Binding of plasminogen to cultured human endothelial cells

Endothelial cells are known to release the two major forms of plasminogen activator, tissue plasminogen activator (TPA) and urokinase. We have previously demonstrated that plasminogen (PLG) immobilized on various surfaces forms a substrate for efficient conversion to plasmin by TPA (Silverstein, R. L., Nachman, R. L., Leung, L. L. K., and Harpel, P. C. (1985) J. Biol. Chem. 260, 10346-10352). We now report the binding of human PLG to cultured human umbilical vein endothelial cell (HUVEC) monolayers, utilizing a newly devised cell monolayer enzyme-linked immunosorbent assay system. PLG binding to HUVEC was concentration dependent and saturable at physiologic PLG concentration (2 microM). Binding of PLG was 70-80% inhibited by 10 mM epsilon-aminocaproic acid, suggesting that it is largely mediated by the lysine-binding sites of PLG. PLG bound at an intermediate level to human fibroblasts, poorly to human smooth muscle cells, and not at all to bovine smooth muscle or bovine endothelial cells; unrelated proteins such as human albumin and IgG failed to bind HUVEC. PLG binding to HUVEC was rapid, reaching a steady state within 20 min, and quickly reversible. 125I-PLG bound to HUVEC with an estimated Kd of 310 +/- 235 nM (S.E.); each cell contained 1,400,000 +/- 1,000,000 (S.E.) binding sites. Functional studies demonstrated that HUVEC-bound PLG is activatable by TPA according to Michaelis-Menten kinetics (Km, 5.9 nM). Importantly, surface-bound PLG was activated with a 12.7-fold greater catalytic efficiency than fluid phase PLG. These results indicate that PLG binds to HUVEC in a specific and functional manner. Binding of PLG to endothelial cells may play a pivotal role in modulating thrombotic events at the vessel surface.     

Stimulation and desensitization of tissue plasminogen activator release from human endothelial cells

Tumor-promoting phorbol esters and histamine induce tissue plasminogen activator (tPA) release from human endothelial cells in a dose- and time-dependent manner. Phorbol myristate acetate (PMA) and phorbol dibutyrate (PDBu) increased tPA concentration in the culture medium by eight to 12 times after 24 h with half-maximal stimulation at 13 and 55 nM, respectively. Maximum release by histamine was only half that of the phorbol esters and required 18 microM for half-maximal response. Kinetics of enhanced release was similar with both types of agonists: a 4-h lag period followed by a period of rapid release (4 h in PMA-treated and 10 h in histamine-treated cultures) followed by a decline toward pretreatment rates. The PMA and histamine effects were additive while histamine and thrombin, which also stimulates tPA release in human endothelial cells, were no more effective together than they were alone. Exposure of the cells to PMA, PDBu, or phorbol 12,13-didecanoate caused a loss of responsiveness to second treatment of the homologous agent that was time- and dose-dependent, sustained, and specific to active tumor promoters (half-maximal desensitization = 52 nM PDBu). A partial desensitized state was also established by histamine which resulted in a 60% lower response to a second challenge dose. Histamine-induced desensitization did not interfere with the PMA response. However, PMA-induced desensitization caused a 75% loss of the histamine and a 67% loss of the thrombin effects. These studies indicate that tumor promoters are potent agonists of tPA release from human endothelial cells and establish a desensitized state to further stimulation. Treatment of these cells with histamine has similar effects which may be mediated at least in part by pathways common to phorbol ester stimulation.     

Vascular origin determines plasminogen activator expression in human endothelial cells. Renal endothelial cells produce large amounts of single chain urokinase type plasminogen activator

