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Elevated factor VIII activity (FVIII:C) is often observed in patients with acquired hemophilia A (PwAHA) in remission. However, comprehensive coagulation potentials in this patient group remain to be investigated.To evaluate comprehensive coagulation potentials in PwAHA.We investigated coagulation function in eleven PwAHA with high FVIII:C ( 150 IU/dL) using thrombin generation assay (TGA) and/or rotational thromboelastometry (ROTEM), and compared findings with results obtained from contrived samples generated by spiking recombinant FVIII.The median FVIII:C and FVIII inhibitor titers during remission in enrolled PwAHA were 206 IU/dL and 0.44 BU/mL, respectively. In all patients, lag time and time to peak were either prolonged or normal compared to contrived samples corresponding to their FVIII:C. However, higher values of peak thrombin and endogenous thrombin potentials compared to contrived samples were observed in two patients. ROTEM parameters were within normal ranges in all cases. One patient (FVIII:C 171 IU/dL) developed venous thrombosis and pulmonary embolism, but TGA parameters showed low or normal coagulation potential compared to contrived samples corresponding to his FVIII:C.PwAHA with high FVIII:C could exhibit lower coagulation potentials than those corresponding to their FVIII:C.

Emicizumab treatment may allow patients with hemophilia A without (PwHA) and with inhibitors (PwHA-I) to undergo some minor surgeries, such as tooth extraction, without peri-operative factor infusions. However, criteria for determining the necessity of factor infusions before minor surgeries are unknown.We report the peri-operative hemostatic management and outcomes of emicizumab-treated PwHA and PwHA-I cases who underwent tooth extractions using our institutional protocol.We retrospectively evaluated PwHA and PwHA-I who underwent tooth extraction with emicizumab prophylaxis at our institution. Local bleeding risk was assessed based on the method, number, and site of tooth extraction. Hemostasis was monitored peri-operatively by rotational thromboelastometry (ROTEM). Hemostatic agents and a mouth splint were used.Twenty-nine extractions (17 interventions) were performed in eight PwHA and two PwHA-I. Based on ROTEM, pre-operative factor infusions were used in ten PwHA and four PwHA-I interventions. Among nine low local bleeding risk interventions, three (33.3%) each received no infusions, one dose of factor infusion pre-operatively, and pre- and post-operative factor infusions. All eight high local bleeding risk interventions involved planned factor infusions. Absorbable hemostats were used in all extractions. A mouth splint was used in 21/25 (84.0%) PwHA and in 4/4 (100%) PwHA-I extractions. No post-extraction bleeding or thrombotic events occurred.Use of a systemic hemostatic treatment plan according to the local bleeding risk, peri-operative coagulation status assessment using ROTEM, filling the extraction socket with hemostats, and use of a mouth splint can achieve effective and safe hemostatic management in emicizumab-treated PwHA and PwHA-I.

An 8-year-old Japanese boy with no underlying disease presented with severe intramuscular hematoma of the hip, and was admitted for a disseminated intravascular coagulation-like state with fibrinolytic dominance. Laboratory examinations revealed severe hyper-fibrinolysis with elevated markers, markedly shortened euglobulin clot lysis time, mildly decreased prothrombin, and severely decreased fibrinogen and factor XIII. Natural fibrin precipitates rapidly appeared in citrate-treated, ethylene-diamine-tetra-acetic-treated, and heparin-treated samples, but not in argatroban-treated samples, indicating that the mechanism of thrombin and fibrin formation was Ca

Background; Factor (F)V 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, appear to be associated with deep vein thrombosis. The detailed mechanism(s) between these mutations and thrombophilia remains to be fully explored, however. Aim; To clarify thrombotic mechanism(s) in the presence of these FV abnormalities. Methods and Results; Full-length wild-type (WT) and mutated FV were prepared using stable, human cell lines (HEK293T) with the piggyBac transposon system. The susceptibility of FVa-A2086D to activated protein C (APC) was reduced, resulting in significant inhibition of APC-catalyzed inactivation with little 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 PS with similar affinities to FV-WT. However, deteriorated interaction between FVa-A2086D to phospholipid membranes was evident, by the prothrombinase assay. Moreover, the addition of FVa-A2086D to plasma failed to inhibit tissue factor (TF)-induced thrombin generation and prothrombin times. This inhibitory effect was independent from protein (P)C, PS, and antithrombin. The coagulant and anticoagulant characteristics of FV(a)-W1920R were similar to those of FV(a)-A2086D. Conclusions; 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.

Global coagulation potential was assessed in 59 patients with acquired hemophilia A (PwAHA) by clot waveform analysis (CWA) and/or thrombin and plasmin generation assay. Relationships between factor VIII activity (FVIII:C) and the parameters from CWA and T/P-GA in patients with congenital HA were compared by grading coagulation potential related to FVIII:C: T1 (FVIII:C < 1 IU/dL), T2 (1 ≤ , ≤ 5 IU/dL), T3 (5 < , 12 ≤ IU/dL), and T4 (12 < , ≤ 50 IU/dL). The median FVIII:C and inhibitor titers in PwAHA on admission were 3.3 IU/dL and 63.0 BU/mL, respectively, but global coagulation parameters corresponded to T1 or less. Median FVIII:C levels during follow-up in PwAHA were 1.7-9.6-6.7-40.0-21.7 IU/dL on days 0-14-28-56-93, respectively. CWA-based data corresponded to less than T2 until day 28, but more closely reflected FVIII:C after day 56. Peak thrombin was severely low (near T1) until day 28 and improved modestly after day 56 but remained less than T2. Peak plasmin was lower than T1 until day 56, and returned to T4 on day 93. In conclusion, global coagulation function in PwAHA was impaired to a greater extent than could be anticipated from assays of FVIII:C, until approximately 1 month after immunosuppression and treatment with FVIII-bypassing agents.

