Longistatin, a novel plasminogen activator from vector ticks, is resistant to plasminogen activator inhibitor-1

Thrombo-occlusive diseases are major causes of morbidity and mortality, and tissue-type plasminogen activator (t-PA) is recommended for the treatment of the maladies. However, both t-PA and u-PA are rapidly inactivated by plasminogen activator inhibitor-1 (PAI-1). Here, we show that longistatin, a novel plasminogen activator isolated from the ixodid tick, Haemaphysalis longicornis is resistant to PAI-1. Longistatin was relatively less susceptible to the inhibitory effect of SDS-treated platelet lysate than physiologic PAs. Platelet lysate inhibited t-PA and tcu-PA with the IC₅₀ of 7.7 and 9.1 μg/ml, respectively, whereas for longistatin inhibition IC₅₀ was 20.1 μg/ml (p < 0.01). Similarly, activated PAI-1 (20 nM) inhibited only 21.47% activity of longistatin but almost completely inhibited t-PA (99.17%) and tcu-PA (96.84%). Interestingly, longistatin retained 76.73% initial activity even after 3 h of incubation with 20 nM of PAI-1. IC₅₀ of PAI-1 during longistatin inhibition was 88.3 nM while it was 3.9 and 3.2 nM in t-PA and tcu-PA inhibition, respectively. Longistatin completely hydrolyzed fibrin clot by activating plasminogen efficiently in the presence of 20 nM of PAI-1. Importantly, unlike t-PA, longistatin did not form complex with PAI-1. Collectively, our results suggest that longistatin is resistant to PAI-1 and maybe an interesting tool for the development of a PAI-1 resistant effective thrombolytic agent.     

The plasminogen activator inhibitor-1 binding site in the kringle-2 domain of tissue-type plasminogen activator

We have shown that synthetic peptides containing the amino acid sequence Asn-Arg-Arg-Leu, derived from the amino acid sequence of the inner loop of the kringle-2 domain of tissue-type plasminogen activator (tPA), inhibited complex formation between two chain tPA and plasminogen activator inhibitor-1 (PAI-1) by binding to PAI-1. This binding was reversible and was inhibited by not only tPA but also by enzymatically inactive tPA. Quantitative analyses of the interaction of PAI-1 with the peptide containing the Asn-Arg-Arg-Leu sequence indicated that the PAI-1 binding site residues in the inner loop of the kringle-2 domain and is preferentially expressed in two chain tPA.     

A novel function of plasminogen activator inhibitor-1 in modulation of the AKT pathway in wild-type and plasminogen activator inhibitor-1-deficient endothelial cells

Cell proliferation, an event associated with angiogenesis, involves coordinated activities of a number of proteins. The role of plasminogen activator inhibitor-1 (PAI-1) in angiogenesis remains controversial. Utilizing proliferating PAI-1-/- endothelial cells (EC), the impact of a host PAI-1 deficiency on Akt activation was evaluated. Hyperactivation of Akt(Ser(P)473) was observed in PAI-1-/- EC, and this was probably due to enhanced inactivation of tumor suppressor PTEN, thus rendering the cells resistant to apoptotic signals. Higher levels of inactivated caspase-9 in PAI-1-/- EC led to lower levels of procaspase-3 and cleaved caspase-3, thereby promoting survival. These effects were reversed when recombinant PAI-1 was added to PAI-1-/- EC. Additional studies demonstrated that regulation of proliferation is dependent on its interaction with low density lipoprotein receptor-related protein. Thus, PAI-1 is a negative regulator of cell growth, exerting its effect on the phosphatidylinositol 3-kinase/Akt pathway and allowing controlled cell proliferation.     

On the reversible interaction of plasminogen activator inhibitor-1 with tissue-type plasminogen activator and with urokinase-type plasminogen activator

