Enhanced Plasminogen Activator Research Articles

Enhanced plasminogen activation by staphylokinase in the presence of streptokinase β/βγ domains: Plasminogen kringles play a role

Presence of isolated β or βγ domains of streptokinase (SK) increased the catalytic activity of staphylokinase (SAK)–plasmin (Pm) complex up to 60%. In contrast, fusion of SK β or βγ domains with the C-terminal end of SAK drastically reduced the catalytic activity of the activator complex. The enhancement effect mediated by β or βγ domain on Pg activator activity of SAK–Pm complex was reduced greatly (45%) in the presence of isolated kringles of Pg, whereas, kringles did not change cofactor activity of SAK fusion proteins (carrying β or βγ domains) significantly. When catalytic activity of SAK–microPm (catalytic domain of Pm lacking kringle domains) complex was examined in the presence of isolated β and βγ domains, no enhancement effect on Pg activation was observed, whereas, enzyme complex formed between microplasmin and SAK fusion proteins (SAKβ and SAKβγ) displayed 50–70% reduction in their catalytic activity. The present study, thus, suggests that the exogenously present β and βγ interact with Pg/Pm via kringle domains and elevate catalytic activity of SAK–Pm activator complex resulting in enhanced substrate Pg activation. Fusion of β or βγ domains with SAK might alter these intermolecular interactions resulting in attenuated functional activity of SAK.

Enhanced plasminogen activator inhibitor-1 expression in transgenic mice with hepatocyte-specific overexpression of superoxide dismutase or glutathione peroxidase.

In this study, we developed a double-transgenic mouse model allowing hepatocyte-specific and regulated expression of the redox-modifying enzymes copper/zinc superoxide dismutase (SOD) and glutathione peroxidase (GPX) by using a tetracycline-regulatable gene expression system. Within this system, the SOD and GPX level can be regulated deliberately by addition or removal of doxycycline hydrochloride to the drinking water. As reactive oxygen species (ROS) have been implicated in a number of pathological conditions, such as atherosclerosis, thrombosis, or liver fibrosis, processes that are also frequently associated with enhanced levels of plasminogen activator inhibitor-1 (PAI-1), it was the aim of the present study to investigate the influence of SOD and GPX overexpression on the regulation of PAI-1. PAI-1 mRNA and protein levels in tetracycline transactivator-dependent SOD-overexpressing double-transgenic mice reached values 2.5- to threefold above the normal mRNA level. By applying doxycycline, a deinduction of the PAI-1 levels was observed. By using the same protocol, PAI-1 mRNA and protein levels were enhanced in GPX double-transgenic mice, and again this response was blunted by the addition of doxycycline. These studies provide some new information regarding the role of ROS within the proteolytic processes in hepatocytes that require PAI-1.

Nonlysine-analog plasminogen modulators promote autoproteolytic generation of plasmin(ogen) fragments with angiostatin-like activity.

We recently discovered several nonlysine-analog conformational modulators for plasminogen. These include SMTP-6, thioplabin B and complestatin that are low molecular mass compounds of microbial origin. Unlike lysine-analog modulators, which increase plasminogen activation but inhibit its binding to fibrin, the nonlysine-analog modulators enhance both activation and fibrin binding of plasminogen. Here we show that some nonlysine-analog modulators promote autoproteolytic generation of plasmin(ogen) derivatives with its catalytic domain undergoing extensive fragmentation (PMDs), which have angiostatin-like anti-endothelial activity. The enhancement of urokinase-catalyzed plasminogen activation by SMTP-6 was followed by rapid inactivation of plasmin due to its degradation mainly in the catalytic domain, yielding PMD with a molecular mass ranging from 68 to 77 kDa. PMD generation was observed when plasmin alone was treated with SMTP-6 and was inhibited by the plasmin inhibitor aprotinin, indicating an autoproteolytic mechanism in PMD generation. Thioplabin B and complestatin, two other nonlysine-analog modulators, were also active in producing similar PMDs, whereas the lysine analog 6-aminohexanoic acid was inactive while it enhanced plasminogen activation. Peptide sequencing and mass spectrometric analyses suggested that plasmin fragmentation was due to cleavage at Lys615-Val616, Lys651-Leu652, Lys661-Val662, Lys698-Glu699, Lys708-Val709 and several other sites mostly in the catalytic domain. PMD was inhibitory to proliferation, migration and tube formation of endothelial cells at concentrations of 0.3-10 microg.mL(-1). These results suggest a possible application of nonlysine-analog modulators in the treatment of cancer through the enhancement of endogenous plasmin(ogen) fragment formation.

