Perioperative bleeding is a major concern in patients with factor VII (FVII) deficiency. Evaluating data of 95 FVII-deficient patients undergoing 110 surgical procedures (61 major, 49 minor), we assessed the impact of type of surgery, bleeding phenotype and FVII coagulant activity (FVII:C) levels on perioperative replacement therapy (RT). Compared to those with higher FVII:C levels, patients with <3% FVII:C received a higher number of RT doses (8 vs. 2, P = 0·003) for a longer RT duration (3 days vs. 1 day, P = 0·001), with no difference in RT dose. Similarly, patients with a history of major bleeds received a higher number of RT doses (8·5 vs. 2-3, P = 0·013) for a longer RT duration (2 days vs. 1 day, P = 0·005) as compared to those with a history of minor bleeds or to asymptomatic patients. No difference in RT was found among major and minor surgical procedures. Overall, multivariate analysis showed that history of major bleeding was the only independent predictor of number of RT doses (β = 0·352, P = 0·001) and RT duration (β = 0·405, P = 0·018). Overall, a ≈20 μg/kg perioperative RT was efficacious in 95·5% of cases. The infusion should be repeated ≈8 times in high-risk subsets (i.e. patients with a history of major bleeding).

Twenty-eight patients were followed up for potential specific antibody formation after repeated use of recombinant factor VIIa (rFVIIa). The population included 27 patients with congenital bleeding disorders and 1 nonhemophiliac with an FVIII inhibitor. From 5 to 77 bleeding episodes were treated during a follow-up period of at least 5 months. None of these repeatedly treated patients showed signs of antibody formation against FVII or foreign protein that could be related to the treatment.

BACKGROUND

This study compared the efficacy of Aryoseven with Novoseven to control bleeding episodes in patients with hemophilia A with inhibitors.

METHODS

Sixty-six patients were randomized into 2 groups, with 4 consecutive block randomization. These groups received Aryoseven and Novoseven dosages of 90 to 120 μg/kg intravenously every 2 hours.

RESULTS

Median (interquartile range) level of factor VIII (FVIII) inhibitor in groups A and B was 15.0 and 19.0 Bethesda Unit (BU) preadministration. Bleeding onset in group A was 1246 ± 1104 minutes and in group B was 2301 ± 1693 minutes (P = .311). The Kavakli global response scores and treatment success rate was comparable in both the groups. The side effects in groups A (9.7%) and B (2.9%) were comparable.

CONCLUSIONS

Biosimilar recombinant activated FVII is found to be as effective as Novoseven in the treatment of acute joint bleeding in patients with hemophilia with inhibitors. Its usage will decrease the gaps in hemophilia.

While certain plasma-derived factor VIII/von Willebrand factor (FVIII/vWF) concentrates have proven to be quite useful in preventing or controlling bleeding in persons with von Willebrand disease who are not candidates for DDAVP, most of the information concerning dosage and effectiveness has been anecdotal. Additionally, the laboratory tests used to quantify vWF (the vWF:RCoF assay and collagen binding assay) are not well standardized. Thus, the US Food and Drug Administration (FDA) has been reluctant to grant licensed indication for use of these products in von Willebrand disease. This brief report describes a survey of non-US physicians (from major centres) who are recognized experts in haemophilia and von Willebrand disease. Twenty-four of 27 questionnaires were completed, returned and analysed. Products thought to be effective in von Willebrand disease included Haemate P, Facteur von Willebrand, Alphanate, and the UK's BPL '8Y'. In calculating dosage to stop or prevent bleeding, most aim for a certain level of both FVII and vWF:RCoF. Postoperatively, 16/24 respondents follow both of these laboratory tests once daily. Twenty-two of 24 would follow FVIII levels once daily. It is noteworthy that FVIII assay results are generally the only test results readily available to guide the respondents' clinical decisions. For treatment of significant mucous membrane bleeding, respondents often individualize dosage and monitoring, depending on the type, location and extent of bleeding. Gastrointestinal bleeding is generally treated more aggressively. Patient monitoring varies between merely looking for cessation of bleeding, to a battery of laboratory tests, including haemoglobin and haematocrit. Seven out of 24 would monitor BT as well. In addition to FVIII content, 22/24 respondents noted that they would find it helpful to have vWF:RCoF listed on the label, while 15/24 would like to know the vWF multimeric composition.

