Immunomodulatory effects of plasminogen activators on hepatic fibrogenesis.

Tissue-type plasminogen activators (tPA) and urokinase-type plasminogen activators (uPA) are involved in liver repair. We examined the potential immunomodulatory actions of uPA, tPA and uPA-receptor (uPAR) in carbon-tetrachloride-induced hepatic fibrosis in wild-type (WT), tPA-/-, uPA-/- and uPAR-/- mice. Carbon-tetrachloride treatment increased fibrosis in four groups but significantly less in three knock-out models. Serum cytokines and intrahepatic T cells elevated significantly following fibrosis process in WT animals but not in the knock-out groups. In culture, uPA increased lymphocyte proliferation significantly in WT and uPA-/- but not uPAR-/- animals. Following uPA exposure in vivo, there was CD8 predominance. To isolate uPA's effect on lymphocytes, WT mice were irradiated sublethally and then reconstituted with WT or uPA-/- lymphocytes. In these animals fibrosis was decreased and T cells were reduced in the uPA-/- recipients. Based on these data we postulate that plasminogen activators affect fibrosis in part by liver-specific activation of CD8 subsets that govern the fibrogenic activity of hepatic stellate cells.

The structure of lipoprotein(a) and ligand-induced conformational changes.

Lipoprotein(a) is composed of low-density lipoprotein linked both covalently and noncovalently to apolipoprotein(a). The structure of lipoprotein(a) and the interactions between low-density lipoprotein and apolipoprotein(a) were investigated by electron microscopy and correlated with analytical ultracentrifugation. Electron microscopy of rotary-shadowed and unidirectionally shadowed lipoprotein(a) prepared without glycerol revealed that it is a nearly spherical particle with no large projections. After extraction of both lipoprotein(a) and low-density lipoprotein with glycerol prior to rotary shadowing, the protein components were observed to consist of a ring of density made up of nodules of different sizes, with apolipoprotein(a) and apolipoprotein B-100 closely associated with each other. However, when lipoprotein(a) was treated with a lysine analogue, 6-aminohexanoic acid, much of the apolipoprotein(a) separated from the apolipoprotein B-100. In 6-aminohexanoic acid-treated preparations without glycerol extraction, lipoprotein(a) particles had an irregular mass of density around the core. In contrast, lipoprotein(a) particles treated with 6-aminohexanoic acid in the presence of glycerol had a long tail, in which individual kringles could be distinguished, extending from the ring of apolipoprotein B-100. The length of the tail was dependent on the particular isoform of apolipoprotein(a). Dissociation of the noncovalent interactions between apolipoprotein(a) and low-density lipoprotein as a result of shear forces or changes in the microenvironment may contribute to selective retention of lipoprotein(a) in the vasculature.

Plasminogen binding and activation by Mycoplasma fermentans.

The binding of plasminogen to Mycoplasma fermentans was studied by an immunoblot analysis and by a binding assay using iodine-labeled plasminogen. The binding of 125I-labeled plasminogen was inhibited by unlabeled plasminogen, lysine, and lysine analog epsilon-aminocaproic acid. Partial inhibition was obtained by a plasminogen fragment containing kringles 1 to 3 whereas almost no inhibition was observed with a fragment containing kringle 4. Scatchard analysis revealed a dual-phase interaction, one with a dissociation constant (kd) of 0.5 microM and the second with a kd of 7.5 microM. The estimated numbers of plasminogen molecules bound were calculated to be 110 and 790 per cell, respectively. Autoradiograms of ligand blots containing M. fermentans membrane proteins incubated with 125I-labeled plasminogen identified two plasminogen-binding proteins of about 32 and 55 kDa. The binding of plasminogen to M. fermentans enhances the activation of plasminogen to plasmin by the urokinase-type plasminogen activator (uPA), as monitored by measuring the breakdown of chromogenic substrate S-2251. Enhancement was more pronounced with the low-molecular-weight and the single-chain uPA variants, known to have low plasminogen activator activities. The binding of plasminogen also promotes the invasion of HeLa cells by M. fermentans. Invasion was more pronounced in the presence of uPA, suggesting that the ability of the organism to invade host cells stems not only from its potential to bind plasminogen but also from the activation of plasminogen to plasmin.

