Vitamin K2 (Menaquinone-7) supplementation does not affect vitamin K-dependent coagulation factors activity in healthy individuals.

BACKGROUND: Vitamin K has long been regarded as a procoagulant drug by physicians, and concerns have been raised with regard to its effects on hemostasis. Although many studies have shown that vitamin K supplementation is safe for thrombotic events, the effect of vitamin K supplementation on the activities of vitamin K dependent procoagulation factors in healthy individuals is not available. OBJECTIVES: This study aimed to investigate whether vitamin K2 supplementation at recommended doses affects the activity of vitamin K dependent procoagulation factors in healthy individuals without any anticoagulation treatment. DESIGN: Forty healthy volunteers between 25 and 40 years of age were recruited. Menaquinone-7 (MK-7) was administrated at 90 µg for 30 days. Prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and blood coagulation factors II, VII, IX, and X activities and Protein induced by vitamin K absence or antagonist-II (PIVKA-II) were measured on days 0 and 30 after MK-7 administration. RESULTS: PT, APTT, and TT showed no significant differences on day 30 when compared with baseline. The activities of coagulation factors II, VII, IX, and X on day 30 showed no significant differences with those at baseline. PIVKA-II levels were unchanged after 30 days of MK-7 supplementation. CONCLUSIONS: MK-7 supplementation at recommended dosage does not affect vitamin K-dependent coagulation factors' coagulation activity, and does not enhance the carboxylation of prothrombin in healthy individuals. This indicated that MK-7 administration does not alter hemostatic balance in healthy populations without anticoagulation treatment.

Comparative study of anticoagulant and procoagulant properties of 28 snake venoms from families Elapidae, Viperidae, and purified Russell's viper venom-factor X activator (RVV-X).

Snake venoms consist of numerous molecules with diverse biological functions used for capturing prey. Each component of venom has a specific target, and alters the biological function of its target. Once these molecules are identified, characterized, and cloned; they could have medical applications. The activated clotting time (ACT) and clot rate were used for screening procoagulant and anticoagulant properties of 28 snake venoms. Crude venoms from Daboia russellii siamensis, Bothrops asper, Bothrops moojeni, and one Crotalus oreganus helleri from Wrightwood, CA, had procoagulant activity. These venoms induced a significant shortening of the ACT and showed a significant increase in the clot rate when compared to the negative control. Factor X activator activity was also measured in 28 venoms, and D. r. siamensis venom was 5-6 times higher than those of B. asper, B. moojeni, and C. o. helleri from Wrightwood County. Russell's viper venom-factor X activator (RVV-X) was purified from D. r. siamensis venom, and then procoagulant activity was evaluated by the ACT and clot rate. Other venoms, Crotalus atrox and two Naja pallida, had anticoagulant activity. A significant increase in the ACT and a significant decrease in the clot rate were observed after the addition of these venoms; therefore, the venoms were considered to have anticoagulant activity. Venoms from the same species did not always have the same ACT and clot rate profiles, but the profiles were an excellent way to identify procoagulant and anticoagulant activities in snake venoms.

Platelet tissue factor synthesis in type 2 diabetic patients is resistant to inhibition by insulin.

OBJECTIVE: Patients with type 2 diabetes have an increased risk of cardiovascular disease and show abnormalities in the coagulation cascade. We investigated whether increased synthesis of tissue factor (TF) by platelets could contribute to the hypercoagulant state. RESEARCH DESIGN AND METHODS: Platelets from type 2 diabetic patients and matched control subjects were adhered to different surface-coated proteins, and TF premRNA splicing, TF protein, and TF procoagulant activity were measured. RESULTS: Different adhesive proteins induced different levels of TF synthesis. A mimetic of active clopidogrel metabolite (AR-C69931 MX) reduced TF synthesis by 56 +/- 10%, an aspirin-like inhibitor (indomethacin) by 82 +/- 9%, and the combination by 96 +/- 2%, indicating that ADP release and thromboxane A(2) production followed by activation of P2Y12 and thromboxane receptors mediate surface-induced TF synthesis. Interference with intracellular pathways revealed inhibition by agents that raise cAMP and interfere with phosphatidylinositol 3-kinase/protein kinase B. Insulin is known to raise cAMP in platelets and inhibited collagen III-induced TF premRNA splicing and reduced TF activity by 35 +/- 5 and 47 +/- 5% at 1 and 100 nmol/l. Inhibition by insulin was reduced in type 2 diabetes platelets resulting in an approximately 1.6-fold higher TF synthesis than in matched control subjects. CONCLUSIONS: We characterized the extra- and intracellular mechanisms that couple surface activation to TF synthesis in adhering platelets. In healthy individuals, TF synthesis is inhibited by insulin, but in patients with type 2 diabetes inhibition is impaired. This leads to the novel finding that platelets from type 2 diabetic patients produce more TF than platelets from matched control subjects.

