The selective inhibition of thrombin by peptides of boroarginine

Peptides containing alpha-aminoboronic acids with neutral side chains are highly effective reaction intermediate analog inhibitors of the serine proteases leukocyte elastase, pancreatic elastase, and chymotrypsin. A protocol has been developed for the synthesis of peptides containing alpha-aminoboronic acids with a basic, 3-guanidinopropyl side chain (boroArg) to extend the range of these compounds to trypsin-like proteases. Ac-(D)Phe-Pro-boroArg-OH, Boc-(D)Phe-Pro-boroArg-OH, and H-(D)Phe-Pro-boroArg-OH were prepared as inhibitors of thrombin based on earlier observations that it has a high affinity for this sequence. All three boronic acids are highly effective, slow-binding inhibitors of thrombin, inhibiting it with final inhibition constants and association rates of: 41 pM, 5.5 x 10(6) M-1 s-1; 3.6 pM, 9.3 x 10(6) M-1 s-1; less than 1 pM, 8.0 x 10(6) M-1 s-1, respectively. Comparison of their binding at equilibrium to thrombin, plasma kallikrein, factor Xa, plasmin, and two-chain tissue plasminogen activator has shown that all three inhibitors have at least 2 orders of magnitude greater affinity for thrombin, with the exception of the acetyl derivative which has a 40-fold greater affinity for thrombin than kallikrein. The boroarginine peptides are effective in inhibiting the action of thrombin in rabbit plasma against its physiological substrates. Activated partial thromboplastin time was significantly prolonged in vitro by all of the inhibitors at concentrations of 50-200 nM. Prolongations of activated partial thromboplastin time were also observed in rabbits after intravenous (40-80 micrograms/kg or subcutaneous (0.20-2 mg/kg) injections of Ac-(D)Phe-Pro-boroArg-OH. Results indicate that this new class of synthetic thrombin inhibitors may be clinically useful as antithrombotic agents.     

The effect of heparin and thromboplastin on the half-life of 125I-protein C in the blood flow of rats]

The influence of heparin and thromboplastin on the halflife of 125I-protein C in rat blood was under investigation. It was found that t1/2 of protein C was of 2.3 h. The intravenous administration of heparin resulted in the prolongation of t1/2 to 6.5 h, that could be explained by inhibition of thrombin generation. Upon the 40-min infusion of thromboplastin the rate of 125I-protein C decay in blood enhanced. That could be explained by the generation of the endogenous thrombin and participation of thrombomodulin in the protein C activation as well as in the removal of the endogenous thrombin from blood.     

A coupled amidolytic assay for thromboplastin (tissue factor) using a fluogenic substrate: its application to monkey leukocyte tissue factor

A sensitive and quantitative amidolytic assay for thromboplastin (tissue factor) coupled to thrombin formation was established. A fluogenic peptide substrate, Boc-Val-Pro-Arg-MCA, was found to be suitable for the coupled amidolytic assay. The amidolytic assay was applied to measure TF activity of endotoxin-stimulated mononuclear leukocytes and monocytes. The amidolytic assay showed good correlation of 0.97 with the currently used clotting assay upon measuring TF activity of the cellular samples.     

The correlated actions of vitamins C,K, and Q

Three vitamins (C, K, and Q), two of which are unequivocally established and the existence of the third supported by both experimental and clinical evidence, are needed to prevent hemorrhagic states and therefore can be designated the hemostatic vitamins. The coordinated actions of these vitamins can be epitomized by a diagram. Vitamin K is responsible for the synthesis of four basic clotting factors that function in two distinct pathways for the activation of prothrombin to thrombin. Vitamin Q also functions in these pathways. In the intrinsic, it supplies by means of platelets the Q factor that with factors VIII and IX generates intrinsic (plasma) thromboplastin. In the extrinsic pathway, it is related to tissue thromboplastin which has the Q factor as a part of its structure. It appears to be a phospholipid obtained from exogenous sources. Both vitamins C and K have a potential redox mechanism in their structure which can be hypothecated to function in the synthesis and maintenance of mesenchymal tissue.     

