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.

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.

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.

Comprehensive genetic screening for vascular Ehlers-Danlos syndrome through an amplification-based next-generation sequencing system.

Vascular Ehlers-Danlos syndrome (vEDS) is a hereditary connective tissue disorder (HCTD) characterized by arterial dissection/aneurysm/rupture, sigmoid colon rupture, or uterine rupture. Diagnosis is confirmed by detecting heterozygous variants in COL3A1. This is the largest Asian case series and the first to apply an amplification-based next-generation sequencing through custom panels of causative genes for HCTDs, including a specific method of evaluating copy number variations. Among 429 patients with suspected HCTDs analyzed, 101 were suspected to have vEDS, and 33 of them (32.4%) were found to have COL3A1 variants. Two patients with a clinical diagnosis of Loeys-Dietz syndrome and/or familial thoracic aortic aneurysm and dissection were also found to have COL3A1 variants. Twenty cases (57.1%) had missense variants leading to glycine (Gly) substitutions in the triple helical domain, one (2.9%) had a missense variant leading to non-Gly substitution in this domain, eight (22.9%) had splice site alterations, three (8.6%) had nonsense variants, two (5.7%) had in-frame deletions, and one (2.9%) had a multi-exon deletion, including two deceased patients analyzed with formalin-fixed and paraffin-embedded samples. This is a clinically useful system to detect a wide spectrum of variants from various types of samples.

Anti-idiotype monoclonal antibodies against emicizumab enable accurate procoagulant and anticoagulant assays, irrespective of the test base, in the presence of emicizumab.

INTRODUCTION: 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. AIM: To investigate whether anti-emicizumab mAbs were measured by all of the aPTT-based assays tested. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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.

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.

Emicizumab enhances thrombus formation in vitro under high shear flow conditions in whole blood from patients with type 1 and type 3 von Willebrand disease.

INTRODUCTION: Type 1 and type 3 von Willebrand disease (VWD) are caused by partial and complete, quantitative deficiency of von Willebrand factor (VWF), respectively, and factor (F)VIII/VWF complex concentrates are used for haemostatic treatment. Emicizumab, mimics activated FVIII, reduces bleeding in haemophilia A patients. The effects of emicizumab on haemostasis in both types of VWD remain to be fully established, however. AIM: To examine the effects of emicizumab on thrombogenesis in type 1 and type 3 VWD. PATIENTS/METHODS: Perfusion chamber experiments under high shear conditions (2500 s-1 ) combined with immunostaining were performed using whole blood samples from patients with type 1 (VWF:Ag 25 U/dl) and type 3 VWD (<1.0 U/dl). RESULTS: The addition of FVIII (1 U/ml) to type 1 blood did not affect thrombus formation, whilst supplementation with VWF (1.6 U/ml) or FVIII/VWF (1 U/ml/1.6 U/ml) enhanced thrombogenesis to a similar extent. FVIII/VWF promoted thrombus formation significantly more than VWF alone, however, in type 3 blood. Emicizumab (100 µg/ml) augmented thrombus formation in type 3 blood compared to FVIII, and this potency seemed to be somewhat greater than that of VWF. Surface coverage of formed thrombus in type 3 VWD was less than that in type 1 VWD, but thrombus height was comparable in both. The addition of emicizumab to type 3 blood enhanced thrombin generation and fibrin formation compared to control IgG. CONCLUSION: Emicizumab promoted mechanisms of thrombus formation in vitro in type 3 and type 1 VWD, suggesting the possibility of alternative therapeutic protocols in these patients.

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.

Hemostatic rebalance in neonatal intrahepatic cholestasis with citrin deficiency.