Laparoscopic-type environment enhances mesothelial cell fibrinolytic activity in vitro via a down-regulation of plasminogen activator inhibitor-1 activity

Background Fewer intraperitoneal adhesions have been observed after laparoscopic surgery compared with conventional techniques. The aim of this study is to assess the effect of the pneumoperitoneum on mesothelial cell fibrinolytic activity by use of an in vitro model. Methods Human peritoneal mesothelial cells were seeded onto 24-well plates and incubated in carbon dioxide or helium at 5 mm Hg for 4 hours or standard culture conditions. Supernatant was removed for analysis at 0, 24, 48, and 72 hours after gas incubation and analyzed for plasminogen activator activity, total tissue plasminogen activator (tPA), and total plasminogen activator inhibitor-1 (PAI-1) concentrations by use of an enzyme-linked immunosorbent assay. The effect of different insufflation pressures (0, 7, and 14 mm Hg) was also examined. Results Enhanced plasminogen activator activity was observed at 48 hours and 72 hours from cells exposed to CO 2 ( P<.04 each) and helium ( P<.05 each) compared with control. This was associated with a decrease in PAI-1 concentrations at 48 and 72 hours in both the CO 2 and helium groups compared with control ( P<.03 each, CO 2 vs control; and P<.04 each, helium vs control). No changes in tPA levels were observed. Changes in insufflation pressures did not affect plasminogen activator activity. Conclusions These results suggest that incubation of human mesothelial cells with both CO 2 and helium in the absence of oxygen enhances mesothelial cell fibrinolytic activity because of a reduction in PAI-1 concentrations. These changes may participate in the observed reduction in adhesions after laparoscopic surgery relative to open surgery.

Endogenous plasmin converts Glu-plasminogen to Lys-plasminogen on the monocytoid cell surface

Recently, we showed that localization of Glu-plasminogen on cell surfaces enhances its conversion to Lys-plasminogen by exogenous plasmin. This leads to stimulation of plasminogen activation because Lys-plasminogen is the preferred substrate on cell surfaces. Here, we show that Glu-plasminogen was converted to Lys-plasminogen on monocytoid cells in the absence of exogenous plasmin. Culture of cells under serum-free conditions did not affect this conversion, suggesting that the enzymatic activity was cell-derived. Therefore, we tested whether endogenous monocytoid plasminogen could provide a source of plasmin to convert cell-associated Glu-plasminogen to Lys-plasminogen because plasmin is the only enzyme known to effect this reaction. We used a recombinant human plasminogen mutant, [D(646)E]Pg, which can be cleaved by plasminogen activators, but cannot catalyze the generation of Lys-plasminogen. Upon incubation with either THP-1 or U937 monocytoid cells, 35 and 38%, respectively, of the cell-bound ligand was converted to Lys-[D(646)E]Pg. Trasylol, alpha2-antiplasmin, and an anticatalytic antiplasminogen monoclonal antibody decreased Lys-[D(646)E]Pg formation to < 5% on monocytoid cells, consistent with a plasmin-dependent mechanism. Plasminogen was detected in these cells by Northern blotting and RT-PCR. Our results suggest that plasmin converts cell-bound Glu-plasminogen to Lys-plasminogen and that this enzyme is produced by activation of monocytoid plasminogen by endogenous monocytoid plasminogen activators to enhance plasminogen activation on the monocytoid cell surface.

