Thrombus lysis by uPA, scuPA and tPA is regulated by plasma TAFI

Summary.  The carboxypeptidase, TAFIa or CPU, is known to prolong plasma clot lysis by tissue plasminogen activator (tPA) and to have a role in thrombus stability in vivo. This current study examined lysis by urokinase (uPA) and single chain urokinase (scuPA) in addition to tPA. Further, we investigated the role of TAFIa in a model thrombus system, in which thrombi are formed under conditions of flow. We show that human thrombi, formed in vivo , and model thrombi both contain TAFI . No effect of thrombus TAFIa was observed in thrombus lysis assays, except when thrombi were bathed in plasma, in which case addition of potato tuber carboxypeptidase inhibitor (CPI) resulted in doubling of the rate of lysis. TAFIa inhibited lysis of model thrombi and plasma clots by uPA, scuPA in addition to lysis by tPA. The effect of TAFIa was more evident at high concentrations of plasminogen activator such as those used in thrombolytic therapy. Addition of plasminogen increased lysis and, in its presence, the enhancement by CPI was smaller. Thus the action of TAFIa could be partially overcome by plasminogen, whether lysis was by tPA, uPA or scuPA. This is consistent with TAFIa exerting its effect primarily through modifying the binding of plasminogen to fibrin and to a lesser extent through modification of the binding of tPA to fibrin.     

A proposed reference method for plasminogen activators that enables calculation of enzyme activities in SI units.

A method has been developed for accurately and precisely measuring the activity of a range of plasminogen activators (PAs) used as thrombolytic agents, including streptokinase, tissue plasminogen activator (tPA) and variants, and urokinase (uPA), both single and two chain forms. Plasminogen activation is monitored in a transparent, solid fibrin matrix but uses chromogenic substrate hydrolysis, rather than changes in fibrin, to quantitate the activity of PAs. The method has been tested in two recent international collaborative studies involving tPA and streptokinase where it has been shown to perform very well. Furthermore, the method is based on sound enzymological principles and once correction for the competitive inhibition of fibrin(ogen) is made, the generation of plasmin can be determined in molar terms and hence the activity of PAs can be expressed and compared in SI units (rate of increase in molar concentration of plasmin) as well as International Units. The assay is also arranged in such a way to reflect the behavior of PAs in vivo during thrombolytic therapy and it is shown that the specific activity of streptokinase and tPA in this system reflects plasmin generation capacity of these thrombolytics for doses given in infusions for treatment of myocardial infarction. The method would make a suitable reference method for PAs and provides a rigorous means of studying and modeling the enzymology of fibrinolysis and will be helpful in the rational design of third generation thrombolytic agents.     

Complementary modes of action of tissue-type plasminogen activator and pro-urokinase by which their synergistic effect on clot lysis may be explained.

Tissue plasminogen activator (t-PA) and/or pro-urokinase (pro-UK) induced lysis of standard 125I-fibrin clots suspended in plasma was studied. Doses were kept below the concentration at which a nonspecific effect was seen, i.e., where fibrinogenolysis and major plasminogen consumption were observed. Small amounts of t-PA potentiated clot lysis by pro-UK by attenuating the lag phase characteristic of pro-UK, and causing a much earlier transition to the rapid phase of lysis. Similar promotion of the fibrinolytic effect of pro-UK was obtained when clots were pretreated with UK or with a little plasmin (less than 1% clot lysis). Promotion by plasmin was nullified by a subsequent treatment of the clot with carboxypeptidase B, indicating that the plasmin effect was related to the exposure of carboxy terminal lysine residues on fibrin. These lysine termini, absent in undegraded fibrin, are known to be essential for the high affinity binding of plasminogen to fibrin. In contrast, clot lysis by t-PA was unaffected by plasmin pretreatment and little affected by carboxypeptidase B treatment of the fibrin substrate. Therefore, plasminogen bound to lysine termini on fibrin, although found to be essential for pro-UK, did not appear to serve as a substrate for t-PA. Selective activation of fibrin bound plasminogen has been attributed to the conformational change in Glu-plasminogen that occurs as a result of binding. The present findings suggest that this conformational change occurs when plasminogen is bound to a terminal lysine but not to an internal lysine. Plasminogen bound to the latter site on fibrin was activated by t-PA and therefore is involved in the ternary complex. This initiates lysis of the undegraded clot and exposes the plasminogen binding sites required by pro-UK. By their complementary activation of fibrin bound plasminogen, t-PA followed by pro-UK induces efficient and synergistic fibrinolysis, whereas each is relatively inefficient when used alone.     