Urokinase mediates fibrinolysis in the pulmonary microvasculature.

The role of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in fibrinolysis remains unsettled. The contribution of uPA may depend on the vascular location, the physical properties of the clot, and its impact on tissue function. To study the contribution of urokinase within the pulmonary microvasculature, a model of pulmonary microembolism in the mouse was developed. Iodine 125 ((125)I)-labeled fibrin microparticles injected intravenously through the tail vein lodged preferentially in the lung, distributing homogeneously throughout the lobes. Clearance of (125)I-microemboli in wild type mice was rapid and essentially complete by 5 hours. In contrast, uPA(-/-) and tissue-type plasminogen activator tPA(-/-) mice, but not uPAR(-/-) mice, showed a marked impairment in pulmonary fibrinolysis throughout the experimental period. The phenotype in the uPA(-/-) mouse was rescued completely by infusion of single chain uPA (scuPA). The increment in clot lysis was 4-fold greater in uPA(-/-) mice infused with the same concentration of scuPA complexed with soluble recombinant uPAR. These data indicate that uPA contributes to endogenous fibrinolysis in the pulmonary vasculature to the same extent as tPA in this model system. Binding of scuPA to its receptor promotes fibrinolytic activity in vivo as well as in vitro. The physical properties of fibrin clots, including size, age, and cellular composition, as well as heterogeneity in endothelial cell function, may modify the participation of uPA in endogenous fibrinolysis. (Blood. 2000;96:1820-1826)

The alpha-defensins stimulate proteoglycan-dependent catabolism of low-density lipoprotein by vascular cells: a new class of inflammatory apolipoprotein and a possible contributor to atherogenesis.

Inflammation may contribute to the pathogenesis of atherosclerosis. On the basis of previous reports that human atherosclerotic lesions contain alpha-defensins, a class of cationic proteins released by activated neutrophils, the study was designed to ask whether defensins modulate the binding and catabolism of low-density lipoprotein (LDL) by human vascular cells. The results of the study demonstrated that defensin stimulated the binding of (125)I-LDL to cultured human umbilical vein endothelial cells, smooth muscle cells, and fibroblasts approximately 5-fold in a dose-dependent and saturable manner. Defensin and LDL formed stable complexes in solution and on cell surfaces. Stimulation of LDL binding by defensin was not inhibited by antibodies against the LDL-receptor (LDL-R), or by recombinant receptor-associated protein, which blocks binding of ligands to the alpha(2)-macroglobulin receptor/LDL-R-related protein and other LDL-R family members. Furthermore, defensin stimulated the binding, endocytosis, and degradation of LDL by fibroblasts lacking LDL-R. Stimulation of LDL degradation by defensin was inhibited approximately 75% by low concentrations of heparin (0.2 units/mL) and was similarly reduced in CHO cells lacking heparan-sulfate-containing proteoglycans. The effect of defensin was substantially increased in cells overexpressing the core protein of the syndecan-1 heparan sulfate proteoglycan. The alpha-defensins released from activated neutrophils may provide a link between inflammation and atherosclerosis by changing the pattern of LDL catabolism from LDL-R to the less efficient LDL-R-independent, proteoglycan-dependent pathway. (Blood. 2000;96:1393-1398)

Urokinase-derived peptides regulate vascular smooth muscle contraction in vitro and in vivo.

