP increased as significantly as plasma FDP. In conclusion, the infusion of 960,000 u of UK caused only very early stage of fibrinogenolysis without advanced fibrinogenolysis in normal volunteers, but in thrombotic patients, advanced fibrinolysis was observed.

A comparison of the thrombolytic and hemorrhagic effects of tissue-type plasminogen activator and streptokinase in rabbits.

Tissue-type plasminogen activator (t-PA) is a promising thrombolytic agent because it can produce thrombolysis without inducing a plasma proteolytic state. It is uncertain if this potentially important feature renders t-PA less hemorrhagic than other plasminogen activators. We have compared the hemorrhagic and thrombolytic effects of t-PA and streptokinase in rabbits. Streptokinase, 4000 U/kg/hr over 4 hr, failed to produce significant thrombolysis and 8000 U/kg/hr streptokinase over 4 hr produced only 28 +/- 6% thrombolysis. Both streptokinase regimens were associated with a plasmin-mediated plasma proteolytic state and both streptokinase regimens produced a significant increase in hemorrhage that was evident within 15 min of beginning the infusion and was progressive over the 4 hr of drug administration. In contrast, t-PA in a dose of 7500 U/kg/hr produced 35 +/- 6% thrombolysis, but it did not produce a plasmin-mediated plasma proteolytic state or a significant increase in hemorrhage over the 4 hr of infusion. t-PA in a dose of 15,000 U/kg/hr produced 85 +/- 4% thrombolysis but was associated with a plasmin-mediated proteolytic state and produced significant bleeding which, in contrast to streptokinase-induced bleeding, was delayed in onset. Therefore, t-PA induced less hemorrhage than streptokinase at doses that produced more effective thrombolysis. Bleeding with both thrombolytic agents was associated with a plasmin-mediated proteolytic state.

Alpha 2-plasmin inhibitor inactivation by human granulocyte elastase.

Plasminogen-binding human alpha 2-plasmin inhibitor is converted by human granulocyte elastase into its non-plasminogen-binding and finally into the inactive form of the inhibitor. This degradation of the plasmin inhibitor, described earlier as "spontaneously" occurring conversion, is shown in dodecyl sulfate polyacrylamide gel electrophoresis, in two-dimensional immunoelectrophoresis and by measuring the kinetics of plasmin inhibition. Experiments in the presence of normal human plasma required unphysiologically high concentrations of elastase to inactivate alpha 2-plasmin inhibitor, suggesting a role of elastase in this type of indirect fibrinolysis in a microenvironment only and not in systemic events.

alpha 2-Antiplasmin and alpha 2-macroglobulin--the main inhibitors of fibrinolysis--during the menstrual cycle, pregnancy, delivery, and treatment with oral contraceptives.

alpha 2-Antiplasmin and alpha 2-macroglobulin have been studied during the menstrual cycle, pregnancy and parturition in healthy women, and during use of various types of contraception in both healthy and diabetic women, and compared with a reference group of healthy men and women. alpha 2-Antiplasmin showed a slight sex difference, with higher values in women. The luteal phase of the menstrual cycle showed slightly higher values than the other phases. alpha 2-Antiplasmin increased during pregnancy, decreased (probably due to consumption) during labor and increased again in the puerperium. Treatment with neither combined contraceptive pills nor low dose progestogen pills gave any changes in alpha 2-antiplasmin. alpha 2-Macroglobulin showed low values during menstruation. The increase during pregnancy and treatment with combined contraceptive pills is in accordance with earlier findings. It is concluded that synthesis and metabolism of alpha 2-antiplasmin are under hormonal influence. The role of alpha 2-antiplasmin in the decreased fibrinolysis in pregnancy is discussed.