Analysis of plasminogen activation by the plasmin-staphylokinase complex in plasma of alpha2-antiplasmin-deficient mice.

Staphylokinase (SAK) expresses plasminogen activator (PA) activity by forming a complex with plasmin; this PA activity is inhibited by alpha2-antiplasmin (alpha2-AP) in plasma. However, SAK's activity is protected against inhibition by alpha2-AP in the presence of fibrin because the plasmin-SAK complex binds to fibrin. In the present study, the interaction between SAK and murine plasminogen was investigated in the plasma of alpha2-AP-deficient (alpha2-AP-/-) mice or plasminogen-deficient (Plg-/-) mice. Although the human plasmin-SAK complex was formed in equimolar mixtures of plasmin and SAK, the murine plasmin-SAK complex was not formed. Human plasminogen was activated by the human plasmin-SAK complex, although equimolar mixtures of murine plasmin and SAK did not activate murine plasminogen. These findings suggest that SAK does not react with murine plasmin. However, the murine plasminogen was activated by the human plasmin-SAK complex, although this activation was approximately 100-fold weaker than human plasminogen. Human and wild-type mouse plasminogens were not activated by the human plasmin-SAK complex in their plasma. In alpha2-AP-/- mouse plasma, murine plasminogen was activated by the human plasmin-SAK complex. Human or murine plasminogen, which had been added to Plg-/- mouse plasma, was not activated by the human plasmin-SAK complex. However, plasma clot lysis by the human plasmin-SAK complex was observed in both human and murine plasma. These findings indicate that: (1) murine plasmin does not react with SAK, (2) human plasmin-SAK complex activates murine plasminogen, (3) this activation is inhibited by murine alpha2-AP, but (4) this activation is not inhibited by murine alpha2-AP in the presence of fibrin.     

Plasminogen enhances virulence of group A streptococci by streptokinase-dependent and streptokinase-independent mechanisms

Interactions between host plasminogen (Plg) and streptokinase (SK) secreted by group A streptococci (GAS) have been hypothesized to promote bacterial invasion of tissues. The virulence of GAS strain UMAA2616, after being subcutaneously inoculated into mice, was studied. Skin lesions and mortality were observed after inoculation of 7x106 cfu. Coadministration of human Plg with UMAA2616 markedly increased virulence. SK-deficient UMAA2616 (UMAA2616-SK(-)) was generated. Mean skin-lesion area and mortality, after bacterial inoculation (3x105 cfu), were significantly greater with UMAA2616 in the presence of human Plg than with UMAA2616-SK(-) in the presence of human Plg (P=.0001). Human Plg also enhanced UMAA2616-SK(-) virulence. Exogenous human Plg enhanced the virulence of MGAS166, a human clinical isolate. These findings suggest that SK-Plg interactions are an important determinant of GAS invasiveness in vivo and that both SK and host Plg activators appear to promote virulence of GAS by catalyzing plasmin formation.     

A new life-long hemorrhagic disorder due to excess plasminogen activator

A life-long bleeding disorder is described, characterized by hemorrhage occurring after surgery, injury, or dental extraction, and finally by spontaneous intracerebral bleeding. No abnormality of platelet function or plasma coagulation was demonstrable, but grossly enhanced overall fibrinolytic activity was present. The patient had, additionally, a hyperlipidemia with gross arterial atheroma and a family history of myocardial infarction but not of any hemorrhagic disorder. Laboratory studies led to the conclusion that the enhanced fibrinolysis was due to consistently greatly raised levels of a plasma plasminogen activator physically and immunologically related to that in human tissues and blood vessel endothelium. No deficiency of any known inhibitor of fibrinolysis was detected. Free plasmin was not detectable in functional assays but continuous intravascular plasmin generation clearly occurred as evidenced by presence of plasmin-alpha 2- antiplasmin complexes and of fibrin/fibrinogen-related antigens. Excessive production of plasminogen activator appeared to have occurred throughout life and to be independent of the hyperlipidemia. The pathologically increased fibrinolytic activity may have accounted for the complete absence of detectable thrombotic vascular occlusion at autopsy despite extensive arterial disease with severe narrowing of coronary and cerebral arteries.     