The reaction between plasminogen activators and plasminogen activator inhibitor-1 is characterized by an initial rapid formation of an inactive reversible complex. The second-order association rate constant (k1) of complex formation of recombinant two-chain tissue-type plasminogen activator (rt-PA) or recombinant two-chain urokinase-type plasminogen activator (rtcu-PA) by recombinant plasminogen activator inhibitor-1 (rPAI-1) is 2.9 +/- 0.4 x 10(7) M-1 s-1 (mean +/- S.D., n = 30) and 2.0 +/- 0.6 x 10(7) M-1 s-1 (n = 12), respectively. Different molecular forms of tissue- or urokinase-type plasminogen activator which do not form covalent complexes with rPAI-1, including rt-PA-Ala478 (rt-PA with the active-site Ser478 mutagenized to Ala) and anhydro-urokinase (rtcu-PA with the active-site Ser356 converted to dehydroalanine) reduced k1 in a concentration-dependent manner, compatible with 1:1 stoichiometric complex formation between rPAI-1 and these ligands. The apparent dissociation constant (KD) of the complex between rPAI-1 and rt-PA-Ala478, determined as the concentration of rt-PA-Ala478 which reduced k1 to 50% of its control value, was 3-5 nM. Corresponding concentrations of active-site-blocked two-chain rt-PA were 150-250-fold higher. The concentration of anhydro-urokinase which reduced k1 to 50% was 4-6 nM, whereas that of active-site-blocked rtcu-PA was 100-250-fold higher. Recombinant single-chain urokinase-type plasminogen activator had an apparent KD of about 2 microM. These results suggest that inhibition of rt-PA or rtcu-PA by rPAI-1 proceeds via a reversible high affinity interaction which does not require a functional active site but which is markedly reduced following inactivation of the enzymes with active-site titrants.     

Decreased plasminogen activator inhibitor-1 secretion in hypoxic corneal epithelial cells is associated with increased urokinase plasminogen activator activity

The aim of this study was to determine the effects of hypoxia on mRNA levels, cell-associated and -secreted protein concentration, activity, and protein complex formation of urokinase-type plasminogen activator, its receptor, and plasminogen activator inhibitor type-1 in corneal epithelium. Non-transformed human corneal epithelial cells were cultured in 20% oxygen (normoxic conditions) or 2% oxygen (hypoxic conditions) for 1, 3, 5, or 7 days. Relative changes in mRNA levels of plasminogen activator, receptor, and plasminogen activator inhibitor-1 were determined using a cDNA expression array, chemiluminescence, and densitometry. Protein concentrations were determined using enzyme linked immunosorbent assays. Activity assays were also used. Protein complex formation was assayed using cell surface biotinylation, immunoprecipitation, and Western blot analysis. Hypoxic corneal epithelial cells demonstrated no significant differences in plasminogen activator or receptor mRNA. Cell-associated plasminogen activator and membrane-associated receptor protein levels were unchanged. In contrast decreases in mRNA and secreted plasminogen activator inhibitor-1 protein were observed in hypoxic cells. Concurrently, increased cell-associated plasminogen activator activity was observed in hypoxic cells. The formation of plasminogen activator/receptor/plasminogen activator inhibitor-1 complex at the cell surface was not inhibited by hypoxia. However, in hypoxic cells less plasminogen activator inhibitor-1 was associated with receptor. It is concluded that in corneal epithelium cultured in 2% oxygen plasminogen activator inhibitor-1 may be an important regulatory factor of the plasminogen activator system resulting in increased urokinase plasminogen activator activity.     

The importance of helix F in plasminogen activator inhibitor-1

Plasminogen activator inhibitor-1 (PAI-1) is the only functionally labile serpin, as it converts spontaneously into a non-reactive 'latent' conformation. Several studies have suggested an important role for helix F in the functional behavior and stability of the serpins, especially for PAI-1. We constructed a mutant of PAI-1 (PAI-1-delhF) in which residues 127-158 (hF-thFs3A) were deleted. Whereas wild-type PAI-1 (wtPAI-1) exhibits inhibitory properties towards t-PA and u-PA to an extent of 60-80% of the theoretical maximum, PAI-1-delhF did not exert any detectable inhibitory properties, but behaved as a stable substrate. Prolonged incubation at 37 degrees C did not change its functional properties in contrast to wtPAI-1 that under those conditions converts to the latent conformation. In contrast to active wtPAI-1 and other substrate-type PAI-1 mutants, PAI-1-delhF showed a 3000-fold decreased binding to vitronectin. The obtained results clearly show the importance of helix F in the inhibitory activity of PAI-1. The absence of helix F apparently leads to an impaired kinetics of insertion of the reactive site loop upon interaction with its target proteinase resulting in the inability to form a stable covalent complex. Moreover, removal of helix F strongly affects the binding of PAI-1 to vitronectin.     

