The insulin resistance syndrome (IRS) is a clustering of atherothrombotic traits associated with increased vascular risk. We investigated the degree to which the phenotypic correlations between these traits are due to shared genetic and environmental factors. A multivariate genetic analysis was performed in 537 adults from 89 healthy white north European families. All traits showed significant heritability. BMI had significant genetic correlations with fasting insulin, systolic blood pressure (sBP), plasminogen activator activator inhibitor-1 (PAI-1) and fibrinogen and triglyceride. Fasting insulin had a significant genetic correlation with fibrinogen and triglyceride and Factor VII (FVII). Significant genetic correlations were shown between triglyceride and PAI-1, fibrinogen and FVII. PAI-1 and tissue plasminogen activator (t-PA) showed significant genetic correlation with sBP and with each other. Pleiotropy was demonstrated between fibrinogen and PAI-1, t-PA and FVII. Significant environmental correlations were also demonstrated. This study demonstrates pleiotropy between coagulation and fibrinolytic factors. Shared genetic and environmental factors influencing haemostatic, metabolic and anthropometric traits underlie the atherothrombotic nature of the IRS.

Three endothelial cysts and five hemorrhagic cysts (pseudocysts) arose in the adrenal glands of seven patients aged 23-73 years. Four patients were male and three were female. Five were symptomatic and gave abdominal pain as their chief complaint. Endothelial cysts were collapsed or filled with serous fluid, multiloculated, had an endothelial lining, and often contained adrenal cortex in their outer walls. The endothelial lining reacted only weakly for Factor VIII-related antigen (FVII-RAg), but it stained strongly for collagen type IV (C-IV). The lack of hemorrhage and the FVIII-RAg/C-IV staining pattern in endothelial cysts suggest lymphatic differentiation. Hemorrhagic cysts were spherical, firm masses containing clotted blood and hyalinized thrombus with attenuated adrenal cortex in the outer fibrous wall. Islands of intact cortical cells were present deep within the thrombi of four hemorrhagic cysts. Three of five hemorrhagic cysts stained strongly for FVIII-RAg and C-IV in irregular vascular channels of the attenuated cortex and within the cyst contents. These channels suggest that at least some hemorrhagic cysts arise when hemorrhage occurs in a preexisting blood vascular anomaly. Entrapment of cortical islands by extravasated blood in hemorrhagic cysts may be misdiagnosed as necrotic cortical neoplasm. To avoid confusion, one must recognize the normality of the entrapped cortical cells, identify an intrinsic vascular anomaly, and distinguish thrombus from necrotic tumor.

The relationship between coagulation factor VII (FVII) levels in plasma and FVII genotypes, determined by three polymorphisms (5'F7, IVS7, and 353R/Q), were studied in 500 control subjects enrolled in European multicenter study. The selection of particular FVII genotypes and the analysis of variance clearly indicated the independent contribution of a single 5'F7 insertion (A2) or 353Q (M2) allele to lowering plasma levels of activated FVII (FVIIa) (by a mean 25%). The M2 allele alone was found to make a major contribution to the genetically determined component of the FVIIa levels. Genotypes associated with low FVII levels were significantly rarer in the northern part of Europe (Oslo) than in the southern part (Rome, Murcia). The contribution made by the FVII genotype to the total variance of FVIIa levels was higher (30%) than that made to either FVII activity (25%) or FVII antigen (12%). Subjects with different FVII genotypes showed up to fivefold differences in mean FVIIa values, thus allowing attribution of a substantial part of the considerable interindividual variation to genetic variation, which may be of assistance in the interpretation of FVIIa levels on an individual basis. When FVII levels were adjusted by age and by triglyceride levels, the contribution of FVII genotypes to the FVII phenotypic variance was virtually unchanged. Taken together, these data indicate that in healthy control subjects the FVII genotype is a major predictor of plasma FVIIa levels and would support further study on the role of FVII genetic components in the development of cardiovascular disease.

