H.R. Lijnen

New insights into the molecular mechanisms of the fibrinolytic system

Summary.  Fibrinolysis is regulated by specific molecular interactions between its main components. Activation of plasminogen by tissue‐type plasminogen activator (t‐PA) is enhanced in the presence of fibrin or at the endothelial cell surface. Urokinase‐type plasminogen activator (u‐PA) binds to a specific cellular u‐PA receptor (u‐PAR), resulting in enhanced activation of cell‐bound plasminogen. Inhibition of fibrinolysis occurs at the level of plasminogen activation or at the level of plasmin. Assembly of fibrinolytic components at the surface of fibrin results in fibrin degradation. Assembly at the surface of cells provides a mechanism for generation of localized cell‐associated proteolytic activity. This review includes novel proteins such a thrombin‐activatable fibrinolysis inhibitor (TAFI) and discusses new insights into molecular mechanisms obtained from the rapidly growing knowledge of crystal structures of proteins.

Pleiotropic functions of plasminogen activator inhibitor-1

Summary.  Plasminogen activator inhibitor‐1 (PAI‐1), a 45‐kDa serine proteinase inhibitor with reactive site peptide bond Arg345‐Met346, is the main physiological plasminogen activator inhibitor. It occurs in human plasma at an antigen concentration of about 20 ng mL−1. Besides the active inhibitory form of PAI‐1 that spontaneously converts to a latent form, also a substrate form exists that is cleaved at the P1‐P1′ site by its target enzymes, but does not form stable complexes. Besides its role in regulating hemostasis, PAI‐1 plays a role in several biological processes dependent on plasminogen activator or plasmin activity. Studies with transgenic mice have revealed a functional role for PAI‐1 in wound healing, atherosclerosis, metabolic disturbances such as obesity and insulin resistance, tumor angiogenesis, chronic stress, bone remodeling, asthma, rheumatoid arthritis, fibrosis, glomerulonephritis and sepsis. It is not always clear if these functions depend on the antiproteolytic activity of PAI‐1, on its binding to vitronectin or on its intereference with cellular migration or matrix binding.

On the specific interaction between the lysine-binding sites in plasmin and complementary sites in α2-antiplasmin and in fibrinogen

Plasminogen and plasminogen derivatives which contain lysine-binding sites were found to decrease the reaction rate between plasmin and alpha2-antiplasmin by competing with plasmin for the complementary site(s) in alpha2-antiplasmin. The dissocwation constant Kd for the interaction between intact plasminogen (Glu-plasminogen) and alpha2-antiplasmin is 4.0 microM but those for Lys-plasminogen or TLCK-plasmin are about 10-fold lower indicating a stronger interaction. The lysine-binding site(s) which is situated in triple-loops 1--3 in the plasmin A-chain is mainly responsible for the interaction with alpha2-antiplasmin. The interaction between Glu-plasminogen and alpha2-antiplasmin furthermore enhances the activation of Glu-plasminogen by urokinase to a comparable extent as 6-aminohexanoic acid, suggesting that similar conformational changes occur in the proenzyme after complex formation. Fibrinogen, fibrinogen digested with plasmin, purified fragment E and purified fragment D interfere with the reaction between plasmin and alpha2-antiplasmin by competing with alpha2-antiplasmin for the lysine-binding site(s) in the plasmin A-chain. The Kd obtained for these interactions varied between 0.2 microM and 1.4 microM; fragment E being the most effective. Thus the fibrinogen molecule contains several complementary sites to the lysine-binding sites located both in its NH2-terminal and COOH-terminal regions; these sites are to a large extent.

Studies on the mechanism of the antifibrinolytic action of tranexamic acid.

