MicroRNA-26a systemic administration attenuates tumor formation in hepatocellular carcinoma mouse model.

MicroRNA (miRNA)-26a is one of the tumor suppressor genes that has been down regulated during the development of hepatocellular carcinoma (HCC). This work was conducted to evaluate the possible preventive effect of exogenous miRNA-26a administration on diethylnitrosamine (DEN)-mediated HCC. Balb/C mice were intraperitoneally injected with saline (Normal group), DEN (HCC group) or miRNA-26a (HCC+miRNA-26a group). On week 8, 12, 16 and 20, the concentrations of alpha-fetoprotein (AFP), des-gamma carboxyprothrombin (DCP), the levels of helper T cells-associated cytokines, and the vascular endothelial growth factor (VEGF), were measured. Flow cytometry determined the frequencies of regulatory T (Treg) cells. The concentrations of AFP, DCP and VEGF, as well as the frequency of Treg cells showed significantly lower values following miRNA-26a administration than in HCC group. miRNA-26a administration has reduced the levels of IL (interleukin)-2 and TNF (tumor necrosis factor)-α, in contrast, IL-10 level was markedly elevated in comparison to HCC model at all experimental time points. The restore of miRNA-26a function significantly (P<0.001) down regulated the expression levels of survivin & caspase-3 compared to HCC group. The obtained data introduce an evidence for the suppressive impact of miRNA-26a on liver tumor formation and its possible manipulation as a therapeutic design for HCC.

Modulation of the Coagulation Cascade Using Aptamers.

As a novel class of therapeutics, aptamers, or nucleic acid ligands, have garnered clinical interest because of the ease of isolating a highly specific aptamer against a wide range of targets, their chemical flexibility and synthesis, and their inherent ability to have their function reversed. The following review details the development and molecular mechanisms of aptamers targeting specific proteases in the coagulation cascade. The ability of these anticoagulant aptamers to bind to and inhibit exosite function rather than binding within the active site highlights the importance of exosites in blocking protein function. As both exosite inhibitors and reversible agents, the use of aptamers is a promising strategy for future therapeutics.

Hemostatic effects of two desogestrel-containing combined oral contraceptive regimens: a multinational, multicenter, randomized, open-label study.

PURPOSE OF INVESTIGATION: To compare the effects of desogestrel (DSG) 150 mcg/ethinyl estradiol (EE) 20 mcg for 21 days followed by either seven days of EE ten mcg (21/7-active) or no treatment (DSG/EE+no Tx) on hemostatic markers. MATERIALS AND METHODS: This was a randomized, multicenter, open-label study that enrolled healthy premenopausal women. Non-inferiority of 21/7-active to DSG/EE+no Tx was determined if the upper limit of the two-sided 95% CI of the mean treatment difference in prothrombin fragment 1+2 (F1+2) over 24 weeks between groups was < 130 pmol/L. RESULTS: 246 subjects (n = 125, 21/7-active; n = 121, DSG/EE+no Tx) comprised the primary analysis. Mean F1+2 levels increased in both 21/7-active and DSG/EE+no Tx regimens (least square [LS] mean changes +45 pmol/L and +56.8 pmol/L, respectively). LS mean treatment difference was -11.8 pmol/L (95% CI: -54.8, 31.2). CONCLUSION: The effect of adding EE ten mcg to the seven-day hormone-free interval of DSG/EE on F1+2 levels was non-inferior to traditional DSG/EE.

Properties of biological and biochemical effects of the Iranian saw-scaled viper (Echis carinatus) venom.

