Site-directed mutagenesis of tissue factor pathway inhibitor-binding exosite D60A on factor VII results in a new factor VII variant with lower coagulant activity.

Background: Recombinant factor (F)VIIa (rFVIIa) has been approved by the US Food and Drug Administration for the treatment of hemophilia A and B with inhibitors and congenital FVII deficiency. Moreover, the investigational uses of rFVIIa are becoming of interest since it can be used to treat various clinical bleeding conditions. However, there is evidence showing that rFVIIa is a potent procoagulant agent that potentially leads to an increased risk of thrombotic complications. Objectives: To design a new rFVII with lower coagulant activity that could potentially be used as an alternative hemostatic agent aiming to minimize the risk of thrombogenicity. Methods: D60A was introduced into the F7 sequence by polymerase chain reaction-based mutagenesis. Wild type (WT) and D60A were generated in human embryonic kidney 293T cells by stable transfection. FVII coagulant activities were determined by amidolytic cleavage of the FVIIa-specific substrate, 2-step FXa generation, thrombin generation (TG), and clot-based assays. Results: WT and D60A demonstrated similar FVIIa amidolytic activity. However, D60A showed approximately 50% activity on FX activation and significantly longer lag time in the TG assay than that shown by WT. The clotting time produced by D60A spiked in FVII-deficient plasma was significantly prolonged than that of WT. Additionally, the ex vivo plasma half-lives of WT and D60A were comparable. Conclusion: D60A demonstrated lower coagulant activities, most likely due to the weakening of FX binding, leading to impaired FX activation and delayed TG and fibrin formation. Considering that a plasma FVII level of 15% to 25% is adequate for normal hemostasis, D60A is a molecule of interest for future development of an rFVII with a lesser extent of thrombogenicity.

The Finding of New In Vivo Metabolite Triptorelin (5-10) in Human Urine Using Liquid Chromatography Coupled with Ion Trap/Time-of-Flight Mass Spectrometry with Dimethyl Sulfoxide Additives in the Mobile Phase.

Triptorelin and leuprorelin are synthetic gonadotrophin-releasing hormones (GnRH) that are on the World Anti-Doping Agency (WADA) list of prohibited substances. To investigate the possible in vivo metabolites of triptorelin and leuprorelin in humans compared to previously reported in vitro metabolites, excreted urine from five patients treated with either triptorelin or leuprorelin was analyzed by liquid chromatography coupled with ion trap/time-of-flight mass spectrometry (LC/MS-IT-TOF). The addition of dimethyl sulfoxide (DMSO) to the mobile phase was found to enhance the detection sensitivity of certain GnRH analogs. The method was validated, and the limit of detection (LOD) was found at 0.02-0.08 ng/mL. Using this method, a novel new metabolite of triptorelin was discovered in the urine of all subjects up to 1 month after triptorelin administration, but it was not observed in the urine of subjects before drug administration. The limit of detection was estimated to be 0.05 ng/mL. The structure of the metabolite, triptorelin (5-10), is proposed from bottom-up mass spectrometry analysis. The discovery of in vivo triptorelin (5-10) can possibly be used as supporting evidence of triptorelin misuse in athletes.

C1 inhibitor deficiency enhances contact pathway-mediated activation of coagulation and venous thrombosis.

C1 inhibitor (C1INH) is a multifunctional serine protease inhibitor that functions as a major negative regulator of several biological pathways, including the contact pathway of blood coagulation. In humans, congenital C1INH deficiency results in a rare episodic bradykinin-mediated swelling disorder called hereditary angioedema (HAE). Patients with C1INH deficiency-associated HAE (C1INH-HAE) have increased circulating markers of activation of coagulation. Furthermore, we recently reported that patients with C1INH-HAE had a moderate but significant increased risk of venous thromboembolism. To further investigate the impact of C1INH deficiency on activation of coagulation and thrombosis, we conducted studies using patient samples and mouse models. Plasmas from patients with C1INH-HAE had significantly increased contact pathway-mediated thrombin generation. C1INH-deficient mice, which have been used as a model of C1INH-HAE, had significantly increased baseline circulating levels of prothrombin fragment 1+2 and thrombin-antithrombin complexes. In addition, whole blood from C1INH-deficient mice supported significantly increased contact pathway-mediated thrombin generation. Importantly, C1INH-deficient mice exhibited significantly enhanced venous, but not arterial, thrombus formation. Furthermore, purified human C1INH normalized contact pathway-mediated thrombin generation and venous thrombosis in C1INH-deficient mice. These findings highlight a key role for endogenous C1INH as a negative regulator of contact pathway-mediated coagulation in humans and mice. Further, this work identifies endogenous C1INH as an important negative regulator of venous thrombus formation in mice, complementing the phenotype associated with C1INH-HAE.