The regulation by fibrinogen and fibrin of tissue plasminogen activator kinetics and inhibition by plasminogen activator inhibitor 1

BACKGROUND: Tissue plasminogen activator (tPA) is unusual in the coagulation and fibrinolysis cascades in that it is produced as an active single-chain enzyme (sctPA) rather than a zymogen. Two chain tPA (tctPA) is produced by plasmin but there are conflicting reports in the literature on the behaviour of sc- and tctPA and little work on inhibition by the specific inhibitor plasminogen activator inhibitor-1 (PAI-1) under physiological conditions. OBJECTIVES: To perform a systematic study on the kinetics of sctPA and tctPA as plasminogen activators and targets for PAI-1. METHODS: Detailed kinetic studies were performed in solution and in the presence of template stimulators, fibrinogen and fibrin, including native fibrin and partially digested fibrin. Numerical simulation techniques were utilized to cope with the challenges of investigating kinetics of activation and inhibition in the presence of fibrin(ogen). RESULTS: Enzyme efficiency (k(cat)/K(m)) was higher for tctPA than sctPA in solution with chromogenic substrate (3-fold) and plasminogen (7-fold) but in the presence of templates, such as fibrinogen and native or cleaved fibrin, the difference disappeared. sctPA was more susceptible to PAI-1 in buffer solution and in the presence of fibrinogen; however, in the presence of fibrin, PAI-1 inhibited more slowly and there was no difference between sc and tctPA. CONCLUSIONS: Fibrinogen and fibrin modulate the activity of tPA differently in regard to their activation of plasminogen and inhibition by PAI-1. Fibrinogen and fibrin stimulate tPA activity against plasminogen but fibrin protects tPA from PAI-1 to promote fibrinolysis.     

Thrombin induction of plasminogen activator-inhibitor in cultured human endothelial cells.

We have examined the effect of thrombin on the activity of plasminogen activator (PA) and plasminogen activator-inhibitor (PA-I) in medium conditioned by primary cultures of human umbilical vein endothelial cells. PA activity was measured by fibrinolytic and esterolytic assays, and total tissue-type PA (tPA) antigen by radioimmunoassay. Net PA-I activity was assayed by titration of human urokinase esterolytic activity. Incubation of confluent endothelial cell cultures with thrombin for 24 h caused a sixfold increase in PA-I activity. The effect of thrombin was half-maximal at approximately 0.4 U/ml (less than 4 nM), and required concomitant RNA and protein synthesis. The stimulation of PA-I activity required active alpha-thrombin and was not obtained with gamma-thrombin nor with thrombin catalytically inactivated with hirudin. Because of the excess of PA-I, PA activity was not measurable in either control or thrombin-treated cells. Thrombin did, however, increase medium concentration of tPA antigen by approximately fourfold. The thrombin-induced PA-I inhibited both tPA and urokinase, did not lose activity upon acidification, and was stable to sodium dodecyl sulfate and thiol reduction. We conclude that physiologic concentrations of thrombin increase both PA-I activity and tPA antigen in medium conditioned by human umbilical vein endothelial cells. Because there was always a several-fold increase in the net activity of free PA-I, these observations suggest that the net effect of thrombin is to decrease fibrinolytic activity in human endothelial cells. Thus, thrombin, in addition to its role in coagulation, may protect clots from premature lysis by increasing the amount of a specific fibrinolytic inhibitor.     