We examined the effect of urokinase (uPA) and its fragments on vascular smooth muscle cell contraction. Single-chain uPA inhibits phenylepherine (PE) -induced contraction of rat aortic rings, whereas two-chain uPA exerts the opposite effect. Two independent epitopes mediating these opposing activities were identified. A6, a capped peptide corresponding to amino acids 136-143 (KPSSPPEE) of uPA, increased the EC(50) of PE-induced vascular contraction sevenfold by inhibiting the release of calcium from intracellular stores. A6 activity was abolished by deleting the carboxyl-terminal Glu or by mutating the Ser corresponding to position 138 in uPA to Glu. A single-chain uPA variant lacking amino acids 136-143 did not induce vasorelaxation. A second epitope within the kringle of uPA potentiated PE-induced vasoconstriction. This epitope was exposed when single-chain uPA was converted to a two-chain molecule by plasmin. The isolated uPA kringle augmented vasoconstriction, whereas uPA variant lacking the kringle had no procontractile activity. These studies reveal previously undescribed vasoactive domains within urokinase and its naturally derived fragments.

A peptide derived from the nonreceptor binding region of urokinase plasminogen activator (uPA) inhibits tumor progression and angiogenesis and induces tumor cell death in vivo.

Urokinase plasminogen activator (uPA) plays an important role in the progression of several malignancies including breast cancer. We have identified a noncompetitive antagonist of the uPA-uPAR interaction derived from a nonreceptor binding region of uPA (amino acids 136-143). This 8-mer capped peptide (A6) inhibited breast cancer cell invasion and endothelial cell migration in a dose-dependent manner in vitro without altering cell doubling time. Intraperitoneal administration of A6 resulted in a significant inhibition of tumor growth and suppressed the development of lymph node metastases in several models of breast cancer cell growth and metastasis. Large areas of tumor necrosis and extensive positive staining by TUNEL were observed on histological and immunohistochemical analysis of experimental tumor sections from A6-treated animals. A6 treatment also resulted in a decrease in factor VIII-positive tumor microvessel hot-spots. These results identify a new epitope in uPA that is involved in the uPA-uPAR interaction and indicate that an antagonist based on this epitope is able to inhibit tumor progression by modulating the tumor microenvironment in the absence of direct cytotoxic effects in vivo.

A region in domain II of the urokinase receptor required for urokinase binding.

The urokinase receptor is composed of three homologous domains based on disulfide spacing. The contribution of each domain to the binding and activation of single chain urokinase (scuPA) remains poorly understood. In the present paper we examined the role of domain II (DII) in these processes. Repositioning DII to the amino or carboxyl terminus of the molecule abolished binding of scuPA as did deleting the domain entirely. By using alanine-scanning mutagenesis, we identified a 9-amino acid continuous sequence in DII (Arg(137)-Arg(145)) required for both activities. Competition-inhibition and surface plasmon resonance studies demonstrated that mutation of Lys(139) and His(143) to alanine in soluble receptor (suPAR) reduced the affinity for scuPA approximately 5-fold due to an increase in the "off rate." Mutation of Arg(137), Arg(142), and Arg(145), each to alanine, leads to an approximately 100-fold decrease in affinity attributable to a 10-fold decrease in the apparent "on rate" and a 6-fold increase in off rate. These differences were confirmed on cells expressing variant urokinase receptor. suPAR-K139A/H143A displayed a 50% reduction in scuPA-mediated plasminogen activation activity, whereas the 3-arginine variant was unable to stimulate scuPA activity at all. Mutation of the three arginines did not affect binding of a decamer peptide antagonist of scuPA known to interact with DI and DIII. However, this mutation abolished both the binding of soluble DI to DII-III in the presence of scuPA and the synergistic activation of scuPA mediated by DI and wild type DII-DIII. These data show that DII is required for high affinity binding of scuPA and its activation. DII does not serve merely as a spacer function but appears to be required for interdomain cooperativity.

Novel interactions between urokinase and its receptor.