Enhancement of plasminogen activation by surfactin C: augmentation of fibrinolysis in vitro and in vivo

The reciprocal activation of plasminogen and prourokinase (pro-u-PA) is an important mechanism in the initiation and propagation of local fibrinolytic activity. We have found that a bacterial lipopeptide compound, surfactin C (3–20 μM), enhances the activation of pro-u-PA in the presence of plasminogen. This effect accompanied increased conversions of both pro-u-PA and plasminogen to their two-chain forms. Surfactin C also elevated the rate of plasminogen activation by two-chain urokinase (tcu-PA) while not affecting plasmin-catalyzed pro-u-PA activation and amidolytic activities of tcu-PA and plasmin. The intrinsic fluorescence of plasminogen was increased, and molecular elution time of plasminogen in size-exclusion chromatography was shortened in the presence of surfactin C. These results suggested that surfactin C induced a relaxation of plasminogen conformation, thus leading to enhancement of u-PA-catalyzed plasminogen activation, which in turn caused feedback pro-u-PA activation. Surfactin C was active in enhancing [ 125I]fibrin degradation both by pro-u-PA/plasminogen and tcu-PA/plasminogen systems. In a rat pulmonary embolism model, surfactin C (1 mg/kg, i.v.) elevated 125I plasma clot lysis when injected in combination with pro-u-PA. The present results provide first evidence that pharmacological relaxation of plasminogen conformation leads to enhanced fibrinolysis in vivo.

Recombinant adeno-associated virus vector-transduced vascular endothelial cells express the thrombomodulin transgene under the regulation of enhanced plasminogen activator inhibitor-1 promoter.

We were able to facilitate plasminogen activator inhibitor 1 (PAI-1) promoter activity approximately by 14-fold using an enhancer element. This enhanced PAI-1 promoter has a strong basal activity, comparable to CAG promoter activity, and has a response similar to the PAI-1 promoter with respect to TGFbeta 1 and TNFalpha stimulation. The characteristics of the enhanced PAI-1 promoter are thought to be suited to timely and tissue-specific expression of anticoagulant molecules in the vascular cells. Thus, we developed recombinant adeno-associated virus (rAAV) vectors using the enhanced PAI-1 promoter and were successful in transducing vascular endothelial cells to express the thrombomodulin transgene under the regulation of the enhanced PAI-1 promoter in vitro. Thromobomodulin transgene expression driven by the enhanced PAI-1 promoter in rAAV vector-transduced cultured endothelial cells was between 600- and 1000-fold higher than constitutive thrombomodulin gene expression in cultured human umbilical vein endothelial cells and was up-regulated by TGFbeta1 and TNFalpha stimulation which may down-regulate endogenous thrombomodulin gene expression in endothelial cells. The brain vascular endothelial cells of Mongolian gerbils could also be transduced by the same rAAV vector in vivo. Transduction of endothelial cells by rAAV vectors to express enhanced PAI-1 promoter-driven transgenes may be a useful gene therapy approach for vascular diseases.

Recombinant Semliki Forest virus enhanced plasminogen activator inhibitor 1 expression and storage in the megakaryocytic cell line MEG-01.

Platelet plasminogen activator inhibitor I (PAI-1), a trace alpha-granule protein, is a key physiological regulator of fibrinolysis. Because information on the packaging of PAI-1 into alpha-granules during megakaryocytopoiesis may reveal novel approaches for controlling hemostasis, this study investigated basal, plasmid-mediated, and alphavirus-mediated PAI-1 packaging into alpha-granules-like structures in the megakaryocytic cell line MEG-01. Differentiation of MEG-01 cells with phorbol myristate acetate (PMA) was observed to result in a four-fold increase in both secreted and cell-associated PAI-1 antigen over a four day period. Subcellular fractionation of PMA-treated MEG-01 cells on 45% self-forming Percoll gradients was employed to separate low density membrane and Golgi-rich fractions from a high density granule-containing region. A subsequent 30-60% pre-formed Percoll gradient was employed to remove contaminating lysosomes from the PAI-1/glycoprotein IIbIIIa-containing granules. Electron microscopy showed that these MEG-01 granules share a similar size distribution (350-600 nm) and morphology to platelet alpha-granules. PAI-1 (40 ng/mg protein) in isolated MEG-01 storage granules was approximately 10% of the levels present in isolated platelet alpha-granules. To elevate PAI-1 production/storage, two expression systems were investigated. Experiments with plasmids encoding PAI-1 and beta-galactosidase resulted in low transfection efficiency (0.001%). In contrast, Semliki Forest virus (SFV)-mediated gene transfer increased cellular PAI-1 by 31-fold (1,200 ng/10(6) cells at 10 MOI) in comparison to mock-infected cells. Pulse-chase experiments demonstrated that SFV/PAI-1 mediated gene expression could enhance PAI-1 storage 6-9-fold, reaching levels present within platelets. To document the ability of PAI-1 to be stored in a rapidly releasable form in MEG-01 cells, we isolated platelet-like particles from the media conditioned by the cells and examined secretagogue-induced release of PAI-1. Particles from SFV/PAI-1 infected cells display a 5-fold enhanced secretion of PAI-1 following treatment with ADP in comparison to particles incubated in the absence of secretagogue. These results suggest that SFV mediated gene expression in MEG-01 cells provides a useful framework for analyzing the production and storage of alpha-granule proteins.