Design and rationale of the Evaluation of M118 IN pErcutaNeous Coronary intErvention (EMINENCE) trial.

BACKGROUND: Currently recommended anticoagulant agents used in the setting of percutaneous coronary intervention (PCI) inhibit, with varying degrees of intensity, 2 critical targets (factor Xa and/or IIa) of the coagulation cascade, yet they carry significant limitations. M118-a novel, rationally engineered heparin-provides consistent anti-Xa and anti-IIa activity with a constant anti-Xa:anti-IIa ratio over time. M118 also combines the desired anticoagulant effects of unfractionated heparin with the beneficial attributes of low-molecular-weight heparin, and may represent the next generation of heparin therapy in patients diagnosed with acute coronary syndrome. STUDY DESIGN: The EMINENCE trial is a prospective, randomized, open-label, multicenter phase 2 study that will evaluate the safety and feasibility of M118 as an anticoagulant versus unfractionated heparin in subjects with stable coronary artery disease undergoing PCI. The primary end point of the study will be the combined incidence of clinical events defined as the composite of 30-day death, myocardial infarction, repeat revascularization, catheter thrombus, stroke, thrombocytopenia, bailout use of glycoprotein IIb/IIIa inhibitors, and major or minor bleeding. CONCLUSION: The EMINENCE trial will assess the safety and feasibility of M118 as an anticoagulant in the setting of PCI and will provide important information to determine the appropriate therapeutic range of activated clotting time for M118 and the appropriate dose or doses to be explored in a phase 3 clinical trial.

Effect of tecarfarin, a novel vitamin K epoxide reductase inhibitor, on coagulation in beagle dogs.

BACKGROUND AND PURPOSE: Tecarfarin (ATI-5923) is a novel vitamin K epoxide reductase inhibitor that is metabolized by esterase (mainly human carboxylesterase 2) to a single major metabolite, ATI-5900, in rats, dogs and humans. Tecarfarin is not significantly metabolized by CYP450 enzymes. The objective of this study was to test and compare the efficacy of tecarfarin with that of warfarin, when administered either intravenously or once a day orally, to produce stable anticoagulation in beagle dogs. EXPERIMENTAL APPROACH: Effects on coagulation were assessed by measuring the activity levels of Factor VII and Factor X and thromboplastin-induced coagulation times, reported as prothrombin time (PT). KEY RESULTS: Continuous intravenous infusions and oral administration of tecarfarin and warfarin caused a dose-dependent decrease in activity of Factor VII and Factor X, and associated increase in PT. Intravenous fresh frozen canine plasma or subcutaneous vitamin K(1) treatment reversed the anticoagulant effects of orally administered tecarfarin. Consistent with the inhibitory effects of amiodarone on CYP2C9, co-administration of amiodarone significantly increased the anticoagulation effect of warfarin and plasma warfarin concentrations. In contrast, amiodarone had no effect on the anticoagulation induced by tecarfarin or tecarfarin plasma concentrations in this model. CONCLUSIONS AND IMPLICATIONS: Overall, the data presented herein indicate that tecarfarin, via a vitamin K-dependent mechanism, causes changes in key parameters of haemostasis in beagle dogs that are consistent with effective anticoagulation. Compared to warfarin it has a decreased potential to interact metabolically with drugs that inhibit CYP450 enzymes and, therefore, may offer an improved safety profile for patients.

