Structural insights into the exosite-mediated activation of Factor IX by Factor XIa using cryo-EM.

Cryo-EM structures of apo human Factor XIa reveal catalytic-domain flexibility and exposure of the Factor IX-binding site

Substitution-induced allosteric activation of coagulation factor IX as a potential alternative hemophilia A treatment strategy.

Targeted modification of regions surrounding the FIXa active site enables allosteric activation, providing a potential foundation for novel bypassing strategies in hemophilia A therapy.

The role of transglutaminase 2 in stabilizing fibrin clots and supporting hemostasis

Transglutaminases are enzymes with pleiotropic functions in the human body, among them are the A-subunit of coagulation factor XIII (FXIII; FXIII-A) and tissue transglutaminase (TG2). FXIII has a crucial function in clot formation and stabilization. FXIII also contributes to extracellular matrix formation, wound healing and tissue regeneration, a function it shares with TG2. Whether TG2 may also have a role in hemostasis has never been studied in detail. The aim of our project was to study the role of TG2 in clot formation, stabilization and hemostasis. Here, with the use of a microfluidic endothelialized whole blood bleeding model, we detected TG2 and its crosslinking activity at the site of vessel injury, and the addition of exogenous TG2 resulted in its co-localization with the fibrin clot. In turbidimetric clot formation and lysis tests, TG2 delayed clot lysis. Finally, mass-spectrometry and proteomic analyses revealed differences in fibrin chain crosslinking patterns and protein composition of purified and plasma fibrin clots generated in the presence of either FXIII or TG2. Our data suggest that TG2 is present at the site of clot formation upon vessel injury and contributes to fibrin crosslinking and clot stabilization. Hence, TG2 may be able support hemostasis.

Factor VIII Aurora: A Naturally Occurring Gain of Function FVIII Variant with Enhanced FIXa Affinity.

Data demonstrate that the prothrombotic phenotype of FVIII-R571S is driven by increased FIXa affinity, which enhances FVIIIa-FIXa complex assembly and function, providing a unifying explanation for the observed gain-of-function phenotype.

Computational design and evaluation of Factor Xa inhibitory peptides derived from the Tamarind Kunitz inhibitor reactive loop.

Blood coagulation is a crucial process, and its dysregulation can lead to life-threatening conditions. Factor Xa (FXA) is a well-established therapeutic target for the management of vascular diseases, as it acts as a converging point of both intrinsic and extrinsic coagulation pathways. Currently available FXA inhibitors display major side effects and toxicity, necessitating novel inhibitors. We previously reported that the inhibition of FXA from the natural source by the Tamarind Kunitz Inhibitor (TKI), a Kunitz-type protease inhibitor from Tamarindus indica seeds, inhibits FXA through its seven-residue reactive loop. In this study, in-silico mutagenesis of the TKI loop (SRARISH) was performed to design improved peptide inhibitors. A peptide library was constructed and screened using molecular docking, and the top mutants were selected and subjected to MD simulations. The binding free energies calculations, principal component analysis and free energy landscape analysis were also performed to elucidate the conformational dynamics. Selected peptides showed significantly improved binding affinities over the wild-type, with total binding free energies ranging from -16.75 to -10.59 kcal/mol compared to -6.83 kcal/mol for the wild-type. The Mutant 2 (SAWWISH) emerged as the best predicted high-affinity binder with -16.75 kcal/mol binding free. The improvement results from the elimination of the electrostatic desolvation penalty and the formation of an extended aromatic scaffold that effectively interacts with the hydrophobic sub-pockets of the FXA active site. These findings provide a strong computational foundation for the utilization of TKI-derived peptide inhibitors as natural anticoagulants against vascular diseases.

Factor V is an anticoagulant of the extrinsic pathway of coagulation and modifier of thrombin generation in hemophilia A.

FV is an endogenous anticoagulant that inhibits TF-initiated coagulation by limiting FX activation by TF:FVIIa through a membrane-dependent mechanism, which refines models of coagulation initiation and may help explain how FV variation contributes to bleeding and thrombosis.