A novel mouse whole blood thrombin generation assay sensitive to FXI- and FIX-mediated amplification of coagulation.

Thrombin generation (TG) assays serve as a valuable tool to study the amplifying roles of intrinsic pathway factors in human coagulation and provide functional insights into the increased bleeding observed in individuals deficient in factors (F) XI, IX, or VIII. Mice are used extensively in hemostasis research owing to the availability of coagulation factor-deficient mice. However, phenotypic differences between mouse and human TG have become apparent. In this study, we describe a novel, calibrated mouse whole blood (WB) TG assay used to assess the amplifying roles of intrinsic pathway factors in mouse coagulation. WB- and plasma-TG was triggered with either silica or tissue factor (TF) in samples from wild-type mice and mice deficient for FXII, FXI, or FIX. Expectedly, silica-triggered WB-TG and platelet-poor plasma (PPP)-TG were significantly reduced by deficiencies for FXII, FXI, or FIX. FXII deficiency had no effect on WB-TG or PPP-TG when triggered with TF. However, FXI deficiency resulted in significantly reduced WB-TG triggered by low concentrations of TF but had no effect on TF-triggered PPP-TG. FIX deficiency profoundly reduced WB-TG when triggered by low or high concentrations of TF whereas TG in PPP or platelet-rich plasma was only moderately reduced under these conditions. In conclusion, we have developed a novel mouse WB-TG assay with enhanced sensitivity to FXI- and FIX-dependent amplification of coagulation compared with an established plasma-TG assay. The enhanced sensitivity of WB-TG to FXI and FIX-dependent amplification of coagulation suggests an important role of blood cells in this process.

The role of topical capsaicin gel in pain management during microfocused ultrasound treatment for neck laxity.

BACKGROUND: The transient receptor potential vanilloid 1 (TRPV1) provides a heat and pain sensation (nociception). Capsaicin, a TRPV1 agonist, has been shown to induce a refractory period in the nerve terminal expressing TRPV1 and create long-term nerve terminal defunctionalization. OBJECTIVE: To evaluate the efficacy of capsaicin for pain reduction during microfocused ultrasound with visualization (MFU-V) treatment. METHODS AND MATERIALS: A randomized, split-side study including 24 subjects was conducted. A combined 0.025% capsaicin gel and topical anesthetic were randomly applied on one side of the neck, and a topical anesthetic monotherapy was applied on the contralateral side for 30 min before MFU-V treatment. Pain score (visual analog scale, 0-10) was evaluated at T1 (before MFU-V), T2a (after the 4.5-mm transducer treatment), T2b (after the 3.0-mm transducer treatment), and T3 (after the entire treatment). Side effects were recorded. RESULTS: Mean pain scores at T2a for combined and single regimens were 5.19 (±2.26) and 6.91 (±1.72), respectively (p < 0.001). The capsaicin-treated side had a lower pain score at T2b and T3 (p < 0.001). Redness was longer on the capsaicin-treated side (112.67 vs. 10.68 min, p < 0.001). No other adverse events including contact dermatitis were reported. CONCLUSION: A single application of a combined 0.025% capsaicin gel with topical anesthesia produces a significantly lesser pain score during the MFU-V treatment. Defunctionalization of TRPV1 may explain the alleviation of painful sensations caused by heat from MFU-V.

Mutations of TFPI-binding exosites on factor VII cause bleeding phenotypes in factor VII deficiency.

Tissue factor (TF) pathway inhibitor (TFPI) is a Kunitz-type anticoagulation protein that inhibits activated factor VII (FVIIa)/TF complex. Incidentally, many different F7 gene variants, including TFPI-binding exosite mutations, have been reported in patients with congenital FVII deficiency and clinical bleeding variabilities. Here, TFPI-binding exosites (R147 and K192) on FVII zymogen were selectively disrupted to understand their roles in the pathogenesis of bleeding phenotypes. Expression of recombinant FVII variants (R147A, K192A, and R147A/K192A) demonstrated markedly reduced secretion of FVII owing to intracellular retention in the endoplasmic reticulum, as demonstrated by upregulation of the unfolded protein response genes in all FVII variants. FVII variants showed a similar FVII activation pattern and FVIIa amidolytic activity than FVII wild-type (WT). In contrast to FVII activation, R147A and K192A showed a 90% reduction in FX activation relative to WT, whereas the R147A/K192A variant demonstrated a 99% decrease in FX activation. The clotting time was markedly prolonged with R147A and K192A than WT, and no FVII coagulant activity was detected in R147A/K192A. In addition, the thrombin generation assay revealed a significant prolongation of lag time in all FVII variants. Our study explains how mutations of TFPI-binding exosites of FVII can lead to bleeding phenotypes in individuals carrying these aberrancies.

