Development of research into the physiology and pathophysiology of the fibrinolysis system

A modern data review on the importance of fibrinolysis system is given. A considerable success has been scored during the study of molecular parameters of fibrinolysis system: the plasminogen, plasmin, its inhibitors, plasminogen activators and the mechanism of activation system have been characterized. The entrance of A, K, C, P and PP vitamins has been established to be necessary for the normal functioning of the fibrinolysis system; the dependence of the blood fibrinolytic activity upon the initial plasminogen content and concentration of its activators in blood has been revealed. The plasminogen activator depletion in tissues has been shown to be one of the reasons of some pathological states development, especially at cardiovascular diseases. The increase of fibrinolysis level by the active fibrinolytic ferment injection in blood has a medical effect at thrombosis. The ferment fibrinolysin received in the laboratory is successfully used in clinical practice. Some other activators of fibrinolytic system: tricholysine and longolytin from the culture of saprophyte fungi, plasminogen activator from the pig heart and the cells culture of the calf kidney have been received and are being studied.     

Plasminogen activation by tissue plasminogen activator on fibrin clots with different surface structures

Transformation of fibrinogen into fibrin with consequent formation of the fibrin clot trimeric structure is one of the final steps in the blood coagulation system. The plasminogen activation by the tissue plasminogen activator (t-PA) is one of the fibrinolysis system key reactions. The effect of different factors on transformation of plasminogen into plasmin is capable to change essentially the equilibrium between coagulation and fibrinolytic sections of haemostasis system. We have studied the plasminogen activation by tissue plasminogen activator on fibrin clots surface formed on the interface between two phases and in presence of one phase. The t-PA plasminogen activation rate on fibrin clots both with film and without it the latter has been analyzed. These data allow to assume that the changes of fibrin clot structure depend on its formations, as well as are capable to influence essentially on plasminogen activation process by means of its tissue activating agent.     

Fibronectin decreases the stimulatory effect of fibrin and fibrinogen fragment FCB-2 on plasmin formation by tissue plasminogen activator.

Fibronectin is a dimeric glycoprotein (Mr 440,000) involved in many adhesive processes. During blood coagulation it is bound and cross-linked to fibrin. Fibrin binding is achieved by structures (type I repeats) which are homologous to the "finger" domain of tissue plasminogen activator. Tissue plasminogen activator also binds to fibrin via the finger domain and additionally via the "kringle 2" domain. Fibrin binding of tissue plasminogen activator results in stimulation of its activity and plays a crucial role in fibrinolysis. Since fibronectin might interfere with this binding, we studied the effect of fibronectin on plasmin formation by tissue plasminogen activator. In the absence of fibrin, fibronectin had no effect on plasminogen activation. In the presence of stimulating fibrinogen fragment FCB-2, fibronectin increased the duration of the initial lag phase (= time period until maximally stimulated plasmin formation occurs) and decreased the rate of maximal plasmin formation which occurs after that lag phase mainly by increasing the Michaelis constant (Km). These effects of fibronectin were dose-dependent and were similar with single- and two-chain tissue plasminogen activator. They were also observed with plasmin-pretreated FCB-2. An apparent Ki of 43 micrograms/ml was calculated for the inhibitory effect of fibronectin when plasminogen activation by recombinant single-chain tissue plasminogen activator was studied in the presence of 91 micrograms/ml FCB-2. When a recombinant tissue plasminogen activator mutant lacking the finger domain was used in a system containing FCB-2, no effect of fibronectin was seen, indicating that the inhibitory effect of fibronectin might in fact be due to competition of fibronectin and tissue plasminogen activator for binding to fibrin(ogen) via the finger domain.     

Clinical studies on the fibrinolytic action of pentosan polysulfate

The influence on fibrinolysis of the heparin-like substance Polyanion SP 54 is described. In vitro tests showed an increase of fibrinolysis by activation of the endogenous pathway via factor XII and prekallikrein. In vivo an increase of the availability of tissue plasminogen activator was assumed in addition. When different ways of administration of Polyanion SP 54 were tested an enhancement of fibrinolysis was also found after oral application of the substance. In a long term test the stimulation of fibrinolysis after oral therapy did not diminish within 12 months. A trial on patients with cerebral ischemic attacks and diminished fibrinolysis is not yet concluded but permits the assumption of a considerable diminution of ischemic attacks during therapeutic use of Polyanion SP 54.     

Cell density-dependent secretion of plasminogen activator by 3T3 cells.

