凝血系统相关基因突变及表达异常与高血凝

摘要高血凝是动脉粥样硬化(As)的危险因子,在As的发展中具有重要作用。凝血系统、抗凝系统、纤溶系统及其它相关基因的突变及表达异常导致高血凝的产生。凝血系统的凝血因子V基因、凝血酶原基因、组织因子基因,抗凝系统的血栓调节蛋白基因、抗凝血酶Ⅲ基因,纤溶系统的纤溶酶原激活物抑制剂-1基因,均与高血凝密切相关。     

Biological control of tissue plasminogen activator-mediated fibrinolysis

Fibrinolysis, the body's ability to degrade fibrin, is an integrated part of hemostasis. Overactivity in the fibrinolytic system causes bleeding and underactivity causes thrombosis. Tissue plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1), alpha 2-antiplasmin (alpha 2-AP) and plasminogen are definitely involved in fibrinolysis because: (1) these components can be assigned a fibrinolytic role in purified systems, i.e. in vitro, and (2) abnormal structural variants and abnormal levels of these components give rise to bleeding or to thrombosis. The biological control of tPA-mediated fibrinolysis is both cellular and humoral. The cellular regulation compasses synthesis of tPA and PAI-1 and release/uptake of these components. The humoral regulation involves: (1) the reaction between tPA and PAI-1; (2) the fibrin-stimulated plasminogen activation; (3) the reaction between plasmin and alpha 2-AP and (4) plasmin degradation of fibrin. The highly developed biological control of tPA-mediated fibrinolysis is indicative of its physiological importance.     

Clinical disorders of fibrinolysis: a critical review

Much progress has recently been made in understanding the biochemistry and physiology of endogenous fibrinolysis. As a result, a better understanding of the mechanisms and clinical consequences of disordered fibrinolysis has emerged. Increased fibrinolytic activity is an uncommon but important cause of hemorrhagic disease. Congenital disorders of fibrinolysis which cause bleeding include increased plasma plasminogen activator activity and deficiency of alpha-2 antiplasmin. Acquired disorders associated with increased fibrinolytic activity and bleeding include liver cirrhosis, amyloidosis, acute promyelocytic leukemia, some solid tumors, and certain snake envenomation syndromes. Increased fibrinolysis is important to recognize because epsilon-aminocaproic acid (EACA) may be required to prevent or control bleeding. Diminished fibrinolytic activity has been associated with a variety of thrombotic disorders, but a direct cause-and-effect relationship has yet to be established. Congenital abnormalities of fibrinolysis associated with thrombosis include plasminogen deficiency, decreased endothelial generation of plasminogen activator activity, and certain abnormal fibrinogens. Thrombosis in these disorders is effectively managed with warfarin. Diminished fibrinolysis has also been reported in "idiopathic" venous thrombosis, oral contraceptive-induced and post-operative venous thrombosis, coronary artery disease, cerebrovascular disease, systemic lupus erythematosus, and thrombotic thrombocytopenic purpura, but the significance of abnormal fibrinolysis in these disorders is uncertain. Large, prospective studies of fibrinolytic variables as risk factors for vascular and thrombotic disease are needed to determine whether pharmacologic augmentation of impaired fibrinolysis could be useful in the prevention or treatment of these disorders.     

Two different mechanisms in patients with venous thrombosis and defective fibrinolysis: low concentration of plasminogen activator or increased concentration of plasminogen activator inhibitor.