Positioned at the boundary between intra- and extravascular compartments, endothelial cells may influence many processes through their production of plasminogen activators (PA). Available data have shown that tissue-type plasminogen activator (t-PA) is the major form produced by human endothelial cells. We have compared the molecular forms of PA produced by human endothelial cells from different microvascular and large vessel sources including two different sites within the circulation of the kidney. Using combined immunoactivity assays specific for u-PA and t-PA activity and antigen, we found that both human renal microvascular and renal artery endothelial cells produced high levels of u-PA antigen (60.48 ng/10(5) cells/24 h and 50.42 ng/10(5) cells/24 h, respectively) and corresponding levels of u-PA activity after activation with plasmin. Activity was not evident before plasmin activation, showing that the u-PA produced is almost exclusively as single chain form U-PA. In contrast, human omental microvascular endothelial cells and human umbilical vein endothelial cells produced exclusively t-PA (8.80 ng/10(5) cells/24 h and 2.17 ng/10(5) cells/24 h, respectively). Neither endothelial cell type from human kidney produced plasminogen activator inhibitor, as determined by reverse fibrin autography and titration assays. Agents including phorbol ester, thrombin, and dexamethasone were shown to regulate the renal endothelial cell production and mRNA expression of both u-PA and t-PA. Among the macro- and microvascular endothelial cells tested, only those from the renal circulation produced high levels of single chain form U-PA, suggesting the vascular bed of origin determines the expression of plasminogen activators.     

The endothelial cell tissue plasminogen activator receptor. Specific interaction with plasminogen.

Human endothelial cells (EC) assemble plasmin-generating proteins on their surface. We have previously identified an EC membrane protein (Mr approximately 40,000) which specifically binds tissue plasminogen activator (t-PA) but not urokinase (Hajjar, K.A., and Hamel, N. M. (1990) J. Biol. Chem. 265, 2908-2916). In the present study, t-PA receptor protein (t-PA-R) was purified to apparent homogeneity from a detergent extract of human placental tissue by diisopropyl fluorophosphate-t-PA affinity chromatography and preparative gel electrophoresis. In a solid phase binding assay wells coated with t-PA-R bound both 125I-t-PA and 125I-Lys-plasminogen (PLG), but not 125I-urokinase in a specific, reversible, and noncompetitive fashion. Binding of 125I-Lys-PLG, but not 125I-t-PA, to t-PA-R was 80% inhibited by a 20-100-fold molar excess of the PLG-like lipoprotein(a), or by the lysine analog, epsilon-aminocaproic acid (50 mM). A polyclonal anti-t-PA-R antibody inhibited 66 and 79% of the specific 125I-t-PA and 125I-Lys-PLG binding, respectively, to EC monolayers. Biosynthetically labeled 40-kDa protein coprecipitated with t-PA- or Lys-PLG-Sepharose beads, but not with unconjugated Sepharose. In a functional assay, t-PA associated with immobilized t-PA-R generated 6.4 times more plasmin than an equivalent amount of t-PA in the fluid phase. These results suggest that t-PA-R can bind both t-PA and Lys-PLG in a manner that mimics the EC surface. This protein may play a role in modulating plasmin generation on cell surfaces.     

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.     

Interaction of single-chain urokinase-type plasminogen activator with human endothelial cells

The interaction of urokinase-type plasminogen activators with receptors on the surface of endothelial cells may play an important role in the regulation of fibrinolysis and cell migration. Therefore, we investigated whether human umbilical vein endothelial cells (HUVEC) express receptors for single-chain urokinase (scu-PA) on the cell surface and examined the effect of such binding on plasminogen activator activity. Binding of 125I-labeled scu-PA to HUVEC, performed at 4 degrees C, was saturable, reversible, and specific (k+1 4 +/- 1 X 10(6) min-1 M-1, k-1 6.2 +/- 1.4 X 10(-3) min-1, Kd 2.8 +/- 0.1 nM; Bmax 2.2 +/- 0.1 X 10(5) sites/cell; mean +/- S.E.). Binding of radiolabeled scu-PA was inhibited by both natural and recombinant wild-type scu-PA, high molecular weight two-chain u-PA (tcu-PA), catalytic site-inactivated tcu-PA, an amino-terminal fragment of u-PA (amino acids 1-143), and a smaller peptide (amino acids 4-42) corresponding primarily to the epidermal growth factor-like domain. Binding was not inhibited by low molecular weight urokinase or by a recombinant scu-PA missing amino acids 9-45. Cell-bound scu-PA migrated at its native molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of plasminogen, scu-PA bound to endothelial cells generated greater plasmin activity than did scu-PA in the absence of cells. In contrast, when tcu-PA was added directly to HUVEC, sodium dodecyl sulfate-stable complexes formed with cell or matrix-associated plasminogen activator inhibitors with a loss of plasminogen activator activity. These studies suggest that endothelial cells in culture express high affinity binding sites for the epidermal growth factor domain of scu-PA. Interaction of scu-PA with these receptors may permit plasminogen activator activity to be expressed at discrete sites on the endothelial cell membrane.     