Emicizumab reduces bleeding episodes in patients with severe hemophilia A (PwHA). Little information is available on hemostatic management of severe traumatic hemorrhages in emicizumab-treated pediatric PwHA. We assessed therapeutic efficacy and global coagulation potentials in two pediatric cases of emicizumab-treated pediatric PwHA with intracranial or retroperitoneal/iliopsoas hemorrhage. A modified clot waveform analysis (CWA) triggered by mixtures of tissue factor and ellagic acid was used to assess coagulant potentials, and maximum coagulant velocity (Ad|min1|) was calculated. One patient with intracranial hemorrhage was treated with continuous infusions of recombinant factor VIII (rFVIII) at a dose of 4-4.6 IU/kg/hr for 9 days, followed by bolus infusion at 66 IU/kg/day for 2 days and 33 IU/kg/day for an additional 2 days. The Ad|min1| was increased from 5.5 (at baseline) to 7.0-8.1 under concomitant treatment and maintained within or near normal range (IQR; 6.9-7.7). The other patient with retroperitoneal/iliopsoas hemorrhage received bolus infusions of rFVIII at 50 IU/kg/day for 20 days and every-other-day infusion of rFVIII for 8 days. The Ad|min1| was increased from 5.2 (at baseline) to 5.8-6.8 under concomitant treatment and maintained within the normal range. We successfully managed a treatment plan for severe traumatic bleeding in emicizumab-treated pediatric PwHA using modified CWA.

Disturbances in the balance between coagulation, anticoagulation and fibrinolysis may lead to thrombosis or haemorrhage. Simultaneous assessments of thrombin and plasmin facilitate overall understandings of pathological haemostasis, especially for thrombophilia. Here, we characterized coagulation-fibrinolysis potentials in plasmas with thrombophilia using anticoagulants-mediated thrombin-plasmin generation assay (T/P-GA). T/P-GA was initiated by adding tissue factor, tissue-type plasminogen activator and anticoagulants [recombinant-thrombomodulin (rTM), activated protein (P)C (APC) and antithrombin (AT)], followed by simultaneous thrombin generation and plasma generation monitoring. Patients' plasmas with PC-deficiency (PC-def), PS-deficiency (PS-def), AT-deficiency (AT-def), factor VLeiden (FVL) and antiphospholipid syndrome (APS) were evaluated. A ratio of peak-thrombin (or peak-plasmin) with and without anticoagulants was calculated as anticoagulants (+)/anticoagulants (-). First, TG, in rTM-mediated, PC-def, PS-def and FVL showed higher peak-thrombin ratios than the controls, whereas AT-def and APS exhibited no differences from the controls. In APC-mediated, PC-def, PS-def and AT-def showed low peak-thrombin ratios, similar to the controls, but immune-depleted PS-def (1%) showed the higher ratio than the controls. FVL and APS showed higher peak-thrombin ratios than the controls. In AT-mediated, peak-thrombin ratios in PS-def, PC-def and APS were lower than in controls, but those in AT-def and FVL was not significantly different from the controls. Second, PG, in rTM-mediated, all thrombophilia plasmas showed low peak-plasmin ratios (∼0.5), but no significant difference was observed, relative to the controls. In APC and AT-mediated, peak-plasmin ratios in thrombophilia-related plasmas were similar to the controls (∼1.0). Anticoagulants-mediated T/P-GA may classify thrombin generation characteristics in thrombophilia-related plasmas upon adding anticoagulants.

Emicizumab markedly shortens the activated partial thromboplastin time (aPTT), resulting in inaccurate measurements of procoagulant and anticoagulant factor activities. We have recently reported that mixtures of two different anti-idiotype monoclonal antibodies against emicizumab (anti-emicizumab-mAbs) allow measurement of factor (F)VIII activity (FVIII:C) and FVIII inhibitor in emicizumab-containing plasmas. It is unknown whether anti-emicizumab mAbs can work for other aPTT-based procoagulant and anticoagulant assays.To investigate whether anti-emicizumab mAbs were measured by all of the aPTT-based assays tested.Two anti-emicizumab-mAbs (300 μg/mL each) were preincubated with emicizumab (200 μg/mL)-spiked FVIII-deficient plasma; we then measured FVIII:C, FIX:C, FXI:C, FXII:C, protein (P)C:C, PS:C, global PC-FV (aPTT-based), and prothrombin time (PT), diluted Russel's viper venom time (dRVVT), chromogenic-based FVIII:C, FIX:C and PC:C (non-aPTT-based). Emicizumab (100 μg/mL)-spiked haemophilia (H)A plasmas from patients (n = 23) were also measured.Emicizumab shortened the clotting time in all aPTT-based assays, resulting in high levels of FVIII:C, FIX:C, FXI:C and FXII:C; low levels of PC:C and PS:C; and false-positive results for activated PC resistance. The addition of anti-emicizumab-mAbs to emicizumab-added plasma restored all factors to the initial levels without emicizumab. Chromogenic FVIII:C measurement by human FIXa/FX was affected by emicizumab, but anti-emicizumab mAbs cancelled this effect. PT-based assays and dRVVT, chromogenic FIX:C and PC:C assays showed no effect with emicizumab. Twenty-three plasma samples from HA patients also showed similar patterns.Anti-emicizumab mAbs in vitro could cancel the effect of emicizumab, irrespective of the test base, resulting in accurate measurements of procoagulant and anticoagulant factor activity.