The expression of extracellular fibrinolytic activity in untransformed 3T3 cell cultures depends on the growth state of the cells. Actively growing 3T3 cultures exhibit a relatively high level of fibrinolysis, which decreases progressively as the cells become confluent and density-inhibited. The low level of fibrinolytic activity in confluent 3T3 cultures is due to a diminution in secretion of plasminogen activator since the intracellular level of plasminogen activator remains high. The amount of plasminogen activator observed in growing 3T3 cultures varies depending upon whether the cells are passaged with trypsin/EDTA solution, or with Ca++ selective chelating agent, ethylene-bis (oxyethylenenitrilo) tetraacetic acid (EGTA). However, in cells passaged using either agent, the amount of plasminogen activator secreted is always greatest when the cells are actively growing and decreases thereafter. In contrast to confluent 3T3 cultures, dense cultures of SV40-virus transformed 3T3 cells continued to secrete relatively large amounts of plasminogen activator. The ability to decrease secretion of plasminogen activator as cells become dense may be an important characteristic of cells which demonstrate density-dependent inhibition of cell multiplication in vitro.     

Effects of thiol reagents on glucose transport in thymocytes

We have investigated the factors governing the plasminogen-dependent fibrinolysis catalyzed by the serine proteinase, plasminogen activator (EC 3.4.21.-), under physiologic conditions. We found that live rabbit fibroblasts digested much less fibrin than predicted by cell-free assay of the secreted plasminogen activator. The reduced catalytic activity of plasminogen activator expressed by cells growing on fibrin was regulated by the salt concentration of culture medium. The plasminogen activators of cells from several mammalian species were inhibited by physiologic salt concentrations (0.15 M NaCl) in cell-free assays. CaCl2 and KCl, but not D-glucose, were also effective inhibitors. The catalytic activity of purified human urokinase and of plasmin was unaffected by increased ionic strength. Plasminogen activators secreted both spontaneously and in response to stimulation by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate, were inhibited by 0.15 M NaCl. Physiologic salt concentration appeared to function by interacting with plasminogen activator, or plasminogen, and a third component, possibly a reversible inhibitor. One consequence of this regulation of plasminogen activator under physiologic conditions is the limitation of plasminogen-dependent fibrin degradation by living cells.     

Regulation of extracellular plasminogen activator by human fibroblasts. The role of protease nexin

When the plasminogen activator urokinase was radioiodinated and incubated at 40 ng/ml in medium conditioned by human foreskin (HF) cells, within 30 min over 80% of the added plasminogen activator was complexed to cell-released protease nexin (PN). The urokinase complexed to PN had little if any activity. Incubation of purified PN with urokinase confirmed that PN is an inhibitor of this plasminogen activator. However, a widely used plasminogen-dependent fibrinolysis assay for plasminogen activator indicated that abundant endogenous plasminogen activator activity co-existed with PN in HF cell-conditioned medium. The source of this activity was electrophoretically and immunologically indistinguishable from urokinase. Furthermore, gel exclusion chromatography showed that about 90% of the urokinase antigen detected in conditioned medium had a molecular weight similar to that of free active urokinase. These paradoxical findings are resolved by evidence that this "PN-resistant urokinase-like" plasminogen activator is actually urokinase proenzyme that is activated by plasmin or conditions in the fibrinolysis assay for plasminogen activator. It is shown that the activated form of HF cell plasminogen activator is sensitive to inhibition by PN. PN may thus be an important component in the cellular regulation of endogenous plasminogen activator activity.     

Kinetic characteristics of the activation of various structural forms of plasminogen by tissue activator in the presence of fibrin

It was shown that activation of two native plasminogen and miniplasminogen forms by the tissue activator in the presence of fibrin obeys the Michaelis-Menten kinetics. The kinetic parameters of activation of both plasminogen native forms differ insignificantly. For miniplasminogen whose molecule contains no lysine-binding sites, a marked decrease of activation power was observed. The Km value of activator for miniplasminogen is 10 times that of plasminogen form I and 20 times that of plasminogen form II. The kcat/Km value of activator for miniplasminogen is 7 times less than that of plasminogen form I and by one order of magnitude more than that of plasminogen form II. These results testify to the importance of lysine-binding sites in the native plasminogen molecule during the activation of fibrinolysis by the major physiological activator.     