Urokinase-type plasminogen activator (uPA) binds to its receptor (uPAR) with a K(d) of about 1 nm. The catalytic activity of the complex is apparent at uPA concentrations close to K(d). Other functions of the complex, such as signal transduction, are apparent at much higher concentrations (35-60 nm). In the present study, we show that uPA and recombinant soluble uPAR (suPAR), at concentrations that exceed the K(d) and the theoretical saturation levels (10-80 nm), establish novel interactions that lead to a further increase in the activity of the single-chain uPA (scuPA)/suPAR and two-chain uPA (tcuPA)/suPAR complexes. Experiments performed using dynamic light scattering, gel filtration, and electron microscopy techniques indicate that suPAR forms dimers and oligomers. The three techniques provide evidence that the addition of an equimolar concentration of scuPA leads to the dissociation of these dimers and oligomers. Biacore data show that suPAR dimers and oligomers bind scuPA with decreased affinity when compared with monomers. We postulate that uPAR is present in equilibrium between oligomer/dimer/monomer forms. The binding of uPA to suPAR dimers and oligomers occurs with lower affinity than the binding to monomer. These novel interactions regulate the activity of the resultant complexes and may be involved in uPA/uPAR mediated signal transduction.

Defensin promotes the binding of lipoprotein(a) to vascular matrix.

Retention of lipoproteins within the vasculature is a central event in the pathogenesis of atherosclerosis. However, the signals that mediate this process are only partially understood. Prompted by putative links between inflammation and atherosclerosis, we previously reported that alpha-defensins released by neutrophils are present in human atherosclerotic lesions and promote the binding of lipoprotein(a) [Lp(a)] to vascular cells without a concomitant increase in degradation. We have now tested the hypothesis that this accumulation results from the propensity of defensin to form stable complexes with Lp(a) that divert the lipoprotein from its normal cellular degradative pathways to the extracellular matrix (ECM). In accord with this hypothesis, defensin stimulated the binding of Lp(a) to vascular matrices approximately 40-fold and binding of the reactants to the matrix was essentially irreversible. Defensin formed stable, multivalent complexes with Lp(a) and with its components, apoprotein (a) and low-density lipoprotein (LDL), as assessed by optical biosensor analysis, gel filtration, and immunoelectron microscopy. Binding of defensin/Lp(a) complexes to matrix was inhibited (>90%) by heparin and by antibodies to fibronectin (>70%), but not by antibodies to vitronectin or thrombospondin. Defensin increased the binding of Lp(a) (10 nmol/L) to purified fibronectin more than 30-fold. Whereas defensin and Lp(a) readily traversed the endothelial cell membranes individually, defensin/Lp(a) complexes lodged on the cell surface. These studies demonstrate that alpha-defensins released from activated or senescent neutrophils stimulate the binding of an atherogenic lipoprotein to the ECM of endothelial cells, a process that may contribute to lipoprotein accumulation in atherosclerotic lesions.

Lysis of plasma clots by urokinase-soluble urokinase receptor complexes.

Single-chain urokinase plasminogen activator (scuPA), the unique form secreted by cells, expresses little intrinsic plasminogen activator activity. scuPA can be activated by proteolytic cleavage to form a two-chain enzyme (tcuPA), which is susceptible to inhibition by plasminogen activator inhibitor type I (PAI-1). scuPA is also activated when it binds to its cellular receptor (uPAR), in which case the protein remains as a single chain molecule with less susceptibility to PAIs. Fibrin clots are invested with PAI-1 derived from plasma and from activated platelets. Therefore, we compared the fibrinolytic activity of complexes between scuPA and recombinant soluble uPAR (suPAR) to that of scuPA, tcuPA, and tcuPA/suPAR complexes. scuPA/suPAR complexes mediated the lysis of plasma-derived fibrin clots 14-fold more extensively than did equimolar concentrations of scuPA and threefold more extensively than did tcuPA or tcuPA/suPAR, respectively. The enhanced catalytic activity of scuPA/suPAR required that all three domains of the receptor be present, correlated with its PAI-1 resistance, was not dependent on fibrin alone, and required a plasma cofactor that was identified as IgG. Human IgG bound specifically to suPAR and scuPA/suPAR as determined by using affinity chromatography and immunoprecipitation. Plasma depleted of IgG lost most of its capacity to promote the fibrinolytic activity of scuPA/suPAR, and the activity of the complex was restored by adding plasma concentrations of purified IgG. These studies indicate that scuPA/suPAR can function as a plasminogen activator in a physiological milieu.