Anticoagulant activities of oleanolic acid via inhibition of tissue factor expressions

Oleanolic acid (OA), a triterpenoid known for its anti-inflammatory and anti-cancer properties, is commonly present in several medicinal plants but its anticoagulant activities have not been studied. Here, the anticoagulant properties of OA were determined by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrin polymerization as well as cell-based thrombin and activated factor X (FXa) generation activities. Data showed OA prolonged aPTT and PT significantly and inhibited thrombin catalyzed fibrin polymerization. In addition, OA inhibited the activities of thrombin and FXa and inhibited the generation of thrombin or FXa in human endothelial cells. OA also inhibited TNF-α-induced tissue factor expression on human endothelial cells. In accordance with these anticoagulant activities, OA showed an anticoagulant effect in vivo. These results indicate that OA possesses antithrombotic activities and suggest that daily consumption of a herb containing OA may be preventing thrombosis in pathological states.     

Fractionation of plasma globulin for prothrombin,thrombokinase, and accessory thromboplastin

1. Crude globulin from more than 1,000 liters of citrated bovine plasma has been used in developing a procedure for moderately large scale separation of clotting factors. Fraction A, prothrombin, kinase, and thrombin fractions were prepared. Fraction A contained both kinase and accessory thromboplastin, the latter predominating when fraction A was diluted. 2. When prothrombin was activated by kinase, the rate of thrombin production was enhanced by the addition of platelets, or brain lipid, or dilute fraction A. These accessory thromboplastins caused this acceleration only when calcium chloride was added. Even with calcium, they were not effective unless kinase was present. 3. In contrast, the action of kinase was not entirely dependent on either ionic calcium or accessory thromboplastin. The concentrated kinase fraction activated prothrombin in the presence of excess oxalate. Although kinase often contaminates highly purified thrombins, it is probably distinct from thrombin. The ratio of kinase to thrombin was 100 times as great in the kinase fraction as in the thrombin fraction. 4. The kinase fraction, diluted 45,000-fold, to protein-nitrogen concentrations as low as 0.02 microgram per ml., accelerated the conversion of crude prokinase in three-stage tests. 5. The findings are consistent with the following concept of the basic enzymatic mechanism: See PDF for Structure It is now added that calcium and accessory thromboplastin exert their effects by impinging on the basic mechanism, in a chemically secondary or indirect manner.     

Enzyme-linked coagulation assay. II. A sensitive assay for tissue factor and factors II, VII, and X

We have developed a solid-phase clotting assay which uses peroxidase-fibrinogen in solution and fibrinogen bound to microtiter plates as a substrate for the thrombin generated from the clotting cascade. We have developed this assay for measurement of the extrinsic pathway factors thromboplastin (tissue factor, factor III), VII and VIIa, X, and II. Using long incubation times (40-90 min), thromboplastin could be measured in extracts of human brain at very low concentrations. Specificity for thromboplastin was demonstrated by showing a requirement for factors II, V, X, and VII but not for VIII, IX, XI, or XII; both substrate plasmas monodeficient in single factors and mixtures of the pure factors were used in demonstrating this specificity. The assay was modified to measure factors II, VII, VIIa, and X using appropriate deficient plasmas. The limit of detection was 2-3 orders of magnitude lower than a one-stage clotting test for all factors assayed. This assay has the advantages of convenience, specificity comparable to standard clotting tests, and high sensitivity.     

The antiheparin thrombocyte factor 4 and its interaction with heparin

A rat platelet factor has a high antiheparin activity. It also decreases nonenzymatic fibrinolytic activity of normal rat plasma and antithrombin III-heparin complex. The platelet factor 4 formed inactive complexes with heparin in molar ratios of 1:1 and 2:1. Intravenous injection of the platelet factor 4 before injection of albino rats with tissue thromboplastin prevented the reaction of anticoagulation system inactivated the synthesis of endogenous thrombin. This effect is accompanied by high hypercoagulation and depression of nonenzymatic fibrinolysis in blood.     

