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.

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.

A novel factor V compound heterozygous mutation associated with thrombosis (Y1961C; FV-Kanazawa, together with 1982_1983del).

BACKGROUND: Factor (F)V is pivotal in both procoagulant and anticoagulant mechanisms. The present report describes a novel F5 mutation in a FV-deficient patient (FV activity, 6 IU/dL; FV antigen, 32 IU/dL) complicated by recurrent deep vein thrombosis. The patient demonstrated activated protein C resistance (APCR) with compound heterozygous mutations consisting of FV-Y1961C (FVKanazawa) and FV-1982_1983del. OBJECTIVES: To clarify thrombotic mechanisms associated with this FV abnormality. METHODS AND RESULTS: Levels of FV-1982_1983del were below the detection sensitivity in our expression experiments using human embryonic kidney 293T cells, and analyses were targeted, therefore, on the FV-Y1961C mutation. Activated partial thromboplastin time-based clotting assays demonstrated that FV-Y1961C exhibited APCR and that the reduced activated protein C (APC) susceptibility in FVa-Y1961C resulted in a marked depression of APC-catalyzed inactivation with delayed cleavage at Arg506 and little cleavage at Arg306 with or without protein S. The APC cofactor activity of FV-Y1961C in APC-catalyzed FVIIIa inactivation promoted by Arg336 cleavage in FVIII was impaired. The binding affinity of FVa-Y1961C to phospholipid membranes was reduced in reactions involving APC/protein S-catalyzed inactivation and in prothrombinase activity. Furthermore, the addition of FVa-Y1961C to plasma failed to inhibit tissue factor-induced procoagulant function. These characteristics were similar to those of FV-W1920R (FVNara) and FV-A2086D (FVBesançon). CONCLUSION: We identified a compound heterozygous FV-Y1961C mutation in the C1 domain representing a novel FV mutation (FVKanazawa) resulting in not only APCR due to impaired FVa susceptibility and FV cofactor activity for APC function but also impaired inhibition of tissue factor-induced procoagulant function. These defects in anticoagulant function associated with FV in FV-Y1961C contributed to a prothrombotic state.

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.

Force-sensing treadmill gait analysis system can detect gait abnormalities in haemophilia patients without arthropathy.

BACKGROUND: Joint damage in patients with haemophilia (PwH) is commonly assessed by imaging, but few reports have described how structural changes in joints, for example, haemophilic arthropathy (HA)-affect gait ability. OBJECTIVES: We evaluated gait function among PwH with HA, PwH without HA, and people without haemophilia (non-PwH) using a Zebris FDM-T treadmill (FDM-T), an easy-to-use gait assessment instrument with a force sensor matrix. METHODS: The following gait parameters were collected: centre of pressure trajectory intersection (COPi) anterior/posterior variability, COPi lateral variability, COPi anterior/posterior symmetry, COPi lateral symmetry, single-limb support line (SLSL) length, and SLSL variability. Participants walked at their typical gait speed. The physical function of the PwH was assessed by the Hemophilia Joint Health Score (HJHS). Parameters were compared among the three groups. RESULTS: Twelve PwH with HA, 28 PwH without HA, and 12 non-PwH were enrolled. Gait speed significantly differed between groups (non-PwH, 3.1 ± 0.7; PwH without HA, 2.0 ± 0.7; PwH with HA; 1.5 ± 0.4). The COPi anterior/posterior variability, COPi lateral variability, SLSL length, and SLSL variability were greater in the PwH groups than in the non-PwH group. The COPi lateral symmetry differed between PwH with HA and the other groups. The HJHS was not correlated with gait parameters among PwH with HA. CONCLUSIONS: Gait parameters and speed were abnormal in both PwH with HA and PwH without HA. The FDM-T can be used to identify early stages of physical dysfunction that cannot be detected by conventional functional assessments such as the HJHS.

Reduced plasma factor X is associated with a lack of response to recombinant activated factor VII in patients with hemophilia A and inhibitor, but does not impair emicizumab-driven hemostasis in vitro.