Effect of dietary omega-3 and omega-6 fatty acids on clotting activities of Factor V, VII and X in fatty liver haemorrhagic syndrome-susceptible laying hens.

1. The relationship between concentrations of omega-3 and omega-6 fatty acids in plasma and Factor V, VII and X clotting activities was determined using a crossover feeding trial with diets supplemented with either soy oil or flax oil. 2. Laying hens on the soy diet, which is high in omega-6 fatty acids, had substantially higher clotting activity for all three factors compared to laying hens on the flax diet that was high in omega-3 fatty acids. 3. Positive associations were seen between liver haemorrhage score and the percentage of liver weight and between the percentage of liver weight and the severity of haemorrhagic and fatty changes seen on histology. 4. These results support the hypothesis that concentrations of omega-6 and omega-3 fatty acids in plasma affect clotting activity; however, there was no relationship between the extent of liver haemorrhages and the composition of plasma fatty acids.

Direct analysis reveals an absence of gamma-carboxyglutamic acid in cancer procoagulant from human tissues.

Additional carboxylation of glutamic acid by vitamin K-dependent gamma-carboxylase is a common posttranslational modification of many proteins, including some of blood clotting factors. Vitamin K-antagonists, such as warfarin, are often included in the therapy of malignant disease, decreasing the blood coagulation potential. Cancer procoagulant, a direct blood coagulation factor X activator from malignant tissue, is considered as a vitamin K-dependent protein, so it could serve as one of possible targets for the therapy with warfarin. However, there is still no experimental data demonstrating directly the presence of gamma-carboxyglutamic acid (Gla) in a cancer procoagulant molecule. The presence of Gla in cancer procoagulant isolated from human amnion-chorion membranes and from human malignant melanoma WM 115 cell line was analyzed directly, using specific anti-Gla monoclonal antibodies. There was no detectable amount of Gla in cancer procoagulant isolated from fetal or malignant tissue. Cancer procoagulant from human tissues does not contain Gla-rich domain. The finding indicates that cancer procoagulant is rather a poor target for warfarin therapy of malignant disease.

Effect of phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine on the activated factor X-prothrombin system.

Membrane phospholipids are essential in blood coagulation reactions. The importance of negatively changed phosphatidylserine has been shown. The roles of other phospholipids in the blood coagulation system, however, are not clear. This study examined the effects of phosphatidylcholine on the blood coagulation system using liposomes containing varying concentrations of phosphatidylcholine in the presence of phosphatidylserine at a constant concentration. In addition, with phosphatidylserine and phosphatidylcholine at constant concentrations, the effects of phosphatidylethanolamine and lysophosphatidylcholine on the blood coagulation system were examined. Using an in vitro reconstructed system of the activated factor X-prothrombin system, blood coagulation was measured by the rate of thrombin formation after the addition of liposome preparations. The results showed suppression of the system by phosphatidylcholine and phosphatidylethanolamine and acceleration by lysophosphatidylcholine. The results of the present study suggest that the cell membrane, the 'location' of blood coagulation, is one of the regulatory factors, and that changes in phosphatidylcholine content and phospholipid composition of the cell membrane regulate the coagulation reaction.

[The comparison of simvastatin and atorvastatin effects on hemostatic parameters in patients with hyperlipidemia type II]. / Porównanie wplywu simwastatyny i atorwastatyny na wybrane parametry ukladu krzepniecia u chorych z hiperlipidemia typu II.

