Understanding the enzymology of fibrinolysis and improving thrombolytic therapy

Cardiovascular disease is responsible for 17 million deaths per year but acute myocardial infarction and stroke can be treated with thrombolytics ("clot busters"), which are plasminogen activators. However, despite many years of study and huge investment from the pharmaceutical industry, clinical trials of new drugs have often been disappointing. Part of the problem may be our incomplete understanding of the regulation of plasminogen activation in vivo. We have developed precise in vitro methods and with the application of computer simulations, we hope to improve our understanding of plasminogen activation to facilitate improvements in thrombolytic therapy.     

Molecular mechanisms of fibrinolysis and their application to fibrin-specific thrombolytic therapy

The fibrinolytic system comprises a proenzyme, plasminogen, which can be converted to the active enzyme, plasmin, which degrades fibrin. Plasminogen activation is mediated by plasminogen activators, which are classified as either tissue-type plasminogen activators (t-PA) or urokinase-type plasminogen activators (u-PA). Inhibition of the fibrinolytic system may occur at the level of the activators or at the level of generated plasmin. Plasmin has a low substrate specificity, and when circulating freely in the blood it degrades several proteins including fibrinogen, factor V, and factor VIII. Plasma does, however, contain a fast-acting plasmin inhibitor, alpha 2-antiplasmin, which inhibits free plasmin extremely rapidly but which reacts much slower with plasmin bound to fibrin. A systemic fibrinolytic state may, however, occur by extensive activation of plasminogen and depletion of alpha 2-antiplasmin. Clot-specific thrombolysis therefore requires plasminogen activation restricted to the vicinity of the fibrin. Two physiological plasminogen activators, t-PA and single-chain u-PA (scu-PA) induce clot-specific thrombolysis, via entirely different mechanisms, however. t-PA is relatively inactive in the absence of fibrin, but fibrin strikingly enhances the activation rate of plasminogen by t-PA. This is explained by an increased affinity of fibrin-bound t-PA for plasminogen and not by alteration of the catalytic rate constant of the enzyme. The high affinity of t-PA for plasminogen in the presence of fibrin thus allows efficient activation on the fibrin clot, while no significant plasminogen activation by t-PA occurs in plasma. scu-PA has a high affinity for plasminogen (Km = 0.3 microM) but a low catalytic rate constant (kcat = 0.02 sec-1). However, scu-PA does not activate plasminogen in plasma in the absence of a fibrin clot, owing to the presence of (a) competitive inhibitor(s). Fibrin-specific thrombolysis appears to be due to the fact that fibrin reverses the competitive inhibition. The thrombolytic efficacy and fibrin specificity of natural and recombinant t-PA has been demonstrated in animal models of pulmonary embolism, venous thrombosis, and coronary artery thrombosis. In all these studies intravenous infusion of t-PA at sufficiently high rates caused efficient thrombolysis in the absence of systemic fibrinolytic activation. The efficacy and relative fibrinogen-sparing effect of t-PA was recently confirmed in three multicenter clinical trials in patients with acute myocardial infarction.(ABSTRACT TRUNCATED AT 400 WORDS)     

30 years of thrombolytic therapy

The present state of streptokinase treatment at a "moderately high dose" was presented. Based on the experience accumulated, recommendations were given for improvement of the thrombolytic therapy.     

Ultrasound enhancement of thrombolytic therapy: observations and mechanisms

Fibrinolytic therapy is a proven approach for achieving reperfusion of occluded coronary arteries during myocardial infarction, resulting in reduced mortality and preservation of ventricular function. The amount of myocardial muscle loss is proportional to the duration of ischemia. Bleeding complications are not infrequent. Adjuvant therapy by ultrasound might enhance the rate of fibrinolysis and reduce the concentrations of lytic agents required to achieve an equivalent degree of clot lysis. Noninvasive ultrasound at low intensities and high frequencies, parameters that potentially could be applied and tolerated in vivo, have been proven to significantly accelerate the rate of fibrinolysis in both in vitro and in vivo models, in pure fibrin as well as whole blood clots. Such enhancement is not drug-specific. These effects were achieved by nonthermal mechanism. Ultrasound exposure did not cause mechanical fragmentation of the clot, did not alter the size of plasmatic derivates and degradation products. Ultrasound caused increased flow rate through thrombi, probably by cavitation-induced changes in fibrin ultrastructure; disaggregation of uncrosslinked fibrin fibers into smaller fibers has been shown. This resulted in increased transport of the lytic agent into the clot, alteration of binding affinity and increased maximum binding. Presence of echo-contrast agent induced further acceleration of thrombolysis by ultrasound.     

Redesigning t-PA for improved thrombolytic therapy.