Exaggerated postprandial lipemia is believed to be atherogenic and to influence risk of thrombosis. The postprandial effects on plasma triacylglycerol concentration, factor VII coagulant activity (FVII(c)) and activated FVII concentration (FVII(a)) of five high fat meals (5.2 MJ, 90 g fat) enriched with medium triacylglycerols (MCT, 8:0+10:0), palmitate(16:0), stearate (18:0), elaidate(18:1 trans) and oleate(18:1 cis) were compared with those following a low fat meal (5.2 MJ,10 g fat) in 16 healthy subjects using a randomized crossover design. Postprandial lipemia measured as the area under the curve (AUC arbitrary units) for plasma triacylglycerol concentration (mean+/-SE) was greater following the oleate (5.8+/-1. 05), elaidate (4.3+/-0.79) and palmitate (4.1+/-0.64) meals compared with stearate (2.0+/-0.45) and MCT (1.1+/-0.47) meals. Fatty acid analyses of the chylomicron lipids suggested that approximately one fifth of the dietary stearate was not absorbed. FVII(c) increased following the oleate, elaidate and palmitate meals and fell following the low fat meal; the increase in FVII(c) was correlated with the AUC for plasma TAG (r=0.34; P=0.001). FVII(a) concentration increased following all high fat meals but not following the low fat meal. The increase in FVII(a) at 7 h was greater after the oleate meal than after the stearate and MCT meals. These results do not support the hypothesis that dietary stearate and elaidate are responsible for the postprandial increases in FVII associated with high fat intakes.

BACKGROUND

Elevated fibrinogen, activated factor XII (FXIIa), and factor VII coagulant activity (FVIIc) are associated with higher risk of fatal ischemic heart disease. This study tested the hypothesis that lowering the dietary ratio of n-6 to n-3 polyunsaturated fatty acids (n-6:n-3) would modify these risk factors in older men and women.

OBJECTIVE

The objective of the study was to measure fasting hemostatic risk factors and postprandial changes in activated FVII (FVIIa) concentrations after a 6-mo alteration in dietary n-6:n-3.

METHODS

In a randomized, parallel design in 258 subjects aged 45-70 y, we compared 4 diets providing 6% of energy as polyunsaturated fatty acids at an n-6:n-3 between 5:1 and 3:1 with a control diet that had an n-6:n-3 of 10:1. The diets were enriched in alpha-linolenic acid, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid, or both.

RESULTS

Fasting and 3-h plasma triacylglycerol concentrations were 11.1% and 7.2% lower with the diet that had an n-6:n-3 of approximately 3:1 and that was enriched with EPA and DHA than with the other diets. Fasting fibrinogen, FXIIa, FVIIc, FVIIa, and FVII antigen and postprandial FVIIa were not influenced by the diets. Avoiding foods high in fat the day before measurement decreased FVIIc and FVIIa by 8% and 19.2%, respectively. A test meal containing 50 g fat resulted in a mean 47% (95% CI: 42%, 52%) increase in FVIIa 6 h later, but the response did not differ by n-6:n-3.

CONCLUSIONS

Decreasing the n-6:n-3 to approximately 3:1 by increasing the intake of EPA and DHA lowers fasting and postprandial plasma triacylglycerol concentrations in older persons but does not influence hemostatic risk factors.

Safe and effective delivery is required for siRNA and mRNA-based therapeutics to reach their potential. Here, we report on the development of poly(glycoamidoamine) brush nanoparticles as delivery vehicles for siRNA and mRNA. These polymers were capable of significant delivery of siRNA against FVII and mRNA-encoding erythropoietin (EPO) in mice. Importantly, these nanoparticles were well-tolerated at their effective dose based on analysis of tissue histology, systemic cytokine levels, and liver enzyme chemistry. The polymer brush nanoparticles reported here are promising for therapeutic applications.

BACKGROUND

Fresh-frozen plasma (FFP) requires thawing, which delays availability. We investigated clotting factor activity and bacterial contamination of FFP when stored at 4 degrees C +/- 2 degrees C for 6 days.

METHODS

Plasma of 20 healthy plasma donors was sampled, frozen, and analyzed at baseline and repeatedly over a period of 6 days after thawing. The activity of fibrinogen, Factor (F)II, FV, FVII, FVIII, F IX, FX, XI, FXII, FXIII, antithrombin III (ATIII), von Willebrand factor antigen (VWF-Ag), protein C (PC), and free protein S (FPS) were determined and analyzed over time.