BACKGROUND: The hemostatic effect of recombinant (r) factor (F)VIIa after repetitive intermittent administration may be attenuated in patients with hemophilia A (PwHA) with inhibitors (PwHAwI) creating a clinically unresponsive status, although mechanism(s) remain to be clarified. In patients receiving prophylaxis treatment with emicizumab, concomitant rFVIIa is sometimes utilized in multiple doses for surgical procedures or breakthrough bleeding. AIM AND METHODS: We identified 'unresponsiveness' to rFVIIa, based on global coagulation function monitored using rotational thromboelastometry (ROTEM) in 11 PwHAwI and 5 patients with acquired HA, and investigated possible mechanisms focusing on the association between plasma FX levels and rFVIIa-mediated interactions. RESULTS: Our data demonstrated that FX antigen levels were lower in the rFVIIa-unresponsive group than in the rFVIIa-responsive group (0.46 ± 0.14 IU/mL vs. 0.87 ± 0.15 IU/mL, p < 0.01). This relationship was further examined by thrombin generation assays using a FX-deficient PwHAwI plasma model. The addition of FX with rFVIIa was associated with increased peak thrombin (PeakTh) generation. At low levels of FX (<0.5 IU/mL), rFVIIa failed to increase PeakTh to the normal range, consistent with clinical rFVIIa-unresponsiveness. In the presence of emicizumab (50 µg/mL), PeakTh was increased maximally to 80 % of normal, even at low levels of FX (0.28 IU/mL). CONCLUSIONS: Unresponsiveness to rFVIIa was associated with reduced levels of FX in PwHAwI. Emicizumab exhibited in vitro coagulation potential in the presence of FX at concentrations that appeared to limit the clinical response to rFVIIa therapy.

Anticoagulant effects of protein C, protein S, and antithrombin levels on the protein C pathway in young children.

The protein C (PC) pathway involves physiological anticoagulant factors (PC, protein S [PS], and factor V) and performs major anticoagulant functions in adults. Variations in overall PC pathway function due to dynamic changes in PC and PS in early childhood are poorly understood. We aimed to evaluate the contributions of PC pathway function during early childhood by measuring changes in plasma thrombin generation (TG) after administration of the PC activator protac. We evaluated correlations between anticoagulant factors and percentage of protac-induced coagulation inhibition (PiCi%). Before protac addition, TG in newborns (n = 35), infants (n = 42), young children (n = 35), and adults (n = 20) were 525 ± 74, 720 ± 96, 785 ± 53, and 802 ± 64 mOD/min, and PiCi% were 42.1 ± 9.9, 69.8 ± 11.0, 82.9 ± 4.4, and 86.9 ± 3.4%, respectively. The distribution of PiCi% on the two axes of TG (with or without protac) changed continuously with age and differed from that of warfarin-treated plasma and adult PC- or PS-deficient plasma. PiCi% increased dynamically during infancy and correlated with PS levels in newborns and PC levels in young children. Addition of PC or fresh frozen plasma equivalent to approximately 25% PC to PC-deficient plasma improved PiCi%. This automatic measurement requires only a small sample volume and is useful for analysis of developmental hemostasis.

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.

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.

Emicizumab-mediated hemostatic function assessed by thrombin generation assay in an in vitro model of factor VIII-depleted thrombophilia plasma.

Patients with hemophilia A (PwHA) may have concurrent deficiency of representative anticoagulant proteins, protein (P)C, PS, and antithrombin (AT), which reduces bleeding frequency. However, emicizumab-driven hemostasis in PwHA with such thrombophilic potential remains unclarified. This study investigated the influence of natural anticoagulants on emicizumab-driven coagulation in HA model plasma. Various concentrations of PS and AT were added to PS-deficient plasma and AT-deficient plasma in the presence of anti-FVIII antibody (FVIIIAb; 10BU/mL). PC-deficient plasma was mixed with normal plasma at various concentrations in the presence of FVIIIAb. Emicizumab (50 µg/mL) was added to these thrombophilic HA model plasmas, prior to tissue factor/ellagic acid-triggered thrombin generation assays. Co-presence of emicizumab increased peak thrombin values (PeakTh) dependent on PS, AT, and PC concentrations. Maximum coagulation potentials in the PS-reduced HA model plasmas remained normal in the presence of emicizumab. PeakTh were close to normal in the presence of 50%AT irrespective of emicizumab, but were higher than normal in the presence of 25%AT. Addition of recombinant FVIIa (corresponding to an administered dose of 90 µg/kg) enhanced coagulation potential to normal levels. Our findings provide novel information on hemostatic regulation in emicizumab-treated PwHA with a possible thrombophilic disposition.

Hemostatic potential of recombinant von Willebrand factor and standard or pegylated extended half-life recombinant factor VIII on thrombus formation under high shear flow.