Autoimmune thrombocytopenic purpura (ITP) is a disease that presents with skin and mucous membrane bleeding due to thrombocytopenia. In the literature, there are a few studies about the effect of high-dose steroid therapy on coagulation tests in different diseases, but their results are still controversial. In this study, coagulation parameters were investigated that might have a role in hemostatic compensation in childhood acute ITP before and after high-dose methylprednisolone (HDMP) treatment. The study includes 21 children age 1.5 to 14 years with acute ITP and 21 healthy age-matched control subjects. All patients with acute ITP received HDMP for 7 days. Before and after HDMP treatment (0 and 8 days) prothrombin time, partial thromboplastin time, fibrinogen, Protein C, Protein S, antithrombin III, and the levels of factor II (FII), FV, FVII, FVIII, FIX, FX, FXI, and FXII were studied in all subjects. The results were compared with those of the control group. Pre-treatment Protein C and Protein S levels in the patient group were significantly lower than those in the control groups (p<0.05). Protein S and Protein C levels were significantly improved after HDMP treatment in patient group. There were lower FV, FVII, FX values in the patient group compared to the control groups on admission. There was no difference in AT III and fibrinogen levels before and after treatment. As a result, some changes in the coagulation system associated with thrombocytopenia were observed in patients with acute ITP. These changes may be accepted as compensatory mechanisms to maintain hemostasis.

An adequate classification of congenital bleeding disorders is of great importance in clinical practice. This is true also for factor X (FX) deficiency. This defect is classified in two forms: type I (cases with low activity and antigen) and type II (cases with low activity and variable levels of antigen). The introduction of molecular biology techniques has allowed a classification based on the site of mutation (propeptide, Gla-domain, catalytic domain etc.) or on the type of mutation (missense, nonsense, deletion etc.). However, with a partial exception for defects in the Gla-domain, no site or type of mutation yields a constant and/or typical phenotype. Due to these difficulties, a classification based on clotting, chromogenic or immunological assays is still the most suited for clinical purposes. A satisfactory classification that takes into account recent advances of FX deficiency could read today as follows: • Type I (cross-reacting material (CRM) negative) (Stuart like) • Type II (CRM positive with inert protein) (Prower like) • Type III (CRM positive with disreactive protein) 1. Defects in all activity systems but for RVV activation (Friuli like) 2. Defects only or predominantly in the extrinsic-Xase system (Padua like) 3. Defects only or predominant in the intrinsic-Xase system (Melbourne like) 4. Defects with discrepant (high) chromogenic assays. Finally, type IV should be added to include cases of FX deficiency associated with FVII deficiency usually due to chromosome 13 abnormalities. By using this nosographic approach, all reported cases of FX deficiency can be adequately allocated to one of these groups.

OBJECTIVE

Despite aggressive measures to miniaturize the cardiopulmonary bypass (CPB) circuit in neonates and infants, the CPB prime volume is often at least as large as the patients' blood volume. We conducted an observational study to characterize the hemostatic consequences of a CPB prime consisting of either non-fresh or reconstituted whole blood.

METHODS

Hematocrit, fibrinogen, platelet count, plasminogen, anti-thrombin III (AT-III), and factors (F) II, V, VII, IX, and X of 30 neonates and infants undergoing cardiac surgery with CPB utilizing either a non-fresh or reconstituted whole blood prime were prospectively evaluated at eight time points. Following protamine administration, microvascular bleeding was treated by protocol.

