A Zymogen-like Factor Xa Improves Hemostasis in a Murine Bleeding Model

Factor XI (FXI) deficiency is an autosomal bleeding disorder characterized by low FXI levels, resulting in bleeding after trauma or surgery. Genetic variants affecting FXI structure and function often result in bleeding diatheses.This study aimed to estimate the variant detection rate (VDR), and assess its correlation with FXI activity (FXI:C) in a large cohort of FXI-deficient patients.Genetic defects in the F11 gene were analyzed in 316 index patients (IPs) using Sanger or next-generation sequencing. Multiplex ligation-dependent probe amplification or copy number variation analysis was used to detect duplications and deletions.Genetic defects were identified in 249 IPs (VDR of 79%). A strong negative correlation (Pearson coefficient: -0.891) was found between FXI:C levels and VDRs: higher FXI:C levels corresponded to a lower likelihood of detecting genetic alterations, with a significant decline in VDR beyond 60 IU/dL. A total of 286 genetic variants were identified in F11 gene: 56% missense, 24% nonsense, 11% small deletions/insertions, and 6% splice-site variants. Large deletions were rare (3%). A total of 48 novel variants were detected. Ashkenazi Jewish founder variants were the most frequent (14.3%). Variants p.Gln134Ter, p.Ile215_Asp216del, and p.Glu315Lys (27% of cases) were recurrent. In four cases, large deletions extended beyond the F11 gene and included the neighboring KLKB1 gene, encoding prekallikrein.This study demonstrated a significant negative correlation between FXI:C levels and VDRs, underscoring the importance of genetic testing. Findings included combined deficiencies in FXI and prekallikrein due to large deletions affecting both F11 and KLKB genes.

Effects of Multimerin 1 on Platelet and Plasma FV as Determined by Calibrated Automated Thrombography.

Cardiac Myosin Acts Is a Potent Procoagulant in Vitro and In Vivo

To analyze the clinical characteristics and causative variation sites in a pedigree with hereditary coagulation factor Ⅴ (FⅤ) deficiency. A pedigree with hereditary FⅤ deficiency who visited Xijing Hospital on March 2, 2025, was selected as the study subject. Clinical data of the pedigree were collected using a retrospective analysis method. Venous blood samples were collected from the proband and family members to obtain platelet-poor plasma and genomic DNA. FⅤ activity (FⅤ:C) was measured by coagulation assay, and FⅤ antigen (FⅤ:Ag) was measured by ELISA for all members. Sanger sequencing was used to detect all exons and flanking sequences of the F5 gene in all members to screen for variations. Thrombin generation assay was performed to evaluate the overall coagulation potential of the proband and parents. Multiple online software tools, including ClustalX-2.1-win, PolyPhen-2, PROVEAN, I-Mutant v2.0, SIFT, Mutation Taster, and Swiss-Pdb Viewer, were used to assess the conservation of the variation sites and their impact on protein structure and function. The pathogenicity of the variation sites was evaluated according to the American College of Medical Genetics and Genomics (ACMG) standards and guidelines for the interpretation of sequence variants. This study was approved by the Medical Ethics Committee of Xijing Hospital (Ethics No.: KY20252367). The proband's FⅤ:C level was only 1.3%, and the FⅤ:Ag level was only 2.4%. The proband was found to carry the c.653T>C (p.Phe218Ser) variant in exon 5 and the c.5660A>T (p.Glu1887Val) variant in exon 18 of the F5 gene. According to the ACMG guidelines, these two variants were classified as "Pathogenic" (PS4+PM2_Supporting+PP1+PP3+PP4+PP5) and "Likely Pathogenic" (PM1+PM2_Supporting+PM3+PP1+PP2+PP3+PP4), respectively. The proband's father and grandfather carried the p.Phe218Ser variant in heterozygous state, and the mother and maternal grandmother carried the p.Glu1887Val variant in heterozygous state. The paternal grandmother and maternal grandfather were wild-type. Thrombin generation assay showed delayed thrombin generation and reduced thrombin output in the proband and parents, indicating impaired coagulation function. Bioinformatics analysis revealed that Phe218 and Glu1887 are highly conserved. Both the p.Phe218Ser and p.Glu1887Val variants were predicted to be deleterious, potentially disrupting local protein conformation and stability, which may lead to functional defects. The compound heterozygous variants p.Phe218Ser and p.Glu1887Val may cause a significant decrease in FⅤ:C and FⅤ:Ag by disrupting the structure and function of the FⅤ protein.