Angiogenin and Its Functions in Angiogenesis

The review is devoted to angiogenin, one of the factors that induce formation of blood vessels, which is unique in that it is a ribonuclease. Consideration is given to the tertiary structure of human angiogenin; the catalytic and cell receptor binding sites, their significance for angiogenic activity; the human angiogenin gene structure, chromosomal localization, and expression; the specificity of angiogenin as a ribonuclease and abolishment of protein synthesis; the nuclear localization of angiogenin in proliferating endothelial cells and its significance for angiogenic activity; angiogenin binding to cell surface actin as a plausible mechanism of inducing neovascularization (enhancement of plasminogen activation by actin, stimulation of the cell-associated proteolytic activity; promotion of the cultured cell invasiveness); modulation of mitogenic stimuli in endothelial, smooth muscle, and fibroblast cells by angiogenin. The importance of angiogenin as an adhesive molecule for endothelial and tumor cells is discussed too, as well as the modulation of tubular morphogenesis by bovine angiogenin, prevention of tumor growth in vivoby angiogenin antagonists, prospects of the use of angiogenin and angiogenin-encoding recombinant plasmids and vaccinia virus in therapeutic practice.

Purification, Cloning, and Characterization of a Profibrinolytic Plasminogen-binding Protein, TIP49a

The plasminogen receptors responsible for enhancing cell surface-dependent plasminogen activation expose COOH-terminal lysines on the cell surface and are sensitive to proteolysis by carboxypeptidase B (CpB). We treated U937 cells with CpB, then subjected membrane fractions to two-dimensional gel electrophoresis followed by ligand blotting with (125)I-plasminogen. A 54-kDa protein lost the ability to bind (125)I-plasminogen after treatment of intact cells and was purified by two-dimensional gel electrophoresis and then sequenced by mass spectrometry. Two separate amino acid sequences were obtained and were identical to sequences contained within human and rat TIP49a. The cDNA for the 54-kDa protein matched the human TIP49a sequence, and encoded a COOH-terminal lysine, consistent with susceptibility to CpB. Antibodies against rat TIP49a recognized the plasminogen-binding protein on two-dimensional Western blots of U937 cell membranes. Human (125)I-Glu-plasminogen bound specifically to TIP49a protein, and binding was inhibited by epsilon-aminocaproic acid. A single class of binding sites was detected, and a K(d) of 0.57 +/- 0.14 microm was determined. TIP49a enhanced plasminogen activation 8-fold compared with the BSA control, and this was equivalent to the enhancement mediated by plasmin-treated fibrinogen. These results suggest that TIP49a is a previously unrecognized plasminogen-binding protein on the U937 cell surface.

Importance of GlcUAβ1-3GalNAc(4 S,6 S) in chondroitin sulfate E for t-PA- and u-PA-mediated Glu-plasminogen activation

Chondroitin sulfate E (CSE) markedly enhanced plasminogen activation by tissue plasminogen activators (t-PAs) and urinary plasminogen activator (u-PA) in vitro; in the presence of 10 μg/ml of CSE, the potentiation factors of single chain of t-PA, two chain of t-PA and u-PA were 400, 140 and 130, respectively. Though the potentiation activity of CSE gradually decreased when it was depolymerized by chondroitinase ABC, the specific disaccharide from CSE still showed significant activity. Glycosaminoglycan (GAG) from sea cucumber, which possesses a very similar core structure to CSE, but has additional sulfated fucose branches exhibit very weak activity. These results suggested that the minimal structural requirement in CSE to enhance plasminogen activation by plasminogen activators is GlcUAβ1-3GalNAc(4 S,6 S) and that additional branching sugars abolish the activity.

