Plasminogen and plasminogen activator assembly on the human endothelial cell

Through assembly of plasminogen and its activators, the endothelial cell surface may provide a favorable environment for constitutive generation of plasmin. This system may be regulated at multiple levels. Abundant expression of a 40-kDa protein with dual ligand-binding capacity may promote cell surface plasmin formation by colocalizing t-PA and plasminogen in a catalytically favorable configuration. Conversion of Glu-PLG to the preactivated form Lys-PLG, in the vicinity of the cell surface, may also precede plasmin formation. Physiologic concentrations of Lp(a), furthermore, may serve to modulate plasminogen activation at the cell surface by competing for binding sites, whereas elevated levels of Lp(a) might suppress this mechanism and lead to a subclinical prothrombotic state. Finally, cell surface binding sites for both plasmin and t-PA appear to protect these molecules from their physiologic antagonists, alpha 2-plasmin inhibitor and plasminogen activator inhibitor, type-1, respectively. Plasmin formation may contribute to the nonthrombogenicity of the blood vessel wall.     

Molecular basis of fibrinolysis, as relevant for thrombolytic therapy

The fibrinolytic system comprises an inactive proenzyme, plasminogen, that is converted by plasminogen activators to the active enzyme, plasmin, that degrades fibrin. Two physiological plasminogen activators have been identified: tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). Plasminogen activation for clot lysis is regulated by specific molecular interactions between tissue-type plasminogen activator (t-PA), plasminogen and fibrin, whereby the lysine-binding sites of the plasminogen molecule play a crucial role by mediating its binding to fibrin, and by controlling the inhibition rate of plasmin by alpha 2-antiplasmin. The recognition that thrombosis within the infarct related coronary artery plays a major role in the pathogenesis of acute myocardial infarction and the observation that early administration of thrombolytic agents results in recanalization of occluded coronary arteries, have provided the basis for the development of thrombolytic therapy in acute myocardial infarction. The elucidation of the biochemical mechanism of fibrin-specific plasminogen activation has fueled the hope that specific and efficacious thrombolytic agents might become available. Comparative studies between the non-fibrin-selective streptokinase and fibrin-selective recombinant t-PA (rt-PA) have shown a difference in efficacy for early coronary artery recanalization, whereas the GUSTO trial has established that clinical benefit in patients with acute myocardial infarction is indeed correlated with the rapidity and frequency of sustained recanalization and that effective thrombolysis requires adequate anticoagulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

A pathological role of increased expression of plasminogen activator inhibitor-1 in human or animal disorders

Plasminogen activator inhibitor-1 (PAI-1) is a specific inhibitor of plasminogen activators and may be the principal regulator of plasminogen activation in vivo. Abnormal expression of PAI-1 has been reported in various types of human disorders and in animal models for the diseases in relation to thrombosis. For example, plasma PAI-1 activity was elevated in patients with endotoxemia, and a dramatic induction of PAI-1 mRNA was observed in tissues of endotoxin-treated animals, resulting in tissue microthrombosis. It has also been demonstrated that PAI-1 expression levels are increased in the kidneys of mice with glomerulonephritis, in the adipose tissue of obese subjects or mice, and in human atherosclerotic arteries. This PAI-1 induction may be relevant to pathological processes in these diseases because PAI-1 not only regulated fibrin dissolution in vivo but also inhibited degradation of extracellular matrix by reducing plasmin generation. The responsible cells for abnormal expression of PAI-1 have been identified in each tissue under pathological conditions and PAI-1 synthesis appears to be regulated in a tissue-specific manner. These observations suggest that PAI-1 could play an important role in the progression of tissue pathologies in a variety of human diseases by controlling the rate of plasmin formation.     