RESULTS

The hemostatic composition of the CPB prime was the same following the use of either non-fresh or reconstituted whole blood. The CPB prime platelet count (mean +/- SD) was 5.87 +/- 2.84 x 10(3) microl(-1) when compared to a preoperative platelet count of 298 +/- 142 x 10(3) microl(-1) (P < 0.0001). Twenty patients received 17.3 +/- 9.2 ml x kg(-1) (0.86 +/- 0.46 units x kg(-1)) of platelets with significant improvement in platelet count. Nine patients received 16.7 +/- 13.4 ml x kg(-1) (0.84 +/- 0.67 units x kg(-1)) of cryoprecipitate with significant improvements in FVIII and fibrinogen.

CONCLUSIONS

Non-fresh or reconstituted whole blood as a component of a small volume CPB prime in neonates and infants induces clinically significant dilutional thrombocytopenia in conjunction with less significant reductions in fibrinogen, FII, FV, FVII, FVIII, FIX, FX, plasminogen, and AT-III.

Colon cancer is the third most common cancer in the world. The overexpression of tissue factor (TF) in colon cancer cells makes it an ideal target for colon cancer therapy. The purpose of the present study was to develop a TF-targeting energized fusion protein, mlFVII-LDP-AE, which is composed of a mouse Factor VII light chain (mlFVII) as the targeting domain conjugated to the highly cytotoxic antibiotic lidamycin (LDM, LDP-AE) as the effector domain. The potential efficacy of mlFVII-LDP-AE for mouse colon cancer therapy was tested in a mouse colon cancer subcutaneous xenograft model and a live metastasis model in BALB/c mice. mlFVII-LDP-AE showed a tumor growth inhibition rate of 91.2% (at a dose of 0.8 mg/kg) and a tumor metastasis inhibition rate of 84.7% (at a dose of 0.6 mg/kg). The results showed that mlFVII-LDP-AE was able to effectively inhibit the growth and metastasis of mouse colon cancer. As human TF and FVII have features similar to those of mice, human FVII light chain (hlFVII)-targeted LDM (hlFVII-LDP-AE) may be expected to have therapeutic potential for human colon cancer.

BACKGROUND

Fresh Frozen Plasma (FFP) is a blood component whose clinical use is widespread worldwide. Transfusion safety of this product is ensured by legally obligatory tests. Although these tests are carried out on each plasma donation, safety levels can be further improved by using some technical procedures, such as, among others, methylene blue (MB) and solvent-detergent (SD) viral inactivation methods. The DMTE (Blood Transfusion Unit) in Mantova has used the pharmaceutical-like SD virally inactivated plasma since 2007 (Plasmasafe, Kedrion) as replacement of the PFC by each single donor. Guidelines for the usage of both products are the same.

METHODS

With the main aim of assessing the therapeutic effectiveness and safety of Plasmasafe, we decided to clinically monitor transfusions performed with this product on patients of the Intensive Care Unit at the city hospital in Mantova. In addition, we controlled some coagulation parameters (PT, aPTT, ATIII, Fibrinogen, PC, PS, FV, FVII, FVIII) before and 24 hours after the Plasmasafe infusion.

RESULTS

From a clinical point of view, the use of Plasmasafe always led to a significant reduction, or complete stop, of the bleeding. No transfusion-related adverse events were recorded. As regards, the most relevant laboratory results, a marked increase in the above mentioned hemostatic parameters was detected. Furthermore, patients transfused with this product received a mean volume significantly lower than an historical cohort of patients treated with FFP (503 mL with Plasmasafe versus 1549 mL with FFP, P<0.001).

CONCLUSIONS

The results of our study clearly document that Plasmasafe, a virally inactivated pharmaceutical-like product with a standardized content of coagulation factors, is a safe and cost-effective treatment, able to rapidly correct hemostatic abnormalities, for critical patients.