Biochemical and biological aspects of the plasminogen activation system

Plasminogen activators (PAs) are specific proteolytic enzymes which convert the inactive proenzyme plasminogen to plasmin. The plasmin formed is a potent and nonspecific protease which cleaves blood fibrin clots and several other extracellular proteins. In addition to their primary role in the initiation of fibrinolysis, PAs are implicated in a variety of basic biological processes, such as, degradation of the extracellular matrix, tumor invasiveness, tissue remodelling, and cellular differentiation. This review describes recent observations on the biochemical and biophysical characteristics of the different components of the plasminogen activation system. This complex system includes: the proenzymes of tissue type PA (tPA) and urokinase type PA (uPA); the active enzymes tPA, uPA and plasmin; the substrate plasminogen; several natural inhibitors of PA and plasmin activity; and the cellular receptors that bind the proenzymes, enzymes, and inhibitor-enzyme complexes. Through the coordinated interactions of these components, the location, timing, and extent of potent proteolytic activity is controlled. Recent findings on the structure, properties, biological functions, and regulation of the different components of the plasminogen activation cascade are reviewed. Current methods for assay of the amount and activity of the enzymes, inhibitors, and receptors are described. Observations implying specific functions of the system in health and disease, and its potential utilization for diagnosis are examined. Specifically, the potential application of PAs as laboratory markers of neoplasia, as diagnostic tools in diseases of the blood clotting system, their use for monitoring of thrombolytic therapy, and their possible relevance in certain disease states are described.     

Mechanism of ancrod anticoagulation: A direct proteolytic effect on fibrin

Fibrin formed in response to ancrod, reptilase, or thrombin was reduced by beta-mercaptoethanol and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was found that ancrod progressively and totally digested the alpha-chains of fibrin monomers at sites different than plasmin; however, further digestion of fibrin monomers by either reptilase or thrombin was not observed. Highly purified ancrod did not activate fibrin-stabilizing factor (FSF); however, the reptilase preparation used in these experiments, like thrombin, activated FSF and thereby promoted cross-link formation. Fibrin, formed by clotting purified human fibrinogen with ancrod, reptilase, or thrombin for increasing periods of time in the presence of plasminogen, was incubated with urokinase and observed for complete lysis. Fibrin formed by ancrod was strikingly more vulnerable to plasmin digestion than was fibrin formed by reptilase or thrombin. The lysis times for fibrin formed for 2 hr by ancrod, reptilase, or thrombin were 18, 89, and 120 min, respectively. Evidence was also obtained that neither ancrod nor reptilase activated human plasminogen. These results indicate that fibrin formed by ancrod is not cross-linked and has significantly degraded alpha-chains: as expected, ancrod-formed fibrin is markedly susceptible to digestion by plasmin.     

A comparative study of the promotion of tissue plasminogen activator and pro-urokinase-induced plasminogen activation by fragments D and E-2 of fibrin.

