Various abnormalities of coagulation-fibrinolytic system have been reported in patients with thyroid dysfunction. Several studies indicate that coagulation and fibrinolytic system is disturbed in the patients with hypothyroidism. Also, the influence of hypothyroidism on hemostasis is controversial; both hypocoagulable and hypercoagulable states have been reported. The levels of plasma thrombin-activatable fibrinolysis inhibitor (TAFI) antigen and tissue factor pathway inhibitor (TFPI) have been investigated only once in patients with hypothyroidism. Therefore, the main purpose of this study was to evaluate the profile of coagulation and fibrinolytic parameters including TAFI and TFPI in patients with hypothyroidism. Fifteen patients with untreated hypothyroidism and 15 age-matched healthy controls were included in the study. Factors V(FV), VII (FVII), VIII (FVIII) activities, von Willebrand factor (vWF), protein C, protein S, thrombomodulin (TM), TFPI, and TAFI were measured. The relationships between serum thyroid hormones and these hemostatic parameters were examined. Compared with the control subjects, FVII activity, and TM Ag and TAFI Ag levels were significantly increased in patients with hypothyroidism, whereas FV, FVIII, vWF, protein C and protein S activities, and TFPI Ag levels were significantly decreased. We did not find any significant correlation between serum thyroid hormones and the hemostatic parameters that we measured. In conclusion, we found some important differences in the hemostatic parameters between the patients with hypothyroidism and healthy controls. Increased FVII, TM, and TAFI and decreased FV, FVIII, vWF, protein C, protein S, and TFPI in these patients represent a potential hypercoagulable and hypofibrinolytic state, possible endothelial dysfunction, which might augment the risk for atherosclerotic and atherothrombotic complications. Thus, disturbances of the hemostatic system may contribute to the excess mortality due to cardiovascular disease seen in patients with hypothyroidism.

Deficiencies of coagulation factors other than factor VIII and factor IX (afibrinogenemia, FII, FV, FV+FVIII, FVII, FX, FXI, FXIII) that cause bleeding disorders (RBDs) are inherited as autosomal recessive traits and are rare, with prevalences in the general population varying between 1 in 500,000 and 1 in 2 million for the homozygous forms. As a consequence of the rarity of these deficiencies, the type and severity of bleeding symptoms, the underlying molecular defects, and the actual management of bleeding episodes are not as well established as for hemophilia A and B. The study of the genetic basis of these disorders could represent an important tool for prevention through prenatal diagnosis. Treatment of patients with RBDs during bleeding episodes or surgery is a challenge because of the lack of experience and the paucity of data. For some deficiency factor concentrates are still non available and severe complications can occur. These complications can be minimized by assessment of risks of bleeding and thrombosis, use of haemostatic means other than blood components or no therapy at all. The RBDs pose a problem for guideline writers because there are no suitable clinical trials to supply good evidence for how these people are best treated. The lack of adequate information on clinical manifestations, treatment and genetic basis of RBDs could be improved by the collection of data in an International Database (http://www.rbdd.org), linkable to others previously published. This could be a useful tool to fill the gap between clinical data and clinical practice. This article reviews the genetic basis of RBDs, problems and complications of treatment, problems in the preparation of suitable guidelines for treatment and the future perspectives of the International Registry on RBDs.

OBJECTIVE

Tissue factor (TF) is the main initiator of the extrinsic coagulation pathway through factor VII (FVII) activation, which is physiologically inhibited by tissue factor pathway inhibitor (TFPI). Alteration of this pathway has been described in Type 2 diabetes mellitus (T2DM). The aim of this study is to assess TF and TFPI plasma levels and FVII coagulant activity (FVIIa) in T2DM in relation to cardiothrombotic disease and their correlation to metabolic and clinical behavior of the patients.

METHODS

The study was conducted on 80 T2DM patients divided to accordingly; groupI: 40 patients without a history or clinically detected heart disease, and groupII: 40 patients with a history of myocardial infarction compared to 30 controls. The patients were recruited from Ain Shams University diabetes clinic from September 2007 to February 2009 after informed consent was obtained. Peripheral blood samples were taken for measurement of plasma TF and TFPI levels using ELISA technique and quantitative FVIIa using FVII deficient plasma.