Engineered transcription factors (TF) have expanded our ability to modulate gene expression and hold great promise as bio-therapeutics. The first-generation TF, based on Zinc Fingers or Transcription-Activator-like Effectors (TALE), required complex and time-consuming assembly protocols, and were indeed replaced in recent years by the CRISPR activation (CRISPRa) technology. Here, with coagulation F7/F8 gene promoters as models, we exploited a CRISPRa system based on deactivated (d)Cas9, fused with a transcriptional activator (VPR), which is driven to its target by a single guide (sg)RNA. Reporter gene assays in hepatoma cells identified a sgRNA (sgRNA_F7.5) triggering a ~35-fold increase in the activity of F7 promoter, either wild-type, or defective due to the c.-61T>G mutation. The effect was higher (~15-fold) than that of an engineered TALE-TF (TF4) targeting the same promoter region. Noticeably, when challenged on the endogenous F7 gene, the dCas9-VPR/sgRNA_F7.5 combination was more efficient (~6.5-fold) in promoting factor VII (FVII) protein secretion/activity than TF4 (~3.8-fold). The approach was translated to the promoter of F8, whose reduced expression causes hemophilia A. Reporter gene assays in hepatic and endothelial cells identified sgRNAs that, respectively, appreciably increased F8 promoter activity (sgRNA_F8.1, ~8-fold and 3-fold; sgRNA_F8.2, ~19-fold and 2-fold) with synergistic effects (~38-fold and 2.7-fold). Since modest increases in F7/F8 expression would ameliorate patients' phenotype, the CRISPRa-mediated transactivation extent might approach the low therapeutic threshold. Through this pioneer study we demonstrated that the CRISPRa system is easily tailorable to increase expression, or rescue disease-causing mutations, of different promoters, with potential intriguing implications for human disease models.

The assessment of factor VIII coagulant activity (FVIII:C) in recently available highly purified and concentrated FVIII therapeutic products calls for careful evaluation of assay methodologies. We assayed more than 130 batches of a concentrate with a specific activity of about 150 FVIII:C units/mg protein, using one-stage and two-stage clotting and chromogenic methods. There was good agreement between the potency estimates obtained with the different methods. We also compared the FVIII:C potencies obtained after predilution in buffer or FVIII-deficient plasma using either calibrated plasma or FVIII concentrate as references. With the one-stage assay we found a marked discrepancy between the potency values obtained with buffer and with FVII-deficient plasma used as prediluents. In order to validate our "in vitro" data we performed 6 "in vivo" analyses in severe haemophilia A patients. On the basis of the overall data obtained we chose to label FVIII potency by using FVIII-deficient plasma as prediluent, reference plasma as standard and the chromogenic assay method.

BACKGROUND

Congenital factor VII (FVII) deficiency is a rare coagulation deficiency caused due to defects in the FVII gene.

METHODS

We analyzed 14 unrelated Indian patients with congenital FVII deficiency for mutations in FVII gene by conformation sensitive gel electrophoresis (CSGE) followed by DNA sequencing.

RESULTS

A total of 11 different missense mutations were identified, of which 5 were novel (Ala191Pro, Asp338Glu, Ile138Thr, Leu263Arg and Trp284Arg) and 6 had been previously reported (Cys22Arg, Arg152Gln, Cys310Phe, Thr324Met, Gly117Arg and His348Arg). Six of the 11 mutations were located in the catalytic serine protease domain, 3 in the activation domain and 1 each in the Gla and the second epidermal growth factor domain respectively. Multiple sequence alignment using ClustalW2 analysis showed that all the mutations were found in residues that are highly conserved across species. Implications of mutations on the structural stability and function of human factor VIII (hFVII) using Swiss-Pdb Viewer and the intra-molecular interactions of the mutant residues using PIC showed that there is a structural instability in all the mutants either by steric hindrance or instability in the protein molecule folding.

CONCLUSIONS

A wide spectrum of mutations was detected in the FVII gene; the presence of 6 out of 11 mutations in the serine protease domain suggests the crucial role of catalytic domain in FVII functional activity.