Phorbol ester and mitogens stimulate human fibroblast secretions of plasmin-activatable plasminogen activator and protease nexin, an antiactivator/antiplasmin

Tumor-promoting phorbol esters have been reported to greatly increase plasminogen activator (PA) activity produced in numerous cell types. Many of these studies have employed a widely used fibrinolysis assay for PA activity that involves large-scale dilution of cell lysates or conditioned medium (CM) into buffer containing plasminogen and the plasmin substrate 125I-fibrin. This assay indicates that phorbol ester and the mitogens epidermal growth factor (EGF) and thrombin all stimulate secretion of PA activity in our human foreskin fibroblast cultures. However, these effects are not observed in a modified fibrinolysis assay employing undiluted conditioned culture medium unless the medium is first treated at pH 3, which inactivates the secreted protease inhibitor, protease nexin (PN). Moreover, a direct assay for plasminogen activator activity based on cleavage of 125I- plasminogen indicates that conditioned culture medium contains little if any active plasminogen activator either before or after treatment of the cultures with phorbol ester or EGF. Phorbol ester and mitogens do stimulate secretion of (a) an inactive PA that can be activated by plasmin and (b) PN, which inhibits both the activated form of the PA and plasmin. Secretions of the inactive PA and PN are further correlated in that release of both is stimulated most by phorbol ester, somewhat less by EGF, and least by thrombin. Significantly, these effects are not accompanied by increases in total protein secretion. We propose that fibroblasts secrete PA in an inactive form in the presence of PN to confine PA activity to an as yet undefined location or event.     

Effects of tissue-type plasminogen activator from a culture of calf kidney cells on hemostasis and fibrinolysis in experimental nephritis

The plasminogen activator 960 IU/mg protein activity isolated from cultured fluid of the calf kidney cells was introduced to albino rats (180-200 g) with experimental Heynmann nephritis every day during 4 days. Nephritis caused activation of haemostasis and inhibition of fibrinolysis in the blood. There was increased excretion of the fibrin, fibrinogen degradation products in urine as a results of the local fibrin deposition in diseased kidneys. The fibrinolytic activity of the cortical zone of kidney was markedly decreased. The plasminogen activator, infused to experimental animals, resulted in normalization of the altered indexes.     

Expression of the catalytic activity of plasminogen activator under physiologic conditions

We have investigated the factors governing the plasminogen-dependent fibrinolysis catalyzed by the serine proteinase, plasminogen activator (EC 3.4.21.-), under physiologic conditions. We found that live rabbit fibroblasts digested much less fibrin than predicted by cell-free assay of the secreted plasminogen activator. The reduced catalytic activity of plasminogen activator expressed by cells growing on fibrin was regulated by the salt concentration of culture medium. The plasminogen activators of cells from several mammalian species were inhibited by physiologic salt concentrations (0.15 M NaCl) in cell-free assays. CaCl₂ and KCl, but not D-glucose, were also effective inhibitors. The catalytic activity of purified human urokinase and of plasmin was unaffected by increased ionic strength. Plasminogen activators secreted both spontaneously and in response to stimulation by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate, were inhibited by 0.15 M NaCl. Physiologic salt concentration appeared to function by interacting with plasminogen activator, or plasminogen, and a third component, possibly a reversible inhibitor. One consequence of this regulation of plasminogen activator under physiologic conditions is the limitation of plasminogen-dependent fibrin degradation by living cels.     

From Cerius² based stereoview to mouse and enzyme: the model systems for discovery of novel urokinase inhibitors

Antifibrinolytic therapy during major complex surgery could reduce blood loss and allogeneic transfusion. Novel antagonists of the plasminogen activator and the corresponding model system are of clinical importance. In this paper (1S,2'S,3S)-1-[2-(S)-carboxylindolemethylenemethineaminoeth-1-yl]-2,3,4,5-tetrahydropyrolo[1,2:1,6]pyrazino[3,4:2,3]-1,2,3,4-tetrahydrocarboline-2,5-dione (CIPPC) was presented as a novel antagonist of plasminogen activator, and its blood coagulation and action mechanism were investigated by using a model system which consisted of a mouse-tail bleeding assay, in vitro and in vivo fibrinolysis inhibition assays, a thrombus formation assay and a plasminogen (PLG) electrophoresis assay.     

Plasminogen activator from cells transformed by an oncogenic virus: inhibitors of the activation reaction.

