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.

Severe thrombotic tendency associated with a type I plasminogen deficiency.

We report a 45-year-old female patient with recurrent spontaneous deep vein thrombosis associated with an isolated hypoplasminogenemia (plasminogen activity and antigen level of 42% and 37%, respectively). The plasminogen molecule was normal as demonstrated by a normal activation by tissue plasminogen activator, electrophoretic mobility on crossed immunoelectrophoresis, molecular weight, and binding to lysine sepharose. All other hemostatic parameters predisposing to recurrent thrombosis were normal. A stimulation test with desmopressin acetate (DDAVP) showed a normal plasma rise of both tissue plasminogen activator and factor VIIIR:WF. This isolated plasminogen deficiency apparently is due to a decreased synthesis of a normal plasminogen molecule and is associated with a severe thrombotic tendency.

The German multicenter trial of anisoylated plasminogen streptokinase activator complex versus heparin for acute myocardial infarction.

A multicenter randomized trial of anisoylated plasminogen streptokinase activator complex (APSAC) versus heparin in patients with acute myocardial infarction of less than 4 hours' duration was undertaken in 19 hospitals. Of the 313 patients, 151 received heparin and 162 APSAC (30 U as intravenous injection). Within 28 days of hospital stay, 19 deaths (12.6%) occurred in the heparin group and 9 deaths (5.6%) in the APSAC group (p = 0.032). After 24 hours, patients in the APSAC group had a significantly lower incidence of cardiogenic shock (3.2 vs 9.5%, p = 0.031), asystole (3.8 vs 10.8%, p = 0.015) and need for resuscitation (5.1 vs 11.5%, p = 0.039). There was no difference in global and infarct-related ejection fraction between the 2 groups. Thus, APSAC favorably influences prognosis and clinical course in hospital.

Impaired fibrinolysis in coronary artery disease.

We assessed endogenous endothelial-dependent fibrinolysis in 99 subjects with coronary artery disease (CAD) documented by angiography and in 28 control subjects with normal coronary arteries on angiography. We used specific, sensitive assays for plasma tissue-type plasminogen activator (t-PA) antigen, t-PA activity, plasminogen activator inhibitor (PAI) activity, plasminogen, and alpha-2 plasmin inhibitor (alpha-2 PI). Mean PAI activity was significantly higher, and mean t-PA activity after venous occlusion of the upper arm (a standard test of the capacity of vascular endothelium to release t-PA) was significantly lower in subjects with CAD than in subjects with normal coronary arteries. The mean increment in t-PA antigen after venous occlusion was significantly lower than normal in subjects with CAD with onset of symptoms before age 45 years. Subjects with CAD had a significantly increased mean plasma fibrinogen level compared with control subjects, and a significant positive correlation was observed between PAI activity and plasma fibrinogen in subjects with CAD. No significant abnormalities of plasminogen or alpha-2 PI were observed in any subset of subjects with CAD. These data support an association between impaired fibrinolysis and CAD, with contributions from both increased PAI activity and in younger subjects from reduced endothelial t-PA release.

Relationship between postsurgical fibrinolytic parameters and deep vein thrombosis in surgical patients treated with compression devices.

This study consisted of 52 patients admitted for orthopedic surgery and 28 patients admitted for general surgery, who were treated with Sequential Compression Devices (SCD) and Thromboembolic Deterrent Stockings (TEDS) and monitored for the development of deep vein thrombosis (DVT). Coagulation and fibrinolytic profiles were carried out on these patients preoperatively, and on days one, three, and six postoperatively. All patients were followed by I-125-Fibrinogen scanning, Venous Doppler, and Impedance Plethysmography studies for clot detection. In the orthopedic surgery group, six (11.5%) developed DVT, and in the general surgery group, one (3.6%) developed DVT. No patients developed pulmonary embolism. The combined incidence of DVT was 8.8 per cent. A variety of parameters was measured in order to determine whether compression devices prevent a fibrinolytic shut-down commonly seen in the postsurgical patient. A combination of three assays was found to be significant in demonstrating a fibrinolytic response. These parameters were a post-surgical decrease in the plasminogen level, an increase in the level of free protease activity postoperatively, and an increase in the level of tissue plasminogen activator after surgery. 56.3 per cent of all patients treated with SCD and TEDS showed a fibrinolytic response on postoperative day one by a combination of all three of these parameters. In the group of patients that developed DVT none showed an increase in free protease activity, and five of seven showed no significant decrease in plasminogen and no increase in tissue plasminogen activator. Patients who developed thrombosis had measurable differences in their fibrinolytic system compared to those without postoperative thrombosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Urokinase-like plasminogen activator increased in plasma after alcohol drinking.