Effect of thrombomodulin on plasminogen activation

Thrombomodulin (TM), a thrombin receptor on the endothelial cell surface, plays an important role in the regulation of blood coagulation. In this study, recombinant TM containing six epidermal growth factor-like structures (D2), and serine and threonine (Ser/Thr)-rich domain (D3), TMD23 (corresponding to Ala224-Ser497), was prepared by a recombinant baculovirus expression system and purified to apparent homogeneity by DEAE-Sepharose CL-6B and affinity nickel-chelating column chromatographies. TMD23 in combination with thrombin could effectively activate protein C. TMD23 alone could enhance Glu-plasminogen activation by single-chain urokinase-type plasminogen activator in a dose-dependent manner. The specific binding of plasminogen to TMD23 was also demonstrated and the binding was inhibited by ε-aminocaproic acid. In conclusion, our results suggest that TMD23 could specifically bind to plasminogen and effectively enhance plasminogen activation.

Angiotensin II has multiple profibrotic effects in human cardiac fibroblasts.

Angiotensin II (Ang II) is implicated in cardiac remodeling and is increasingly recognized for its profibrotic activity. Because little is known about the direct cellular effects of Ang II on human cardiac fibroblasts, we isolated fibroblasts from ventricles of explanted human hearts and added Ang II (100 nmol/L) to determine its role in growth, extracellular matrix accumulation, and adhesion. To assess which Ang II receptor is involved, Ang II was added in the presence of irbesartan (10 micromol/L), a specific AT(1) receptor antagonist; PD 123319 (10 micromol/L), a specific AT(2) receptor antagonist, or divalinil (100 nmol/L), an AT(4) receptor inhibitor. In human ventricles (n=13), message levels of atrial natriuretic peptide and AT(1) receptor were inversely correlated, which suggests a decrease in AT(1) receptor expression with progressive heart failure. Northern analysis and fluorescence-activated cell sorting demonstrated AT(1) receptor in cultured human cardiac fibroblasts. Ang II increased mitogen-activated protein kinase activity and in DNA synthesis (5-fold, P<0.01) stimulated a 2-fold increase in transforming growth factor-beta(1) (P<0.05) mRNA levels at 2 hours and a 2-fold increase in laminin (P<0.05) and fibronectin (P<0.05) mRNA levels at 24 hours. Ang II also enhanced plasminogen activator inhibitor-1 expression, which inhibits metalloproteinases that degrade the extracellular matrix. All of these effects were inhibited by irbesartan but not PD 123319 or divalinil. In addition, Ang II increased cardiac fibroblast attachment to collagens I and III, which was associated with an increase in focal adhesion kinase activity. Activation of the AT(1) receptor in human heart promotes fibrosis. Ang II plays a novel role in stimulation of plasminogen activator inhibitor-1 expression and adhesion of cardiac fibroblasts to collagen.

Discriminating between Cell Surface and Intracellular Plasminogen-binding Proteins: Heterogeneity in Profibrinolytic Plasminogen-binding Proteins on Monocytoid Cells

When plasminogen binds to cell surfaces, its activation is markedly enhanced compared to soluble plasminogen. Although several distinct molecules may contribute to plasminogen binding to a given cell type, the subset of plasminogen receptors responsible for enhancing plasminogen activation expose a carboxyl-terminal lysine on the cell surface and are sensitive to proteolysis by carboxypeptidase B (CpB). To distinguish this subset of plasminogen receptors from plasminogen-binding proteins that are not profibrinolytic, we treated intact U937 monocytoid cells and peripheral blood monocytes with CpB to remove exposed carboxyl-terminal lysines, and subjected the membrane proteins to two-dimensional gel electrophoresis followed by ligand blotting with 125I-plasminogen. Western blotting was performed with antibodies against previously characterized candidate plasminogen receptors to identify plasminogen-binding proteins on the two-dimensional ligand blots. Densitometry of autoradiograms of the 125I-plasminogen ligand blots of U937 cell membranes revealed that membrane-associated alpha-enolase, actin and annexin II showed minimal changes in 125I-plasminogen binding following CpB treatment of intact cells, suggesting that these proteins are not accessible to CpB on the U937 cell surface and most likely do not serve as profibrinolytic plasminogen receptors on U937 cells. In contrast, densitometry of autoradiograms of 125I-plasminogen ligand blots of monocyte membranes revealed that 125I-plasminogen binding to alpha-enolase was reduced 71% by treatment of intact cells with CpB, while binding to annexin II was reduced 14%. Thus, a portion of membrane-associated alpha-enolase and annexin II expose carboxyl terminal lysines that are accessible to CpB on the peripheral blood monocyte surface, suggesting that these molecules may serve as profibrinolytic plasminogen receptors on monocytes. Our data suggest that U937 cells and peripheral blood monocytes have distinct sets of molecules that constitute the population of cell surface profibrinolytic plasminogen-binding proteins. Furthermore, our data suggest that while several plasminogen-binding proteins with carboxyl terminal lysines are associated with cell membranes, only a small subset of these proteins expose a carboxyl terminal lysine that is accessible to CpB on the cell surface.

