Blockade by ruthenium red of tissue factor-initiated coagulation.

The ability of ruthenium red (RuR) to inhibit tissue factor (TF)-initiated blood coagulation was demonstrated at the protein and cellular levels as well as in human plasma. In a single-stage clotting assay, RuR concentration-dependently inhibited rabbit brain thromboplastin (rbTF)-induced coagulation and offset bacterial endotoxin (LPS)-induced monocytic TF (mTF) hypercoagulation; the IC(50)s were estimated at 7.5 and 12.3 microM, respectively. A 15-min preincubation of RuR with rbTF or monocyte suspension resulted in the pronounced inhibition with a significantly lowered IC(50) at 1.8 or 7.7 microM for rbTF or mTF procoagulation, respectively. The differences in IC(50)s between rbTF and mTF without or with the preincubation indicated that TF was a primary target for RuR action. The effect of RuR on the physiological function of TF in FVII activation was demonstrated by the proteolytic cleavage of FVII zymogen to its active forms of serine protease on Western blotting analyses. RuR readily blocked TF-catalyzed FVII activation (diminished FVIIa formation), thus down regulating the initiation of blood coagulation. Inclusion of RuR into human plasma samples in vitro significantly prolonged prothrombin time, indicating the depressed coagulation. FVII activity was inhibited by 30 - 60% depending on the dose; as a result, FX activity also decreased. However, RuR showed no effect on thrombin time. Thus, RuR inhibited FVII activation to block the initiation of coagulation.     

IV. Anticoagulant activity of compound 48/80: inhibition of factor VII activation in leukemia THP-1 monocytes

Our previous study described a novel biologic function of compound 48/80 (48/80) in the downregulation of monocytic tissue factor (TF)-initiated hypercoagulation in response to bacterial endotoxin (lipopolysaccharide; LPS). The inhibition was not due to the blockade of LPS cell signaling, as evidenced by the unaffected LPS-induced TF synthesis. We herein determined the mechanism by which 48/80 inhibits the extrinsic coagulation in agonist-challenged THP-1 monocytes. LPS as well as A23187 substantially induced TF activity. TF synthesis was enhanced by LPS but not by A23187. However, the elevated FVII binding to monocytes accompanying the upregulation of factor VII (FVII) activation was uniformly observed in both cases. A 5-min preincubation of the cells with a sheep anti-humanTF antibody (anti-hTF Ab) showed the downregulation of FVII activation, indicating a regulatory role of FVII binding in the modulation of the extrinsic coagulation. The 48/80 blocked FVII binding to monocytes, leading to the preferential inhibition of FVII activation. As the result of the diminished FVIIa formation, monocytic TF-initiated extrinsic coagulation was downregulated in agonist-challenged THP-1 monocytes.     

Polycations selectively blocking tissue factor-dependent FVII activation: collective in vitro anticoagulation studies

Tissue factor (TF) is an initiator of the extrinsic blood coagulation, which is often susceptible to upregulation by tissue injury, advanced glycation end-product, or diverse inflammation. TF hypercoagulability is accompanied by elevated generation of clotting factors (e.g., FVIIa, FXa, and thrombin) and fibrin production, all of which are proinflammatory. In this laboratory, our in vitro experimental results show that polycationic anticoagulants (compound 48/80, ruthenium red, polybrene, protamine, Buforin I, and cationic polyamino acids) intervene TF hypercoagulability at posttranslational level. Polycations preferentially suppress TF-dependent FVII activation with diminished FVIIa formation shown on Western blotting, resulting in non- or un-competitive inhibition on FVIIa amidolytic activity. In contrast, polycations have no effect on FVIIa catalysis, FXa activity, or thrombin activity per se. Polycations could present a new class of anticoagulants with such unique upstream downregulation of blood coagulation. In view of coagulation-dependent inflammation and the new paradigm of blood coagulation-inflammation-thrombosis circuit, the polycations as a new class of anticoagulants could effectively contribute to antiinflammation, antithrombosis, and cardioprotection. Further development of effective anticoagulants is of biopharmaceutical significance in broadly easing disease conditions.     

