A bi-functional anti-thrombosis protein containing both direct-acting fibrin(ogen)olytic and plasminogen-activating activities.

UNLABELLED: Direct-acting fibrin(ogen)olytic agents such as plasmin have been proved to contain effective and safety thrombolytic potential. Unfortunately, plasmin is ineffective when administered by the intravenous route because it was neutralized by plasma antiplasmin. Direct-acting fibrin(ogen)olytic agents with resistance against antiplasmin will brighten the prospect of anti-thrombosis. As reported in 'Compendium of Materia Medica', the insect of Eupolyphaga sinensis Walker has been used as traditional anti-thrombosis medicine without bleeding risk for several hundreds years. Currently, we have identified a fibrin(ogen)olytic protein (Eupolytin1) containing both fibrin(ogen)olytic and plasminogen-activating (PA) activities from the beetle, E. sinensis. OBJECTIVES: To investigate the role of native and recombinant eupolytin1 in fibrin(ogen)olytic and plasminogen-activating processes. METHODS AND RESULTS: Using thrombus animal model, eupolytin1 was proved to contain strong and rapid thrombolytic ability and safety in vivo, which are better than that of urokinase. Most importantly, no bleeding complications were appeared even the intravenous dose up to 0.12 µmol/kg body weight (3 times of tested dose which could completely lyse experimental thrombi) in rabbits. It is the first report of thrombolytic agents containing both direct-acting fibrin(ogen)olytic and plasminogen-activating activities. CONCLUSIONS: The study identified novel thrombolytic agent with prospecting clinical potential because of its bi-functional merits containing both plasmin- and PA-like activities and unique pharmacological kinetics in vivo.

Fibrinolytic efficacy of Amediplase, Tenecteplase and scu-PA in different external plasma clot lysis models: sensitivity to the inhibitory action of thrombin activatable fibrinolysis inhibitor (TAFI).

In this study, the in-vitro fibrinolytic efficacy of Tenecteplase, Amediplase and scu-PA was investigated in different external lysis models by measuring the lysis of human plasma clots after the addition of the plasminogen activators (PAs) to the surrounding plasma. The effect of TAFI was examined for each PA by neutralising TAFIa with potato carboxypeptidase inhibitor (PCI). The lytic efficacy of Amediplase was lower than that of Tenecteplase at low PA concentrations but slightly higher at therapeutic concentrations. The activity of scu-PA was clearly lower than that of either Tenecteplase or Amediplase. The TAFI system inhibited external clot lysis mediated by all the PAs when thrombomodulin was present in the model. In the therapeutic range (5-10 mug/ml) however, the TAFIa effect was negligible for both Amediplase and Tenecteplase. At lower PA concentrations the effect of TAFI on Amediplase was slightly stronger than that on Tenecteplase. Under static conditions the lysis rates were lower than with stirring. The role of TAFI was similar under both conditions. In conclusion, at therapeutic concentrations Amediplase was slightly more active than Tenecteplase and scu-PA under all conditions used. Therefore, Amediplase might possibly be a more potent thrombolytic agent at these concentrations and increase the efficacy of thrombolysis. The potential of TAFI for inhibiting thrombolytic therapy is probably low. However in conditions where the local PA concentrations are sub-optimal TAFI might affect the lysis rate.

Ganglioside GM3 promotes carcinoma cell proliferation via urokinase plasminogen activator-induced extracellular signal-regulated kinase-independent p70S6 kinase signaling.