The venom of Echis carinatus is rich in proteins and peptides effective on the hemostatic system. This venom is contains metalloproteinase which convert prothrombin to meizothrombin. The prothrombin activator which leads to the formation of small blood clots inside the blood vessels throughout the body. To understand the mechanism of the effects of Iranian Echis carinatus venom, the effects of E. carinatus on human and Wistar rat plasma, plasma proteins (prothrombin and fibrinogen) and blood coagulation were studied. Proteolytic activity of the crude venom on blood coagulation factors such as prothrombin, partial thromboplastin and fibrinogen times were studied. In the present study the PT test for human plasma was reduced from 13.4 s (±0.59) to 8.6 s (±0.64) when human plasma was treated with crude venom (concentration of venom was 1 mg/ml) and for rat plasma PT was reduced from 14.5 s (±0.47) to 8 s (±0.49). Some possible biological and biochemical effects of IEc crude venom were investigated. The blood coagulation in human and in rat were investigated in vivo and in-vitro. In this paper, we show that the procoagulant action of Echis carinatus venom is due in part to a protein component that activates prothrombin and the procoagulant activity on human and rat plasma was evaluated (Tab. 2, Fig. 2, Ref. 31).

Proteomic profiling of k-11706 responsive proteins.

Erythropoietin promotes the production of red blood cells. Recombinant human erythropoietin is illicitly used to improve performance in endurance sports. Expression of the ERYTHROPOIETIN gene is negatively controlled by the transcription factor GATA-binding protein (GATA). Specific GATA inhibitors have recently been developed as novel drugs for the management of anemia. These drugs could, therefore, be illicitly used like recombinant human erythropoietin to improve performance in sports. To examine alterations in levels of plasma protein after administration of GATA inhibitors, proteomic analyses were conducted on mouse plasma samples treated with the potent GATA inhibitor K-11706. The analysis based on gel electrophoresis identified 41 protein spots differentially expressed when compared with normal plasma. Each spot was identified with liquid chromatography coupled to tandem mass spectrometry and 2 of them, fetuin-B and prothrombin, were verified by Western blotting. The results showed that the expression of fetuin-B in mice plasma was increased by K-11706, but not by recombinant human erythropoietin or hypoxia. These results suggest the potential of proteomic-based approaches as tools to identify biomarkers for the illegal use of novel drugs (e.g., GATA inhibitors). Also, fetuin-B could be a sensitive marker for the detection of abuse of GATA inhibitors.

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.

Use of warfarin therapy at a target international normalized ratio of 3.0 for cutaneous polyarteritis nodosa.

BACKGROUND: Cutaneous polyarteritis nodosa (CPN) is an uncommon disorder that can be difficult to manage effectively. We have previously suggested that CPN might be associated with the presence of anti-phosphatidylserine-prothrombin complex (anti-PS/PT) antibodies, members of the antiphospholipid antibody family. OBJECTIVE: To evaluate clinical manifestations and effective treatments of CPN. METHODS: We conducted a retrospective analysis of three patients with CPN who responded to warfarin therapy. IgG and IgM anti-PS/PT antibodies were measured with a specific enzyme-linked immunosorbent assay. RESULTS: There was a dramatic improvement in our three CPN patients following warfarin therapy adjusted to a target international normalized ratio (INR) of about 3.0. Active disease progression was halted by sustained warfarin therapy during which the patients experienced resolution of their skin manifestations. LIMITATIONS: A small number of cases were studied and the study design was retrospective. CONCLUSION: We propose that warfarin therapy at a target INR of roughly 3.0 could be effective for treating patients with CPN. We further believe that treatment with warfarin led to the effective attenuation of anti-PS/PT antibodies related to prothrombin, and improved the symptoms in our CPN patients.

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.

Vitamin K2 inhibits the growth of hepatocellular carcinoma via decrease of des-gamma-carboxy prothrombin.

