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The role of urokinase plasminogen activator (uPA) in idiopathic pulmonary fibrosis (IPF) remains unclear. uPA-generated plasmin has potent fibrogenic actions involving protease activated receptor-1 (PAR-1) and interleukin-6 (IL-6). Here we characterize uPA distribution or levels in lung tissue and sera from IPF patients to establish the mechanism of its fibrogenic actions on lung fibroblasts (LFs). uPA immunoreactivity was detected in regions of fibrosis including fibroblasts of lung tissue from IPF patients (n = 7). Serum uPA levels and activity were also higher in IPF patients (n = 18) than controls (n = 18) (P < 0.05), being negatively correlated with lung function as measured by forced vital capacity (FVC) %predicted (P < 0.05). The culture supernatants of LFs from IPF patients, as compared to controls, showed an increase in plasmin activity after plasminogen incubation (5-15 μg/mL), corresponding with increased levels of uPA and IL-6 (n = 5-6, P < 0.05). Plasminogen-induced increases in plasmin activity and IL-6 levels were attenuated by reducing uPA and/or PAR-1 expression by RNAi. Plasmin(ogen)-induced mitogenesis was also attenuated by targeting uPA, PAR-1 or IL-6. Our data shows uPA is formed in active regions of fibrosis in IPF lung and contributes to LF plasmin generation, IL-6 production and proliferation. Urokinase is a potential target for the treatment of lung fibrosis., Competing Interests: The authors declare no competing financial interests.

Plasminogen and plasminogen activators play important roles in liver regeneration. Previously, we found that plasminogen potentiates hepatocyte proliferation in the primary culture of rat hepatocytes. Here, we examined how exogenous plasminogen affects the downstream events leading to cell proliferation. The addition of plasminogen to hepatocytes increased urokinase-type plasminogen activator (uPA) activity, but did not affect matrix metalloproteinase (MMP)-9 or MMP-2 activities. To increase uPA activity, plasminogen was required to bind the hepatocyte surface through the lysine-binding site of plasminogen molecule, but neither uPA mRNA nor uPA receptor (uPAR) mRNA was affected by the exogenous plasminogen. In addition, treatment of hepatocytes with an uPA inhibitor, p-aminobenzamidine, inhibited the plasminogen-induced and even EGF-induced hepatocyte proliferation. These results suggest that plasminogen-related control of hepatocyte proliferation is exerted topically by producing a hyperfibrinolytic state on the cellular surface involving the activation of uPA.

The aim of this study was to investigate the effects of different doses of exogenous recombinant human tissue plasminogen activator (rt-PA) on the endogenous cerebral plasminogen-plasmin system in focal ischemia in rats. Ischemia was induced using the suture model. Each group of rats (n = 6) received either treatment (0.9, 9 or 18 mg rt-PA/kg body weight) or saline (control group) at the end of ischemia; a sham-operated group was added. The activity of the plasminogen activators was measured by casein-dependent plasminogen zymography. In the cortex urokinase (u-PA) rose from sham (no ischemia), 91 +/- 7% to ischemia, 176 +/- 10% (P < 0.005). Increasing rt-PA doses led to further significant (P < 0.001) cortical u-PA activation which was maximal at 18 mg: 249 +/- 13%. An extreme increase in the u-PA activity was observed in the basal ganglia to 1019 +/- 22% (P < 0.001). This increase was further aggravated by higher rt-PA doses (18 mg, 1236 +/- 15%; P < 0.001). The t-PA level did not change I3R24 during (3 h ischemia followed by reperfusion for 24 h); however, during low and moderate doses of rt-PA, endogenous t-PA was reduced. In conclusion, while ischemia leads to a significant increase in u-PA, mainly in the basal ganglia, t-PA is not altered. Increasing doses of rt-PA lead to a further elevation of u-PA. Thus, u-PA seems to play a major role in the endogenous plasminogen activator system following focal cerebral ischemia.

The carboxypeptidase, TAFIa or CPU, is known to prolong plasma clot lysis by tissue plasminogen activator (tPA) and to have a role in thrombus stability in vivo. This current study examined lysis by urokinase (uPA) and single chain urokinase (scuPA) in addition to tPA. Further, we investigated the role of TAFIa in a model thrombus system, in which thrombi are formed under conditions of flow. We show that human thrombi, formed in vivo, and model thrombi both contain TAFI. No effect of thrombus TAFIa was observed in thrombus lysis assays, except when thrombi were bathed in plasma, in which case addition of potato tuber carboxypeptidase inhibitor (CPI) resulted in doubling of the rate of lysis. TAFIa inhibited lysis of model thrombi and plasma clots by uPA, scuPA in addition to lysis by tPA. The effect of TAFIa was more evident at high concentrations of plasminogen activator such as those used in thrombolytic therapy. Addition of plasminogen increased lysis and, in its presence, the enhancement by CPI was smaller. Thus the action of TAFIa could be partially overcome by plasminogen, whether lysis was by tPA, uPA or scuPA. This is consistent with TAFIa exerting its effect primarily through modifying the binding of plasminogen to fibrin and to a lesser extent through modification of the binding of tPA to fibrin.