RESULTS

Plasma levels of TF, TFPI and FVIIa were significantly higher in T2DM patients compared to the controls (p<0.001). TF (236.50±79.23)and TFPI (242.33±85.84)were significantly higher in group II, compared to group I (150.33±81.16), (152.8± 82.46), (p<0.001). TF and TFPI were significantly correlated to body mass index and glycemic control. Also, TF and TFPI were significantly higher in hypertensives (p=0.001) and dyslipidemics (p=0.006) but not in smokers (p=0.64), (p=0.11) respectively.

CONCLUSIONS

There was a correlation between high TF, TFPI plasma levels, FVIIa activity and cardiothrombotic complications in T2DM especially in the presence of high risk factors such as poor glycemic control, dyslipidemia and obesity. Future target therapy against TF may be beneficial for T2DM patients.

BACKGROUND

Current transfusion-related acute lung injury reduction strategies include avoiding transfusion of plasma products collected from female donors or female donors that have been pregnant to reduce transfusion of plasma-containing HLA antibodies. Such a policy considerably decreases the number of donors available for generation of fresh-frozen plasma (FFP). To increase the supply of FFP, substitution of 24-hour plasma (FP24) and thawed plasma (TP) derived from either FFP or FP24 may be viable substitutes. To justify such a policy the coagulation factor content of FFP, FP24, and TP derived from both product types was assessed.

METHODS

Coagulation factor (F)II, FV, FVII, FVIII, F IX, and FX; protein C (PC) and protein S (PS); von Willebrand factor antigen and ristocetin cofactor; fibrinogen; and antithrombin activities were analyzed in nonpaired FFP and FP24 at the time of product thaw and again after 120 hours of 1 to 6°C storage.

RESULTS

At thaw, mean FVIII and PC activities were lower in FP24 than FFP. Mean PC and PS activities were lower in FP24- than FFP-derived 120-hour-old TP. No other differences in mean activity reached significance. Activity levels were generally lower in TP; FVIII, FV, and FVII showed the largest changes. However, prestorage leukoreduction appears to improve the stability of FV.

CONCLUSIONS

FFP, FP24, and the derived TP all contain adequate coagulation factor activities to maintain hemostatic activity. As FFP becomes less available, increased use of FP24 and TP are viable alternatives for most clinical situations.

An increasing amount of evidence suggests that coagulation factors VIII and IX play a role not only in the intrinsic but also in the extrinsic pathway of coagulation. In this context the influence of the Extrinsic Pathway Inhibitor (EPI) on the coagulation time of hemophilia plasma lacking FVIII or FIX has been investigated. The coagulation time was measured in a dilute thromboplastin assay. Addition of recombinant EPI (rEPI) prolonged the coagulation time of normal plasma while the addition of an inhibitory antibody against EPI shortened the coagulation time. At low concentrations of thromboplastin the coagulation time of hemophilia plasma was prolonged and at all dilutions of thromboplastin, addition of anti-EPI IgG normalized the coagulation time of a hemophilia plasma. Analysis of 10 individual donor plasma samples and 8 individual hemophilia samples showed that addition of anti-EPI IgG shortened the coagulation time more in hemophilia plasma than in normal plasma. This illustrates the importance of a powerful extrinsic FVII dependent pathway to achieve hemostasis in the case of FVIII or FIX deficiency (hemophilia A and B).

The concept of using factor VIIa (FVIIa) to induce hemostasis in hemophilia is new and takes advantage of the normal tissue factor/FVII-dependent coagulation pathway which is enhanced by the administration of extra exogenous recombinant FVIIa. Since this is a pharmacological treatment and not a simple substitution therapy the current dosis recommendations are based on preclinical data (in vitro and in animals) as well as on clinical experience. It is concluded that the initial dose should be high enough to maintain a plasma level of FVII-coagulant activity (FVII:C) at>> 6 U/ml for several hours corresponding roughly to a dose of 90-120 mu g/kg.