After taking the local Japanese spirit 'Shochu', 'Sake' and beer, increased plasma fibrinolysis was confirmed in normal persons at about 1 hr after for both the pyro-Glu-Gly-Arg-pNA amidolytic and Glu-plasminogen activating activities in eluates from [N alpha-(epsilon-aminocaproyl)-DL-homoarginine hexylester;)]-Sepharose affinity column. The activity was highest in the Shochu group, followed by the Sake and beer groups with an approximately 2.1-, 1.7- and 1.5-fold increase in fibrinolytic activity, respectively, compared to the control. The enzyme could be further purified using urokinase-IgG-Sepharose immunoadsorbent column from the Shochu group and it reacted with and was inhibited by urokinase specific antibody. The main molecular weight of the active enzyme was about 30,000, and those of minor components were about 50,000 and 100,000, as determined by zymography. These findings suggest that some of the enzymes appearing in the plasma after drinking are related to 'urokinase-like plasminogen activator', being endogenously produced.

[Fibrinolytic activity of rat blood during transient coronary insufficiency]. / Fibrinoliticheskaia aktivnost' krovi krys pri tranzitornoi koronarnoi nedostatochnosti.

Local 10-min and 40-min myocardial ischemia was accompanied by a significant depression in the rat blood fibrinolytic activity. During 40-min postischemic myocardial reperfusion after short-term (10-min) myocardial ischemia fibrinolytic reactions were activated and after prolonged (40-min) myocardial ischemia they were suppressed.

Modulatory effect of the gastrointestinal tract (gut) on fibrinolysis and fibrinogen derivatives.

We have investigated some components of the fibrinolytic system and fibrinogen derivatives in blood samples taken simultaneously from the human portal and cubital veins. In the first series the blood was drawn during laparotomy from 12 cholecystectomized, otherwise healthy patients. The mean value of euglobulin lysis time was significantly lower in the portal than in the cubital vein. The values of the fibrinogen, plasminogen, serial dilution protamine sulfate, and ethanol gelation tests in both samples were quite similar. In the second series blood was taken from eight patients with cancer of the gut and from six cholecystectomized patients. The staphylococcal clumping test (SCT) in serum was performed for fibrin degradation products (FDP) in samples from both veins. In patients with cancer the mean FDP level was significantly higher in the portal than in the cubital vein. In cholecystectomized patients the SCT was negative in samples from both veins. We conclude that fibrinogen pathway metabolism does not differ in the portal and cubital veins under normal conditions, whereas in neoplastic disease fibrinogen degradation is greater in the portal vein. We also suggest that the gut probably modulates fibrinolytic activity in normal conditions.

Effects of moderate alcohol consumption on platelet aggregation, fibrinolysis, and blood lipids.

To investigate the effect of moderate alcohol consumption on blood constituents related to cardiovascular disease, 12 male volunteers consumed (instead of their usual alcoholic drinks) four different standardized amounts of red wine in addition to their habitual diet. Each dose was given to the subjects during a period of 5 weeks in a randomized order, all subjects receiving the four doses. They consisted of 0, 2, and 4 glasses/d, providing 0, 23, and 46 g alcohol/d as well as in "binge drinking" (14 glasses in the weekend, comparable to an average of 2 glasses/d). The results showed a clear dose-related response to the drinking for several blood constituents. Most marked was a decrease in the tissue-type plasminogen activator activity and to a lesser degree an increase in plasminogen levels. Collagen-induced platelet aggregation was reduced, affecting all parameters measured. Levels of HDL3-cholesterol, gammaglutamyltransferase, and urate showed a small but significant increase. No change was noted in the levels of alkaline phosphatase, alanine-aminotransferase, aspartate-aminotransferase, bile acids, folate, fibrinogen, the ADP-induced platelet aggregation, platelet secretion, or in hematologic values. The results are only partially in accordance with the presumed protective action of moderate drinking on the cardiovascular system and show a stronger response to the consumption of alcohol in coagulation and fibrinolysis factors than in blood lipids.

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.

Heat stability of plasmin (milk proteinase) and plasminogen.