BACKGROUND: Des-gamma-carboxy prothrombin (DCP) is a serum protein produced by hepatocellular carcinoma (HCC) cells in the absence of vitamin K. Serum and tissue DCP expressions are thought to reflect the biological malignant potential of HCC. Hence, we aimed to examine the efficacy of vitamin K(2) on the production of DCP as well as tumor cell growth and invasion. METHODS: Cell growth and viability were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The in vivo efficacy of vitamin K(2) was examined in nude mice bearing HCC cells. A 24-well transwell chamber was used to evaluate the motility and invasive ability of HCC cells. Levels of DCP in supernatant of cultures and in serum of mice were measured using an electrochemiluminescence immunoassay method. Western blot and immunohistochemical analysis were employed to evaluate the expression of DCP in HCC. RESULTS: Vitamin K(2) (2-40 muM) significantly decreased the levels of DCP production in supernatant of PLC/PRF/5 and HepG2 cells and in serum of nude mice bearing HCC xenografts. The inhibition of DCP was also observed using the assays of Western blot analysis in HCC cultures and immunohistochemical analysis in HCC xenografts in mice. As a result of administration of vitamin K(2), the capacity of HCC growth was inhibited and the invasion and migration of tumor cells were decreased. Furthermore, the inhibitory effects of HCC growth were also observed in vivo and the sensitivity was well correlated with the decrease of DCP in the serum of mice. CONCLUSION: Vitamin K(2) might suppress the growth and invasion of HCC cells via decrease of DCP.

Effects of prothrombin complex concentrate and recombinant activated factor VII on vitamin K antagonist induced anticoagulation.

INTRODUCTION: Warfarin and its derivatives are widely used for prevention of thrombotic incidents. Prothrombin complex concentrate (PCC) and recombinant activated factor VII (rFVIIa) have been used clinically for the acute reversal of this agent but there is a paucity of data on comparative efficacies of these hemostatic interventions. MATERIALS AND METHODS: Using in vivo rat and in vitro human models of anticoagulation, we compared PCC and rFVIIa on the recovery of endogenous thrombin generation. For in vivo reversal of anticoagulation, saline (control), PCC 50 U ml(-1), or rFVIIa100 mug ml(-1) was given to rats which received phenprocoumon (2.5 mg kg(-1)) orally. For in vitro model, plasma samples from warfarin-treated individuals with INR values of 2.1-6.7 were spiked with PCC (0.2, 0.4, or 0.72 U ml(-1)) or rFVIIa (3.0 mug/ml). The treatments were evaluated using prothrombin time (PT) and thrombin generation (Thrombinoscope). RESULTS: Administration of phenprocoumon to rats prolonged PT (14.7+/-0.5 to 50.43+/-0.7 s) and decreased peak thrombin generation by 89+/-2.3%. Administration of PCC dose dependently reversed the anticoagulation effects both in warfarin-treated human plasma and in phenprocoumon-treated rats by shortening PT and increasing peak thrombin levels. However, rFVIIa only reversed PT, but had minimal effects on peak thrombin levels. CONCLUSION: Both PCC and rFVIIa reverse warfarin anticoagulation based on PT, but only PCC restores overall thrombin generation.

Two coagulation factor X activators from Vipera a. ammodytes venom with potential to treat patients with dysfunctional factors IXa or VIIa.

Two activators of coagulation factor X, 58kDa VAFXA-I and 70kDa VAFXA-II, were purified from the venom of long-nosed viper (Vipera ammodytes ammodytes) by chromatography on gel filtration, affinity, ion-exchange and hydroxyapatite media. Both enzymes are glycoproteins composed of a heavy chain and two C-type lectin-like light chains all joined by disulphide bonds. LC-MS and LC-MS/MS analysis of their tryptic fragments demonstrated that the heavy chain consists of three domains, metalloproteinase, disintegrin-like and cysteine-rich domains. The partial amino acid sequences of VAFXAs are very similar to those of the known factor X activators, RVV-X from Vipera russelli and VLFXA from Vipera lebetina venoms, as well as to other members of the reprolysin family of metalloproteinases. The VAFXAs activate factor X in a Ca(2+)-dependent manner with the same specificity as physiological activators. The activators weakly hydrolyzed insulin B-chain, fibrinogen and some components of the extracellular matrix in vitro, but did not activate prothrombin or plasminogen. VAFXAs inhibit collagen-induced platelet aggregation in vitro. They activate coagulation factor X to Xa without toxic effects. Their application in treating patients with dysfunctional factors IXa or VIIa to restore the normal blood coagulation process is thus promising.