Overexpression of NeuAcalpha2-3Galbeta1-4Glcbeta1-Cer (GM3), a major ganglioside of cutaneous tumor cell membranes, inhibits ligand-dependent and ligand-independent activation of the epidermal growth factor (EGF) receptor in normal and neoplastic epithelial cells. This leads to the suppression of Ras/extracellular signal-regulated kinase (ERK) activation and, in the presence of EGF or fibronectin, inhibits cell proliferation. However, some tumor cells show increased levels of GM3, and vaccines that target GM3 can inhibit the growth of neoplastic cells in vivo, especially melanomas. We report that in the presence of urokinase plasminogen activator (uPA), overexpression of GM3 paradoxically increases the proliferation of carcinoma cells by augmenting ERK-independent p70S6 kinase activation. Functional blockade of uPA receptor (uPAR) or inhibition of p70S6 kinase, but not inhibition of Ras/ERK signaling, suppresses this GM3-induced stimulation of cell proliferation. The ERK-independent activation of p70S6 kinase involves phosphorylation at threonine-389, threonine-421/serine-424, and serine-411 sites with intermediate phosphatidylinositol 3 kinase and protein kinase C-zeta activation. These studies implicate gangliosides as enhancers of uPAR-related signaling and suggest that the response to GM3 depends on the local concentration of uPA. Therapeutic modalities that target or supplement gangliosides may require concomitant treatment that suppresses EGFR or uPAR signaling, respectively, to control neoplastic cell proliferation.

[Test for urokinase-type plasminogen activator inhibitor of edible plants in vitro].

OBJECTIVE: To study the effect of anti-progression activity of edible plants using urokinase as the biomarker. METHODS: Based on the assay of urokinase activity with peptide of Glu-Gly-Arg as the reaction substrate, extract of 25 fruits and 37 vegetables and water extract of tea were reacted against urokinase activity after Spectrozyme UK [carbobenzyl-1-gamma-Glu(alpha-t-BuO)-Gly-Arg-rho-nitroanilide. 2C2H5OH] was added and the residual urokinase activity was measured by the microplate photometer. RESULTS: About half of the fruit and vegetable samples showed urokinase inhibitory activity (UIA) at 20% or more and among them lemon, kiwi-fruit, peas, spinach and pumpkin showed effects over 80%, while garlic, radish, Japanese butterbur, garland chrysanthemum, celery, plum, pineapple and grape ranged between 50% and 79%. Average UIA of 51 kinds of tea was 83.2% and among them 28 kinds showed UIA over 90% and other 20 kinds of tea ranged from 70% to 89%. Green tea showed inhibitory effects on urokinase activity more powerful than black tea and Oolong tea. On the other hand, vegetable showed various UIA with different ways of processing. Comparing the effects of various solvents, garlic extracted with methanol, spinach with ethyl acetate, and pumpkin and radish with water showed highest UIA. CONCLUSION: Popular fruits, vegetables and tea in daily life could inhibit urokinase activity and may be helpful in the prevention of malignant tumor formation.

Modulation of PAI-1 and tPA activity and thrombolytic effects of corilagin.

In this study, Charlton's and Tomihisa's methods were modified to investigate the thrombolytic effect of corilagin from the Chinese herbal plant Phyllanthus urinaria L., as well as its effect on carotid artery patency status. The activity of type 1 plasminogen activator inhibitor (PAI-1) in rat plasma or platelet-released substances and tissue-type plasminogen activator (tPA) in rat plasma was assayed by use of a chromogenic substrate. The results showed that corilagin had a dose-dependent thrombolytic effect in rats. 5 mg/kg of corilagin produced a nearly similar reperfusion rate to that of 20000 U/kg of urokinase, whereas it produced a lower reocclusion rate than urokinase. Corilagin significantly inhibited PAI-1 activity in rat plasma or platelet-released substances while it elevated plasma tPA activity, in a concentration-dependent manner. Corilagin, however, had no influence on rabbit platelet aggregation. It is indicated that corilagin inhibited PAI-1 activity and increased tPA activity, and this property of corilagin is assumed to be responsible for the thrombolytic effect. Abbreviations. PO:persistent occlusion CR:cyclic reflow PP:persistent patency PAI-1:type 1 plasminogen activator inhibitor tPA:tissue-type plasminogen activator PBS:phosphate buffer solution IC (50):50 % of inhibitory concentration PRP:platelet-rich plasma ADP:adenosine diphosphate AA:arachidonic acid PAF:platelet-activating factor

Effects of inhaled plasminogen activator on the balance between coagulation and fibrinolysis in traumatized pigs.