Introduction Emicizumab prophylaxis improves coagulation function in congenital hemophilia A, regardless of inhibitor presence. We recently reported that emicizumab enhanced the coagulant potentials, ex vivo, in plasmas from patients with acquired hemophilia A (PwAHAs) at diagnosis. However, coagulant effects of emicizumab in PwAHAs during the clinical course remain unclear. Aim To assess ex vivo coagulant effects of emicizumab in PwAHAs during the clinical course. Methods/Results Blood samples were obtained from 14 PwAHAs on (median) days 0 and 6 during a severe-bleeding phase, and days 27 and 59 during a reduced-bleeding phase with elevated endogenous factor VIII (FVIII) and decreased inhibitor titers. If administered a single dose of 3 or 6 mg/kg, or two doses at 6 mg/kg followed by 3 mg/kg, estimated plasma emicizumab concentrations (10/5/2.5, 20/10/5, and 30/15/7.5 µg/mL on days 0–7/30/60, respectively) could be used to represent potential changes, based on the half-life (T 1/2: ∼30 days). Emicizumab concentrations that covered maximum plasma concentrations of each dosage were used for spiking on day 0. Ex vivo addition of estimated emicizumab to PwAHA's plasma containing endogenous FVIII and/or inhibitor, without and with recombinant (r)FVIIa administration during immunosuppressive therapy, increased the calculated Ad|min1| values, assessed by clot waveform analysis, and their coagulant potentials were below normal levels. Rotational thromboelastometry revealed that ex vivo emicizumab addition resulted in the further improvement of coagulant potentials in whole bloods when combined with rFVIIa administration. Conclusion Based on ex vivo and in vitro data, emicizumab has the potential to be effective in clinical situations for PwAHAs.

Emicizumab prophylaxis reduces bleeding in hemophilia A (HA) patients. However, there are few data on emicizumab treatment in neonates with HA (neonate-HA), and the procoagulant effects of emicizumab in these patients are unknown.To investigate the coagulation activity of emicizumab in vitro in a plasma model of neonate-HA.Plasmas from 84 neonates with non-HA were enrolled. However, due to the limited plasma volumes in some cases, 50 plasmas were assigned to two different assay groups. To prepare the neonate-HA model, plasma was first preincubated with an antifactor (F) VIII A2 monoclonal antibody (mAb). After further incubation with emicizumab, global coagulation activity was measured: adjusted maximum coagulation velocity (Ad|min1|) in clot waveform analysis (CWA) and peak thrombin in thrombin generation assay (TGA).Because the addition of anti-FVIII mAb to 22 of 43 samples showed little decrease in Ad|min1|, the remaining 21 samples were analyzed by CWA. The addition of emicizumab increased Ad|min1| in 18 of the 19 cases (effective group) but not in the remaining 3 cases (noneffective group). Similarly, TGA found that emicizumab (effective group) improved peak thrombin in seven of the nine samples tested, but two cases did not respond (noneffective group). Although the effective group had lower levels of FX, there was no significant difference between the effective and noneffective groups in terms of FIX, protein S, protein C, antithrombin, and fibrinogen.The in vitro coagulant potentials of emicizumab in the neonate-HA model were more heterogeneous than those recorded in the adult-HA model.

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.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.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 (FVrTM-mediated TF-triggered CWA might be useful for screening PC pathway abnormality-related thrombophilia.

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.

When patients with hemophilia A develop factor VIII (FVIII) inhibitors, FVIII replacement therapy becomes ineffective. Although immune-tolerance induction (ITI) therapy has been used to eradicate inhibitors, treatment is unsuccessful in approximately 30% of cases. However, the mechanism behind treatment failure remains unclarified. We retrospectively examined the longitudinal profiles of immunoglobulin G (IgG) subclasses and/or the inhibitory activities of FVIII in plasma samples from 14 Japanese patients with congenital hemophilia A during hemostatic, FVIII replacement, and/or ITI therapies. In five patients, an increase in IgG4 was observed simultaneously with a decrease in IgG1 when the patient had a history of relatively high FVIII inhibitor titers, reflecting an apparent change in humoral immunity. In addition, we examined the reactivity and specificity of the patients' anti-FVIII IgG1 and IgG4 to FVIII domains by immunoblotting. Under our experimental conditions, plasma from three patients with historically higher inhibitor titers appeared to have high titers of antibodies against the A2-a2 domain, which did not necessarily correlate with ITI failure. These observations may improve scientific understanding of the immune response to infused FVIII in patients with hemophilia A.

Coagulation and fibrinolytic mechanisms are enhanced in patients with coronavirus (COVID-19), but disturbances in the balance of both functions in COVID-19 patients remain unclear. We assessed global coagulation and fibrinolysis in plasma from 167 COVID-19 patients (mild/moderate/severe: 62/88/17, respectively) on admission using clot-fibrinolysis waveform analysis (CFWA). Maximum coagulation velocity (|min1|) and maximum fibrinolysis velocity (|FL-min1|) were expressed as ratios relative to normal plasma. Ten patients (6.0%) developed thrombosis, 5 (3.0%) had bleeding tendency, and 13 (7.8%) died during admission. FDP levels increased with severity of COVID-19 symptoms (mild/moderate/severe; median 2.7/4.9/9.9 μg/mL, respectively). The |min1| ratios were elevated in all categories (1.27/1.61/1.58) in keeping with enhanced coagulation potential, with significant differences between mild cases and moderate to severe cases. The |FL-min1| ratios were also elevated in all groups (1.19/1.39/1.40), reflecting enhanced fibrinolytic potential. These data identified coagulation dominance in moderate to severe cases, but balanced coagulation and fibrinolysis in mild cases. There were significant differences in FDP and TAT, but no significant differences in |min1| or |FL-min1| ratios, between patients with and without thrombosis. CFWA monitoring of coagulation and fibrinolysis dynamics could provide valuable data for understanding hemostatic changes and disease status in COVID-19 patients.