Tissue factor regulates plasminogen binding and activation.

Tissue factor (TF) has been implicated in several important biologic processes, including fibrin formation, atherogenesis, angiogenesis, and tumor cell migration. In that plasminogen activators have been implicated in the same processes, the potential for interactions between TF and the plasminogen activator system was examined. Plasminogen was found to bind directly to the extracellular domain of TF apoprotein (amino acids 1-219) as determined by optical biosensor interaction analysis. A fragment of plasminogen containing kringles 1 through 3 also bound to TF apoprotein, whereas isolated kringle 4 and miniplasminogen did not. Expression of TF on the surface of a stably transfected Chinese hamster ovary (CHO) cell line stimulated plasminogen binding to the cells by 70% more than to control cells. Plasminogen bound to a site on the TF apoprotein that appears to be distinct from the binding site for factors VII and VIIa as judged by a combination of biosensor and cell assays. TF enhanced two-chain urokinase (tcuPA) activation of Glu-plasminogen, but not of miniplasminogen, in a dose-dependent, saturable manner (half maximal stimulation at 59 pmol/L). TF apoprotein induced an effect similar to that of relipidated TF, but a relatively higher concentration of the apoprotein was required (half maximal stimulation at 3.8 nmol/L). The stimulatory effect of TF on plasminogen activation was confirmed when plasmin formation was examined directly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In accord with this, TF inhibited fibrinolysis by approximately 74% at a concentration of 14 nmol/L and almost totally inhibited the binding of equimolar concentrations of plasminogen to human umbilical vein endothelial cells and human trophoblasts. Further, CHO cells expressing TF inhibited uPA-mediated fibrinolysis relative to a wild-type control. TF apoprotein and plasminogen were found to colocalize in atherosclerotic plaque. These data suggest that plasminogen localization and activation may be modulated at extravascular sites through a high-affinity interaction between kringles 1 through 3 of plasminogen and the extracellular domain of TF.

Regulation of single chain urokinase binding, internalization, and degradation by a plasminogen activator inhibitor 1-derived peptide.

The internalization and degradation of cell-associated urokinase type plasminogen activator (uPA) through the alpha2-macroglobulin receptor/low density lipoprotein-related receptor (alpha2MR/LRP) represent important steps in the control of plasmin formation. Complexes between two chain urokinase (tcuPA) and plasminogen activator type 1 are degraded rapidly whereas single chain urokinase (scuPA) is not, suggesting that alpha2MR/LRP requires specific epitopes in the serpin for effective function. We report an alternative mechanism that may contribute to this process. The binding of scuPA to LM-TK- cells that lack the uPA receptor was stimulated by the hexapeptide EEIIMD, corresponding to amino acids 350-355 of plasminogen activator type 1, which contacts the sequence RHRGGS, corresponding to amino acids 179-184 in uPA. EEIIMD increased the Bmax of scuPA binding 4-fold with the half-maximal effect achieved at a peptide concentration of 50 microM. Stimulation was dependent on the charge on the COOH-terminal amino acid but not on the NH2 terminus of the peptide. EEIIMD also stimulated the internalization and degradation of scuPA. Both the binding and internalization of scuPA in the presence of EEIIMD were blocked by recombinant, 39-kDa alpha2MR/LRP-associated protein as well as by an anti-alpha2MR/LRP antibody. EEIIMD also stimulated the binding of scuPA to purified alpha2MR/LRP. EEIIMD had no effect on the binding of tcuPA or of complexes between scuPA and its receptor. These results suggest that EEIIMD regulates the binding of scuPA with alpha2MR/LRP. These findings also suggest a potential mechanism by which scuPA can be cleared which is independent of activation by plasmin or binding to uPA receptor.

Defensin stimulates the binding of lipoprotein (a) to human vascular endothelial and smooth muscle cells.