Activated factor X (FXa) and thrombin can up-regulate gene expression of connective tissue growth factor (CTGF/CCN2) on fibroblasts. Since tissue factor (TF) is expressed on these cells, we hypothesized that they may assemble the prothrombinase complex leading to CTGF/CCN2 upregulation. In addition, the effect of thrombospondin-1 (TSP1) on this reaction was evaluated. Human foreskin fibroblasts were incubated with purified factor VII (FVII), factor X (FX), factor V (FV), prothrombin and calcium in the presence and absence of TSP1. Generation of FXa and of thrombin were assessed using chromogenic substrates. SMAD pathway phosphorylation was detected via Western-blot analysis. Pre-incubation of fibroblasts with FVII led to its auto-activation by cell-surface expressed TF, which in turn in the presence of FX, FVa, prothrombin and calcium led to FXa (9.7±0.8nM) and thrombin (7.9±0.04 U/mL×10-3) generation. Addition of TSP1 significantly enhanced thrombin (23.3±0.7 U/mL×10-3) but not FXa (8.5±0.6nM) generation. FXa and thrombin generation leads to upregulation of CTGF/CCN2. TSP1 alone upregulated CTGF/CCN2, an effect mediated via activation of transforming growth factor beta (TGFβ) as shown by phosphorylation of the SMAD pathway, an event blunted by using a TGFβ receptor I inhibitor (TGFβRI). FXa- and thrombin-induced upregulation of CTGF/CCN2 was not blocked by TGFβRI. In summary, assembly of the prothrombinase complex occurs on fibroblast's surface leading to serine proteases generation, an event enhanced by TSP1 and associated with CTGF/CCN2 upregulation. These mechanisms may play an important role in human diseases associated with fibrosis.

We have used recombinant mammalian expression and purification of the factor VII (FVII) variant Gln100 ->> Arg (Q100RFVII) to study FVII deficiency in subjects with this mutation. Q100RFVII was secreted poorly in comparison with wild-type FVII (WTFVII) in a stable mammalian expression system, and purified variant protein was found to have undetectable clotting activity. Following activation by immobilized factor Xa, Q100RFVIIa had amidolytic activity similar to WTFVIIa in the absence of soluble tissue factor (sTF); however, unlike WTFVIIa no typical increase in activity was seen after addition of sTF. In a factor X activation assay using relipidated transmembrane truncated tissue factor (residues 1-243), Q100RFVIIa showed less than 5% of the ability of WTFVIIa to activate factor X. We performed direct binding analysis of WT and Q100RFVII/FVIIa to immobilized sTF using surface plasmon resonance, and severely reduced binding of both non-activated and activated Q100RFVII to sTF was seen, indicating a pronounced defect in tissue factor (TF) interaction with this variant. In the sTF-FVIIa crystal structure the candidate residue Gln100 is not in contact with TF but is at the epidermal growth factor 2-protease domain interface. We suggest that the mutation results in a global fold change severely reducing tissue factor interaction; mutation of FVII residues not directly involved in the interaction with TF may still result in variant FVII unable to take part in the initiation of coagulation.

Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

Tissue factor (TF) initiates the extrinsic pathway of blood coagulation through sequential binding and activation of coagulation factors VII (FVII) and X (FX). In addition, through activation of G-protein-coupled protease activated receptors (PARs) TF induces cell signaling that is related to cancer, angiogenesis and inflammation. Monoclonal antibodies (mAbs) proved to be a useful tool for studying the interplay between TF signaling and coagulation. MAb 10H10 is unique in that it blocks the signaling pathway and thus inhibits angiogenesis and tumor growth without interfering with coagulation. It was also presumed that mAb 10H10 recognizes the cryptic pool of TF devoid of procoagulant activity. The crystal structure of the 10H10 Fab was determined in the absence and in the presence of the TF extracellular domain (ECD). The structures show that the antibody operates by the key-and-lock mechanism causing no conformational changes in either Fab or TF. The TF:10H10 interface is extensive and includes five segments of TF in both the N-terminal and C-terminal domains of the ECD. Neither the known epitope of FVII, nor the putative epitope of FX overlaps with the 10H10 binding site. The 10H10 epitope points to the likely location of the PAR2 exosite. It is also the hypothetical site of TF interaction with integrins that may play a major role in the encryption-decryption process.