Plasmin generation by equimolar concentrations of tissue plasminogen activator (t-PA), pro-urokinase (pro-UK), and urokinase (UK), and a twofold higher concentration of a plasmin-resistant mutant rpro-UK (Ala-158-pro-UK) was measured on a microtiter plate reader. The promoting effects on this reaction of equimolar concentrations of fibrinogen, soluble fibrin (Desafib), CNBr fragment FCB-2 (an analogue of fragment D), or purified fragment E-2 were compared. Plasmin generation by t-PA was moderately promoted by fibrinogen, substantially promoted by Desafib and FCB-2, but not at all promoted by fragment E-2. By contrast, plasmin generation by pro-UK or by Ala-158-pro-UK was not promoted either by fibrinogen, Desafib, or FCB-2, but was significantly promoted by fragment E-2. Plasmin generation by UK was not significantly promoted by any of the fibrin(ogen) preparations. Treatment of fragment E-2 by carboxypeptidase-B (CPB), eliminated its promotion of pro-UK and Ala-158-pro-UK-induced plasmin generation. Pretreatment of FCB-2 with plasmin slightly potentiated its promotion of t-PA activity. This effect of plasmin pretreatment of FCB-2 was reversed by CPB treatment. Plasmin pretreatment of FCB-2 did not induce any promotion of activity in pro-UK or Ala-158-pro-UK. The findings show that the intrinsic activity of pro-UK and the activity of t-PA are promoted by different regions of the fibrin(ogen) molecule. The latter is stimulated primarily by a determinant in the fragment D region, which is available in intact fibrin. By contrast, plasminogen activation by the intrinsic activity of pro-UK was stimulated exclusively by fragment E-2, which is unavailable in intact fibrin. The findings are believed relevant to fibrinolysis and support the concept that t-PA and pro-UK are complementary, sequential, and synergistic in their actions.     

Plasminogen and Plasminogen Activators Protect against Renal Injury in Crescentic Glomerulonephritis

The plasminogen/plasmin system has the potential to affect the outcome of inflammatory diseases by regulating accumulation of fibrin and other matrix proteins. In human and experimental crescentic glomerulonephritis (GN), fibrin is an important mediator of glomerular injury and renal impairment. Glomerular deposition of matrix proteins is a feature of progressive disease. To study the role of plasminogen and plasminogen activators in the development of inflammatory glomerular injury, GN was induced in mice in which the genes for these proteins had been disrupted by homologous recombination. Deficiency of plasminogen or combined deficiency of tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA) was associated with severe functional and histological exacerbation of glomerular injury. Deficiency of tPA, the predominant plasminogen activator expressed in glomeruli, also exacerbated disease. uPA deficiency reduced glomerular macrophage infiltration and did not significantly exacerbate disease. uPA receptor deficiency did not effect the expression of GN. These studies demonstrate that plasminogen plays an important role in protecting the glomerulus from acute inflammatory injury and that tPA is the major protective plasminogen activator.     

Hindered dissolution of fibrin formed under mechanical stress

See also Weisel JW. Stressed fibrin lysis. This issue, pp 977–8. Summary. Background: Recent data indicate that stretching forces cause a dramatic decrease in clot volume accompanied by gross conformational changes of fibrin structure. Objective: The present study attempts to characterize the lytic susceptibility of fibrin exposed to mechanical stress as a model for fibrin structures observed in vivo. Methods and results: The relevance of stretched fibrin models was substantiated by scanning electron microscopic (SEM) evaluation of human thrombi removed during surgery, where surface fibrin fibers were observed to be oriented in the direction of shear forces, whereas interior fibers formed a random spatial meshwork. These structural variations were modeled in vitro with fibrin exposed to adjustable mechanical stress. After two‐ and three‐fold longitudinal stretching (2 × S, 3 × S) the median fiber diameter and pore area in SEM images of fibrin decreased two‐ to three‐fold. Application of tissue plasminogen activator (tPA) to the surface of model clots, which contained plasminogen, resulted in plasmin generation which was measured in the fluid phase. After 30‐min activation 12.6 ± 0.46 pmol mm^?2 plasmin was released from the non‐stretched clot (NS), 5.5 ± 1.11 pmol mm^?2 from 2 × S and 2.3 ± 0.36 pmol mm^?2 from 3 × S clot and this hampered plasmin generation was accompanied by decreased release of fibrin degradation products from stretched fibrins. Confocal microscopic images showed that a green fluorescent protein‐fusion variant of tPA accumulated in the superficial layer of NS, but not in stretched fibrin. Conclusion: Mechanical stress confers proteolytic resistance to fibrin, which is a result of impaired plasminogen activation coupled to lower plasmin sensitivity of the denser fibrin network.     

