Vitamin K Effects on Gas6 and Soluble Axl Receptors in Intensive Care Patients: An Observational Screening Study.

Growth arrest-specific gene 6 protein (Gas6) is avitamin K-dependent tissue bound protein. Gas6 has been shown to promote growth and therapy resistance among different types of cancer as well as thromboembolism. The aim of this prospective screening study: ClinicalTrials.gov; Identifier: NTC3782025, was to evaluate the effects of intravenously administered vitamin K1 on Gas6 and its soluble (s)Axl receptor plasma levels in intensive care patients. Vitamin K1 was intravenously injected in non-warfarin treated patients with prolonged Owren prothrombin time international normalized ratio (PT-INR) > 1.2 and blood samples were retrieved before and 20-28 h after injection. Citrate plasma samples from 52 intensive care patients were analysed for different vitamin K dependent proteins. There was a significant, but small increase in median Gas6. Only one patient had a large increase in sAxl, but overall, no significant changes in sAxl Gas6 did not correlate to PT-INR, thrombin generation assay, coagulation factors II, VII, IX and X, but to protein S and decarboxylated matrix Gla protein (dp-ucMGP). In conclusion, there was a small increase in Gas6 over 20-28 h. The pathophysiology and clinical importance of this remains to be investigated. To verify a true vitamin K effect, improvement of Gas6 carboxylation defects needs to be studied.

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.

HDAC inhibition reduces white matter injury after intracerebral hemorrhage.

Inhibition of histone deacetylases (HDACs) has been shown to reduce inflammation and white matter damage after various forms of brain injury via modulation of microglia/macrophage polarization. Previously we showed that the HDAC inhibitor scriptaid could attenuate white matter injury (WMI) after ICH. To access whether modulation of microglia/macrophage polarization might underlie this protection, we investigated the modulatory role of HDAC2 in microglia/macrophage polarization in response to WMI induced by intracerebral hemorrhage (ICH) and in primary microglia and oligodendrocyte co-cultures. HDAC2 activity was inhibited via conditional knockout of the Hdac2 gene in microglia or via administration of scriptaid. Conditional knockout of the Hdac2 gene in microglia and HDAC inhibition with scriptaid both improved neurological functional recovery and reduced WMI after ICH. Additionally, HDAC inhibition shifted microglia/macrophage polarization toward the M2 phenotype and reduced proinflammatory cytokine secretion after ICH in vivo. In vitro, a transwell co-culture model of microglia and oligodendrocytes also demonstrated that the HDAC inhibitor protected oligodendrocytes by modulating microglia polarization and mitigating neuroinflammation. Moreover, we found that scriptaid decreased the expression of pJAK2 and pSTAT1 in cultured microglia when stimulated with hemoglobin. Thus, HDAC inhibition ameliorated ICH-mediated neuroinflammation and WMI by modulating microglia/macrophage polarization.

Prevention of postoperative bleeding after complex pediatric cardiac surgery by early administration of fibrinogen, prothrombin complex and platelets: a prospective observational study.

BACKGROUND: Postoperative bleeding is a major problem in children undergoing complex pediatric cardiac surgery. The primary aim of this prospective observational study was to evaluate the effect of an institutional approach consisting of early preventive fibrinogen, prothrombin complex and platelets administration on coagulation parameters and postoperative bleeding in children. The secondary aim was to study the rate of re-intervention and postoperative transfusion, the occurrence of thrombosis, length of mechanical ventilation, ICU stay and mortality. METHODS: In fifty children (age 0-6 years) with one or more predefined risk factors for bleeding after cardiopulmonary bypass (CPB), thrombelastography (TEG) and standard coagulation parameters were measured at baseline (T1), after CPB and reversal of heparin (T2), at sternal closure (T3) and after 12 h in the ICU (T4). Clinical bleeding was evaluated by the surgeon at T2 and T3 using a numeric rating scale (NRS, 0-10). RESULTS: After CPB and early administration of fibrinogen, prothrombin complex and platelets, the clinical bleeding evaluation score decreased from a mean value of 6.2 ± 1.9 (NRS) at T2 to a mean value of 2.1 ± 0.8 at T3 (NRS; P <  0.001). Reaction time (R), kinetic time (K), maximum amplitude (MA) and maximum amplitude of fibrinogen (MA-fib) improved significantly (P <  0.001 for all), and MA-fib correlated significantly with the clinical bleeding evaluation (r = 0.70, P <  0.001). The administered total amount of fibrinogen (mg kg- 1) correlated significantly with weight (r = - 0.42, P = 0.002), priming volume as percentage of estimated blood volume (r = 0.30, P = 0.034), minimum CPB temperature (r = - 0.30, P = 0.033) and the change in clinical bleeding evaluation from T2 to T3 (r = 0.71, P <  0.001). The incidence of postoperative bleeding (> 10% of estimated blood volume) was 8%. No child required a surgical re-intervention, and no cases of thrombosis were observed. Hospital mortality was 0%. CONCLUSION: In this observational study of children with an increased risk of bleeding after CPB, an early preventive therapy with fibrinogen, prothrombin complex and platelets guided by clinical bleeding evaluation and TEG reduced bleeding and improved TEG and standard coagulation parameters significantly, with no occurrence of thrombosis or need for re-operation. TRIAL REGISTRATION: German Clinical Trials Register DRKS00018109 (retrospectively registered 27th August 2019).