Fibrinolytic components after venous occlusion and concentrations of tissue plasminogen activator inhibitor were studied in 100 consecutive patients with confirmed recurrent deep vein thrombosis or pulmonary embolism. After 20 minutes of venous occlusion the fibrinolytic response was decreased in 33 patients, as measured both amidolytically with S-2251 and on fibrin plates. Two different mechanisms responsible for the poor fibrinolytic response could be distinguished. Twenty two of the patients in whom the response was poor released normal amounts of tissue plasminogen activator antigen, as assayed by immunoradiometric assay, but had appreciably increased concentrations of tissue plasminogen activator inhibitor. The 11 other patients in whom the response was poor had both low tissue plasminogen activator activities and low tissue plasminogen activator antigen concentrations but normal concentrations of tissue plasminogen activator inhibitor. The results show not only that defective synthesis or release of tissue plasminogen activator may be important in the pathogenesis of venous thrombosis but also that a large group of patients with thrombosis have an increased concentration of the inhibitor to tissue plasminogen activator.     

Plasminogen activation: biochemistry, physiology, and therapeutics

The mammalian serine protease zymogen, plasminogen, can be converted into the active enzyme plasmin by vertebrate plasminogen activators urokinase (uPA), tissue plasminogen activator (tPA), factor XII-dependent components, or by bacterial streptokinase. The biochemical properties of the major components of the system, plasminogen/plasmin, plasminogen activators, and inhibitors of the plasminogen activators, are reviewed. The plasmin system has been implicated in a variety of physiological and pathological processes such as fibrinolysis, tissue remodeling, cell migration, inflammation, and tumor invasion and metastasis. A defective plasminogen activator/inhibitor system also has been linked to some thromboembolic complications. Recent studies of the mechanism of fibrinolysis in human plasma suggest that tPA may be the primary initiator and that overall fibrinolytic activity is strongly regulated at the tPA level. A simple model for the initiation and regulation of plasma fibrinolysis based on these studies has been formulated. The plasminogen activators have been used for thrombolytic therapy. Three new thrombolytic agents--tPA, pro-uPA, and acylated streptokinase-plasminogen complex--have been found to possess better properties over their predecessors, urokinase and streptokinase. Further improvements of these molecules using genetic and protein engineering tactics are being pursued.     

Development of new thrombolytic agents using recombinant DNA technology.

The increasing incidence of thromboembolic diseases has sustained the search for new agents able to stimulate the natural fibrinolytic system. The first generation of antithrombotic agents include bacterial streptokinase and human urine urokinase. Because these molecules lack specificity for the fibrin clot, important efforts have been made to produce, using recombinant DNA technology, agents presenting higher fibrin clot selectivity such as t-PA (tissue-type plasminogen activator) and scu-PA (single chain urokinase-type plasminogen activator). In parallel, several laboratories are presently attempting to create mutants and hybrids plasminogen activators displaying improved thrombolytic properties with respect to the natural molecules. In this paper, we describe briefly the mechanisms of fibrinolysis and the role of the different natural thrombolytic agents. In addition, we review the possibilities of genetic engineering for the production of natural and novel plasminogen activators.     

Deep venous thrombosis of the arm: a study of coagulation and fibrinolysis.

Sixty consecutive patients with phlebographically verified deep venous thrombosis of the upper arm were studied for disorders of coagulation and fibrinolysis. No appreciable increase in abnormalities of the factor VIII complex, antithrombin III, or inhibitors of activators of fibrinolysis were found. A decreased fibrinolytic defence mechanism, evident either as a deficient release capacity of fibrinolytic activators from the vein during stasis or as decreased fibrinolytic activity in the vein wall as determined histochemically, was found in 26 out of 53 patients studied (49%). It is concluded that deep venous thrombosis of the upper arm is a multifactorial disease. An impaired fibrinolytic defence mechanism is one of the factors that may be of pathogenetic importance.     