Biochemical strategies to anticoagulation: a comparative overview

Hypercoagulability is widely associated with sepsis, inflammation, diabetes, cancers, aging, and many pathological conditions, resulting in life-threatening disseminated intravascular coagulation (DIC), venous thrombosis, thromboembolism, cardiovascular complications, or even deadly multiple organ failure. Relieving coagulation dysfunction is not only a task for research scientists but also a challenge for physicians. The development of effective anticoagulants is under way with the basic understanding of the pathophysiology of hypercoagulable state. In this overview, various anticoagulants will be discussed according to the proposed inhibitory target-sites along the extrinsic pathway that is believed to play an integral role in homeostasis. Anticoagulants generally fall into two broad categories as natural or pharmacological ones. Antithrombin (AT), activated protein C (APC), and tissue factor pathway inhibitor (TFPI) mainly constitute the natural anticoagulant system apart from the recently reported physiological components such as lipoproteins, sphingosine, thrombomodulin (TM) or cellular Marcks protein. Pharmacological anticoagulants include warfarin, FVIIa inhibitors, FXa inhibitors, and thrombin inhibition by its direct inhibitors or heparins. In addition, a group of novel compounds inhibiting TF-dependent FVII activation result in anticoagulation; such upstream downregulation in the extrinsic pathway awaits further research to establish their in vivo benefits. The molecular genetic approaches such as developing soluble TF, FVII and thrombin mutants provide unique downregulation. Anticoagulation also extends its significance to anti-inflammation, making broad impacts on the improvement of human health.     

Peptides corresponding to the second epidermal growth factor-like domain of human blood coagulation factor VII: synthesis,folding and biological activity

Factor VIIa (FVIIa) is the enzymatically active constituent of the FVIIa/tissue factor (TF) complex, the initiator of the extrinsic pathway of blood coagulation. The zymogen FVII and FVIIa are composed of discrete domains, two of which are homologous to the epidermal growth factor (EGF). This investigation examined the significance of the FVII EGF-2 domain in the processes leading to activation of factor X (FX). Peptides 47 residues in length and corresponding to the amino acid sequence of the EGF-2 domain of human FVII were prepared by solid-phase synthesis methods. Peptide variants with all six Cys residues replaced by l -2-aminobutyryl residues (1), or containing one (2a-c), two (3a,b) or three (4) disulfide bonds, were obtained by application of various S-protecting groups and oxidation methods. Peptide 4, containing the cystine bridge arrangement corresponding to that found in the native protein, was prepared by a two-step regioselective disulfide bond formation method. An evaluation of the anti-coagulant properties of peptides 1-4 revealed that all peptides, with the exception of the two-cystine isomer containing non-native disulfide pairings (3b), were potent inhibitors of TF/FVIIa-mediated activation of FX. The fully constrained peptide 4 was found to be twice as active as its completely non-constrained counterpart 1, the two peptides showing IC₅₀ values of 1.6 ± 0.5μm (1) and 0.8 ± 0.2 μm (4) with respect to TF/FVIIa-dependent FX activation. The results of this study demonstrate the functional importance of the EGF-2 domain of FVII in the induction of coagulation by the extrinsic pathway.     

急性心肌梗死患者血浆凝血因子Ⅶ和抗凝血酶Ⅲ的变化及其意义

目的 探讨急性心肌梗死（AMI）患者血浆凝血因子/Ⅶ、抗凝血酶Ⅲ（AT-Ⅲ）活性的变化及其意义.方法 对40例AMI患者血浆FVIIag含量、FVIIa水平、FVII;C及AT-Ⅲ水平进行检测,并与20名健康人进行比较,分析其变化,并探讨其在急性心血管事件中的意义.结果 AMI患者血浆FVIIag含量、FVIIa水平及FVII：C高于（P〈0.05或P〈0.01）,且FVIIa水平升高更显著,而血浆AT-Ⅲ活性显著低于健康对照组（P〈0.01）.结论 血浆凝血因子Ⅶ和AT-Ⅲ参与AMI时的凝血过程,血浆活化凝血因子Ⅶ增高及AT-Ⅲ活性降低可能是急性心血管事件敏感预测指标.     