BACKGROUND: Von Willebrand factor (VWF) and factor VIII (FVIII) complex play a pivotal role in hemostasis. A deficiency or defect of VWF causes von Willebrand disease (VWD). Recombinant (r)VWF product has proved to be effective for hemostatic treatment of VWD, but limited information is available on their role in moderating thrombus formation under flow condition. We aimed to assess thrombus formation in the presence of rVWF combined with rFVIII or pegylated-extended half-life rFVIII (peg-EHL-rFVIII) in VWD whole blood under high shear flow. METHODS: Perfusion chamber experiments under high shear (2,500 s- 1) combined with immunostaining were performed using patient's whole blood with type 1 VWD, mixed with rVWF (Vonvendi®; 1.6 IU/mL), rFVIII or peg-EHL-rFVIII (Advate® or Adynovate®; 1.0 IU/mL), or both. Similar experiments were also conducted with clinical medical devices (T-TAS®). RESULTS: The addition of rFVIII did not augment thrombus formation assessed by surface coverage (SC) and thrombus height (TH), whereas rVWF enhanced these parameters (SC 19.1 ± 1.1% vs. 30.1 ± 4.1%, TH 2.2 ± 0.14 µm vs. 3.6 ± 0.40 µm, respectively). The co-presence of rVWF/rFVIII was comparable to plasma-derived VWF/FVIII (Confact®, VWF:FVIII ratio = 1.6:1.0) for increasing thrombogenicity in SC (32.5 ± 4.3% vs. 38.7 ± 5.5%) and in TH (5.0 ± 0.60 µm vs. 5.5 ± 0.64 µm), respectively. The pre-incubation time with rVWF and rFVIII appeared to have a little effect on the size of thrombus. Peg-EHL-rFVIII mediated thrombus formation to similar extent as rFVIII in the co-presence of rVWF. Similar results were obtained even with T-TAS. Immunostaining demonstrated that rFVIII and peg-EHL-rFVIII were similarly co-localized with rVWF in formed thrombi, indicating that pegylation did not interfere with molecular complexes. CONCLUSION: The effects of high-level rVWF and peg-EHL-rFVIII on thrombus formation were comparable to conventional therapeutic products in a patient's whole blood with VWD under high shear flow.

Pediatric mesangial proliferative glomerulonephritis has increased the platelet thrombus formation potentials under high-shear flow condition.

INTRODUCTION: Blood coagulation is associated with glomerulonephritis (GN) pathophysiology. Using whole-blood-based rotational thromboelastometry, we recently reported that the degree of hypercoagulability in pediatric patients with immunoglobulin A nephropathy (IgAN), a GN, might be associated with pathological severity. To further clarify the coagulation status of mesangial proliferative glomerulonephritis (MesPGN), we assessed the platelet thrombus formation (PTF) under high-shear flow using a microchip-based flow chamber system (T-TAS®). METHODS: Thirty-four pediatric patients definitively diagnosed with MesPGN by renal biopsy at Nara Medical University Hospital between 2015 and 2022 were enrolled, and 29 patients (case group; median age, 8.0 years) were assessed. Microchips coated with collagen (PL-chip) were used to assess PTF at high-shear in whole blood. The times to increase by 10 and 30 kPa (T10 and T30) from baseline were calculated and compared with those of the pediatric controls. Changes in the parameters during the treatment course and the relationship between pathological severity and the parameters were evaluated. RESULTS: T10 and T30 parameters in the PL-chip were significantly shorter and the area under the curves were greater in the case group than those in the control group (both p <0.05). Each parameter was enhanced during the 3-week treatment but improved after the end of treatment. No significant relationship was observed between pathological severity and these parameters. Little PTF difference was observed between IgAN and Henoch-Schönlein purpura nephritis. CONCLUSIONS: Pediatric MesPGN increased the potential for PTF under high-shear flow conditions.

Longitudinal dynamic changes in factor VIII inhibitor titers in patients with hemophilia A and inhibitors receiving emicizumab prophylaxis.