The accumulation of fibrillar amyloid-beta protein (A beta) in cerebral blood vessels, a condition known as cerebral amyloid angiopathy (CAA), is a key pathological feature of Alzheimer's disease and certain related disorders and is intimately associated with cerebrovascular cell death both in vivo and in vitro. Moreover, severe CAA leads to loss of vessel wall integrity and cerebral hemorrhage. Although the basis for these latter pathological consequences in CAA remains unresolved alterations in local proteolytic mechanisms may be involved. Here we show that pathogenic forms of A beta stimulate the expression of plasminogen activator activity in cultured human cerebrovascular smooth muscle (HCSM) cells, an in vitro model of CAA. RNase protection assays and plasminogen zymography showed that urokinase-type plasminogen activator (uPA) was responsible for this activity. There was preferential accumulation of uPA on the HCSM cell surface that was mediated through a concomitant increase in expression of the uPA receptor. In the presence of plasminogen there was robust degradation of A beta that was added to the HCSM cells resulting in restoration of cell viability. This suggests that increased expression of uPA may initially serve as a protective mechanism leading to localized degradation and clearance of the pathogenic stimulus A beta. On the other hand, chronic expression of uPA and plasminogen activation led to a profound loss of HCSM cell attachment. This suggests that a similar prolonged effect in vivo in the cerebral vessel wall may contribute to loss of integrity and cerebral hemorrhage in CAA.

Amyloid-beta (Abeta) appears central to Alzheimer's disease (AD), aggregates spontaneously, and is neurotoxic to neurons in vitro. Recently, several groups reported a familial AD locus on chromosome 10. Here, we note that urokinase-type plasminogen activator (uPA) is located within this locus. Previously, we reported that uPA and its functional homolog, tissue-type plasminogen activator, are induced by Abeta treatment of neurons in vitro as well as in a mouse model of Abeta accumulation in vivo. Moreover, the target of plasminogen activators, plasmin, degraded nonaggregated and aggregated Abeta and modulated Abeta toxicity and deposition. Here, we have evaluated the effects of uPA and plasminogen on Abeta fibril formation and neurotoxicity. We report that the combination of uPA and plasminogen, but neither alone, inhibits Abeta toxicity, reduces Abeta deposition in vitro, and inhibits Abeta fibrillogenesis. We interpret these observations as suggesting that uPA represents a possible candidate gene for the chromosome 10 familial AD locus., (Copyright 2002 Wiley-Liss, Inc.)

Purpose: To investigate the toxicity of intravitreal plasminogen, urokinase, and their combination, and to evaluate their efficacy in the production of posterior vitreous detachment (PVD) in the rabbit eye., Methods: Fifty-six albino New Zealand rabbits were examined before and after injection using the indirect ophthalmoscope, slit-lamp biomicroscopy, and electroretinography. Various concentrations of urokinase or recombinant plasminogen or a combination were injected intravitreally into the right eyes of four rabbits for each concentration. The left eyes of the animals served as controls and received 0.1 mL balanced salt solution. Group 1 was injected with pure urokinase (1,000, 5,000, or 10,000 IU); Group 2 with recombinant plasminogen (0.1, 0.4, 1.0, 2.0, 4.0, 8.0, or 16.0 caseinolytic units [CU]); and Group 3 with a combination of 1,000 IU urokinase (highest nontoxic dose) and nontoxic concentrations of plasminogen (0.1, 0.4, 1.0, or 2.0 CU). The animals were killed and the eyes enucleated 15 days after injection. Electron and light microscopy were performed., Results: A concentration of 1,000 IU of urokinase was found to be nontoxic to the retina. Plasminogen concentrations of 2.0 CU or less did not produce retinal toxicity, whereas 4.0, 8.0, and 16.0 CU of plasminogen caused minimal-to-severe inflammatory response in the vitreous without histologic or electroretinographic changes. Neither plasminogen nor urokinase alone was successful in producing PVD. The combination of 1,000 IU of urokinase and 1.0 to 2.0 CU of plasminogen was effective without causing retinal toxicity., Conclusion: Posterior vitreous detachment can be produced in the rabbit eye using a combination of plasminogen and urokinase.