The studies results of statin influence on hemostasis are various. The aim of our study was to evaluate the effects of simvastatin and atorvastatin on hemostatic parameters, such as activity of factor X, antithrombin III, fibrinogen concentration and Lp(a). 72 patients (pts) aged 40-65 were involved in the study; 49 of them suffered from hyperlipidemia II (hlp II) with the initial concentration of total cholesterol (TC) >200 mg/dL, cholesterol LDL (LDL-C) >145 mg/dL, triglycerides (TG) <400 mg/dL. The control group consisted of 20 healthy persons. The pts with hlp II who underwent 4 weeks long lipid lowering diet were randomized into two groups: I--27 pts treated with simvastatin (20 mg/d), II--22 pts treated with atorvastatin (10 mg/d). The active statin therapy lasted 8 weeks. The activity of factor X and antithrombin III (AT III) was estimated by amidolytic methods, fibrinogen concentration (Fb) by the Clauss method, Lp(a)-immunoenzymatic method. The mean values of factor X activity and Fb serum concentration were higher in pts with hip II than in the control group, the AT III activity was lower. The Lp(a) concentration didn't differ between groups. Statin treatment was associated with significant reduction of factor X activity. Both simvastatin and atorvastatin markedly increased AT III (87%, 98%) in comparison to the initial values. No changes of Lp(a) concentration were observed during statin therapy. Atorvastatin therapy significantly increased the Fb concentration (12.3%). Simvastatin treatment didn't influence Fb concentration.

Folate deficiency-induced hyperhomocysteinemia attenuates, and folic acid supplementation restores, the functional activities of rat coagulation factors XII, X, and II.

Hyperhomocysteinemia (HH) constitutes a risk marker for thrombosis, but the pathophysiological mechanisms in thrombus formation are still unresolved. We investigated the influence of HH on single coagulation factor functions and evaluated the platelet GpIIb/IIIa receptor function in HH-induced changes in whole-blood coagulation profiles (WBCP). Three groups of 12 rats were investigated: control rats, folate deficient-HH (FD-HH) rats, and treated rats. Plasma total homocysteine was 7.1 micromol/L in controls, 31.3 micromol/L in FD-HH rats, and 7.6 micromol/L in treated rats. Factor (F) II:C, FX:C, and FXII:C were reduced in FD-HH rats compared with controls and normalized in treated rats (P < 0.05). FVII:C activity did not differ among the groups. Factor VIII:C activity was greater in FD-HH rats than in controls (P < 0.05). Blockage of the platelet GpIIb/IIIa receptor by Integrilin (Schering-Plough A/S) did not abolish the FD-HH-induced increase in whole-blood coagulation velocity, irrespective of the dosage of Integrilin. In conclusion, FD-HH reduced the functional activities of FXII:C, FX:C and FII:C, whereas FVII:C was unchanged and FVIII:C increased. These findings may partially explain the prolonged initiation phase of WBCP in FD-HH rats. The changes in single coagulation factor functions and WBCPs in FD-HH rats were reversed by treatment with folic acid.

[Advances in adjunctive pharmacological therapy for percutaneous coronary interventions]. / Avances en el tratamiento farmacológico coadyuvante en la intervención coronaria.

All percutaneous interventions disrupt atherosclerotic plaque and denude the endothelium. These processes stimulate both platelet aggregation and the coagulation cascade. Therefore, pharmacological treatment during percutaneous intervention is based on the use of antithrombotic agents. In addition to aspirin, whose benefit has been clearly demonstrated in all forms of ischemic heart disease, clopidogrel, given before and after cardiac catheterization, also reduces the rate of thrombosis after stent placement. Moreover, the introduction of glycoprotein IIb/IIIa inhibitors has improved the results of percutaneous revascularization, especially in high-risk patients. On the other hand, anticoagulants are essential for preventing the acute thrombotic complications that result from the invasive nature of the procedure. Low-molecular-weight heparins, direct thrombin inhibitors (e.g., hirudin and its derivatives), and recently developed pentasaccharides, which inhibit factor X, provide new alternatives to classical unfractionated heparin. These novel compounds lead to fewer hemorrhagic complications than unfractionated heparin and do not require such extensive monitoring. Finally, new antiproliferative agents, such as oral rapamycin, have been introduced to reduce the rate of coronary restenosis during follow-up.