The low density lipoprotein receptor-related protein is a motogenic receptor for plasminogen activator inhibitor-1

Although plasminogen activator inhibitor-1 (PAI-1) is known to stimulate cell migration, little is known about underlying mechanisms. We show that both active and inactive (e.g. cleaved) PAI-1 can activate the Jak/Stat signaling system and stimulate cell migration in chemotaxis, haptotaxis, chemokinesis, and wound healing assays. Moreover, antibodies to the LDL receptor-related protein (LRP) and an LRP antagonist (RAP) blocked these motogenic effects of PAI-1, while a PAI-1 mutant that did not bind to LRP failed to activate the Jak/Stat signaling pathway or to stimulate cell migration. PAI-1 had no chemotactic effect on LRP-deficient cells. These results indicate that LRP is a signaling molecule, that it mediates the migration-promoting activity of PAI-1, and that this activity does not require intact, biologically active PAI-1. Activation of this LRP-dependent signaling pathway by PAI-1 may begin to explain how the inhibitor stimulates cell migration in a variety of normal and pathological processes.     

Binding of tissue-type plasminogen activator with human endothelial cell monolayers. Characterization of the high affinity interaction with plasminogen activator inhibitor-1

The formation and release of covalent complexes between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) limits the application of equilibrium radioligand binding analysis to characterize the interaction between t-PA and human umbilical vein endothelial cell (HUVEC) monolayers. To avoid this difficulty, we used a recombinant mutant of t-PA, S478A rt-PA, in which alanine has been substituted for the active-site serine. Although the mutant is incapable of covalently reacting with PAI-1, 125I-labeled S478A rt-PA binding to HUVEC monolayers is specific and reversible and is characterized by a high affinity (Kd of 1.5 nM) and a large number of sites (1.5 x 10(6)/cell). This binding was shown to occur through noncovalent interaction with PAI-1 in the HUVEC monolayer by the fact that a monoclonal anti-PAI-1 antibody (MA-7D4) completely blocked S478A rt-PA binding. Two solution-phase assays with recombinant PAI-1 (rPAI-1) confirmed this noncovalent interaction: complexes between 125I-S478A rt-PA and rPAI-1 could be isolated by immunoprecipitation with anti-PAI-1 antibodies, and S478A rt-PA competed with rt-PA for inactivation by rPAI-1. In contrast diisopropylphosphate rt-PA (in which the active site serine is chemically modified) showed minimal binding to HUVEC monolayers, as a result of impaired interaction with PAI-1, in the two assays. Thus, both wild-type rt-PA and S478A rt-PA interact with the HUVEC monolayer through PAI-1. With rt-PA this results in the formation of covalent rt-PA.PAI-1 complexes that are released from the monolayer into the supernatant. With S478A rt-PA this results in the formation of noncovalent complexes that remain associated with the HUVEC monolayer, thereby identifying a large pool of reactive PAI-1 molecules in the monolayer.     

Aldosterone up-regulates production of plasminogen activator inhibitor-1 by renal mesangial cells

In vivo studies have demonstrated that aldosterone is an independent contributor to glomerulosclerosis. In the present study, we have investigated whether aldosterone itself mediated glomerulosclerosis, as angiotensin II (Ang II) did, by inducing cultured renal mesangial cells to produce plasminogen activator inhibitor-1 (PAI-1), and whether these effects were mediated by aldosterone-induced increase in transforming growth factor beta(1) (TGF-beta(1)) expression and cellular reactive oxygen species (ROS) activity. Quiescent rat mesangial cells were treated by aldosterone alone or by combination of aldosterone and spironolactone, Ang II, neutralizing antibody to TGF-beta(1) or antioxidant Nacetylcysteme (NAC). This study indicate that aldosterone can increase PAI-1 mRNA and protein expression by cultured mesangial cells alone, which is independent of aldosterone-induced increases in TGF-beta(1) expression and cellular ROS. The effects on PAI-1, TGF-beta(1) and ROS generation were markedly attenuated by spironolactone, a mineralocorticoid receptor antagonist, which demonstrate that mineralocorticoid receptor (MR) may play a role in mediating these effects of aldosterone.     

Interactions between the finger and kringle-2 domains of tissue-type plasminogen activator and plasminogen activator inhibitor-1.