The variations of FVII, PAI-1, TAT complexes, fibrinopeptide A, D-Dimers and beta thromboglobulin plasma levels were studied on 30 sedentary men, smokers and non-smokers, who were admitted to a 6 months' program of physical training and smoking cessation. After 3 months of intervention, sustained physical training was associated with the decrease of FVII and PAI-1 levels. Mild exercise performed during a second 3-month period could maintain normal FVII and PAI-1 activities but participants who stopped the training increased their FVII and PAI-1 plasma levels. FVII was not influenced by smoking habits. Smoking cessation seemed to slightly potentiate the decrease of PAI-1 levels associated with mild exercise. Overweight, FVII and PAI-1 levels were correlated and the weight reduction induced by training was related to the changes in the factors. In smokers, physical exercise was associated with a significant increase of hemostatic markers. This exercise-induced variation disappeared after 3 months of intervention in participants who stopped smoking and reappeared in those who smoked again after 6 months of intervention. This finding was not influenced by the physical training program.

The hyaluronan-binding protease (HABP) is a serine protease in human plasma which is structurally related to plasminogen activators, coagulation factor XII and hepathocyte growth factor activator. It can in vitro activate the coagulation factor FVII, kininogen and plasminogen activators. The present study was initiated to gain a more complete picture of the cell-associated activities of this fibrinolysis-related protease. Treatment of lung fibroblasts with HABP lead to a rapid activation of signalling pathways, including the mitogen-activated protein kinase (MAPK) pathway with c-Raf, MEK and ERK1/2. Additionally the activation of the PI3K/Akt pathway and of several translation-related proteins was found. Proliferation assays confirmed the assumption of a strong growth-stimulating effect of HABP on human lung and skin fibroblasts. Intracellular signalling and growth stimulation were strongly dependent on the proteolytic activity of HABP. Stimulation of signalling and proliferation by HABP involved the fibroblast growth factor receptor 1 (FGFR-1). HABP-stimulated proliferation of lung fibroblasts MRC-5 was accompanied by a significant intracellular increase in basic fibroblast growth factor (bFGF), the major ligand of FGFR-1; bFGF could however not be identified in the supernatant of HABP-treated cells. Though, the conditioned medium from HABP-treated cells showed a strong growth-promoting activity on quiescent fibroblasts, indicating the release of a yet unknown growth factor amplifying the initial growth stimulus. In a two-dimensional wound model HABP stimulated the invasion of fibroblasts into a scratch area, adding a strong pro-migratory activity to this plasma protease. In summary, HABP exhibits a significant growth factor-like activity on quiescent human lung and dermal fibroblasts. Our findings suggest that this fibrinolysis-related plasma protease may participate in physiologic or pathologic processes where cell proliferation and migration are pivotal, like tissue repair, vascular remodelling, wound healing or tumor development.

BACKGROUND

Warfarin reversal is a common clinical situation. This is commonly performed using vitamin K and, depending on the urgency, fresh frozen plasma (FFP), prothrombin complex concentrates (PCCs), or activated factor VII. Even though PCCs are widely used, the ideal dosing regimen is far from established.

OBJECTIVE

To verify differences in warfarin reversal patterns using FFP, recombinant FVIIa (rFVIIa), and PCC; and to test the hypothesis that supratherapeutic International Normalized Ratios (INRs) might not correlate with thrombin generation (TG) and identify the ideal concentrations of PCC required to reverse various INR thresholds.

METHODS

We studied the effects of FFP, rFVIIa and Beriplex P/N on the INR and TG, using the calibrated automated thrombography assay in ex vivo warfarinized plasma. Plasmas with different INRs were spiked with different concentrations of Beriplex P/N.

RESULTS

Beriplex P/N was the only agent that completely normalized TG and the INR. The endogenous thrombin potential (ETP) and the peak thrombin showed a significant negative correlation with all INRs. The ETP and velocity of TG reached a plateau at an INR of approximately 4.0. A concentration equivalent to a dose of 30 IU kg(-1) Beriplex P/N normalized the ETP, the INR, FII, FVII, FIX and FX of samples with INRs>> or = 4.0. Higher doses resulted in hypercoagulable TG patterns. A concentration equivalent to a dose of 20 IU kg(-1) was sufficient to reverse warfarin at an INR range of 2.0-3.9, as judged by the same tests.