Urokinase-type plasminogen activator (uPA) activation of plasminogen is an important mediator of cell migration in many cell types. In the developing avian heart, uPA has been implicated as a mediator of atrioventricular (AV) cushion cell migration; however, the role of the plasminogen/plasmin system has not been examined. The purpose of this study was to test the hypothesis that uPA conversion of plasminogen to plasmin mediates AV cushion cell migration in vitro. Stage 17/18 chicken atrioventricular tissue lysates converted plasminogen into plasmin through uPA activity but no tissue-type plasminogen activator activity was detected. Zymograms on living cultured AV explants also activated plasminogen producing plasmin that degraded extracellular protein. The migratory capacity of cushion cells was assessed in the presence or absence of various test reagents known to alter the plasminogen/plasmin system. Addition of either human or chicken plasminogen or aprotinin (an inhibitor of plasmin) had no effect on cell migration. However, an anti-catalytic uPA antibody that blocked AV uPA activity, significantly decreased cell migration at all concentrations tested. These results showed that uPA mediated a portion of cushion cell migration in vitro. Although AV segments activated plasminogen and degraded extracellular proteins, uPA's functional role in cushion cell migration did not involve the plasminogen/plasmin system., (Copyright 1999 Wiley-Liss, Inc.)

Because hepatocyte growth factor (HGF) is a potent mitogen for normal human exocrine pancreas cells (NPCs) in vitro, we have analyzed the expression of HGF and its receptor, Met, in NPC and pancreas cancer cells and studied its effects in vitro. Using immunohistochemistry, Northern blotting, and reverse transcription-polymerase chain reaction, we examined the expression of HGF and Met in normal pancreas and pancreas cancer. Scatter assays, wound-healing assays, and migration through transwell filters were used to study HGF-stimulated motility of IMIM-PC-2 cancer cells. In tumors, HGF is mainly detected in stromal cells, whereas Met is overexpressed in cancer cells with an unpolarized distribution. In vitro, HGF stimulates motogenesis but not proliferation in cancer cells. Cell motility is accompanied by a rapid decrease in the cytoskeleton-bound E-cadherin, an acceleration of cellular adhesion to the substrate, an up-regulation of urokinase plasminogen activator (u-PA) RNA and protein, and a change in the solubility and proteolysis of the u-PA receptor. Cell motility is significantly reduced by inhibitors of u-PA proteolytic activity such as antibodies neutralizing u-PA activity, plasminogen activator inhibitor 1, and amiloride. These results show that a paracrine loop of HGF activation may participate in the development or progression of pancreas cancer. In vitro, the HGF-stimulated motogenesis of pancreas cancer cells involves the activation of the u-PA/u-PA receptor proteolytic system, suggesting its role in the invasive stages of tumor progression.

Staphylococcus aureus commonly causes bovine mastitis, but bovine strains, unlike human isolates of S. aureus, do not produce the bacterial plasminogen activator, staphylokinase. By use of bovine mammary epithelial and myoepithelial cell lines, it was found that bovine S. aureus M60 and its culture filtrates induce a 3- to 10-fold increase in urokinase-type plasminogen activator activity in mammary cell-conditioned media and cellular lysates. Furthermore, transcytosis of S. aureus M60 across a mammary epithelial cell monolayer was significantly enhanced by the addition of bovine plasminogen and inhibited by aprotinin. These findings provide evidence that S. aureus M60 can trigger superactivation of host plasminogen activator production and may then utilize the plasminogen activator-plasmin(ogen) system to facilitate tissue invasion without producing staphylokinase.

Objective: The purpose of this study was to compare the fibrinolytic effect of urokinase (UK) with that of tissue plasminogen activator (tPA) on subarachnoid clots and to investigate the effect of lysine-plasminogen (Lys-plg) on fibrinolytic therapy., Methods: We measured subarachnoid clot lysis rate chronologically for 24 hours during continuous intrathecal irrigation with tPA or UK alone in a canine subarachnoid hemorrhage model. Also, we measured the clot lysis rate for 24 hours during continuous irrigation with UK and Lys-plg or with UK after preadministration of Lys-plg in the same model., Results: The rate of clot lysis caused by UK and tPA dose-dependently increased up to 3,000 IU/ml and 125,000 IU/ml, respectively. With the same molar concentrations, the lytic effect of tPA was significantly higher than that of UK. The slope of the dose-response curve, however, was almost the same with both agents, and the slope of the dose-response curve of UK and tPA for a 24-hour irrigation was 25 and 23% per log 10 concentration, respectively. On the other hand, clot lysis with the combined use of UK and Lys-plg was higher than that with the single use of UK. Particularly, the preadministration of Lys-plg markedly enhanced clot lysis by UK at the early stage of irrigation., Conclusion: Subarachnoid clot lysis can be accelerated by administration of Lys-plg before UK administration and by use of high concentrations of UK or tPA.

