Simultaneous evaluation of coagulation and fibrinolysis facilitates an overall understanding of normal and pathological haemostasis. We established an assay for assessing clot formation and fibrinolysis simultaneously using clot waveform analysis by the trigger of a mixture of activated partial thromboplastin time reagent and an optimized concentration of tissue-type plasminogen activator (0Á63 lg/ml) to examine the temporal reactions in a short monitoring time (<500 s). The interplay between clot formation and fibrinolysis was confirmed by analysing the effects of argatroban, tranexamic acid and thrombomodulin. Fibrinogen levels positively correlated with coagulation and fibrinolytic potential and initial fibrin clot formation was independent of plasminogen concentration. Plasminogen activator inhibitor-1deficient (-def) and a2-antiplasmin-def plasmas demonstrated different characteristic hyper-fibrinolytic patterns. For the specificity of individual clotting factor-def plasmas, factor (F)VIII-def and FIX-def plasmas in particular demonstrated shortened fibrinolysis lag-times (FLT) and enhanced endogenous fibrinolysis potential in addition to decreased maximum coagulation velocity, possibly reflecting the fragile formation of fibrin clots. Tranexamic acid depressed fibrinolysis to a similar extent in FVIII-def and FIX-def plasmas. We concluded that the clot-fibrinolysis waveform analysis technique could sensitively monitor both sides of fibrin clot formation and fibrinolysis, and could provide an easy-to-use assay to help clarify the underlying pathogenesis of bleeding disorders in routine clinical practice.
The link between factor (F)VIII and FX is essential for optimum activity of the tenase complex. The interactive site(s) in FVIII for FX remains to be completely clarified, however. We investigated the FVIII A2 domain-FX association that was speculated from inhibitory mechanism(s) by an anti-A2 autoantibody. SDS-PAGE demonstrated that the purified inhibitor IgG recognizing residues 373-562 blocked FXa cleavage at Arg372 in FVIII, and surface-plasmon resonance (SPR)-based assays showed that intact A2 subunit directly bound to FX (; 63 nM). The FVIII structure model indicated possible FX-binding site(s) in residues 400-429 in A2. One peptide corresponding to residues 400-409 competitively inhibited both the A2-FX binding and FVIIIa/FIXa-dependent FXa generation. Covalent cross-linking was observed between this peptide and FX following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) using SDS-PAGE. K408 and S409 were not evident in N-terminal sequence analysis of the cross-linked product, suggesting that two residues participated in cross-link formation. SPR-based assays using recombinant FVIII mutants with one or both residues substituted to alanine demonstrated that K408A and K408A/S409A had approximately fourfold high values of wild-type (WT-)FVIII. FXa cleavages at Arg372 in both mutants were significantly delayed, suggesting a contribution of K408 for FXa cleavage at Arg372. Furthermore, FXa generation assays with these mutants demonstrated that the values were 1.4- to 1.7-fold greater, and overall catalytic efficiency (/) was 0.49- to 0.89-fold lower than with WT-FVIII, suggesting a significant contribution of K408 for FVIII-FX interaction in tenase assembly. We concluded that the K408 in the A2 domain provided an interactive-site for FX.
Factor (F) VIII functions as a cofactor in the tenase complex responsible for phospholipid surface-dependent conversion of FX to FXa by FIXa. FIXa binding sites were identified on A2, A3, and C2 domains of FVIII. Some earlier studies confirmed the FIXa-binding sites on residues 1811-1818 in FVIII A3 domain. According to the 3-D model of FVIIIa, the residues 1790-1798 in FVIII A3 domain form a loop and exist just close to residues 1811-1818 on the FVIIIa extended surface and might contribute to one of the FIXa binding regions. We, therefore, hypothesized that residues 1790-1798 might be one of the FIXa-binding components, and prepared synthetic peptides (1811-1818 and 1790-1798). The 1811-1818 peptide inhibited the FVIII light chain-EGR FIXa interaction (apparent Ki;10.5 ± 4.0 μM) on ELISA. The 1790-1798 peptide also inhibited these interaction (apparent Ki ;4.2± 1.2 μM), suggesting that the 1790-1798 region contributed to FIXa-interactive site. In general, the binding sites of serine protease in FVIII contain the acidic or basic rich residues. The residues 1790-1798, and 1811-1818 contain basic residues E1793, E1794, D1795, E1811, K1813, and K1818. To investigate the significance of these residues for FIXa-binding, the mutant forms of the A3 domain, converted to alanine, in BHK system and purified. Furthermore, because previous study demonstrated that F1816 might contribute to FIXa binding, we also prepared F1816A. Compared with wild type FVIII (Kd; 6.3± 0.3 nM), the binding affinity of F1816A and E1793A/E1794A/D1795A mutants for EGR FIXa were decreased by 1.4-fold (Kd; 9.1± 0.2 nM) and 1.3-fold (Kd; 8.4± 0.4 nM), respectively, on SPR-based assay, suggesting contribution of F1816, and E1793/E1794/D1795 to the binding interactions. On the other hand, the binding affinities of E1811A (Kd; 6.3± 2.1 nM) and K1818A (Kd; 5.1± 1.8 nM) mutants for FIXa were almost the same as that of wild type. Interestingly, the binding affinity of K1813A (Kd; 3.9± 0.7 nM) for FIXa was increased compared to wild type FVIII. For the functional evaluation of the association with FVIII mutants to FIXa, FVIII mutants were reacted with varying concentrations of FIXa in a FXa generation assay. F1816A and E1793A/E1794A/D1795A mutants decreased the FIXa affinity by 1.6-fold (Km; 10.6± 1.0 nM) and 2.8-fold (Km; 18.5± 4.3 nM), respectively, compared to wild type FVIII (Km; 6.6 ± 1.0 nM). These data suggested that not only F1816A but E1793A/E1794A/D1795A mutations contributed high Kmand low catalytic efficiency in FXase complex. E1811A (Km; 7.2 ± 0.7 nM), K1813A (Km; 4.5 ± 0.7 nM), and K1818A (Km; 4.5 ± 0.2 nM) mutants showed the similar FIXa affinities to wild type FVIII. Furthermore, to confirm that the F1816A and E1793A/E1794A/D1795A mutants were responsible for FIXa association, FIXa cleavages in these mutants were evaluated by SDS-PAGE and Western blot using anti-A1 monoclonal antibody. Compared with wild type FVIII, cleavages at Arg336 were slightly delayed in F1816A. On the other hand, those in E1793A/E1794A/D1795A were significantly delayed, indicative of contribution of these residues for FIXa bindings. After all, these results indicate that the 1790-1798 region in the FVIII A3 domain, and in particular a cluster of basic amino acids at residues 1793-1795, contributes to one of the FIXa-interactive sites. Disclosures Nogami: Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau. Shima:Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees.
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