Microglia express the type 2 plasminogen activator inhibitor in the brain of control subjects and patients with Alzheimer's disease

Localization of type 2 plasminogen activator inhibitor (PAI-2) was investigated immunohistochemically in postmortem brain tissue of control subjects and patients with Alzheimer's disease (AD). Both the mouse monoclonal and the goat polyclonal antibodies to PAI-2 stained microglia. Reactive microglia were stained more intensely than resting microglia. PAI-2 may regulate the plasminogen activators and plasmin system in lesions of AD. It is suggested that PAI-2 plays a role for cell migration and matrix breakdown as well as for aberrant neurite outgrowth in senile plaques where persistent microglial activation is observed.     

A molecular variant of plasminogen activator inhibitor of rat decidual tissue

Artificially induced rat decidual tissue expresses plasminogen activator inhibitor (PAI). This PAI, isolated and purified employing chromatographic techniques, is a low molecular weight protein unlike the known PAIs. The final purified preparation resolves into a single band following SDS-PAGE and has an approximate molecular weight of 29 kDa. The properties studied include specificity for urokinase-type (uPA) and tissue-type (tPA) plasminogen activators, binding to conA and heparin, inhibition of thrombin, plasmin and trypsin. Decidual PAI is immunogenic in rabbit and a monospecific antiserum raised against the decidual inhibitor cross reacts with an extract of human placenta.     

Specificity of serine proteinase/serpin complex binding to very-low-density lipoprotein receptor and alpha2-macroglobulin receptor/low-density-lipoprotein-receptor-related protein

Very-low-density lipoprotein receptor (VLDLR) and alpha2-macroglobulin receptor/low-density-lipoprotein-receptor-related protein (alpha2MR/LRP) are multifunctional endocytosis receptors of the low-density lipoprotein receptor family. Both have been shown to mediate endocytosis and degradation of complex between plasminogen activators and type-1 plasminogen-activator inhibitor (PAI-1) by cultured cells. We have now studied the specificity of binding and endocytosis by VLDLR and alpha2MR/LRP among a variety of serine proteinase/serpin complexes, including various combinations of the serine proteinases urokinase-type and tissue-type plasminogen activators, plasmin, thrombin, human leukocyte elastase, cathepsin G, and plasma kallikrein with the serpins PAI-1, horse leukocyte elastase inhibitor, protein C inhibitor, C1-inhibitor, alpha2-antiplasmin, alpha1-proteinase inhibitor, alpha1-antichymotrypsin, protease nexin-1, heparin cofactor II, and antithrombin III. Binding was estimated with radiolabelled ligands in ligand blotting analysis and microtiter well assays. Endocytosis was estimated by measuring receptor-associated protein (RAP)-sensitive degradation of radiolabelled complexes by Chinese hamster ovary cells transfected with VLDLR cDNA and by COS-1 cells, which have a high endogenous expression of alpha2MR/LRP. We found that the receptors bind with high affinity to some, but not all, combinations of plasminogen activators and thrombin with PAI-1, protease nexin-1, protein C inhibitor, and antithrombin III, while complexes of many serine proteinases with their primary inhibitor, i.e. plasmin/alpha2-antiplasmin complex, do not bind, or bind with a very low affinity. Both the serine proteinase and the serpin moieties contribute to the binding specificity. The binding specificities of VLDLR and alpha2MR/LRP are overlapping, but not identical. The results suggest that VLDLR and alpha2MR/LRP have different biological functions by having different binding specificities as well as by being expressed by different cell types.     

Wound fluid from venous leg ulcers degrades plasminogen and reduces plasmin generation by keratinocytes

Plasminogen, the pro-enzyme of plasmin, aids various processes essential for normal, acute wound healing, such as fibrinolysis and cell migration. We have investigated if plasminogen is available to perform these functions in chronic wounds such as venous leg ulcers. We report that plasminogen is degraded by fluid from venous leg ulcers to a number of fragments, including kringle domains 1-3, an angiostatin-related protein. The enzyme responsible was inhibited by the serine protease inhibitor phenyl-methylsulfonyl fluoride, but was not inhibited by alpha1-anti-trypsin, an inhibitor of neutrophil elastase, by alpha2-anti-plasmin, an inhibitor of plasmin, or by the matrix metalloprotease inhibitor 1,10 phenanthroline. Plasminogen degraded by wound fluid was a weaker substrate than intact plasminogen for plasmin generation by the keratinocyte cell line HaCaT. These results suggest that serine protease activity in leg ulcer fluid degrades plasminogen and support the hypothesis that keratinocyte migration may be impaired in leg ulcers because of a reduced availability of intact plasminogen for plasmin generation.     