Coagulation Factor VII is a vitamin K-dependent serine protease which has a pivotal role in the initiation of the coagulation cascade. The congenital Factor VII deficiency is a recessive hemorrhagic disorder that occurs due to mutations of F7 gene. In the present study C91S (p.C91S) substitution was detected in a patient with FVII deficiency. This mutation has not been characterized by a functional study.

In this study, we aimed to evaluate the impact of C91S substitution on factor VII expression and function.

Materials and Methods

The F7 complete cDNA was isolated from HepG2 cell line and inserted into the pcDNA3.1 mammalian expression vector. The desired mutation was generated by the site-directed mutagenesis and the wild-type and mutated constructs were transfected into CHO-K1 cells. The protein activity and antigen level (antigen concentration) were validated in the culture medium and cell lysate of the transiently transformed cells. An immunocytochemistry procedure was also performed to evaluate the intracellular localization of the mutated and the wild-type FVII, as well.

Results

The present in vitro study has demonstrated that C91S antigen expression was increased in the transfected CHO-K1 cells compared to the wild-type (WT) protein. Despite an increased protein secretion, the factor VII coagulant activity was diminished following C91S substitution when it was assessed by a standard one-stage analysis. In addition, the immunocytochemistry procedure revealed that there was no difference in the intracellular localization of the C91S mutated FVII compared to the WT protein.

Our results present that C91S mutation has an effect on the coagulation activity, secretion, biosynthesis, and probably folding of the FVII leading to the FVII deficiency.

A comparative study on weak anion exchangers was performed to investigate the pH dependence, binding strength, particle size distribution, and static and dynamic capacity of the chromatographic resins. The resins tested included: DEAE Sepharose FF, Poros 50 D, Fractogel EMD DEAE (M), MacroPrep DEAE Support, DEAE Ceramic HyperD 20, and Toyopearl DEAE 650 M. Testing was performed with five different model proteins: Anti-FVII mAb (immunoglobulin G), aprotinin, bovine serum albumin (BSA), Lipolase (Novozymes), and myoglobin. Retention showed an expected increasing trend as a function of pH for proteins with low pI. A decrease in retention was observed for some resins at pH 9 likely due to initiation of deprotonation of the weak anion-exchange ligands. Expected particle size distribution was obtained for all resins compared to previous studies. Binding strength to weak anion-exchange resins as a function of ionic strength depends on the specific protein. Binding and elution at low salt concentration may be performed with Toyopearl DEAE 650 M, while binding and elution at high salt concentration may be performed with MacroPrep DEAE Support. Highest binding capacities were generally obtained with Poros 50 D followed by DEAE Ceramic HyperD 20. A general good agreement was obtained between this study and data obtained by the suppliers. Verification of binding strength trends with model proteins was achieved with human growth hormone (hGH) and a hGH variant on the same resins with different elution salts, sodium chloride, sodium hydrogenphosphate, sodium sulphate, and sodium acetate. Static capacity measurements obtained in the traditional experimental set-up were compared with high-throughput screening (HTS) technique experiments with reasonable agreement. Isotherm data obtained from HTS techniques and pulse experiments were successfully combined with mathematical modelling to simulate, develop and optimise the separation process of two model proteins, Lipolase and BSA. The data presented in this paper may be used for selection of resins for testing in process development.

OBJECTIVE

To demonstrate the efficacy of low or high dose statin treatment on C-reactive protein (CRP), von Willebrand Factor (vWF) and Factor VII (FVII) during the first two weeks of acute coronary syndromes.