Two sandwich enzyme-linked immunosorbent assays (ELISA) have been developed for quantitation of bovine milk plasminogen and plasmin. The assays used two monoclonal antibodies, one specific for plasminogen and the other specific for plasminogen plus plasmin. Plasmin concentration was obtained by subtracting the first concentration from the second. The assays were sensitive (linear range, 5-75 ng/ml), repeatable (CV, 8 and 5% for plasmin and plasminogen titration respectively), specific (no cross reactivity with the major milk proteins) and directly applicable to skimmed milk with no particular pretreatment of the sample. However, ELISA did not permit complete titration of milk plasminogen and plasmin (only 60-90% could be quantified). This lack of accuracy was due to casein interfering with the plasmin-plasminogen titration by ELISA. Results obtained with ELISA or an enzymic technique on 20 milk samples collected from individual cows milked throughout pregnancy showed that the ELISA was particularly suitable for analysis of late lactation or mastitic milk where proteinase inhibitors interfered with enzymic quantitation.

Proper migration and differentiation of neural crest (NC) cells are required for normal development of craniofacial structures, heart and great vessels, sensory and autonomic nervous systems, and other organs with vertebrate embryos. Serine-protease inhibitors reduce NC cell migration in vitro, suggesting the extracellular proteases are important mediators of NC cell migration. While plasminogen activator activity levels are high in NC cells relative to other embryonic tissue, its ability to regulate NC cell migration has not been specifically tested in vivo or in vitro through its ability to convert plasminogen to plasmin. Using a transfilter migration assay, NC cell migration was measured in the presence or absence of plasminogen. Our results showed that plasminogen significantly enhanced NC cell migration. This increase could not be attributed to differences in initial NC cell attachment or cytotoxicity and did not require a chemotactic gradient. The plasminogen-enhanced NC cell migration was blocked by aprotinin (a plasmin inhibitor) and was mimicked by the direct addition of plasmin to the NC cells, indicating that the plasminogen effect was mediated through plasmin generation. Furthermore, anticatalytic-uPA antibody blocked the plasminogen-enhanced NC cell migration showing that NC cell-associated uPA activity was required for this effect. Finally, decreasing NC-uPA activity by treating cells with transforming growth factor-Beta, also blocked the plasminogen-dependent increase in cell migration. These data show that in vitro, NC cell migration is regulated by NC-associated uPA activity suggesting that growth factor-regulation of this activity may play a major role in regulating NC cell migratory capacity in vivo.

Borrelia burgdorferi is transmitted by infected ticks and causes Lyme disease. To infect distant organ sites, B. burgdorferi spirochetes must disseminate from the site of the tick bite. During dissemination from the dermal tissue, they breach tissue barriers, probably by proteolysis. The previous findings that spirochetes bind serum-derived plasminogen and that plasmin favors spirochetal invasiveness and infectivity suggested a role for plasmin in the pathogenicity of B. burgdorferi. Binding of plasminogen to spirochetes and activation into plasmin is favored in a microenvironment that is rich in plasminogen and plasminogen activators. Plasminogen is abundant in plasma and interstitial fluids, and it is increased in inflammatory exudates. Since B. burgdorferi does not express endogenous plasminogen activators, the conversion of spirochete-bound plasminogen depends on host-derived plasminogen activators. In this report, we show that both intact B. burgdorferi organisms and its recombinant outer surface lipoprotein A induce human monocytes to express and secrete urokinase-type plasminogen activator in its zymogen form (pro-uPA). Moreover, we demonstrate that the presence of B. burgdorferi accelerates the interaction between (pro-)uPA and plasmin(ogen), leading to spirochete-bound plasmin. In a pro-uPA-serum mixture, spirochete-bound plasmin activity is generated. Taken together, the data suggest that B. burgdorferi may induce pro-uPA in a monocyte-containing inflammatory site and that the spirochetal surface provides an appropriate milieu for subsequent interactions between (pro-)uPA and plasmin(ogen), which result in spirochete-bound plasmin even in the presence of inhibitors for plasminogen activators and plasmin.

