Blood coagulation factor IX: structural insights impacting hemophilia B therapy.

Coagulation factor IX plays a central role in hemostasis through interaction with factor VIIIa to form the factor X-activating complex at the site of injury. The absence of factor IX activity results in the bleeding disorder hemophilia B. This absence of activity can arise either from a lack of circulating factor IX protein or from mutations that decrease the activity of factor IX. This review focuses on analyzing the structure of factor IX with respect to molecular mechanisms that are at the basis of factor IX function. Proteolytic activation of factor IX to activated factor IX(a) and subsequent structural rearrangements are insufficient to generate fully active factor IXa. Multiple specific interactions between factor IXa, the cofactor VIIIa, and physiological substrate factor X further alter the factor IXa structure to realize the full enzymatic activity of factor IXa. Factor IXa also interacts with inhibitors, extravascular proteins, and cellular receptors that clear factor IX(a) from circulation. Hemophilia B is treated by replacement of the missing factor IX by plasma-derived protein, a recombinant bioequivalent, or via gene therapy. An understanding of how the function of factor IX is tied to structure is leading to modified forms of factor IX that have increased residence time in circulation, higher functional activity, protection from inhibition, and even activity in the absence of factor VIIIa. These modified forms of factor IX have the potential to significantly improve therapy for patients with hemophilia B.

ptFVa (Pseudonaja Textilis Venom-Derived Factor Va) Retains Structural Integrity Following Proteolysis by Activated Protein C.

OBJECTIVE: The Australian snake venom ptFV (Pseudonaja textilis venom-derived factor V) variant retains cofactor function despite APC (activated protein C)-dependent proteolysis. Here, we aimed to unravel the mechanistic principles by determining the role of the absent Arg306 cleavage site that is required for the inactivation of FVa (mammalian factor Va). APPROACH AND RESULTS: Our findings show that in contrast to human FVa, APC-catalyzed proteolysis of ptFVa at Arg306 and Lys507 does not abrogate ptFVa cofactor function. Remarkably, the structural integrity of APC-proteolyzed ptFVa is maintained indicating that stable noncovalent interactions prevent A2-domain dissociation. Using Molecular Dynamics simulations, we uncovered key regions located in the A1 and A2 domain that may be at the basis of this remarkable characteristic. CONCLUSIONS: Taken together, we report a completely novel role for uniquely adapted regions in ptFVa that prevent A2 domain dissociation. As such, these results challenge our current understanding by which strict regulatory mechanisms control FVa activity.

Effect of prepropeptide replacement on γ-carboxylation and activity of recombinant coagulation factor IX.

Based on observations indicating that the γ-carboxylase enzyme has a lower affinity for the protein C (PC) propeptide and that the γ-carboxylase region in the PC propeptide has a higher net charge, expression of recombinant chimeric factor IX (FIX) equipped with the PC propeptide was studied. The prepropeptide of FIX was replaced with that of PC by SOEing PCR and after cloning, recombinant pMT-prepro PC/FIX was transfected into insect Drosophila S2 cells. The expression and activity of expressed FIX were analyzed employing antigen and activity analyses 72 h of post-induction with copper. Higher secretion (1.2 fold) and activity (1.6 fold) levels were observed for chimeric prepro- PC/FIX in relation to wild-type FIX. Furthermore, after barium citrate precipitation, the evaluation of fully γ-carboxylated FIX indicated that more than 51% of the total FIX produced with the PC prepropeptide was fully γ-carboxylated, representing a substantial improvement (twofold) over a system employing the native FIX propeptide in which 25% of the protein is fully γ-carboxylated. The data illustrated that the expression of FIX using the PC propeptide led to much higher fully γ-carboxylated material, which is preferred to FIX constructs tolerating the sequence for the native FIX propeptide expressed in heterologous S2 systems.

Association Between Hepatic Triglyceride Content and Coagulation Factors: The Netherlands Epidemiology of Obesity Study.

OBJECTIVE: Whether hepatic triglyceride content (HTGC) contributes to hypercoagulability beyond total body fat (TBF) and visceral adipose tissue (VAT) is unclear. We, therefore, aimed to investigate the association between HTGC and coagulation factors (F)I (fibrinogen), VIII, IX, and XI while adjusting for TBF and VAT. Approach and Results: In this cross-sectional analysis of the NEO study (Netherlands Epidemiology of Obesity; n=6671), a random subset of participants underwent magnetic resonance imaging and magnetic resonance spectroscopy to assess VAT and HTGC (n=2580). We excluded participants without complete imaging and coagulation assessment, and with history of liver disease, venous thrombosis, or on anticoagulation. Mean differences in coagulation factor levels across HTGC quartiles were estimated by linear regression adjusted for age, sex, ethnicity, education, alcohol intake, physical activity, smoking, estrogen, and menopause, in addition to TBF and VAT. Among the 1946 participants included, median HTGC was 2.66% (interquartile range: 1.34%-6.27%). Coagulation factor levels increased dose-dependently across HTGC quartiles. Mean differences between the fourth and first quartiles were 14.7 mg/dL (95% CI, 2.1-27.2) for fibrinogen, 6.7 IU/dL (95% CI, 0.5-12.9) for FVIII, 26.1 IU/dL (95% CI, 22.4-29.8) for FIX, and 8.6 IU/dL (95% CI, 4.6-12.6) for FXI. With further adjustment for TBF and VAT, the dose-response association of HTGC with FIX persisted, whereas associations with other factors disappeared. CONCLUSIONS: HTGC was associated with various coagulation factors, of which FIX remained associated with HTGC after adjustment for TBF and VAT. HTGC might contribute to venous thrombosis risk beyond total body and visceral fat through FIX levels.