Cyclic AMP potentiates phorbol ester stimulation of tissue plasminogen activator release and inhibits secretion of plasminogen activator inhibitor-1 from human endothelial cells

We have examined the effect of phorbol esters and cAMP elevating compounds on tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) secretion. Phorbol esters induce a time- and dose-dependent increase in tPA release from endothelial cells, while forskolin, isobutylmethylxanthine, dibutyryl cAMP, and 8-bromo-cAMP had no significant stimulatory effect on tPA secretion. However, elevation of cAMP simultaneously with phorbol ester treatment potentiated the phorbol ester-induced release of tPA 6 times from 22.2 ng/ml with phorbol myristate acetate (PMA) alone to 122.1 ng/ml (PMA and forskolin). Potentiation was dose-dependent (half-maximal potentiation = 4 microM forskolin), and tPA release was enhanced at all stimulatory concentrations of PMA with no change in the PMA concentrations causing half-maximal or maximum tPA release. The kinetics of release was also similar in PMA versus PMA-forskolin-treated cells. A 4-h delay was observed, enhanced release was transient, and was followed by the onset of a refractory period. In contrast, elevation of cAMP reduced constitutive secretion of PAI-1 by 30-40% and prevented the increase in PAI-1 secretion stimulated by PMA. Elevated cAMP also decreased the rate of PAI-1 deposition into the endothelial substratum. These studies indicate that activation of a cAMP-dependent pathway(s) in coordination with phorbol ester-induced responses plays a central role in modifying the tPA and PAI-1 secretion from endothelial cells, leading to a profibrinolytic state in the endothelial environment.     

Binding of plasminogen and tissue plasminogen activator to plasmin-modulated factor X and factor Xa

Previous work in our laboratory has suggested that the fibrinolytic enzyme plasmin (Pn) inactivates coagulation factors X (FX) and Xa (FXa) in the presence of Ca(2+) and anionic phospholipid (aPL), producing fragments which bind plasminogen (Pg) and accelerate tissue plasminogen activator (t-PA). Our goals here were to determine if the Pn-mediated fragments of FX or FXa remain associated, whether they directly bind t-PA, and to quantify their interaction with Pg. Binding to aPL, benzamidine-Sepharose, or the active-site inhibitor dansyl-Glu-Gly-Arg-chloromethyl ketone demonstrated that Pn cleavage yielded noncovalent heterodimers of a fragment containing the aPL-binding domain (FXgamma(47) or FXagamma(33)) and a 13-kDa fragment (FXgamma(13) or FXagamma(13)). Both ligand blotting and surface plasmon resonance (SPR) showed that Pn-cleaved FX and FXa bound t-PA directly when Pn-treatment was effected in the presence of aPL and Ca(2+). Using SPR, apparent K(d) values of 1-3 microM and 0.3-0.4 microM were measured directly and by competition for the FXgamma(47/13)-Pg and FXagamma(33/13)-Pg interactions, respectively. For the first time, Pg-binding to a receptor was shown to be Ca(2+) enhanced, although primarily mediated by C-terminal lysine residues. Mathematical modeling of kinetic data suggesting two Pg per FXgamma(47/13) or FXagamma(33/13) was consistent with our conclusion that each subunit of FXgamma(47/13) or FXagamma(33/13) contains a C-terminal lysine. Earlier X-ray structures show that these Lys residues are distal from each other and the membrane, supporting the model where each interacts with a separate Pg. t-PA acceleration by FXgamma(47/13) or FXagamma(33/13) may therefore involve simultaneous presentation of two substrate molecules.     