Emicizumab prophylaxis dramatically reduces bleeding events in patients with hemophilia A (PwHA) with or without factor VIII (FVIII) inhibitors. However, long-term dynamic changes in FVIII inhibitor titers during emicizumab prophylaxis remain to be investigated. We conducted a retrospective follow-up study of FVIII inhibitor titers after initiation of emicizumab prophylaxis in 25 PwHA carrying current or historical FVIII inhibitors. Nineteen PwHA had FVIII inhibitors at initiation of emicizumab prophylaxis (age: median 22 [range 4-60] years and inhibitor titer: 30 [1.0-1450] BU/mL). In 17 of the 19 patients, the inhibitor titers markedly decreased to a median of 1.2 (< 0.6-58) BU/mL at a median follow-up of 71 (38-111) months. In two patients, titers were slightly elevated after initiation of emicizumab but decreased in the long term. The remaining six patients had negative inhibitor status (< 0.6 BU/mL) when switched to emicizumab from FVIII prophylaxis. Five patients maintained negative titers. One patient had inhibitor recurrence, with a peak titer of 1.6 BU/mL that decreased to 0.9 BU/mL. In most cases, FVIII inhibitor titers can be expected to decrease spontaneously during emicizumab prophylaxis, but regular follow-up is necessary to manage breakthrough bleeds.

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.

The spleen is the major site for the development and expansion of inhibitor producing-cells in hemophilia A mice upon FVIII infusion developing high-titer inhibitor.

BACKGROUND: Hemophilia A (HA) is a hereditary bleeding disorder caused by defects in endogenous factor (F)VIII. Approximately 30 % of patients with severe HA treated with FVIII develop neutralizing antibodies (inhibitors) against FVIII, which render the therapy ineffective. The managements of HA patients with high-titter inhibitors are especially challenging. Therefore, it is important to understand the mechanism(s) of high-titer inhibitor development and dynamics of FVIII-specific plasma cells (FVIII-PCs). AIMS: To identify the dynamics of FVIII-PCs and the lymphoid organs in which FVIII-PCs are localized during high-titer inhibitor formation. METHODS AND RESULTS: When FVIII-KO mice were intravenously injected with recombinant (r)FVIII in combination with lipopolysaccharide (LPS), a marked enhancement of anti-FVIII antibody induction was observed with increasing FVIII-PCs, especially in the spleen. When splenectomized or congenitally asplenic FVIII-KO mice were treated with LPS + rFVIII, the serum inhibitor levels decreased by approximately 80 %. Furthermore, when splenocytes or bone marrow (BM) cells from inhibitor+ FVIII-KO mice treated with LPS + rFVIII were grafted into immune-deficient mice, anti-FVIII IgG was detected only in the serum of splenocyte-administered mice and FVIII-PCs were detected in the spleen but not in the BM. In addition, when splenocytes from inhibitor+ FVIII-KO mice were grafted into splenectomized immuno-deficient mice, inhibitor levels were significantly reduced in the serum. CONCLUSION: The spleen is the major site responsible for the expansion and retention of FVIII-PCs in the presence of high-titer inhibitors.

Impaired factor V-related anticoagulant mechanisms and deep vein thrombosis associated with A2086D and W1920R mutations.

Factor V (FV) plays pivotal roles in both procoagulant and anticoagulant mechanisms. Genetic mutations, FV-W1920R (FVNara) and FV-A2086D (FVBesançon), in the C1 and C2 domains of FV light chain, respectively, seem to be associated with deep vein thrombosis. However, the detailed mechanism(s) through which these mutations are linked to thrombophilia remains to be fully explored. The aim of this study was to clarify thrombotic mechanism(s) in the presence of these FV abnormalities. Full-length wild-type (WT) and mutated FV were prepared using stable, human cell lines (HEK293T) and the piggyBac transposon system. Susceptibility of FVa-A2086D to activated protein C (APC) was reduced, resulting in significant inhibition of APC-catalyzed inactivation with limited cleavage at Arg306 and delayed cleavage at Arg506. Furthermore, APC cofactor activity of FV-A2086D in APC-catalyzed inactivation of FVIIIa through cleavage at Arg336 was impaired. Surface plasmon resonance-based assays demonstrated that FV-A2086D bound to Glu-Gly-Arg-chloromethylketone active site-blocked APC and protein S (P) with similar affinities to that of FV-WT. However, weakened interaction between FVa-A2086D and phospholipid membranes was evident through the prothrombinase assay. Moreover, addition of FVa-A2086D to plasma failed to inhibit tissue factor (TF)-induced thrombin generation and reduce prothrombin times. This inhibitory effect was independent of PC, PS, and antithrombin. The coagulant and anticoagulant characteristics of FV(a)-W1920R were similar to those of FV(a)-A2086D. FV-A2086D presented defects in the APC mechanisms associated with FVa inactivation and FV cofactor activity, similar to FV-W1920R. Moreover, both FV proteins that were mutated in the light chain impaired inhibition of TF-induced coagulation reactions. These defects were consistent with congenital thrombophilia.