Modifications of the Prothrombin Active Site S4 Subpocket Confer Resistance to Dabigatran.

Direct anticoagulants inhibit coagulation serine proteases by reversibly engaging their active site with high affinity. By modifying the S4 active site subpocket of factor (F)Xa, we previously introduced inhibitor-resistance while preserving catalytic activity. Given the homology between FXa and thrombin in active site architecture and direct anticoagulant binding, we have targeted the S4 subsite to introduce inhibitor resistance in (pro)thrombin.

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 a 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 mutations that decrease the activity of factor IX.

Minimally modified human blood coagulation factor X to bypass direct factor Xa inhibitors.

Background: Direct oral factor (F)Xa inhibitors are widely used as alternatives to conventional vitamin K antagonists in managing venous thromboembolism and nonvalvular atrial fibrillation. Unfortunately, bleeding-related adverse events remain a major concern in clinical practice. In case of bleeding or emergency surgery, rapid-onset reversal agents may be required to counteract the anticoagulant activity.

Genetic polymorphisms and major bleeding risk during vitamin K antagonists treatment: The BLEEDS case-cohort.

Background: Major bleeding occurs annually in 1%-3% of patients on vitamin K antagonists (VKAs), despite close monitoring. Genetic variants in proteins involved in VKA response may affect this risk.

Aim: To determine the association of genetic variants (cytochrome P450 enzymes 2C9 [CYP2C9] and 4F2 [CYP4F2], gamma-glutamyl carboxylase [GGCX]) with major bleeding in VKA users, separately and combined, including vitamin K epoxide reductase complex subunit-1 (VKORC1).

A factor IX variant that functions independently of factor VIII mitigates the hemophilia A phenotype in patient plasma.

Background: Recombinant factor (F)IX-FIAV has previously been shown to function independently of activated FVIII (FVIIIa) and ameliorate the hemophilia A (HA) phenotype in vitro and in vivo.

Objectives: The aim of this study was to assess the efficacy of FIX-FIAV in plasma from HA patients using thrombin generation (TG) and intrinsic clotting activity (activated partial thromboplastin time [APTT]) analyses.

Methods: Plasma obtained from 21 patients with HA (>18 years; 7 mild, 7 moderate, and 7 severe patients) was spiked with FIX-FIAV.

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.

Lower-leg injury and knee arthroscopy have distinct effects on coagulation.

It is unknown how lower-leg injury and knee arthroscopy, both associated with venous thromboembolism (VTE), affect coagulation. To study the effect of (1) lower-leg trauma and (2) knee arthroscopy on coagulation, plasma samples of the Prevention of Thrombosis following CAST immobilization (POT-CAST, #NCT01542762) and Prevention of Thrombosis following Knee Arthroscopy (POT-KAST, #NCT01542723) trials were used, which were collected shortly after lower-leg trauma and before/after (<4 hours) knee arthroscopy. For aim 1, 1204 lower-leg injury patients were compared with preoperative samples of 1001 controls.

Thrombin in complex with dabigatran can still interact with PAR-1 via exosite-I and instigate loss of vascular integrity.

Background: Atrial fibrillation (AF) can lead to the loss of microvascular integrity thereby enhancing AF progression. Mechanistically, the pro-coagulant state that drives the risk of stroke in patients with AF may also play a causal role in microvascular loss. Direct oral anticoagulants (DOACs), the preferred anticoagulants for AF, can target factors upstream (factor Xa [FXa]) or downstream (thrombin) in the coagulation cascade and mediate differential vascular effects through interaction with protease-activated receptors (PARs).

Major bleeding during oral anticoagulant therapy associated with factor V activation by factor Xa.

Objective: Plasma thrombin generation (TG) provides important information on coagulation status; however, current TG output parameters do not predict major bleeding of patients on anticoagulants. We recently reported that factor V (FV) activation by factor X (FX)a contributes importantly to the initiation phase of TG. Here we investigated how this pathway varies in the normal population and whether FXa-mediated activation of FV is associated with major bleeding in patients on anticoagulant therapy.

Pharmacological Characterisation of and 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 and 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.

ptFVa ( 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.

Structure-function of anticoagulant TIX-5, the inhibitor of factor Xa-mediated FV activation.

Background: The prothrombinase complex consists of factors Xa (FXa) and Va (FVa) on an anionic phospholipid surface and converts prothrombin into thrombin. Both coagulation factors require activation before complex assembly. We recently identified TIX-5, a unique anticoagulant tick protein that specifically inhibits FXa-mediated activation of FV.

Elevated anti-human factor Xa activity in rabbit and rodent plasma: Implications for preclinical assessment of human factor X in animal models of hemostasis.

A wide variety of animal models on thrombosis and hemostasis are used in thrombosis and hemostasis research for the preclinical assessment of hemostatic agents. While the vertebrate coagulome is highly conserved, human and animal plasmas differ considerably when evaluated in coagulation assays such as prothrombin time (PT), activated partial thromboplastin time (APTT), and calibrated automated thrombography (CAT). Here, we have aimed to provide a reference framework for the evaluation of coagulation assays and inhibition of activated human FXa (hFXa) in various animal plasmas.

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).

High Soluble Thrombomodulin Is Associated with an Increased Risk of Major Bleeding during Treatment with Oral Anticoagulants: A Case-Cohort Study.

Background:  Major bleeding occurs in 1 to 3% of patients treated with oral anticoagulants per year. Biomarkers may help to identify high-risk patients. A proposed marker for major bleeding while using anticoagulants is soluble thrombomodulin (sTM).

Evolutionary Adaptations in Pseudonaja Textilis Venom Factor X Induce Zymogen Activity and Resistance to the Intrinsic Tenase Complex.

The venom of the Australian snake comprises powerful prothrombin activators consisting of factor X (v-ptFX)- and factor V-like proteins. While all vertebrate liver-expressed factor X (FX) homologs, including that of , comprise an activation peptide of approximately 45 to 65 residues, the activation peptide of v-ptFX is significantly shortened to 27 residues. In this study, we demonstrate that exchanging the human FX activation peptide for the snake venom ortholog impedes proteolytic cleavage by the intrinsic factor VIIIa-factor IXa tenase complex.

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.

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.

Enhanced functional recombinant factor IX production by human embryonic kidney cells engineered to overexpress VKORC1.

Replacement therapy with recombinant drugs is the main therapeutic strategy for hemophilia B patients. To reduce the production costs of recombinant coagulation factors, improvement of their expression and activity by enhancement of γ-carboxylation might be of interest. The expression and functional activity of vitamin K-dependent (VKD) coagulation proteins rely, in part, on the VKD process of γ-carboxylation that is mediated by the enzymes γ-carboxylase and vitamin K epoxide reductase (VKOR).

Improved activity and expression of recombinant human factor IX by propeptide engineering.

Purpose: The main therapeutic strategy for Hemophilia B patients involves the administration of recombinant coagulation factors IX (rFIX). Although there are various approaches to increasing the activity of rFIX, targeted protein engineering of specific residues could result in increased rFIX activity through enhanced γ-carboxylation. Specific amino acids in the propeptide sequence of vitamin K-dependent proteins are known to play a role in the interaction with the enzyme γ-carboxylase.