Binding of type 1 plasminogen activator inhibitor to the extracellular matrix of cultured bovine endothelial cells

The binding of type 1 plasminogen activator inhibitor (PAI-1) to the extracellular matrix (ECM) of cultured bovine aortic endothelial cells was investigated using purified 125I-labeled or L-[35S]methionine-labeled PAI-1 as probes. Little specific binding of latent PAI-1 to ECM previously depleted of endogenous PAI-1 could be demonstrated. In contrast, the guanidine-activated form of PAI-1 bound to ECM in a dose- and time-dependent manner, and binding was saturable. The dissociation constant (Kd) for this interaction was estimated to be 60 nM by Scatchard analysis, and approximately 6 pmol of activated PAI-1 was bound per cm2 of ECM. Binding was relatively specific since unlabeled, activated PAI-1 competed with 35S-labeled PAI-1 for binding to ECM, but latent PAI-1 did not. Moreover, PAI-2, protein C inhibitor (i.e. PAI-3), protease nexin-1, and alpha 2-antiplasmin were not able to compete. Tissue-type plasminogen activator (tPA) also inhibited binding, but diisopropyl fluorophosphate-inactivated tPA did not. Pretreatment of ECM with tPA, urokinase-type PA, or thrombin had no effect on its ability to subsequently bind PAI-1, whereas trypsin, plasmin, and elastase pretreatment greatly reduced its ability to bind PAI-1. Guanidine-activated, radiolabeled PAI-1 resembled active endogenous PAI-1 since it was unstable in solution but stable when bound to ECM. In addition, it formed complexes with tPA that had a relatively low affinity for ECM. These data suggest that ECM of bovine aortic endothelial cells contains a protease-sensitive structure that binds active PAI-1 tightly and relatively selectively and that this association stabilizes PAI-1 against the spontaneous loss of activity that occurs in solution.     

Analysis of binding protein for tissue-type plasminogen activator in human endothelial cells.

The specific binding sites for tissue-type plasminogen activator (t-PA) were investigated in human umbilical vein endothelial cells. After adding 125I-t-PA (M.W. 70 kDa) to endothelial cells in suspension culture, the ligand was recovered from the cell extract after disuccinimidyl suberate treatment as a high molecular complex with M.W. of 90 kDa on SDS-PAGE. The complex reacted to only anti-t-PA IgG but not to anti-PAI-1 IgG immunoblot analysis, indicating a t-PA specific binding protein. 125I-t-PA ligand blotting of the cell extract revealed that the binding protein had M.W. 20 kDa. The binding of 125I-t-PA to endothelial cells was reduced in the presence of an excess amount of t-PA, plasminogen and 6-aminohexanoic acid, indicating that the binding sites were also recognized by plasminogen, and that t-PA and plasminogen were bound via lysine binding sites in the molecule. These findings suggest that human endothelial cells have specific t-PA binding molecules which may be expressed on the cell surface as t-PA receptors.     

Tissue-type plasminogen activator binding to human endothelial cells. Evidence for two distinct binding sites

The endothelium may contribute to fibrinolysis through the binding of plasminogen activators or plasminogen activator inhibitors to the cell surface. Using a solid-phase radioimmunoassay, we observed that antibodies to recombinant tissue-type plasminogen activator (rt-PA) and plasminogen activator inhibitor type 1 (PAI-1) bound to the surface of cultured human umbilical vein endothelial cells (HUVEC). HUVEC also specifically bound added radiolabeled rt-PA with apparent steady-state binding being reached by 1 h at 4 degrees C. When added at low concentrations (less than 5 nM), rt-PA bound with high affinity mainly via the catalytic site, forming a sodium dodecyl sulfate-stable 105-kDa complex which dissociates from the cell surface over time and which could be immunoprecipitated by a monoclonal antibody to PAI-1. rt-PA bound to this high affinity site retained less than 5% of its expected plasminogen activator activity. At higher concentrations, binding did not require the catalytic site and was rapidly reversible. rt-PA initially bound to this site retained plasminogen activator activity. These studies suggest that tissue-type plasminogen activator and PAI-1 are expressed on the surface of cultured HUVEC. HUVEC also express unoccupied binding sites for exogenous tissue-type plasminogen activator. The balance between the expression of plasminogen activator inhibitors and these unoccupied binding sites for plasminogen activators on the endothelial surface may contribute to the regulation of fibrinolysis.     