Targeting exosites on blood coagulation proteases

A alta especificidade das proteases da coagulação tem sido atribuída não somente aos resíduos que cercam o sítio ativo, mas também a outros domínios de superfície que estão envolvidos no reconhecimento e interação com substratos macromoleculares e inibidores. Inibidores específicos da coagulação sanguínea obtidos de fontes exógenas como glândulas salivares de animais hematófagos e venenos de serpentes têm sido identificados. Alguns desses inibidores interagem com os exosítios das enzimas da coagulação. Dois exemplos são discutidos nesta curta revisão. A Botrojaracina é uma proteína derivada de veneno de serpente que se liga aos exosítios 1 e 2 da trombina. A formação do complexo impede várias atividades da trombina dependentes do exosítio 1 incluindo a clivagem do fibrinogênio e a ativação plaquetária. A Botrojaracina também interage com o proexosítio 1 da protrombina diminuindo a ativação do zimogênio pelo complexo protrombinase (FXa/FVa). O ixolaris é um inibidor com dois domínios Kunitz obtido da glândula salivar de carrapato, homólogo ao inibidor da via do fator tecidual. Recentemente foi demonstrado que o ixolaris se liga ao exosítio de ligação à heparina do FXa, impedindo o reconhecimento da protrombina pela enzima. Além disso, o ixolaris interage com o FX, possivelmente através do proexosítio de ligação à heparina. Diferentemente do FX, o complexo ixolaris-FX não é reconhecido como substrato pelo complexo tenase intrínseco (FIXa/FVIIIa). Nós concluimos que esses inibidores podem servir como ferramentas para o estudo dos exosítios da coagulação, assim como protótipos para novas drogas anticoagulantes.

Targeting exosites on blood coagulation proteases.

The high specificity of blood coagulation proteases has been attributed not only to residues surrounding the active site but also to other surface domains that are involved in recognizing and interacting with macromolecular substrates and inhibitors. Specific blood coagulation inhibitors obtained from exogenous sources such as blood sucking salivary glands and snake venoms have been identified. Some of these inhibitors interact with exosites on coagulation enzymes. Two examples are discussed in this short revision. Bothrojaracin is a snake venom-derived protein that binds to thrombin exosites 1 and 2. Complex formation impairs several exosite-dependent activities of thrombin including fibrinogen cleavage and platelet activation. Bothrojaracin also interacts with proexosite 1 on prothrombin thus decreasing the zymogen activation by the prothrombinase complex (FXa/FVa). Ixolaris is a two Kunitz tick salivary gland inhibitor, that is homologous to tissue factor pathway inhibitor. Recently it was demonstrated that ixolaris binds to heparin-binding exosite of FXa, thus impairing the recognition of prothrombin by the enzyme. In addition, ixolaris interacts with FX possibly through the heparin-binding proexosite. Differently from FX, the ixolaris-FX complex is not recognized as substrate by the intrinsic tenase complex (FIXa/FVIIIa). We conclude that these inhibitors may serve as tools for the study of coagulation exosites as well as prototypes for new anticoagulant drugs.

Use of the chromogenic factor X assay to predict the international normalized ratio in patients transitioning from argatroban to warfarin.

STUDY OBJECTIVE: To determine the clinical utility of the chromogenic factor X level for conversion from argatroban to warfarin in hospitalized patients. DESIGN: Prospective observational study. PATIENTS: Sixty-two hospitalized patients with indications for anticoagulation in whom the chromogenic factor X assay was used for conversion from argatroban to warfarin. SETTING: University-affiliated hospital. INTERVENTION: From December 2003-May 2004, data for all patients in whom the chromogenic factor X assay was used for conversion from argatroban to warfarin were screened for inclusion. When the chromogenic factor X level was satisfactory, the clinician discontinued the argatroban and a confirmatory international normalized ratio (INR) was obtained. MEASUREMENTS AND MAIN RESULTS: To determine the ability of the chromogenic factor X level to predict the INR free of argatroban influence, we calculated the sensitivity and specificity by using a cutoff chromogenic factor X level of 45% or less, or greater than 45%, which corresponded to an INR of 2 or greater, or less than 2, respectively. We constructed a receiver operating characteristic curve to illustrate various cutoff levels of chromogenic factor X. Of 146 patients screened, 62 had data that met criteria for analysis. An average of 6 +/- 3 doses of warfarin were administered before the confirmatory coagulation studies were obtained. The average time from the chromogenic factor X measurement to obtainment of confirmatory coagulation studies was 9 +/- 4 hours. Use of a chromogenic factor X level of 45% or less to predict an INR of 2 or greater absent of argatroban influence had a sensitivity of 93%, a specificity of 78%, and an accuracy of 89%. The area under the receiver operating characteristic curve was 0.91 (95% confidence interval 0.81-0.99, p<0.0001). CONCLUSION: The chromogenic factor X level is an accurate alternative when converting hospitalized patients from argatroban to warfarin. A chromogenic factor X level of 45% or less is a reliable predictor that the INR will be therapeutic when argatroban therapy is discontinued.