RESULTS

Immediately after thawing there was a significant decrease of fibrinogen (-9%), FII (-7%), FV (-14%), FVII (-12%), FX (-11%), FXIII (-20%), PC (-7%), and ATIII (-4%), whereas FVIII (+8%), F IX (+1%), FXI (+11%), FXII (-1%), FPS (-1%), and VWF-Ag (-6%) remained stable without significant change. Over 6 days after thawing fibrinogen, ATIII (+2%) and VWF-Ag (+2%) remained stable whereas FXII (+2%), FXIII (+6%), and PC (+3%) changed significantly over time and increased at the end. FII (-8%), FV (-16%), FVII (-31%), FVIII (-47%), F IX (-12%), FX (-10%), FXI (-25%), and FPS (+/-0%) changed also significantly over time and decreased at the end. All clotting factors and inhibitors remained within the reference range requested by quality assurance regulations. No FFP bag showed bacterial contamination.

CONCLUSIONS

This provides evidence for maintaining quality of thawed FFP and may improve rapid availability in emergency situations and reduce cost for health care givers.

Advanced chronic heart failure (CHF) is associated with abnormal haemostasis and inflammation, but it is not known how these abnormalities are related, whether they are modified by oral anticoagulants (OAT), or if they persist after successful heart transplantation. We studied 25 patients with CHF (New York Heart Association class IV, 10 of whom underwent heart transplantation) and 25 age- and sex-matched healthy controls by measuring their plasma levels of prothrombin fragment 1 + 2 (F1 + 2), thrombin-antithrombin (TAT) complexes, tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), D-dimer, factor VII (FVII), fibrinogen, von Willebrand factor (VWF), tumour necrosis factor (TNF), soluble TNF receptor II (sTNFRII), interleukin 6 (IL-6), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), endothelial-selectin (E-selectin) and thrombomodulin. CHF patients had higher plasma levels of TAT, D-dimer, t-PA, fibrinogen, VWF, TNF, IL-6, sTNFRII, sVCAM-1 (P = 0.0001), sICAM-1 (P = 0.003) and thrombomodulin (P = 0.007) than controls. There were significant correlations (r = 0.414-0.595) between coagulation, fibrinolysis, endothelial dysfunction and inflammation parameters, which were lower in those patients treated with OATs. Heart transplantation led to reductions in fibrinogen (P = 0.001), VWF (P = 0.05), D-dimer (P = 0.05) and IL-6 levels (P = 0.05), but all the parameters remained significantly higher (P = 0.01-0.0001) than in the controls. Advanced CHF is associated with coagulation activation, endothelial dysfunction and increased proinflammatory cytokine levels. Most of these abnormalities parallel each other, tend to normalize in patients treated with OATs and, although reduced, persist in patients undergoing successful heart transplantation, despite the absence of clinical signs of CHF.

Recently developed lipid nanoparticle (LNP) formulations of siRNA have proven to be effective agents for hepatocyte gene silencing following intravenous administration with at least three LNP-siRNA formulations in clinical trials. The aim of this work was to develop LNP-siRNA systems for hepatocyte gene silencing that can be administered subcutaneously (s.c.). Three parameters were investigated, namely LNP size, residence time of the polyethylene glycol (PEG)-lipid coating and the influence of hepatocyte-specific targeting ligands. LNP sizes were varied over the range of 30 to 115 nm in diameter and PEG-lipid that dissociates rapidly (PEG-DMG) and slowly (PEG-DSG) were employed. In mice, results show that large (~80 nm) LNP exhibited limited accumulation in the liver and poor Factor VII (FVII) gene silencing at 1mg siRNA/kg body weight. Conversely, small (~30 nm) LNP systems showed maximal liver accumulation yet still had minimal activity. Interestingly, intermediate size (~45 nm) LNP containing PEG-DSG exhibited nearly equivalent liver accumulation as the smaller systems following s.c. administration but reduced FVII levels by 80% at 1mg siRNA/kg body weight. Smaller systems (~35 nm diameter) containing either PEG-DMG or PEG-DSG were less active; however addition of 0.5 mol.% of a GalNAc-PEG lipid to these smaller systems improved activity to levels similar to that observed for the ~45 nm diameter systems. In summary, this work shows that appropriately designed LNP-siRNA systems can result in effective hepatocyte gene silencing following s.c administration.

Thromboembolic events are the major factor affecting the prognosis of patients with chronic kidney disease (CKD). Haemostatic alterations are possible causes of these complications, but their roles remain poorly characterised. In the prospective observational study, we investigated the entire coagulation process in patients with CKD to elucidate the mechanisms of their high thromboembolic risk.