Regulation of the single-chain urokinase-urokinase receptor complex activity by plasminogen and fibrin: novel mechanism of fibrin specificity

Activation of plasminogen by urokinase plasminogen activator (uPA) plays important roles in several physiologic and pathologic conditions. Cells secrete uPA as a single-chain molecule (scuPA). scuPA can be activated by proteolytic cleavage to a 2-chain enzyme (tcuPA). scuPA is also activated when it binds to its receptor (uPAR). The mechanism by which the enzymatic activity of the scuPA/suPAR complex is regulated is only partially understood. We now report that the plasminogen activator activity of the scuPA/suPAR complex is inhibited by Glu- and Lys-plasminogen, but not by mini-plasminogen. In contrast, neither Glunor Lys-plasminogen inhibits the activation of plasminogen by 2-chain uPA. Inhibition of scuPA/suPAR activity was evident at a Glu-plasminogen concentration of approximately 100 nM, and at physiologic plasma concentrations inhibition was nearly complete. A plasminogen fragment containing kringles 1-3 inhibited the enzymatic activity of scuPA/suPAR with an inhibition constant (Ki) equal to 1.9 microM, increased the Michaelis constant (Km) of scuPA/suPAR from 18 nM to 49 nM, and decreased the catalytic constant (Kcat) approximately 3-fold from 0.035 sec(-1) to 0.011 sec(-1). Inhibition of scuPA/suPAR by plasminogen was completely abolished in the presence of fibrin clots. These studies provide insight into the regulation of uPA-mediated plasminogen activation and identify a novel mechanism for its fibrin specificity.     

Depressed plasma activity of plasminogen or alpha2 plasmin inhibitor is not due to consumption coagulopathy in septic patients with disseminated intravascular coagulation.

We have attempted to determine whether depressed plasma plasminogen and alpha2 plasmin inhibitor (or alpha2 antiplasmin) activity is, as a result of consumption coagulopathy, a specific finding of disseminated intravascular coagulation (DIC) in septic patients. The hemostatic parameters of 139 septic patients (68 with DIC and 71 without DIC) were analyzed. Among the group as a whole, plasma activities of plasminogen and alpha2 plasmin inhibitor were significantly depressed in septic patients with DIC relative to those without DIC (P < 0.01 and P < 0.05, respectively). Notably, a significant correlation was observed between plasma levels of albumin and plasminogen activity, as well as between plasma levels of albumin and alpha2 plasmin inhibitor activity both in septic patients with DIC and those without DIC. However, no significant correlation was observed between plasma levels of plasmin-alpha2 plasmin inhibitor complex (PIC) and plasminogen activity, nor between PIC and alpha2 plasmin inhibitor activity either in septic patients with DIC or those without DIC. We concluded that depressed activity of plasminogen or alpha2 plasmin inhibitor is not as a result of consumption coagulopathy, but rather a result of low synthetic function of the liver in septic patients with DIC.     

Regulation of streptokinase-human plasmin complex by the plasma proteinase inhibitors alpha 2-antiplasmin and alpha 2-macroglobulin is species specific and temperature dependent

Streptokinase-plasmin complex (SkPl) was prepared with human plasminogen. Regulation of SkPl and plasmin by the plasma proteinase inhibitors, alpha 2-antiplasmin (alpha 2AP) and alpha 2-macroglobulin (alpha 2M), was studied as a function of temperature in plasminogen- depleted human plasma, mouse plasma, and solutions of purified proteins. The reaction of plasmin with proteinase inhibitors in human plasma was complete. alpha 2AP was the predominant inhibitor. The fraction of alpha 2M-plasmin recovered was not affected significantly by incubation temperature. In contrast, the reaction of SkPl with human proteinase inhibitors was markedly temperature dependent. The apparent second-order rate constant for the reaction of SkPl with purified alpha 2AP at 37 degrees C (1.5 x 10(2) mol/L-1 s-1) was greater than 150-fold higher than the constant derived at 4 degrees C. In human plasma and in solutions containing mixtures of purified human proteins, alpha 2AP was the principal inhibitor of SkPl. Elevating the temperature enhanced the reaction of SkPl with alpha 2AP and alpha 2M comparably. Equivalent results were obtained when incubations were performed in platelet-rich plasma (PRP) or whole blood. In murine plasma, SkPl reacted readily with the proteinase inhibitors. The principal inhibitor of SkPl was alpha 2M. Maximum reaction between SkPl and murine alpha 2M was observed at 37 degrees C; however, significant reaction also occurred at 4 degrees C. alpha 2 AP was the predominant inhibitor of plasmin in mouse plasma. Reaction of alpha 2AP with SkPl in murine plasma was significant only after the alpha 2M was inactivated with methylamine. These results were not affected by platelets or whole blood cells. We conclude that the thrombolytic efficacy of streptokinase reflects not only the nature of the plasminogen activator complex but also the function of the proteinase inhibitors.     