This paper describes an assay for direct measurement of plasminogen activation and its application for determining the kinetic constants and for screening potential inhibitors of the reaction. The assay is based on the conversion of the single chain of 125I-labelled plasminogen to the two chains of 125I-labelled plasmin (EC 3.4.21.7), the latter then being separated from each other and from the plasminogen substrate by electrophoresis under reducing conditions in SDS-polyacrylamide gels. The Km of activator from transformed murine cells for human plasminogen was 180 nM. A broad range of compounds was tested as potential inhibitors of plasminogen activation and of plasmin-catalyzed fibrinolysis respectively, and the two reactions differed qualitatively and quantitatively in their response to previous agents. The principal qualitative difference was in the susceptibility of the reactions to a spectrum of naturally-occurring macromolecular inhibitors: all of the macromolecular inhibitors that blocked the action of plasmin were without effect on murine activator or human urokinase (EC 3.4.99.26). A variety of small molecules inhibited both of the reactions tested, and showed significant quantitative differences; some of these were active at micron concentrations. The exacting specificity of plasminogen activators for macromolecules, both substrates and inhibitors, encourages the expectation that effective inhibitors of great specificity may be isolated from as yet undiscovered natural sources.     

Plasminogen activator inhibitor type-1-deficient mice have an enhanced IFN-gamma response to lipopolysaccharide and staphylococcal enterotoxin B

Plasminogen activator inhibitor type-1 (PAI-1) is a major inhibitor of fibrinolysis by virtue of its capacity to inhibit urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Systemic inflammation is invariably associated with elevated circulating levels of PAI-1, and during human sepsis plasma PAI-1 concentrations predict an unfavorable outcome. Knowledge about the functional role of PAI-1 in a systemic inflammatory response syndrome is highly limited. In this study, we determined the role of endogenous PAI-1 in cytokine release induced by administration of LPS or staphylococcal enterotoxin B (SEB). Both LPS and SEB elicited secretion of PAI-1 into the circulation of normal wild-type (Wt) mice. Relative to Wt mice, PAI-1 gene-deficient (PAI-1(-/-)) mice demonstrated strongly elevated plasma IFN-gamma concentrations after injection of either LPS or SEB. In addition, PAI-1(-/-) splenocytes released more IFN-gamma after incubation with LPS or SEB than Wt splenocytes. Both PAI-1(-/-) CD4+ and CD8+ T cells produced more IFN-gamma upon stimulation with SEB. LPS-induced IFN-gamma release in mice deficient for uPA, the uPA receptor, or tPA was not different from IFN-gamma release in LPS-treated Wt mice. These results identify a novel function of PAI-1 during systemic inflammation, where endogenous PAI-1 serves to inhibit IFN-gamma release by a mechanism that does not depend on its interaction with uPA/uPA receptor or tPA.     

Mechanism of action of θ-amino acids on plasminogen activation and fibrinolysis induced by staphylokinase

Stimulation of Lys-plasminogen (Lys-Pg) and Glu-plasminogen (Glu-Pg) activation under the action of staphylokinase and Glu-Pg activation under the action of preformed plasmin-staphylokinase activator complex (Pm-STA) by low concentrations and inhibition by high concentrations of omega-amino acids (>90-140 mM) were found. Maximal stimulation of the activation was observed at concentrations of L-lysine, 6-aminohexanoic acid (6-AHA), and trans-(4-aminomethyl)cyclohexanecarboxylic acid 8.0, 2.0, and 0.8 mM, respectively. In contrast, the Lys-Pg activation rate by Pm-STA complex sharply decreased when concentrations of omega-amino acids exceeded the above-mentioned values. It was found that formation of Pm-STA complex from a mixture of equimolar concentrations of staphylokinase and Glu-Pg or Lys-Pg is stimulated by low concentrations (maximal at 10 mM) of 6-AHA. Negligible increase in the specific activities of plasmin and Pm-STA complex was detected at higher concentrations of 6-AHA (to maximal at 70 and 50 mM, respectively). Inhibitory effects of omega-amino acids on the rate of fibrinolysis induced by staphylokinase, Pm-STA complex, and plasmin were compared. It was found that inhibition of staphylokinase-induced fibrinolysis by omega-amino acids includes blocking of the reactions of Pm-STA complex formation, plasminogen activation by this complex, and lysis of fibrin by forming plasmin as a result of displacement of plasminogen and plasmin from the fibrin surface. Thus, the slow stage of Pm-STA complex formation plays an important role in the mechanism of action of omega-amino acids on Glu-Pg activation and fibrinolysis induced by staphylokinase. In addition to alpha-->beta change of Glu-Pg conformation, stimulation of Pm-STA complex formation leads to increase in Glu-Pg activation rate in the presence of low concentrations of omega-amino acids. Inhibition of Pm-STA complex formation on fibrin surface by omega-amino acids is responsible for appearance of long lag phases on curves of fibrinolysis induced by staphylokinase.     