The effect of leech extracts on endothelial cell coagulation-related factors and endothelial dysfuction-related molecules.

OBJECTIVE: This study was focused on screening leech extracts to identify those with little or no anti-coagulation effect or with significant anti-endothelial dysfunction activity. METHODS: Different leech extracts were prepared by enzymolysis and microbial transformation and their cytotoxicity were measured by MTT assay. The effect of different leech extracts on mRNA expression of coagulation-related factors (PAI, vWF, tPA, PS, TFPI, TM) was quantified by RT-PCR. After identifying a leech extract with little anti-coagulatory effect, RT-PCR was then used to assess the effect of this extract on the mRNA expression of endothelial dysfunction-related molecules (ET-1, iNOS, MCP-1, IL-6). RESULTS: 8 leech extracts were obtained, including 4 enzymatic extracts (LP, PHL, PTHL, CEHL) and 4 Lactobacillus metabolites (MRS, MRS-1, MRS-2, and MRS-3). Following optimization of conditions using MTT assays, we treated EA.hy926 cells with 0, 12.5, 25, 50 µg/mL of LP, PTHL, CEHL, MRS, MRS-1 or MRS-3 extract for 24 h. We found that PHL and MRS-1 had no significant effect on coagulation-related factors. Furthermore, treatment with 50 µg/mL PHL resulted in significant decreases in ET-1, iNOS, MCP-1, and IL-6 mRNA expression by 28.06%, 33.30%, 19.80%, and 52.34%, respectively. CONCLUSIONS: In the present study, we found that PHL, a pepsin hydrolysate of leech with little anti-coagulatory effect, could significantly suppress TNF-α induced mRNA overexpression of endothelial dysfunction-related molecules (ET-1, iNOS, MCP-1, and IL-6). These results provide a reliable experimental basis for identifying new anti-atherosclerosis therapeutics for long term use and with minimal bleeding side effects.

Prothrombotic response to norepinephrine infusion, mimicking norepinephrine stress-reactivity effects, is partly mediated by α-adrenergic mechanisms.

BACKGROUND: Stress-induced prothrombotic changes are mediated by the sympathetic nervous system and critically involved in mental triggering of acute coronary syndromes, but the underlying psychobiology is not fully understood. We tested the hypothesis that a norepinephrine (NE) infusion to mimic effects of stress-induced NE release on blood coagulation elicits prothrombotic changes and examined to what extent these would be mediated by an alpha-adrenergic mechanism. METHODS AND RESULTS: In a single-blind placebo-controlled within-subjects design, 24 middle-aged, non-smoking, non-obese and normotensive men participated in three experimental trials with an interval between one and two weeks. Each trial applied two sequential infusions of 1 and 15 min duration with varying substances [i.e., saline as placebo, the non-specific α-blocker phentolamine (2.5 mg/min), and NE (5 µg/min)]: trial 1=saline + saline; trial 2=saline + NE, and trial 3=phentolamine + NE. Plasma levels of clotting factor VIII activity (FVIII:C), fibrinogen, and D-dimer were assessed from blood samples collected immediately before and 1 min and 20 min after infusion procedures. Compared to saline + saline, saline + NE induced increases over time in FVIII:C, fibrinogen, and D-dimer levels. With phentolamine + NE, fibrinogen levels remained increased compared to saline + saline, but changes in FVIII:C and D-dimer levels were no more different. Coagulation changes did not differ between saline + NE and phentolamine + NE. CONCLUSIONS: NE infusion activates blood coagulation. The resulting prothrombotic state could be one psychobiological mechanism underlying mental triggering of acute coronary syndromes. Blockade of α-adrenergic receptors partly attenuated NE effects on coagulation and could be implied to have preventive potential in susceptible individuals.