Tissue-type plasminogen activator increases the binding of glu-plasminogen to clots.

Porcine tissue-type plasminogen activator (t-PA) increases the binding of 125I-glu-plasminogen to clots made from human plasma or purified fibrinogen in a time and t-PA concentration dependent fashion. The accumulation of plasminogen was faster and greater on noncrosslinked plasma clots than on clots which had been crosslinked by Factor XIIIa. Furthermore, the uptake of plasminogen to crosslinked fibrin clots occurred at a slower rate in the presence of alpha 2-plasmin inhibitor (alpha 2 PI) than in its absence. The kinetics of the uptake of 125I-plasminogen were analyzed using SDS-polyacrylamide gel electrophoresis and radioautography of solubilized plasma clots formed in the presence of t-PA. During the initial phase there was a decrease of clot-bound glu-plasminogen; simultaneously, there was a slight increase in clot-bound glu-plasmin and in plasmin complexed to alpha 2 PI that was crosslinked to alpha-chain polymers of fibrin. This was followed by a marked increase in clot-bound plasminogen having glutamic acid as NH2-terminal (glu-plasminogen) and gluplasmin. t-PA-induced enhancement of glu-plasminogen uptake appears to be mediated by plasmin but does not require the conversion of glu-plasminogen to plasminogen having lysine or methionine as NH2-terminal. The described mechanism assures an adequate supply of clot-bound plasmin, which is the enzyme ultimately involved in the degradation of fibrin.     

A turbidimetric method of determining plasminogen]

Plasminogen level in euglobulin plasma fraction was determined from the rate of the sample transparency changes in the course of fibrin clot formation and lysis. Natural plasmin substrate, fibrin is used for measuring plasminogen this improving the measurement specificity and accuracy. Errors due to high concentrations of fibrinogen and/or its degradation products in the sample influencing the amidolytic activity of streptokinase-plasminogen complex are ruled out. The method is available and does not involve the use of chromogenic substrate.     

Soluble fibrin degradation products potentiate tissue plasminogen activator-induced fibrinogen proteolysis.

Despite its affinity for fibrin, tissue plasminogen activator (t-PA) administration causes systemic fibrinogenolysis. To investigate the mechanism, t-PA was incubated with plasma in the presence or absence of a fibrin clot, and the extent of fibrinogenolysis was determined by measuring B beta 1-42. In the presence of fibrin, there is a 21-fold increase in B beta 1-42 levels. The potentiation of fibrinogenolysis in the presence of fibrin is mediated by soluble fibrin degradation products because (a) the extent of t-PA induced fibrinogenolysis and clot lysis are directly related, (b) once clot lysis has been initiated, fibrinogenolysis continues even after the clot is removed, and (c) lysates of cross-linked fibrin clots potentiate t-PA-mediated fibrinogenolysis. Fibrin degradation products stimulate fibrinogenolysis by binding t-PA and plasminogen because approximately 70% of the labeled material in the clot lysates binds to both t-PA- and plasminogen-Sepharose, and only the bound fractions have potentiating activity. The binding site for t-PA and plasminogen is on the E domain because characterization of the potentiating fragments using gel filtration followed by PAGE and immunoblotting indicates that the major species is (DD)E complex, whereas minor components include high-molecular weight derivatives containing the (DD)E complex and fragment E. In contrast, D-dimer is the predominant species found in the fractions that do not bind to the adsorbants, and it has no potentiating activity. Thus, soluble products of t-PA-induced lysis of cross-linked fibrin potentiate t-PA-mediated fibrinogenolysis by providing a surface for t-PA and plasminogen binding thereby promoting plasmin generation. The occurrence of this phenomenon after therapeutic thrombolysis may explain the limited clot selectivity of t-PA.     

