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.
Abstract
Factor VIII Aurora (FVIII-R571S) is the first described naturally occurring enhanced-potency FVIII variant identified in a patient with recurrent thrombosis and early mortality. The patient's plasma exhibited increased procoagulant activity and reduced responsiveness to activated protein C (APC). To define the mechanism, we generated recombinant FVIII-R571S and performed in vitro and in vivo studies. Consistent with the clinical phenotype, FVIII-R571S demonstrated a 6-fold increase in one-stage assay activity, while chromogenic substrate assay activity was comparable to wild-type FVIII (FVIII-WT). This discrepancy was explained by biochemical studies showing that activated FVIII-R571S (FVIIIa-R571S) has 10-20-fold higher affinity for FIXa; notably, the chromogenic assay is insensitive to differences in FVIIIa-FIXa affinity. Additional analyses demonstrated that FVIII-R571S is inactivated by APC and protein S analogous to FVIII-WT, indicating that the variant is not intrinsically APC-resistant. However, the increased affinity of FVIIIa-R571S for FIXa confers FIXa-dependent reduced A2-domain dissociation and APC-mediated inactivation in purified and plasma-based studies. These enhanced biochemical properties translated in vivo to a more potent procoagulant phenotype in hemophilia A mice. In the tail clip assay, FVIII-R571S exhibited a 4-5-fold increase in potency compared to FVIII-WT. In a thrombosis model, FVIII-R571S promoted significantly increased platelet and fibrin accumulation relative to FVIII-WT at equivalent antigen levels. Collectively, these data demonstrate that the prothrombotic phenotype of FVIII-R571S is driven by increased FIXa affinity, which enhances FVIIIa-FIXa complex assembly and function. This same mechanism confers reduced A2 dissociation and functional APC resistance, providing a unifying explanation for the observed gain-of-function phenotype.
Targeted modification of regions surrounding the FIXa active site enables allosteric activation, providing a potential foundation for novel bypassing strategies in hemophilia A therapy.
Viola J. F. Strijbis, K. Cheung, D. Gobbo et al.· Thrombosis Research· 0 citations
Data indicate that platelets support greater activity of FVIII than PLV through stabilization against dissociation of the A2 domain and protection from degradation by APC.
Valerie A Novacovic, Jialan Shi, G. Gilbert· Blood Advances· 0 citations
Hemophilia A is an X-linked bleeding disorder caused by mutations in the F8 gene, leading to a deficiency of coagulation factor VIII (FVIII). However, FVIII infusions for the treatment of bleeding triggers the development of neutralizing FVIII antibodies in about 30% of patients with severe hemophilia A, rendering FVIII replacement therapy ineffective. Although several studies have demonstrated that various immune cells, such conventional dendritic cells (cDCs), macrophages, monocytes, and marginal zone B cells, co-localize or interact with FVIII, the primary immune cell population mediating FVIII immune recognition remains poorly explored. DCs endocytose and process FVIII into peptides, which are then recognized by CD4+ T cells that activate B cells to produce anti-FVIII antibodies. We hypothesize that type 2 cDCs (cDC2) mediate FVIII recognition and CD4+ T cell activation that enhances FVIII immunity.
Batf3⁻/⁻ mice (cDC1 knockout [KO]), Zeb2-Delta3⁻/⁻ mice (cDC2 KO), and Zbtb46-DTR mice without diphtheria toxin depletion (WT) were utilized to isolate bone marrow-derived dendritic cells (BMDC).
Immature BMDC endocytosed more FVIII than mature and intermediate-mature BMDC as expected. However, immature BMDC from cDC2 KO mice exhibited reduced FVIII uptake compared to those from WT and cDC1 KO mice. Although BMDC from cDC2 KO mice enhanced CD4+ T cell proliferation compared to WT mice, cDC2 KO mice treated with FVIII exhibited reduced anti-FVIII antibodies than WT mice.
Together, these data suggest that cDC2 predominantly contributes to FVIII-specific adaptive immunity through FVIII uptake and promotion of CD4+ T cell proliferation.
K99
Immune Mechanisms of Human Disease (HUM)
Mengjie Kong, G. Batsuli· Journal of Immunology· 0 citations
Despite significant insights into factor VIII (FVIII) biosynthesis, the mechanisms involved in FVIII clearance remain poorly defined. To investigate genetic modifiers of FVIII clearance, we studied 106 SNPs across 43 different loci previously reported to influence plasma FVIII and/or von Willebrand (VWF) levels and reviewed their association with FVIII pharmacokinetic (PK) parameters in 52 patients with hemophilia A. Marked inter-individual variability in FVIII clearance was observed. Plasma VWF levels, ABO blood group and age all influenced FVIII PK parameters. Collectively however, linear regression analysis demonstrated that these explained less than 40% of the observed variance in FVIII clearance. Consistent with previous reports, SNPs mapped to the FVIII/VWF clearance receptors STAB2, ASGR2, and CLEC4M were associated with FVIII clearance. Importantly however, we identified four additional genetic signals marked by SNPs rs4904820 (TC2N), rs3124768 (ADAMTS13), rs12979891 (RASIP1/MAMSTR/FUT2) and rs6494314 (C2CD4B) that were also significantly associated with FVIII PK parameters. Of particular interest, the FUT2 gene on chromosome 19q13 determines Secretor blood group status and thus determines secretion of ABO(H) glycan determinants into plasma. In addition to the association between the rs12979891 SNP and FVIII clearance, we further observed an effect of Secretor genotype on FVIII PK parameters, with prolonged FVIII half-life in non-secretor individuals compared to homozygous secretors. Furthermore, plasma FVIII levels were significantly increased in Fut2 knockout mice. Cumulatively, these data identify novel modifiers of FVIII clearance in vivo. Given that plasma FVIII levels constitute a dose-dependent risk factor for cardiovascular disease, these findings are of direct clinical importance.
A. Hulshof, Lilian Antunes Heck, F. Atiq et al.· Blood· 0 citations
The first cryo-EM structures of FXIa in complex with its substrate, FIX, and activated FIX (FIXaβ) define the full FXIa:FIX interface providing a structural template for understanding the sequential activation of FIX and for developing a new class of selective allosteric antithrombotic agents.
Bassem M Mohammed, Samantha Deavila, Tristan Friet et al.· Journal of Thrombosis and Ha...· 0 citations
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.
M. Jewell, Christine H Baird, D. Thornhill et al.· Blood· 0 citations
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