Alpha 2-antiplasmin Consumption Research Articles

Coronary thrombolysis with K1K2PU, a chimeric tissue-type and urokinase-type plasminogen activator

K1K2Pu is a recombinant chimeric tissue-type and urokinase-type plasminogen activator consisting of the two kringle domains (K1 and K2) of human tissue-type plasminogen activator (t-PA) and the serine proteinase domain (Pu) of single-chain urokinase-type plasminogen activator (scu-PA). In experimental animal models of thrombosis, its thrombolytic potency has been shown to be five- to 10-fold greater than that of its parent molecules. The effect of a bolus injection of K1K2Pu on coronary thrombolysis over 30 min was evaluated in six patients with acute myocardial infarction of less than 5 h duration in whom total occlusion of the infarct-related artery was confirmed using angiography. In two patients given an intravenous bolus of 10 mg over 5 min, persistent coronary artery recanalization was not observed within 30 min. In two out of four patients given a second bolus of 10 mg K1K2Pu, 15 min after the first, persistent coronary recanalization occurred within 30 min. The four patients without recanalization within 30 min were immediately given 100 mg t-PA over 90 min. In all patients the infarct-related artery was patent after 24 h, and the hospital course was uneventful. The bolus injections did not produce significant fibrinogen breakdown or alpha 2-antiplasmin consumption within 30 min. The plasma K1K2Pu level increased to 2-3 micrograms/ml after the first bolus injection and to 4-5 micrograms/ml after the second. K1K2Pu disappeared from the plasma with an initial half-life of 9 min and a clearance of approximately 50 ml/min. A bolus injection of 20 mg K1K2Pu is well tolerated and can induce clot-selective coronary thrombolysis in patients with acute myocardial infarction.

Thrombolytic and pharmacokinetic properties of an immunoconjugate of single-chain urokinase-type plasminogen activator (u-PA) and a bispecific monoclonal antibody against fibrin and against u-PA in baboons

Targeting of plasminogen activators to the fibrin component of a thrombus with the use of monoclonal antibodies (MA) directed against human fibrin may enhance their thrombolytic potency and fibrin- specificity. The thrombolytic and pharmacokinetic properties of rscu- PA/MA-FU1–74, an immunoconjugate of recombinant single-chain urokinase- type plasminogen activator (rscu-PA) and a bispecific MA directed against u-PA and against the beta-chain of human fibrin (MA-FU1–74), were investigated in baboons with a [125I]fibrin-labeled autologous blood clot in the femoral vein. Continuous intravenous infusion of rscu- PA/MA-FU1–74 (1:1.2 molar ratio) over 2 hours showed a fivefold increased thrombolytic potency (lysis per unit dose) over that of unconjugated rscu-PA, as evidenced both by a higher maximal rate of lysis (380% +/- 68% v 78% +/- 25% lysis per mg u-PA equivalent of compound administered per kg body weight, P less than .001), and by a lower dose at which the maximal rate of lysis occurs (0.19 +/- 0.03 v 0.82 +/- 0.10 mg compound per kg body weight, P less than .001). The specific thrombolytic activity (percent lysis per unit steady-state plasma u-PA antigen level) was lower for rscu-PA/MA-FU1–74 than for rscu-PA, as shown by both a lower maximal rate of lysis (60% +/- 13% v 220% +/- 22% lysis per microgram/mL u-PA antigen level in plasma, P less than .001) and a higher plasma antigen level at which maximal lysis is achieved (1.2 +/- 0.17 v 0.20 +/- 0.01 microgram/mL, P less than .001). The thrombolytic potency of rscu-PA/MA-UK1–3, an immunoconjugate of rscu-PA with the parental anti-u-PA antibody was similar to that of unconjugated rscu-PA. Clot lysis was achieved without systemic fibrinogen or alpha 2-antiplasmin consumption, and with a minor transient prolongation of the bleeding time. After the end of the infusions, u-PA-related antigen disappeared from plasma in a biphasic manner, with an initial half-life of 3.3 +/- 0.4 minutes for rscu-PA, 13 +/- 1 minutes for rscu-PA/MA-FU1–74, and 13 +/- 1 minutes for rscu-PA/MA-UK1–3, with corresponding plasma clearances of 340 +/- 28, 10 +/- 1, and 37 +/- 4 mL/min, respectively (mean +/- SEM). rscu- PA/MA-FU1–74 has a fivefold higher thrombolytic potency than unconjugated rscu-PA, as a result both of fibrin targeting by the specific idiotype of the antibody and of a slower plasma clearance.