CONCLUSIONS

Warfarin reversal algorithms could be simplified with the adoption of this strategy utilizing two doses of PCC, depending on the INR of the patient. This would also lead to cost reductions and, possibly, a reduction in thrombotic risk.

Recombinant factor VIIa (rFVIIa; NovoSeven) is a recent addition to the hemostatic alternatives for the treatment of hemophiliacs with inhibitors. A drawback in the use of rFVIIa has been its half-life of only about 2 h, which necessitates very frequent and punctual injections. We evaluated the stability of reconstituted, but not further diluted, rFVIIa in 3 infusion systems (WalkMed 350 and CADD-Plus minipumps and Meddex 2001 syringe pump). The factor VII (F VII) activity was maintained for at least 3 days at room temperature with only a minor and clinically insignificant increase in oxidized forms of rFVIIa and minimal leaching of the plastic softeners dibutylphthalate and di-octylphthalate after 24-48 h. Addition of heparin, 5-10 U/ml, to reconstituted rFVIIa caused a loss of about 50% of the activity within 4 h of storage in the infusion system, whereas low molecular weight heparin had no such effect. Repeated samples showed that the infusion systems maintained sterility. Reconstituted rFVIIa did not support bacterial growth when inoculated with Staphylococcus aureus or Escherichia coli to any greater extent than did reconstituted factor VIII, lidocaine in saline or heparin in saline. Two patients were treated with continuous infusion of rFVIIa on 4 occasions (total knee arthroplasty, wound revision, and twice straightening of a 90 degrees contracture of the knee under general anaesthesia). A preoperative pharmacokinetic evaluation was performed, and the clearance was used to calculate the maintenance dose, aiming at a FVII level of 10 U/ml, which proved to be a hemostatic level. The first patient had no change in the clearance during the two treatment episodes. He suffered from repeated thrombophlebitis at the infusion site. The second patient had a progressive decrease of the clearance from 86.4 to 24.7 ml/h/kg. He received during the first treatment a parallel infusion with heparin (approximately 250 U/24 h) to the same venous access and did not develop thrombophlebitis during 3.5 days of therapy. For the second episode low molecular weight heparin was added directly to the infusion bag, and no adverse effects were observed. Continuous infusion with rFVIIa is thus feasible with the minipumps used by us, eliminates the need for 2 h injections and reduces the total dose of rFVIIa by 50-75%, depending on the behaviour of the clearance.

OBJECTIVE

FVII:C has been shown to be an independent risk factor for myocardial infarction and is related to environmental and genetic factors. This study sought to investigate FVII:C levels and factor VII (FVII) gene polymorphisms in relation to stroke and disease outcome.

METHODS

To examine the association of FVII:C and the Msp I and promoter insertion polymorphisms of the FVII gene in acute stroke, 317 patients and 198 age-matched control subjects were studied.

RESULTS

FVII:C levels were significantly lower in patients at onset than 3 months later (119% versus 135%, respectively; P < .0005). Levels were significantly lower in patients at onset than in control subjects (124% [95% confidence interval, 120% to 129%] versus 141% [95% confidence interval, 135% to 148%], respectively; P < .0005) but were not significantly different at 3 months (135% [95% confidence interval, 128% to 141%] versus 141% [95% confidence interval, 135% to 148%], respectively). We found no difference in genotype distribution for either polymorphism between patients and control subjects, no difference in FVII:C level or genotype distribution between pathological types of stroke, and no relationship with poststroke mortality. Both polymorphisms were significantly associated with FVII:C levels in patients and control subjects. In a multiple regression model for patients, Msp I genotype, cholesterol, and smoking remained as independent predictors of FVII:C levels, accounting for 32% of interindividual variation.

CONCLUSIONS

These results suggest that neither FVII:C levels nor FVII gene polymorphisms are associated with cerebrovascular disease. There were no genotype-specific correlations of environmental factors with FVII:C, but there was evidence of an acute-phase or consumptive fall in FVII:C levels at the time of stroke, whereas levels increased to those similar for healthy age-matched control subjects by 3 months, when the acute phase had presumably subsided.