Sphingosine inhibits monocyte tissue factor-initiated coagulation by altering factor VII binding

Tissue factor is a lipoprotein, expressed on the surface of cells, which binds coagulation Factor VII or VIIa, leading to activation of Factors X and IX with subsequent fibrin generation. Cellular tissue factor activity is important in pathophysiologic processes such as inflammation and disseminated intravascular coagulation. In this study, the long-chain base sphingosine inhibited coagulation initiated by lipopolysaccharide-stimulated intact human monocytes. Sphingosine (5-100 microM) also profoundly inhibited thromboplastin-initiated coagulation (greater than 90% decrease in thromboplastin activity). This inhibition was dose- and time-dependent. Sphingosine inhibited neither the intrinsic pathway of coagulation nor thrombin generation of fibrin. The sphingosine analogues sphingomyelin, ceramide, or N-acetylsphingosine did not affect thromboplastin activity, suggesting that the polar head of sphingosine was necessary for interaction of the molecule with the coagulation system. Investigation of the biochemical mechanism revealed that sphingosine (5-50 microM), but neither sphingomyelin nor ceramide, inhibited specific binding of radiolabeled Factor VII to lipopolysaccharide-stimulated intact monocytes. The results suggest that sphingosine may regulate monocyte tissue factor-initiated coagulation by modulating Factor VII binding to tissue factor. Sphingosine may represent a new class of inhibitors of hemostasis.     

组织因子膜外区融合蛋白基因的表达及产物的分离纯化与活性分析

 为构建表达组织因子 (TF)膜外区的融合载体 ,制备组织因子膜外区 ,抽提人胎盘组织的总RNA,通过 RT- PCR法扩增出 TF的 c DNA克隆至 p UC1 8并测定全序列 .然后以此为模板 ,再次PCR扩增出 TF膜外区 (soluble TF,s TF) c DNA,并将其插入到谷胱甘肽巯基转移酶融合表达载体 p GEX4T- 1 ,构建了 tac启动子控制下的 GST- s TF融合蛋白的表达载体 .表达的融合蛋白经亲和层析、凝血酶切得到纯化的 s TF.表达产物经 ELISA验证 ,能特异性地与 TF抗体结合 .重新脂化后 ,该产物具有较大凝血活性 .以上说明采用融合蛋白表达系统可以大量制备组织因子膜外区 ,为研制国产重组凝血活酶试剂和研究 s TF的结构和功能创造条件 .     

Alterations in the apparent tissue factor (thromboplastin) expression in HeLa cells by a cellular factor Xa inhibitor

HeLa cells have undetectable tissue factor (thromboplastin) activity when measured by a one-stage coagulation assay. In contrast, these cells accelerated the factor VII-catalyzed cleavage of factor X. The two assays gave similar results after either heating the samples to 100°C for 2 min or exposure to thrombin. Neither of these treatments altered the tissue factor activity of human foreskin fibroblasts, a cell type with high tissue factor activity. HeLa cells contain an inhibitors(s) directed against factor X_a but not thrombin. The inhibitor(s) was inactivated by exposure to thrombin or by heat treatment. Inhibition of factor X_a-catalyzed cleavage of a synthetic peptide was blocked by ethyleneglycol bis(β-maminoethyl ether)-N,N′-tetraacetic acid (EGTA) so the inhibition was apparently dependent on divalent cations. Inhibition was not accelerated by heparin. The inhibitor(s) was not protein C or other serine proteases since it was not inactivated by diisopropylfuorophosphate. The factor X_a inhibitors(s) has been isolated from HeLa cells with an approximate 500-fold increase in specific activity. After SDS-polyacrylamide gel electrophoresis factor X_a-inhibitory activity was recovered from a region corresponding to the major Coomassie-staining band at 43 kDa and in lesser amounts from regions corresponding to 26 and 17 kDa. Cellular inhibitors of coagulation may partially explain the low apparent tissue factor observed in some in vitro cells and may serve a regulatory role in limiting the expression of tissue factor.     

Circadian changes in anticoagulant effect of heparin infused at a constant rate.