Regulation of plasminogen activation on cell surfaces and fibrin

Summary

The fibrinolytic system dissolves fibrin and maintains vascular patency. Recent advances in imaging analyses allowed visualization of the spatiotemporal regulatory mechanism of fibrinolysis, as well as its regulation by other plasma hemostasis cofactors. Vascular endothelial cells (VECs) retain tissue‐type plasminogen activator (tPA) after secretion and maintain high plasminogen (plg) activation potential on their surfaces. As in plasma, the serpin, plasminogen activator inhibitor type 1 (PAI‐1), regulates fibrinolytic potential via inhibition of the VEC surface‐bound plg activator, tPA. Once fibrin is formed, plg activation by tPA is initiated and effectively amplified on the surface of fibrin, and fibrin is rapidly degraded. The specific binding of plg and tPA to lytic edges of partly degraded fibrin via newly generated C‐terminal lysine residues, which amplifies fibrin digestion, is a central aspect of this pathophysiological mechanism. Thrombomodulin (TM) plays a role in the attenuation of plg binding on fibrin and the associated fibrinolysis, which is reversed by a carboxypeptidase B inhibitor. This suggests that the plasma procarboxypeptidase B, thrombin‐activatable fibrinolysis inhibitor (TAFI), which is activated by thrombin bound to TM on VECs, is a critical aspect of the regulation of plg activation on VECs and subsequent fibrinolysis. Platelets also contain PAI‐1, TAFI, TM, and the fibrin cross‐linking enzyme, factor (F) XIIIa, and either secrete or expose these agents upon activation in order to regulate fibrinolysis. In this review, the native machinery of plg activation and fibrinolysis, as well as their spatiotemporal regulatory mechanisms, as revealed by imaging analyses, are discussed.     

Fibrinolysis inhibitors.

The fibrinolytic system involves a series of enzymatic reactions that results in the conversion of the proenzyme, plasminogen, into the trypsin-like lytic enzyme, plasmin. The major physiologic target of plasmin is fibrin. Free plasmin in plasma is a nonspecific lytic enzyme that will degrade other proteins such as fibrinogen and coagulation factors V and VIII. Plasmin and activators of plasminogen also play a role in ovulation, embryo implantation, tissue remodeling, and inflammation.     

Fibrinolysis in health and disease: severe abnormalities in systemic lupus erythematosus

Methods are described to measure fibrinolysis in healthy persons and in patients with systemic lupus erythematosus. Using the fibrin plate method, total fibrinolytic activity and vascular plasminogen activator were measured. (Total fibrinolytic activity expresses the fibrinolytic potential and consists of both the intrinsic [factor XII-dependent and independent] activities and the extrinsic activities [vascular or tissue type]. Vascular plasminogen activator, assessed in a separate assay, refers to the endothelium-derived component only.) In addition, the degree of inhibition by plasma of both urokinase-induced and of plasmin-induced fibrinolysis were analyzed. Vascular plasminogen activator levels were low in 63% of plasma samples from 55 patients with systemic lupus erythematosus. The level of an inhibitor of plasminogen activation was significantly elevated in 87% of patients and levels of an inhibitor of plasmin were significantly elevated in 29%. The nonspecific serine protease inhibitors, including alpha 2-macroglobulin, were within the normal range in all patients. The natures of inhibitor of plasminogen activation and plasmin inhibitor were studied further. Using both the fibrin plate and the lysis time methods, the data indicated that the urokinase-inhibiting activity increased with time of incubation of plasma-enzyme mixtures, whereas the plasmin inhibiting activity did not. Elevated levels of plasmin inhibitor measured with the fibrin plate method correlated well with prolonged lysis times. Results using the chromogenic substrate S-2251, commonly used as a simple and specific assay for antiplasmin, agreed reasonably well with those using the fibrin plate method, but elevated plasmin inhibitor levels could be quantitated with greater accuracy and sensitivity by the fibrin plate method. Studies with an antiserum directed against alpha 2-antiplasmin showed that inhibitor of plasminogen activation and plasmin inhibitor were different inhibitors, and that plasmin inhibitor was identical to alpha 2-antiplasmin. The abnormalities are discussed in the light of current knowledge on fibrinolysis and as possible mediators in the pathogenesis and perpetuation of lupus glomerulonephritis.     