Emicizumab reduces bleeding events in patients with severe hemophilia A (HA). The coagulation potential of emicizumab at a clinical dose appears to correspond to about 15 IU/dL of factor VIII activity (FVIII:C), the equivalent of converting from a severe to mild phenotype. However, the clinical and laboratory characteristics of HA patients receiving emicizumab (Emi-PwHA) compared with patients with mild HA (PwMHA) remain to be determined. We reviewed clinical data from Emi-PwHA (n = 63) and PwMHA (n = 15) and evaluated comprehensive coagulation function using Ca

Type 2N von Willebrand disease (VWD) is characterized by impaired factor VIII (FVIII) binding to von Willebrand factor (VWF). Type 2N VWD patients generally exhibit mild bleeding tendency, but some exhibit a more severe hemorrhagic pattern. An assay for assessing hemostatic potential and predict clinical severity could significantly improve clinical management in these patients. We examined the relationship between bleeding score (BS) and the potential for thrombus formation in whole blood from type 2N VWD patients with various BS using rotational thromboelastometry (ROTEM) and microchip flow-chamber system (T-TAS®). Collagen-coated PL-chips, or thromboplastin- and collagen-coated AR-chips, were utilized in the T-TAS to assess platelet thrombus formation at high shear flow, or fibrin-rich platelet thrombus formation at low shear flow, respectively. Neither ROTEM nor the T-TAS using PL-chips reflected the BS. The AR-chip parameters in the T-TAS, however, were highly sensitive to different BS levels among these patients, despite similar FVIII/VWF-related measurements including FVIII/VWF binding. Additionally, the results with AR-chip assay were restored to normal after infusions of FVIII/VWF concentrates in the most severe patients. The data indicate that T-TAS using AR-chips may be a useful assay for predicting clinical severity and assessing therapeutic efficiency in type 2N VWD patients.

Emicizumab is a bispecific antibody to factor (F) IX/IXa and FX/FXa, which mimics FVIIIa cofactor function. Emicizumab prophylaxis significantly decreases bleeding events for patients with hemophilia A (PwHA). However, global hemostatic monitoring in emicizumab-treated PwHA remains poorly investigated. Using rotational thromboelastometry (ROTEM), we evaluated coagulation potentials of whole blood samples from seven emicizumab-treated PwHA who participated in ACE001JP/ACE002JP studies. Dose-dependent coagulation-enhancing effects of emicizumab to whole blood from PwHA mixed with an anti-FVIII C2 antibody in vitro were evident by non-activated ROTEM analysis (NATEM). The relationship between FVIII levels and NATEM parameters in PwHA not participating in the clinical trials demonstrated that CT + CFT inversely correlated with FVIII levels. These parameters were defined as NATEM-based grading of coagulation potential; ‘T1’ (FVIII

Essentials Emicizumab mimics factor (F)VIIIa cofactor function, augments the intrinsic tenase activity. We assessed the emicizumab-driven hemostatic function in FXI-deficient plasmas. Emicizumab improved the coagulation potentials in severe FXI-deficient plasma. Emicizumab may provide a possibility for clinical application in patients with FXI deficiency. SUMMARY: Background Patients with factor (F)XI deficiency commonly present with markedly prolonged activated partial thromboplastin times (APTT), although bleeding phenotypes are heterogeneous. Emicizumab, a bispecific monoclonal antibody to FIX/FIXa and FX/FXa, mimics FVIIIa cofactor function on phospholipid (PL) surfaces. Antibody reactions were designed, therefore, to augment mechanisms during the propagation phase of blood coagulation. Aim To assess emicizumab-driven hemostatic function in FXI-deficient plasmas. Methods and Results Standard ellagic acid (Elg)/PL-based APTTs of different FXI-deficient plasmas (n = 13; FXI activity, < 1 IU dl-1 ) were markedly shortened dose dependently by the presence of emicizumab. To further analyze the effects of emicizumab, clot waveform analysis (CWA) in FXI-deficient plasmas with emicizumab, triggered by tissue factor (TF)/Elg demonstrated improvements in both clot times, reflecting the initiation phase, and coagulation velocity, which represents the propagation phase. Emicizumab also enhanced the TF/Elg-triggered thrombin generation in FXI-deficient plasmas dose-dependently although the degree of enhancement varied in individual cases. Thrombin generation with either FVII-deficient plasma or FIX-deficient plasma treated with anti-FXI antibody showed little or no increase by the co-presence of emicizumab, suggesting that the accelerated thrombin generation in FXI-deficient plasmas by emicizumab should depend on the FIXa-involved coagulation propagation initially triggered by FVIIa/TF. The ex vivo addition of emicizumab to whole blood from three patients with severe FXI deficiency demonstrated modest, dose-dependent improvements in Ca2+ -triggered thromboelastograms (NATEM mode). Conclusion Emicizumab appeared to improve coagulation function in severe FXI-deficient plasma, and might provide possibilities for clinical application in patients with FXI deficiency.