Recombinant factor VIIa (rFVIIa) improves haemostasis and eliminates the risk of transmission of blood-borne infection in haemophilia patients with inhibitors, acquired inhibitors to factor VIII or IX or FVII deficiency. rFVIIa has been available on a compassionate use basis since 1988 and has been administered to 111 patients for a total of 494 joint and muscle bleeding episodes. Seventy-nine percent of joint and 65% of muscle bleeding episodes were evaluated by the investigator as having an excellent/effective response. rFVIIa is also an effective treatment for joint and muscle bleeding episodes in patients undergoing immune tolerance regimens and does not affect the inhibitor titre level.

OBJECTIVE

Inherited factor VII (FVII) deficiency is a rare bleeding disorder characterized by a poor relationship between reported FVII clotting activity (FVII:C) and bleeding tendency. Our study was aimed at defining biological parameters that are possibly predictive for bleeding risk in this condition.

METHODS

Forty-two FVII-deficient patients (FVII:C <30%) were classified into two opposite clinical groups defined as severe and non-or-mild bleeders. For each patient, plasma samples were collected and then investigated for FVII:C (using a sensitive method and human recombinant thromboplastin as the reagent), FVII antigen, activated FVII coagulant activity (FVIIa:C) and the free-form of tissue factor pathway inhibitor.

RESULTS

None of these tests could be used as highly accurate predictors of bleeding. Nevertheless, both FVII:C and FVIIa:C differed significantly between the two clinical groups. Using ROC-curve analysis, two critical values of 8% and 3mIU/mL for FVII:C and FVIIa:C, respectively, could be proposed to discriminate between severe bleeders and non-or-mild bleeders.

CONCLUSIONS

A highly accurate diagnostic test for predicting bleeding tendency in inherited FVII deficiency still eludes definition, highlighting the fact that factors other than FVII itself interfere with the expression of bleeding phenotypes in this condition. Nevertheless, potential critical values using sensitive FVII:C and FVIIa:C methods may be useful in clinical laboratories for FVII-deficient patients. Those patients with FVII:C levels higher than 8% FVII:C or FVIIa:C higher than 3 mIU/mL, with no other hemostatic defect, seem to have a minimal risk of severe bleeding. Extended clinical studies are needed to support these findings.

OBJECTIVE

Factor VII (FVII) deficiency is a rare coagulation disorder, historically treated with prothrombin complex concentrates or plasma-derived FVII concentrates. We treated such patients (n = 17) with a recombinant, activated FVII preparation.

METHODS

Twenty-seven spontaneous bleeding episodes were treated and 7 major and 13 minor surgical interventions were carried out. The dosages employed ranged from 8.08 to 70.5 Ig/kg body weight.

RESULTS

A mean dose between 22 and 26 Ig/kg was sufficient to normalise the prothrombin time. Fifteen haemarthroses were treated with single doses and results were excellent in 13 cases. In 5/6 bleeding episodes of other types, the treatment gave either excellent or at least effective results. Haemostasis was secured in the 7 major and 13 minor surgical interventions. One patient developed antibodies 4-5 weeks after an extremely high dose. Otherwise, there were no side effects and no evidence of a thrombotic tendency.

CONCLUSIONS

This recombinant concentrate is efficacious in FVII-deficient patients. It is safe since any risk of transmission of blood-borne viruses is eliminated.

BACKGROUND

Clopidogrel (Plavix®) therapy, although effective for minimizing risk of thrombotic events, is also associated with potential bleeding risk. Recombinant activated FVII (rFVIIa, NovoSeven®) induces hemostasis in hemophilia patients with inhibitors (alloantibodies) and has been proposed as potential treatment for mitigating clopidogrel therapy-mediated bleeding.

METHODS

In this single-center, randomized, placebo-controlled, double-blind, dose-escalation, exploratory phase I trial, we assessed the safety and effects of rFVIIa in reversing clopidogrel-enhanced bleeding in an experimentally induced punch biopsy in healthy subjects. Efficacy assessments included the reversal of bleeding characteristics (bleed duration [BD], the primary end point and blood loss volume [BV] induced by punch biopsy, and thromboelastograph [TEG®] parameters) with rFVIIa or placebo after clopidogrel treatment.