Thromboelastographic Characterization of the Activated Clotting System in Children with Sickle Cell Trait or Sickle Cell Disease

Residual platelet factor V ensures thrombin generation in patients with severe congenital factor V deficiency and mild bleeding symptoms

Prothrombin Membrane Binding and Gla-Dependent Function Are Not Required for Effective Hemostasis In Vivo

Measurement of procoagulant platelet subpopulations in whole blood: Development of an assay for population-based studies

Objective To investigate the gene defects of a pedigree with inherited coagulation factor Ⅺ (FⅪ) deficiency by analyzing its phenotype and molecular genetic characteristics. Methods A pedigree with inherited FⅪ deficiency was enrolled in this study. The activated partial thromboplastin time (APTF), prothrombin time (PT), FⅪ activity (FⅪ: C) and FⅪ antigen (FⅪ: Ag) were determined for phenotype diagnosis. Fifteen exons and their flanks of F11 gene from the proband's genomic DNA were amplified by polymerase chain reaction (PCR), and the PCR products were directly sequenced to analyze the F11 gene mutation. The PCR products amplified from genomic DNA from the proband, her parents and 100 healthy donors were digested with restriction enzyme BssSI to exclude gene polymorphism and confirm the mutation site. The cleavage site in the signal peptide was predicted by the SignalP software. Results The values of APTT, PT, FⅪ: C and FⅪ: Ag of the proband were 69.5 s, 12.3 s, 2.6% and 2.5%, respectively, indicating that this case was cross-reacting material (CRM) negative. The same values of healthy controls were 35 s, 13 s, 100% and 100%, respectively. As compared with Genbank AY191837 sequence, four variants in F11 exons were found. G3733C heterozygous mutation in exon 2 causod Gly to Arg substitution at-1 amino acid position in signal peptide (G-1R). The G3733C mutation in exon 2 introduced a new BssSI enzyme digestion site. Further analysis of the 100 randomly collected DNA samples from the normal population excluded the possibility of G3733C as a polymorphism. CI6642T heterozygous mutation in exon 8 introduced a premature stop codon at 263 amino acid position (Q263Term). Conclusions G-1R mutation and Q263Term compound heterozygous mutation in F11 gene are the mechanism of FⅪ deficiency for the proband. G-1R mutation is a novel F11 gene mutation causing inherited FⅪ deficiency. Key words: Factor Ⅺ deficiency; Blood coagulation disorders, inherited; Prothrombin; Mutation; Pedigree

Characterization of a Novel Human Factor VIIa Chimera with Increased Tissue Factor-Independent Activity for Emergency Hemostasis

Thrombelastography (TEG) and Thrombin Generation Assay (TGA) in Severe Hemophilia Following Factor Replacement Therapy

Protein Z-dependent protease inhibitor (ZPI) is an anticoagulant serpin that targets factor Xa (FXa) in the presence of protein Z (PZ), and factor XIa (FXIa). In factor-VIII-deficient mice, PZ or ZPI gene knock-out mitigates the bleeding phenotype, and pharmacological inhibition of PZ enhances thrombin generation in plasma from patients with hemophilia. To develop a single-domain antibody (sdAb) directed against ZPI to inhibit its anticoagulant activity. We screened for anti-ZPI sdAbs in a llama-derived phage display immune library of sdAbs. The sdAbs that bound ZPI were produced and purified for characterization. The binding of sdAbs to ZPI or other serpins was evaluated using ELISAs, and ZPI inhibition was measured in an anti-FXa or anti-FXIa chromogenic assay. The sdAbs's procoagulant activity was assessed in a thrombin generation assay in normal plasma, factor VIII- and FXI-deficient plasma. Of the four sdAbs found to bind to ZPI, one (referred to as ZPI-sdAb2) dose-dependently inhibited ZPI's anti-FXa and anti-FXIa activities with a mean half-maximal inhibitory concentration of 1.8 and 1.3 µM, respectively. ZPI-sdAb2 did not cross-react with other plasma serpins, such as antithrombin and α1-antitrypsin. ZPI-sdAb2 induced a significant increase in thrombin generation in plasma samples from healthy donors, patients with severe hemophilia A, and patients with FXI deficiency. ZPI-sdAb2 is the first specific, direct ZPI inhibitor found to exhibit procoagulant activity in plasma. This sdAb might have potential as a treatment for hemophilia or other bleeding disorders.