A total of 95 patients with CKD and 20 healthy controls who met the inclusion criteria were consecutively recruited from September 2015 to March 2016. The platelet count, platelet aggregation, von Willebrand factor antigen (vWF:Ag), vWF ristocetin cofactor activity (vWF:RCo), fibrinogen, factor V (FV), FVII, FVIII, antithrombin III, protein C, protein S, D-dimer, standard coagulation tests and thromboelastography were measured in patients with CKD and controls. Associations between the estimated glomerular filtration rate (eGFR) and haemostatic biomarkers were tested using multivariable linear regression.

The adjusted and unadjusted levels of vWF:Ag, vWF:RCo, fibrinogen, FVII, FVIII and D-dimer were significantly higher in patients with CKD than that in the healthy controls, and were elevated with CKD progression. However, after adjustment for baseline differences, platelet aggregation and thromboelastography parameters showed no significant differences between patients with CKD and healthy controls. In the correlation analysis, vWF:Ag, vWF:RCo and FVIII were inversely associated with eGFR (r=-0.359, p<0.001; r=-0.391, p<0.001; r=-0.327, p<0.001, respectively). During the 1-year of follow-up, one cardiovascular event occurred in patients with CKD 5 stage, whereas no thromboembolic event occurred in the CKD 3 and 4 and control groups.

Patients with CKD are characterised by endothelial dysfunction and increased coagulation, especially FVIII activity. The abnormal haemostatic profiles may contribute to the elevated risk of thrombotic events but further longer-term study with large samples is still required to more precisely determine the relationship between the elevation of procoagulant factors and clinical outcomes.

OBJECTIVE

Although the strong association between hyperprolactinaemia and platelet aggregation is well recognized, there are no studies on changes in coagulation and fibrinolytic status in patients with prolactinoma. To our knowledge, tissue plasminogen activator inhibitor-1 (PAI-1), plasma tissue factor pathway inhibitor (TFPI) and thrombin-activatable fibrinolysis inhibitor (TAFI) levels in these patients have not been investigated. Therefore, the main purpose of this study was to evaluate the markers of endogenous coagulation/fibrinolysis, including TFPI and TAFI, and to investigate the relationships between prolactin (PRL) and these haemostatic parameters and serum lipid profile in patients with prolactinoma.

METHODS

Twenty-two patients with untreated, newly diagnosed prolactinoma and 20 age-matched healthy controls were included in the study. Platelet count, mean platelet volume, prothrombin time, activated partial thromboplastin time, fibrinogen, factors V, VII, VIII, IX and X activities, von Willebrand factor, antithrombin III (AT-III), protein C, protein S, tissue plasminogen activator (t-PA), PAI-1, TFPI and TAFI, as well as common lipid variables, were measured. The relationships between serum PRL and these haemostatic parameters were evaluated.

RESULTS

Compared with the control subjects, total cholesterol, low density lipoprotein cholesterol, apolipoprotein B, platelet count, fibrinogen, AT-III, PAI-1 and PAI-1/t-PA ratio were significantly increased in patients with prolactinoma (P < 0.0001, P < 0.001, P < 0.05, P < 0.05, P < 0.0001, P < 0.05, P < 0.0001 and P < 0.0001, respectively), whereas TFPI levels were significantly decreased (P < 0.01). Plasma TAFI Ag levels were not significantly different in patients with prolactinoma compared with the controls. In patients with prolactinoma, serum PRL was positively correlated with plasma FVII levels and apo B (r: 0.679, P < 0.05; r: 0.548, P < 0.05, respectively).

CONCLUSIONS

We found some important differences in the haemostatic parameters between the patients with prolactinoma and healthy controls. Increased platelet count, fibrinogen, PAI-1 and decreased TFPI in patients with prolactinoma may represent a potential hypercoagulable and hypofibrinolytic state, which might augment the risk for atherosclerotic and atherothrombotic complications. Thus, disturbances of the haemostatic system and dyslipidaemia may lead to the excess mortality in patients with prolactinoma.