There is evidence to suggest that elevated plasma levels of lipoprotein (a) [Lp(a)] represent a risk factor for the development of atherosclerotic vascular disease, but the mechanism by which this lipoprotein localizes to involved vessels is only partially understood. In view of studies suggesting a link between inflammation and atherosclerosis and our previous finding that leukocyte defensin modulates the interaction of plasminogen and tissue-type plasminogen activator with cultured human endothelial cells, we examined the effect of this peptide on the binding of Lp(a) to cultured vascular endothelium and vascular smooth muscle cells. Defensin increased the binding of Lp(a) to endothelial cells approximately fourfold and to smooth muscle cells approximately sixfold. Defensin caused a comparable increase in the amount of Lp(a) internalized by each cell type, but Lp(a) internalized as a consequence of defensin being present was not degraded, resulting in a marked increase in the total amount of cell-associated lipoprotein. Abundant defensin was found in endothelium and in intimal smooth muscle cells of atherosclerotic human cerebral arteries, regions also invested with Lp(a). These studies suggest that defensin released from activated or senescent neutrophils may contribute to the localization and persistence of Lp(a) in human vessels and thereby predispose to the development of atherosclerosis.

Soluble human urokinase receptor is composed of two active units.

The mechanism by which single-chain urokinase (scuPA) binds to its receptor (uPAR) is incompletely understood. We report that a fragment comprising the first domain of recombinant soluble uPAR (sDI) as well as a fragment comprising the remaining domains (sDII-DIII) competes with the binding of recombinant full-length soluble uPAR (suPAR) to scuPA with an IC50 = 253 nM and an IC50 = 1569, respectively. sDII-III binds directly to scuPA with Kd = 238 nM. Binding of scuPA to each fragment also induces the expression of plasminogen activator activity. sDI and sDII-DIII (200 nM each) induced activity equal to 66 and 36% of the maximum activity induced by full-length suPAR (5 nM), respectively. Each fragment also stimulates the binding of scuPA to cells lacking endogenous uPAR. Although scuPA binds to sDI and to sDII-DIII through its amino-terminal fragment, the fragments act synergistically to inhibit the binding of suPAR and to stimulate plasminogen activator activity. Furthermore, sDII-DIII retards the velocity and alters the pattern of cleavage of sDI by chymotrypsin. These results suggest that binding of scuPA to more than one epitope in suPAR is required for its optimal activation and association with cell membranes.

Thermotaxis of human trophoblastic cells.

The objective of this study was to determine whether cultured human trophoblasts migrate in response to changes in oxygen tension or temperature. Human trophoblastic cells distributed homogenously within individual wells of standard culture plates were subjected to oxygen and thermal gradients. The redistribution of cells was determined 90 min to 18 h after these gradients had been established. Trophoblastic cells did not migrate in response to gradients of oxygen or carbon dioxide applied in this manner. In contrast, the cells migrated in response to thermal gradients of less than 1 degree C in the direction of the warmer temperature. The response began within minutes, was reversed by a change in the direction of the thermal gradient, and was inhibited at high cell concentrations. Migration was independent of proliferation or protein synthesis, but required microfilament assembly. The capacity of trophoblasts to migrate in response to small difference of temperature within the physiologic range may contribute to the initiation of placental development before contact with the maternal circulation has been established.

Interaction of single-chain urokinase with its receptor induces the appearance and disappearance of binding epitopes within the resultant complex for other cell surface proteins.