We have shown that plasminogen activator inhibitor-1 (PAI-1) inhibits the fibrin binding of both the single chain and two chain forms of tissue-type plasminogen activator (tPA) through two different mechanisms. PAI-1 inhibits the finger domain-dependent fibrin binding of diisopropylfluorophosphate-inactivated single chain tPA and the kringle-2 domain-dependent fibrin binding of diisopropylfluorophosphate-inactivated two chain tPA. In accordance with the data, preformed complexes of single chain tPA/PAI-1 and of two chain tPA/PAI-1 lost the fibrin binding abilities mediated by the finger and kringle-2 domains, respectively. These effects of PAI-1 appear to be mediated by steric hindrance of the fibrin binding sites after PAI-1 binding to adjacent regions in the functional domains of tPA. We thus propose a model in which a PAI-1 binding site resides in the finger domain of a single chain, and plays a role in the reversible association of single chain tPA and PAI-1. Conformational changes may take place during the conversion of single chain tPA to two chain tPA, resulting in burying of the original PAI-1 binding site and exposure of an alternate PAI-1 binding site on the surface of the kringle-2 domain.     

Characterization of interaction of active-site serine mutants of tissue-type plasminogen activator with plasminogen activator inhibitor-1

To define determinants of interactions of tissue-type plasminogen activator (t-PA) with plasminogen activator inhibitor type-1 (PAI-1), we utilized site-directed mutagenesis to substitute either threonine or glycine for the active-site serine of tissue-type plasminogen activator. Assays of conditioned media of transfected cells demonstrated that the threonine substitution markedly decreased but did not entirely abolish plasminogen activating activity. In contrast, the glycine substitution yielded a mutant with absolutely no detectable plasminogen activating activity. Wild-type t-PA formed stable complexes with PAI-1. However, even when exogenous inhibitor was present in the medium or purified mutant was added to plasma that had been rendered PAI-1-rich in vivo, the mutants were present in the free form exclusively judging from results of fibrin autography and Western blot analysis. Thus, despite maintenance of some residual plasminogen-activating activity associated with preservation of the hydroxyl group at the active site, the threonine mutant did not form stable complexes with inhibitor. The glycine mutant, developed so that steric hindrance or other unfavorable interactions at the modified active site would be minimal, was similarly incapable of forming complexes with PAI-1. These results show that the presence of an active site serine residue is necessary for formation of stable complexes between t-PA and PAI-1.     

Kinetic analysis of the interaction between plasminogen activator inhibitor-1 and tissue-type plasminogen activator.

The kinetics of inhibition of tissue-type plasminogen activator (t-PA) by the fast-acting plasminogen activator inhibitor-1 (PAI-1) was investigated in homogeneous (plasma) and heterogeneous (solid-phase fibrin) systems by using radioisotopic and spectrophotometric analysis. It is demonstrated that fibrin-bound t-PA is protected from inhibition by PAI-1, whereas t-PA in soluble phase is rapidly inhibited (K1 = 10(7) M-1.s-1) even in the presence of 2 microM-plasminogen. The inhibitor interferes with the binding of t-PA to fibrin in a competitive manner. As a consequence the Kd of t-PA for fibrin (1.2 +/- 0.4 nM) increases and the maximal velocity of plasminogen activation by fibrin-bound t-PA is not modified. From the plot of the apparent Kd versus the concentration of PAI-1 a Ki value of 1.3 +/- 0.3 nM was calculated. The quasi-similar values for the dissociation constants between fibrin and t-PA (Kd) and between PAI-1 and t-PA (Ki), as well as the competitive type of inhibition observed, indicate that the fibrinolytic activity of human plasma may be the result of an equilibrium distribution of t-PA between both the amount of fibrin generated and the concentration of circulating inhibitor.     

Stimulation of human synovial fibroblast plasminogen activator production by mononuclear cell supernatants

Conditioned medium from concanavalin A-stimulated human peripheral blood mononuclear cells (c-MCCM) stimulates the plasminogen activator (PA) production of nonrheumatoid human synovial fibroblasts obtained from explant cultures. The effect of this synovial fibroblast-stimulating activity is observed within 2 to 4 hr and requires RNA and protein synthesis. Reversible morphological changes in the synovial cells can be observed as a result of c-MCCM action. These enzymatic and morphologic changes are similar to some of the effects of transforming viruses and tumor promoters on target cells. The possible significance of these data for an understanding of the cellular interactions involved in the formation and function of the rheumatoid "pannus" is discussed.     