The management of bleeding in haemophilia patients with inhibitors can be challenging when using monotherapy with either activated prothrombin complex concentrate (APCC) or recombinant activated FVII (rFVIIa) fail. The antifibrinolytic agent tranexamic acid (TXA) increases clot stability and is used concomitantly with coagulation factor replacement to improve haemostasis in haemophilia patients without inhibitors in many countries in Europe. Combined treatment with TXA and rFVIIa is not contraindicated in haemophilia patients with inhibitors. However, the combined approach of TXA and APCC has not been investigated due to safety concerns of increased risk of thrombosis or disseminated intravasal coagulation (DIC). The aim of this study is to report our experience of concomitant use of APCC and TXA in haemophilia A patients with inhibitor and in patients with acquired haemophilia A with respect to safety and efficacy. Seven (n = 6) haemophilia A patients with inhibitors and one (n = 1) with acquired haemophilia A from Oslo (Norway) and Stockholm (Sweden) were included in the study. The APCC was given at doses consistent to the manufacturers' recommendation. TXA was administered concomitantly either 10 mg kg(-1) every 6-8 h intravenously or 20 mg kg(-1) every 6-8 h orally. Haemostatic response was assessed by thromboelastography (TEG) and thrombin generation assay (TGA) in three of the patients. A total number of three bleeding episodes and two minor and six major surgical procedures were performed under the coverage with APCC and TXA. Haemostatic outcome was rated excellent or good in 10 of 11 (91%) treatment episodes. One episode was rated with poor effect. No episodes of arterial, venous thrombosis or DIC occurred during or after the treatment. Data from TEG and TGA analysis showed no signs of hypercoagulability following the combined treatment. This report demonstrates that, in a limited number of patients, combined treatment with APCC and TXA seemed to be safe, tolerated and relatively effective in management of bleeding episodes and in preventing haemorrhage during surgery in haemophilia patients with inhibitors and in a patient with acquired haemophilia A. Further studies should be performed to confirm these data.

Inherited factor VII (FVII) deficiency is considered to be a haemorrhagic disease. Nonetheless, some patients paradoxically present with venous thrombosis. We assessed whether there was a link between phenotype and genotype in seven patients with inherited FVII deficiency and thrombosis (eleven venous thrombotic events). For each patient (FVII:C < 50%), clinical data were collected, aetiological assessment of risk factors for thrombosis was investigated, and direct sequencing of the nine exons and promoter of the FVII gene (F7) was performed. We present the second series ever published on FVII patients with thrombosis. In nine of the eleven thrombotic events, there was at least one classical triggering risk factor; clinical (n = 4), familial antecedent (n = 2), or biological, defined by phospholipid-binding antibodies or elevated FVIII:C levels (n = 7). In contrast to a previous series, only two events occurred after surgery, performed both with and without replacement therapy. The thrombotic event remained unexplained in one young patient, highlighting the lack of 'protection' against venous thrombosis by low FVII:C levels. Genetic mutations were found to be heterogeneous. Among the seven F7 sequence alterations identified in the present study, only two (p.Ala354Val and p.Arg364Gln) have previously been reported in FVII-deficient patients presenting with venous thrombosis. Our genetic analyses of the F7 mutations in these patients show the complexity of FVII deficiency associated with thrombosis. These data justify a holistic, clinical and biological approach for patients with these specific symptoms. This series also strongly suggest that mild FVII deficiency should not prevent physicians from using antithrombotic prophylaxis in FVII-deficient patients.

Inflammation and thrombosis are important mechanisms in cardiovascular disease, as illustrated by the consistent association between inflammatory and hemostatic variables and the risk of cardiovascular events in epidemiological studies. However, the relationship between plasma concentrations of inflammatory and hemostatic markers and the severity of atherosclerosis is not yet well studied. We have evaluated 325 men and 370 women of 60 years, participating in the Danish Glostrup study. We diagnosed atherosclerosis by ultrasonographic measurement of intima-media thickness (IMT) of the right carotid artery and the assessment of plaque occurrence. Plasma samples were analyzed for the concentration of C-reactive protein (CRP), fibrinogen, d-dimer, plasminogen activator inhibitor type-1 (PAI-1) antigen and activity, tissue-type plasminogen activator (t-PA) antigen and activity, factor VII (FVII) antigen, FVII coagulant activity (FVII:C) and activated FVII (FVIIa). DNA variations were determined for fibrinogen, PAI-1, t-PA, FVII, factor XIII and methylene tetrahydrofolate reductase (MTHFR). Subjects with high IMT (upper 10% of distribution, n = 63) had higher CRP levels [2.2 mg L-1 (SE 0.3)] than subjects with IMT in the lowest tertile (n = 217) [1.7 mg L-1 (SE 0.1), P = 0.04], whereas there was no association between the hemostatic variables and IMT. There was an association between fibrinogen and d-dimer concentrations and number of plaques (P < 0.01), whereas there were no associations between CRP and the other hemostatic variables and the number of plaques. Genetic variation in the t-PA and MTHFR gene was associated with IMT. In conclusion, in the Glostrup population study, thrombosis and inflammation are associated with the severity of atherosclerosis, as reflected by IMT and plaque occurrence.

