Hybrid human-porcine factor VIII proteins partially escape the inhibitory effects of anti-factor VIII inhibitor alloantibodies having A2 or C2 domain specificity.

INTRODUCTION: Porcine factor (pF)VIII has low cross-reactivity with anti-human (h)FVIII inhibitor alloantibodies. Clinical trials of pFVIII in congenital haemophilia A patients with inhibitor (PwHA-I) are in progress. Most polyclonal anti-hFVIII inhibitors recognize its A2 and/or C2 domain(s), and recombinant human-porcine hybrid (hp)FVIII proteins may escape neutralization by these inhibitors. AIM: To evaluate the ability of hpFVIII to limit the anti-FVIII activity of inhibitor alloantibodies. METHODS: Three hybrid proteins were created by substituting the hFVIII A2, C2 domain or both with the corresponding domains of pFVIII [termed hp(A2), hp(C2) and hp(A2/C2), respectively]. The reactivity of these hybrids was assessed by one-stage clotting assays (OSA), thrombin generation assays (TGA) and rotational thromboelastometry (ROTEM) by adding them to FVIII-deficient samples. RESULTS: OSA demonstrated that the hybrid proteins avoided neutralization by anti-FVIII A2 or C2 monoclonal antibodies (mAb) and polyclonal inhibitor-antibodies (polyAb) from PwHA-I. In TGA, thrombin generation with hp(A2) and hp(A2/C2) was not attenuated in the presence of patient IgG recognizing anti-A2 domain. In contrast, that with hFVIII and hp(C2) was suppressed by this IgG to levels equivalent to those of FVIII-deficient plasma. With anti-A2/C2 polyAb, the activity of hp(A2/C2) was unaffected. ROTEM demonstrated that the addition of hp(A2) or hp(A2/C2) to anti-A2 polyAb shortened clot times/clot formation times, whilst hFVIII or hp(C2) were ineffective. Similarly with anti-A2/C2 polyAb, hp(A2/C2) restored coagulation potential to a greater extent than hp(A2) and hp(C2). CONCLUSION: Hybrid FVIII proteins containing porcine FVIII A2 and/or C2 domain(s) could support effective therapy in PwHA-I by avoiding neutralization.

ROTEM could be useful for lupus anticoagulant hypoprothrombinemia syndrome.

BACKGROUND: Lupus anticoagulant-hypoprothrombinemia syndrome (LAHPS) is a rare disease caused by acquired factor II (FII) deficiency and lupus anticoagulant. Patients with LAHPS typically present with thrombosis and bleeding. However, little information is available on the evaluation of coagulation potential in patients with LAHPS. We examined global coagulation potentials in patients with LAHPS during the clinical course in this study. METHODS: Coagulation potentials in two pediatric patients with LAHPS were assessed by measuring clotting time (CT) and clot formation time using Ca2+-triggered rotational thromboelastometry (ROTEM), CT and maximum coagulation velocity using clot waveform analysis (CWA), and lag time and peak thrombin using the thrombin generation assay (TGA). The day of admission was defined as day 0. RESULTS: In case 1, the bleeding symptoms disappeared by day 5. However, the TGA and CWA results were markedly lower than normal, although FII activity (FII:C) returned to within the normal range by day 14. In contrast, ROTEM revealed a recovery to near-normal levels (day 14). All coagulation parameters (day 80) were within normal ranges. In case 2, coagulation potential was severely depressed until day 12, although FII:C returned to normal levels. Bleeding symptoms disappeared on day 19, and the ROTEM data revealed that the parameters were close to the normal range. The coagulation parameters in all assays were normalized on day 75. CONCLUSIONS: Recovery of coagulation potential in patients with LAHPS was slower than the recovery of FII:C. Moreover, ROTEM appeared to be clinically useful for assessing coagulation potential in patients with LAHPS.

Coagulant potentials of emicizumab in the plasmas from infant and toddler patients with hemophilia A.