Inhibitors of Tissue Factor. Factor VIIa for anticoagulant therapy

Factor VIIa (FVIIa) is a key serine protease involved in the initiation of the coagulation cascade. It is a glycosylated disulfide-linked heterodimer comprised of an amino-terminal gamma-carboxyglutamic acid-rich (Gla) domain and two epidermal growth factor (EGF)-like domains in the light chain, and a chymotrypsin-like serine protease domain in the heavy chain. FVIIa requires tissue factor (TF), a membrane bound protein, as an essential cofactor for maximal activity towards its biological substrates Factor X, Factor IX and Factor VII (FVII). Inhibition of TF.FVIIa activity may prevent the formation of fibrin clots and thus be useful in the management of thrombotic disease. The development of TF.FVIIa inhibitors to validate this target has been of great interest. A wide array of strategic approaches to inhibiting the biochemical and biological functions of the TF.FVIIa complex has been pursued. This has been greatly aided from our understanding of the structures for TF, FVII, FVIIa, and the TF.FVIIa complex. These approaches have resulted in inhibitors directed specifically towards either FVIIa or TF. Antagonists include active site inhibited FVIIa, TF mutants, anti-TF antibodies, anti-FVII/FVIIa antibodies, naturally-occurring protein inhibitors, peptide exosite inhibitors, and protein and small molecule active site inhibitors. These antagonists can inhibit catalysis directly at the active site as well as impair function by binding to exosites that may interfere with substrate, membrane, or cofactor binding. The rationale of TF.FVIIa as a target and the development, characteristics and biological uses of TF.FVIIa inhibitors are discussed.     

Non-hemostatic activity of coagulation factor Xa: potential implications for various diseases.

Because of its unique position at the convergence point of the intrinsic (contact) and extrinsic (tissue factor/factor VIIa) pathways in the coagulation system, coagulation factor Xa (FXa) has been a theoretically interesting therapeutic target for antithrombotic drugs for many years. More recently, the discovery of naturally occurring FXa inhibitors, such as tick anticoagulant peptide and antistasin, has helped substantiate FXa as a desirable target by demonstrating the efficacy and potential safety advantages of FXa inhibition over conventional antithrombotic therapy. These discoveries led to the design and development of many small-molecule inhibitors of FXa, which have provided potent and selective tools for evaluating the potential role of FXa in various diseases. In addition, these advances have been instrumental in defining the biology of FXa and have aided in the discovery of specific receptors and intracellular signaling pathways for FXa that may be important in the progression of, or the response to, various diseases.     

Recent advances in serine protease inhibitors as anticoagulant agents

The drawbacks and limitations of existing anticoagulant therapy which may result in serious adverse effects and a high mortality rate, have given rise to many anticoagulant development programmes in the last decade, focusing mainly at development of thrombin and FXa low-molecular weight inhibitors. A detailed understanding of blood coagulation pathways, functioning of the serine proteases thrombin, FXa, FVIIa and FIXa and elucidation of their crystal structures resulted in many potent compounds, among which some have entered the clinical phase or have been approved for use in clinical practice. Recently, the focus of anticoagulant research turned to inhibition of the TF:FVIIa complex, with some promising clinical candidates on the horizon. This article provides an overview of the current development status of serine protease inhibitors as anticoagulants, including new trends such as dual coagulation factor inhibitors.     