Factor VII (FVII) deficiency is a rare inheritable bleeding disorder affecting 1/500 000 individuals. Clinical manifestations are heterogeneous, from asymptomatic to severe and potentially fatal bleeding. These clinical manifestations do not correlate well with FVII plasma levels. For this reason, FVII-deficient patient management during surgery or for long-term prophylaxis remains challenging. Laboratory testing for FVII activity is, however, the first-line method for FVII deficiency diagnosis and is helpful for managing patients in combination with clinical history. Additional testing consists of FVII immunoassay and genetic testing. Genetic abnormalities on the FVII gene are heterogeneous and can translate into quantitative or qualitative defects. Some of the latter can react differently with different thromboplastins; this can be misleading for the laboratory as no consensus exists at present on an FVII deficiency diagnosis methodology. Indeed, no single test is able to predict accurately the bleeding risk. This review provides a broad picture of inherited and acquired FVII deficiency with a particular focus on laboratory diagnosis.

Health economic evaluations provide valuable information for healthcare providers, facilitating the treatment decision-making process in a climate where demand for healthcare exceeds the supply. Although an uncommon disease, haemophilia is a life-long condition that places a considerable burden on patients, healthcare systems and society. This burden is particularly large for patients with haemophilia with inhibitors, who can develop serious bleeding complications unresponsive to standard factor replacement therapies. Hence, bleeding episodes in these patients are treated with bypassing agents such as recombinant activated FVII (rFVIIa) and plasma-derived activated prothrombin complex concentrates (pd-APCC). With the efficacy of these agents now well established, a number of health economic studies have been conducted to compare their cost-effectiveness for the on-demand treatment of bleeding episodes in haemophiliacs with inhibitors. In a cost-utility analysis, which assesses the effects of treatment on quality of life (QoL) and quantity of life, the incremental cost per quality-adjusted life-year (QALY) gained (US $44,834) indicated that rFVIIa was cost-effective. Similarly, eight of 11 other economic modelling evaluations found that rFVIIa was more cost-effective than pd-APCC in the on-demand treatment of bleeding episodes. These findings indicate that treating patients with haemophilia promptly and with the most effective therapy available may result in cost savings.

Adenoviruses (Ads) are promising vectors for therapeutic interventions in humans. When injected into the bloodstream, Ad vectors can bind several vitamin K-dependent blood coagulation factors, which contributes to virus sequestration in the liver by facilitating transduction of hepatocytes. Although both coagulation factors FVII and FX bind the hexon protein of human Ad serotype 5 (HAdv5) with a very high affinity, only FX appears to play a role in mediating Ad-hepatocyte transduction in vivo. To understand the discrepancy between efficacy of FVII binding to hexon and its apparently poor capacity for supporting virus cell entry, we analyzed the HAdv5-FVII complex by using high-resolution cryo-electron microscopy (cryo-EM) followed by molecular dynamic flexible fitting (MDFF) simulations. The results indicate that although hexon amino acids T423, E424, and T425, identified earlier as critical for FX binding, are also involved in mediating binding of FVII, the FVII GLA domain sits within the surface-exposed hexon trimer depression in a different orientation from that found for FX. Furthermore, we found that when bound to hexon, two proximal FVII molecules interact via their serine protease (SP) domains and bury potential heparan sulfate proteoglycan (HSPG) receptor binding residues within the dimer interface. In contrast, earlier cryo-EM studies of the Ad-FX interaction showed no evidence of dimer formation. Dimerization of FVII bound to Ad may be a contributing mechanistic factor for the differential infectivity of Ad-FX and Ad-FVII complexes, despite high-affinity binding of both these coagulation factors to the virus.