BACKGROUND: Emicizumab significantly reduces bleedings in patients with hemophilia A (PwHA). A clinical study (HAVEN 7; NCT04431726) for PwHA aged less than or equal to 12 months is ongoing, but emicizumab-driven coagulation potential in PwHA in early childhood remains to be clarified. AIM: To investigate the in vitro or in vivo coagulation potential of emicizumab in plasmas obtained from infant and toddler PwHA. METHODS: Twenty-seven plasma samples from 14 infant/toddler PwHA (aged 0-42 months, median 19 months) who received emicizumab (n = 9), factor (F)VIII products (n = 8), or no treatment (n = 10) were obtained. FVIII activity in FVIII-treated plasmas was cancelled by the addition of anti-FVIII monoclonal antibody (mAb). Emicizumab-treated plasmas (in vivo) and emicizumab-spiked plasmas (in vitro) were analyzed. Emicizumab-untreated plasma or emicizumab-treated plasma supplemented with two anti-emicizumab mAbs were used as references. Adjusted maximum coagulation velocity (Ad|min1|) by clot waveform analysis and peak thrombin (Peak-Th) by thrombin generation assay was assessed. RESULTS: Ad|min1| values in 24 samples were improved by the presence of emicizumab. Values did not improve in the three remaining samples (aged 1, 23, and 31 months). Although the presence of emicizumab showed an age-dependent increase in Peak-Th in 20 samples, this increase was not observed in seven samples (aged 0, 1, 1, 2, 8, 19, and 36 months). Emicizumab-dependent increases in both Ad|min1| and Peak-Th were shown in 18 samples, and increases in either parameter were shown in eight samples. One sample (from patient aged 1 month) showed no increase in both, however. CONCLUSION: Emicizumab could improve coagulant potential in plasmas from infant/toddler patients with hemophilia A.

Size-Dependent Geometrical Structures of Platinum Oxide Cluster Cations Studied by Ion Mobility-Mass Spectrometry.

Structures of platinum oxide cluster cations (PtnOm+) were studied by ion mobility-mass spectrometry in combination with theoretical calculations. Structures of oxygen-equivalent PtnOn+ (n = 3-7) clusters were discussed from the comparison between their collision cross sections (CCSs) obtained by mobility measurement and simulated CCSs of their structural candidates from structural optimization calculations. Assigned structures of PtnOn+ were found to be composed of Pt frameworks and bridging O atoms, which follows the previous theoretical prediction on the neutral clusters. The structures change from planar (n = 3 and 4) to three-dimensional (n = 5-7) with increasing cluster size by deforming platinum frameworks. Comparison with other group-10 metal oxide cluster cations (MnOn+; M = Ni and Pd) showed that the PtnOn+ structures have a similar tendency to PdnOn+ rather than NinOn+.

Screening of the protein C pathway abnormality-related thrombophilia by using thrombomodulin-mediated tissue factor-triggered clot waveform analysis.

OBJECTIVES: Absolute or relative protein (P)C pathway abnormalities (PC deficiency, PS deficiency, antiphospholipid syndrome (APS), factor (F)V-abnormality, and high FVIII level) cause thrombophilia. Although screening assays for these thrombophilias are available, one utilizing clot waveform analysis (CWA) remains unknown. We aimed to establish a CWA-based screening assay to distinguish PC pathway abnormality-related thrombophilia. METHODS: Samples were reacted with tissue factor (TF)/phospholipids and recombinant thrombomodulin (rTM; optimal 20 nM), followed by CWA measurement. The peak ratio (with/without rTM) of the first derivative curve of clot waveform was calculated. RESULTS: The peak ratio in healthy plasmas (n = 35) was 0.36 ± 0.13; hence, the cutoff value was set to 0.49. The peak ratios in plasmas with PC deficiency, PS deficiency, high-FVIII (spiked 300 IU/dl), and APS were higher than the cutoff values (0.79/0.97/0.50/0.93, respectively). PC-deficient plasma or PS-deficient plasma mixed with normal plasma (25%/50%/75%/100% PC or PS level) showed dose-dependent decreases in the peak ratios (PC deficient: 0.85/0.64/0.44/0.28; PS deficient: 0.69/0.53/0.40/0.25), suggesting that the peak ratio at ≤50% of PC or PS level exceeded the cutoff value. The peak ratio in FV deficiency with FV ≤25% was higher than the cutoff value. FV-deficient plasma spiked with 40 IU/dl rFV-R506Q (FVLeiden ) or rFV-W1920R (FVNara ) showed >90% peak ratios. CONCLUSIONS: rTM-mediated TF-triggered CWA might be useful for screening PC pathway abnormality-related thrombophilia.