Coagulant activity of recombinant human factor VII produced by lentiviral human F7 gene transfer in immortalized hepatocyte-like cell line.

Human mesenchymal stem cells (hMSCs) have the potential to differentiate into hepatocyte-like cells, indicating that these cells may be the new target cell of interest to produce biopharmaceuticals. Our group recently established a hMSC-derived immortalized hepatocyte-like cell line (imHC) that demonstrates several liver-specific phenotypes. However, the ability of imHC to produce coagulation factors has not been characterized. Here, we examined the potential for imHC as a source of coagulation protein production by investigating the ability of imHC to produce human factor VII (FVII) using a lentiviral transduction system. Our results showed that imHC secreted a low amount of FVII (~22 ng/mL) into culture supernatant. Moreover, FVII from the transduced imHC (0.11 ± 0.005 IU/mL) demonstrated a similar coagulant activity compared with FVII from transduced HEK293T cells (0.12 ± 0.004 IU/mL) as determined by chromogenic assay. We demonstrate for the first time, to the best of our knowledge, that imHC produced FVII, albeit at a low level, indicating the unique characteristic of hepatocytes. Our study suggests the possibility of using imHC for the production of coagulation proteins.

Elevated Plasma Factor IXa Activity in Premenopausal Women on Hormonal Contraception.

OBJECTIVE: Combined oral contraceptives induce a reversible hypercoagulable state with an enhanced risk of venous thromboembolism, but the underlying mechanism(s) remain unclear. Subjects on combined oral contraceptives also demonstrate a characteristic resistance to APC (activated protein C) in the thrombin generation assay. Here, we report the potential role of plasma factor IXa (FIXa) as a mechanism for hormone-induced systemic hypercoagulability. APPROACH AND RESULTS: A novel assay was used to determine FIXa activity in plasma samples from volunteer blood donors. Plasma from 36 premenopausal females on hormonal contraception and 35 not on hormonal contraception, 35 postmenopausal females, and 10 males were analyzed for FIXa activity, total PS (protein S), total tissue factor pathway inhibitor (TFPI), and TFPI-α antigen. Premenopausal females on hormonal contraception demonstrated significantly increased FIXa activity and decreased TFPI-α compared with the other groups. Remarkably, FIXa values were not normally distributed in the hormonal contraception group, but skewed toward the high end. Plasma FIXa activity inversely correlated with both TFPI-α and total PS antigen. Ex vivo determination of TF-dependent FIX activation in FV-deficient plasma demonstrated that inhibitory anti-TFPI antibodies enhanced FIXa generation by 2- to 3-fold, whereas addition of 75 nmol/L PS reduced FIXa generation by ≈2-fold. Further, increasing FIXa concentration enhanced APC resistance during TF-triggered plasma thrombin generation. CONCLUSIONS: Elevation of plasma FIXa activity in association with reductions in TFPI-α and PS is a potential mechanism for systemic hypercoagulability and resistance to APC in premenopausal females on hormonal contraception.

Risk of venous thromboembolism in patients with celiac disease: A systematic review and meta-analysis.

BACKGROUND AND AIM: Patients with celiac disease (CD) might be at an increased risk of developing venous thromboembolism (VTE) due to chronic inflammation and vitamin deficiency. However, epidemiologic studies attempting to investigate this risk have yielded inconsistent results. We conducted this meta-analysis with the aims to better characterize this possible association. METHODS: We conducted a systematic review and meta-analysis of observational studies that reported odds ratio, relative risk, hazard ratio, or standardized incidence ratio comparing the risk of VTE in patients with CD versus participants without CD. Generic inverse variance method of DerSimonian and Laird was used to calculate the pooled risk ratio. RESULTS: Out of 279 potentially relevant articles, four studies met the inclusion criteria and were included in the meta-analysis. We found a statistically significant increased risk of VTE among patients with CD with the pooled risk ratio of 1.25 (95% confidence interval, 1.02-1.53). The statistical heterogeneity was moderate with an I(2) of 69%. CONCLUSIONS: A significantly increased risk of VTE among patients with CD was demonstrated in this meta-analysis. Further studies are required to clarify how this risk should be addressed in clinical practice.