A profibrinolytic state is normal in the alveoli, but this may change as a result of trauma, possibly leading to fibrin deposition, a characteristic of acute lung injury/acute respiratory distress syndrome. Therefore, the present study investigated in a double-blind, placebo-controlled manner the effect of severe trauma on the alveolar fibrinolytic/coagulation balance, and the effect here-upon of inhalation of single-chain urokinase plasminogen activator (scu-PA) in pigs. The study shows an increased concentration of scu-PA in the bronchoalveolar lavage fluid of the treated animals in association with an increased plasmin-dependent fibrinolytic activity without increased systemic fibrinolytic activity, the transient increase in the concentration of scu-PA in the plasma being minimal. In conclusion, the study shows that activatable scu-PA can be nebulized to the lower respiratory tract and can increase the alveolar fibrinolysis without any significant systemic effects.

[Characterization of urokinase type plasminogen activator modified by phenylglyoxal]. / Svoistva aktivatora plazminogena urokinaznogo tipa, modifitsirovannogo fenilglioksalem.

A chemical modification of single-chain urokinase-type plasminogen activator (scu-PA) with phenylglyoxal under mild conditions resulted in the scu-PA derivatives with various numbers of the modified Arg residues. The study of properties of the resulting derivatives demonstrated that the modification of 4-12 Arg residues did not cause any loss of the activator, fibrinolytic, and potential amidase activities of the activator. The scu-PA with four modified Arg residues was found to be the most stable derivative in human blood plasma; it causes a more efficient lysis of plasma clots than the native activator. Three of four modified Arg residues are supposed to be within the 178RRHRGGS184 cluster, which was localized in the superficial loop of the scu-PA globule and was shown to interact with the complementary series of negatively charged residues in the molecule of the main plasma inhibitor PAI-1. The neutralization of positively charged Arg residues in this cluster decreases the affinity of scu-PA and the double chain urokinase-type plasminogen activator for PAI-1, which results in an enhancement of the stability in plasma and the fibrinolytic efficiency of the activator. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.

Prourokinase mutant that induces highly effective clot lysis without interfering with hemostasis.

Prourokinase (proUK) is a zymogenic plasminogen activator that at pharmacological doses is prone to nonspecific activation to urokinase. This has handicapped therapeutic exploitation of its fibrin-specific physiological properties. To attenuate this susceptibility without compromising specific activation of proUK on a fibrin clot, a Lys300-->His mutation (M5) was developed. M5 had a lower intrinsic activity and, therefore, remained stable in plasma at a 4-fold higher concentration than did proUK. M5 had a higher 2-chain activity and induced more rapid plasminogen activation and fibrin-specific clot lysis in vitro. Sixteen dogs embolized with radiolabeled clots were infused with saline, proUK, tissue plasminogen activator, or M5. The lower intrinsic activity allowed a higher infusion rate with M5, which induced the most rapid and efficient clot lysis (50% clot lysis by approximately 600 microg/kg M5 versus approximately 1200 microg/kg proUK). In association with this, M5 caused neither a significant increase in the primary bleeding time nor secondary bleeding (total blood loss). By contrast, these measurements increased 4-fold and 5-fold, respectively, with proUK and >5-fold and 8-fold, respectively, with tissue plasminogen activator. Clot lysis by M5 and hemostasis were further evaluated in 6 rhesus monkeys. M5 again induced rapid clot lysis without a significant increase in the primary bleeding time, and secondary bleeding did not occur. In conclusion, a site-directed mutation designed to improve the stability of proUK in blood at therapeutic concentrations induced superior clot lysis in vitro and in vivo without causing significant interference with hemostasis.

Survival and function of baboon RBCs released from clotted blood and washed before autologous transfusion.