Endothelin-1 enhances plasminogen activator inhibitor-1 production by human brain endothelial cells via protein kinase C-dependent pathway.

The effects of endothelin-1 (ET-1) on the production of plasminogen activator inhibitor 1 (PAI-1) and tissue plasminogen activator (t-PA) by human brain-derived endothelial cells in culture were studied. At 100 nmol/L, ET-1 increased PAI-1 production by 88+/-6% within 72 hours, and increased PAI-1 mRNA expression within 1 hour of stimulation; there was no significant effect on t-PA production. PAI-1 activity was also examined and found to increase with ET-1 treatment. Suboptimal concentrations of ET-1 and tumor necrosis factor-alpha (TNF-alpha) acted synergistically to increase PAI-1 production. ET-1 activated protein kinase C and cAMP-dependent protein kinase pathways within 3 to 5 minutes of treatment, with the peak at 10 minutes. Activation of protein kinase C by phorbol-12-myristate-13-acetate (PMA) resulted in increased PAI-1 production, whereas activation of the cAMP-dependent protein kinase by forskolin or dibutyryl cAMP (dBu-cAMP) significantly decreased PAI-1 production. However, simultaneous activation of protein kinase C by PMA and cAMP-dependent protein kinase by dBu-cAMP only slightly attenuated PMA-induced PAI-1 increase. Inhibition of protein kinase C by GF-109213X abolished the effects of ET-1. These results demonstrate that ET-1 and TNF-alpha function synergistically to induce procoagulant activity of brain endothelial cells in a process that involves a protein kinase C-dependent pathway.

Regulation of Tissue Plasminogen Activator Activity by Cells: DOMAINS RESPONSIBLE FOR BINDING AND MECHANISM OF STIMULATION

A number of cell types have previously been shown to bind tissue plasminogen activator (tPA), which in some cases can remain active on the cell surface resulting in enhanced plasminogen activation kinetics. We have investigated several cultured cell lines, U937, THP1, K562, Molt4, and Nalm6 and shown that they bind both tPA and plasminogen and are able to act as promoters of plasminogen activation in kinetic assays. To understand what structural features of tPA are involved in cell surface interactions, we performed kinetic assays with a range of tPA domain deletion mutants consisting of full-length glycosylated and nonglycosylated tPA (F-G-K1-K2-P), DeltaFtPA (G-K1-K2-P), K2-P tPA (BM 06.022 or Reteplase), and protease domain (P). Deletion variants were made in Escherichia coli and were nonglycosylated. Plasminogen activation rates were compared with and without cells, over a range of cell densities at physiological tPA concentrations, and produced maximum levels of stimulation up to 80-fold with full-length, glycosylated tPA. Stimulation for nonglycosylated full-length tPA dropped to 45-60% of this value. Loss of N-terminal domains as in DeltaFtPA and K2P resulted in a further loss of stimulation to 15-30% of the full-length glycosylated value. The protease domain alone was stimulated at very low levels of up to 2-fold. Thus, a number of different sites are involved in cell interactions especially within finger and kringle domains, which is similar to the regulation of tPA activity by fibrin. A model was developed to explain the mechanism of stimulation and compared with actual data collected with varying cell, plasminogen, or tPA concentrations and different tPA variants. Experimental data and model predictions were generally in good agreement and suggest that stimulation is well explained by the concentration of reactants by cells.

