Journal of Thrombosis and Haemostasis最新文献

Background: Hemostasis is maintained through a delicate balance between procoagulant and anticoagulant mechanisms. Protein S (PS), a multidomain, vitamin K-dependent glycoprotein, contributes to this balance not only as a cofactor for activated protein C and tissue factor pathway inhibitor but also by directly inhibiting activated factor IX (FIXa). However, the structural determinants underlying FIXa inhibition remain unclear.

Objectives: This study aimed to (1) identify the FIXa binding interface on PS and (2) investigate the role of its laminin G (LG) domains in mediating FIXa binding and inhibition.

Methods: Molecular docking was used to predict the FIXa binding interface on PS. Fluorescence-based binding assays were performed to determine binding affinities, and functional coagulation assays were conducted to measure inhibitory constants. Finally, site-directed mutagenesis was carried out to generate specific PS mutants.

Results: Molecular docking and in vitro binding assays demonstrated that both the LG1 and LG2 domains interact with FIXa. Quantitative fluorescence-based binding analyses revealed that the LG1+2 tandem domains exhibited the highest affinity for FIXa (K_d ≈ 52.15 nM). Functional coagulation assays showed that LG1+2 effectively blocked FIXa-mediated factor X activation and suppressed thrombin generation. Furthermore, site-directed mutagenesis confirmed that residues E435 and E437 within the LG domains are critical for FIXa binding and inhibition.

Conclusion: These findings identify the LG domains of PS as essential structural elements for direct FIXa inhibition, independent of its cofactor function. By elucidating this domain-specific mechanism, our work provides a structural framework for the rational design of selective FIXa inhibitors and FIXa-binding peptides as novel antithrombotic strategies.

Background: Inversions involving intron 22 (Inv22) of the F8 gene are detected in approximately 45% of all severe hemophilia A (HA) patients. Digital droplet polymerase chain reaction using milepost assays and multiplex ligation-dependent probe amplification (MLPA) allows robust diagnosis of inversions, copy number variations, and more complex rearrangements in F8.

Objectives: A total of 350 HA patients and 280 of their mothers from the Hemophilia Inhibitor Genetics Study cohort were analyzed to identify additional F8 mutations.

Methods: Digital droplet polymerase chain reaction and MLPA were used to investigate archival samples.

Results: Of 350 patients analyzed, 13 were found to harbor previously unidentified mutations: 3 with inversions (Inv22 type 1) and 10 with large deletions. In addition, 7 patients had complex rearrangements with duplications, in addition to Inv22 type 1. Of 138 mothers of patients with inversions, 7 were noncarriers. Five of these were found to have the same duplications as those detected in their 7 sons, indicating that most, if not all, noncarrier mothers have other rearrangements in the F8 region. MLPA showed that these duplications had breakpoints in intron 22, with either duplication of the first part of the gene (exons 1-22) or the last part of the gene (exons 23-26).

Conclusion: Mutation detection in F8 can be improved by dedicated analysis for inversions and duplications/deletions using milepost assays. Noncarrier mothers of HA patients with Inv22 often have other rearrangements.

Background: Several illicit drugs have been reported to be associated with ischemic stroke and myocardial infarction. However, their underlying mechanisms remain poorly investigated.

Objectives: In this study, we focused on N-ethylpentylone (NEP), a synthetic cathinone with potent hallucinogenic properties, and explored its role in platelet activation.

Methods: Platelet function assays and flow cytometry were used to evaluate the effects of NEP on platelet aggregation, spreading, clot retraction, and integrin α_IIbβ₃ activation. The role of 5-hydroxytryptamine2A receptor (5-HT2AR) in NEP-mediated platelet responses was investigated using the selective 5-HT2AR antagonist M100907 in both ex vivo and in vivo experiments. Phosphoproteomics identified NEP-regulated phosphorylation sites, and Western blotting validated mitogen-activated protein kinase pathway activation by targeting key phosphorylation nodes.

Results: NEP significantly potentiated platelet aggregation, spreading, clot retraction, and integrin α_IIbβ₃ activation in a concentration-dependent manner. The NEP-potentiated platelet responses were suppressed by M100907 in both ex vivo and in vivo models, confirming the critical regulatory role of 5-HT2AR. Phosphoproteomics revealed NEP-triggered upregulation of phosphorylation, which was enriched in mitogen-activated protein kinase pathways. Western blotting confirmed selective extracellular signal-regulated kinase and p38 phosphorylation, with no effect on C-Jun NH2-terminal kinase.

Conclusion: NEP directly enhances agonist-induced platelet activation by targeting 5-HT2AR on platelet membranes. The findings of this study provide valuable insights for elucidating the hematotoxic mechanisms of NEP and other illicit drugs.

Thrombosis, when considering all its manifestations including myocardial infarction, stroke, and venous thromboembolism, constitutes the leading cause of death world-wide. Treatment of thrombotic diseases has been revolutionized by direct oral anticoagulants (DOACs) targeting thrombin and factor (F)Xa. However, the therapeutic or prophylactic use of DOACs is not without limitations, including persistent and significant bleeding risks. In this review, we summarize and discuss current state-of-the-art of novel and experimental anticoagulation and fibrinolytic/thrombolytic therapies beyond DOACs. In particular, we review studies investigating contact pathway inhibition, including in vitro and in vivo studies of FXIIa and FXIa inhibition, and clinical trials of contact pathway inhibition. We review in vitro and in vivo studies investigating inhibition of common pathway coagulation targets, including FV, FVIII, and FIX. This is followed by analysis of options for the therapeutic targeting of fibrin or fibrinogen and FXIII. Next, we explore opportunities for the therapeutic harnessing of naturally occurring anticoagulant pathways as well as fibrinolytic mechanisms. The current state-of-the-art research for each of these mechanisms is summarized, including whether studies have progressed from in vitro to in vivo experimentation and whether clinical trials have been performed. We highlight particularly novel areas of interest and include evaluation of the relative preclinical and clinical trial progress for the selected targets. The increased understanding of mechanisms driving thrombosis holds promise for future developments in novel anticoagulants that may contribute to reducing the impact and burden of thrombotic diseases while improving safety of current therapeutic options.