Background Emicizumab prophylaxis is a promising treatment that reduces bleeding events in severely affected patients with hemophilia A (PwHA). It is anticipated that emicizumab could be similarly effective in mild/moderate PwHA (PwMHA) although this effect has not been investigated. Aim We evaluated ex vivo coagulant effects of emicizumabin PwMHA. Methods Clot waveform analysis (CWA) triggered by prothrombin time/activated partial prothrombin time-mixed reagents was utilized to examine coagulant effects of emicizumabin factor (F)VIII-deficient plasma mixed with recombinant (r)FVIIIand in native plasmas from 16 PwMHA. The CWA parameter, adjusted-|min1| (Ad|min1|), was used. Increases in Ad|min1| (ΔAd|min1|) mediated by emicizumab were calculated from the slopes of regression lines in the presence of rFVIII. Results Ad|min1| in FVIII-deficient plasma with various concentrations of rFVIII negatively correlated with ΔAd|min1|by adding emicizumab, and these data were defined as standard reference values. Ad|min1| (4.57 ± 0.50) in 16 PwMHA increased to 5.05 ± 0.54 and 5.37 ± 0.60 by adding emicizumab at 50 and 100 μg/mL, respectively, but remained lower than the normal range (7.22 ± 0.21). ΔAd|min1| levels were 1.5 to 2-fold higher in five cases and 0.4 to 0.6-fold lower in four cases, compared with reference values determined by rFVIII. In some cases, genetic analyses suggested that specific point mutations could have contributed to these findings. Further studies using rFVIII mutants indicated, however, that the differences in ΔAd|min1| were not related to individual FVIII gene defects. Conclusion Emicizumab enhances coagulation potential in PwMHA. Assessment of ex vivo coagulant activity of emicizumab could be helpful for predicting coagulant potentials prior to treatment in these patients.

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.

Background and aims Oxidized low-density lipoprotein (oxLDL) causes the development of atherosclerosis and kidney injury. Although circulating oxLDL levels were reportedly increased in type 2 diabetic patients with macroalbuminuria, it remains unclear whether albuminuria or the reduced glomerular filtration rate (GFR) is independently associated with the circulating oxLDL level. This study aimed to elucidate the association between the stage of diabetic nephropathy and serum malondialdehyde-modified LDL (MDA-LDL) and the ratio of MDA-LDL to LDL-cholesterol (MDA-LDL/LDL). Methods and results This retroactive cross-sectional study used data from 402 patients with type 2 diabetes. Patients undergoing hemodialysis were excluded. Serum MDA-LDL levels were significantly increased with increases in severity of albuminuria (103 ± 44 U/L, 109 ± 54 U/L, and 135 ± 72 U/L for normoalbuminuria, microalbuminuria, and macroalbuminuria, respectively; P for trend = 0.020) but not according to the estimated GFR (eGFR). An increased MDA-LDL/LDL ratio was significantly associated with both increased albuminuria (35 ± 13, 37 ± 14, and 40 ± 15 for normoalbuminuria, microalbuminuria, and macroalbuminuria, respectively; P for trend = 0.003) and reduced eGFR (34 ± 13, 36 ± 13, 38 ± 12, and 51 ± 28 for grade 1, 2, 3 and 4, respectively; P for trend = 0.002). Multiple linear regression analysis showed that neither the albumin excretion rate nor eGFR but ln-transformed triglycerides and LDL-C levels were independent determinants of both serum MDA-LDL levels and MDA-LDL/LDL ratios. Conclusion Serum MDA-LDL levels and MDA-LDL/LDL ratios were increased in those with dyslipidemia associated with diabetic kidney disease.

Bypassing therapy is essential for the haemostatic management of patients with haemophilia A with inhibitor (PWHA-inh), but the therapeutic effects are inconsistent. We previously reported that activated prothrombin complex concentrates (aPCC) activated factor (F)VIIIin vitro, and was mediated mainly by the activated FVII (FVIIa) contained in aPCC. We have extended those studies to assess global coagulation in whole blood from 18 PWHA-inh in the co-presence of aPCC and FVIII using Ca2+ -triggered rotational thromboelastometry. The clot times (CTs) in the presence of both aPCC (0·05 iu/ml) and recombinant (r)FVIII (1 iu/ml) ex vivo were shortened compared to the aPCC alone (P

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.To investigate the relationship between WB-RIPA, bleeding scores (BS) and VWF-related measurements in type 1 VWD.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.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 BS4 (defined as no abnormal bleeding tendency), respectively. However, no significant difference was observed in WB-RIPA between the BS ≥4 group and BS4 group. Overall, VWD patients with a WB-RIPA level70 U did not seem to have an abnormal bleeding tendency, but low levels of WB-RIPA did not correlate with BS.WB-RIPA did not reflect clinical severity in type 1 VWD patients.

We investigated how Sasa kurilensis ramet longevity differs under three light conditions. Ramet longevity is an important factor affecting the abundance of ramet populations and their biomass. The objectives of this study were to clarify: (1) whether ramet longevity varies spatially along a gradient of light conditions; (2) whether physiological functions, including water transport in culms, change with ontogenesis; (3) whether ramet longevity is associated with the most effective turnover for gaining a carbon profit within a ramet following a cost-benefit model or is affected by other factors such as death caused by a decline in physiological function. We analyzed S. kurilensis ramet longevity, hydraulic resistance, photosynthetic rate, and carbon content. We then estimated the ramet carbon budget. The longevity of S. kurilensis ramets was 2.8–4.5 years in Beneath-patch plots, 5.8–8.7 years in Edge-patch plots, and 1.6–2.2 years in Open-patch plots. Although leaf photosynthetic activity was stable, the instantaneous photosynthetic rate decreased during clear days in the open area. This may have been due to increased ramet hydraulic resistance. The rotation period of the most efficient carbon budget quantified by ecophysiological measurements was consistent with ramet longevity in Open-patch (2 years) and Edge-patch (5 years) sites. Meanwhile, the longevity of ramets grown under canopies was inconsistent with the cost-benefit model rules for a carbon budget because the carbon gain was low throughout the ramet life span.