Comprehensive coagulation and fibrinolytic potential in the acute phase of pediatric patients with idiopathic nephrotic syndrome evaluated by whole blood-based rotational thromboelastometry.

BACKGROUND: Venous thromboembolism is a rare, serious complication of idiopathic nephrotic syndrome (INS) in childhood. The mechanisms responsible for the hypercoagulable state in the acute phase of INS are poorly understood, however. This study aimed to assess overall coagulation and fibrinolytic function in pediatric patients with INS. METHODS: Global coagulation and fibrinolysis were examined in whole blood samples from 22 children with initial onset INS (initial-group), 22 children with relapsed INS (relapse-group), and 15 control pediatric patients using rotational thromboelastometry (ROTEM®). In the initial-group, blood samples were obtained before (week 0) and 1-4 weeks after initiation of corticosteroid therapy. EXTEM and FIBTEM were used to assess coagulation and fibrinolysis, respectively. Clot time (CT), clot formation time (CFT), maximum clot firmness (MCF), and α-angle were determined as coagulation parameters, and lysis index at 30 and 60 min (LI30 and LI60, respectively) were assessed as fibrinolytic parameters. RESULTS: CT was significantly shortened, and MCF and α-angle were significantly greater than controls at week 0 and week 1 both in the initial-group and the relapse-group. MCF correlated with serum albumin (r = 0.70, p < 0.001) and fibrinogen level (r = 0.68, p < 0.001). The fibrinolytic parameters (LI30 and LI60) in the initial-group were stable and higher than those in controls at all time points (p < 0.01). CONCLUSIONS: We have shown that the hypofibrinolytic defect did not improve with effective NS treatment at the early 4-week time-point. Additionally, a likely pre-thrombotic state was evident in the period before initial onset and 1 week after corticosteroid therapy in pediatric INS.

Coagulation potential in pediatric patients with immunoglobulin A nephropathy.

BACKGROUND: Immunoglobulin A nephropathy (IgAN) is the most prevalent primary chronic glomerular disease in children. Understanding the changes in coagulability caused by IgAN is important for clarifying pathophysiology and choice of treatment. The coagulation potential in patients with IgAN remains to be investigated, however. We aimed to assess comprehensive coagulation potential in pediatric patients with IgAN and explore its relationship with pathological disease severity. METHODS: Fourteen children with IgAN diagnosed by renal biopsy, who were admitted at Nara Medical University Hospital between 2015 and 2020, were analyzed. Rotational thromboelastometry was used to evaluate coagulation potential. Values of rotational thromboelastometry parameters in patients with IgAN were compared with those in control children. RESULTS: In patients with IgAN (aged median 9.5 year), clotting time plus clot formation time (CT + CFT) was shortened (P = 0.003) and α angle was greater (P < 0.001) than those in controls, indicating a hypercoagulable state. The rate of mesangial hypercellularity of glomeruli correlated with CT + CFT, α, and maximum clot firmness (MCF) (rs = -0.79, 0.56, and 0.37). The rate of cellular/fibrocellular crescent of glomeruli correlated with CT + CFT, α, and MCF (rs = -0.41, 0.60, and 0.50). Patients with mesangial hypercellularity ≥80% of glomeruli showed reduced CT + CFT and increased α angle (P = 0.007 and 0.03). Patients with cellular/fibrocellular crescent ≥10% of glomeruli showed decreased CT + CFT and increased α angle (both P = 0.02). CONCLUSIONS: The hypercoagulable state in pediatric patients with IgAN may be associated with the pathological severity of their disease.

Protein C system in preterm babies with chronic lung disease: Prospective study.

BACKGROUND: Chronic lung disease (CLD) is a major neonatal pulmonary disorder associated with inflammation. Recent studies have shown that protein C anticoagulant pathways, such as those for protein C (PC), protein S (PS), and thrombomodulin (TM), could be useful indices for reflecting pulmonary injury. However, the involvement of these factors in preterm infants with very low birthweight (VLBW) who have developed CLD remains to be investigated. Here, we investigated whether PC pathway-related factors could predict the development of CLD in preterm infants with VLBW. METHODS: We collected plasma samples from 26 preterm infants with VLBW (13 each from those with and without CLD) at the time of birth and measured TM, PC, and PS levels in their plasmas. We analyzed prospectively the relationship between these factors in infants with and without CLD. RESULTS: There were significant differences in gestational age, birthweight, Apgar score (5 min), and duration of mechanical ventilation between the CLD and non-CLD groups. No significant differences in the PC and PS levels at birth were observed between the two groups, whereas the TM levels in the CLD group were significantly higher than those in the non-CLD group (P = 0.013). The TM levels correlated with gestational age and duration of mechanical ventilation. However, covariance analysis demonstrated that gestational age was significantly associated with TM levels, and consequently, development of CLD was not associated with TM level at birth. CONCLUSIONS: Thrombomodulin, PC, and PS levels at birth could not predict the development of CLD in preterm infants with VLBW.