BACKGROUND: Neonatal intrahepatic cholestasis with citrin deficiency (NICCD) results in coagulopathy due to decreased levels of vitamin (V)K-dependent clotting factors, similar to biliary atresia (BA). However, the involvement of VK-independent coagulant and anticoagulant factor(s) remains unknown. We examined relationships between coagulant and anticoagulant potential before and after nutritional treatment in NICCD. METHODS: Three cases (aged 12, 21, and 45 days) with NICCD-associated coagulopathy were evaluated with standard coagulation/anticoagulation tests and comprehensive coagulation assays, rotational thromboelastometry, and protein C/protein S (PC/PS) pathway function assay (ThromboPath® ), before and after nutritional treatment. RESULTS: In all cases, activated partial thromboplastin time and prothrombin time were significantly prolonged, which is associated with very low levels of VK-independent fibrinogen and antithrombin. The initiation of nutritional treatment of medium-chain triglycerides oil improved these levels within the normal range, although low levels of other clotting factors were modestly increased. Whole blood- rotational thromboelastometry analysis revealed near-normal coagulation potential, even before treatment, comparable to healthy adults, and supportive of their non-bleeding symptoms. The introduction of nutritional treatment had further improved comprehensive coagulation potential. The global PC/PS-pathway function assay demonstrated the absence of the features of this function associated with the pathogenesis of NICCD. Compared to BA, the plasma levels of fibrinogen and antithrombin in all cases were markedly low, whilst those after treatment improved, especially to similar level of BA. CONCLUSIONS: Neonatal intrahepatic cholestasis with citrin deficiency has the characteristic of rebalancing hemostatic mechanisms associated with coagulant and anticoagulant potential involving low levels of fibrinogen and antithrombin, suggesting a pathophysiological coagulopathy distinct from BA.

Genetic determinants of VWF clearance and FVIII binding modify FVIII pharmacokinetics in pediatric hemophilia A patients.

Factor VIII (FVIII) pharmacokinetic (PK) properties show high interpatient variability in hemophilia A patients. Although previous studies have determined that age, body mass index, von Willebrand factor antigen (VWF:Ag) levels, and ABO blood group status can influence FVIII PK, they do not account for all observed variability. In this study, we aim to describe the genetic determinants that modify the FVIII PK profile in a population of 43 pediatric hemophilia A patients. We observed that VWF:Ag and VWF propeptide (VWFpp)/VWF:Ag, but not VWFpp, were associated with FVIII half-life. VWFpp/VWF:Ag negatively correlated with FVIII half-life in patients with non-O blood type, but no correlation was observed for type O patients, suggesting that von Willebrand factor (VWF) half-life, as modified by the ABO blood group, is a strong regulator of FVIII PK. The FVIII-binding activity of VWF positively correlated with FVIII half-life, and the rare or low-frequency nonsynonymous VWF variants p.(Arg826Lys) and p.(Arg852Glu) were identified in patients with reduced VWF:FVIIIB but not VWF:Ag. Common variants at the VWF, CLEC4M, and STAB2 loci, which have been previously associated with plasma levels of VWF and FVIII, were associated with the FVIII PK profile. Together, these studies characterize the mechanistic basis by which VWF clearance and ABO glycosylation modify FVIII PK in a pediatric population. Moreover, this study is the first to identify non-VWF and non-ABO variants that modify FVIII PK in pediatric hemophilia A patients.

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.

Emicizumab improves the stability and structure of fibrin clot derived from factor VIII-deficient plasma, similar to the addition of factor VIII.

INTRODUCTION: Emicizumab is an antifactor (F)IXa/FX bispecific antibody, mimicking FVIIIa cofactor function. Emi prophylaxis effectively reduces bleeding events in patients with haemophilia A. The physical properties of emicizumab-induced fibrin clots remain to be investigated, however. AIM: We have investigated the stability and structure of emicizumab-induced fibrin clots. METHODS: Coagulation was initiated by activated partial thromboplastin time (aPTT) trigger and prothrombin time (PT)/aPTT-mixed trigger in FVIII-deficient plasma with various concentrations of emicizumab or recombinant FVIII. The turbidity and stability of fibrin clots were assessed by clot waveform and clot-fibrinolysis waveform analyses, respectively. The resulting fibrin was analysed by scanning electron microscopy (SEM). RESULTS: Using an aPTT trigger, the turbidity was decreased and the fibrinolysis times were prolonged in the presence of emicizumab dose-dependently. Scanning electron microscopy imaging demonstrated that emicizumab improved the structure of fibrin network with thinner fibres than in its absence. Although emicizumab shortened the aPTT dramatically, the nature of emicizumab-induced fibrin clots did not reflect the hypercoagulable state. Similarly, using a PT/aPTT-mixed trigger that could evaluate potential emicizumab activity, emicizumab improved the stability and structure of fibrin clot in a series of experiments. In this circumstance, fibrin clot properties with emicizumab at 50 and 100 µg/mL appeared to be comparable to those with FVIII at ~12 and ~24-32 IU/dL, respectively. CONCLUSION: Emicizumab effectively improved fibrin clot stability and structure in FVIII-deficient plasma, and the physical properties of emicizumab-induced fibrin clots were similar to those with FVIII.