Human coagulation FXa (Factor Xa) plays a key role in blood coagulation by activating prothrombin to thrombin on 'stimulated' platelet membranes in the presence of its cofactor FVa (Factor Va). PS (phosphatidylserine) exposure on activated platelet membranes promotes prothrombin activation by FXa by allosterically regulating FXa. To identify the structural basis of this allosteric regulation, we used FRET to monitor changes in FXa length in response to (i) soluble short-chain PS [C6PS (dicaproylphosphatidylserine)], (ii) PS membranes, and (iii) FVa in the presence of C6PS and membranes. We incorporated a FRET pair with donor (fluorescein) at the active site and acceptor (Alexa Fluor® 555) at the FXa N-terminus near the membrane. The results demonstrated that FXa structure changes upon binding of C6PS to two sites: a regulatory site at the N-terminus [identified previously as involving the Gla (γ-carboxyglutamic acid) and EGFN (N-terminus of epidermal growth factor) domains] and a presumptive protein-recognition site in the catalytic domain. Binding of C6PS to the regulatory site increased the interprobe distance by ~3 Å (1 Å=0.1 nm), whereas saturation of both sites increased the distance by a further ~6.4 Å. FXa binding to a membrane produced a smaller increase in length (~1.4 Å), indicating that FXa has a somewhat different structure on a membrane from when bound to C6PS in solution. However, when both FVa2 (a FVa glycoform) and either C6PS- or PS-containing membranes were bound to FXa, the overall change in length was comparable (~5.6-5.8 Å), indicating that C6PS- and PS-containing membranes in conjunction with FVa2 have comparable regulatory effects on FXa. We conclude that the similar functional regulation of FXa by C6PS or membranes in conjunction with FVa2 correlates with similar structural regulation. The results demonstrate the usefulness of FRET in analysing structure-function relationships in FXa and in the FXa·FVa2 complex.

IntroductionPlasma factor X (FX) levels may affect the therapeutic effects of bypass hemostatic therapy among patients with hemophilia with inhibitors. This study aimed to reproduce low and high FX level conditions in vitro and analyze changes in coagulation capacity. Materials and methodsTo achieve low FX concentrations, FX-deficient plasma was preincubated with anti-factor VIII (FVIII) or anti-factor IX (FIX) antibody. Following this, it was incubated with activated factor VII (FVIIa) or FVIIa/FX mixture in the presence of FX (0.13–0.64 IU/mL). To achieve high FX concentrations, FVIII- or FIX-deficient plasma was preincubated with anti-FVIII or anti-FIX antibody. Next, FX (4–20 IU/mL) was added in the presence of FVIIa (140 IU/mL). Under both conditions, changes in coagulation capacity were assessed by evaluating thrombin generation (TG) and activated partial thromboplastin time (aPTT). ResultsFX at low concentrations induced concentration-dependent changes in TG. In the presence of FX (0.13 IU/mL), adding FVIIa inadequately restored TG. Further, TG in the plasma was normalized after adding FVIIa/FX (62.5/2 IU/mL), and the FVIIa/FX-added group had a stable TG and aPTT, regardless of FX concentrations (0.13–0.64 IU/mL). The FVIIa-added group exhibited a FX concentration-dependent increase in TG and a decrease in aPTT. Furthermore, TG increased with FX concentrations under high FX concentrations (up to 10 IU/mL). ConclusionsSimultaneous FX supplementation in addition to FVIIa may be effective in promoting hemostasis under low plasma FX levels. Moreover, the risk of overcoagulation might be low even under high plasma FX levels.

Thromboelastographic characterization of the activated clotting system in children with sickle cell trait or sickle cell disease