Reward magnitude, but not time of day, influences the trial-spacing effect in autoshaping with rats

An important regulator of the initiation of blood coagulation is the plasma glycoprotein, tissue factor pathway inhibitor (TFPI). TFPI inhibits factor Xa and factor VIIa/tissue factor complex, thereby dampens the proteolytic cascade of the tissue factor pathway. Plasma clot lysis is primarily mediated by the fibrinolytic enzyme, plasmin, which is generated through limited proteolysis of plasminogen by endogenous or exogenously administered plasminogen activators. In this study, the interaction of plasmin with recombinant E. coli-derived TFPI (rTFPI) was examined. Plasmin was found to cause a time and concentration dependent proteolysis of rTFPI, resulting in the decrease of anti-factor Xa (measured by chromogenic substrate assay) and anticoagulant (measured by tissue factor-induced clotting assay) activities. Amino-terminal sequencing of the proteolytic fragments revealed that plasmin cleaved rTFPI at K86-T87, R107-G108, R199-A200, K249-G250, and K256-R257. Western blot analysis showed that proteolysis of exogenously added rTFPI also occurred in plasma supplemented with urokinase, and this is accompanied by decrease of anticoagulant activity. These changes were abolished by addition of aprotinin, an inhibitor of plasmin. These data indicate that TFPI is susceptible to proteolysis when plasma fibrinolytic system is activated. The results taken together suggest that plasmin degradation of TFPI may contribute to rethrombosis after thrombolysis, and may contribute to the variability of the efficacy of TFPI in various thrombolysis/reocclusion studies reported previously.     

Phosphorylation of vitronectin on Ser362 by protein kinase C attenuates its cleavage by plasmin

Vitronectin, found in the extracellular matrix and in circulating blood, has an important role in the control of plasminogen activation. It was shown to be the major protein substrate in human blood fluid for a protein kinase A (PKA) released from platelets upon their physiological stimulation with thrombin. Since vitronectin was shown to have only one PKA phosphorylation site, but to contain 2-3 mol covalently bound phosphate, it was reasonable to assume that other protein kinases might phosphorylate vitronectin at other sites in the protein. We have reported earlier that human serum contains at least three protein kinases, one of which was found to be cAMP independent and to phosphorylate a repertoire of plasma proteins that was very similar to that obtained upon phosphorylation of human plasma with protein kinase C (PKC). Since there are now several examples of proteins with extracellular functions that are phosphorylated by PKC, we undertook to study the phosphorylation of vitronectin by PKC. Here, we show that vitronectin is a substrate for PKC, and characterize the kinetic parameters of this phosphorylation (Km approximately tenfold lower than the concentration of vitronectin in blood), indicating that, from the biochemical point of view, this phosphorylation can occur at the locus of a hemostatic event. We also identify Ser362 as the major PKC phosphorylation site in vitronectin, and confirm this localization by means of synthetic peptides derived from the cluster of basic amino acids in vitronectin surrounding Ser362. We show that the PKC phosphorylation at Ser362 alters the functional properties of vitronectin, attenuating its cleavage by plasmin at Arg361-Ser362. This phosphorylation has the potential to regulate plasmin production from plasminogen by a feedback mechanism involving the above-mentioned plasmin cleavage, a loosening of the vitronectin grip on inhibitor 1 of plasminogen activators, and a subsequent latency of this regulatory inhibitor.     