Fibrinogen counteracts the antiadhesive effect of fibrin‐bound plasminogen by preventing its activation by adherent U937 monocytic cells

Summary. Background: Fibrinogen and plasminogen strongly reduce adhesion of leukocytes and platelets to fibrin clots, highlighting a possible role for these plasma proteins in surface‐mediated control of thrombus growth and stability. In particular, adsorption of fibrinogen on fibrin clots renders their surfaces non‐adhesive, while the conversion of surface‐bound plasminogen to plasmin by transiently adherent blood cells results in degradation of a superficial fibrin layer, leading to cell detachment. Although the mechanisms whereby these proteins exert their antiadhesive effects are different, the outcome is the same: the formation of a mechanically unstable surface that does not allow firm cell attachment. Objectives: Since fibrin clots in circulation are exposed to both fibrinogen and plasminogen, their combined effect on adhesion of monocytic cells was examined. Methods: Fibrin gels were coated with plasminogen and its activation by adherent U937 monocytic cells in the presence of increasing concentrations of fibrinogen was examined by either measuring ¹²⁵I‐labeled fibrin degradation products or plasmin amidolytic activity. Results: Unexpectedly, the antiadhesive effects of two fibrin binding proteins were not additive; in fact, in the presence of fibrinogen, the effect of plasminogen was strongly reduced. An investigation of the underlying mechanism revealed that fibrinogen prevented activation of fibrin‐bound plasminogen by cells. Confocal microscopy showed that fibrinogen accumulates in a thin superficial layer of a clot, where it exerts its blocking effect on activation of plasminogen. Conclusion: The results point to a complex interplay between the fibrinogen‐ and plasminogen‐dependent antiadhesive systems, which may contribute to the mechanisms that control the adhesiveness of a fibrin shell on the surface of hemostatic thrombi.     

Thrombolysis with human extrinsic (tissue-type) plasminogen activator in dogs with femoral vein thrombosis.

Extrinsic (tissue-type) plasminogen activator (plasminogen activator) was isolated either as a single-chain or as a two-chain molecule from the culture medium of a human melanoma cell line. The thrombolytic activity of both molecular forms of activator was investigated in beagle dogs with an experimental femoral vein thrombosis and compared with that of urokinase. The 125I-fibrinogen-labeled thrombus was formed in an isolated 4-cm segment of the vein, aged for 30 min, and the thrombolytic substances were infused over a 4-h period. The degree of thrombolysis was measured 2 h later as the difference between the injected and recovered 125I. In six control animals with a saline infusion the extent of thrombolysis was 16.3 +/- 3.8% (mean +/- SEM), in five dogs receiving 100,000 IU urokinase, 17.4 +/- 3.7% (P less than 0.4) and in four dogs with 1,000,000 IU urokinase 40.6 +/- 4.8% (P less than 0.001). Infusion of 100.000 IU single-chain plasminogen activator in five dogs resulted in 3.5 +/- 7.8% lysis (P less than 0.05) and of 100,000 IU two-chain plasminogen activator in five dogs in 60.1 +/- 10.8% (P less than 0.001). Infusion of 300,000 IU one-chain plasminogen activator yielded 57.5% lysis and of the same amount of two-chain plasminogen activator 72.9%. Significant activation of plasminogen, consumption of alpha 2-antiplasmin, and fibrinogen breakdown in plasma was only observed in animals receiving the high doses of urokinase but not in the saline, plasminogen activator, or the low-dose urokinase groups. It is thus concluded that in this thrombosis model human extrinsic plasminogen activator has a higher specific thrombolytic effect that urokinase. Plasminogen activator also appears to induce thrombolysis without systemic fibrinolytic activation and fibrinogen breakdown.     