Experimental Pulmonary Embolus in the Rat

Currently, the effects of the thrombolytic drugs are tested in vivo in dog or rabbit models that require a relatively large amount of the drug. The goal of the present study was to describe a new model that would allow one to test the in vivo thrombolytic effect of drugs with a limited amount of compound. For this purpose, we have induced a pulmonary embolus in anesthetized rats by injecting I125 radiolabeled clots into the venous circulation and we have measured the lysis of these clots occurring either spontaneously or induced by increasing doses of wild-type tissue plasminogen activator (tPA) (from 0.125 to 2 mg/kg i.v.) or by streptokinase (750,000 I.U./kg i.v.) or urokinase (750,000 I.U./kg i.v.). In the plasma of these rats, we have also measured the plasminogen activation activity of tPA as well as the concentrations of plasminogen, fibrinogen, and alpha 2-antiplasmin. Spontaneously, there was a time dependent thrombolysis (33%/h) that could be partially inhibited by aprotinin. Wild-type tPA induced a dose-related thrombolysis with a limited decrease of plasma fibrinogen concentration with doses over 0.25 mg/kg. Streptokinase and to a smaller extent urokinase induced a larger hemostatic breakdown (as indicated by systemic fibrinogenolysis, plasminogen activation, and alpha 2-antiplasmin consumption). We conclude that the rat pulmonary embolus model is suitable for testing the thrombolytic efficacy, potency, and the fibrin specificity of thrombolytic drugs and requires a smaller amount of drug than the previously described in vivo models.

Fibrinolysis during liver transplantation in humans: role of tissue- type plasminogen activator

Human liver transplantation is frequently associated with a coagulopathy and bleeding diathesis developing during the anhepatic phase of surgery. The hemostatic defect has been attributed in part to accelerated fibrinolysis. In this study we evaluated changes in specific blood fibrinolytic parameters occurring in eight adult patients undergoing first-time orthotopic liver transplantation. Five of the eight patients experienced moderate to severe systemic fibrinolysis as reflected by alpha 2-antiplasmin consumption and fibrinogen degradation with the concomitant appearance of fibrin(ogen) degradation products. In association with these changes, an increase in tissue-type plasminogen activator (t-PA) activity and t-PA antigen levels was also observed. Fibrinolysis was most pronounced during the anhepatic phase of surgery and decreased after revascularization of the grafted liver. Three additional patients who underwent the same procedure manifested much less evidence of systemic fibrinolytic activation and had minimal elevation of t-PA antigen levels or activity. Urokinase-type plasminogen activator levels, although elevated in three patients, were disassociated from increased t-PA levels and concomitant systemic fibrinolysis. The operative course of those patients developing t-PA-associated fibrinolysis was characterized by shock, acidosis, generalized bleeding, and a need for substantially greater blood product support during surgery. These findings suggest that the observed fibrinolytic defect is related to increased circulating plasma levels of t-PA, presumably resulting from a combination of increased intravascular release and decreased hepatic clearance of t-PA. These observations may have implications for intraoperative therapy for the transplant-related coagulopathy and its associated bleeding.

Fibrinolysis During Liver Transplantation in Humans: Role of Tissue-Type Plasminogen Activator

Human liver transplantation is frequently associated with a coagulopathy and bleeding diathesis developing during the anhepatic phase of surgery. The hemostatic defect has been attributed in part to accelerated fibrinolysis. In this study we evaluated changes in specific blood fibrinolytic parameters occurring in eight adult patients undergoing first-time orthotopic liver transplantation. Five of the eight patients experienced moderate to severe systemic fibrinolysis as reflected by alpha 2-antiplasmin consumption and fibrinogen degradation with the concomitant appearance of fibrin(ogen) degradation products. In association with these changes, an increase in tissue-type plasminogen activator (t-PA) activity and t-PA antigen levels was also observed. Fibrinolysis was most pronounced during the anhepatic phase of surgery and decreased after revascularization of the grafted liver. Three additional patients who underwent the same procedure manifested much less evidence of systemic fibrinolytic activation and had minimal elevation of t-PA antigen levels or activity. Urokinase-type plasminogen activator levels, although elevated in three patients, were disassociated from increased t-PA levels and concomitant systemic fibrinolysis. The operative course of those patients developing t-PA-associated fibrinolysis was characterized by shock, acidosis, generalized bleeding, and a need for substantially greater blood product support during surgery. These findings suggest that the observed fibrinolytic defect is related to increased circulating plasma levels of t-PA, presumably resulting from a combination of increased intravascular release and decreased hepatic clearance of t-PA. These observations may have implications for intraoperative therapy for the transplant-related coagulopathy and its associated bleeding.

Comparative thrombolytic properties of single-chain forms of urokinase- type plasminogen activator