BACKGROUND: One alternative to an allogeneic transfusion is the salvaging of the patient's own shed blood. In this study, baboon blood was allowed to clot and the RBCs that were released from the clotted blood lysed with and without urokinase were washed before autologous transfusion. STUDY DESIGN AND METHODS: Forty-four studies were done in 13 baboons (Papio cynocephalus or Papio anubis) over a 3-year period. In 24 studies, a 50-mL volume of blood was collected without an anticoagulant and stored at 22 degrees C for as long as 72 hours before washing and autologous transfusion. In 20 other studies, a 50-mL volume of blood was collected without an anticoagulant and allowed to clot for 30 to 60 minutes. Urokinase, ranging from 2,500 to 10,000 units per mL, was added, and the blood was stored at 22 degrees C for 24 hours before washing and autologous transfusion. RESULTS: RBCs that were stored at 22 degrees C without urokinase for 24 hours exhibited an in vitro recovery value of 45 percent, a (51)Cr 24-hour posttransfusion survival of 86 percent, and an index of therapeutic effectiveness of 39 percent. The (51)Cr T(50) value was normal at 14 days, and RBC oxygen-transport function was slightly reduced. RBCs that were stored at 22 degrees C for 24 hours with 10,000 units per mL of urokinase exhibited an in vitro recovery value of 89 percent, a (51)Cr 24-hour posttransfusion survival value of 86 percent, and an index of therapeutic effectiveness of 76 percent. The (51)Cr T(50) value was normal at 14 days, and the RBC oxygen-transport function was only slightly reduced. CONCLUSION: Autologous baboon RBCs isolated from clotted blood treated or not treated with urokinase and washed before transfusion have excellent survival and normal or only slightly reduced oxygen-transport function.

Functional domains of the very low density lipoprotein receptor: molecular analysis of ligand binding and acid-dependent ligand dissociation mechanisms.

The very low density lipoprotein (VLDL) receptor is closely related in structure to the low density lipoprotein receptor. The ectodomain of these endocytic receptors is composed of modules which include clusters of cysteine-rich class A repeats, epidermal growth factor (EGF)-like repeats, tyrosine-tryptophan-threonine-aspartic acid (YWTD) repeats and an O-linked sugar domain. To identify important functional regions within the ectodomain of the VLDL receptor, we produced a mutant receptor in which the EGF, YWTD and O-linked sugar domains were deleted. Cells transfected with the mutant receptor were able to bind and internalize (125)I-labeled receptor associated protein (RAP). In contrast to the wild-type receptor, however, RAP did not dissociate from the mutant receptor and consequently was not degraded. Immunofluoresence data indicated that once bound to the mutant receptor, fluorescent-labeled RAP co-localized with markers of the endosomal pathway, whereas, in cells expressing the wild-type receptor, RAP fluorescence co-localized with lysosomal markers. Thus this deleted region is responsible for ligand uncoupling within the endosomes. To identify regions responsible for ligand recognition, soluble receptor fragments containing the eight cysteine-rich class A repeats were produced. (125)I-RAP and (125)I-labeled urokinase-type plasminogen activator:plasminogen activator inhibitor type I (uPA:PAI-1) complexes bound to the soluble fragment with K(D, app) values of 0.3 and 14 nM, respectively. Deletion analysis demonstrate that high affinity RAP binding requires the first four cysteine-rich class A repeats (L1-4) in the VLDL receptor while the second repeat (L2) appears responsible for binding uPA:PAI-1 complexes. Together, these results confirm that ligand uncoupling occurs via an allosteric-type mechanism in which pH induced changes in the EGF and/or YWTD repeats alter the ligand binding properties at the amino-terminal portion of the molecule.

Coagulation-dependent inhibition of fibrinolysis: role of carboxypeptidase-U and the premature lysis of clots from hemophilic plasma.