METHODS

Patients with acute coronary syndromes (n=60) were randomly and prospectively allocated in three different groups. They received 10 mg (low dose), 80 mg (high dose) of atorvastatin and placebo for two weeks. Plasma levels of CRP, vWF and FVII were compared at baseline, first and second weeks of treatment.

RESULTS

Low dose therapy resulted in non-significant elevation of CRP at first week, although high dose therapy significantly lowered its level (7.75+/-3.57 vs 7.13+/-2.95; p=.04). Both low and high dose therapies effectively suppressed the production and elevation of vWF in contrast to placebo (121.15+/-31.99 vs 139.7+/-28.53; p=.04).

CONCLUSIONS

High dose atorvastatin significantly decreased CRP during the early days of acute coronary syndromes. Although vWF significantly increased in placebo group, both low and high dose atorvastatin treatments effectively suppressed its increased production.

Hypercoagulation with upregulated monocytic tissue factor (TF) activity often occurs under a variety of inflammatory conditions including endotoxemia. The antagonism to bacterial endotoxin (LPS) signaling often results in the depression in TF upregulation. We herein report that compound 48/80 (48/80) significantly depressed LPS-induced TF activity in human and cebus monkey peripheral blood monocytes. Employing a model monocyte-like cell line (THP-1), we explored the regulatory mechanism to identify the inhibitory site(s) of 48/80. We determine whether the inhibition results from the blockade of LPS signaling. 48/80 dose-dependently inhibited LPS-induced TF activity. Chase of LPS-challenged cells with 48/80 also significantly offset TF upregulation. In immunofluorescent approaches, FACScan analysis revealed that 48/80 had no effect on either LPS recognition or the expression of its receptors (CD14 and CD11b). Moreover, LPS-induced TF expression as well as synthesis remained unaffected in the presence of 48/80. Consistent with the independence of LPS action, 48/80 was also able to inhibit TF activity induced by A23187, ionomycin, or Quin-2 AM. Interestingly, 48/80 significantly decreased the FVII binding to either resting or LPS-challenged cells. In conclusion, our results elucidate that the inhibitory action of 48/80 was independent of LPS signaling including recognition, receptor expression, and the induced TF expression/ synthesis. However, 48/80 was able to directly block FVII binding to monocytic TF, thereby resulting in such antagonism to LPS-induced TF-initiated extrinsic coagulation.

BACKGROUND

We evaluated plasminogen activator inhibitor-1 (PAI-1), factor VII activity (FVII), and fibrinogen in a sample of octo-nonagenarians. Furthermore, we investigated the relationship of these fibrinolytic and coagulation parameters with lipoprotein profile and anthropometric variables in the absence or presence of disability.

METHODS

We enrolled a population of 162 octo-nonagenarians, divided in two groups on the basis of presence or absence of disability in the activity of daily living (ADL). All the anthropometric determinations were carried out according to standardized methods. Blood samples for hemostatic and lipid determinations were collected after overnight fasting and resting.

RESULTS

PAI-1 activity and fibrinogen levels were significantly higher in disabled (DIS) compared to free-living (FL) adults, whereas FVII did not show differences in the two groups. PAI-1 activity and FVII positively correlated to anthropometric parameters (body mass index, subscapular and tricipital skinfold thickness) in both DIS and FL. No correlations were found between fibrinogen and other variables in FL, whereas a negative relation with high density lipoprotein-cholesterol levels emerged in DIS. FVII was positively related with total cholesterol low density lipoprotein-cholesterol, and apolipoprotein B in both FL and DIS.

CONCLUSIONS

In a sample of octo-nonagenarians, PAI-1 activity and FVII show a significant correlation with several anthropometric and lipoprotein parameters, suggesting that these variables are strongly associated with body composition and lipid metabolism independent from age and disability. DIS presented higher PAI-1 and fibrinogen levels; this observation may take in account the high prevalence of vascular diseases and also occult inflammation, which are known to affect these parameters.