Six patients with venous thromboembolism were treated with heparin, administered intravenously by a constant infusion pump. The initial daily dose of heparin was adjusted to keep the activated partial thromboplastin time, sampled at 0800, between 1.5 and 2.5 times the control level. Once that level was obtained, this dose was kept constant. Anticoagulation was thereafter measured, every four hours for 48 hours, by activated partial thromboplastin time, thrombin time, and coagulation factor Xa inhibition assay. The results of all three coagulation tests showed a circadian variation in the six patients. Maximum values were achieved at night and minimum values in the morning. These circadian variations were reproduced for two consecutive days. Differences between night and morning values reached almost 50% for activated partial thromboplastin time, 60% for thrombin time, and 40% for factor Xa inhibition assay. This circadian variation resulted from two rhythms, a circadian rhythm lasting 24 hours and an ultradian rhythm lasting 12 hours, which were detected by cosinor analysis for each coagulation test (p less than 0.01). A circadian rhythm was detected individually in most of the patients for each coagulation test (p less than 0.05). All patients had a nocturnal peak in activated partial thromboplastin time on both days. In four patients this peak exceeded the upper desired limit of activated partial thromboplastin time. These rhythms should be taken into account when evaluating the dosage of heparin to be administered.     

Prevention of intravascular blood coagulation in rats by DIP-alpha-thrombin administration

The possibility of prevention of intravascular blood coagulation in rats by DIP-alpha-thrombin devoid of proteolytic activity and capable of stimulating the reaction of anticoagulation system was studied. The injection of lethal thromboplastin dose was shown to produce a sharp increase in soluble fibrin blood content, total disappearance of fibrinolytic activity and intravascular blood coagulation. The animals died of thrombosis in 90% of cases. It was established that the injection of lethal thromboplastin dose 5 min after DIP-alpha-thrombin injection caused a 13% lethality from thrombosis. No reliable changes in fibrinolytic activity and soluble fibrin content were observed. A significant increase in thrombin and recalcification time was recorded. It is suggested that DIP-alpha-thrombin prevents intravascular blood coagulation induced by lethal thromboplastin dose due to mobilization of the reserve capacities of neuro-humoral anticoagulation system.     

Hypocoagulable state of human preovulatory ovarian follicular fluid: role of sulfated proteoglycan and tissue factor pathway inhibitor in the fluid

Ovulation accompanied by tissue damage can cause an increase in the level of tissue factor (TF) in the follicular fluid, triggering the extrinsic coagulation pathway. However, follicular fluid must block fibrin formation and maintain fluidity until the release of the oocyte at ovulation. The combination of sulfated proteoglycan, antithrombin, and TF pathway inhibitor (TFPI) appears to play a critical role in the hypocoagulability of human follicular fluid. When compared with plasma, folicular fluid differs markedly in the levels of a number of important coagulation proteins. Principal among these are 15-fold, 13-fold, and 3.7-fold increases in free TFPI, thrombin-antithrombin complex, and TF, respectively. The excessively prolonged activated partial thromboplastin time (APTT) and prothrombin time (PT) of human ovarian follicular fluid appear to be primarily due to high concentrations of sulfated proteoglycans, which accelerate the inactivation of thrombin and the anti-Xa activity of TFPI. Thus, heparitinase treatment shortened the clotting times of follicular fluid and reduced the inhibition of thrombin by the proteoglycan fraction combined with a fraction containing antithrombin. The remaining prolongation of APTT and PT may be caused by high levels of free TFPI in follicular fluid, which were confirmed by Northern blotting analysis, demonstrating TFPI mRNA expression by granulosa cells.     

Anticoagulant activities of a monoclonal antibody that binds to exosite II of thrombin.

A monoclonal IgG isolated from a patient with multiple myeloma has been shown to bind to exosite II of thrombin, prolong both the thrombin time and the activated partial thromboplastin time (aPTT) when added to normal plasma, and alter the kinetics of hydrolysis of synthetic peptide substrates. Although the IgG does not affect cleavage of fibrinogen by thrombin, it increases the rate of inhibition of thrombin by purified antithrombin approximately 3-fold. Experiments with plasma immunodepleted of antithrombin or heparin cofactor II confirm that prolongation of the thrombin time requires antithrombin. By contrast, prolongation of the aPTT requires neither antithrombin nor heparin cofactor II. The IgG delays clotting of plasma initiated by purified factor IXa but has much less of an effect on clotting initiated by factor Xa. In a purified system, the IgG decreases the rate of activation of factor VIII by thrombin. These studies indicate that binding of a monoclonal IgG to exosite II prolongs the thrombin time indirectly by accelerating the thrombin-antithrombin reaction and may prolong the aPTT by interfering with activation of factor VIII, thereby diminishing the catalytic activity of the factor IXa/VIIIa complex.     