Coagulation is initiated by the binding of factor VIIa to tissue factor, with resultant limited factor IX and X activation and thrombin production. Owing to the feedback inhibition of the factor VIIa/tissue factor complex by tissue factor pathway inhibitor (TFPI), additional factor X activation and thrombin generation must proceed through a pathway involving factors VIII, IX, and XI. Experiments designed to elucidate the requirement for amplified factor Xa and thrombin generation in normal hemostasis show that the resistance of plasma clots to tissue plasminogen activator (tPA)- and urokinase-induced fibrinolysis is related to the extent of thrombin generation. Inhibition of fibrinolysis is mediated in part by plasma carboxypeptidase-U ([CPU] carboxypeptidase-R, procarboxypeptidase-B, thrombin-activatable fibrinolysis inhibitor), a proenzyme that is proteolytically activated by thrombin in a process enhanced dramatically by the cofactor thrombomodulin. A clot induced in factor IX-deficient plasma with limited amounts of tissue factor in the presence of urokinase (100 U/mL) lyses prematurely, and this defect is corrected by supplementation of the deficient plasma with factor IX (5 micrograms/mL) or thrombomodulin (20 ng/mL). These additions enhance the rate and extent of CPU activation: in the case of factor IX, presumably by permitting amplified generation of factor Xa and thrombin, and in the case of thrombomodulin, presumably by increasing the degree of CPU activation produced by the low levels of thrombin generated in the absence of factor IX. Pretreatment of the factor IX-deficient plasma with specific anti-CPU antibodies prevents the increased resistance to fibrinolysis produced by addition of factor IX and thrombomodulin. Likewise, when coagulation is induced by thrombin (2 U/mL) in the presence of tPA (60 U/mL), clots formed from plasmas deficient in factors VIII, IX, X, or XI lyse prematurely unless the missing factor is replaced or thrombomodulin (20 ng/mL) is added.

Mac-1 (CD11b/CD18) and the urokinase receptor (CD87) form a functional unit on monocytic cells.

The leukocyte integrin Mac-1 (CD11b/CD18) and the urokinase receptor (uPAR, CD87) mediate complementary functions in myelomonocytic cells. Both receptors promote degradation of fibrin(ogen) and also confer adhesive properties on cells because Mac-1 and uPAR bind fibrin and vitronectin, respectively. Staining of lung biopsy specimens from patients with acute lung injury indicated that fibrin and vitronectin colocalize at exudative sites in which macrophages bearing these receptors accumulate. Because of the parallel roles and physical proximity of Mac-1 and uPAR, the capacity of these receptors to functionally interact was explored. Induction of Mac-1 and uPAR expression on monocytic cell lines by transforming growth factor- beta 1 and 1.25-(OH)2 vitamin D3 conferred urokinase and uPAR-dependent adhesion to vitronectin, which was further promoted by engagement of Mac-1. Vitronectin attachment promoted subsequent Mac-1-mediated fibrinogen degradation threefold to fourfold. In contrast, enhancement of uPAR occupancy by exogenous urokinase or receptor binding fragments thereof inhibited Mac-1 function. Addition of urokinase progressively inhibited Mac-1-mediated fibrinogen binding and degradation (maximal inhibition, 91% +/- 14% and 72% +/- 15%, respectively). Saturation of uPAR with urokinase also inhibited binding of the procoagulant Mac-1 ligand, Factor X. These inhibitory effects of uPAR were reproduced in fresh monocytes, cultured monocytic cells, and in Chinese hamster ovary (CHO) cells transfected with both human Mac-1 and human uPAR. These data show that the procoagulant and fibrinolytic potential of monocytic cells is co-ordinately regulated by ligand binding to both Mac-1 and uPAR and identify uPAR as a regulator of integrin function. Vitronectin-enhanced fibrin(ogen) turnover by Mac-1 may operate as a salvage pathway in the setting of urokinase and plasmin inhibitors to promote clearance of the provisional matrix and subsequent healing.