RESULTS

A significant number of subjects (56%) had limited response to clopidogrel (defined as ≤30% platelet aggregation inhibition) and were discontinued from study. The remaining subjects continued and had 4 biopsies. Of 40 subjects randomized, 37 were evaluated for efficacy. Clopidogrel treatment increased BD and BV compared with the baseline biopsy. Recombinant FVIIa (10 and 20 μg/kg) significantly mitigated the clopidogrel-induced effects on BV (P = 0.007 and P = 0.001, respectively). Early trial termination limited the evaluation of effects of higher rFVIIa doses. Subgroup analyses of subjects biopsied by the same physician demonstrated significant reduction of clopidogrel-induced BD with 20 μg/kg rFVIIa (P = 0.048). Ex vivo analysis of rFVIIa demonstrated clotting dynamics presented by parameters time to clot onset (TEG®-R) and clot angle (TEG®-A) (P < 0.005).

CONCLUSIONS

In this clinical study, rFVIIa (10 and 20 μg/kg) reversed the effect of clopidogrel on blood loss.

Data is presented from two compassionate-use clinical trials using recombinant factor VIIa (rFVIIa) to treat central nervous system haemorrhage in 18 haemophilia A and B patients with inhibitors, and in 3 patients with FVII deficiency. Prior to rFVIIa treatment 78% of the haemophilia patients had inhibitor titres greater than 10 Bethesda units/ml. Sixty-two percent of the bleeding episodes were treated with a mean dose of 80-100 mu g/kg of rFVIIa administered repeatedly until cessation of bleeding. The overall efficacy was 84% with only one fatality and there were no major adverse events or laboratory indicators of disseminated intravascular coagulation.

Changes of the tissue factor (TF) pathway of blood coagulation have been described in diabetes and could be involved in its vascular complications. In order to evaluate the influence of the type of diabetes and of the obesity index and age on these changes, factor VII coagulant activity, factor VII antigen, activated factor VII, monocyte TF expression, and plasma Tissue Factor Pathway Inhibitor (TFPI) were examined in 18 Type 1 and 16 Type 2 diabetic patients compared to non-diabetic control subjects matched for age, sex, and obesity index (Types 1 and 2 controls, respectively). Multicomplicated patients were excluded. FVIIc, FVIIAg, and FVIIa were higher in Type 2 diabetic patients and controls than in Type 1 diabetic patients and controls (P< .03). However, FVIIc and FVIIAg were lower in diabetic patients than in their matched controls (P< .03). Monocyte expression of TF was not different between Types 1 and 2 diabetic patients and their matched controls except for LPS-stimulated monocyte TF activity which was lower in Type 2 diabetic patients than in Type 2 controls (P< .05). Plasma TFPI was slightly but significantly higher in Type 1 diabetic patients than in Type 1 controls (P= .01) and was correlated to glycemia. However, both in Type 2 diabetic patients and controls, TFPI was higher than in Type 1 controls and was correlated with BMI (P< .0003). These results indicate that in not multicomplicated patients, the increase of FVII and TFPI was highly dependent on obesity index and age rather than on diabetes by itself.

Factor VII (FVII) consists of an N-terminal gamma-carboxyglutamic acid domain followed by two epidermal growth factor-like (EGF1 and EGF2) domains and the C-terminal protease domain. Activation of FVII results in a two-chain FVIIa molecule consisting of a light chain (Gla-EGF1-EGF2 domains) and a heavy chain (protease domain) held together by a single disulfide bond. During coagulation, the complex of tissue factor (TF, a transmembrane glycoprotein) and FVIIa activates factor IX (FIX) and factor X (FX). FVIIa is structurally "zymogen-like" and when bound to TF, it is more "active enzyme-like." FIX and FX share structural homology with FVII. Three structural biology aspects of FVIIa/TF are presented in this review. One, regions in soluble TF (sTF) that interact with FVIIa as well as mapping of Ca2+, Mg2+, Na+ and Zn2+ sites in FVIIa and their functions; two, modeled interactive regions of Gla and EGF1 domains of FXa and FIXa with FVIIa/sTF; and three, incompletely formed oxyanion hole in FVIIa/sTF and its induction by substrate/inhibitor. Finally, an overview of the recognition elements in TF pathway inhibitor is provided.