A 27‐year‐old woman with panic disorder taking 20 mg olanzapine daily for 4 months was admitted to Mito Kyodo General Hospital, Mito, Ibaraki, Japan, because of disturbed consciousness with fever, hyperglycemia, hyperosmolarity and elevated creatine phosphokinase. She was diagnosed with a hyperosmolar hyperglycemic state and neuroleptic malignant syndrome. Brain magnetic resonance imaging showed transiently restricted diffusion in the splenium of the corpus callosum, with a high signal intensity on diffusion‐weighted imaging. The neurological abnormalities disappeared along with improvement of metabolic derangements, and the follow‐up magnetic resonance imaging carried out on the 26th day of admission showed complete resolution of the lesions in the splenium of the corpus callosum. These clinical and radiological features are highly suggestive of clinically mild encephalitis/encephalopathy with a reversible splenial lesion. The first case of mild encephalitis/encephalopathy with a reversible splenial lesion caused by olanzapine‐induced hyperosmolar hyperglycemic state and neuroleptic malignant syndrome is reported.

Pre-zygotic interspecies incompatibility in angiosperms is a male-female relationship that inhibits the formation of hybrids between two species. Here, we report on the identification of STIGMATIC PRIVACY 1 (SPRI1), an interspecies barrier gene in Arabidopsis thaliana. We show that the rejection activity of this stigma-specific plasma membrane protein is effective against distantly related Brassicaceae pollen tubes and is independent of self-incompatibility. Point-mutation experiments and functional tests of synthesized hypothetical ancestral forms of SPRI1 suggest evolutionary decay of SPRI1-controlled interspecies incompatibility in self-compatible A. thaliana. Hetero-pollination experiments indicate that SPRI1 ensures intraspecific fertilization in the pistil when pollen from other species are present. Our study supports the idea that SPRI1 functions as a barrier mechanism that permits entrance of pollen with an intrinsic signal from self species.

Factor VIIa/tissue factor (FVIIa/TF) initiates blood coagulation by promoting FXa generation (extrinsic-Xa). Subsequent generation of intrinsic FXa (intrinsic-Xa) amplifies thrombin formation. Previous studies suggested that FVIIa/TF activates FVIII rapidly in immediate coagulation reactions, and FVIIa/TF/FXa activates FVIII prior to thrombin-dependent feedback. We investigated FVIII/FVIIa/TF/FXa relationships in early coagulation mechanisms. Total FXa generated by FVIIa/TF and FVIIa/TF-activated FVIII (FVIIIaVIIa/TF) was 22.6 ± 1.7 nM (1 min); total FXa with FVIIa-inhibitor was 3.4 ± 0.7 nM, whereas FXa generated by FVIIa/TF or FVIII/TF was 10.4 ± 1.1 or 0.74 ± 0.14 nM, respectively. Little Xa was generated by FVIII alone, suggesting that intrinsic-Xa mechanisms were mediated by FVIIIaVIIa/TF and FVIII/TF in the initiation phase. Intrinsic-Xa was delayed somewhat by von Willebrand factor (VWF). FVIII activation by FXa with FVIIa/TF was comparable to activation with Glu-Gly-Arg-inactivated-FVIIa/TF. TF counteracted the inhibitory effects of VWF on FXa-induced FVIII activation mediated by Arg372 cleavage. The FVIII-C2 domain bound to cytoplasmic domain-deleted TF (TF1−243), and VWF blocked this binding by > 80%, indicating an overlap between VWF- and TF1−243-binding site(s) on C2. Overall, these data suggest that FVIII-associated intrinsic-Xa, governed by both FVIIa/TF-induced and FXa-induced FVIII activation mediated by FVIII-TF interactions, together with FVIIa-dependent extrinsic-Xa mechanisms, may be central to the initiation phase of coagulation.

Changes in the levels of amino-acid enantiomers are associated with some serious diseases; consequently, amino acid monitoring in peripheral blood can be used to diagnose and predict the onset of disease. Herein, we report the design and synthesis of a new chiral derivatization reagent, namely succinimidyl (4S)-(3-[(benzyloxy)carbonyl]-5-oxo-1,3-oxazolidin-4-yl)acetate ((S)-COXA-OSu), for the separation of dl -amino-acid enantiomers. The usefulness of (S)-COXA-OSu was examined as a derivatization reagent for LC-MS/MS following certification of its total optical purity (>99%). The enantiomeric separations of amino-acid derivatives tagged with the reagent were examined using a triazole-bonded phase. (S)-COXA-OSu enabled the simultaneous enantiomeric separation of more than 40 α-amino acids. (S)-COXA-amino-acid derivatives were efficiently converted into their product ions, from which formaldehyde (CH2O) was eliminated [M-30] from the oxazolidinone moiety of COXA by collision-induced dissociation during LC-MS/MS. Limits of detection were in the 0.0138–0.518 pmol/injection range. For precise and accurate quantitation, we synthesized and used a stable-isotope-labeled (S)-COXA-OSu that was used as an internal standard in LC-MS/MS-determination experiments. Finally, changes in plasma amino-acid levels in rats, following administration of S-methyl- l -cysteine, an alanine-serine-cysteine transporter-1 (Asc-1) inhibitor, were successfully detected by LC-MS/MS using (S)-COXA-OSu.

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 (K d; 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 K d 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 K m values were 1.4- to 1.7-fold greater, and overall catalytic efficiency (k cat/K m) 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.