Evaluation of the Factors Contributing to Fibrin–dependent Plasminogen Activation

Polymerized fibrin strongly enhances tissue plasminogen activator (tPA)-mediated plasminogen activation, concomitant with exposure of 'fibrin-specific' epitopes at 'Aalpha148-160' and 'gamma312-324'. To investigate which aspects of polymerization are involved in these activities, we explored the fibrin polymerization process by evaluating the ability of factor XIIIa-crosslinked fibrinogen polymers to expose 'fibrin-specific' epitopes and enhance plasminogen activation. Crosslinked normal fibrinogen, fibrinogen with deficient [des Bbeta1-42] or defective [Birmingham (AalphaR16H)] fibrin 'D:E' assembly sites ('E(A)'), or with defective end-to-end self-association sites ('D:D') [Cedar Rapids (gammaR275C)], exposed both 'fibrin-specific' epitopes and enhanced tPA-dependent plasminogen activation, whereas non-crosslinked fibrinogens showed minimal or no such activities. Epitope expression in crosslinked fibrinogen was retained in the presence of the fibrin E(A) site peptide homolog, gly-pro-arg-pro (GPRP), which inhibits fibrin D:E association, except for the Aalpha148-160 epitope in des Bbeta1-42 fibrinogen, which was not expressed. Fibrin prepared from crosslinked normal or abnormal fibrinogen, except for the des Bbeta1-42 fibrin epitopes, which were reduced or absent, expressed 'fibrin-specific' epitopes even in the presence of GPRP, which otherwise impairs such expression in non-crosslinked fibrin. Epitope exposure in fibrin prepared from non-crosslinked fibrinogen was nearly normal in Cedar Rapids fibrin (heterozygous D:D defect), but reduced in Birmingham fibrin (heterozygous E(A) defect), nil in des Bbeta1-42 fibrin (E(A) deficient), and absent in all cases in the presence of GPRP. In contrast, plasminogen activation stimulatory activity that had been exposed in crosslinked normal fibrinogen or in crosslinked des Bbeta1-42 or Cedar Rapids fibrin, was preserved to a large extent in the presence of GPRP, suggesting that once enhanced stimulatory activity and epitopes are exposed, they are not completely reversible. The findings indicate that end-to-end intermolecular associations (D:D) are not critical for 'fibrin-specific' epitope exposure, but that polymerization brought about in fibrinogen through factor XIIIa crosslinking, or in fibrin through 'D:E' interactions, is necessary for 'fibrin-specific' (more correctly, 'polymerization-specific') epitope exposure and enhancement of plasminogen activation.

Binding of Human Single Chain Urokinase to Chinese Hamster Ovary Cells and Cloning of Hamster u-PAR

The plasminogen activator, urokinase (u-PA), interacts with the u-PA receptor (u-PAR) which results in enhanced plasminogen activation on cell surfaces. The u-PAR is comprised of three homologous domains of approximately 90 amino acids, defined by the pattern of disulfide bonds. Domain 1 (amino acids 1-87) binds the ligand. Within this domain, Y57, and a site between residues 47 and 53, have been suggested as ligand contact points. Intradomain interactions also contribute to the interaction of u-PA and u-PAR. The interaction of u-PA with its receptor exhibits some species specificity. Previous studies have shown that human u-PA does not bind to the murine u-PAR and murine u-PA does not recognize human u-PAR. However, human u-PA does interact with bovine cells with high affinity. To further examine the interaction of the human ligand with the u-PAR of a different species, we characterized the binding of human 125I single chain u-PA (scu-PA) to hamster cells. Chinese Hamster Ovary (CHO) cells bound human scu-PA with high affinity and capacity (Kd = 1.13 +/- 0.8 nM; Bmax = 5.45 +/- 0.98 x 10(4) sites/cell). In ligand blotting with human 125I-scu-PA, major bands migrating with apparent Mr's of 74, 49 and 38 kDa were observed. The cDNA of hamster u-PAR was cloned and a single 1.4 kb mRNA species identified in Northern blots of CHO cell RNA. For comparison, we also cloned u-PAR cDNA from human THP-1 cells. Our human sequence was identical to those published for U937 and endothelial cells. These sequences were aligned with the published sequences for the murine, bovine and rat u-PAR's to obtain a consensus sequence for five species. The cysteine residues could be aligned for all species. Y57, which has been suggested as a ligand contact point was also conserved across species. In addition, 5 of the 7 amino acids between amino acids 47 and 53 were conserved in all species. Gly283, the most likely glycosyl-phosphatidyl inositol attachment site, was also conserved in all species. The conservation of these amino acid residues across all five species, attests to their importance in u-PAR function. In addition, the results of our studies suggest that the hamster may be a useful small animal model for studies of human urokinase function.