Anticoagulant low molecular weight heparin does not enhance the activation of plasminogen by tissue plasminogen activator

The activity of tissue plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) is stimulated by heparin. Heparin binds tightly to t-PA, u-PA, and plasminogen and decreases the usual stimulatory effect of fibrin on t-PA activity. In the present study we have found that low molecular weight heparin (LMW-heparin) preparations obtained by nitrous acid depolymerization or heparinase treatment of standard heparin have different properties with respect to their interaction with the fibrinolytic system. LMW-heparin prepared by either method does not stimulate plasmin formation by t-PA. However, these preparations of heparin still efficiently accelerate the inhibition of thrombin by antithrombin III. Binding data show that LMW-heparin does not bind t-PA and Glu-plasminogen and only binds very weakly to Lys-plasminogen. These results illustrate that it is possible to selectively destroy the fibrinolytic stimulating properties of heparin while leaving the classical anticoagulant characteristics intact.     

嵌合体纤溶酶的研究进展

嵌合体纤溶酶是采用基因重组技术或化学偶联的方法将纤溶酶与其它的功能多肽(舍有酶原结构域,具有抗凝血酶、抗血小板聚集活性,能特异识别纤维蛋白的多肽)结合起来所形成的嵌合体蛋白质．它尽可能地保留了分子中每一组分的生物活性,增强了纤溶酶的溶栓功效,较大程度地克服了临床溶栓药物在某些方面的不足,是目前溶栓药物研究领域中的热点。     

Highly potent fibrinolytic serine protease from Streptomyces

We introduce a highly potent fibrinolytic serine protease from Streptomyces omiyaensis (SOT), which belongs to the trypsin family. The fibrinolytic activity of SOT was examined using in vitro assays and was compared with those of known fibrinolytic enzymes such as plasmin, tissue-type plasminogen activator (t-PA), urokinase, and nattokinase. Compared to other enzymes, SOT showed remarkably higher hydrolytic activity toward mimic peptides of fibrin and plasminogen. The fibrinolytic activity of SOT is about 18-fold higher than that of plasmin, and is comparable to that of t-PA by fibrin plate assays. Furthermore, SOT had some plasminogen activator-like activity. Results show that SOT and nattokinase have very different fibrinolytic and fibrinogenolytic modes, engendering significant synergetic effects of SOT and nattokinase on fibrinolysis. These results suggest that SOT presents important possibilities for application in the therapy of thrombosis.     

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.     

Effects of extracellular DNA on plasminogen activation and fibrinolysis

The increased levels of extracellular DNA found in a number of disorders involving dysregulation of the fibrinolytic system may affect interactions between fibrinolytic enzymes and inhibitors. Double-stranded (ds) DNA and oligonucleotides bind tissue-(tPA) and urokinase (uPA)-type plasminogen activators, plasmin, and plasminogen with submicromolar affinity. The binding of enzymes to DNA was detected by EMSA, steady-state, and stopped-flow fluorimetry. The interaction of dsDNA/oligonucleotides with tPA and uPA includes a fast bimolecular step, followed by two monomolecular steps, likely indicating slow conformational changes in the enzyme. DNA (0.1-5.0 μg/ml), but not RNA, potentiates the activation of Glu- and Lys-plasminogen by tPA and uPA by 480- and 70-fold and 10.7- and 17-fold, respectively, via a template mechanism similar to that known for fibrin. However, unlike fibrin, dsDNA/oligonucleotides moderately affect the reaction between plasmin and α(2)-antiplasmin and accelerate the inactivation of tPA and two chain uPA by plasminogen activator inhibitor-1 (PAI-1), which is potentiated by vitronectin. dsDNA (0.1-1.0 μg/ml) does not affect the rate of fibrinolysis by plasmin but increases by 4-5-fold the rate of fibrinolysis by Glu-plasminogen/plasminogen activator. The presence of α(2)-antiplasmin abolishes the potentiation of fibrinolysis by dsDNA. At higher concentrations (1.0-20 μg/ml), dsDNA competes for plasmin with fibrin and decreases the rate of fibrinolysis. dsDNA/oligonucleotides incorporated into a fibrin film also inhibit fibrinolysis. Thus, extracellular DNA at physiological concentrations may potentiate fibrinolysis by stimulating fibrin-independent plasminogen activation. Conversely, DNA could inhibit fibrinolysis by increasing the susceptibility of fibrinolytic enzymes to serpins.     