Apposition-dependent induction of plasminogen activator inhibitor type 1 expression: a mechanism for balancing pericellular proteolysis during angiogenesis

Plasminogen-activator inhibitor type I (PAI-1), the primary inhibitor of urinary-type plasminogen activator, is thought to play an important role in the control of stroma invasion by both endothelial and tumor cells. Using an in vitro angiogenesis model of capillary extension through a preformed monolayer, in conjunction with in situ hybridization analysis, we showed that PAI-1 mRNA is specifically induced in cells juxtaposed next to elongating sprouts. To further establish that PAI-1 expression is induced as a consequence of a direct contact with endothelial cells, coculture experiments were performed. PAI-1 mRNA was induced exclusively in fibroblasts (L-cells) contacting endothelial cell (LE-II) colonies. Reporter gene constructs driven by a PAI-1 promoter and stably transfected into L-cells were used to establish that both mouse and rat PAI-1 promoters mediate apposition-dependent regulation. This mode of PAI-1 regulation is not mediated by plasmin, as an identical spatial pattern of expression was detected in cocultures treated with plasmin inhibitors. Because endothelial cells may establish direct contacts with fibroblasts only during angiogenesis, we propose that focal induction of PAI-1 at the site of heterotypic cell contacts provides a mechanism to negate excessive pericellular proteolysis associated with endothelial cell invasion.     

Plasminogen activation in lesional skin of Pemphigus vulgaris type Neumann

Zymographic and immunological studies revealed that primarily tissue-type plasminogen activator and to a lesser extent urokinase-type plasminogen activator were present in fluids of pemphigus vulgaris (type Neumann) skin blisters. Furthermore, plasmin activity was detected in pemphigus blister fluids using chromogenic peptide substrate assays. In pemphigus, but not in control, suction blister fluids plasmin/alpha 2-antiplasmin and plasmin/alpha 2-macroglobulin complexes were found by immunoprecipitation or by testing in immunoassays after fractionation by molecular-sieve chromatography. Plasmin activity, detected by a low molecular weight chromogenic peptide assay, was ascribed to plasmin/alpha 2-macroglobulin complexes. Since formation of plasmin/inhibitor complexes requires active plasmin, the finding indicates previous activation of plasminogen in pemphigus lesions.     

Reduced metastasis of Polyoma virus middle T antigen-induced mammary cancer in plasminogen-deficient mice

To investigate the role of plasmin(ogen) in mammary tumor development and progression, plasminogen-deficient mice were crossed with transgenic mice expressing Polyoma middle T antigen under the control of the mouse mammary tumor virus long terminal repeat. Virgin females carrying the Polyoma middle T antigen uniformly developed multiple, bilateral mammary tumors, regardless of the presence or absence of circulating plasminogen. Both the age at which these tumors became palpable and subsequent tumor growth were indistinguishable between plasminogen-deficient mice and plasminogen-expressing littermates. However, plasminogen was found to greatly modify the metastatic potential in this model system; lung metastasis in plasminogen-deficient mice was significantly reduced as compared to littermate controls with respect to frequency of occurrence, total number of metastases, and total metastatic tumor burden. Plasminogen activators, as well as other key factors that govern the conversion of plasminogen to plasmin, were expressed within the mammary tumors, suggesting that the plasminogen/plasmin system may promote metastasis by contributing to tumor-associated extracellular proteolysis. The data provide direct evidence that plasmin(ogen) is a tumor progression factor in PymT-induced mammary cancer, and support the hypothesis that hemostatic factors play an important role in tumor biology.     