We aimed to monitor changes in the levels of amino acid neurotransmitters or neuromodulators simultaneously at the synaptic clefts of experimental animals. We developed a method for the simultaneous determination of the levels of amino acids, such as D-Ser, Gly, and L-Glu, which were involved in neurotransmission via the N-methyl-D-aspartate (NMDA) receptor, and other protein-constituted amino acids in a rat brain microdialysis (MD) sample. We used a liquid chromatography-mass spectrometry (LC-MS)/MS device equipped with a triazole-bonded column. The determination was achieved without using stable isotope-labeled compounds. We instead used suitable amino acid analogues as internal standards (ISs). We examined various analyte-IS combinations to improve reproducibility. We found a positive correlation (r = 0.720, **p

The patient was a 25-year-old woman whose paternal family was Japanese, maternal grandfather was Filipino, and maternal grandmother was Chinese. Eleven days after delivery, she presented with excessive thirst and disturbed consciousness due to diabetic ketoacidosis. She was diagnosed as having fulminant type 1 diabetes associated with pregnancy (PF). The antibody concentration against glutamic acid decarboxylase was 1.2 (

Essentials von Willebrand factor levels don't always reflect the severity of von Willebrand disease (VWD). Relationship between new flow system (T-TAS®) and bleeding score (BS) in type 1 VWD was studied. Patients with PL-T10 > 10 min had higher BS and particularly, PL-T10 > 8 min correlated best. T-TAS could be a useful tool for discriminating and predicting the BS in VWD type 1 patients. SummaryBackground The clinical phenotype of von Willebrand disease (VWD) is heterogeneous, and von Willebrand factor ristocetin cofactor activity (VWF:RCo) does not always reflect clinical severity, especially in VWD type 1. We have reported the potential of a microchip flow-chamber system (Total-Thrombus Formation Analysis System [T-TAS®]) for assessing physiologic hemostasis in VWD. Aim To evaluate the relationship between T-TAS, bleeding score (BS) and laboratory test results in type 1 VWD patients. Methods Microchips coated with collagen (platelet chip [PL-chip]) or collagen/thromboplastin (AR-chip) were used to assess platelet thrombus formation (PTF) at high shear rates or fibrin-rich PTF at low shear rates, respectively, in whole blood from 50 patients. The times needed for the flow pressure to increase by 10 kPa and 30 kPa (T10 and T30) from baseline were calculated from flow pressure curves. BS was determined by the use of a standardized questionnaire. Results PL-T10 values correlated with BS (R2 ~ 0.45) better than VWF:RCo (R2 ~ 0.36), irrespective of the flow rate, whereas AR-T10 showed only a weak correlation with BS (R2 ~ 0.18). Patients with PL-T10 > 10 min or AR-T10 > 30 min had lower VWF levels and higher BS than those with PL-T10 ≤ 10 min or AR-T10 ≤ 30 min, and the greatest differences were observed with PL-T10. Clinical severity appeared to correlate best with PL-T10 > 8 min. BS was significantly higher in patients with VWF:RCo of 8 min than in those with PL-T10 ≤ 8 min. Conclusion T-TAS could be a useful technique for discriminating and predicting BS in VWD type 1 patients.

Platelet storage pool disease (SPD) is a platelet function disorder characterized by a reduction in the number or content of α-granules, dense granules, or both, and is diagnosed by specialized tests. Patients with SPD often present with prolonged bleeding time (BT), but the sensitivity and reproducibility of this test have limitations, often resulting in false negatives. It has recently been reported that an automated microchip flow-chamber system (T-TAS(®)) is useful in the assessment of anti-platelet therapy, and could have potential as a screening test for SPD. We examined the utility of T-TAS in three individuals from one family diagnosed with δ-SPD. The propositus had a mildly prolonged BT, and the standard tests for platelet function were close to the normal range. Whole blood samples were anti-coagulated with hirudin and applied to T-TAS microchips coated with collagen (PL-chips) at shear rates of 1000 and 2000 s(-1). Platelet thrombus formation (PTF) was monitored with a pressure sensor. Markedly depressed PTF was observed in all cases at both shear rates. These findings indicate that T-TAS is highly sensitive to the defect in these patients with SPD, and may represent a good candidate screening test for a wide range of platelet function disorders.

Summary Background The management of hemophilia A (HA) patients with inhibitors on bypassing therapy remains challenging. In particular, the monitoring of treatment is restricted by the limited reliability and lack of standardization of currently available methods to evaluate the physiological effects of various hemostatic agents. Accurate monitoring of these patients is particularly important in surgical situations. The recently developed comprehensive coagulation assays, including rotational thromboelastometry (ROTEM), may be useful in these circumstances. Objective We have attempted to establish a systematic monitoring protocol using ROTEM (NATEM triggered by CaCl2) to evaluate the choice and effectiveness of different bypassing agents in the perioperative period. Methods and Results The hemostatic effects of recombinant factor VIIa (rFVIIa) and activated prothrombin complex concentrates (aPCC) were determined using a three-step procedure (spike, preoperative and perioperative) in eight patients with HA inhibitor admitted for elective surgery and assessed for individually tailored therapy. The ROTEM parameters demonstrated similar improvement to approximately normal levels at each stage after treatment with rFVIIa. Results in the presence of aPCC showed a marked improvement in the spike data, although this appeared to be different from those in the preoperative and perioperative assessments. The information derived from the spike and preoperative findings provided a useful guide for establishing an effective dose of therapeutic material, and facilitated good hemostatic control during and after surgery in all cases. Conclusion The findings suggest that this systematic analysis using ROTEM could provide a promising strategy for the use of bypassing therapy in HA patients with inhibitor.