A novel simultaneous clot-fibrinolysis waveform analysis for assessing fibrin formation and clot lysis in haemorrhagic disorders.

Simultaneous evaluation of coagulation and fibrinolysis facilitates an overall understanding of normal and pathological haemostasis. We established an assay for assessing clot formation and fibrinolysis simultaneously using clot waveform analysis by the trigger of a mixture of activated partial thromboplastin time reagent and an optimized concentration of tissue-type plasminogen activator (0·63 µg/ml) to examine the temporal reactions in a short monitoring time (<500 s). The interplay between clot formation and fibrinolysis was confirmed by analysing the effects of argatroban, tranexamic acid and thrombomodulin. Fibrinogen levels positively correlated with coagulation and fibrinolytic potential and initial fibrin clot formation was independent of plasminogen concentration. Plasminogen activator inhibitor-1-deficient (-def) and α2-antiplasmin-def plasmas demonstrated different characteristic hyper-fibrinolytic patterns. For the specificity of individual clotting factor-def plasmas, factor (F)VIII-def and FIX-def plasmas in particular demonstrated shortened fibrinolysis lag-times (FLT) and enhanced endogenous fibrinolysis potential in addition to decreased maximum coagulation velocity, possibly reflecting the fragile formation of fibrin clots. Tranexamic acid depressed fibrinolysis to a similar extent in FVIII-def and FIX-def plasmas. We concluded that the clot-fibrinolysis waveform analysis technique could sensitively monitor both sides of fibrin clot formation and fibrinolysis, and could provide an easy-to-use assay to help clarify the underlying pathogenesis of bleeding disorders in routine clinical practice.

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.

Hemostatic function in hyperfibrinolytic disseminated intravascular coagulation.

BACKGROUND: Global hemostatic mechanism(s) in patients with disseminated intravascular coagulation (DIC) are poorly understood. There are few diagnostic criteria of DIC based on overall or global hemostatic mechanisms. METHODS: We have assessed in detailed the dynamic global hemostatic changes using thrombin and plasmin generation assay (T/P-GA), clot fibrinolytic waveform analysis (CFWA) and not-activated rotational thromboelastometry (NATEM), in a young girl with DIC associated with acute myeloid leukemia (AML). The ratios of endogenous thrombin potential (T-EP) and plasmin lag time (P-LT) relative to normal plasma was sourced from pooled normal plasma from healthy volunteers on T/P-GA. RESULTS: The inverse P-LT ratio prior to tranexamic acid (TXA) treatment was greater than the T-EP ratio (1.1-2.8 and 0.83-1.2, respectively). Significant reduction in inverse P-LT ratio (0.084-1.3) was observed after TXA treatment. The interval from clotting to the initiation of fibrinolysis (fibrinolysis lag time: FLT) in CFWA was significantly shorter than the control at onset (74.2-91.6 s vs 109 s), indicating enhanced fibrinolysis. Data from an adult with acute promyelocytic leukemia-associated DIC also supportively showed a high inverse P-LT ratio (2.1) and shortened FLT (83.7 s). The clotting time in patient whole blood using NATEM-mode during an episode of severe epistaxis markedly shortened beyond control, but returned to normal after the addition of an anti-tissue factor (TF) monoclonal antibody. CONCLUSION: The release of intravascular TF contributed to sustained activation of coagulation and subsequent fibrinolytic activity in this patient with AML-associated DIC, and T/P-GA could provide better quantitative data than conventional assays in these circumstances.

Factor (F)VIII/VIIa enhances global haemostatic function in the co-presence of bypassing agents and FVIII among patients with haemophilia A with inhibitor.

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 < 0·01). These enhancing effects of rFVIII were observed, irrespective of recognizing inhibitor epitopes; however, the clot formation time and 'α'-angle were not significantly different. In samples from 7 PWHA-inh post-infusion of aPCC (70-80 iu/kg), only the CTs were shortened in the presence of rFVIIIex vivo compared to its absence (P < 0·05), indicating that the enhanced activity centred on the initiation phase of coagulation. Furthermore, experiments in the co-presence of rFVIIa and rFVIII demonstrated that FVIII accelerated only the CTs. We concluded that FVIII/FVIIa-related coagulation mechanism enhanced global haemostatic function by the co-presence of bypassing agents and FVIII in PWHA-inh.