The effect of the antifibrinolytic agent tranexamic acid on the solubilization of 125I-labeled fibrin by plasmin or by mixtures of plasminogen and plasminogen activator (tissue activator or urokinase) was studied. The time required to solubilize half of the radioactivity (S50) decreased curvilinearly with the logarithm of the concentration of plasmin or plasminogen. Tranexamic acid caused a concentration-dependent retardation of fibrinolysis. When corresponding S50 values were converted to apparent concentrations of plasmin or plasminogen in the absence of tranexamic acid, sigmoidal relationships were obtained between the apparent plasmin(ogen) concentration and the logarithm of the concentration of tranexamic acid. When tissue activator (with a high affinity for fibrin) was used, the shape of these curves was compatible with single association reactions between plasminogen and tranexamic acid. A 50% decrease of the apparent plasminogen concentration was obtained at 1.2 μM tranexamic acid for native plasminogen (Glu-plasminogen) and 2.3 μM for proteolytically degraded plasminogen (Lys-plasminogen). The dissociation constants of the interaction between tranexamic acid and the high affinity lysine-binding site of Glu-plasminogen or Lys-plasminogen have previously been estimated at 1.1 and 2.2 μM, respectively. Direct measurements of the binding of 125I-labeled plasminogen to fibrin clots revealed that tranexamic acid displaced plasminogen from the fibrin surface; a 50% displacement was obtained at 1.3 μM tranexamic acid for Glu-plasminogen and 5.0 μM for Lys-plasminogen. These observations are compatible with the interpretation that saturation of the high affinity lysine-binding site of plasminogen with tranexamic acid results in its displacement from the fibrin surface and abolishes its activation by fibrin-bound plasminogen activator. When native or degraded plasminogen was activated with urokinase (with a low affinity for fibrin), tranexamic acid also caused a concentration-dependent retardation of fibrinolysis but of a much more complex nature. Saturation of the high affinity lysine-binding site in plasminogen with tranexamic acid had no significant influence on the solubilization rate of fibrin, indicating that binding of plasminogen to fibrin is of little importance for its activation by urokinase. At higher tranexamic acid concentrations an enhancement of Glu-plasminogen activation was observed, followed by interference of tranexamic acid with fibrinolysis by plasmin. These effects have already been described previously. Tranexamic acid caused a concentration-dependent retardation of fibrinolysis by plasmin; a 50% reduction of the apparent plasmin concentration was obtained at 45 μM tranexamic acid.

Tissue-type plasminogen activator. A review of its pharmacology and therapeutic use as a thrombolytic agent