A study of the mechanism of inhibition of fibrinolysis by activated thrombin-activable fibrinolysis inhibitor

TAFI (thrombin-activable fibrinolysis inhibitor) is a recently described plasma zymogen that, when exposed to the thrombin-thrombomodulin complex, is converted by proteolysis at Arg92 to a basic carboxypeptidase that inhibits fibrinolysis (TAFIa). The studies described here were undertaken to elucidate the molecular basis for the inhibition of fibrinolysis. When TAFIa is included in a clot undergoing fibrinolysis induced by tissue plasminogen activator and plasminogen, the time to achieve lysis is prolonged, and free arginine and lysine are released over time. In addition, TAFIa prevents a 2.5-fold increase in the rate constant for plasminogen activation which occurs when fibrin is modified by plasmin in the early course of fibrin degradation. The effect is specific for the Glu- form of plasminogen. TAFIa prevents or at least attenuates positive feedback expressed through Lys-plasminogen formation during the process of fibrinolysis initiated by tissue plasminogen activator and plasminogen. TAFIa also inhibits plasmin activity in a clot and prolongs fibrinolysis initiated with plasmin. We conclude that TAFIa suppresses fibrinolysis by removing COOH-terminal lysine and arginine residues from fibrin, thereby reducing its cofactor functions in both plasminogen activation and the positive feedback conversion of Glu-plasminogen to Lys-plasminogen. At relatively elevated concentrations, it also directly inhibits plasmin.     

Hemoptysis caused by pulmonary arterial aneurysm, disclosing Beh?et disease. Apropos of 2 cases]

MCF7 and ZR75-1 breast cancer cells grow as adherent monolayers in tissue culture. Treatment with the serum serine protease plasmin causes them to detach and to grow as floating multicellular spheroids. Two plasmin activators, urokinase plasminogen activator and streptokinase, induce the same growth pattern changes in the presence of plasminogen. Serum contains also plasminogen activator inhibitors. Aged serum, deficient in plasminogen activator inhibitors, converts spontaneously monolayer breast cancer cells into multicellular spheroids which readily revert to monolayer growth after addition of fresh serum. Urokinase blocks the reversion. The formation of multicellular spheroids does not affect the proliferative rate of breast tumor cells but endows tumor cells with increased resistance to the chemotherapeutic drugs, doxorubicin and paclitaxel.     

Production of plasminogen activator inhibitor-1 by human saphenous vein endothelial cells seeded onto endarterectomy surfaces in vitro

PURPOSE: Endothelial cells produce many biologically important factors that may be used as functional markers, including plasminogen activators and their inhibitors (PAI). PAI-1 is a common peptide with a central role in the balance of thrombosis and fibrinolysis in vivo, and its production by vascular endothelial cells has been demonstrated for many in vitro cell lines. METHODS: The basal rate of PAI-1 release from cultured human adult endothelial cells was studied in both a well-plate-seeding model and after seeding onto human endarterectomy specimens. The effect of nonspecific stimulation with thrombin on PAI-1 release was examined in well-plate cultures. PAI-1 was measured by enzyme-linked immunosorbent assay. RESULTS: Cultured human saphenous endothelial cells release PAI-1 constitutively at a steady rate, which can be increased in the short term by the addition of thrombin. CONCLUSION: After seeding onto endarterectomy specimens, seeded endothelial cells release significant amounts of PAI-1, which suggests that they retain functional integrity and may potentially influence thrombosis in vivo after seeding.     

Signaling via the CD2 receptor enhances HTLV-1 replication in T lymphocytes

Three (0140J, C197C and EF20) out of four strains of Streptococcus uberis exhibited high levels of bound plasmin activity following growth in the presence of bovine plasminogen. The remaining strain (C197) bound considerably less plasmin following growth in the same medium. In contrast to the others, this strain was unable to activate bovine plasminogen. Following growth of strain C197 in the presence of bovine plasminogen and a source of plasminogen activator (urokinase or culture filtrate from strain 0140J) high levels of bacterially associated plasmin were detected. None of the strains was able to activate human plasminogen and only trace levels of plasmin activity were detected in association with the S. uberis following growth in the presence of human plasminogen. All strains were able to bind plasmin activity following incubation in the presence of either bovine or human plasmin. However, in each case the level of activity detected following incubation in human plasmin was approximately five-fold less than that observed following incubation with bovine plasmin. None of the strains bound detectable levels of either human or bovine plasminogen. It is concluded that activation of plasminogen is required prior to binding of plasmin by S. uberis.     