Urokinase-type plasminogen activator-induced monocyte adhesion requires a carboxyl-terminal lysine and cAMP-dependent signal transduction.

Urokinase-type plasminogen activator (u-PA) or its amino-terminal fragment (ATF) containing the u-PA receptor (u-PAR) binding domain, is known to promote monocyte adhesion. In the present study, U937 monocyte adhesion to a plastic surface was used to investigate the mechanism of its promotion by u-PA and ATF. Adhesion was found to be inhibited by cycloheximide or actinomycin D, implicating protein synthesis and gene expression in u-PA-induced monocyte adhesion. Adhesion was prevented by 2'-deoxyadenosine 3'-monophosphate, indicating that a cAMP-dependent pathway of signal transduction was involved. This concept was supported by the complementary finding that u-PA-induced adhesion was greatly promoted by forskolin, cholera toxin, or 8-bromo-cAMP, which by themselves induced little adhesion. Furthermore, similar to many other cAMP-dependent activities, cGMP diminished u-PA-induced adhesion. When u-PA or ATF was treated with immobilized carboxypeptidase B, its proadhesive effect was abolished, implicating the involvement of carboxyl-terminal lysine residues (Lys158 on u-PA and Lys135 on ATF). Moreover, when a carboxyl-terminal lysine analog was added, the proadhesive effect of carboxypeptidase B-treated u-PA or ATF was restored. In conclusion, the present study indicates that u-PA- or ATF-induced monocyte adhesion involves cAMP-dependent signal transduction, which is triggered by u-PAR binding. It is also critically dependent on the presence of a carboxyl-terminal lysine.

Thrombolytic therapy in peripheral arterial occlusive disease: mechanisms of action and drugs available.

Catheter-directed thrombolytic therapy has become an important part of the treatment of patients with acute arterial and graft occlusion. The underlying pharmacologic principle is the activation of plasminogen, bound to fibrin within the thrombus. Guide-wire passage reliably predicts success of catheter-directed thrombolysis. The underlying disease process leading to thrombosis should be accurately identified and promptly corrected to reduce the probability of recurrent occlusion. Streptokinase (SK), urokinase (UK) and recombinant tissue plasminogen activator (rt-PA) are the three agents used to treat peripheral arterial occlusive disease. The evolution from SK to UK and rt-PA and improvements in techniques and delivery systems have led to improved success rates and lower complication rates. Patient selection, basic technical considerations and overall results are discussed here. The currently available thrombolytic agents, as well as those being developed, are reviewed to provide background information for current and future applications.

The beneficial effect of lys-plasminogen upon the thrombolytic efficacy of urokinase in a dog model of peripheral arterial thrombosis.

The efficacy of thrombolytic therapy may be limited by local availability of plasminogen near a poorly perfused thrombus. The purpose of this study was to determine if the local (i.e., clot site) administration of 0.5 mg glu-plasminogen (glu-plg) or 0.5 mg lysplasminogen (lys-plg) could safely increase the thrombolytic efficacy of a 30-min intraarterial injection of 3,500 U kg-1 of two-chain urokinase plasminogen activator (UK) in a dog model of arterial thrombosis. Thrombolysis was measured by monitoring the continuous decrement of 125I-gamma emissions from a radiolabeled thrombus. Reflow was evaluated by a distally placed flowmeter and by direct visual examination. Forty-two dogs (mean weight 10.1 +/- 1.9 kg) were randomly sorted into six groups of 7 each. The dogs in each group were given either saline plus saline (group 1), saline plus UK (group 2), glu-plg plus saline (group 3), glu-plg plus UK (group 4), lys-plg plus saline (group 5), or lys-plg plus UK (group 6) by selective arterial catheterization 60 min after formation of an occlusive thrombus. Ninety minutes following drug administration, all groups which received UK (groups 2, 4, and 6) showed greater lysis (p less than 0.05) than the groups which received only saline or either glu- or lys-plg plus saline. Group 6, which received lys-plg plus UK, showed significantly greater lysis (34 +/- 4%) than both group 2 (23 +/- 2%), which received saline plus UK, and group 4 (19 +/- 3%), which received glu-plg plus UK (p less than 0.05). All dogs (7/7) in group 6 had reflow at 90 min whereas only 3/7 dogs had reflow in both groups 2 and 4.(ABSTRACT TRUNCATED AT 250 WORDS)

Complementary modes of action of tissue-type plasminogen activator and pro-urokinase by which their synergistic effect on clot lysis may be explained.

