Koji Yada

Correlations between global clotting function tests, duration of operation, and postoperative chest tube drainage in pediatric cardiac surgery.

Background:  Systemic coagulation disorders after cardiac surgery represent serious postoperative complications. There have been few reports, however, identifying preoperative coagulation tests that predict postoperative bleeding. The aim of the present study was to investigate the relationship between postoperative hemorrhage and coagulation parameters determined by global coagulation assays, to define potential predictive markers.

Systematic monitoring of hemostatic management in hemophilia A patients with inhibitor in the perioperative period using rotational thromboelastometry.

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.

The mild phenotype in severe hemophilia A with Arg1781His mutation is associated with enhanced binding affinity of factor VIII for factor X

The clinical severity in some patients with haemophilia A appears to be unrelated to the levels of factor (F)VIII activity (FVIII:C), but mechanisms are poorly understood. We have investigated a patient with a FVIII gene mutation at Arg1781 to His (R1781H) presenting with a mild phenotype despite FVIII:C of 0.9 IU/dl. Rotational thromboelastometry using the patients whole blood demonstrated that the clot time and clot firmness were comparable to those usually observed at FVIII:C 5-10 IU/dl. Thrombin and FXa assays using plasma samples also showed that the peak levels of thrombin formation and the initial rate of FXa generation were comparable to those observed at FVIII:C 5-10 IU/dl. The results suggested a significantly greater haemostatic potential in this individual than in those with severe phenotype. The addition of incremental amounts of FX to control plasma with FVIII:C 0.9 IU/dl in clot waveform analyses suggested that the enhanced functional tenase assembly might have been related to changes in association between FVIII and FX. To further investigate this mechanism, we prepared a stably expressed, recombinant, B-domainless FVIII R1781H mutant. Thrombin generation assays using mixtures of control plasma and FVIII revealed that the coagulation function observed with the R1781H mutant (0.9 IU/dl) was comparable to that seen with wild-type FVIII:C at ~5 IU/dl. In addition, the R1781H mutant demonstrated an ~1.9-fold decrease in Km for FX compared to wild type. These results indicated that relatively enhanced binding affinity of FVIII R1781H for FX appeared to moderate the severity of the haemophilia A phenotype.

Activated prothrombin complex concentrate (APCC)‐mediated activation of factor (F)VIII in mixtures of FVIII and APCC enhances hemostatic effectiveness

Activated prothrombin complex concentrates (APCCs), utilized in bypassing therapy for hemophiliacs with inhibitor, contain factors (Fs) VII, FII, FIX and FX, and their active forms. A recent report has demonstrated that mixtures of APCC and FVIII potentiated thrombin generation, in vitro, in plasma from patients with severe hemophilia A, but the mechanism(s) involved remains unknown.

A Neonate with Umbilical Arteriovenous Malformation Showing Hemorrhagic Shock from Massive Umbilical Hemorrhage

We describe herein the case of a 3-day-old male neonate with umbilical arteriovenous malformation showing umbilical hemorrhage. The patient was born after 38 weeks and 3 days of gestation with a birth weight of 2784 g. Sudden massive umbilical hemorrhage occurred on day 3. Cardiopulmonary arrest developed, but the patient was successfully rescued by immediate cardiopulmonary resuscitation. An umbilical venous catheter was inserted for blood access. However, umbilical hemorrhage continued and hemostasis was difficult. Congenital bleeding disorders were excluded based on laboratory findings. Ultrasonography on day 15 revealed a mass with rich blood supply directly under the umbilicus. Umbilical arteriovenous malformation was suspected from abdominal contrast-enhanced computed tomography on day 17. Excision of the arteriovenous malformation was performed on day 29. The mass was connected to three arteries including the umbilical arteries, with the umbilical vein flowing out from the mass. Umbilical arteriovenous malformation was diagnosed from evidence during the operation and pathological findings. Umbilical arteriovenous malformations are rare and often discovered by heart failure symptoms, but rare cases present with umbilical bleeding, as in this report. Umbilical arteriovenous malformation must be taken into consideration as along with congenital bleeding disorders when massive umbilical hemorrhage is identified.

Effects of anti-factor VIII inhibitor antibodies on factor VIIa/tissue factor-catalysed activation and inactivation of factor VIII