A microchip flow-chamber assay screens congenital primary hemostasis disorders.

BACKGROUND: Von Willebrand disease (VWD) and platelet function disorders (PFDs) are congenital bleeding disorders caused by primary hemostasis defects. Platelet function tests are time-consuming and require considerable amounts of blood sample, and there have been no easy-to-use assays for assessing platelet function quickly and sensitively. We report the usefulness of a microchip flow-chamber system (T-TAS® ) for detecting and/or predicting clinical severity in patients with VWD type 1 and type 2N and platelet storage pool disease. Here, we developed an application of a screening assay for primary hemostasis disorders. METHODS: Microchips coated with collagen (PL-chip) and collagen/thromboplastin (AR-chip) were utilized to evaluate platelet thrombus formation (PTF) at high shear and fibrin-rich PTF at low shear, respectively, in whole blood samples from 22 patients with VWD (16 type 2A, four type 2B, two type 3) and four patients with PFDs (two BSS, two Glanzmann thrombasthenia). The time-to-increase by 10 kPa (T10 ) was calculated from flow pressure curves. Also, whole blood-induced platelet aggregation was assessed using Multiplate® analysis. RESULTS: PL-chip T10 values ≥10 min successfully distinguished patients with all types of VWD and PFDs from healthy controls, irrespective of age, bleeding scores, and von Willebrand factor levels. However, AR-chip assay incompletely distinguished between type 2A patients and healthy ones. Multiplate analysis permitted screening of PFDs and type 3 VWD, but values in type 2A partially overlapped with those in controls. PL-chip assay did not reflect the clinical severity in these patients. CONCLUSIONS: T-TAS with PL-chip could be a quick screening tool for congenital primary hemostasis disorder, VWD, and PFDs.

Prediction of the haemostatic effects of bypassing therapy using comprehensive coagulation assays in emicizumab prophylaxis-treated haemophilia A patients with inhibitors.

In emicizumab prophylaxis, the concomitant therapy using bypassing agents (BPAs) is required for breakthrough bleeding and invasive procedures with attention to thrombotic complications. To predict coagulant effects of BPAs in emicizumab-treated patients with haemophilia A (PwHA) with inhibitor (PwHAwI), blood samples from emicizumab-treated PwHAwI (n = 8) and PwHA without inhibitor (n = 2) in phase 1/2 and HAVEN 1 study, spiked with activated prothrombin complex concentrates (aPCC) or recombinant factor VIIa (rFVIIa) ex vivo, and blood samples from emicizumab-treated PwHAwI-receiving BPAs were analysed by Ca2+ -triggered rotational thromboelastometry (ROTEM) and ellagic acid/tissue factor-triggered clot waveform analysis (CWA). Spiked aPCC, corresponded to 10-100 U/kg, markedly shortened ROTEM parameters beyond the normal range, while spiked rFVIIa, corresponded to 90-270 µg/kg, shortened them within near-normal range. Each of the spiked BPA-improved adjusted maximum coagulation velocity of CWA to within or near the normal range. In blood samples at post-infusion of aPCC (44-73 U/kg) or rFVIIa (79-93 µg/kg), the parameters of both assays improved to approximately the normal range. Taken together, ex vivo results of spiking tests in ROTEM and CWA, except aPCC spiking test in ROTEM, were relatively consistent with in vivo ones, and could usefully predict the coagulant effects of concomitant bypassing therapy for emicizumab-treated PwHAwI.

Whole blood ristocetin-induced platelet impedance aggregometry does not reflect clinical severity in patients with type 1 von Willebrand disease.