Coagulation factor VII (FVII) is a key enzyme of the extrinsic coagulation cascade that is predominantly produced by hepatocytes. The F7 gene mutations cause FVII deficiency with considerable molecular and phenotypic heterogeneity. We characterized the molecular alterations of the F7 gene and their corresponding mRNA transcripts in Iranian patients from eight unrelated families. The mutations were detected by polymerase chain reaction (PCR)-sequencing of all F7 gene exons, their flanking intronic sequences, as well as their corresponding cDNA fragments. Homozygous P303T, C91S and R304Q mutations were detected in patient 2, patient 5, and patient 6, respectively. Patient 7 was a compound heterozygote for S282R and H348R and patient 8 was a compound heterozygote for R304Q and IVS7+7A>G mutations. Furthermore, our investigation revealed three heterozygous individuals, patient 1 and patient 3 with the A244V mutation who were symptomatic and patient 4 with V(-39)I mutation who was also asymptomatic. The F7 mRNA expression analysis revealed that, except the transcript of V(-39)I, other mutation-harboring transcripts were expressed at detectable levels. In conclusion, this report reinforces the genetic and phenotypic heterogeneity of FVII deficiency. The findings of the mRNA study implied that decreased FVII protein activity subsequent to missense mutations does not completely reflect the degradation of mutation-harboring mRNA.

Breast cancer disease and as well as CMF-chemotherapy are associated with an increased risk for thromboembolic complications. There is evidence that effects on the hemostatic system may play an important role. To minimize the impact of tumor associated hypercoagulability, we choose to study CMF-associated effects on the hemostatic system within an adjuvant setting. Blood coagulation and fibrinolysis were examined before and 24 hours after intravenous application of CMF-therapy at 17 patients with breast cancer. 16 parameters of coagulation and fibrinolysis were studied. In a longitudinal analysis covering the complete 6 month treatment period we found a decrease of thromboplastin time (TPZ) factor VII (FVII) and protein C antigen (PC Ag) and activity (PC Act). Clinically relevant pathological results and cumulative effects were observed only for PC Ag and Act, while the mean values of TPZ and FVII returned to pre-treatment levels after each course of treatment. These data suggest a potential impact of CMF-chemotherapy on synthesis and activation of vitamin-K-dependent coagulation factors thus providing a possible explanation for the increased risk for thrombosis during CMF-chemotherapy.

As compared to haemophilia, although the clinical features and the management strategies for rare coagulation factor deficiencies are discussed, little is known about them. This study was undertaken to assess the distribution, clinical presentation and treatment of patients with rare coagulation factor deficiency disorders in a cross-sectional population of India. Blood samples and other clinical details from patients suspected of rare coagulation factor deficiencies were collected by the Haemophilia Treatment Centers across India and were diagnosed at National Institute of Immunohaematology, Mumbai. A total of 321 cases of rare clotting factor deficiencies were diagnosed, of which 88% were severe, 10% moderate and 2% mild. Commonest deficiency encountered was factor XIII (FXIII) (30%) followed by FX (15.6%), FVII (15%), fibrinogen (12.1%), FXI (9%), combined V and VIII deficiency (5.6%) and congenital multiple vitamin K-dependent coagulation factor deficiency (MCFD, 2.1%). Major representation of these deficiencies was from Southern and Western India (82%). Mucocutaneous bleeding was the commonest clinical presentation (59%); intracranial (IC) haemorrhage was seen in 18% of the patients; menorrhagia was an important clinical pointer in women in the reproductive age group (78%); 8% of the severe cases had no history of bleeding and 73% of the FXIII deficiency cases had umbilical stump bleeding. The major therapeutic products used was fresh frozen plasma (64%), cryoprecipitate (15%), whole blood (15%), antifibrinolytics (5%) and recombinant FVIIa (1%). A distinct pattern in the distribution of rare clotting factor deficiencies was observed which was based on multiple factors that include ethnicity and the available diagnostic facilities in different regions of this vast country.