Evaluation of the changes in hemostatic parameters induced by etoricoxib in rat model.

The effects of etoricoxib on blood coagulation parameters were evaluated, along with acetylsalicylic acid, in male Wistar rats. Blood samples were collected at the end of the first, second, third and fourth weeks of the administration period and the plasma concentrations of etoricoxib determined by liquid chromatography-tandem mass spectrometry; the samples were also used for the analysis of the hematological parameters. There were no significant changes in the platelet count and fibrinogen levels, and a decrease by 1.9% of the prothrombin time was detected at the third week. The activated partial thromboplastin time assay showed a nonsignificant shortening. The antiactivated factor X and antiactivated factor II activities showed reductions lower than 2.8 and 1.3%, respectively. These findings and the comparison with acetylsalicylic acid can be helpful to support the benefit-to-risk profile, contributing to the safe therapeutic use.

Prolonged exposure to inhaled nitric oxide does not affect haemostasis in piglets.

OBJECTIVE: To examine possible adverse effects on haemostasis from prolonged exposure to inhaled nitric oxide (iNO). DESIGN AND SETTING: Blinded, randomised, experimental animal study in a university animal laboratory. INTERVENTIONS: Anaesthetised and intubated piglets received central venous, arterial, and transabdominal urinary catheters. Twelve piglets were studied with triggered pressure support ventilation breathing with an air-oxygen mixture for 30 h with nitric oxide (NO), 40 parts per million (ppm) (n = 6) or without NO gas (n = 6) added. The tests of platelet function were assessed in a separate 1-h experiment in which 12 additional animals were blindly randomised to receive intravenous acetylsalicylic acid (ASA) (n = 7) or placebo (n = 5). MEASUREMENTS AND RESULTS: All 12 animals were clinically stable during the study period of 30 h. Haemostasis was assessed in terms of bleeding time and platelet function by Adeplat-S, reflecting platelet adhesion. Prothrombin fragment 1 + 2, fibrin D-dimer, tissue plasminogen activator and prothrombin complex were measured to investigate whether inhaled NO (iNO) had any effects on thrombin formation, fibrin formation, fibrinolysis or coagulation. All parameters including bleeding time and Adeplat-S were unaffected by iNO. ASA significantly increased bleeding time, but did not affect Adeplat-S. Nitrate in plasma and NOx (nitrate and nitrite) in urine increased significantly in pigs receiving iNO compared with controls. CONCLUSIONS: Prolonged exposure to iNO at 40[Symbol: see text]ppm did not affect bleeding time or coagulation parameters in healthy piglets. The findings do not support the hypothesis that iNO increases the risk of bleeding in humans.

Efficacy of antivenom against the procoagulant effect of Australian brown snake (Pseudonaja sp.) venom: in vivo and in vitro studies.

Snake venom induced consumption coagulopathy (VICC) is a common complication of snake bite due to prothrombin activators or thrombin-like enzymes in the venom. This study aimed to determine the efficacy and dose of antivenom for treating VICC in patients envenomed by brown snakes (Pseudonaja spp.), including in vitro coagulation studies. In serial blood samples from patients with brown snake envenoming, venom and antivenom concentrations were measured using enzyme immunoassays. In vitro mixtures of brown snake venom and antivenom were used to investigate antivenom binding, neutralisation of prothrombin activity, prevention of venom-mediated clotting and effect on thrombin generation parameters using a thrombinoscope. In 27 envenomed patients the median venom concentration was 20 ng/mL (Interquartile range[IQR]:12-44 ng/mL) prior to antivenom and was not detected after antivenom administration, including 9 patients given one vial. In vitro, 200 microg/mL of antivenom bound all free venom at venom concentrations seen in patients. In vitro prothrombinase activity of the venom (using a chromogenic substrate) was not neutralised by antivenom. However, for venom concentrations seen in humans, 100 microg/mL of antivenom prevented venom clotting activity in human plasma and 479 microg/mL neutralised procoagulant venom activity measured by triggering thrombin generation. One vial of antivenom appears to be sufficient to bind and neutralise all venom in patients with severe brown snake envenoming.