Tissue plasminogen activator (t-PA) and/or pro-urokinase (pro-UK) induced lysis of standard 125I-fibrin clots suspended in plasma was studied. Doses were kept below the concentration at which a nonspecific effect was seen, i.e., where fibrinogenolysis and major plasminogen consumption were observed. Small amounts of t-PA potentiated clot lysis by pro-UK by attenuating the lag phase characteristic of pro-UK, and causing a much earlier transition to the rapid phase of lysis. Similar promotion of the fibrinolytic effect of pro-UK was obtained when clots were pretreated with UK or with a little plasmin (less than 1% clot lysis). Promotion by plasmin was nullified by a subsequent treatment of the clot with carboxypeptidase B, indicating that the plasmin effect was related to the exposure of carboxy terminal lysine residues on fibrin. These lysine termini, absent in undegraded fibrin, are known to be essential for the high affinity binding of plasminogen to fibrin. In contrast, clot lysis by t-PA was unaffected by plasmin pretreatment and little affected by carboxypeptidase B treatment of the fibrin substrate. Therefore, plasminogen bound to lysine termini on fibrin, although found to be essential for pro-UK, did not appear to serve as a substrate for t-PA. Selective activation of fibrin bound plasminogen has been attributed to the conformational change in Glu-plasminogen that occurs as a result of binding. The present findings suggest that this conformational change occurs when plasminogen is bound to a terminal lysine but not to an internal lysine. Plasminogen bound to the latter site on fibrin was activated by t-PA and therefore is involved in the ternary complex. This initiates lysis of the undegraded clot and exposes the plasminogen binding sites required by pro-UK. By their complementary activation of fibrin bound plasminogen, t-PA followed by pro-UK induces efficient and synergistic fibrinolysis, whereas each is relatively inefficient when used alone.

A comparative study of the efficacy and specificity of tissue plasminogen activator and pro-urokinase: demonstration of synergism and of different thresholds of non-selectivity.

Clot lysis and non-specific plasminogen activation in human plasma by tissue tissue plasminogen activator (t-PA) and/or pro-urokinase (pro-UK) were studied. The fibrinolytic activity of pro-UK was expressed as latent units, i.e. measured after activation with plasmin on a fibrin plate against the reference standard. The t-PA unitage was assigned on a weight basis of a similar equivalence of 100,000 IU/mg. To simplify comparison, both activators were expressed in IU (1 IU = approximately 10 ng). At low concentration (1-50 IU/ml), t-PA induced more effective and more linear clot lysis, whereas pro-UK induced lysis was preceded by a lag phase. The two activators were equivalently effective at higher concentrations and saturated at the same lysis rate. Clots made from platelet rich plasma or whole blood were more responsive to lysis by pro-UK but not t-PA than corresponding platelet poor clots. At very low concentrations (2.5-5 IU/ml) of t-PA combined with moderate concentrations (25-50 IU/ml) of pro-UK, a synergistic effect on clot lysis, which was fibrin-specific, was observed. Plasminogen and fibrinogen and the appearance of plasmin-inhibitor complexes in plasma were measured after incubation with either activator with and without a clot present. Non-specific plasminogen activation occurred above a certain concentration of either activator but was found at lower concentrations of t-PA than pro-UK. In the absence of a clot, plasmin generation occurred with t-PA at about 30% of the concentration at which pro-UK induced a corresponding effect. It is concluded that there are important differences in the fibrinolytic and clot selective properties of t-PA and pro-UK, and that some of these properties may be complementary resulting in a fibrin specific, synergistic fibrinolytic effect.