The specific thrombolytic properties of urokinase and three molecular forms of single-chain urokinase-type plasminogen activator (scu-PA) were compared in a human plasma milieu in vitro and in an experimental thrombosis model in rabbits. These scu-PA molecules included Mr 54,000 scu-PA from human urine (urinary scu-PA), scu-PA from conditioned media of a human lung adenocarcinoma cell line (CALU-3,ATCC,HTB-55) (cellular scu-PA) and an Mr 32,000 proteolytic derivative of cellular scu-PA (scu- PA-32k). All four molecular forms induced significant lysis of a 125I- labeled human plasma clot immersed in citrated human plasma at concentrations between 50 and 200 IU/mL. None of the four showed absolute fibrin-specificity, but at equivalent lytic dose the three single-chain forms appeared to cause less fibrinogen degradation and alpha 2-antiplasmin consumption than two-chain urokinase. In addition, the fibrinolytic potential of the three single-chain forms was largely maintained during pre-incubation in plasma for up to 48 hours whereas that of urokinase was completely inhibited. Intravenous (IV) infusion of cellular scu-PA or scu-PA-32k into rabbits with a 125I-labeled thrombus in the jugular vein caused significant dose-dependent lysis at concentrations ranging from 8,700 to 35,000 and from 9,000 to 36,000 IU/kg respectively. Clot lysis was accompanied by minor alpha 2- antiplasmin consumption or fibrinogen breakdown. In contrast, urokinase induced lysis at doses between 20,000 and 200,000 IU/kg, but at higher doses was associated with significant systemic activation of the fibrinolytic system. It is concluded that scu-PA obtained from CALU-3 cell cultures has identical thrombolytic properties to that obtained from urine. In addition, the scu-PA-32k proteolytic derivative has the same fibrin-specific thrombolytic properties as the intact molecule. Cellular scu-PA and scu-PA-32k may therefore constitute more readily available alternatives for clot-selective thrombolytic therapy in man.

Comparative Thrombolytic Properties of Single-Chain Forms of Urokinase-Type Plasminogen Activator

The specific thrombolytic properties of urokinase and three molecular forms of single-chain urokinase-type plasminogen activator (scu-PA) were compared in a human plasma milieu in vitro and in an experimental thrombosis model in rabbits. These scu-PA molecules included Mr 54,000 scu-PA from human urine (urinary scu-PA), scu-PA from conditioned media of a human lung adenocarcinoma cell line (CALU-3,ATCC,HTB-55) (cellular scu-PA) and an Mr 32,000 proteolytic derivative of cellular scu-PA (scu-PA-32k). All four molecular forms induced significant lysis of a 125I-labeled human plasma clot immersed in citrated human plasma at concentrations between 50 and 200 IU/mL. None of the four showed absolute fibrin-specificity, but at equivalent lytic dose the three single-chain forms appeared to cause less fibrinogen degradation and alpha 2-antiplasmin consumption than two-chain urokinase. In addition, the fibrinolytic potential of the three single-chain forms was largely maintained during pre-incubation in plasma for up to 48 hours whereas that of urokinase was completely inhibited. Intravenous (IV) infusion of cellular scu-PA or scu-PA-32k into rabbits with a 125I-labeled thrombus in the jugular vein caused significant dose-dependent lysis at concentrations ranging from 8,700 to 35,000 and from 9,000 to 36,000 IU/kg respectively. Clot lysis was accompanied by minor alpha 2-antiplasmin consumption or fibrinogen breakdown. In contrast, urokinase induced lysis at doses between 20,000 and 200,000 IU/kg, but at higher doses was associated with significant systemic activation of the fibrinolytic system. It is concluded that scu-PA obtained from CALU-3 cell cultures has identical thrombolytic properties to that obtained from urine. In addition, the scu-PA-32k proteolytic derivative has the same fibrin-specific thrombolytic properties as the intact molecule. Cellular scu-PA and scu-PA-32k may therefore constitute more readily available alternatives for clot-selective thrombolytic therapy in man.

Effects of Pro-Urokinase and Urokinase on the Fibrinolytic System in Man

The fibrinolytic and fibrinogenolytic properties of kidney cell pro-urokinase (PUK) were compared with those of natural urinary urokinase in human volunteers. Comparable degrees of fibrinolysis were obtained at a concentration of 500,000 U UK and a concentration of 500,000 IU natural urokinase. Natural urokinase showed a strong activation of the fibrinolytic system in plasma, evidenced by plasminogen activation, alpha 2-antiplasmin consumption, and the rise in fibrinogen-fibrin degradation products. In contrast to UK, there was no fall in plasminogen, no consumption of alpha 2-antiplasmin, and only a slight amount of fibrinogen-fibrin degradation products produced with PUK. These findings with PUK reveal a high affinity for fibrin and may result in better clot selectivity.

Synergism of thrombolytic agents in vivo.

The existence of significant synergism between tissue-type plasminogen activator (t-PA) and single-chain urokinase-type plasminogen activator (scu-PA), and between t-PA and urokinase in thrombolysis in vivo is described. In a quantitative preparation of thrombolysis, consisting of rabbits in which a blood clot was induced in the jugular vein with 125I-labeled fibrin, intravenous infusion over 4 hr of t-PA, scu-PA, or urokinase in amounts of 0.5, 1.0, or 2.0 mg/kg body weight resulted in significant thrombolysis (30% to 60%). The simultaneous infusion of t-PA and scu-PA or of t-PA and urokinase had a significantly greater (p less than .001) thrombolytic effect than could be anticipated on the basis of the added effects of each agents alone. However, no synergism was observed between scu-PA and urokinase. The observed alpha 2-antiplasmin consumption and fibrinogen breakdown after urokinase at higher doses did not occur with the equivalent thrombolytic combinations of t-PA and urokinase. The combined use of synergic thrombolytic agents in patients may permit a significant reduction in total administered doses, probably with elimination of the systemic activation of the fibrinolytic system and the concomitant fibrinogen breakdown that is unavoidable with the currently used thrombolytic doses of each agent.