In vitro effects of poly-N-acetyl glucosamine on the activation of platelets in platelet-rich plasma with and without red blood cells.

BACKGROUND: This study was performed to assess the effect of poly-N-acetyl glucosamine fiber slurry on plasma clotting proteins, platelets, and red blood cells in the clotting of the blood. METHODS: Citrate phosphate dextrose whole blood was stored at 22degreesC for 48 hours to prepare platelet-poor plasma, platelet-rich plasma (PRP), and PRP plus red blood cells with hematocrit values of 20%, 35%, and 45% with and without an equal volume of poly-N-acetyl glucosamine fibers (1 mg/mL 0.9% NaCl). RESULTS: Thromboelastogram data show that poly-N-acetyl glucosamine fibers (p-GlcNAc) significantly reduced the R time in platelet-poor plasma, PRP, and PRP supplemented with red blood cells. Poly-N-acetyl glucosamine fibers increased, but not significantly, Annexin V and factor X binding to platelets, platelet microparticles, and red blood cell Annexin V binding. Poly-N-acetyl glucosamine fibers increased the production of thromboxane B2 by PRP. CONCLUSION: Poly-N-acetyl glucosamine slurry activates platelets.

The predictability of factor V Leiden (FV:Q(506)) gene mutation via clotting-based diagnosis of activated protein C resistance.

After the discovery of activated protein C resistance (APCR) due to factor V Leiden mutation and the causal relationship of the phenomenon with clinical thromboembolism, a wide variety of functional clotting-based assays were developed for testing of APCR in relation to the specific DNA-based analysis of FV:Q(506) Leiden. The aim of this study is to assess a clotting-based APCR assay using procoagulant crotalidae snake venom with respect to the sensitivity, specificity, and predictability for the presence of the factor V Leiden mutation. APCR testing and factor V DNA analyses have been performed concurrently on 319 patient specimens. APCR values of the patients with homozygous factor V Leiden mutation (70.4+/-13.5 s) were significantly lower (p<0.001) in comparison to the subjects with the heterozygous mutation (87.6+/-13.4 s). The assay is highly sensitive (98.7%) and specific (91.9%) for the screening of factor V Leiden mutation. The sensitivity and specificity of the APCR testing reached to 100% below the cut-off value of 120 s among the patients with homozygous factor V Leiden mutation. Therefore, this method could help the desired effective optimal screening strategy for the laboratory search of hereditary thrombophilia focusing on the diagnosis of APCR due to FV:Q(506).

Effect of the metalloproteinase inhibitor batimastat in the systemic toxicity induced by Bothrops asper snake venom: understanding the role of metalloproteinases in envenomation.

The peptidomimetic hydroxamate metalloproteinase inhibitor batimastat (BB-94) was assessed for its ability to neutralize the systemic effects (lethality, hemorrhage and coagulopathy) induced by the venom of Bothrops asper, the most important snake from a medical standpoint in Central America. Batimastat inhibited lethality when a venom challenge dose of two LD(50)s was used by intraperitoneal and intravenous routes, with ED(50)s of 250 and 22 microM, respectively. With a challenge dose of three LD(50)s, lethality was not abrogated, but a conspicuous and dose-dependent delay in the time of death was observed in mice injected with mixtures of venom plus batimastat. Upon incubation with 500 microM batimastat, venom LD(50) increased 2.86-fold (intraperitoneal route) and 2.37-fold (intravenous route), when compared with LD(50) of venom alone. Batimastat also inhibited the hemorrhagic effect induced by venom in the lungs after intravenous injection. Moreover, batimastat exerted a significant inhibition of in vitro coagulant and in vivo defibrinogenating effects of venom, evidencing that metalloproteinases play a key role in the coagulopathy characteristic of B. asper envenomation. The remaining uninhibited coagulant effect is due to serine proteinases, i.e. thrombin-like enzymes, since this effect was completely abrogated by the combination of batimastat and PMSF. Our results stress the view that metalloproteinases play a relevant role in the systemic pathophysiology of B. asper envenomation and that metalloproteinase inhibitors may become a therapeutic alternative in this pathology.