Factor (F)VIIa/tissue factor (TF) rapidly activates FVIII activity by proteolysis at Arg372 and Arg740, and subsequently inactivates FVIIIa activity by proteolysis at Arg336, although this activation is weaker than that by thrombin. The effects of anti-FVIII inhibitor antibodies on these reactions remain unknown, however. In this study, 13 of anti-FVIII inhibitor antibodies recognising the A2 or C2 domain were prepared. None of them, irrespective of epitope specificity, significantly affected FVIIa/TF-catalysed FVIII activation in one-stage clotting assays. Anti-A2 and anti-C2 type 2 antibodies had little effect on the inactivation phase. Anti-C2 type 1 antibodies, however, modulated inactivation by 40-60% of that seen with control IgG, suggesting that the activity of FVIIIa generated by FVIIa/TF persisted in the presence of this specific type of inhibitor. SDS-PAGE analysis demonstrated that all antibodies had little effect on FVIIa/TF-catalyzed proteolysis at Arg372 and Arg740. Anti-C2 type 1, however, significantly delayed cleavage at Arg336 in dose- dependent manners. Neither anti-A2 nor anti-C2 type 2 affected this reaction, and the findings were consistent with the results of the functional assays. In addition, anti-C2 monoclonal antibodies with type 1 and 2 demonstrated similar patterns of reaction as the anti-C2 polyclonal antibodies in FVIIa/TF-mediated FVIII mechanisms. We demonstrated that FVIIa/TF activated FVIII even in the presence of anti-FVIII antibodies, but inactivation patterns appeared to depend on inhibitor type. It could be important to determine the characteristic of these inhibitor antibodies for prediction of their effects on FVIIa-related FVIII reactions, and the results could have significant therapeutic implications.

Possible assessment of coagulation function and haemostasis therapy using comprehensive coagulation assays in a patient with acquired haemophilia A

MVR designed the study, obtained funding, enrolled subjects, interpreted the data and wrote the manuscript. NCJ enrolled study subjects, contributed to study design and reviewed the manuscript. PFF, CMK, ATN and LR contributed to study design and reviewed the manuscript. JGY performed data analysis, interpreted the data and reviewed the manuscript. CGM contributed to the design, interpreted the data and reviewed the manuscript. KBZ performed the assays, interpreted the data and edited the manuscript. Disclosures

Clot waveform analysis using CS-2000i™ distinguishes between very low and absent levels of factor VIII activity in patients with severe haemophilia A

A recently developed method to assess comprehensive coagulation function, clot waveform analysis (CWA), accurately detect low levels (<1 IU/dL) of factor VIII activity (FVIII:C) in haemophilia A patients (HA‐pts). Improvements are needed, however, to differentiate patients with very low from absent levels of FVIII:C.

Different factor VIII neutralizing effects on anti‐factor VIII inhibitor antibodies associated with epitope specificity and von Willebrand factor

Inhibitor neutralization therapy based on factor (F)VIII replacement is used for haemostatic treatment in haemophilia A patients with inhibitors on low responder, but effects appear to depend on various properties of inhibitors. We investigated this nature by evaluating the global coagulation function in timed‐reactions after mixing FVIII (1 U/ml) with anti‐FVIII alloantibodies containing distinct epitopes (2·5 Bethesda units/ml). Thrombin generation assays showed that peak thrombin and mean velocity to peak thrombin were depressed by anti‐C2 type 1 inhibitors to significantly greater extents than by anti‐A2 type 1 and anti‐C2 type 2 (2‐ to 6‐fold and 10‐ to 20‐fold, respectively). In the presence of FVIII‐von Willebrand Factor (VWF) complex, the anti‐C2 type 1‐mediated decreased thrombin generation was reduced by 20–40%, reflecting the protective function of VWF. However, the activities of anti‐A2 type 1 were little affected, and that of anti‐C2 type 2 was rather enhanced by c. 2·5‐fold, relative to FVIII. Clot waveform analysis also showed similar patterns. Anti‐FVIII monoclonal antibodies with well‐defined characteristics demonstrated similar reactions to those with polyclonal inhibitors. In conclusion, the neutralizing effects of FVIII(‐VWF) depending on epitopes could have significant therapeutic implications, and it could be important to determine inhibitor properties in order to predict the effects of infused FVIII in neutralization therapy.

Emicizumab-mediated haemostatic function in patients with haemophilia A is down-regulated by activated protein C through inactivation of activated factor V

Activated protein C (APC) inactivates activated factor V (FVa) and moderates FVIIIa by restricting FV cofactor function. Emicizumab is a humanized anti‐FIXa/FX bispecific monoclonal antibody that mimicks FVIIIa cofactor function. In recent clinical trials in haemophilia A patients, once‐weekly subcutaneous administration of emicizumab was remarkably effective in preventing bleeding events, but the mechanisms controlling the regulation of emicizumab‐mediated haemostasis remain to be explored. We investigated the role of APC‐mediated reactions in these circumstances. APC dose‐dependently depressed thrombin generation (TG) initiated by emicizumab in FVIII‐deficient plasmas, and in normal plasmas preincubated with an anti‐FVIII antibody (FVIII‐depleted). FVIIIa‐independent FXa generation with emicizumab was not affected by the presence of APC, protein S and FV. The results suggested that APC‐induced down‐regulation of emicizumab‐dependent TG was accomplished by direct inactivation of FVa. The addition of APC to emicizumab mixed with FVIII‐depleted FV‐deficient plasma in the presence of various concentrations of exogenous FV demonstrated similar attenuation of TG, irrespective of specific FV concentrations. Emicizumab‐related TG in FVIII‐depleted FVLeiden plasma was decreased by APC more than that observed with native FVLeiden plasma. The findings indicated that emicizumab‐driven haemostasis was down regulated by APC‐mediated FVa inactivation in plasma from haemophilia A patients without or with FV defects.