BACKGROUND: The haemorrhagic phenotype in patients with von Willebrand disease (VWD) is heterogeneous, and assays of von Willebrand factor ristocetin cofactor activity (VWF:RCo) do not always reflect clinical severity, especially in those individuals classed as type 1 VWD. Recent studies have shown that whole blood ristocetin-induced platelet agglutination (WB-RIPA) using an easy-to-use analyzer, Multiplate® platelet impedance technique, could be informative as a diagnostic test in VWD, although inconsistencies were evident in patients with the type 1 disorder, possibly associated with clinical symptoms. AIM: To investigate the relationship between WB-RIPA, bleeding scores (BS) and VWF-related measurements in type 1 VWD. METHODS: WB-RIPA assay using the Multiplate® was performed using whole blood from 55 patients with type 1 VWD. BS was determined using a standardized questionnaire. RESULTS: WB-RIPA values were significantly lower in type 1 VWD than in healthy controls (P < 0.0001). Weak correlations were apparent between WB-RIPA and VWF:RCo or VWF antigen (VWF:Ag; r = 0.22 or 0.28, respectively). There were significant differences in VWF:RCo (P = 0.036) and VWF:Ag (P = 0.0013) between patients with BS ≥4 (defined as abnormal bleeding tendency) and BS <4 (defined as no abnormal bleeding tendency), respectively. However, no significant difference was observed in WB-RIPA between the BS ≥4 group and BS <4 group. Overall, VWD patients with a WB-RIPA level >70 U did not seem to have an abnormal bleeding tendency, but low levels of WB-RIPA did not correlate with BS. CONCLUSION: WB-RIPA did not reflect clinical severity in type 1 VWD patients.

The role of proteolytic cleavage at Arg336 and Arg372 of the A1 domain in factor VIIa/tissue factor-catalyzed reactions of B domain-deleted factor VIII.

BACKGROUND: We previously demonstrated that factor (F)VIII was rapidly activated through proteolytic cleavage at Arg372 and Arg740 by activated FVII (FVIIa)/tissue factor (TF) in very early coagulation phase, followed by inactivation by cleavage at Arg336. The influence of the absence of FVIII B domain in this series of FVIIa/TF-catalyzed reaction remains unclear, however. AIM: To examine the FVIIa/TF-catalyzed reaction of B domain-deleted (BDD-)FVIII. METHODS AND RESULTS: The FVIII activity (FVIII:C) of commercial full-length (FL-)FVIII and BDD-FVIII increased by ∼1.7-fold within 0.5 min after addition of FVIIa/TF (1 nM/0.1 nM). FVIII: C decreased to initial levels with inactivation rate constant (k; ∼0.035) within 15 min of FL-FVIII activation, but decreased gradually to initial levels (k; ∼0.017) within 30 min of BDD-FVIII activation. SDS-PAGE analyses demonstrated that the FVIIa/TF-catalyzed cleavage of BDD-FVIII occurred at Arg336 within 0.5 min in parallel with elevation of FVIII:C, but cleavage at Arg372 was not evident. FVIIa/TF-catalyzed activation of both recombinant BDD-FVIII R336A and R372A mutants that were prepared, were similar to that of wild-type (WT) BDD-FVIII. However, FVIII:C returned to initial levels (k; ∼0.046) within 30 min of R336A mutant activation, but little reduction of FVIII:C was observed with R372A mutant (k; ∼0.0046). SDS-PAGE analysis indicated that FVIIa/TF-catalyzed cleavage of WT and R372A mutant was predominant at Arg336, whereas that of R336A mutant was observed at Arg372. CONCLUSIONS: FVIIa/TF-catalyzed activation of BDD-FVIII was initiated by cleavage at Arg336, and the FVIII B domain appeared to control FVIIa/TF-catalyzed reactions by altering pattern of cleavage at Arg336 and Arg372.

Changes in coagulation potential over time after administration of recombinant activated factor VII in an emicizumab-treated hemophilia A patient with inhibitors.