Stimulation of radiation-impaired plasminogen activator release by phorbol ester in aortic endothelial cells

Ionizing radiation has been reported to affect the fibrinolytic activity of exposed tissue. With cultured bovine aortic endothelial cells, radiation suppresses the release of plasminogen activator to the conditioned media, with a concomitant increase in intracellular plasminogen activator. Thus study was undertaken to determine whether radiation-impaired plasminogen activator release can be modified by phorbol ester. We exposed cultured bovine aortic endothelial cells to a sterilizing dose of 10 Gy of gamma-rays and found the treatment led to cell injury, as evidenced by an increased release of prelabeled chromium, and to a reduction of plasminogen activator in the conditioned media with elevated intracellular plasminogen activator in irradiated cells. Phorbol ester enhanced plasminogen activator activity in both sham-irradiated and irradiated endothelial cells. It was interesting to note that the increased plasminogen activator in phorbol ester-stimulated sham-irradiated cells was largely retained inside the cell, while it was released to the conditioned media in irradiated cells. Apparently, altered plasminogen activator activity of radiation-sterilized endothelial cells can be modified by exogenous stimuli.     

Plasmin and the regulation of tissue-type plasminogen activator biosynthesis in human endothelial cells.

Plasmin inhibited the biosynthesis of tissue-type plasminogen activator (tPA) antigen by human umbilical vein endothelial cells (HUVEC) in a dose-dependent manner. The amount of tPA antigen found in the 24-h conditioned medium of cells treated with 100 nM plasmin for 1 h was 20-30% of that in the control group. However, in contrast to tPA, such treatment led to a 3-fold increase in plasminogen activator inhibitor (PAI) activity, whereas the amount of PAI type 1 antigen was unchanged. The effects of plasmin on HUVEC were binding- and catalytic activity-dependent and were specifically blocked by epsilon-aminocaproic acid. Microplasmin, which has no kringle domains, was less effective in reducing tPA antigen biosynthesis or enhancing PAI activity in HUVEC. Kringle domains of plasmin affected neither tPA antigen nor PAI activity of the cells. Other proteases including chymotrypsin, trypsin, and collagenase at comparable concentrations did not have a significant effect on the biosynthesis of tPA antigen or PAI activity of HUVEC. Thrombin stimulated the biosynthesis of tPA and PAI-1 antigens by HUVEC. Thrombin also stimulated an increase in the protein kinase activity in HUVEC, whereas plasmin inhibited the protein kinase activity of the cells. It is possible that plasmin regulates the biosynthesis of tPA in HUVEC through the signal transduction pathway involving protein kinase.     

人组织型纤溶酶原激活剂突变体微小基因的构建

tPA基因全长约36kb,至少由13个内含子分隔为14个外显子。根据tPA的第一、二外显子的编码情况,考虑建立从第二至第六外显子序列在内的tPA微小基因。即将tPA的部分基因组序列与LAtPA cDNA的序列在第六外显子的NarI位点处相连。     

Binding of human thrombin to cultured human endothelial cells.

Binding of thrombin to monolayer cultures of human umbilical vein endothelium is studied. Binding is measured as inhibition by unlabeled ligand of the binding of 125I-thrombin to the cells. Radioactivity bound to cultures at equilibrium is measured after draining but not washing the cells. To correct for unremoved supernatant, 131I-albumin is included as a second label in the medium. Equilibrium between bound and free thrombin is attained within 1 min, and Scatchard analysis indicates a population of approximately 3 x 10(3) sites/cell with a dissociation constant of 10(-10) M, and a larger population with a dissociation constant greater than 10(-8) M. The two populations of sites are also indicated by a biphasic dissociation of bound label. Thrombin inactivated with diisopropyl fluorophosphate binds to the same receptor, with an affinity similar to that of active thrombin. Binding is unaffected by albumin (an acidic protein) and cytochrome c (a basic protein). Cultures of umbilical cord smooth muscle and fibroblasts bind thrombin at least 100 times more weakly than endothelium, and no binding to erythrocytes or a monolayer culture of mouse neuroblastoma is detected.