Recombinant Factor VIIa-Mediated Activation of Prothrombin Complex Concentrates.

Recombinant factor VIIa (rFVIIa) is used in the management of bleeding in patients with hemophilia. A generic biosimilar version of NovoSeven is also developed (AryoSeven). To compare the activation profile of NovoSeven and AryoSeven, 2 commercially available protein complex concentrates (PCCs) were used. Profilnine activated by RecombiPlasTin 2G resulted in conversions of prothrombin to prethrombin and thrombin at 5 to 30 minutes. However, addition of rFVIIa at final concentration range of 0.25 to 0.5 µg/mL to the same mixture resulted in total conversion of prothrombin to thrombin with a doublet at 36 kDa. Recombinant factor VIIa alone did not generate thrombin in native Beriplex, and the addition of rFVIIa to Beriplex failed to generate thrombin. Beriplex activated by RecombiPlasTin 2G resulted in complete conversion of prothrombin to thrombin. Both NovoSeven and AryoSeven exhibited similar activation profiles. These studies indicate that the activation of PCCs by both rFVIIa preparations results in comparable generation of thrombin.

Age-related differences in heparin response.

INTRODUCTION: Major physiological differences in the coagulation system throughout childhood, compared to adults, are well documented. However, the impact of this on anticoagulant drugs is unknown. This study aimed to determine whether heparin therapeutic range determination is affected by the age of plasma donors and whether age-specific therapeutic ranges for heparin therapy may need to be considered in the clinical setting. MATERIALS AND METHODS: Plasma samples were obtained from healthy children and adults, and pooled into age-specific pools. These were spiked with different concentrations of heparin and APTT; Anti-Factor Xa and Anti-Factor IIa were measured using standard techniques. The experiments were repeated using three separate plasma pools, and results expressed as means with standard deviations. RESULTS AND CONCLUSIONS: The results show clear age-related differences in APTT for the same Anti-Factor Xa heparin concentration. The differences were more pronounced in younger children, with higher APTT for same Anti-Factor Xa. The Anti-Factor IIa activity of heparin for a given Anti-Factor Xa activity also differed between age-specific plasma pools. This study suggests that when using heparin in children, basic assumptions about the drug mechanism of action and implications for therapeutic ranges need to be considered.

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.

A prospective randomized trial of aspirin-clopidogrel combination therapy and dose-adjusted warfarin on indices of thrombogenesis and platelet activation in atrial fibrillation.

OBJECTIVES: This study was designed to investigate whether or not combination aspirin-clopidogrel therapy would reduce markers of thrombogenesis and platelet activation in atrial fibrillation (AF), in a manner similar to warfarin. BACKGROUND: Dose-adjusted warfarin is beneficial as thromboprophylaxis in AF, but potentially serious side effects and regular monitoring leave room for alternative therapies. METHODS; We randomized 70 patients with nonvalvular AF who were not on any antithrombotic therapy to either dose-adjusted warfarin (international normalized ratio 2 to 3) (Group I) or combination therapy with aspirin 75 mg and clopidogrel 75 mg (Group II). Plasma indices of thrombogenesis (fibrin D-dimer, prothrombin fragment 1+2) and platelet activation (beta-thromboglobulin [TG] and soluble P-selectin) were quantified, along with platelet aggregation responses to standard agonists, at baseline (pretreatment) and at six weeks posttreatment. RESULTS; Pretreatment levels of fibrin D-dimer (p = 0.001), beta-TG (p = 0.01) and soluble P-selectin (p = 0.03) were raised in patients with AF, whereas plasma prothrombin fragment 1+2 levels and platelet aggregation were not significantly different compared with controls. Dose-adjusted warfarin reduced plasma levels of fibrin D-dimer, prothrombin fragment 1+2 and beta-thromboglobulin levels at six weeks (all p < 0.001), enhanced plasma levels of soluble P-selectin (p < 0.001) and had no significant effect on platelet aggregation. Aspirin-clopidogrel combination therapy made no difference to the plasma markers of thrombogenesis or platelet activation (all p = NS), but the platelet aggregation responses to adenosine diphosphate (p < 0.001) and epinephrine (p = 0.02) were decreased. CONCLUSIONS: Aspirin-clopidogrel combination therapy failed to reduce plasma indices of thrombogenesis and platelet activation in AF, although some aspects of ex vivo platelet aggregation were altered. Anticoagulation with warfarin may be superior to combination aspirin-clopidogrel therapy as thromboprophylaxis in AF.