These studies were conducted to examine the lytic efficacy of recombinant urokinase (r-UK) and pro-urokinase (r-proUK) in the presence and absence of truncated forms of plasminogen. Due to differences in their structures, these modified proteins are more readily activated to plasmin than the circulating form of plasminogen. Use of such modified substrates for plasminogen activators may improve the clinical outcome in patients treated for a variety of thrombotic diseases. Lys-plasminogen (46 units) or mini-plasminogen (in units of equivalent chromogenic activity), in conjunction with r-UK (7,500 units), were administered in the absence of heparin to dogs (9-11 kg) in which a radiolabelled thrombus was formed in a femoral artery. Fibrinolysis was measured as a loss of radioactivity from the clot. After intra-arterial administration of the agents, clot lysis was 48 +/- 8%, 50 +/- 9% and 75 +/- 2% in the presence of r-UK + vehicle, r-UK + lys-plasminogen, and r-UK + mini-plasminogen, respectively. When these treatment groups were examined in the presence of heparin (500 units + 350 units/hour) in a second study, r-UK (2,000 units) produced clot lysis of 54 +/- 3%; addition of lys- or mini-plasminogen to the regimen resulted in lysis of 62 +/- 9% and 46 +/- 10%, respectively. A third phase of the study examined r-proUK (1,000 units) with heparin; in this case, lysis was 51 +/- 9% in the presence of vehicle, but 55 +/- 17% and 10 +/- 5% when lys- and mini-plasminogen were administered, respectively. Flow restoration, measured in the femoral artery in each experiment, generally paralleled the lytic profile. The results indicate that supplementation with mini-plasminogen is only useful when added to a lytic regimen in the absence of heparin, and that lys-plasminogen, in conjunction with either of the lytic agents, does not improve clot lysis in this canine model.

Background: Intracranial bleeding is the most catastrophic potential complication of treatment with thrombolytic agents. To identify potential factors that may contribute to this problem, we characterized elaboration by human brain endothelial cells of plasminogen activator inhibitor-1 (PAI-1) and measured PAI-1 mRNA levels., Methods and Results: When human cerebral microvascular endothelial cells (HCMEC), pial arterial endothelial cells, and middle meningeal arterial endothelial cells were exposed to 10 to 1000 ng/mL recombinant tissue-type plasminogen activator (RTPA), urokinase-type plasminogen activator (UPA), or streptokinase/ plasminogen (37 U streptokinase plus 2 mumol/L plasminogen) for 24 hours, they exhibited concentration-dependent decreases in elaboration of PAI-1 of 65 +/- 3%, 48 +/- 3%, and 59 +/- 8%. UPA and streptokinase/plasminogen elicited decreases of 33 +/- 8% and 35 +/- 4%, respectively, that were specific with respect to the protease agonists as to total protein synthesis and cell type; ie, neither human umbilical vein endothelial cells nor cerebral pericytes exhibited inhibition of PAI-1 elaboration. No decrease in HCMEC PAI-1 elaboration was induced by coagulation factor XB (10 nmol/L). A 2.7 +/- 0.5-fold increase was induced by alpha-thrombin (10 nmol/L). PAI-1 secretion from HCMEC decreased within 4 hours of exposure to 100 ng/mL RTPA. In HCMEC exposed to RTPA for 8 hours, PAI-1 mRNA decreased from 176 +/- 20 to 43 +/- 2.2 pg/microgram RNA., Conclusions: These results indicate that brain endothelial cells exposed to RTPA exhibit paradoxically diminished elaboration of PAI-1. This property may render brain vasculature vulnerable to attack by serine proteases, thereby predisposing to injury and initiating an underlying subsequent intracerebral hemorrhage in patients given plasminogen activators for treatment of coronary thrombosis.

Urokinase-type plasminogen activator (u-PA) was found to induce monocyte adhesion through a u-PA receptor (u-PAR)-mediated cAMP-dependent signal transduction pathway (J. Biol. Chem. 270, 30282-30285, 1995). In the present study, the effects of kininogen, kallikrein, factor XII, and plasminogen on u-PA-induced monocyte adhesion were examined since these proteins are abundant in plasma and closely related to u-PA in fibrinolysis and inflammation. Monocyte adhesion to a standard plastic surface by u-PA was shown to be inhibited by the activated, two-chain forms of kininogen (HKa) and kallikrein. The latter occurred only at higher, though physiological, concentrations and was dependent on its catalytic activity. Monocyte adhesion was promoted by factor XII and plasminogen via a noncatalytic mechanism. The findings indicated that u-PA-induced monocyte adhesion was downregulated by HKa and kallikrein and upregulated by factor XII and plasminogen at physiological concentrations. Therefore, these contact system proteins may be important modulators of u-PA-induced monocyte adhesion, a process which is involved in many pathophysiological events.

