Wenman Wu

Background: Predictors for the successful eradication of neutralizing anti-FVIII alloantibodies (inhibitors) in hemophilia A patients receiving immune tolerance induction (ITI) therapy remain limited. Maternal microchimerism (MMc) showed potential to protect hemophilia A patients from inhibitor development. Objectives: To investigated the role of MMc in ITI therapy. Patients/Methods: This observational study enrolled 121 pediatric with hemophilia A and inhibitors. MMc was determined using droplet digital PCR. Low-dose ITI (FVIII ~50IU/kg every other day) was administered, with adjunctive rituximab given to patients with higher risk clinical features. Results: Of the 101 patients evaluable for MMc, 88 completed ITI, 18 were MMc positive (MMc+) and 70 were MMc negative (MMc-). Success was achieved in 75 (85.2%) patients, including 16 of 18 MMc+ patients (88.9%) and 59 of 70 MMc- patients (84.3%). Compared with MMc- patients, MMc+ patients had a lower peak inhibitor during ITI (median, 5.3 vs. 37.8 BU/ml, p = 0.029), less frequent rituximab use (27.8% vs. 61.4%; p = 0.011), and a shorter time to ITI success (median, 3.0 vs 9.9 months; p = 0.009). Multivariate Cox regression identified MMc+ (HR = 2.770), pre-ITI inhibitor F8 mutations (HR = 2.344) as independent predictors of shorter time to ITI success. Conclusion: MMc was associated with more rapid ITI success in children with hemophilia A and inhibitors receiving low-dose ITI regimen, suggesting the potential role of MMc in facilitating the eradication of FVIII inhibitors.

Elevated FXI levels are associated with increased risk of venous thromboembolism, yet genetic mutations contributing to elevated FXI levels have not been reported yet. We described a patient with a history of deep venous thrombosis (DVT) who tested negative on routine laboratory screening but was found to carry a novel pathogenic FXI mutation, Gly397Ser (G397S). FXI mutant protein was expressed, and functional assays were conducted in vitro to explore the underlying thrombotic mechanism. Consistent with FXI activity (FXI:C) and antigen (FXI:Ag) determined in the patient, the FXI G397S mutant exhibited a 2-fold increase of FXI:C/FXI:Ag. FXIa hydrolysis assay demonstrated that the Km value for G397S mutant was comparable to the wild-type FXIa (186.2~281.9 μM V.S. 141.6~242.6 μM), whereas the mutant FXIa displayed approximately 3-fold increases in kcat values. FXI activation by polyanions was more pronounced in the G397S mutant. Notably, this activation was more significant when triggered by thrombin compared with FXIIa in the presence of heparin. Otherwise, the G397S FXI mutant exhibited similar hydrolytic activity of FXIa against FIX, and equivalent inhibition of FXIa by Protease nexin 2 (PN2). Clot lysis assay revealed that the lysis time was longer in G397S mutant than the wild-type and the enhanced fibrinolytic resistance was thrombin-activatable fibrinolysis inhibitor-dependent. In summary, the FXI G397S mutation exhibited higher FXIa activity mainly because of the enhanced activation of FXI, which sustained thrombin generation and manifested resistance to fibrinolysis, ultimately leading to the development of DVT. ^‡ These authors contributed equally to this article. Received: 19 February 2025 Accepted after revision: 05 November 2025 Article published online: 08 December 2025 © 2025. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany

The human coagulation pathway orchestrates a complex series of events vital for maintaining vascular integrity, in which the intrinsic pathway plays a pivotal role in amplifying and propagating the coagulation response. Dysregulation of this pathway can lead to various bleeding disorders and thrombotic complications, posing significant health risks. In this pathway, the activation of Factor (F) X zymogen is catalyzed by the FVIIIa-FIXa binary complex, but knowledge about this is still incomplete. Understanding the structural and functional intricacies of the FVIIIa-FIXa-FX (zymogen) complex is imperative for unraveling the molecular mechanisms underlying coagulation regulation and guiding the development of targeted therapeutic interventions. In this study, utilizing Alphafold-Multimer and molecular dynamics (MD) simulations, we provide insights into factor interactions within the ternary complex and propose novel functional mechanisms contributing to the functional defects inflicted by their cross-reactive material (CRM) positive mutations. The amino acid residue replacement impairs the coagulation function by interfering with structure elements, including the following: (1) a knot-like structure between Arg-562 of FVIIIa’s 558-Loop (residue 555–571) and the 333-Loop of FIXa (residue 333–346) contributes to FVIIIa-FIXa binding; (2) the a2 region of FVIIIa (residue 716–740) opens the lid of active site (FIXa’s 266-Loop, residue 256–270) and facilitates substrate binding; (3) the activation peptide (AP) of FX zymogen (residue 143–194) not only assists in the activation of itself but also adheres the interface of the three factors like a double-sided tape. Our work provides novel insights for the pathogenesis of a number of reported clinical CRM-positive mutations and may lay the groundwork for the structure-based development of therapeutic interventions.