Streptokinase-producing streptococci grown in human plasma acquire unregulated cell-associated plasmin activity.

Group A streptococci grown in the presence of human plasma generated plasmin from plasminogen and captured the functional enzyme to a specific cell-surface receptor. Bacteria-bound plasmin was not regulated by alpha 2-antiplasmin present in the medium. The ability of the bacteria to acquire cell-associated plasmin activity was dependent on both the presence of plasminogen in the culture medium and the production of a bacterial plasminogen activator, streptokinase. The ability of group A streptococci to produce a plasminogen activator and capture resulting plasmin in an unregulatable form could provide the organism with a mechanism for invasion of normal tissue barriers.     

Inhibition of renin by plasma linoleic acid

The authors have demonstrated previously that human plasma contains an inhibitor(s) of the enzymatic activity of renin. The purpose of this study is to identify the circulating renin inhibitor. Plasma initially was fractionated with preparative sephacryl S-300 chromatography. A fraction with low protein content inhibited the in vitro enzymatic activity of human renin by 76%. The inhibitory activity of this fraction was not altered by boiling and/or acidification. This fraction was applied to an affinity column, using purified mouse submaxillary renin as ligand. The inhibitor was recovered after elution with hypertonic NaCl. Linoleic acid, a previously identified renin inhibitor, was present in this fraction. The authors conclude that circulating linoleic acid inhibits renin. Conceivably, the overall activity of the renin-angiotensin-aldosterone cascade may be modified by alterations of plasma fatty acid concentrations.     

Regulation and secretion of plasminogen activators and their inhibitors in a human leukemic cell line (K562)

The secretion of tissue plasminogen activator (t-PA), urokinase (u-PA) and their inhibitors by the human leukemia cell line K562 was examined. K562 cells normally secrete both t-PA and u-PA in a ratio of 3:1. After addition of 10 or 1 ng/mL phorbol myristate acetate (PMA) to K562 cells, a marked decrease in enzymatic activity is observed in the medium. However, when t-PA antigen rather than activity is measured, an increased amount is found in the medium under these conditions. PMA also induces secretion of the two inhibitors of plasminogen activator: plasminogen activator inhibitor 1 (PAI-1) and plasminogen activator inhibitor 2 (PAI-2). This accounts for the decrease in total enzymatic activity under conditions when production of t-PA antigen is increased. A study of the time course of induction revealed that the synthesis of plasminogen activator occurred before that of its inhibitors. Low concentrations of PMA (0.1 ng/mL) induce t-PA antigen primarily and not the inhibitors. This results in an increase in total enzymatic activity, with 94% of the secreted activity being t-PA. Thus, the secretion of plasminogen activators and their inhibitors can be manipulated in certain leukemic cells by inducers such as PMA.     

Fibrin-dependent activation of plasminogen by a proteolytic digest of streptokinase

Among four enzymatic digests of streptokinase (SK), the smallest peptide with plasminogenolytic activity was in a tryptic digest; it had a molecular weight of 29,000. A complex of this peptide, SK29, and human plasminogen hydrolysed human fibrin, but a complex of native streptokinase and human plasminogen hydrolysed both human and bovine fibrin. The complex with SK29 caused amidolysis of the synthetic substrate S-2251 in the presence of human fibrin, but was inactive in the presence of human fibrinogen, bovine fibrinogen or bovine fibrin. Analysis of the amino terminal sequence of SK29 indicated that cleavage by trypsin was on the carboxyl side of lysine, the 59th amino acid of streptokinase. These results suggest that the conformational changes caused by human fibrin formation resulted in the generation of an active site of human plasminogen by SK29.     

Extracellular deoxyribonuclease made by group A Streptococcus assists pathogenesis by enhancing evasion of the innate immune response