Both in cell culture and in vivo, keratinocytes that are migrating in response to a wound express enhanced levels of both urokinase-type plasminogen activator (uPA) and the uPA cell surface receptor (uPA-R). To explore the mechanism of this up-regulation, keratinocyte cultures were treated proir to wounding with a variety of metabolic and growth factor inhibitors in order to evaluate their effect on uPA and uPA-R expression. Actinomycin D and cycloheximide inhibited the up-regulation of both uPA and uPA-R, as determined by immunohistochemistry, indicating that RNA and protein syntheses are required for their induction in migrating keratinocytes. Neither removal of protein growth factors from the medium nor addition of inhibitory antibodies to a number of growth factors depressed uPA or uPA-R induction; these findings suggest that a variety of exogenous or endogenous growth factors [i.e., basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), amphiregulin, and tumor necrosis factor-alpha (TNF-alpha) do not have a critical role in the induction of uPA or uPA-R. In contrast, when protein kinase C (PKC) was either down-regulated with bryostatin 5 or inhibited with Ro31-8220 or staurosporine, the expression of both uPA and uPA-R was greatly decreased in migrating keratinocytes. Furthermore, pharmacologic activation of PKC enhanced uPA levels in non-wounded cultures. These data suggest that the enhanced expression of uPA and uPA-R in migrating keratinocytes is mediated by selective activation of PKC in these cells, perhaps secondary to alterations in the cytoskeleton induced by wounding. To test the requirement for uPA during keratinocyte migration in vitro, the extent of migration was quantified in the presence and absence of a variety of inhibitors in the wounded culture model. Migration was not altered by actinomycin D, cycloheximide, any of the above growth factor inhibitors, anti-uPA antibodies, a variety of inhibitors of uPA or plasmin enzymatic activity, or exogenous uPA. The independence of keratinocyte migration in vitro from uPA was further suggested by experiments which combined the phagokinetic assay of migration and the zymographic assay for pericellular uPA activity; no relationship was observed between pericellular uPA activity and the motility of individual cells.

We introduce a new fibrin plate assay performed in microtiter plates. By means of spectroscopic studies we optimized the structure of the fibrin gel and then used the optimized fibrin gel to determine plasminogen activator activity. Plasminogen activator solutions were applied on top of the fibrin gel, and the absorbance of the gel was recorded at 405 nm. After incubation for 17 h at 25 degrees C, the absorbance was measured again. The difference in absorbance was proportional to the concentration of plasminogen activator, such that the dose-response curves were linear when the difference in absorbance was plotted as a function of the logarithmic concentration of plasminogen activator. We assayed both tissue-type and urokinase-type plasminogen activator activity. The intraassay CV was < 4.7% (n = 20); the interassay CV was < 3.1% (n = 15). Using the optimized procedure, we modified the assay for determination of plasma-coagulum lysis time in human plasma. We established a reference interval for lysis time in apparently healthy subjects of 75 to 201 ks. Patients with deep vein thrombosis showed significantly (P = 0.013) higher values.

Clots formed from platelet rich plasma were found to be lysed more readily by low concentrations of pro-urokinase (pro-UK) than clots formed from platelet poor plasma. This was not a non-specific effect since the reverse occurred with tissue plasminogen activator. A mechanical explanation due to platelet-mediated clot retraction was excluded by experiments in which retraction was inhibited with cytochalasin B. Therefore, a platelet-mediated enzymatic mechanism was postulated to explain the promotion of fibrinolysis. Casein autography of isolated platelets revealed a approximately 90 kDa band of activity which comigrated with plasma prekallikrein (PK)/kallikrein, a known activator of pro-UK. Furthermore, treatment of platelets with plasma PK activator (PPA), consisting essentially of factor XIIa, induced activation of pro-UK and of chromogenic substrate for kallikrein (S-2302). This activity corresponded to approximately 40-200 pM kallikrein per 10(8) washed and gel filtered platelets per ml. The activation of pro-UK by PPA-pretreated platelets was dose-dependent and inhibited by soybean trypsin inhibitor but not by bdellin, a specific inhibitor of plasmin, nor by the corn inhibitor of factor XIIa. Kinetic analysis of pro-UK activation by kallikrein showed promotion of the reaction by platelets. The KM of the reaction was reduced by platelets by approximately 7-fold, while the kcat was essentially unchanged. In conclusion, PK was shown to be tightly associated with platelets where it can be activated by factor XIIa during clotting. The activation of pro-UK by platelet-bound kallikrein provides an explanation for the observed platelet mediated promotion of pro-UK-induced clot lysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Purpose: A relative deficiency of plasminogen within the thrombus may be the rate limiting factor in clot lysis., Methods: To investigate this hypothesis, we used an in vitro perfusion system and expanded polytetrafluoroethylene graft segments filled with radiolabeled human thrombus. Three groups of five perfusions were compared: (1) urokinase infusion (333 IU/min) into clots laced with buffer, (2) urokinase infusion (333 IU/min) into clots laced with plasminogen (44 CU), and (3) control, D5W infusion into clots laced with buffer. Two end points were measured over time: the amount of lysed thrombus and the flow through the graft., Results: Urokinase infusion resulted in augmented flow through the graft when compared with control (p < 0.05). Lacing with plasminogen resulted in more rapid restoration of flow when compared with urokinase infusion alone (p < 0.05). Similarly, the rate of clot dissolution was significantly greater in plasminogen-laced thrombi (p < 0.05) when compared with the control and urokinase groups. Embolization of particles of thrombus was uniformly observed in the urokinase group, resulting in a temporary decrease in flow through the thrombosed graft. This event characteristically occurred after 60 minutes of infusion but was never seen in the urokinase/plasminogen treatment group., Conclusions: These results suggest that plasminogen supplementation of urokinase thrombolysis may result in significant clinical benefits with respect to the rate of clot lysis and the uniformity of clot dissolution with a lower likelihood of secondary embolization.