Recombinant coagulation factor VIIa labelled with the fac‐99 mTc(CO)3‐core: synthesis and in vitro evaluation of a putative new radiopharmaceutical for imaging in acute bleeding lesion

Coagulation in blood is initiated when coagulation factor VII (FVII) binds to exposed TF and is activated to FVIIa, and the TF/FVIIa complex may therefore provide a marker of vascular injury potentially applicable in diagnostic imaging of acute gastrointestinal (GI) bleeding. Methods: Recombinant FVIIa (rFVIIa) was radiolabeled with technetium‐99 m in a direct labeling reaction using the ‘carbonyl approach’ using the IsoLink^® carbonyl labeling agent. The properties of ^{99 m}Tc(CO)₃‐rFVIIa complex was analyzed by TCA precipitation, HPLC and FVIIa functional integrity was tested in in vitro assays. Results: Labeling of rFVIIa was possible without tagging with a chelater. Incorporation of radioactivity depended strongly on rFVIIa concentration and temperature. More than 95% incorporation was achieved after 30 min at 45°C with 0.76 mg/ml rFVIIa. ^{99 m}Tc(CO)₃‐rFVIIa was obtained in 46% radiochemical yield and in >95% radiochemical purity. Pull down experiments showed that the biological activity (binding to tissue factor and to anti‐FVII antibody) of the radiolabelled product remained intact in the formulation mixture as well as in human serum. By computer modeling analysis, two candidate sites for stabilizing the ^{99 m}Tc(CO)‐ligand structure in FVIIa were identified. Conclusion: Radiolabelled rFVIIa derivatives may represent a novel tool for the diagnosis of acute gastrointestinal bleeding lesions. Copyright © 2010 John Wiley & Sons, Ltd.     

New insights into the coagulation system and implications for new therapeutic options with recombinant factor VIIa

The classical model of the coagulation cascade is to be replaced by a new, cell based model of coagulation emphasizing the interaction of coagulation proteins with cell surfaces of platelets subendothelial cells and the endothelium. According to current knowledge hemostasis is initiated by the formation of a complex between tissue factor (TF) exposed as a result of a vessel wall injury, and already activated factor (F) VII (FVIIa) normally present in the circulating blood. The TF-FVIIa complexes convert FX into FXa on the TF bearing cell. FXa then activates prothrombin (FII) into thrombin (FIIa). This limited amount of thrombin activates FVIII, FV, FXI and platelets. Thrombin-activated platelets change shape and as a result will expose negatively charged phospholipids, which form the perfect template for full thrombin generation involving FVIIIa and FIXa. Thrombin also converts fibrinogen into fibrin, it activates the fibrin stabilizing FXIII, as well as the thrombin activatable fibrinolysis inhibitor (TAFI). The fibrin structure has been found to be dependent on the amount of thrombin formed and the rate of thrombin generation. Full thrombin generation is necessary for the formation of a tight, stable fibrin hemostatic plug resistant to premature fibrinolysis which is required for full and sustained hemostasis. Since thrombin has such a crucial role in providing hemostasis, any agent that enhances thrombin generation in situations with impaired thrombin formation may be characterized as a 'general hemostatic agent' - a term that has been applied to recombinant activated FVII. Recombinant coagulation factor VIIa (rFVIIa; NovoSeven(R)) was originally developed and approved for the treatment of bleeding episodes and the prevention of bleeding during surgery in hemophilia patients with inhibitors and in patients with auto-antibodies against FVIII or FIX (acquired hemophilia). As rFVIIa in pharmacological doses enhances thrombin generation on activated platelets, it has been suggested that rFVIIa may also help to improve hemostasis in other situations involving impaired thrombin generation. This is substantiated by the accumulation of published data indicating that rFVIIa is able to control bleeding in patients with thrombocytopenia or platelet function deficiencies as well as in patients without pre-existing coagulopathies.     

Small size fullerenol nanoparticles inhibit thrombosis and blood coagulation through inhibiting activities of thrombin and FXa

Thrombus is one of main causes of death in the world and also a vital trouble of biomaterials application in vivo. Recently, effect of fullerenol nanomaterials on anticoagulation was found in our research through extension of bleeding times in treated Sprague-Dawley rats via intravenous injection. Inhibiting of fullerenols on thrombosis was ascertained further by thromboembolism model. Effects of fullerenols on intrinsic and extrinsic pathway were distinct in prolonging activated partial thromboplastin time and prothrombin time, which supported that fullerenols induced defects in both pathways. Inhibited activities of activated coagulation factor X (FXa) and thrombin were verified by experiments in vitro and AutoDock Vina. The results suggest that fullerenols depending on small size and certainly surface property occupied the active domain of FXa and thrombin to block their activity; further, thrombosis was inhibited. This putative mechanism offers an insight into how fullerenol NPs were utilized further in biomedical applications.     