Are factor Xa inhibitors superior to thrombin inhibitors in anticoagulation?

Protease inhibitors are useful tools for increasing the inhibitor potential of plasma. In this context, thrombin inhibitors attracted special interest. However, other clotting enzymes, especially factor Xa, are target enzymes of protease inhibitors besides thrombin. Our studies on structure-activity relationships of benzamidine derivatives resulted in selective inhibitors of thrombin and factor Xa. The use of these inhibitors enabled us to clarify whether the antithrombin activity or the anti-factor Xa activity of a compound is more efficient in anticoagulation. We assessed the concentration-dependent inhibition of the activated partial thromboplastin time by these compounds. If one correlates the inhibitor concentration, which prolonged the clotting time by 60 s, with the dissociation constants one will realize that thrombin inhibition is significantly more efficient in anticoagulation than inhibition of factor Xa.     

Meizothrombin formation during factor Xa-catalyzed prothrombin activation. Formation in a purified system and in plasma

Meizothrombin and thrombin formation were quantitated during factor Xa-catalyzed activation of human prothrombin in reaction systems containing purified proteins and in plasma. In the purified system considerable amounts of meizothrombin accumulated when prothrombin was activated by factor Xa (with or without accessory components) under initial steady state conditions. The ratio of the rates of meizothrombin and thrombin formation was not influenced by variation of the pH, temperature, or ionic strength of the reaction medium. When 2 microM prothrombin was activated by the complete prothrombinase complex (factor Xa, factor Va, Ca2+, and phospholipid) 80-90% of the initially formed reaction product was meizothrombin. Lowering the prothrombin concentration from 2 to 0.03 microM caused a gradual decrease in the ratio of meizothrombin/thrombin formation from 5 to 0.6. When the phosphatidylserine content of the phospholipid vesicles was varied between 20 and 1 mol % and prothrombin activation was analyzed at 2 microM prothrombin the relative amount of meizothrombin formed decreased from 85 to 55%. With platelets, cephalin, or thromboplastin as procoagulant lipid, thrombin was the major reaction product and only 30-40% of the activation product was meizothrombin. We also analyzed complete time courses of prothrombin activation both with purified proteins and in plasma. In reaction systems with purified proteins substantial amounts of meizothrombin accumulated under a wide variety of experimental conditions. However, little or no meizothrombin was detected in plasma in which coagulation was initiated via the extrinsic pathway with thromboplastin or via the intrinsic pathway with kaolin plus phospholipid (cephalin, platelets, or phosphatidylserine-containing vesicles). Thus, thrombin was the only active prothrombin activation product that accumulated during ex vivo coagulation experiments in plasma.     

Effect of polyanetholesulphonic acid and xylan sulphate on antithrombin III activity.

Both polyanetholesulphonic acid and xylan sulphate prolonged the partial thromboplastin clotting time of plasma. The anticoagulant effect of both compounds was reduced following pre-incubation of plasma with antiserum specific for antithrombin III. Polyanetholesulphonic acid was more effective than xylan sulphate in inhibiting thrombin-initiated clotting of plasma, and potentiated antithrombin III inhibition of both thrombin and Xa. Xylan sulphate was more effective in potentiating antithrombin III inhibition of Xa than of thrombin. These differential effects of xylan sulphate on different blood serine proteases are discussed in terms of the antithrombin III-mediated anticoagulant activity of heparin.     

Chemical characteristic and anticoagulant activity of the sulfated polysaccharide isolated from Monostroma latissimum (Chlorophyta)

A polysaccharide was isolated from marine green algae Monostroma latissimum, and its chemical characteristic and anticoagulant activity were investigated. The results demonstrated that the polysaccharide was high rhamnose-containing sulfated polysaccharide, and was mainly composed of 1,2-linked l-rhamnose residues with sulfate groups substituted at positions C-3 and/or C-4. The sulfated polysaccharide exhibited high anticoagulant activities by assays of the activated partial thromboplastin time (APTT) and thrombin time (TT). The anticoagulant property of the sulfated polysaccharide was mainly attributed to powerful potentiation thrombin by heparin cofactor II.