Background Venous thromboembolism (VTE) is predisposed by thrombotic mutations in patients with hereditary thrombophilia. Although prothrombin deficiencies caused by homozygous or compound heterozygous mutations are associated with bleeding diathesis, rare cases have shown a correlation between heterozygous prothrombin mutations and thrombosis. Materials and Methods We surveyed genetic variants involved in thrombosis and hemostasis in 347 patients with unprovoked VTE or having a positive family history of thrombosis. For patients identified with heterozygous prothrombin mutations, we conducted family investigations and performed a thrombin generation test (TGT) to elucidate the thrombotic risk. Novel mutants were expressed and subjected to functional assays to clarify the underlying thrombotic mechanisms. Results Heterozygous prothrombin mutations were identified in 3.5% of patients (12/347), including three novel mutations Phe382Ser, Phe382Leu, and Asp597Tyr found in one patient each, as well as previously reported Arg541Trp mutation in four patients and Arg596Gln mutation in five patients. A total of 42 mutation carriers were identified within the 12 pedigrees, among whom 64.3% (27/42) had experienced thrombotic events. TGT results demonstrated hypercoagulability for carriers of the five mutations, with Arg596Gln showing the highest thrombin generation potential followed by Arg541Trp. The Phe382-associated mutations severely impaired thrombomodulin-binding ability of thrombin, resulting in obviously reduced protein C (PC) activation. The Asp597Tyr mutation exhibited a mild reduction in both inactivation by antithrombin and PC activation reactions. Conclusion The presence of heterozygous prothrombin mutations represents a potential genetic predisposition for VTE. All thrombosis-associated mutations potentiate coagulation activity by either conferring antithrombin resistance and/or impairing PC pathway activity.

Aim To structurally and functionally characterize three newly identified F9 missense mutations, C268Y, I316F, and G413V, in Chinese hemophilia B patients. Methods FIX mutants were expressed in vitro by transient transfection of Chinese hamster ovary (CHO) cells. One-stage activated partial thromboplastin time (APTT) based assay and enzyme-linked immunosorbent assay (ELISA) were used to measure the coagulation activity and antigen level of FIX in conditioned medium. Western blot analysis was also used to evaluate interference of the mutations with synthesis and secretion of FIX. A structural model of the FIX G413V mutant was constructed and structural disturbance caused by the mutation was determined by molecular dynamics simulations. Results Both C268Y and I316F impaired expression of FIX. However, the I316F mutant degraded quickly, whereas the C268Y mutant mostly accumulated intracellularly. The G413V mutant could be synthesized and secreted normally, but procoagulant activity was almost completely lost. This loss is likely mostly due to the impact on the catalytic residue cS195. Conclusion The three FIX mutations identified in Chinese hemophilia B patients either impaired the expression of FIX, as was seen with the I316F and C268Y mutants, or impaired the function of FIX, as was seen with the G413V mutant.

Summary: Hereditary factor VII (FVII) deficiency is a rare recessive bleeding disorder with an estimated prevalence of 1/500 000. We had investigated 50 unrelated Chinese patients with FVII deficiency and identified, in total, 25 mutations, including 18 missense mutations and 5 splicing mutations, on the F7 gene. The nucleotide transition c.1224T>G (p.His408Gln) in exon 9 constitutes a hotspot of mutation, with 19 patients harbouring this genetic variance. Few patients were homozygous or compound heterozygous for deleterious mutations, such as non‐sense mutations, large insertion or deletions, indicating that complete deficiency of FVII may not be compatible with life. The eight novel mutations identified in the study, including one small deletion (p.Glu49GlyfsTer101), three type I missense mutations, p.Cys238Phe, p.Gly420Asp, p.Ala252Val and four type II missense mutations, p.Val336Met, p.Ser342Gly, p.Gly432Ser and p.Ile213Asn, were further analysed by in vitro expression and functional studies. The laboratory phenotype and structural analysis confirmed the functional consequence of p.Ile213Asn mutation involving cleavage and activation site. The molecular dynamic simulations and binding energy calculations along with functional probing of p.Gly432Ser mutation revealed the critical role of residue Gly432 in the binding between activated factor VII (factor VIIa) and tissue factor.

Approximately 10% of von Willebrand factor (VWF) gene variants are suspected to disrupt messenger RNA (mRNA) processing, the number of which might be underestimated due to the lack of transcript assays. In the present study, we provided a detailed strategy to evaluate the effects of nine putative splice site variants (PSSVs) of VWF on mRNA processing as well as protein properties and establish their genotype-phenotype relationships. Eight of nine PSSVs affected VWF splicing: c.322A>T, c.1534-13_1551delinsCA, and c.8116-2del caused exon skipping; c.221-2A>C, c.323+1G>T, and c.2547-13T>A resulted in the activation of cryptic splice sites; c.2684A>G led to exon skipping and activation of a cryptic splice site; c.2968-14A>G created a new splice site. The remaining c.5171-9del was likely benign. The efficiency of nonsense-mediated mRNA decay (NMD) was much higher in platelets compared to leukocytes, impairing the identification of aberrant transcripts in 4 of 8 PSSVs. The nonsense variant c.322A>T partially impaired mRNA processing, leaking a small amount of correct transcripts with c.322T (p.Arg108*), while the missense variant c.2684A>G totally disrupted normal splicing of VWF, rather than produced mutant protein with the substitution of Gln895Arg. The results of this study would certainly add novel insights into the molecular events behind von Willebrand disease.