Binding of urokinase-type plasminogen activator (uPA) to its glycosylphosphatidylinositol-anchored receptor (uPAR) initiates signal transduction, adhesion, and migration in certain cell types. To determine whether some of these activities may be mediated by associations between the uPA/uPAR complex and other cell surface proteins, we studied the binding of complexes composed of recombinant, soluble uPA receptor (suPAR) and single chain uPA (scuPA) to a cell line (LM-TK- fibroblasts) that does not express glycosylphosphatidylinositol (GPI)-anchored proteins to eliminate potential competition by endogenous uPA receptors. scuPA induced the binding of suPAR to LM-TK- cells. Binding of labeled suPAR/scuPA was inhibited by unlabeled complex, but not by scuPA or suPAR added separately, indicating cellular binding sites had been formed that are not present in either component. Binding of the complex was inhibited by low molecular weight uPA (LMW-uPA) indicating exposure of an epitope found normally in the isolated B chain of two chain uPA (tcuPA), but hidden in soluble scuPA. Binding of LMW-uPA was independent of its catalytic site and was associated with retention of its enzymatic activity. Additional cell binding epitopes were generated within suPAR itself by the aminoterminal fragment of scuPA, which itself does not bind to LM-TK- cells. When scuPA bound to suPAR, a binding site for alpha 2-macroglobulin receptor/LDL receptor-related protein (alpha 2 MR/LRP) was lost, while binding sites for cell-associated vitronectin and thrombospondin were induced. In accord with this, the internalization and degradation of cell-associated tcuPA and tcuPA-PAI-1 complexes proceeded less efficiently in the presence of suPAR. Further, little degradation of suPAR was detected, suggesting that cell-bound complex dissociated during the initial stages of endocytosis. Thus, the interaction of scuPA with its receptor causes multiple functional changes within the complex including the dis-appearance of an epitope in scuPA involved in its clearance from the cell surface and the generation of novel epitopes that promote its binding to proteins involved in cell adhesion and signal transduction.

Defensin modulates tissue-type plasminogen activator and plasminogen binding to fibrin and endothelial cells.

Defensins are naturally occurring antimicrobial peptides that may participate in host defense against microorganisms. We previously reported that the amino acid sequence of leukocyte defensins resembles the lysine-binding site in the kringles of plasminogen and that defensin inhibits fibrinolysis mediated by tissue-type plasminogen activator (tPA) and plasminogen. In the present paper we analyze the mechanisms of this inhibition. Defensin binds specifically to cultured human umbilical vein endothelial cells (HUVEC) (half-maximal binding = 3 microM) as well as to fibrin. At saturating concentrations (5-10 microM), defensin stimulates the maximum binding of plasminogen to HUVEC and to fibrin approximately 10-fold. However, defensin inhibits plasminogen binding to both surfaces at concentrations >10 microM. Defensin also inhibits tPA and plasminogen-mediated fibrinolysis in a dose-dependent manner at all concentrations tested. Fibrinolysis is almost totally inhibited by 6 microM defensin, a concentration that stimulates the binding of plasminogen to fibrin. Discordance between the enhancement of plasminogen binding and its activation cannot be explained by an inhibitory effect of defensin on tPA binding nor by inhibition of plasmin activity, each of which occur only at higher concentrations. Rather, these results suggest that plasminogen bound to fibrin in the presence of defensin is less susceptible to activation by tPA.

Single-chain urokinase-type plasminogen activator bound to its receptor is relatively resistant to plasminogen activator inhibitor type 1.

Urokinase-type plasminogen activator (uPA) is synthesized as single-chain protein (scuPA) with little intrinsic activity. scuPA is activated when it is converted to two-chain urokinase (tcuPA) by plasmin or when it binds as a single-chain molecule to its cellular receptor (uPAR). Previous data indicate that complexes between scuPA and its receptor have somewhat higher affinity for plasminogen than does tcuPA. The current study indicates that plasminogen activator activity of scuPA bound to recombinant, soluble uPAR (suPAR) is also fivefold less sensitive to inhibition by plasminogen activator type 1 (PAI-1) than is soluble or receptor-bound tcuPA. Binding of PaI-1 to suPAR/scuPA complexes is totally reversible and can be overcome by increasing the concentration of plasminogen, suggesting a competitive mechanism of inhibition (Ki = 18 nmol/L). Binding of scuPA to suPAR also retards its cleavage by plasmin. These results indicates that binding of single-chain urokinase to its receptor promotes its activity, retards its inhibition, and protects it from conversion to a two-chain form of the enzyme, a step that may precede its inactivation and clearance from cell surfaces. These results are consistent with a physiologic role for receptor-bound single-chain urokinase as a cellular plasminogen activator.