Activation and inactivation of human factor X by proteases derived from Ficus carica.

We investigated the effect of proteases derived from Ficus carica (common fig) on human blood coagulation. The milky sap (latex) of several Ficus (F.) species contain ficin, which is a mixture of proteases. Ficin derived from Ficus carica shortened the activated partial thromboplastin time and the prothrombin time of normal plasmas and plasmas deficient in coagulation factors, except plasma deficient in factor X (FX) and generated activated FX (FXa) in defibrinated plasma. Chromatographic separation of ficin from Ficus carica yielded six proteolytic fractions with a different specificity towards FX. We isolated two factor X activators with molecular masses of 23.2 and 23.5 kDa, and studied their action on purified human FX. Factor X was converted to activated FXbeta by consecutive proteolytic cleavage in the heavy chain between Leu178 and Asp179, Arg187 and Gly188, and Arg194and Ile195 (FX numbering system) with concomitant release of a carboxy-terminal peptide. The cleavage pattern of FXa degradation products in the light chain was influenced by Ca2+ and Mn2+. These data suggest the haemostatic potency of Ficus proteases is based on activation of human coagulation factor X.

Oral anticoagulation reduces activated protein C less than protein C and other vitamin K-dependent clotting factors.

Oral anticoagulant therapy, which is used for prophylaxis and management of thrombotic disorders, causes similar reductions in plasma levels of vitamin K-dependent procoagulant and anticoagulant clotting factor zymogens. When we measured levels of circulating activated protein C, a physiologically important anticoagulant and anti-inflammatory agent, in patients on oral anticoagulant therapy, the results unexpectedly showed that such therapy decreases levels of activated protein C substantially less than levels of protein C, prothrombin, and factor X, especially at lower levels of prothrombin and factor X. Thus, we suggest that oral anticoagulant therapy results in a relatively increased expression of the protein C pathway compared with procoagulant pathways not only because there is less prothrombin to inhibit activated protein C anticoagulant activity, but also because there is a disproportionately higher level of circulating activated protein C.

Hemoglobin binds melanoma cell tissue factor and enhances its procoagulant activity.

Tissue factor (TF), the membrane-bound glycoprotein that normally initiates the coagulation pathway, is expressed on the surface of various cells including endothelial cells, fibroblasts, monocytes and tumor cells. We recently reported that hemoglobin (Hb) enhances TF expression and procoagulant activity on TF-bearing human A375 malignant melanoma cells. To elucidate the mechanism of Hb-induced TF expression, we studied the interaction between purified TF from human A375 malignant melanoma cells and Hb. Selective binding of highly purified melanoma cell TF-apoprotein to Hb was demonstrated under native conditions using a dot-immunobinding assay and under denaturing conditions by Western blotting. The complex formation between purified melanoma cell TF-apoprotein and Hb was also demonstrated by the binding of fluid-phase Hb to immobilized TF-apoprotein (0-2.0 microg/ml) in an enzyme-linked immunosorbent assay. The binding was specific, concentration-dependent, saturable and inhibited significantly (60%) by Concanavalin-A. Hb enhanced the factor X-activating procoagulant activity of melanoma cell TF in a concentration-dependent manner, but had no effect on recombinant human TF. Concanavalin-A and wheat germ agglutinin significantly (60%) inhibited the Hb-induced procoagulant activity of malignant cell TF. We conclude that TF-apoprotein selectively binds Hb, most probably via the carbohydrate moieties (alpha-d-glucosyl; alpha-d-mannosyl and N-acetyl-beta-d-glucosaminyl residues) of TF, and enhances its procoagulant activity. The physiological significance of this interaction remains to be established.