Apolipoprotein(a) inhibits the conversion of Glu‐plasminogen to Lys‐plasminogen: a novel mechanism for lipoprotein(a)‐mediated inhibition of plasminogen activation

Summary. Background: Elevated plasma concentrations of lipoprotein(a) [Lp(a)] are associated with an increased risk for thrombotic disorders. Lp(a) is a unique lipoprotein consisting of a low‐density lipoprotein‐like moiety covalently linked to apolipoprotein(a) [apo(a)], a homologue of the fibrinolytic proenzyme plasminogen. Several in vitro and in vivo studies have shown that Lp(a)/apo(a) can inhibit tissue‐type plasminogen activator‐mediated plasminogen activation on fibrin surfaces, although the mechanism of inhibition by apo(a) remains controversial. Essential to fibrin clot lysis are a number of plasmin‐dependent positive feedback reactions that enhance the efficiency of plasminogen activation, including the plasmin‐mediated conversion of Glu‐plasminogen to Lys‐plasminogen. Objective: Using acid–urea gel electrophoresis to resolve the two forms of radiolabeled plasminogen, we determined whether apo(a) is able to inhibit Glu‐plasminogen to Lys‐plasminogen conversion. Methods: The assays were performed in the absence or presence of different recombinant apo(a) species, including point mutants, deletion mutants and variants that represent greater than 90% of the known apo(a) isoform sizes. Results: Apo(a) substantially suppressed Glu‐plasminogen conversion. Critical roles were identified for the kringle IV types 5–9 and kringle V; contributory roles for sequences within the amino‐terminal half of the molecule were also observed. Additionally, with the exception of the smallest naturally‐occurring isoform of apo(a), isoform size was found not to contribute to the inhibitory capacity of apo(a). Conclusion: These findings underscore a novel contribution to the understanding of Lp(a)/apo(a)‐mediated inhibition of plasminogen activation: the ability of the apo(a) component of Lp(a) to inhibit the key positive feedback step of plasmin‐mediated Glu‐plasminogen to Lys‐plasminogen conversion.     

Increased Plasminogen Activator (Urokinase) in Tissue Culture After Fibrin Deposition

Lysis of fibrin in tissue culture has been shown to be due to plasminogen activator identified immunologically as urokinase. The present study examines fibrinolytic events in culture, particularly mechanisms leading to increased urokinase levels and accelerated fibrinolysis.Deposition of fibrin on cells in culture was followed by a two- to six-fold increase in urokinase in the supernates and rapid disappearance of the fibrin. Investigation of factors that might be responsible for these events (including fibrin, fibrinogen, vasoactive stimuli, and the enzymes thrombin and plasmin) indicated that the enhanced urokinase yields were mediated through plasmin and thrombin.Study of the possible modes of action of thrombin and plasmin indicated that these enzymes are capable of acting on the cells themselves as well as on cell-produced material. The effect on cells was manifested by mitotic activity or, occasionally, cell injury and death. Although these effects influenced urokinase levels, enhanced yields were explained best by the action of enzymes on cellproduced material. Studies with plasmin and thrombin, and also trypsin, indicated that proteolytic enzymes may act in various ways-affect the stability of urokinase, interfere with inhibition of urokinase by naturally occurring inhibitor(s), and induce urokinase activity from inactive material. Plasma and thrombin appeared to act primarily through the latter mechanism.Inactive material, which gave rise to urokinase upon exposure to proteolytic enzymes and which may represent urokinase precursor, was found in cultures of kidney, lung, spleen, and thyroid. Urokinase in such inactive state appears to be readily accessible to activation by enzymes, particularly plasmin and thrombin, thus facilitating removal of fibrin and possibly also providing pathways to excessive fibrinolysis.