SummarySynopsisCoronary arterial thrombolysis is becoming an established treatment of acute myocardial infarction. If given early enough, it recanalises occluded coronary arteries, salvages myocardial function and reduces mortality. A reduction of mortality in patients with acute myocardial infarction has now been demonstrated for streptokinase, anisoylated plasminogen streptokinase activator complex (APSAC; anistreplase) and recombinant tissue-type plasminogen activator (rt-PA)From the biochemical point of view, rt-PA has several attractive properties. It is similar to or identical with the physiological plasminogen activator in blood, it does not induce an antibody response, and it is more fibrin-specific than most or all other currently known thrombolytic agents. The rate of recanalisation of occluded coronary arteries with rt-PA is about 60 to 80% in non-comparative and placebo-controlled trials. rt-PA was similar in efficacy to urokinase in the only trial to compare the 2 agents. In 2 comparative trials evaluated by meta-analysis, rt-PA appeared more effective than streptokinase for the early recanalisation of occluded arteries. Both agents were comparable in their effects on left ventricular function in 2 comparative trials, but further study is needed to conclusively evaluate this parameter. Moreover, both agents reduce inhospital mortality, but much larger direct comparative trials are required before scientifically valid statements can be made on the relative clinical efficacy of available thrombolytic agents in terms of their effects on both morbidity and mortality.Thus, rt-PA constitutes a notable contribution of recombinant DNA technology to the treatment of thromboembolic disease, the main cause of death and disability in Western societies.Pharmacodynamic PropertiesRecombinant tissue-type plasminogen activator (rt-PA) for clinical use is produced by bulk fermentation of a Chinese hamster ovary cell line transfected with the cDNA for the naturally occurring human product. The subsequently purified proteinase is a single polypeptide chain of 527 amino acids which is fully glycosylated and identical to the naturally occurring human protein.The mechanism of action of rt-PA is similar to that of naturally occurring t-PA. t-PA has a high affinity to fibrin in a thrombus. In turn, t-PA has a high affinity and specificity towards fibrin-bound plasminogen, where it causes enzymatic degradation of the latter into plasmin and consequently thrombolysis. t-PA has only low affinity for plasminogen in the absence of fibrin. Thus, the fibrinolytic process induced by t-PA is fibrin-specific and causes only limited systemic plasminogen activation and fibrinogenolysis.Various in vitro studies have demonstrated the fibrinolytic activity of t-PA against clots, while causing only minor systemic activation of the fibrinolytic system. The in vivo thrombolytic properties of t-PA have been confirmed in numerous and varied animal models of thrombolysis, including pulmonary emboli, thrombosis of jugular and femoral veins and coronary and femoral arteries. t-PA was more potent than u-PA (urokinase-type plasminogen activator), and it produced more rapid and more effective lysis. It was also more rapid and more effective than streptokinase. In addition, t-PA caused less extensive systemic breakdown of fibrinogen than u-PA and streptokinase. Preliminary animal studies suggest a use for t-PA in stroke and some ophthalmological conditions.Pharmacokinetic PropertiesThe disposition of t-PA in plasma can be represented by a 2-compartment model composed of 1 central (plasma) compartment and 1 peripheral compartment. t-PA is rapidly cleared from circulation by the liver, with an initial half-life of only a few minutes in animals. However, fibrin-bound t-PA remains pharmacologically active at the clot site for several hours after withdrawal of the systemic infusion of rt-PA and its clearance from circulation. In healthy subjects intravenous infusion of commercially available rt-PA (8.3 μg/kg/min) yields a mean steady-state plasma concentration of about 1 to 1,5 mg/L. Other mean pharmacokinetic parameters in subjects were: initial half-life 3 to 4 minutes; terminal half-life about 30 minutes; plasma clearance about 40 L/h; and volumes of distribution of the central compartment and at steady-state of 3.9 and 7.2L, respectively. Estimation of t-PA may vary significantly depending on the assay system used and on the method of blood collection and storage.Therapeutic StudiesUsing coronary artery reperfusion or patency as end-points, intravenous infusion of rt-PA is superior to placebo in the treatment of acute myocardial infarction. At total doses of 40 to 100mg administered over 1 to 3 hours, rt-PA produces reperfusion in about 60 to 80% of infarct-related arteries, as demonstrated in non-comparative trials. Compared with placebo, rt-PA improves regional wall motion of the infarcted zone and enhances left ventricular function. The earlier rt-PA is administered after the onset of symptoms the better the chance of reperfusion and salvage of left ventricular function. Overall inhospital mortality after rt-PA therapy was low (4 to 7%), but there were no control groups in most studies. However, 2 recent placebo-controlled studies have shown that mortality is significantly reduced, especially if rt-PA is administered within 3 hours of the onset of symptoms. Reocclusion of the coronary arteries can occur in about 7 to 15% of patients after rt-PA therapy.Cumulated results of non-comparative patency or reperfusion trials using comparable end-points demonstrate rt-PA to reperfuse coronary arteries more efficiently than streptokinase. Meta-analysis of patency data from 2 trials directly comparing the efficacies of rt-PA and streptokinase indicates a significantly higher frequency of patent arteries with rt-PA at 90 minutes after starting treatment. Reocclusion rates have not been evaluated in directly comparable trials, but appear to be similar. Bleeding complications in comparative trials appear somewhat, but not dramatically, lower with rt-PA than with streptokinase. The relative impact of streptokinase and rt-PA on preservation of left ventricular function has been evaluated in 2 comparative trials, which showed comparable effects for the 2 agents. However, further studies are needed to arrive at conclusive results. In small comparative trials not designed with mortality as end-points, the combined inhospital mortality was 5.4% for rt-PA and 7.7% for streptokinase. Large directly comparative trials designed to specifically address mortality are required to establish the relative impact of each drug on mortality. rt-PA and urokinase were of similar efficacy in the only trial to compare these 2 agents.Several small studies and case reports have indicated that rt-PA may be useful in the treatment of a variety of other indications including pulmonary embolism, unstable angina pectoris, deep vein thrombosis, peripheral arterial occlusion, stroke and some ophthalmological conditions. However, controlled studies in larger numbers of patients will be required before any definite conclusions can be drawn concerning its efficacy in these indications.Adverse EffectsMinor adverse effects such as nausea, vomiting, hypotension and fever have been reported with rt-PA, but these effects may not be attributable to the drug as they are frequent sequelae of myocardial infarction. No serious immunogenic reactions have been noted with rt-PA, unlike some other thrombolytic treatments, although mild hypersensitivity reactions such as urticaria have occasionally been observed.Most interest concerning the tolerability of thrombolytic therapy has centred on the relative risk of systemic fibrinolytic activation and consequent bleeding complications. At usual therapeutic doses rt-PA induces little systemic fibrinolytic activation, in particular compared with streptokinase. However, in clinical practice rt-PA therapy remains associated with a residuai bleeding tendency. The type of bleeding associated with thrombolytic therapy may be divided into 2 kinds: superficial and internal. Superficial bleeding (e.g. venous cutdowns, arterial punctures, sites of recent surgical intervention) are relatively more frequent but not critical compared with the rare, more serious cases of internal bleeding involving the gastrointestinal tract, genitourinary tract, retroperitoneal and intracranial sites. Life-threatening intracranial bleeding occurred in some clinical studies of rt-PA when total doses up to 150mg were used, but there appears to be no increased risk of fatality from this complication since a total dose restriction to a maximum of 100mg has been instituted. In a controlled clinical trial in over 5000 patients who did not receive concomitant aspirin the stroke rates in rt-PA and control groups were similar.Dosage and Administrationrt-PA is indicated for use in adults with acute myocardial infarction. Treatment should be initiated as soon as possible after the onset of symptoms (at the latest 6 hours after the onset of pain). In the US the recommended total dose is 100mg administered by intravenous infusion as 60mg in the first hour (of which 6 to 10mg is administered as a bolus over the first 1 to 2 minutes) followed by 20mg in each of the subsequent 2 hours. For patients weighing less than 65kg, a total dose of 1.25 mg/kg administered over 3 hours, as described above, may be used. In European countries the recommended total dose is 70 to 100mg in 90 minutes with a 10mg bolus, to a total dosage of 1 mg/kg. Dose regimens adjusted for bodyweight appear to result in an improvement in coronary patency, with a lower rate of haemorrhagic complications. A higher total dose of rt-PA must not be used as 150mg has been associated with an increased risk of intracranial bleeding. In most patients treated to date, heparin has been administered concomitantly for 48 hours or more. Aspirin and/o