Many pathogenic bacteria produce extracellular DNase, but the benefit of this enzymatic activity is not understood. For example, all strains of the human bacterial pathogen group A Streptococcus (GAS) produce at least one extracellular DNase, and most strains make several distinct enzymes. Despite six decades of study, it is not known whether production of DNase by GAS enhances virulence. To test the hypothesis that extracellular DNase is required for normal progression of GAS infection, we generated seven isogenic mutant strains in which the three chromosomal- and prophage-encoded DNases made by a contemporary serotype M1 GAS strain were inactivated. Compared to the wild-type parental strain, the isogenic triple-mutant strain was significantly less virulent in two mouse models of invasive infection. The triple-mutant strain was cleared from the skin injection site significantly faster than the wild-type strain. Preferential clearance of the mutant strain was related to the differential extracellular killing of the mutant and wild-type strains, possibly through degradation of neutrophil extracellular traps, innate immune structures composed of chromatin and granule proteins. The triple-mutant strain was also significantly compromised in its ability to cause experimental pharyngeal disease in cynomolgus macaques. Comparative analysis of the seven DNase mutant strains strongly suggested that the prophage-encoded SdaD2 enzyme is the major DNase that contributes to virulence in this clone. We conclude that extracellular DNase activity made by GAS contributes to disease progression, thereby resolving a long-standing question in bacterial pathogenesis research.     

Effects of fibrin and α2-antiplasmin on plasminogen activation by staphylokinase

Staphylokinase obtains plasminogen activating activity by forming a complex with plasminogen. Although the enzymatic activity of staphylokinase is enhanced by fibrin, how fibrin enhances enzymatic activity has not been determined yet. The effects of fibrin, or fibrinogen fragments, on the activation of plasminogen by staphylokinase was investigated using CNBr-digested fibrinogen fragments (FCB-2 and FCB-5) and plasmin-degraded cross-linked fibrin fragments ((DD)E complex, DD fragments and E fragments). Kinetic analysis of the activity of staphylokinase revealed that its plasminogen activating activity, which was expressed as kcat/Km, was enhanced by FCB-2 (10-fold) and FCB-5 (5-fold). These fibrin fragments caused 38-, 30-, and 8.5-fold increases in activity for the DD fragment, (DD)E complex and E fragment, respectively. Although α₂-antiplasmin inhibited the activation of plasminogen by staphylokinase, FCB-2 abolished its inhibitory effects, and the plasminogen activating activity of staphylokinase was restored. The inhibitory effects of a₂-antiplasmin on the activation of mini-plasminogen by staphylokinase were less than for Glu-or Lys-plasminogen, and the inhibitory effect of α₂-antiplasmin was not altered by fibrin or EACA. These findings indicate that the staphylokinase/plasmin-(ogen) complex reacts with fibrin even in the presence of α₂-antiplasmin, and efficient plasminogen activation takes place on the surface of fibrin. © 1996 Wiley-Liss, Inc.     

Correlations between fibrinolytic function and acute myocardial infarction

The diurnal variation of tissue plasminogen activator and plasminogen activator inhibitor--2 important factors in regulation of fibrinolysis--is well established, but its clinical implications are not yet fully understood. Fibrinolytic function was measured in a group of 133 patients less than 45 years of age, 3 months after they presented with myocardial infarction, and they were compared with an equal group of carefully matched control subjects. A sub-sample of 71 patients and 50 control subjects was examined 3 years later. Tissue plasminogen activator inhibitor plasma levels were higher in patients versus control subjects at 3 months and at 3 years after myocardial infarction, with a high degree of correlation between the 2 determinations (r = 0.87). Of all hemostatic parameters studied, only low tissue plasminogen activator activity and increased tissue plasminogen activator inhibitor levels were significantly different between patients with and without recurrent infarction during the 3-year period. In those patients with a second infarction greater than 3 years later, however, tissue plasminogen activator inhibitor concentration was not significantly related to reinfarction.     

Plasminogen-activator inhibitor type 1 is a potent natural inhibitor of extracellular matrix degradation by fibrosarcoma and colon carcinoma cells.

We have analyzed the role of plasminogen-activator inhibitor type 1 (PAI-1) in the regulation of tumor cell-mediated extracellular matrix degradation. Immunocytochemical analysis revealed PAI-1 associated with microgranular and fibrillar material of the extracellular matrix and demonstrated the presence of PAI-1 as a cell surface-associated antigen. Transforming growth factor beta significantly reduced matrix degradation mediated by HT-1080 human fibrosarcoma cells. This inhibition was correlated with an increase in PAI-1 antigen expression, whereas urinary-type plasminogen activator (u-PA) secretion was unaffected. In this experimental system, PAI-1 regulated extracellular matrix breakdown, as added PAI-1 inhibited matrix solubilization, whereas monoclonal antibodies to PAI-1 increased it. A cell line (LPAI) producing high levels of biologically active PAI-1 was established by transfection of a human PAI-1 cDNA clone into mouse L cells. Coculture experiments demonstrated that LPAI cells prevented matrix degradation by Lu-PA cells (L cells expressing high levels of u-PA) or Co-115 human colon carcinoma cells (expressing tissue-type plasminogen activator). These results indicate that PAI-1 may play a critical role in the regulation of extracellular matrix degradation during tumor cell invasion.     