Autoimmune hemolytic anemia and venous thromboembolism: A systematic review and meta-analysis.

OBJECTIVE: To investigate the risk of venous thromboembolism (VTE) among patients with autoimmune hemolytic anemia (AIHA). METHODS: We conducted a systematic review and meta-analysis of observational studies that reported odds ratio, relative risk, hazard ratio or standardized incidence ratio comparing the risk of VTE in patients with AIHA versus participants without AIHA. Pooled risk ratio and 95% confidence intervals were calculated using a random-effect, generic inverse variance method of DerSimonian and Laird. RESULTS: Out of 592 potentially relevant articles, four studies (three cohort studies and one cross-sectional study) met our inclusion criteria and were included in the data analysis. The pooled risk ratio of VTE in patients with AIHA was 2.63 (95% CI, 1.37-5.05). The statistical heterogeneity of this study was high with an I(2) of 97%. CONCLUSIONS: Our study demonstrated a significantly increased risk of VTE among patients with AIHA.

Trend of human rabies prophylaxis in developing countries: toward optimal rabies immunization.

Rabies is a fatal infectious disease. Because prevention is the key management for rabies, many vaccination regimens have been developed and used worldwide. The aims for developing rabies vaccination regimens include decreasing the number and amount of dosages, decreasing the duration and the number of clinical visits, and reducing cost. Interestingly, some intradermal (ID) regimens have proved to be as effective as the standard intramuscular (IM) regimens, and have been increasingly used in developing countries because they are less expensive. In this article, we reviewed rabies vaccines based on results obtained from clinical trials and international treatment guidelines for post-exposure prophylaxis, pre-exposure prophylaxis for the high risk group, and booster vaccination.

Decreased nitrite levels in erythrocytes of children with ß-thalassemia/hemoglobin E.

Nitrite anion is bioactive nitric oxide (NO) species circulating in blood, and represents the NO bioavailability and endothelial function. In this study, we aimed to investigate the nitrite levels and the correlation with hemolysis and severity in ß-thalassemia/hemoglobin E (ß-thal/HbE). 38 Children (12.0±1.9 years of age) with a diagnosis of mild, moderate and severe ß-thalassemia were enrolled in the study. The blood nitrite levels and potential plasma NO consumption were measured by the chemiluminescence method. The nitrite levels in whole blood and erythrocytes of the severe thalassemia subjects were lower than those of the control subjects. At day 7 after transfusion of packed erythrocytes, the nitrite levels in erythrocytes increased. The plasma hemoglobin and NO consumption increased in the severe thalassemia subjects. The nitrite levels in erythrocytes inversely correlated with plasma hemoglobin, lactate dehydrogenase activity, potential NO consumption, and lipid peroxidation. Our studies demonstrate the decreased NO bioavailability in thalassemia, which could result from endothelial dysfunction, the increased potential NO consumption in plasma by cell-free hemoglobin and oxidative stress.

Oxidative modification and poor protective activity of HDL on LDL oxidation in thalassemia.

Oxidative modification of low-density lipoprotein (LDL) has been reported in thalassemia, which is a consequence of oxidative stress. However, the levels of oxidized high-density lipoprotein (HDL) in thalassemia have not been evaluated and it is unclear whether HDL oxidation may be linked to LDL oxidation. In this study, the levels of total cholesterol, iron, protein, conjugated diene (CD), lipid hydroperoxide (LOOH), and thiobarbituric acid reactive substances (TBARs) were determined in HDL from healthy volunteers and patients with beta-thalassemia intermedia with hemoglobin E (beta-thal/Hb E). The protective activity of thalassemic HDL on LDL oxidation was also investigated. The iron content of HDL(2) and HDL(3) from beta-thal/HbE patients was higher while the cholesterol content was lower than those in healthy volunteers. Thalassemic HDL(2) and HDL(3) had increased levels of lipid peroxidation markers i.e., conjugated diene, LOOH, and TBARs. Thalassemic HDL had lower peroxidase activity than control HDL and was unable to protect LDL from oxidation induced by CuSO(4). Our findings highlight the oxidative modification and poor protective activity of thalassemic HDL on LDL oxidation which may contribute to cardiovascular complications in thalassemia.