Analysis of coagulation and fibrinolysis during intravenous infusion of recombinant human tissue-type plasminogen activator in patients with acute myocardial infarction.

Coagulation and fibrinolysis were studied in patients with acute myocardial infarction during intravenous infusion of recombinant human tissue-type plasminogen activator (rt-PA) (0.75 mg/kg over 90 min, n = 101), streptokinase (1,500,000 IU over 60 min, n = 61), or placebo (n = 40). In the rt-PA group, the plasma level of rt-PA antigen was 1.2 +/- 0.6 micrograms/ml (mean +/- SD) and the euglobulin fibrinolytic activity (EFA) was 910 +/- 735 IU t-PA/ml. In the streptokinase group, the EFA was equivalent to 430 +/- 435 IU t-PA/ml. At the end of the infusion, the plasma fibrinogen level measured with a coagulation rate assay was decreased to 57 +/- 33% of the preinfusion value in the rt-PA group, to 7 +/- 10% in the streptokinase group, and remained unchanged in the placebo group. Fibrinogen-fibrin degradation products increased to 0.75 +/- 0.54 mg/ml in the streptokinase group but to only 0.10 +/- 0.13 mg/ml in the rt-PA group. The plasma levels of alpha 2-antiplasmin, plasminogen, and factor V decreased to between 30% and 45% in the rt-PA group but significantly more in the streptokinase group (to between 15% and 25%). Thus rt-PA induced much less systemic fibrinolytic activation than streptokinase. In the patients who received rt-PA, a weak correlation (r = .21, n = 89, .1 greater than p greater than .05) was found between the extent of fibrinogen breakdown at 90 min and the plasma rt-PA concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of PAI-1 on Adipose Tissue Growth and Metabolic Parameters in a Murine Model of Diet-Induced Obesity

An increased plasma plasminogen activator inhibitor-1 (PAI-1) level is a risk factor for myocardial infarction, particularly when associated with visceral obesity. Although the link between PAI-1 and obesity is well documented, little is known about the physiological relevance of PAI-1 production by adipose tissue. Therefore, we have compared adipose tissue development and insulin resistance plasma parameters in PAI-1-deficient mice (PAI-1(-/-)) and wild-type littermates (PAI-1(+/+)) in a model of nutritionally induced obesity. After 17 weeks of consuming a high-fat diet (HFD), PAI-1(+/+) mice showed marked obesity, with a 52% increase in body weight compared with mice that were kept on a standard fat diet (P<0.0001). This weight gain was accompanied by adipocyte hypertrophy and an increase in the number of stroma cells in the gonadal fat pad, expressed as stroma cells/adipocytes (0.67+/-0.05 versus 0.43+/-0. 02; P<0.001). In plasma, the HFD induced a marked increase in PAI-1 antigen (5.1+/-0.56 versus 2+/-0.22 ng/mL; P<0.001), fasting insulinemia (1.1+/-0.21 versus 0.21+/-0.04 ng/mL; P<0.001), and glycemia (7.4+/-0.5 versus 5+/-0.3 mmol/L; P<0.001), whereas plasma triglyceride levels were not affected. When we compared PAI-1(-/-) and PAI-1(+/+) mice on the HFD, PAI-1(-/-) mice gained weight faster than did PAI-1(+/+) mice, with a significant difference in body weight between 3 and 8 weeks of the diet (32+/-1.7 versus 26+/-1.6 g at 6 weeks; P<0.05). After 17 weeks of the HFD, its effect on weight gain and the number and size of adipocytes was similar in PAI-1(+/+) and PAI-1(-/-) mice. By contrast, the increase in the number of stroma cells presented by PAI-1(+/+) mice was not observed in PAI-1(-/-) mice. In obese PAI-1(-/-) mice, tissue-type PA activity and antigen levels in the gonadal fat pad were significantly higher than in obese PAI-1(+/+) mice (230+/-50 versus 47+/-20 arbitrary units/g, P<0.01; 40+/-13 versus 17+/-13 ng/g, P<0.05, respectively), whereas urokinase-type PA activity and antigen levels were similar in both groups. In plasma, nonobese PAI-1(-/-) mice displayed 62% higher insulin levels (P<0.05) than did PAI-1(+/+) mice. Obese PAI-1(-/-) mice displayed 68% higher triglyceride levels (P<0.01) and 21% lower glucose levels (P<0.05) than did PAI-1(+/+) mice. These data support an effect of PAI-1 on weight gain and adipose tissue cellularity in the induction of obesity in mice. Moreover, PAI-1 influences glucidolipidic metabolism. The elevated expression of PAI-1 observed in human obesity could be involved in mechanisms that control adipose tissue development.