Pro-urokinase: a study of its stability in plasma and of a mechanism for its selective fibrinolytic effect

Highly purified pro-urokinase (pro-UK) or single-chain urokinase-type plasminogen activator (scu-PA) was treated with diisopropylfluorophosphate (1 mmol/L) to eliminate traces of two-chain UK activity. This preparation was found to retain a low activity against a chromogenic substrate (S2444), equivalent to 0.1% to 0.5% of the activity of its plasmin-activated derivative. Evidence is presented that the intrinsic activity of pro-UK (scu-PA) was sufficient to activate plasminogen on a fibrin plate or in buffer and was far more reactive against Lys-plasminogen than against Glu-plasminogen. The relative resistance of Glu-plasminogen to activation was overcome by the addition of lysine (25 mmol/L) to the reaction mixture. By contrast, in plasma, pro-UK (scu-PA) was stable and nonreactive for greater than 72 hours when incubated (37 degrees C). Pro-UK (scu-PA) did not form sodium dodecyl sulfate-stable inhibitor complexes, whereas complexation occurred rapidly with UK. Only at high concentrations of pro-UK (scu-PA) (greater than or equal to 250 IU/mL) did plasminogen activation in plasma occur. The relative inertness of pro-UK (scu-PA) in plasma, in contrast to its low-grade enzymatic activity in buffer, was attributed to the effect of inhibitors. The addition of EDTA or the removal of divalent cations by dialysis was associated with a lower threshold for nonspecific plasminogen activation by pro-UK (scu-PA) in plasma. Replacement of Ca++ but not other cations restored baseline conditions. In the presence of a clot, fibrin-selective plasminogen activation and clot lysis were triggered. Lysis was accompanied by less than 10% conversion of pro-UK (scu-PA) to two-chain UK, suggesting that the intrinsic activity of pro-UK (scu-PA) itself may have been responsible for fibrinolysis, although a contribution by the small amount of UK generated could not be excluded. Similarly, pro-UK (scu- PA) supported clot lysis for several days in the same plasma before the effect dissipated as a result of degradation to UK. When Glu- plasminogen in plasma was replaced by Lys-plasminogen, or when lysine was added to normal plasma, nonselective plasminogen activation and fibrinogenolysis occurred. It was concluded that under the experimental conditions, the fibrin specificity of pro-UK (scu-PA) can be explained by its selective activation of fibrin-bound plasminogen and is due to the latter's Lys-plasminogen-like conformation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Estrogen-stimulated uptake of plasminogen by the mouse uterus

Administration of a single low dose of estradiol to the immature (4- to 5-week-old) female mouse caused a rapid, uterine-specific increase in the uptake of radiolabeled plasminogen from plasma. A significant increase in uptake was detectable within 30 min and reached a maximum 2-4 h after administration of the hormone. After 4 h, a substantial amount (42%) of the newly taken up plasminogen was found in the uterine lumen. Half-maximal stimulation of uptake occurred at a dose of 0.20 microgram estradiol/animal. Estrogen stimulation of uptake was not blocked by puromycin, indicating that new protein synthesis was not required. Similar results were obtained with mouse plasma albumin. Estrogen-stimulated uptake was not blocked by indomethacin (10 micrograms/g BW, iv), but was blocked by prednisolone. Approximately 50% inhibition of the stimulation induced by 0.5 microgram estradiol in these 10-g animals was accomplished with 50 micrograms prednisolone. This study extends our initial findings on the estrogen-stimulated uptake of plasma proteins by the mouse uterus and provides a mechanism by which uterine plasminogen levels can be elevated before implantation.     

Plasma plasminogen activator inhibitor-1 activity in normoglycemic hypertriglyceridemic north Asian Indian subjects: a preliminary case-control study.