Murine leukemic cells from the WEHI-3B line, present in the cell surface a latent collagenase activity which is activated proteolytically. In this paper we show that this enzyme is activated by plasmin generated by the activity of a urokinase-like plasminogen activator (u-PA) also present in the surface of these cells. Using a reverse fibrin autography method we found that u-PA is the major proteolytic activity present in the cell membranes. This fact suggests that u-PA could represent a normal activating system for this collagenase.

We have determined whether macrophage derived-foam cells, a prominent component of the atherosclerotic lesion, express more urokinase-type plasminogen activator (uPA) and whether their ability to generate plasmin stimulates the release of matrix-bound growth factors. Steady state levels of uPA mRNA and both membrane and intracellular uPA activities were significantly increased in foam cells. When cultured on cell-derived matrices containing bound 125I-basic fibroblast growth factor (bFGF), both macrophage and foam cells released intact 125I-bFGF into their media. The release of 125I-bFGF by either cell was significantly enhanced in the presence of plasminogen. However, foam cells, which expressed more membrane uPA, released more 125I-bFGF than control cells. The release of matrix-bound bFGF was independent of heparanase activity, since neither macrophage nor foam cells degraded 35SO4-labeled heparan sulfate proteoglycans. In addition, media derived from foam cells cultured on cell-derived matrices in the presence of plasminogen had increased levels of transforming growth factor (TGF) beta activity as compared to cells grown in the absence of plasminogen. In contrast, plasminogen had no effect on TGF-beta activity recovered in the media of foam cells grown on plastic. Moreover, when macrophage were cultured on matrices containing bound 125I-TGF-beta, the release of labeled TGF-beta was increased in the presence of plasminogen. This is the first demonstration that foam cells can release two important growth regulators, bFGF and TGF-beta, from the extracellular matrix, and provides a mechanism by which macrophage and foam cells can stimulate atherosclerotic lesion development.

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)

A venous clot model in rats was used to compare the ability of locally infused thrombolytic agents to lyse clots. Urokinase and a combination of streptokinase and human plasminogen (Thrombolysin) proved to be equally effective in lysing clots. In comparison, streptokinase alone was not as effective a thrombolytic agent in this model.

Several tumor cells secrete significantly increased amounts of the plasminogen activator urokinase, a trypsinlike serine protease, whose biological function in tumor biology is unclear. In this study we report that cells of the human epidermal tumor cell line CCL 20.2 express about 80,000 high-affinity urokinase receptors per cell that bind active as well as diisopropylfluorophosphate-treated high-molecular-weight (HMW) urokinase. Low-molecular-weight (LMW) urokinase is not bound to the receptor. Occupation of these receptors by active HMW urokinase stimulates cell proliferation independently in the presence of plasminogen in the culture medium. LMW urokinase has again no effect on cell proliferation. Calculated on a molar basis, this effect is about 28% of that of epidermal growth factor. Active HMW urokinase might therefore provide an autocrine receptor-mediated growth-promoting mechanism for tumor cells similar to those described for other growth factors.

The role and effect of added lys-plasminogen (lys-PLG) on urokinase-induced thrombolysis in an in vitro biphasic system were investigated. The kinetics of lysis of whole blood thrombi was followed in perfusion mediums of normal plasma, PLG-deficient plasma and normal saline using a high and a low concentration of urokinase (UK). The lysis of standard whole blood thrombi in whole blood perfusion mediums to which had been added UK alone or UK plus lys-PLG was compared to whole blood thrombi enriched with lys-PLG by incorporation during thrombus formation or by adsorption during perfusion. In addition, the kinetics of lysis of PLG-deficient fibrin thrombi perfused in PLG-deficient plasma or normal saline was studied when lys-PLG had been added to the thrombus, to the perfusion medium or to both thrombus and medium. In PLG-deficient plasma from which plasmin inhibitors had not been removed, thrombolysis was minimal even at a high concentration of UK. This effect could be neutralized, and to some extent, regulated, by lys-PLG enrichment of the medium. Both PLG-incorporated and PLG-adsorbed whole blood thrombi gave initial and sustained acceleration of UK-induced lysis in comparison with standard nontreated thrombi. It is concluded that in a blood-thrombus biphasic thrombolytic system induced by UK, there is interaction between the phases, and that PLG in both phases influences thrombolysis.