Rosuvastatin use improves measures of coagulation in patients with venous thrombosis.

Aims: Observational studies indicate that statins reduce the risk of recurrent venous thrombosis (VT). However, trials have not been performed and the mechanism is unknown. We aimed to determine whether statin therapy improves the coagulation profile in patients with prior VT. Methods and results: Randomized clinical trial (NCT01613794). Patients were randomized to rosuvastatin 20 mg/day for 4 weeks or no intervention. Blood was drawn at baseline and at end of study. The primary outcome was factor (F) VIII:C. In total, five coagulation factors were measured: FVIII:C, von Willebrand factor:Ag, FVII:C, FXI:C, and D-dimer. Among 247 randomized participants, mean age was 58 years, 62% were women and 49% had unprovoked VT. For all tested coagulation factors, mean levels were clearly decreased at end of study in rosuvastatin users, whereas they hardly differed in non-statin users. Results were most consistent for FVIII:C where mean FVIII:C levels were 7.2 IU/dL [95% CI (confidence interval) 2.9-11.5] lower in rosuvastatin users, while among non-users, no change in FVIII:C was observed (mean difference -0.1; 95% CI -3.0 to 2.9). The mean age and sex adjusted difference in FVIII:C change was -6.7 IU/dL (95% CI -12.0 to -1.4) in rosuvastatin users vs. non-users. Subgroup analyses revealed that the decrease in coagulation factors by rosuvastatin was more pronounced in participants with unprovoked VT and in those with cardiovascular risk factors. Conclusion: Rosuvastatin 20 mg/day substantially improved the coagulation profile among patients with prior VT. These results suggest that statin therapy might be beneficial in patients at risk of recurrent VT.

Effect of Thrombomodulin Alfa on Disseminated Intravascular Coagulation in Patients with Lung Cancer.

Objective The mortality rate due to disseminated intravascular coagulation (DIC) is higher in patients with lung cancer than in those without. We examined the effect of treatment with thrombomodulin alfa (TM-α) for DIC in lung cancer patients. Methods Subjects were 57 patients with DIC (43 men, 14 women; mean age, 71.7 years), comprising 31 with lung cancer and 26 without. DIC patients with or without lung cancer did not differ significantly in their background characteristics. Results No significant difference was noted in the mortality rate between patients with lung cancer (61.3%) and those without (57.7%). However, the dose of TM-α was higher for survivors with lung cancer than for non-survivors (473.1 U/kg/day vs. 380.6 U/kg/day; p<0.01). Although no significant difference was noted in the DIC score between these four groups, the serum C-reactive protein level (6.9 mg/dL vs. 11.6 mg/dL; p<0.05) and prothrombin time-international normalized ratio (PT-INR; 1.10 vs. 1.52; p<0.05) were lower in survivors with lung cancer than in the non-survivors with lung cancer. The initial body temperature in non-survivors without lung cancer was lower than that in survivors without lung cancer (37.2°C vs. 37.9°C, p<0.01), and the platelet count and the time to recovery from DIC in patients without lung cancer showed a significant negative correlation (r2=0.438, p<0.05). Conclusion Our findings suggest that although 380 U/kg/day of TM-α is the recommended dose for DIC treatment, a higher dose may reduce the mortality rate of lung cancer patients with DIC. Furthermore, TM-α should be initiated before worsening of DIC parameters.

A Joint Model for Vitamin K-Dependent Clotting Factors and Anticoagulation Proteins.

BACKGROUND: Warfarin acts by inhibiting the reduction of vitamin K (VK) to its active form, thereby decreasing the production of VK-dependent coagulation proteins. The aim of this research is to develop a joint model for the VK-dependent clotting factors II, VII, IX and X, and the anticoagulation proteins, proteins C and S, during warfarin initiation. METHODS: Data from 18 patients with atrial fibrillation who had warfarin therapy initiated were available for analysis. Nine blood samples were collected from each subject at baseline, and at 1-5, 8, 15 and 29 days after warfarin initiation and assayed for factors II, VII, IX and X, and proteins C and S. Warfarin concentration-time data were not available. The coagulation proteins data were modelled in a stepwise manner using NONMEM® Version 7.2. In the first stage, each of the coagulation proteins was modelled independently using a kinetic-pharmacodynamic model. In the subsequent step, the six kinetic-pharmacodynamic models were combined into a single joint model. RESULTS: One patient was administered VK and was excluded from the analysis. Each kinetic-pharmacodynamic model consisted of two parts: (1) a common one-compartment pharmacokinetic model with first-order absorption and elimination for warfarin; and (2) an inhibitory E max model linked to a turnover model for coagulation proteins. In the joint model, an unexpected pharmacodynamic lag was identified and the estimated degradation half-life of VK-dependent coagulation proteins were in agreement with previously published values. The model provided an adequate fit to the observed data. CONCLUSION: The joint model represents the first work to quantify the influence of warfarin on all six VK-dependent coagulation proteins simultaneously. Future work will expand the model to predict the influence of exogenously administered VK on the time course of clotting factor concentrations after warfarin overdose and during perioperative warfarin reversal procedures.