The effect of intravenous infusion of urokinase on coronary artery thrombolysis in the dog.

The effect of intravenous administration of urokinase on the rate and time of thrombolysis was investigated in 22 dogs with an occlusive coronary artery thrombus induced by placement of a copper coil in the left anterior descending artery. One hour after angiographic confirmation of complete coronary artery occlusion, an intravenous bolus of urokinase was administered in a dose of either 1.2 X 10(6) units (Group 1, n = 7); 0.6 X 10(6) units (Group 2, n = 9) or 0.3 X 10(6) units (Group 3, n = 6). Reperfusion occurred in all 7 (100%) Group 1 dogs, in 7 of 9 (78%) group 2 dogs and in 3 of 6 (50%) group 3 dogs (p = 0.07 for Group 1 vs Group 3). There was no residual intra-arterial thrombus in any group 1 (high dose) dog but 4 of the 10 reperfused Group 2 or Group 3 (intermediate or low dose) dogs had small thrombus in the distal left anterior descending artery (3.5 +/- 4.0 mg) and all 5 non-reperfused dogs had thrombus in the coil (26.1 +/- 6.2 mg). The frequency of residual thrombus was significantly less in Group 1 compared to Group 2 and 3 (p less than 0.01). We conclude that intravenous administration of urokinase has a dose dependent efficacy for lysing intracoronary thrombus in the canine model.

New approaches to thrombolytic therapy.

Tissue-type plasminogen activator (t-PA), purified from the culture fluid of a stable human melanoma cell line, is a serine protease, different from urokinase, with a molecular weight of about 70,000. It is composed of one polypeptide chain, which is converted to a two-chain molecule by limited plasmic action. Activation of plasminogen to plasmin occurs by cleavage of the Arg 560-Val 561 peptide bond. Kinetic analysis has shown that the activation obeys Michaelis-Menten kinetics and that the presence of fibrin strikingly enhances the activation rate by increasing the affinity of plasminogen for fibrin-bound t-PA. The directed action of plasmin toward fibrin in vivo, might be explained by the low Michaelis constant in the presence of fibrin (0.16 microM), which allows efficient plasminogen activation on a fibrin clot, while its high value in the absence of fibrin (65 microM) prevents efficient activation in plasma. Plasmin formed on the fibrin surface would then be protected from rapid inactivation by alpha 2-antiplasmin. An important consequence of this molecular model for physiological fibrinolysis is that specific thrombolysis is only expected with the use of a specific plasminogen activator, which confines activation to the fibrin surface. Studies on the thrombolytic properties of purified t-PA in various animal species and in humans have revealed a higher specific thrombolytic activity than urokinase. Thrombolysis could be achieved without causing significant plasminogen activation, alpha 2-antiplasmin consumption, or fibrinogen breakdown. Alternatively, pro-urokinase, the zymogen precursor of urokinase, also displays a certain degree of fibrin specificity. Its mechanism of action and potential therapeutic value remain to be established.

Ditazole activity and its interaction with urokinase on experimental thrombosis.

The effect of ditazole, a new antiaggregant oxazole derivative as well as its possible interaction with urokinase on the formation of electrically induced thrombus, was assayed in rabbits. The activity of ditazole in reducing thrombus weight was comparable to that of aspirin. In the ditazole- or aspirin-treated animals, the microscopical examination of the thrombus showed a reduction in the fibrin component, and well-isolated platelets not undergoing a viscous metamorphosis were present. Urokinase, administered in combination with these antiaggregant drugs, did not induce a further reduction in thrombus weight. However, this additional treatment did induce clearly visible lytic areas and histological modifications as observed with the antiaggregant drugs. These data suggest that the antiplatelet drug ditazole may be an effective antithrombotic agent in man and could facilitate the penetration of urokinase into the thrombus.