Prothrombotic Consequences of the Oxidation of Fibrinogen and their Inhibition by Aspirin.

Oxidant stress leads to covalent oxidative modification of several plasma proteins, chief among which is fibrinogen. Aspirin can nonenzymatically acetylate fibrinogen's lysine residues, the functional groups most susceptible to oxidative modification. Because oxidation of fibrinogen may occur in the atheromatous environment, we studied the effects of oxidative modification on fibrinogen function and the consequences of acetylation by aspirin on fibrinogen's susceptibility to oxidation and functional properties. We exposed fibrinogen to Fe(3+) ascorbate for 1 hour and showed that the carbonyl/protein molar ratio increased from 0.71 +/- 0.18 to 2.86 +/- 0.50 mol carbonyl/mol protein (P < 0.02) with an accompanying reduction in the alpha-helical content of the protein from 34% to 29%. Exposure of fibrinogen to aspirin led to acetylation of lysine residues and inhibition of oxidation. Oxidized fibrinogen was more readily able to form fibrin, and acetylation prevented this enhancement of clot formation. Oxidized fibrinogen also supported platelet aggregation better than did native, unoxidized fibri ogen, and aretylation of fibrinogen prior to oxidation prevented the enhanced platelet aggregation. Oxidized fibrinogen was less effective in stimulating plasminogen activation by tissue-type plasminogen activator (tPA), with a catalytic efficiency that was reduced by 88% compared with native, unoxislized fibrinogen; metylated fibrinogen, by contrast, enhanced plasminogen activation by t-PA with a catalytic efficiency that was increased by 18% compared with native, anoxidized fibrinogen (P < 0.05) and was increased by 51% compared with oxidized fibrinogen (P < 0.05). Acetylation prevented the reduction in catalytic efficiency induced by oxidation. These data show that oxidized fibrinogen manifests prothrombotic effects that can be prevented by acetylation and suggest that inhibition of fibrinogen oxidation may be an additional antithrombotic benefit of aspirin therapy.

Denatured Proteins as Cofactors for Plasminogen Activation

Activation of covalently intact plasminogen by tissue-type plasminogen activator (tPA) is facilitated by a majority of proteins subjected to denaturing conditions. Except for heat-denatured apoferritin, the denatured proteins examined require partial proteolysis by plasmin for cofactor activity. The same proteins in their native state are resistant to proteolysis with plasmin and develop no activity. Denatured preparations of apoferritin, antithrombin, α1-protease inhibitor, α2-macroglobulin, and albumin also accelerate des1–77-plasminogen activation by tPA. The rate enhancements are comparable with that of the fibrin(ogen) fragments on a w/w basis. The cofactor activities are inhibited by 6-aminohexanoate and inactivated by pepsin. Analysis of heat-denatured apoferritin and albumin preparations by ultracentrifugation and gel chromatography indicates that cofactor is associated predominately with aggregates, which have binding capacity for both tPA and zymogen. Heat-denatured albumin pretreated with plasmin decreasesKMand increaseskcatfor both intact plasminogen and des1–77-plasminogen activation by tPA, yielding catalytic efficiencies in excess of 8 × 103m−1s−1and 2 × 104m−1s−1, respectively. Because of enhanced plasmin-catalyzed proteolysis of plasminogen to des1–77-plasminogen, activation by urokinase-type plasminogen activator is also facilitated by denatured proteins; activation of des1–77-plasminogen is not affected. It is concluded that denatured proteins serve as both cofactors and substrates in the fibrinolytic system, and that enhancement of plasminogen activation by denatured proteins is mechanistically indistinguishable from that observed with fibrin.