凝血因子Ⅹa抑制剂研究进展

凝血因子Ⅹa是一个丝氨酸蛋白酶,在凝血级联反应中,因子Ⅹa处于外源性和内源性凝血级联反应的交汇点,也就是凝血瀑布共同通路的起始位置。由于其在凝血途径中位置的重要性,Ⅹa因子已经成为新型抗凝药物开发的重要靶点。本文从凝血因子Ⅹa的功能和结构、已上市Ⅹa因子抑制剂以及天然来源的Xa因子抑制剂等方面进行综述。     

A novel fibrinogenase from Agkistrodon acutus venom protects against DIC via direct degradation of thrombosis and activation of protein C

The incidence of disseminated intravascular coagulation (DIC), which leads to multiple organ dysfunction and high mortality, has remained constant in recent years. At present, treatments of DIC have focused on preventing cytokine induction, inhibiting coagulation processes and promoting fibrinolysis. Recent clinical trials have supported the use of antithrombin and activated protein C supplementation in DIC. To better understand the mechanism of treatment on DIC, we here report a novel fibrinogenase from Agkistrodon acutus (FIIa) that effectively protected against LPS-induced DIC in a rabbit model, and detected the tissue factors expression in HUVE cells after using FIIa. In vivo, administration of FIIa reduced hepatic and renal damage, increased the concentration of fibrinogen, the activities of protein C, the platelet count, APTT, PT, FDP, the level of AT-III and t-PA, decreased the level of PAI-1, and increased survival rate in LPS-induced DIC rabbits. In vitro experiments, we further confirmed that FIIa up-regulated the expression of t-PA and u-PA, down-regulated the expression of PAI-1, and directly activated protein C. Our findings suggest that FIIa could effectively protect against DIC via direct degradation of microthrombi and activation of protein C as well as provide a novel strategy to develop a single proteinase molecule for targeting the main pathological processes of this disease.     

Synthesis, characterization, and structure-activity relationships of amidine-substituted (bis)benzylidene-cycloketone olefin isomers as potent and selective factor Xa inhibitors

Factor Xa (FXa) is a trypsin-like serine protease that plays a key role in blood coagulation linking the intrinsic and extrinsic pathways to the final common pathway of the coagulation cascade. During our initial studies, we observed facile photochemical conversion of the known FXa/tPA inhibitor, BABCH ?(E,E)-2, 7-bis(4-amidinobenzylidene)cycloheptan-1-one, 1a, to the corresponding (Z,Z) olefin isomer, 1c (FXa K(i) = 0.66 nM), which was over 25,000 times more potent than the corresponding (E,E) isomer (1a, FXa K(i) = 17 000 nM). In order to determine the scope of this observation, we expanded on our initial investigation through the preparation of the olefin isomers in a homologous series of cycloalkanone rings, 4-substituted cyclohexanone analogues, and modified amidine derivatives. In most cases the order of potency of the olefin isomers was (Z,Z) > (E,Z) > (E,E) with the cycloheptanone analogue (1c) showing the most potent factor Xa inhibitory activity. In addition, we found that selectivity versus thrombin (FIIa) can be dramatically improved by the addition of a carboxylic acid group to the cycloalkanone ring as seen with 8c (FXa K(i) = 6.9 nM, FIIa K(i) > 50,000 nM). Compounds with one or both of the amidine groups substituted with N-alkyl substituents or replaced with amide groups led to a significant loss of activity. In this report we have demonstrated the importance of the two amidine groups, the cycloheptanone ring, and the (Z,Z) olefin configuration for maximum inhibition of FXa within the BABCH template. The results from this study provided the foundation for the discovery of potent, selective, and orally active FXa inhibitors.     