Endothelial protein C receptor-dependent antichemotactic effects of canine protein C.

OBJECTIVE To determine whether canine protein C (CnPC) had antichemotactic effects on canine neutrophils, whether endothelial protein C receptor (EPCR) was expressed on canine neutrophils, and the role of EPCR in neutrophil chemotaxis. SAMPLE Neutrophils isolated from blood samples from healthy dogs (n = 6) and sick dogs with (2) or without (3) an inflammatory leukogram. PROCEDURES Neutrophils were analyzed by reverse transcriptase PCR assay and flow cytometry for detection of EPCR mRNA and protein expression, respectively. Neutrophils were incubated with CnPC zymogen or canine activated protein C (CnAPC), with or without RCR-379 (an anti-human EPCR antibody). Neutrophils were then allowed to migrate through a filter membrane toward a chemokine. Untreated neutrophils served as positive control samples. Migration was quantified by fluorescence measurement, and chemotaxis index (Chx) values (fluorescence of test sample/fluorescence of positive control sample) were computed. RESULTS The cDNA for EPCR was amplified, and EPCR expression was detected on neutrophil surfaces. Obtained Chx values were significantly higher in cells treated with RCR-379 than in cells treated with CnPC or CnAPC alone. The Chx values for neutrophils treated with RCR-379 were not significantly different from 1, whereas those for neutrophils treated without RCR-379 were significantly less than 1. The effects of RCR-379 on neutrophil migration were independent of concentration or activation status of protein C. CONCLUSIONS AND CLINICAL RELEVANCE Canine neutrophils expressed EPCR, and inhibition of neutrophil chemotaxis by CnPC and CnAPC depended on EPCR. Interventions with EPCR signaling may have therapeutic application in dogs.

Protein disulfide isomerase inhibition blocks thrombin generation in humans by interfering with platelet factor V activation.

BACKGROUND: Protein disulfide isomerase (PDI) is required for thrombus formation. We previously demonstrated that glycosylated quercetin flavonoids such as isoquercetin inhibit PDI activity and thrombus formation in animal models, but whether extracellular PDI represents a viable anticoagulant target in humans and how its inhibition affects blood coagulation remain unknown. METHODS: We evaluated effects of oral administration of isoquercetin on platelet-dependent thrombin generation in healthy subjects and patients with persistently elevated anti-phospholipid antibodies. RESULTS: Following oral administration of 1,000 mg isoquercetin to healthy adults, the measured peak plasma quercetin concentration (9.2 µM) exceeded its IC50 for inhibition of PDI by isoquercetin in vitro (2.5 ± 0.4 µM). Platelet-dependent thrombin generation decreased by 51% in the healthy volunteers compared with baseline (P = 0.0004) and by 64% in the anti-phospholipid antibody cohort (P = 0.015) following isoquercetin ingestion. To understand how PDI affects thrombin generation, we evaluated substrates of PDI identified using an unbiased mechanistic-based substrate trapping approach. These studies identified platelet factor V as a PDI substrate. Isoquercetin blocked both platelet factor Va and thrombin generation with an IC50 of ~5 µM. Inhibition of PDI by isoquercetin ingestion resulted in a 53% decrease in the generation of platelet factor Va (P = 0.001). Isoquercetin-mediated inhibition was reversed with addition of exogenous factor Va. CONCLUSION: These studies show that oral administration of isoquercetin inhibits PDI activity in plasma and diminishes platelet-dependent thrombin generation predominantly by blocking the generation of platelet factor Va. These pharmacodynamic and mechanistic observations represent an important step in the development of a novel class of antithrombotic agents targeting PDI. TRIAL REGISTRATION: Clinicaltrials.gov (NCT01722669) FUNDING: National Heart, Lung, and Blood Institute (U54 HL112302) and Quercegen Pharma.