The relevance of urokinase receptors to urokinase-mediated laminin degradation was investigated in cultured colon cancer. Six colon cancer cell lines degraded laminin in a plasminogen-dependent manner. The ability of the individual cell lines to cleave the glycoprotein correlated well (r2 = 0.9242) with the amount of urokinase recovered from the cell surface by a mild acid treatment. A radioreceptor assay indicated that colon cancer cells most active in degrading the laminin, possessed the largest number of urokinase receptors. Moreover, acid treatment which depletes the receptors of endogenous plasminogen activator augmented the specific binding of radioactive urokinase to the colon cancer cells by 12-200%. A cell line (HCT 116) which displayed 1.1 x 10(5) receptors/cell the majority of which were occupied with endogenous urokinase was selected and the effects of a urokinase receptor antagonist on laminin degradation determined. The peptide antagonist reduced laminin turnover by 60-80%. Morphological observations were consistent with these findings. Plasminogen-treated HCT 116 cells retracted from the culture dish and many cells were observed in the culture medium. This effect could be largely reversed by simultaneous treatment with the peptide antagonist. A poor correlation was found between laminin degradation and soluble urokinase (r2 = 0.1074). These data strongly argue for a role of the urokinase receptor in facilitating the action of the plasminogen activator in colon cancer.

Influence of heparin, chondroitin sulfate C and dextran sulfate (MW 3,500 and 7,000) on plasmin catalyzed conversion of single-chain (scu-PA) to two-chain (u-PA) urokinase-type plasminogen activator and generation of plasmin in mixtures of scu-PA and Glu-plasminogen (Glu-plg) was investigated. Conversion of scu-PA to u-PA catalyzed by plasmin was enhanced by chondroitin sulfate C and heparin, maximally by 10-fold and 3-fold, respectively. Sulfated polysaccharides potentiated generation of plasmin in mixtures of Glu-plg and scu-PA, as well as enhanced plasminogenolytic activity of u-PA. The possible role of these interactions is discussed.

Cellular migration is an essential component of invasive biological processes, many of which have been correlated with an increase in plasminogen activator production. Endothelial cell migration occurs in vivo during repair of vascular lesions and angiogenesis, and can be induced in vitro by wounding a confluent monolayer of cells. By combining the wounded monolayer model with a substrate overlay technique, we show that cells migrating from the edges of an experimental wound display an increase in urokinase-type plasminogen activator (uPA) activity, and that this activity reverts to background levels upon cessation of movement, when the wound has closed. Our results demonstrate a direct temporal relationship between endothelial cell migration and uPA activity, and suggest that induction of uPA activity is a component of the migratory process.

Study has been made of the influence of addition of human NH2 terminal glutamic acid plasminogen (Glu-Plg) or human NH2 terminal lysine plasminogen (Lys-Plg) to normal citrated plasma upon the rate of lysis of fully crosslinked plasma clots in the presence of single or two chain urokinase type plasminogen activator (scu-PA/tcu-PA) or tissue plasminogen activator (t-PA). The specificity of any thrombolytic property was evaluated by measurement of plasma fibrinogen levels. Lys-Plg added to a concentration of 20% of normal plasma plasminogen caused 5 to 6 fold increase in the extent of lysis observed at 6 hours by 100 units/ml of scu-PA and with a small increase in fibrinogenolysis. Glu-Plg added at 20% of normal level had no influence on thrombolysis but at 50% of normal caused increased thrombolysis with rapid depletion of plasma fibrinogen. An apparently synergistic effect of addition of tcu-PA on scu-PA activity was increased by addition of plasminogen (e.g. addition of 20% Lys-Plg increased the lysis rate 4 to 5 fold over the first hour equivalent to an increase of potency of approximately three to four fold). Addition of plasminogen up to double the normal plasma concentration was observed to have no influence on clot lysis in the presence of t-PA. Plasminogen potentiated the rate of lysis by scu-PA/t-PA synergic mixtures with an approximately 1.5 to 1.9 fold increase in potency. Potentiation occurred without increase in the depletion of plasma fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Urokinases of different molecular weights are now commerically available. An international collaborative study (eight laboratories) has been conducted to investigate the effect of type and concentration of plasminogen on the assay of two different urokinase preparations against the International Reference Preparation (IRP). Considerable inter-laboratory variation in relative potency estimation was found, and a small effect of plasminogen concentration, independent of type, was apparent.