The effect of heating on plasmin activity in various media, including phosphate buffer pH 7.0, skim milk, blood plasma, solutions of casein and solutions of whey proteins were investigated. Plots of log residual activity v. heating time were linear at all temperatures from 63 to 143 degrees C. In buffer solutions the presence of casein led to substantial substrate protection, the Arrhenius plots being linear both in the presence and absence of casein. The activation energy, Ea, for the inactivation reaction, was 62.4 kJ/mol in buffer alone and 58.4 kJ/mol with casein present at 25 mg/ml. In skim milk, despite the presence of casein at a similar concentration, plasmin was no more stable to heat than in buffer alone, and a curved Arrhenius plot was obtained indicating a more complex inactivation mechanism. Heating in the presence of proteins having free -SH groups accelerated the inactivation of plasmin. The role of -SH groups was confirmed by experiments with added alpha-lactalbumin, in which no free -SH groups occur, and reduced carboxymethylated beta-lactoglobulin, both of which were without effect. In blood plasma, plasmin was less stable to heat than in buffer (pH 7.0) or in skim milk. Plasminogen behaved very similarly to plasmin either when activated to plasmin with urokinase before heating or when activated afterwards. A hypothesis is presented to describe the heat inactivation and denaturation of plasmin. Technologically important findings are that in skim milk plasmin was largely unaffected by pasteurization conditions and 30-40% of its activity remained even after ultra high temperature processing conditions.

Plasma factor XIII and some other haemostasis parameters in patients with diabetic angiopathy.

Since intravascular and endoparietal fibrin deposition is thought to be involved in the development of atherosclerosis, we measured factor XIII activity and its subunit 'a' and 'b' concentrations against a background of other haemostasis parameters in diabetics with angiopathy and in 2 control groups (healthy subjects and diabetics without vascular complications). Diabetics with angiopathy revealed a significant increase of factor XIII activity as well as its subunit concentrations. They also had significantly elevated anti-thrombin III, alpha 2 macroglobulin, alpha 1 antitrypsin, C1 inhibitor, fibrinogen, FDP concentrations and prolongation of euglobulin lysis time. The highest factor XIII levels were found in diabetics with renal failure. We suppose that increased factor XIII level and other observed changes of haemostasis in patients with diabetic angiopathy might promote intravascular and endoparietal fibrin deposition and contribute to the development of atherosclerotic complications of diabetes.

Plasma histidine-rich glycoprotein and plasminogen in patients with liver disease.

Histidine-rich glycoprotein (HRG) has been reported to be a fibrinolysis regulating protein due to its capacity to bind to the high affinity lysine binding sites of plasminogen. Using immunological methods we have measured the concentrations in plasma of HRG and total plasminogen and calculated the amounts of plasminogen not bound to HRG (free plasminogen) in 28 patients with moderate to severe liver disease. All three parametres showed wide individual variations, but with decreasing functional capacity of the liver the individual levels of plasminogen were reduced earlier than those of HRG leading to decreased amounts of free plasminogen. Simultaneous determinations of HRG and total plasminogen combined with a calculation of free plasminogen might yield valuable information when evaluating patients for the availability of plasminogen.

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.

Histidine-rich glycoprotein and changes in the components of the fibrinolytic system after streptokinase therapy in patients with pulmonary thromboembolism.

Although the biological function of histidine-rich glycoprotein (HRG) is unknown, it may serve as an antifibrinolytic agent by interfering with the binding of plasminogen to fibrin. To define the role of HRG, plasma titers were measured by single radial immunodiffusion in eleven patients with thromboembolism before and after streptokinase (SK) therapy and were found unchanged (84.7 +/- 6.2%, M +/- SEM before, and 99.5 +/- 6.3% after 12 hr of SK therapy). The HRG peaks on crossed immunoelectrophoresis before and after SK infusion were also unchanged. Alpha 2-plasmin inhibitor fell during SK infusion as measured immunologically (102.0 +/- 15.0% before and 28.0 +/- 1.6% after 12 hr of therapy) and fibrinogen-fibrin degradative products appeared (mean titer of 1:2,048 after 12 hr of therapy), indicating that the infused SK was biologically active. Plasminogen levels before therapy were normal, as measured functionally and immunologically (105.4 +/- 4.9% and 96.0 +/- 5.6%, respectively), and both decreased after 12 hr of SK therapy (15.2 +/- 5.6% and 50.8 +/- 4.3%). No changes in functional antithrombin III titer, Hageman factor antigen level, or fibrinogen concentration, as measured turbidimetrically, were observed. Thus, although these data do not allow one to make any firm conclusions regarding the physiologic role of this protein in fibrinolysis, they do not exclude its increased catabolism, compensated by increased production, in patients undergoing fibrinolytic therapy.