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.     

Stimulation of tPA-dependent provisional extracellular fibrin matrix degradation by human recombinant soluble melanotransferrin

Tissue-type plasminogen activator (tPA) and its substrate plasminogen (Plg) are key components in the fibrinolytic system. We have recently demonstrated, that truncated human recombinant soluble melanotransferrin (sMTf) could stimulate the activation of Plg by urokinase plasminogen activator and inhibit angiogenesis. Since various angiogenesis inhibitors were shown to stimulate tPA-mediated plasminogen activation, we examined the effects of sMTf on tPA-dependent fibrinolysis. This study demonstrated that sMTf enhanced tPA-activation of Plg by 6-fold. sMTf also increased the release of [125I]-fibrin fragments by tPA-activated plasmin. Moreover, we observed that the interaction of sMTf with Plg provoked a change in the fibrin clot structure by cleaving the fibrin alpha and beta chains. Overall, the present study shows that sMTf modulates tPA-dependent fibrinolysis by modifying the clot structure. These results also suggest that sMTf properties could involve enhanced dissolution of the provisional extracellular fibrin matrix.     

Antithrombotic therapy in patients with known risk factors for thromboembolism

In about 50% of the cases of spontaneous deep vein thrombosis a congenital deficiency of an inhibitor of coagulation or an insufficient fibrinolytic mechanism can be detected. In arterial thromboembolism a connection with hyperactive platelets or with a diminished availability of tissue plasminogen activator can be found in about 70%. However, in these cases the defect which provokes thrombosis is mostly acquired and is connected with hyperlipidemia and/or with atherosclerotic alterations of the vessel wall. A study on patients with thromboembolic tendency and detectable risk factors was carried out. A total of 470 patients could be observed for 2 years under an adequate antithrombotic prophylaxis. The occurrence of thromboembolic episodes 2 years prior to prophylaxis and 2 years under prophylaxis was compared. In venous cases thrombosis could be controlled almost completely by coumarins when the underlying cause was a deficient plasmatic inhibitor. In patients with diminished fibrinolysis there was only a partial effect of oral anticoagulants. A better result could be obtained when pentosan polysulfate was administered. In arterial thromboembolism the results of prophylaxis were less convincing. The efficacy of ASA in patients with an increased platelet function was only moderate. In addition, ASA hat to be discontinued in about 20% of the patients because of gastrointestinal problems. Pentosan polysulfate in patients with a diminished fibrinolytic capacity had a fairly good effect and resulted in a 60% reduction of thromboembolic manifestations. It is shown that an exact diagnosis of the underlying deficiency which is likely to cause thrombosis can also improve the efficacy and the specificity of prophylaxis.     

Fibrinolysis in Cholestatic Jaundice

The fibrinolytic system was studied in primary biliary cirrhosis (16 patients) and large bile duct obstruction (10 patients, nine of whom had carcinoma). Plasma fibrinolysis (plasminogen activator activity) was decreased and fibrinogen increased in both groups of patients, particularly in those with large duct obstruction. These changes were related to the degree of cholestasis. Plasminogen activator activity was inversely related to serum triglyceride levels in patients with primary biliary cirrhosis. Urokinase inhibitors were decreased in both groups and antiplasmins increased in patients with large duct obstruction; fibrin/fibrinogen degradation products were normal in primary biliary cirrhosis and moderately increased in large duct obstruction. None of these fibrinolytic indices was related to the degree of cholestasis. Fibrinolytic activity and fibrinogen returned almost to normal levels after palliative surgery in the three patients with large duct obstruction who were studied. The decreased plasma fibrinolysis and increased fibrinogen may be due to altered lipid metabolism in cholestatic jaundice. In patients undergoing surgery for large duct obstruction there may be an increased risk of thrombosis.     