Inhibition of thrombin generation by simvastatin and lack of additive effects of aspirin in patients with marked hypercholesterolemia.

OBJECTIVES: To assess the effects of aspirin compared with simvastatin on thrombin generation in hypercholesterolemic men, and to establish whether the reduction of elevated blood cholesterol by simvastatin would affect the action of aspirin on thrombin formation. BACKGROUND: Aspirin inhibits thrombin formation, but its performance is blunted in hypercholesterolemia. By virtue of altering lipid profile, statins could be expected to influence thrombin generation. METHODS: Thirty-three men, aged 34 to 61 years, with minimal or no clinical symptoms, serum total cholesterol >6.5 mmol/liter and serum triglycerides <4.6 mmol/liter, completed the study consisting of three treatment phases. First, they received 300 mg of aspirin daily for two weeks (phase I), which was then replaced by simvastatin at the average dose of 24 mg/d for three months (phase II). In phase III, aspirin, 300 mg/day, was added for two weeks to simvastatin, the dose of which remained unchanged. Thrombin generation was assessed: 1) in vivo, by measuring levels of fibrinopeptide A (FPA) and prothrombin fragment 1+2 (F1+2) in venous blood; and 2) ex vivo, by monitoring the rates of increase of FPA and F1+2 in blood emerging from standardized skin incisions of a forearm. A mathematical model was used to describe the kinetics of thrombin formation at the site of microvascular injury. RESULTS: Two-week treatment with aspirin had no effect on thrombin markers in vivo, while ex vivo it depressed the total amount of thrombin formed, though not the reaction rate. After simvastatin treatment, serum cholesterol decreased by 31% and LDL cholesterol by 42%, while thrombin generation became markedly depressed. In venous blood, FPA was significantly reduced. Concomitantly, the initial thrombin concentration and total amount of thrombin generated decreased significantly. Addition of aspirin to simvastatin (phase III) had no further effect on any of these parameters. CONCLUSIONS: In men with hypercholesterolemia, lowering serum cholesterol level by a three-month simvastatin treatment is accompanied by a marked reduction of thrombin generation both at basal conditions in venous blood and after activation of hemostasis by microvascular injury. Once blood cholesterol became reduced, adding aspirin to simvastatin did not enhance dampening of thrombin formation.

Structure and function comparison of Micropechis ikaheka snake venom phospholipase A2 isoenzymes.

Comparison of the crystal structures of three Micropechis ikaheka phospholipase A2 isoenzymes (MiPLA2, MiPLA3 and MiPLA4, which exhibit different levels of pharmacological effects) shows that their C-terminus (residues 110-124) is the most variable. M-Type receptor binding affinity of the isoenzymes has also been investigated and MiPLA4 binds to the rabbit M-type receptor with high affinity. Examination of surface charges of the isoenzymes reveals a trend of increase in positive charges with potency. The isoenzymes are shown to oligomerize in a concentration-dependent manner in a semi-denaturing gel. The C-termini of the medium (MiPLA4) and highly potent (MiPLA2) isoenzyme molecules cluster together, forming a highly exposed area. A BLAST search using the sequence of the most potent MiPLA2 results in high similarity to Staphylococcus aureus clotting factor A and cadherin 11. This might explain the myotoxicity, anticoagulant and hemoglobinuria effects of MiPLA2s.