Attempts are being made to redesign the structure of tissue-type plasminogen activator (t-PA) in order to increase its plasma half-life, increase its fibrin affinity or decrease its rate of interaction with plasma inhibitors. The principal strategies employed so far have been to construct hybrid enzymes, to mutate the polypeptide sequence of t-PA or to add extra fibrin-binding elements. It has been relatively easy to alter the half-life of t-PA but more difficult to do this with retention of the full specific activity of the molecule; the most promising molecules will have to be evaluated in the clinic before we know whether the redesign of t-PA has been truly successful.     

Mechanism of inhibition of non-enzymatic fibrinolysis by a spleen factor

Some data on the mechanism of inhibition of non-enzymatic fibrinolysis by a spleen factor are presented. It is demonstrated that the spleen protein factor which interacts with heparin at certain ratios forms a complex with the latter. As a result, the anticoagulating activity and properties of the spleen factor as a non-enzymatic fibrinolysis inhibitor are blocked.     

Mechanism of fibrin-specific fibrinolysis by staphylokinase: participation of alpha 2-plasmin inhibitor

When the extent of plasminogen activation by staphylokinase (SAK) or streptokinase (SK) was measured in human plasma, SAK barely induced plasminogen activation, whereas SK activated plasminogen significantly. When the plasma was clotted with thrombin, the plasminogen activation by SAK was markedly enhanced, but that of SK was little enhanced. Similarly, in a purified system composed of plasminogen, fibrinogen and alpha 2-plasmin inhibitor (alpha 2-PI, alpha 2-antiplasmin), such a fibrin clot increased the activity of SAK significantly. However, when alpha 2-PI was removed from the reaction system, enhancement of the SAK reaction was not observed. In addition, SAK as distinct from SK, showed very little interference with the action of alpha 2-PI. Plasminogen activation by SAK is thus essentially inhibited by alpha 2-PI, but this reaction is not inhibited in fibrin clots. These results suggest that SAK forms a complex with plasminogen, which binds to fibrin and induces fibrinolysis.     

Prospective evaluation of eligibility for thrombolytic therapy in acute myocardial infarction.

OBJECTIVES--To determine the proportion of patients presenting with acute myocardial infarction who are eligible for thrombolytic therapy. DESIGN--Cohort follow up study. SETTING--The four coronary care units in Auckland, New Zealand. SUBJECTS--All 3014 patients presenting to the units with suspected myocardial infarction in 1993. MAIN OUTCOME MEASURES--Eligibility for reperfusion with thrombolytic therapy (presentation within 12 hours of the onset of ischaemic chest pain with ST elevation > or = 2 mm in leads V1-V3, ST elevation > or = 1 mm in any other two contiguous leads, or new left bundle branch block); proportions of (a) patients eligible for reperfusion and (b) patients with contraindications to thrombolysis; death (including causes); definite myocardial infarction. RESULTS--948 patients had definite myocardial infarction, 124 probable myocardial infarction, and nine ST elevation but no infarction; 1274 patients had unstable angina and 659 chest pain of other causes. Of patients with definite or probable myocardial infarction, 576 (53.3%) were eligible for reperfusion, 39 had definite contraindications to thrombolysis (risk of bleeding). Hence 49.7% of patients (537/1081) were eligible for thrombolysis and 43.5% (470) received this treatment. Hospital mortality among patients eligible for reperfusion was 11.7% (55/470 cases) among those who received thrombolysis and 17.0% (18/106) among those who did not. CONCLUSIONS--On current criteria about half of patients admitted to coronary care units with definite or probable myocardial infarction are eligible for thrombolytic therapy. Few eligible patients have definite contraindications to thrombolytic therapy. Mortality for all community admissions for myocardial infarction remains high.     

Successes, failures and complications in thrombolytic therapy in peripheral, arterial and venous occlusions]

Thrombocytic therapy in peripheral arterial and venous vessel occlusion represents a clearly described alternative towards the surgery of vessels. A success rate of 36.5% can be found in subacute peripheral arterial thrombosis and 46.3% in subacute thrombotic occlusion of a bypass-graft. Contrary to that, a rate of 29.8% can be found in complications or side-effects respectively. In cases of peripheral deep venous thrombosis, a partial or full success can be found in 72%. However, the rate of complication amounting to 44.2% is comparatively high. The longer thrombolytic therapy with streptokinase or urokinase will last, the more frequently and more serious will be the complications, such as bleedings of different kind as well as increase of temperature to mention the most frequent ones. The application of urokinase is absolutely possible today, however, the use of urokinase seems to be only justified, if a thrombolytic therapy with streptokinase was carried out successfully and a subsequent surgical therapy was not possible. The present costs of this preparation are far too high for urokinase to be applied routinely. A thrombolytic therapy with SK as well as with UK has to be followed by an anticoagulant treatment.