Production of plasminogen activator in a melanoma cell line

A melanoma cell line (Bowes) was found to produce plasminogen activator even on its growing phase, and the rate of plasminogen activator production was rather constant. The production of plasminogen activator was proportional to the cell number. Morphologically, no specific features for plasminogen activator production were seen. Plasminogen activator was observed in the lysate of this cell line only when the cell number was large. The extracellular plasminogen activator activity was higher than the intracellular plasminogen activator activity, suggesting the existence of a secretion mechanism for the plasminogen activator.     

The conversion of active to latent plasminogen activator inhibitor-1 is an energetically silent event

PAI-1 is a proteinase inhibitor, which plays a key role in the regulation of fibrinolysis. It belongs to the serpins, a family of proteins that behave either as proteinase inhibitors or proteinase substrates, both reactions involving limited proteolysis of the reactive center loop and insertion of part of this loop into beta-sheet A. Titration calorimetry shows that the inhibition of tissue-type plasminogen and pancreatic trypsin are exothermic reactions with DeltaH = -20.3, and -22.5 kcal.mol(-1), respectively. The Pseudomonas aeruginosa elastase-catalyzed reactive center loop cleavage and inactivation of the inhibitor is also exothermic (DeltaH = -38.9 kcal.mol(-1)). The bacterial elastase also hydrolyses peptide-bound PAI-1 in which acetyl-TVASSSTA, the octapeptide corresponding to the P(14)-P(7) sequence of the reactive center loop is inserted into beta-sheet A of the serpin with DeltaH = -4.0 kcal.mol(-1). In contrast, DeltaH = 0 for the spontaneous conversion of the metastable active PAI-1 molecule into its thermodynamically stable inactive (latent) conformer although this conversion also involves loop/sheet insertion. We conclude that the active to latent transition of PAI-1 is an entirely entropy-driven phenomenon.     

Prostaglandin E2 regulates production of plasminogen activator isoenzymes,urokinase receptor,and plasminogen activator inhibitor-1 in primary cultures of rat calvarial osteoblasts

The bone resorbing agent, prostaglandin E₂ (PGE₂), was found to alter several components of the plasminogen activator (PA)/plasmin pathway in primary cultures of rat neonatal osteoblast-like cells. The mRNA and activities of both urokinase-type PA (uPA) and tissue-type PA (tPA) were enhanced by PGE₂ treatment. The presence of mRNA for the uPA receptor (uPAR) has been demonstrated in these cells and steady-state levels shown to be greatly enhanced, the response being rapid and sustained for at least 24 hours. mRNA for plasminogen activator inhibitor 1 (PAI-1) was modulated in a biphasic manner, with inhibition of the constitutive level apparent at 4 hours of treatment and stimulation apparent at 12 hours and longer, while PAI-1 protein, measured by an ELISA assay for rat PAI-1, was diminished over this period. Neither PAI-2 mRNA nor mRNA for the broad spectrum protease inhibitor, protease nexin-1 (PN-1), was found to be modulated by PGE₂. Therefore, PGE₂ is likely to stimulate cell surface proteolytic activity, since uPA mRNA and cell-associated activity were elevated, as was mRNA for the cellular receptor for uPA. Although it was not possible to measure uPAR number and affinity it seems likely that elevated uPAR mRNA would translate into increased uPARs which would localize the increased uPA activity to the pericellular region. tPA mRNA and activity were also increased transiently with the activity inhibited with prolonged incubations, apparently by PAI-1. Elevation of tPA mRNA and activity may result in elevated activity within the extracellular matrix as tPA has been reported to associate with several matrix proteins. Thus the early effect of PGE₂ would be to promote proteolysis, both pericellularly and in the extracellular matrix. The inhibition of PAI-1 mRNA and protein, which would contribute to the elevation of activity, is due to PGE₂, but the later stimulatory effect on PAI-1 mRNA may be due to feedback regulation by transforming growth factor beta (TGFβ), secreted by osteoblasts and activated by elevated levels of PA. © 1995 Wiley-Liss Inc.     