BACKGROUND: Recent evidence suggests that increased activity of plasma plasminogen activator inhibitor-1, an important component of the insulin resistance syndrome, plays a crucial role in the pathogenesis of atherosclerosis. METHODS AND RESULTS: In this case-control study, relationships between plasma plasminogen activator inhibitor-1 activity, serum triglyceride levels and hyperinsulinemia were explored in 40 non-diabetic patients with primary hypertriglyceridemia (Group 1) and 40 non-diabetic normotriglyceridemic controls (Group 2) matched for potential confounders like smoking and physical activity. Mean values of fasting serum insulin levels were increased in Group 1 (p>0.05). Hyperinsulinemia was observed in 14 (17.5%) individuals in Group 1 and 11 (13.8%) individuals in Group 2. Mean plasma plasminogen activator inhibitor-I activity in Group 1 (9.8+/-8.4 IU) was higher than in Group 2 (7.0+/-7.7 IU), though the difference was not significant (p>0.05). However, when only subjects with elevated levels of plasma plasminogen activator inhibitor-1 activity were taken into account, mean values were significantly higher in Group 1 (p<0.05). The plasma plasminogen activator inhibitor-1 activity was higher in subjects with body mass index >25 in both the groups, significantly so in males (p=0.05). Hyperinsulinemic subjects with a body mass index >25 and raised serum triglyceride levels had higher mean values of plasma plasminogen activator inhibitor-1 activity (18.42+/-11.15 IU) than subjects with similar characteristics and normal triglyceride levels (14.22+/-8.20 IU, p<0.05). CONCLUSIONS: Though in the current study a trend for hyperinsulinemia and high plasma plasminogen activator inhibitor-1 activity was observed in hypertriglyceridemic subjects, a larger study is needed to achieve significant differences and correlations. Obese male subjects, irrespective of their lipid profile, are at risk for thrombotic events in view of their significantly higher plasma plasminogen activator inhibitor-1 values. Procoagulant tendency is further enhanced if hypertriglyceridemia and hyperinsulinemia are added on to obesity.     

Profibrinolytic effect of Enzamin,an extract of metabolic products from <Emphasis Type="Italic">Bacillus subtilis AK</Emphasis> and <Emphasis Type="Italic">Lactobacillus</Emphasis>

Fibrinolytic system impairment contributes to the development of thrombotic disease such as cardiovascular disease and stroke. Therefore, an agent that increases fibrinolytic activity may be useful for the prevention of these diseases. In this study, to explore novel profibrinolytic agents, we examined the profibrinolytic effect of Enzamin, an extract of metabolic products from Bacillus subtilis AK and Lactobacillus in vitro and in vivo. Enzamin directly enhanced plasmin activity generated by tissue-type plasminogen activator (t-PA) by twofold but not by urokinase-type plasminogen activator (u-PA) in vitro, which was measured employing both the chromogenic substrate H-d-Val-Leu-Lys-pNA (S-2251) and fibrin plate. Enzamin also increased plasmin activity generated by t-PA in the cell lysate and culture medium of endothelial cells, measured by fibrin zymography. Furthermore, the oral administration of a 1% concentration of Enzamin increased plasmin activity generated by t-PA by 1.7-fold but not by u-PA in the euglobulin fraction of mouse plasma. In conclusion, Enzamin has a unique ability to enhance the fibrinolytic activity through an increase in endogenous plasmin activity generated by t-PA released from endothelial cells, and may be a beneficial supplement for the prevention of thrombotic episodes.     

Prourokinase activation on the surface of human rhabdomyosarcoma cells: localization and inactivation of newly formed urokinase-type plasminogen activator by recombinant class 2 plasminogen activator inhibitor.

Recombinant class 2 plasminogen activator inhibitor (PAI-2) was used in an approach to probe the formation and location of enzymatically active urokinase-type plasminogen activator (u-PA) sites on the surface of cultured human rhabdomyosarcoma cells (RD cells). Activation of pro-u-PA on the cell surface and consequent binding of PAI-2 was dependent on the addition of native plasminogen to serum cultures of the cells. Inhibition of the enzyme activity of surface-bound u-PA by the added PAI-2 resulted in a 79% reduction in the capacity of the RD cells to generate cell surface-associated plasmin activity from bound plasminogen. Under these conditions, the PAI-2 probe was localized at focal adhesions of RD cells, where it colocalized with both extracellular u-PA and intracellular vinculin antigens in double immunofluorescence labeling. Specificity of the probe's interaction with cell surface-bound u-PA was confirmed by blocking with a monoclonal antibody to human u-PA, which could also inhibit the formation of bound plasmin activity. These results showed the assembly of the plasmin-generating system at focal adhesions and the accessibility of bound u-PA on which it depends to added PAI-2. Therefore, PAI-2 has the potential both to localize at sites of tumor expression of functionally active u-PA and simultaneously to inhibit cell surface plasminogen activation.