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.

Drospirenone as estrogen-free pill and hemostasis: coagulatory study results comparing a novel 4 mg formulation in a 24 + 4 cycle with desogestrel 75 µg per day.

INTRODUCTION: A novel estrogen free contraceptive pill, with drospirenone 4 mg in a dosing regimen of 24 + 4, has been developed with a pearl-index of 0.51 (95% CI 0.1054; 1.4922). The aim of the following study was to determine if 4 mg DRSP has an impact on coagulation factors and thrombotic risks in comparison with desogestrel 75 µg. PATIENTS AND METHODS: Thirty-nine patients received 4 mg DRSP 24 + 4 d and 29 desogestrel 75 µg per day continuously during nine complete cycles. Following hemostatic parameters were evaluated: Apc resistance, Antithrombin III, Protein C reactivity, Factor VII, Factor VIII, and d-Dimer. RESULTS: Factor VII decreased from 1.123 to 1.066 in the DRSP group and from 1.241 to 1.034 in the desogestrel group (p = 0.0088). The difference in change of mean Protein C activity from baseline to endpoint was -0.0332 in the DRSP versus -0.157 in the desogestrel group (p = 0.0249). d-Dimer values dropped in the DRSP group from baseline values of 264.9-215.0 ng/mL, whereas in the desogestrel group there was a rise from 201.4 ng/mL to 281.5 ng/mL. DISCUSSION: DRSP 4 mg was not associated with any meaningful changes on hemostatic parameters, indicating a lack of effect on hemostasis.

Patient Management with Metallic Valve Prosthesis during Pregnancy and Postpartum Period.

Prosthetic thrombosis is a rare complication, but it has high mortality and morbidity. Young women of childbearing age that have prosthetic heart valves are at increased risk of thrombosis during pregnancy due to changes in coagulation factors. Anticoagulation with adequate control and frequent follow-up if pregnancy occurs must be performed in order to prevent complications related to anticoagulant use. Surgery remains the treatment of choice for prosthetic heart valve thrombosis in most clinical conditions. Patients with metallic prosthetic valves have an estimated 5% risk of thrombosis during pregnancy and maternal mortality of 1.5% related to the event. Anticoagulation with vitamin K antagonists during pregnancy is related to varying degrees of complications at each stage of the pregnancy and postpartum periods. Warfarin sodium crosses the placental barrier and when used in the first trimester of pregnancy is a teratogenic agent, causing 1-3% of malformations characterized by fetal warfarin syndrome and also constitutes a major cause of miscarriage in 10-30% of cases. In the third trimester and at delivery, the use of warfarin is associated with maternal and neonatal bleeding in approximately 5 to 15% of cases, respectively. On the other hand, inadequate anticoagulation, including the suspension of the oral anticoagulants aiming at fetal protection, carries a maternal risk of about 25% of metallic prosthesis thrombosis, particularly in the mitral valve. This fact is also due to the state of maternal hypercoagulability with activation of coagulation factors V, VI, VII, IX, X, platelet activity and fibrinogen synthesis, and decrease in protein S levels. The Registry of Pregnancy and Cardiac Disease (ROPAC), assessing 212 pregnant women with metal prosthesis, showed that prosthesis thrombosis occurred in 10 (4.7%) patients and maternal hemorrhage in 23.1%, concluding that only 58% of patients with metallic prosthesis had a complication-free pregnancy.

New Insights into Nonvitamin K Antagonist Oral Anticoagulants' Reversal of Intracerebral Hemorrhage.

The nonvitamin K antagonist oral anticoagulants (NOACs) dabigatran, rivaroxaban, apixaban, and edoxaban are associated with an equal or lower incidence of stroke and systemic embolism and a much lower incidence of intracranial hemorrhage and hemorrhagic stroke than warfarin is, without the need for routine laboratory monitoring. However, reversal strategies are not currently established in the case of NOAC-related hemorrhagic stroke. In emergency situations, well-defined management for NOAC-related hemorrhagic stroke may improve clinical outcomes. Thus, in this chapter, general measures initially required to prevent the expansion of intracerebral hematomas, charcoal administration to reduce NOAC absorption from the gastrointestinal tract, application of hemodialysis to remove dabigatran, and coagulation factor therapy including 4-factor prothrombin complex concentrate and recombinant activated factor VII are reviewed. The specific reversal agents idarucizumab, which is a monoclonal antibody against dabigatran; andexanet alfa, a recombinant human factor Xa decoy for Xa inhibitors; and PER977, a small synthetic molecule for reversal of both Xa and thrombin inhibitors, are currently under development. These agents will facilitate the clinical management of NOAC-associated hemorrhagic stroke and other severe bleeding.