Interactions between tissue-type plasminogen activator and extracellular matrix-associated plasminogen activator inhibitor type 1 in the human hepatoma cell line HepG2

Hepatic parenchymal cells contribute to the clearance of circulating tissue-type plasminogen activator (t-PA) in vivo. The hepatocyte extracellular matrix is interposed between the endothelial-lined sinusoids and the parenchymal cell surface and thus may influence t-PA clearance. To test this hypothesis, the well differentiated human hepatoma cell line HepG2 was used to characterize the role of extracellular matrix in t-PA clearance in vitro. Previous studies with these cells demonstrated their capacity for specific catabolism of t-PA in a system modulated by plasminogen activator inhibitor type 1 (PAI-1). In the present study the extracellular matrix growth substratum of HepG2 cells is shown to contain active PAI-1. PAI-1 is distributed in a punctuate pattern throughout the substratum. Components of the substratum confer stability to active PAI-1 for intervals of at least 24 h. Exposing substratum to 125I-t-PA leads rapidly to the formation and release of a sodium dodecyl sulfate-stable 95-kDa 125I-t-PA.PAI-1 complex. In comparison, cell monolayers have the additional capacity for specific binding of the complex. However, PAI-1 is not detected at the surface of HepG2 cells in suspension, suggesting that 125I-t-PA.PAI-1 complexes form in substratum and subsequently bind to cells. Specific binding of performed 125I-t-PA.PAI-1, but not 125I-t-PA, was demonstrated for HepG2 cells in suspension. These results suggest that components of extracellular matrix participate in the clearance of t-PA by hepatocytes.     

Inhibition of angiogenesis in vivo by plasminogen activator inhibitor-1

The process of angiogenesis is important in both normal and pathologic physiology. However, the mechanisms whereby factors such as basic fibroblast growth factor promote the formation of new blood vessels are not known. In the present study, we demonstrate that exogenously added plasminogen activator inhibitor-1 (PAI-1) at therapeutic concentrations is a potent inhibitor of basic fibroblast growth factor-induced angiogenesis in the chicken chorioallantoic membrane. By using specific PAI-1 mutants with either their vitronectin binding or proteinase inhibitor activities ablated, we show that the inhibition of angiogenesis appears to occur via two distinct but apparently overlapping pathways. The first is dependent on PAI-1 inhibition of proteinase activity, most likely chicken plasmin, while the second is independent of PAI-1's anti-proteinase activity and instead appears to act through PAI-1 binding to vitronectin. Together, these data suggest that PAI-1 may be an important factor regulating angiogenesis in vivo.     

Structural determinants of the noncatalytic chain of tissue-type plasminogen activator that modulate its association rate with plasminogen activator inhibitor-1

In order to identify the regions of recombinant (r) tissue plasminogen activator (tPA) that mediate its kinetically relevant interaction with r-plasminogen activator inhibitor-1 (rPAI-1), we have determined the second-order association rate (k1) constants of domain-altered variants of tPA with rPAI-1, at 10 degrees C. With two-chain, wild-type recombinant tPA (tcwt-rtPA), obtained by expression of the human cDNA for tPA in five different cell systems (viz. insect cells, human kidney 293 cells, Chinese hamster ovary cells, human melanoma cells, and mouse C127 cells), the average k1 was 1.45 x 10(7) M-1 s-1 (range, 1.34 10(7) M-1 s-1-1.68 x 10(7) M-1 s-1). Since this value was not significantly different for the different tcwt-rtPA preparations, it appears as though the nature of the glycosylation of tPA plays little role in its initial interaction with PAI-1. The k1 determined for tcwt-rtPA was slightly higher than that of 0.87 x 10(7) M-1 s-1, obtained for a similar inhibition of human urokinase by rPAI-1. The k1 value obtained for single-chain (sc) wt-rtPA was approximately 6-fold lower than that of the two-chain molecules, results consistent with previous conclusions on this matter. The k1 value for tcwt-rtPA was not influenced by the presence of epsilon-aminocaproic acid, suggesting that the lysine-binding site associated with the kringle 2 (K2) region of tPA does not modulate the rate of its initial interaction with rPAI-1. Removal of the K2 domain from tPA, by recombinant DNA technology, results in a protein, F-E-K1-P (tc-r delta K2-tPA), containing only the finger (F), growth factor (E), kringle 1 (K1), and serine protease (P) domains. This variant protein was more rapidly inhibited by rPAI-1 (k1 = 3.00 x 10(7) M-1 s-1) than its wild-type counterparts. Deletion of both the K1 and K2 domains resulted in a variant molecule, F-E-P (tc-r delta K1 delta K2-tPA), that was slightly more rapidly inhibited by rPAI-1 (k1 = 2.01 x 10(7) M-1 s-1).(ABSTRACT TRUNCATED AT 400 WORDS)