Pharmacological Characterisation of Pseudocerastes and Eristicophis Viper Venoms Reveal Anticancer (Melanoma) Properties and a Potentially Novel Mode of Fibrinogenolysis.

Venoms are a rich source of potential lead compounds for drug discovery, and descriptive studies of venom form the first phase of the biodiscovery process. In this study, we investigated the pharmacological potential of crude Pseudocerastes and Eristicophis snake venoms in haematological disorders and cancer treatment. We assessed their antithrombotic potential using fibrinogen thromboelastography, fibrinogen gels with and without protease inhibitors, and colourimetric fibrinolysis assays. These assays indicated that the anticoagulant properties of the venoms are likely induced by the hydrolysis of phospholipids and by selective fibrinogenolysis. Furthermore, while most fibrinogenolysis occurred by the direct activity of snake venom metalloproteases and serine proteases, modest evidence indicated that fibrinogenolytic activity may also be mediated by selective venom phospholipases and an inhibitory venom-derived serine protease. We also found that the Pseudocerastes venoms significantly reduced the viability of human melanoma (MM96L) cells by more than 80%, while it had almost no effect on the healthy neonatal foreskin fibroblasts (NFF) as determined by viability assays. The bioactive properties of these venoms suggest that they contain a number of toxins suitable for downstream pharmacological development as candidates for antithrombotic or anticancer agents.

Reversal Agents for the Direct Factor Xa Inhibitors: Biochemical Mechanisms of Current and Newly Emerging Therapies.

The direct oral anticoagulants targeting coagulation factor Xa or thrombin are widely used as alternatives to vitamin K antagonists in the management of venous thromboembolism and nonvalvular atrial fibrillation. In case of bleeding or emergency surgery, reversal agents are helpful to counteract the anticoagulant therapy and restore hemostasis. While idarucizumab has been established as an antidote for the direct thrombin inhibitor dabigatran, reversal strategies for the direct factor Xa inhibitors have been a focal point in clinical care over the past years. In the absence of specific reversal agents, the off-label use of (activated) prothrombin complex concentrate and recombinant factor VIIa have been suggested as effective treatment options during inhibitor-induced bleeding complications. Meanwhile, several specific reversal agents have been developed. In this review, an overview of the current state of nonspecific and specific reversal agents for the direct factor Xa inhibitors is provided, focusing on the biochemistry and mechanism of action and the preclinical assessment of newly emerging therapies.

Coagulopathy after hemorrhagic traumatic brain injury, an observational study of the incidence and prognosis.

BACKGROUND: Traumatic brain injury is associated with high rates of mortality and morbidity. Trauma patients with a coagulopathy have a 10-fold increased mortality risk compared to patients without a coagulopathy. The aim of this study was to identify the incidence of coagulopathy and relate early coagulopathy to clinical outcome in patients with traumatic intracranial hemorrhages. METHODS: Between September 2015 and December 2016, 108 consecutive cranial trauma patients with traumatic intracranial hemorrhages were included in this study. To assess the relationship between patients with a coagulopathy and outcome, a chi-squared test was performed. RESULTS: A total of 29 out of the 108 patients (27%) with a traumatic intracranial hemorrhage developed a coagulopathy within 72 h after admission. Overall, a total of 22 patients (20%) died after admission of which ten were coagulopathic at emergency department presentation. Early coagulopathy in patients with traumatic brain injury is associated with progression of hemorrhagic injury (odds ratio 2.4 (95% confidence interval 0.8-8.0)), surgical intervention (odds ratio 2.8 (95% confidence interval 0.87-9.35)), and increased in-hospital mortality (odds ratio 23.06 (95% confidence interval 5.5-95.9)). CONCLUSION: Patients who sustained a traumatic intracranial hemorrhage remained at risk for developing a coagulopathy until 72 h after trauma. Patients who developed a coagulopathy had a worse clinical outcome than patients who did not develop a coagulopathy.

Lipid levels and risk of venous thrombosis: results from the MEGA-study.

The relationship between lipid levels and risk of venous thrombosis is not well established. We aimed to assess the association between several lipids and risk of venous thrombosis using data from a population-based case-control study, and to evaluate the underlying mechanism, considering confounding by common risk factors and mediation via hemostatic factors and C-reactive protein. From the Multiple Environmental and Genetic Assessment of risk factors for venous thrombosis (MEGA) study, 2234 patients with a first venous thrombosis and 2873 controls were included. Percentile categories of total/low-density lipoprotein/high-density lipoprotein cholesterol, triglycerides, and apolipoproteins B and A1 were established in controls (<10th, 10th-25th, 25th-75th [reference], 75th-90th, >90th percentile). In age- and sex-adjusted models, decreasing levels of apolipoproteins B and A1 were dose-dependently associated with increased thrombosis risk, with odds ratios of 1.35 (95% confidence interval 1.12-1.62) and 1.50 (95% confidence interval 1.25-1.79) for the lowest category versus the reference category, respectively. The dose-response relation remained with further adjustment for body mass index, estrogen use, statin use, and diabetes. Although apolipoproteins B and A1 were associated with several hemostatic factors and C-reactive protein, none explained the increased risk in mediation analyses. The other lipids were not associated with venous thrombosis risk. In conclusion, decreasing levels of apolipoproteins B and A1 were associated with increased risk of venous thrombosis. Our findings are consistent with experimental data on the anticoagulant properties of apolipoproteins B and A1. These findings need to be confirmed and the underlying mechanism further investigated.