OBJECTIVE

Age-related changes in blood coagulation and fibrinolysis are associated with increased risk of thrombotic events. Inherited deficiencies of coagulation proteins, such as factor V (FV) Leiden and prothrombin G20210A, explain a small fraction of venous thromboembolic disease (VTE). Additional genetic factors are likely to underlie the etiology of VTE, some of which may become manifest at older ages.

METHODS

We tested 290 common SNPs within 51 thrombosis and inflammation genes for association with VTE in the Cardiovascular Health Study, a large, prospective cohort of older adults followed for up to 12 years.

RESULTS

There were 184 VTE events that occurred at mean age of 78 years. TagSNPs within four genes encoding FXIII subunit A (F13A), FVII activating protease (HABP2), protease activated receptor-1 (F2R) and the urokinase receptor (PLAUR) showed the strongest evidence for association with VTE, with each gene having a global P-value < 0.05 and at least one tagSNP false discovery rate (FDR) q-value < 0.05. The rs3024409 variant allele of F13A1 was associated with 1.66-fold increased risk of VTE, while the minor alleles of HABP2 rs6585234 and rs3862019, F2R rs253061 and rs153311, and PLAUR rs344782 were each associated with lower risk of VTE (hazard ratios in the range of 0.49-0.66). Consistent with the observed protective association for VTE risk, the HABP2 rs3862019 variant allele was also associated with lower activity levels of coagulation factors FVIII, FIX, FX and plasminogen. We also confirm previously reported associations between common variants of the coagulation FII, FV, FVIII, FXI, alpha-fibrinogen and protein C genes and risk of VTE.

CONCLUSIONS

These findings suggest that several novel common coagulation gene variants may be related to risk of VTE in older adults. Further studies in older adults are needed to validate these findings and assess functional molecular mechanisms.

We have developed a cell-based model of hemostasis. This model suggests that the defect in hemophilia is specifically a failure of platelet-surface factor Xa (FXa) generation, leading to a failure of platelet surface thrombin generation. Activation of FX by FVIIa/tissue factor (TF) does not compensate for a lack of FXa activation on the platelet surface by the FVIIIa/FIXa complex. This is because plasma protease inhibitors prevent FXa from moving through the fluid phase from the TF-bearing cell to the platelet surface. We have previously proposed a platelet-dependent mechanism of action for high-dose factor VIIa (FVIIa; Novoseven, Novo Nordisk, Copenhagen, Denmark). Our data suggest that, when present at high levels, FVIIa binds to activated platelets and activates small amounts of FX independent of TF. This platelet-surface FXa can partially restore platelet-surface thrombin generation in hemophilia. Recently, van't Veer and colleagues reported results from an in vitro model in which coagulation reactions were initiated by relipidated TF. The authors concluded that high-dose FVIIa may exert a hemostatic effect in hemophilia by overcoming inhibition of FVIIa/TF activity by zymogen FVII. By contrast, we found that plasma levels of FVII did not slow thrombin generation in a model system initiated with cell-associated TF. This discrepancy highlights the potential differences between the studies of the coagulation reactions assembled on living cells compared to phospholipid vesicles. Our data suggest that in a cellular system high-dose FVIIa acts primarily by enhancing the rate of thrombin generation on platelet surfaces and not by overcoming inhibition by zymogen FVII of TF-dependent activation of FX.

Recombinant factor VIIa (rFVIIa) is used for treatment of hemophilia patients with inhibitors, as well for off-label treatment of severe bleeding in trauma and surgery. Effective bleeding control requires supraphysiological doses of rFVIIa, posing both high expense and uncertain thrombotic risk. Two major competing theories offer different explanations for the supraphysiological rFVIIa dosing requirement: (1) the need to overcome competition between FVIIa and FVII zymogen for tissue factor (TF) binding, and (2) a high-dose-requiring phospholipid-related pathway of FVIIa action. In the present study, we found experimental conditions in which both mechanisms contribute simultaneously and independently to rFVIIa-driven thrombin generation in FVII-deficient human plasma. From mathematical simulations of our model of FX activation, which were confirmed by thrombin-generation experiments, we conclude that the action of rFVIIa at pharmacologic doses is dominated by the TF-dependent pathway with a minor contribution from a phospholipid-dependent mechanism. We established a dose-response curve for rFVIIa that is useful to explain dosing strategies. In the present study, we present a pathway to reconcile the 2 major mechanisms of rFVIIa action, a necessary step to understanding future dose optimization and evaluation of new rFVIIa analogs currently under development.