Factor (F)VIII functions as a cofactor in the tenase complex responsible for phospholipid (PL) surface-dependent conversion of FX to FXa by FIXa. On the other hand, protein S (PS) functions as a cofactor of activated protein C that inactivates FVIII(a) and FV(a). We have reported a new regulatory mechanism on coagulation that PS directly impaired the FXase complex by competing the FIXa-FVIIIa interaction (Takeyama, Br J Haematol. 2008;143:409), and identified the PS-interactive site on the FVIII A2 domain (Takeyama, Thromb Haemost. 2009;102:645). However, the contribution of FVIII light chain (LC) to PS-binding has not been determined. In this study, several approaches were employed to assess a PS-FVIII LC interaction. The binding of FVIII LC to active site-modified FIXa (EGR-FIXa) was inhibited by PS dose-dependently ( K i ; 4.6 nM) on ELISA. Because FVIII C2 domain has a binding site for FIXa, we examined whether FVIII C2 domain bound to PS. PS bound to FVIII C2 domain ( K d app ; 283 nM) by the ELISA. A SPR-based assay also revealed that FVIII C2 domain bound to PS ( K d ; 62 nM). We have reported that a FIXa-interactive site exits on FVIII C2 domain (residues 2228-2240) (Soeda, J Biol Chem. 2009;284:3379). Therefore, we assessed the interaction of the synthetic peptide spanning the residues (2228-2240) with PS. ELISA showed that the peptide bound to PS ( K d app ; 104 µM). SPR-based assay also revealed that the peptide bound to PS ( K d ; 31.4 µM), although scrambled peptide failed to bind to PS. Covalent cross-linking was observed between the biotynated 2228-2240 peptide and PS following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) using SDS-PAGE. This cross-linking formation was blocked by the addition of the unlabeled peptide. Furthermore, N-terminal sequence analysis of the peptide-PS product showed that one basic residue (K2239) could not be detected, supporting that this residue participates in cross-link formation. These results indicate that the 2228-2240 residues in the FVIII C2 domain, in particular K2239, may contribute to a PS-interactive site. To inquire further into the interactive region of FVIII C2 domain to PS, we prepared a stably expressed recombinant B-domainless FVIII mutants (E2228A, D2233A, K2236A, and K2239A), which were polar amino acids in the 2228-2240 residues, subsequently compared interaction with PS of the mutants with B-domainless FVIII wild type. To evaluate the binding affinity to PS, we performed SPR-based assay with the wild type FVIII and the FVIII mutants. The assay showed K2239A bound to PS with ~1.9-fold higher K d value (28.1 nM) than that of wild type ( K d ; 14.7 nM), although E2228A, D2233A, or K2236A bound to PS ( K d ; 12.7, 16.3, and 10.3 nM, respectively) with no significant difference compared with wild type. In addition, to evaluate the effect of mutants for FXa generation, we assessed FXa generation assay with wild type or K2239A in the presence or absence PS, because K2239 might contribute to a PS interaction of FVIII. After each FVIII (1 nM) was reacted with various concentrations of PS in the presence of PL (20 µM) for 30 minutes, FVIII was activated by thrombin (30 nM), followed by the reaction with FIXa (40 nM) and FX (300 nM) for 1 minute. Although generated FXa with both wild type and K2239A were reduced in the presence of PS dose-dependently, compared with its absence, the inhibition ratio of K2239A was less than that of wild type (18.2% and 31%, respectively in the presence of 500 nM PS). On the other hand, the K m value on FXa generation of K2239A for various concentrations of FIXa was ~1.8-fold higher than that of wild type (5.5±0.9 and 3.1±0.2 nM, respectively), suggesting that K2239 residue, involved in the FIXa-interactive site, might contribute to the inhibition of FVIII function by PS. In conclusion, FVIII C2 domain, in particular K2239, was possible to play an important role of the inhibitory mechanism to FVIII function by PS, due to the binding to PS. Disclosures Shima: Sysmex Corporation: Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Patents & Royalties, Research Funding. Nogami: Sysmex Corporation: Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Abstract 2237 Introduction: von Willebrand disease (VWD) has clinically heterogeneous phenotype. Routine measurements of von Willebrand factor (VWF) antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo) and FVIII activity (FVIII:C) do not always reflect clinical severity, especially in type 1 VWD. These assays evaluate VWF function under non-physiological static condition - lacking blood flow. We had reported that a new microchip flow chamber system, total-thrombus-formation analysis system (T-TAS®, Fujimori Kogyo, Tokyo) would be a clinically useful flow assay for VWD (ASH 2011). In this study, we extended this study for application to evaluation and hemostatic monitoring for type 1 VWD. Methods: Citrated or hirudin-added blood from 15 patients with type 1 VWD was utilized. Re-calcified citrated blood added corn trypsin inhibitor was injected to a microchip in T-TAS at a constant flow rate (240 s−1), which flow surface was coated by collagen and tissue factor (AR chip). Hirudin-added blood was injected to a microchip in T-TAS at higher shear rate (1,000 s−1) which surface was coated by collagen (PL chip). Flow pressure curve was visualized and time until reach to 10 kPa (T10) was evaluated. AR chip promoted thrombus formation by both platelet aggregation and fibrin generation, whilst PL chip promoted thrombus formation by platelet alone. Standard laboratory tests for VWD were also performed. Clinical severities of VWD patients were evaluated by using a quantitative bleeding score (BS, from −3 without any symptoms to +45 with all major symptoms) previously reported by Tosetto (JTH, 2006). Results: Fifteen patients with type 1 VWD showed low levels of VWF:Ag [median 14% (range 1.3–51%)], VWF:RCo [8% (1.6–32%)], and FVIII:C [31% (3.0–68%)]. T10 in AR chip or PL chip was 17.7 min (11->30) or 7.1 min (3.3->10) [normal control (n=20); 12.2 min (8.6–16.6) or 3.5 min (2.4–6.6), respectively], showing delayed thrombus formation in type 1 VWD. Correlations between VWF:Ag and VWF:RCo (r2=0.80, p Conclusion: Traditional standard assays did not always reflect the clinical phenotype of patients with VWD. In contrast, T-TAS parameters in PL chip showed good correlations to BS, indicating its usefulness for distinguishing clinical phenotype and for monitoring of treatment for patients with type 1 VWD. Disclosures: Hosokawa: Fujimori Kogyo: Employment.