We describe a 67-year-old patient with hemophilia A and inhibitors (PwHA-I) receiving emicizumab prophylaxis who underwent surgical treatment for pseudoaneurysm. He was treated with a bolus infusion of recombinant factor VIIa (rFVIIa; 79 µg/kg) immediately before surgery, and a second dose of rFVIIa after an initial treatment on day 1. A third rFVIIa bolus was infused 17 h after the second dose on day 2, and the treatment was continued every 24 h on day 3 and day 4. Treatment with rFVIIa was discontinued on day 4. No perioperative bleeding or thrombotic events were observed. Coagulation potentials at 8 h after rFVIIa administration determined by clot waveform analysis (CWA) and thrombin generation assay (TGA) were within near-normal ranges, and results at 17 h after rFVIIa administration showed coagulation function comparable to that in the patient without rFVIIa. Our experimental data suggest that the coagulation potential in FVIII-deficient plasma spiked with both 0.28 µg/mL (11.2 µg/kg) rFVIIa and emicizumab was equivalent to or greater than that spiked with 2.2 µg/mL (90 µg/kg) rFVIIa alone. Thus, administration of rFVIIa every 8 h may be feasible for managing perioperative treatment in emicizumab-treated PwHA-I.

Additional Factor X Enhances Emicizumab-Driven Coagulation Function in Patients with Hemophilia A and Hemophilia A Mice.

BACKGROUND: Bypassing agents are used for breakthrough bleedings in patients with hemophilia A with inhibitor (PwHAwI) receiving emicizumab prophylaxis. Previous study demonstrated a weak binding affinity between emicizumab and factor (F)X (K d; 1.85 µM), and that this value was much greater than the plasma FX concentration (∼130 nM). We speculated that increased FX levels could enhance coagulation potential in emicizumab-treated patients with hemophilia A (PwHA). To investigate the relationship between FX concentrations and emicizumab-driven coagulation. METHODS: Plasma FX (up to 1,040 nM) and emicizumab (50 µg/mL) were added to FVIII-deficient plasmas, and plasma-derived FX (520 nM) or recombinant (r)FVIIa (2.2 µg/mL) was added to plasmas from three emicizumab-treated PwHAwI. The adjusted maximum coagulation velocity (Ad|min1|) by clot waveform analysis and peak thrombin (PeakTh) by thrombin generation assay in them were evaluated. Emicizumab (3.0 mg/kg), human (h)FIX (100 IU/kg), and various doses of hFX (100-500 IU/kg) were intravenously administered to HA mice. Clotting time/clot formation time (CT/CFT) were assessed using rotational thromboelastometry, and blood loss was estimated by a tail-clip assay. RESULTS: The addition of FX to FVIII-deficient plasma with emicizumab increased Ad|min1| and PeakTh. The coagulation parameters in emicizumab-treated PwHAwI spiked with additional FX remained within the normal range as well as the additional rFVIIa. In animal models, hFX injection shortened the CT and CT + CFT. The shorter CT and CT + CFT, and the lower blood loss were evident after 200 or 500 IU/kg hFX administration, and those indices were comparable to those in wild-type mice. CONCLUSION: Supplementation with FX may improve emicizumab-driven hemostasis in PwHA.

NXT007-mediated hemostatic potential is suppressed by activated protein C-catalyzed inactivation of activated factor V.

Background: Activated protein C (APC) inactivates activated factor (F) V (FVa) and FVIIIa. NXT007, an emicizumab-based engineered therapeutic bispecific antibody, enhances the coagulation potential of FVIII-deficient plasma (FVIIIdef-plasma) to near normal levels. However, little is known about the effect of APC-induced inactivation in NXT007-mediated hemostatic function. Objectives: To investigate the contribution of APC-mediated reactions to NXT007-driven hemostasis. Methods: In pooled normal plasma (PNP) or FVIIIdef-plasma spiked with NXT007 (10 µg/mL), effects of APC (0-16 nM) were measured using a thrombin generation assay (TGA). The direct effects of APC on cofactor activity of NXT007 or FVIIIa in a FXa generation assay were also measured. The FVdef-plasma and FV Leiden plasma (FVLeiden plasma) were preincubated with 2 anti-FVIII monoclonal antibodies (termed FVIII-depleted), and the APC effect in the presence of NXT007 in FVIII-depleted FVdef-plasma with the addition of exogenous FV (7.5-30 nM) or FVIII-depleted FVLeiden plasma was investigated. Results: The APC dose-dependent suppression effect in TGA of FVIIIdef-plasma spiked with NXT007 was similar to that of PNP. FXa generation with NXT007 was not impaired by the addition of APC, suggesting that the APC-induced reaction in TGA with NXT007 was attributed to the direct inactivation of FVa. The addition of APC to FVIII-depleted FVdef-plasma, along with NXT007 and various FV concentrations, showed a similar attenuation to PNP. The NXT007-driven thrombin generation in FVIII-depleted FVLeiden plasma was suppressed by APC, similar to the reaction in native FVLeiden plasma. Conclusion: NXT007 did not impair APC-mediated downregulation of FVa in FVIIIdef-plasmas, regardless of the presence of FV mutation with APC resistance.