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.     

Polarized secretion of tissue-plasminogen activator in cultured thyroid cells

Summary We studied the polarized secretion of tissue-type plasminogen activator in porcine thyroid cells cultured as a monolayer on porous bottom chambers. The presence of tissue-type plasminogen activator was detected by zymographic analysis on two independent media that were in contact either with the apical surface or with the basolateral membrane. The amount of tissue-type plasminogen activator was determined in both media by ELISA and enzyme assay. Measurable tissue-type plasminogen activator activity was found in the basal but not in the apical medium. However, on zymogram, a lytic zone corresponding to tissue-type plasminogen activator was visible in both media. In addition, a lytic band at 130 kDa suggested presence of a complex formed by tissue-type plasminogen activator and an inhibitor. Preferential basolateral tissue-type plasminogen activator antigen secretion (70%) has been observed, showing the possible relation between tissue-type plasminogen activator and extracellular matrix components. Neither tissue-type plasminogen activator level nor polarized secretion seemed to be regulated by thyrotropin (0.1 mU/ml).     

Depletion of fibrinolysis activators as a result of continuous stress

The dynamics of disorders in haemostasis and fibrinolysis upon continuous stress was studied in model experiments with rats exposed to emotional-pain-stress. It was shown that the plasminogen activator (PA) is released into the blood within the first few minutes of exposure to stress. Four hours after cessation of continuous stress (6 hours) the enzyme depletion and fibrinolysis depression occurred. Such stress-induced changes in the blood system function can be regarded as a risk factor in the development of thrombosis.     

Pitavastatin alters the expression of thrombotic and fibrinolytic proteins in human vascular cells

In addition to lowering blood lipids, clinical benefits of 3-hydroxy-3-methylglutaryl coenzyme A (HMG Co-A; EC 1.1.1.34) reductase inhibitors may derive from altered vascular function favoring fibrinolysis over thrombosis. We examined effects of pitavastatin (NK-104), a relatively novel and long acting statin, on expression of tissue factor (TF) in human monocytes (U-937), plasminogen activator inhibitor-1 (PAI-1), and tissue-type plasminogen activator (t-PA) in human aortic smooth muscle cells (SMC) and human umbilical vein endothelial cells (HUVEC). In monocytes, pitavastatin reduced expression of TF protein induced by lipopolysaccharide (LPS) and oxidized low-density lipoprotein (OxLDL). Similarly, pitavastatin also reduced expression of TF mRNA induced by LPS. Pitavastatin reduced PAI-1 antigen released from HUVEC under basal, OxLDL-, or tumor necrosis factor-alpha (TNF-alpha)-stimulated conditions. Reductions of PAI-1 mRNA expression correlated with decreased PAI-1 antigen secretion and PAI-1 activity as assessed by fibrin-agarose zymography. In addition, pitavastatin decreased PAI-1 antigen released from OxLDL-treated and untreated SMC. Conversely, pitavastatin enhanced t-PA mRNA expression and t-PA antigen secretion in untreated OxLDL-, and TNF-alpha-treated HUVEC and untreated SMC. Finally, pitavastatin increased t-PA activity as assessed by fibrin-agarose zymography. Our findings demonstrate that pitavastatin may alter arterial homeostasis favoring fibrinolysis over thrombosis, thereby reducing risk for thrombi at sites of unstable plaques.     

The role of heparin and tissue-type plasminogen activator in the adaptation of the hemostatic system to high altitude]

The role of endogenous heparin and tissue-type plasminogen activator in the middle-period (25 days) adaptation of haemostasis to high altitude (altitude 3200 m) and formation or "high-altitude hypocoagulation" was studied in the experiments on white rats. It was observed that the formation of "high-altitude hypocoagulation" is connected with an increase of heparin and tissue-type plasminogen activator level due to its release from must and endothelial cells to the bloodstream. Histochemical analysis showed that at the course of adaptation to high altitude the increase in blood heparin level was caused by the stimulation of must cells secretory activity. The endothelium of lung vessels is the main source of tissue-type plasminogen activator release into the blood. The existence of interconnection between the changes in haemostasis and stimulation of angiogenesis at high altitude is proposed.