Factor (F) XII, a key component of the contact system, triggers clotting via the intrinsic pathway, and is implicated in propagating thrombosis. Although nucleic acids are potent activators, it is unclear how the contact system is regulated to prevent uncontrolled clotting. Previously, we showed that histidine-rich glycoprotein (HRG) binds FXIIa and attenuates its capacity to trigger coagulation. To investigate the role of HRG as a regulator of the intrinsic pathway, we compared RNA- and DNA-induced thrombin generation in plasma from HRG-deficient and wild-type mice. Thrombin generation was enhanced in plasma from HRG-deficient mice, and accelerated clotting was restored to normal with HRG reconstitution. Although blood loss after tail tip amputation was similar in HRG-deficient and wild-type mice, carotid artery occlusion after FeCl3 injury was accelerated in HRG-deficient mice, and HRG administration abrogated this effect. To confirm that HRG modulates the contact system, we used DNase, RNase, and antisense oligonucleotides to characterize the FeCl3 model. Whereas DNase or FVII knockdown had no effect, carotid occlusion was abrogated with RNase or FXII knockdown, confirming that FeCl3-induced thrombosis is triggered by RNA in a FXII-dependent fashion. Therefore, in a nucleic acid-driven model, HRG inhibits thrombosis by modulating the intrinsic pathway of coagulation.

The procoagulant subcellular matrix of stimulated endothelial cells that contains tissue factor (TF) was used to investigate the mechanism by which TF pathway inhibitor (TFPI) inhibits thrombin formation initiated by TF/factor VIIa (FVIIa) under flow conditions. Purified coagulation factors VII, X, and V and prothrombin were perfused at a wall shear rate of 100 s-1 through a flow chamber containing a coverslip covered with matrix of cultured human umbilical vein endothelial cells. This resulted in a TF- and FVII-dependent FXa and thrombin generation as measured in the effluent at the outlet of the system. Inhibition of this TF/FVIIa-triggered thrombin formation by TFPI purified from plasma was dependent on the amount of TF present on the endothelial cell matrix. The rate of prothrombinase assembly and steady-state levels of thrombin formation were decreased by TFPI. Because persistent albeit decreased steady-state levels of thrombin formation occurred in the presence of TFPI, we conclude that plasma-TFPI does not inhibit FXa present in the prothrombinase complex. The addition of FIX and FVIII to perfusates containing FVII and FX increased the FXa generation on endothelial matrices, and counteracted the inhibition of thrombin formation on endothelial cell matrices by TFPI. Our data provide further evidence for the hypothesis that the rapid inactivation of TF/FVIIa by TFPI in combination with the absence of either FVIII or FIX causes the bleeding tendency of patients with hemophilia A or B.

Excessive bleeding represents a major complication of surgical interventions and its control is especially relevant in patients with Congenital Bleeding Disorders (CBD). In factor VII (FVII) deficiency, scanty data on surgery is available to guide treatment strategies. The STER (Seven Treatment Evaluation Registry) is a multi-centre, prospective, observational, web-based study protocol providing the frame for a structured and detailed data collection. Inhibitor occurrence was checked in a centralized fashion. Forty-one surgical operations (24 'major' and 17 'minor') were performed in 34 subjects with a carefully characterized FVII deficiency under the coverage of recombinant activated Factor VII (rFVIIa). Bleeding occurred during three major interventions of orthopaedic surgery, but rFVIIa was given at very low dose in each case. An antibody to FVII was observed in one patient who underwent a multiple dental extraction. No thromboses were reported during the 30-d follow up period. Replacement therapy with rFVIIa proved effective when suitable doses were used, which, during the period of maximum bleeding risk (the day of operation), were calculated (Receiver Operated Characteristic analysis) to be of at least 13 μg/kg/body weight per single dose and no less than three administrations. This indication is important especially in the case of major surgery.

Blast cell extracts from patients with acute non lymphoid leukemia (ANLL) express cancer procoagulant (CP). This factor X (FX) activator is distinct from tissue factor (TF) in that it does not require factor VII (FVII) to trigger blood coagulation, it acts as a cysteine proteinase and is not present in normal mononuclear cells. To assess whether there is any relationship between the presence of CP and the status of the disease, ANLL patients have been studied at diagnosis, during remission, at relapse. The procoagulant activity in either the presence or absence of F VII and sensitivity to cysteine proteinase inhibitors were tested on cell extracts. Immunoreactivity was explored with an anti-CP polyclonal antibody. Data obtained in 91 newly-diagnosed ANLL patients (subtypes M1 to M5, FAB classification) confirmed the presence of CP in M1 to M4 groups (mean +/- SE FVII-independent activity: M1 = 2.1 +/- 0.7 unit/mg; M2 = 5.7 +/- 1.7 unit/mg; M3 = 31.5 +/- 8 unit/mg; M4 = 1.6 +/- 1.2 unit/mg); CP was absent in the M5 type. In eight patients analyzed in a subsequent phase of partial remission, specific activity had dropped from 26.9 +/- 7.8 to 10.5 +/- 4.0 unit/mg. Activity was virtually absent (0-0.05 unit/mg) in the bone marrow of 37 patients studied at complete remission. Bone marrow samples from six subjects tested at different intervals after complete remission were repeatedly negative for CP but became positive 2 to 5 months before relapse. Upon relapse, the FVII independent activity rose to 24.2 +/- 8.2 unit/mg.(ABSTRACT TRUNCATED AT 250 WORDS)