This updated safety review summarises the large body of safety data available on the use of recombinant activated factor VII (rFVIIa) in approved indications: haemophilia with inhibitors, congenital factor VII (FVII) deficiency, acquired haemophilia and Glanzmann's thrombasthenia. Accumulated data up to 31 December 2013 from clinical trials as well as post-marketing data (registries, literature reports and spontaneous reports) were included. Overall, rFVIIa has shown a consistently favourable safety profile, with no unexpected safety concerns, in all approved indications. No confirmed cases of neutralising antibodies against rFVIIa have been reported in patients with congenital haemophilia, acquired haemophilia or Glanzmann's thrombasthenia. The favourable safety profile of rFVIIa can be attributed to the recombinant nature of rFVIIa and its localised mechanism of action at the site of vascular injury. Recombinant FVIIa activates factor X directly on the surface of activated platelets, which are present only at the site of injury, meaning that systemic activation of coagulation is avoided and the risk of thrombotic events (TEs) thus reduced. Nonetheless, close monitoring for signs and symptoms of TE is warranted in all patients treated with any pro-haemostatic agent, including rFVIIa, especially the elderly and any other patients with concomitant conditions and/or predisposing risk factors to thrombosis.

Patients with congenital haemophilia with inhibitors are at risk of peri-operative bleeding complications, since replacement of the missing coagulation factor is ineffective, presenting a therapeutic challenge in elective or emergency surgery. Therefore, the management of peri-operative bleeding requires the use of bypassing agents, such as recombinant activated FVII (rFVIIa, NovoSeven(®) ). This article presents an updated evaluation of the safety and effectiveness of rFVIIa in the treatment of peri-operative bleeding in this patient population. Surgical and other medical procedures managed with rFVIIa from two randomized clinical trials, the Hemophilia Research Society/Hemophilia and Thrombosis Research Society (HRS/HTRS) registry databases and the medical literature were analysed. There were 395 rFVIIa-treated procedures (261 surgical, 89 dental and 45 other medical procedures) reported for 263 congenital haemophilia patients with inhibitors. In trials, initial rFVIIa dosing was 35-90 mcg kg(-1) bolus injection or 50 mcg kg(-1) h(-1) continuous infusion. Dosing in the registries and literature was more variable. Recombinant FVIIa effectiveness was comparable across data sources, with an overall rate of 84% (333/395). The incidence of thrombotic events was very low (0.4% of patients and 0.025% of procedures). Prior to the US approval of rFVIIa in 1999, surgical procedures in congenital haemophilia patients with inhibitors were often considered too risky. Recombinant FVIIa has consistently demonstrated effectiveness in treatment of bleeding in these patients during such procedures. Thrombotic events were rare. This analysis confirms the value of corroborating clinical trial results with post-marketing surveillance registries to assess small patient populations with clinically challenging management decisions.

Recombinant human proteins are generally recovered in low yields from mammalian tissue culture following transfection with commercially available vectors. We have constructed a novel vector containing both the neomycin-resistance-encoding gene (neo) as a dominant selectable marker, and the dihydrofolate reductase-encoding gene (DHFR) to enable amplification of transfected DNA followed by stable expression in mammalian cell lines. Levels of 5 micrograms/ml of the coagulation proteins, factor VII (FVII) and factor XI (FXI), have been achieved in serum-free media. N-terminal sequencing of the purified proteins, and of their separated chains after proteolytic activation, demonstrated correct processing of the recombinant products. In addition, the ratios of clotting activity to antigen for each are close to unity, and the recombinant and plasma-derived proteins had identical mobilities upon electrophoresis in the presence of SDS. The vector described will be of use for the synthesis of recombinant proteins, both wild-type and variants produced by site-directed mutagenesis, especially where complex post-translational modification of the protein makes it essential to use mammalian cells.