Glucose phosphorylated on carbon 6 suppresses lipopolysaccharide binding to lipopolysaccharide-binding protein and inhibits its bioactivities

Lipid A comprises the active region of lipopolysaccharide (LPS), and its phosphate group is required for LPS activities. Additionally, it is essential for effects of inhibitors of LPS-induced coagulation activity in limulus amebocyte lysate (LAL) tests. Lipid A has phosphorylated glucosamine residues, which are structurally similar to glucose 1-phosphate (G1P) and glucose 6-phosphate (G6P). This study focused on the antagonistic effects of glucose phosphates on the action of protein or non-protein inhibitors against LAL coagulation, LPS–LPS-binding protein (LBP) interaction, and LPS bioactivities. These effects of glucose phosphates were evaluated and compared with those of other charged sugars such as fructose 6-phosphate and glucuronic acid by LAL tests, ELISA-based LPS–LBP binding assay, cell-based assay, and using a mouse endotoxin shock model. G6P neutralized the interfering actions of drug substances and plasma proteins on LPS coagulation activity in LAL tests. Compared to other sugars, G6P more strongly inhibited LPS binding to LBP, leading to significant inhibition of LPS-induced cellular responses in human umbilical vein endothelial cells and in the THP-1 human leukemic line. Consistent herewith, G6P inhibited inflammatory cytokine release and decreased serum alanine aminotransferase and hepatic caspase-3/7 activities and mortality in LPS-stimulated d-galactosamine-sensitized mice. These data indicated that the structural properties of G6P, such as its glucose moiety and phosphorylation on carbon 6, are important for suppressing the interaction of proteins with LPS. Therefore, G6P is useful to improve sensitivity and accuracy of plasma and drug LPS assays, and such structural property is more suitable to antagonize LPS activities.     

Blood coagulation factor Xa as an emerging drug target

INTRODUCTION: Factor (F)Xa is well-known as an important player in the coagulation cascade responsible for thrombin generation. More recently, FXa emerged as an essential player in cell biology via activation of protease-activated receptors (PAR)-1 and -2. This pleiotropic role of FXa forms the basis for its potential contribution to the pathogenesis of several diseases. AREAS COVERED: The role of FXa in pathophysiology is reviewed with special emphasis on its signal transduction properties. To this end, we first discuss the important role of FXa in the coagulation cascade, we continue with recent data on FXa induced signaling in pathophysiology with special emphasis on tissue remodeling and fibrosis and discuss the potential of FXa as an emerging drug target. EXPERT OPINION: FXa is more than a passive intermediate in the coagulation cascade and FXa may in fact orchestrate fundamental processes during pathophysiology. Targeting FXa may be an exciting new therapeutic strategy in the treatment of (fibro)proliferative diseases for which current treatment options are limited.     

Investigational anticoagulants for hematological conditions: a new generation of therapies

Introduction: The introduction of novel anticoagulants has had contrasting effects on the agents in the pipeline, fueling the development of some and sinking the others. The complexity of the coagulation cascade offers interesting inhibition choices that might become valid treatment options.

Areas covered: This review will highlight some of the anticoagulants in the pipeline. Following the success of the direct thrombin and FXa inhibitors already in the market, new agents are being tested. These include AZD0837, betrixaban, letaxaban, darexaban, and LY517717. Targeting other components of the hemostatic pathway might lead to better safety profiles without influencing efficacy. Inhibitors to FVIIa–tissue factor (FVIIa/TF) complex, FIX, FXI, and FXII are being assessed. New inspiring inhibitors are antisense oligonucleotides (ASOs) and aptamers. These are highly specific agents with readily reversible effect and might be engineered to inhibit any coagulation factor. Currently tested ASOs and aptamers are inhibitors of FXI, FXII, thrombin, FIXa, and platelet GPIV.

Expert opinion: Some of the agents in the pipeline offer valid treatment option for long-term therapy, overcoming some of the drawbacks of the novel anticoagulants. Research is being driven by an expanding market in the anticoagulation field that has been unexploited for a long time.