Comparison of production of choriogonadotropin inhibitory protein, prolactin and insulin-like growth factor binding protein-1 by human decidua in vitro

BACKGROUND: Tissue plasminogen activator (tPA) is elevated in cancer patients and is thought to promote tumor angiogenesis by facilitating endothelial cell migration through plasmin-mediated degradation of extracellular matrix. Due to the presence of an epidermal growth factor (EGF)-finger domain in the tPA A-chain and the existence of an endothelial cell (EC) receptor that binds this domain, it was hypothesized that tPA has a direct receptor-mediated effect on EC proliferation, independent of plasmin. METHODS AND RESULTS: Using cultured canine ECs, tPA (7.25 microg/ml, approximately 107 nM) increased proliferation as much as 50 and 170% in the absence and presence of growth factors, respectively. tPA-induced increases in EC proliferation occurred independent of plasmin generation, as the plasmin inhibitor, aprotinin (10 microg/ml) did not inhibit tPA-induced proliferation. However, tPA-induced proliferation was inhibited dose-dependently to a maximum of 78% using a monoclonal antibody against the tPA EGF-finger domain. This antibody, known to inhibit tPA binding to its receptor, did not inhibit tPA-induced plasmin generation. To investigate the role of potential signal transduction pathways, ECs were exposed to lavendustin A, a tyrosine kinase inhibitor, at 33.5 microM (IC50 for basic fibroblast growth factor). Lavendustin A did not inhibit tPA-induced EC proliferation. However, Rp-cAMP, an inhibitor of cAMP-dependent kinases, specifically inhibited tPA-induced EC proliferation in a dose-dependent manner (IC50 = 50.5 microM). Pertussis toxin at maximal concentrations for this system (0.5 ng/ml) did not inhibit tPA-induced EC proliferation. CONCLUSION: These results lend support to the hypothesis that tPA may have a direct receptor-mediated effect on EC proliferation and that this effect occurs independent of plasmin and may be dependent upon protein kinase A activity.     

Delivery system for targeted thrombolysis without the risk of hemorrhage

Cardiovascular diseases that result from thrombosis of critically situated blood vessels remain the leading cause of death in industrialized countries. One primary clinical treatment is dissolution of the thrombus with thrombolytic agents, plasminogen activators (PA). Activation of plasminogen by a PA agent produces plasmin that degrades fibrin. However, plasmin also degrades other circulating clotting factors. Therefore, thrombolytic therapy, which introduces systemic generation of excess plasmin, carries the risk of hemorrhage. We propose a novel approach that could lead to targeted thrombolysis without bleeding risk. The system is comprised of a protein conjugate made of two parts: a fibrin-targeting antibody (Ab) linked with anionic heparin; and a PA derivatized with cationic species. These two parts are linked via an electrostatic interaction. Because the cationic species are relatively small, the derivatized PA would retain its thrombolytic activity, but this activity would be inhibited after binding with the Ab-heparin counterpart because of the blockage of the PA's active site by these appended macromolecules. Because protamine is a clinical heparin antagonist with a much stronger affinity for heparin than the incorporated cations, it can be used safely to dissociate the modified PA from the Ab-heparin counterpart. Therefore, this approach would permit administration of a fibrin-targeting but inactive thrombolytic, and subsequently, a triggered release of the active modified PA drug in close proximity to the fibrin deposit. These features would enhance the potency and the specificity of the thrombolytic agent and alleviate the bleeding risk by avoiding systemic generation of excess plasmin. In this report, we present preliminary results demonstrating the feasibility of the approach. A cationic octapeptide, (Arg)7-Cys, was linked to urokinase (UK) using the N-succinimidyl-3-[2-pyridylidithio]propionate activation method. This UK peptide retained a significant amount of its catalytic activity, as measured by the S-2251 chromogenic assay. However, this activity was almost completely inhibited (approximately 99%) after the addition of heparin, but was fully reversed (100%) after the addition of protamine.