BACKGROUND

Acquired haemophilia A (AHA) is a rare, often severe, auto-immune bleeding disorder caused by the development of inhibitory antibodies (inhibitors) to factor VIII (FVIII). Bypassing agents, recombinant activated FVII or activated prothrombin complex concentrate, are currently recommended as first-line treatments to control bleeding events in patients with AHA.

OBJECTIVE

A plasma-derived porcine FVIII (Hyate:C, Ipsen, UK) was used as a first-line treatment for AHA but was discontinued in 2004 due to viral safety concerns. A recombinant pFVIII (rpFVIII), Obizur (OBI-1; BAX801), which is expected to have a similar efficacy profile to Hyate:C but with a superior safety profile was developed and recently approved by the US Food and Drug Administration for the treatment of AHA.

METHODS

Obizur manufacturing begins with the expression of B domain deleted rpFVIII by genetically modified baby hamster kidney-derived cells. The final purified and lyophilized drug product has a negligible risk of viral contamination and contains no animal-derived plasma proteins. Obizur was evaluated for immunogenicity, tolerability, pharmacokinetics and bleeding times in preclinical models including in haemophiliac dogs, cynomolgus monkeys and FVIII-knockout mice.

RESULTS

Preclinical animal studies show that the efficacy and immunogenicity of Obizur are similar to that of Hyate:C and that Obizur has a more favourable safety profile.

CONCLUSIONS

Obizur is a highly purified recombinant porcine FVIII drug product that has been demonstrated to have a favourable safety and efficacy profile when compared with Hyate:C and can be a valuable treatment option for control of bleeding in AHA patients.

Haemostasis is initiated by the complex formed by tissue factor (TF) and activated factor VII (FVIIa) present in the blood [1% of the factor VII (FVII) protein]. Recombinant FVIIa (rFVIIa), enzymatically active only after complex formation with TF exposed following tissue damage, has been demonstrated to induce haemostasis in haemophilia patients with life- and limb-threatening bleedings with an efficacy rate of 76-84% in patients having failed on other treatment. rFVIIa has been successfully used in patients with congenital FVII deficiency and has been demonstrated to normalize the prolonged prothrombin time in patients with liver disease and in warfarin-treated individuals. In patients with thrombocytopenia, rFVIIa shortened the prolonged bleeding time in 50% of the patients treated and a haemostatic effect on acute bleeds in eight patients was demonstrated. One patient with Glanzmann's thrombasthenia and three with Type III von Willebrand's disease were successfully treated with rFVIIa. By exploiting the binding capacity of FVIIa to platelets, rFVIIa, may also be used to enhance normal haemostasis in patients without coagulation defects but suffering from bleedings in organs or at sites with limited possibilities for mechanical haemostasis.

Hemophilia A is a rare X-linked bleeding disorder caused by lack or dysfunction of coagulation factor VIII (FVIII). Hemophilia A is treated with replacement therapy, but frequent injections of the missing FVIII often lead to the formation of inhibitory antibodies. Patients who develop high levels of inhibitors must be treated with bypassing agents such as activated FVII (FVIIa). Both FVIII and FVIIa have short half-lives and require multiple injections. Long-acting forms of these proteins would therefore reduce the frequency of injections, improve patient compliance and reduce complications. In this article we present a new platform technology that produces long-acting forms of FVIII and FVIIa and improves the efficacy of hemophilia treatment. This technology is based on the binding of proteins/peptides to the outer surface of PEGylated liposomes (PEGLip). Binding is dependent on an amino acid consensus sequence within the proteins and is highly specific. At the same time, binding is non-covalent and does not require any modification of the therapeutic agent or its production process. Association of proteins with PEGLip results in substantial enhancements in their pharmacodynamic properties following administration. These improvements seem to arise from the association of formulated proteins with platelets prior to induction of coagulation.