Maggot excretion products from the blowfly Lucilia sericata contain contact phase/intrinsic pathway-like proteases with procoagulant functions.

For centuries, maggots have been used for the treatment of wounds by a variety of ancient cultures, as part of their traditional medicine. With increasing appearance of antimicrobial resistance and in association with diabetic ulcers, maggot therapy was revisited in the 1980s. Three mechanisms by which sterile maggots of the green bottle fly Lucilia sericata may improve healing of chronic wounds have been proposed: Biosurgical debridement, disinfecting properties, and stimulation of the wound healing process. However, the influence of maggot excretion products (MEP) on blood coagulation as part of the wound healing process has not been studied in detail. Here, we demonstrate that specific MEP-derived serine proteases from Lucilia sericata induce clotting of human plasma and whole blood, particularly by activating contact phase proteins factor XII and kininogen as well as factor IX, thereby providing kallikrein-bypassing and factor XIa-like activities, both in plasma and in isolated systems. In plasma samples deficient in contact phase proteins, MEP restored full clotting activity, whereas in plasma deficient in either factor VII, IX, X or II no effect was seen. The observed procoagulant/intrinsic pathway-like activity was mediated by (chymo-) trypsin-like proteases in total MEP, which were significantly blocked by C1-esterase inhibitor or other contact phase-specific protease inhibitors. No significant influence of MEP on platelet activation or fibrinolysis was noted. Together, MEP provides contact phase bypassing procoagulant activity and thereby induces blood clotting in the context of wound healing. Further characterisation of the active serine protease(s) may offer new perspectives for biosurgical treatment of chronic wounds.

Impact of aliskiren on some hemostatic parameters in experimental arterial thrombosis in rats.

BACKGROUND: Aliskiren is the first orally active inhibitor of renin to be approved for clinical use as an antihypertensive agent. A number of studies show a link between aliskiren and intravascular thrombosis. MATERIALS AND METHODS: The goal of the present study was to investigate the impact of aliskiren on arterial thrombosis in normotensive and renovascular hypertensive rats. The contribution of each coagulation and fibrinolytic parameters in the mode of aliskiren action was determined. Six weeks after clipping of the left renal artery rats developed hypertension which was confirmed by the "tail cuff" method. Animals were treated with aliskiren (10, 30 and 100mg/kg/day) per os for 10 days. Arterial thrombosis was induced by electrical stimulation of the common carotid artery. RESULTS: It was found that aliskiren in a dose-dependent manner decreased weight of the arterial thrombus in normotensive and hypertensive rats. It has been shown that this result was not associated with the effects on blood pressure, TF, PT, APTT, fibrinogen and hematological parameters. It was found that aliskiren caused increase of t-PA activity and decrease of its inhibitor activity. CONCLUSIONS: The presented results indicate that aliskiren inhibits hemostasis in the arterial thrombosis in rats. The antithrombotic effect is related with improvement of the fibrinolytic balance, and also depends on antiplatelet action.

Exendin-4 inhibits endothelial protein C receptor shedding in vitro and in vivo.

Exendin-4 (EX4), a glucagon-like peptide-1 receptor agonist, has been reported to attenuate myocardial ischemia and reperfusion (I/R) injury, inflammatory and oxidative responses. Increasing evidence has demonstrated that beyond its role in activation of protein C, endothelial cell protein C receptor (EPCR) is involved in vascular inflammation. EPCR activity is markedly decreased by ectodomain cleavage and release as the soluble EPCR. EPCR can be shed from the cell surface, which is mediated by tumor necrosis factor-α converting enzyme (TACE). However, little is known about the effects of EX4 on EPCR shedding. Data from this study showed that EX4 induced potent inhibition of phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, and interleukin (IL)-1ß-induced EPCR shedding in human umbilical vein endothelial cells (HUVECs), and cecal ligation and puncture (CLP)-induced EPCR shedding in mice. EX4 also inhibited expression and activity of TACE induced by PMA in HUVECs. In addition, treatment with EX4 resulted in reduced PMA-stimulated phosphorylation of p38, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK). These results demonstrate the potential of EX4 as an anti-sEPCR shedding reagent against PMA and CLP-mediated EPCR shedding.