The combination of Asp519Val/Glu665Val and Lys1813Ala mutations in FVIII markedly increases coagulation potential.

ABSTRACT: A2 domain dissociation in activated factor VIII (FVIIIa) results in reduced activity. Previous studies demonstrated that some FVIII mutants (D519V/E665V and K1813A) with delayed A2 dissociation enhanced coagulation potential. We speculated, therefore, that FVIII encompassing a combination of these mutations might further enhance coagulant activity. The aim was to assess the D519V/E665V/K1813A-FVIII mutation as a gain of function. The FVIII mutants, D519V/E665V/K1813A, D519V/E665V, and K1813A were expressed in a baby hamster kidney cell system, and global coagulation potential of these mutants was compared with wild-type (WT) FVIII in vitro and in hemophilia A mice in vivo. Kinetic analyses indicated that the apparent Kd for FIXa on the tenase assembly with D519V/E665V and D519V/E665V/K1813A mutants were lower, and that the generated FXa for D519V/E665V/K1813A was significantly greater than WT-FVIII. WT-FVIII activity after thrombin activation increased by ∼12-fold within 5 minutes, and returned to initial levels within 30 minutes. In contrast, The FVIII-related activity of D519V/E665V/K1813A increased further with time after thrombin activation, and showed an ∼25-fold increase at 2 hours. The A2 dissociation rate of D519V/E665V/K1813A was ∼50-fold slower than the WT in a 1-stage clotting assay. Thrombin generation assays demonstrated that D519V/E665V/K1813A (0.125 nM) exhibited coagulation potential comparable with that of the WT (1 nM). In animal studies, rotational thromboelastometry and tail-clip assays showed that the coagulation potential of D519V/E665V/K1813A (0.25 µg/kg) was equal to that of the WT (2 µg/kg). FVIII-D519V/E665V/K1813A mutant could provide an approximately eightfold increase in hemostatic function of WT-FVIII because of increased FVIIIa stability and the association between FVIIIa and FIXa.

Activated protein C resistance in the copresence of emicizumab and activated prothrombin complex concentrates.

Background: Venous thromboembolic events have been reported in persons with hemophilia A who received emicizumab and activated prothrombin complex concentrate (APCC) concomitantly, but the relevant mechanism(s) remains unclear. We speculated that activated protein C (APC) and antithrombin (AT) resistance might be associated with these adverse events. Objectives: To investigate APC and AT resistance in factor (F)VIII-deficient (FVIIIdef) plasma in the presence of emicizumab and APCC. Methods: In pooled normal plasma or FVIIIdef plasma samples mixed with emicizumab (50 µg/mL) and FVIII-bypassing agents, including recombinant FVIIa (2.2 µg/mL), APCC (1.3 IU/mL), or plasma-derived FVIIa/FX (1.5 µg/mL), the suppression effect of AT (0-2.4 µM) and APC (0-16 nM) was assessed by tissue factor-triggered thrombin generation assay. The APC effects in FVIIIdef plasma with the copresence of emicizumab, FII (1.3 µM), and/or FIXa (280 pM) were also examined. Results: The AT resistance in emicizumab and each bypassing agent was not observed. Moreover, APC dose-dependent suppression effect was observed in pooled normal plasma or FVIIIdef plasma mixed with emicizumab and recombinant FVIIa or plasma-derived FVIIa/FX. However, APC-catalyzed inactivation had little effect on thrombin generation assay potential in FVIIIdef plasma spiked with emicizumab and APCC. The addition of FIXa to emicizumab in FVIIIdef plasma could lead to partial APC resistance. Furthermore, FVIIIdef plasma spiked with emicizumab, FIXa, and FII was markedly resistant to APC-mediated inactivation. Conclusion: FII and FIXa in APCCs were key clotting factors for APC resistance in FVIIIdef plasma supplemented with emicizumab and APCCs. The APC resistance in persons with hemophilia A receiving emicizumab and APCC may contribute to venous thromboembolic events.