Endothelium in hemostasis and thrombosis.

Endothelial cells synthesize and secrete PA and PAI, and thus provide anticoagulant and procoagulant regulatory mechanisms, respectively. Both plasminogen and plasminogen activators (t-PA and u-PA) bind to specific cellular receptors; assembly of components of the fibrinolytic system at the endothelial cell surface results in stimulation of fibrinolytic activity. Several mechanisms contribute to this stimulation, eg, enhanced plasminogen activation by t-PA or u-PA, enhanced conversion of scu-PA to tcu-PA, and impaired inhibition of plasmin by alpha2-antiplasmin or of PAs by PAIs. Thus, the endothelial cell surface serves as a focal point for plasmin generation.

Stromelysin-1 (MMP-3) is critical for intracranial bleeding after t-PA treatment of stroke in mice

Summary.  Background: Tissue‐type plasminogen activator (t‐PA) is approved for treatment of ischemic stroke patients, but it may increase the risk of intracranial bleeding (ICB). Matrix metalloproteinases (MMPs), which can be activated through the plasminogen/plasmin system, may contribute to ICB after ischemic stroke. Objectives: To explore the contribution of plasminogen, MMP‐3 and MMP‐9 to ICB associated with t‐PA treatment after ischemic stroke. Methods: Using a thrombotic middle cerebral artery occlusion (MCA‐O) model, ICB was studied in mice with genetic deficiencies of plasminogen (Plg−/−), stromelysin‐1 (MMP‐3−/−), or gelatinase B (MMP‐9−/−) and their corresponding wild‐type (WT) littermates. The induction of MMP‐3 and MMP‐9 was also studied in C57BL/6 WT mice. Results: ICB induced by t‐PA (10 mg kg−1) was significantly less than WT in Plg−/− (P < 0.05) and MMP‐3−/− (P < 0.05) but not in MMP‐9−/− mice. Furthermore, administration of the broad‐spectrum MMP inhibitor GM6001 after t‐PA treatment reduced ICB significantly (P < 0.05) in MMP‐3+/+ mice, but had no effect on MMP‐3−/− mice. MMP‐3 expression was significantly enhanced at the ischemic hemisphere; with placebo treatment, it was expressed only in neurons, whereas it was up‐regulated in endothelial cells with t‐PA treatment. Although MMP‐9 expression was also significantly enhanced at the ischemic brain, the amount and the distribution were comparable in mice with and without t‐PA treatment. Conclusions: Our data with gene‐deficient mice thus suggest that plasminogen and MMP‐3 are relatively more important than MMP‐9 for the increased ICB induced by t‐PA treatment of ischemic stroke.

Tissue-type plasminogen activator: a historical perspective and personal account.

Summary.  Over the past two decades tissue‐type plasminogen activator (t‐PA), the main physiological plasminogen activator, has been developed as a fibrin‐specific thrombolytic agent for the treatment of various thromboembolic diseases. Milestones in this development include: first purification of human t‐PA from uterine tissue, elucidation of the interactions regulating physiological fibrinolysis, thus providing a molecular basis for the concept of fibrin‐specific plasminogen activation, first animal models of thrombosis and pilot studies in patients supporting the therapeutic potential of t‐PA, cloning and expression of recombinant t‐PA providing sufficient amounts for large scale clinical use, and demonstration of its therapeutic benefit in large multicenter clinical trials, mainly in patients with acute myocardial infarction (AMI), but also in patients with massive pulmonary embolism, ischemic stroke, deep vein thrombosis and peripheral arterial occlusion. Genetically modified variants of t‐PA have been developed for bolus administration in patients with AMI.