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.     

Interaction of heparin with plasminogen activators and plasminogen: effects on the activation of plasminogen

The amidolytic plasmin activity of a mixture of tissue plasminogen activator (tPA) and plasminogen is enhanced by heparin at therapeutic concentrations. Heparin also increases the activity in mixtures of urokinase-type plasminogen activator (uPA) and plasminogen but has no effect on streptokinase or plasmin. Direct analyses of plasminogen activation by polyacrylamide gel electrophoresis demonstrate that heparin increases the activation of plasminogen by both tPA and uPA. Binding studies show that heparin binds to various components of the fibrinolytic system, with tight binding demonstrable with tPA, uPA, and Lys-plasminogen. The stimulation of tPA activity by fibrin, however, is diminished by heparin. The ability of heparin to promote plasmin generation is destroyed by incubation of the heparin with heparinase, whereas incubation with chondroitinase ABC or AC has no effect. Also, stimulation of plasmin formation is not observed with dextran sulfate or chondroitin sulfate A, B, or C. Analyses of heparin fractions after separation on columns of antithrombin III-Sepharose suggest that both the high-affinity and the low-affinity fractions, which have dramatically different anticoagulant activity, have similar activity toward the fibrinolytic components.     

华广虻(Tabanusamaenus Walker)溶纤活性蛋白的纯化及生物活性分析

经过 ７５％ 饱和度硫酸铵沉淀、 Ｓｅｐｈａｄｅｘ Ｇ ７５ 凝胶过滤层析、 Ｌｙｓ Ｓｅｐｈａｒｏｓｅ ４ Ｂ 亲和层析和电泳制备洗脱,从华广虻（ Ｔａｂａｎｕｓ ａｍ ａｅｎｕｓ Ｗ ａｌｋｅｒ）腹部组织匀浆液中分离纯化出分子量约为 ６７ｋ Ｄ 的溶纤活性蛋白 Ｔ Ａ Ｆ Ｐ经纤维蛋白平板测定表明, Ｔ Ａ Ｆ Ｐ 只具有纤溶酶作用,不具有激活纤溶酶原的作用;但 Ｔ Ａ Ｆ Ｐ 能分解纤溶酶原激活剂的生色底物—— Ｃｈｒｏｍ ｏｚｙｍ Ｕ Ｋ 及 Ｓ ２２８８还能水解胰蛋白酶专一底物 Ｂｚ Ｐｈｅ Ｖａｌ Ａｒｇ Ｎ Ａ 及 Ｃ Ｂ Ｚ Ｇｌｙ Ｐｒｏ Ａｒｇ Ｎ Ａ,表明 Ｔ Ａ Ｆ Ｐ具有类胰蛋白酶活性,专一水解精氨酸形成的酰胺键（或肽键） Ｔ Ａ Ｆ Ｐ无胰凝乳蛋白酶活性      

Prophylactic fibrinolysis through selective dissolution of nascent clots by tPA-carrying erythrocytes

A fibrinolytic agent consisting of a tissue-type plasminogen activator (tPA) coupled to the surface of red blood cells (RBCs) can dissolve nascent clots from within the clot, in a Trojan horse-like strategy, while having minimal effects on preexisting hemostatic clots or extravascular tissue. After intravenous injection, the fibrinolytic activity of RBC-tPA persisted in the bloodstream at least tenfold longer than did that of free tPA. In a model of venous thrombosis induced by intravenously injected fibrin microemboli aggregating in pulmonary vasculature, soluble tPA lysed pulmonary clots lodged before but not after tPA injection, whereas the converse was true for RBC-tPA. Free tPA failed to lyse occlusive carotid thrombosis whether injected before or after vascular trauma, whereas RBC-tPA circulating before, but not injected after, thrombus formation restored blood flow. This RBC-based drug delivery strategy alters the fibrinolytic profile of tPA, permitting prophylactic fibrinolysis.