Yasuaki Shida

Role of red blood cells in the anemia-associated bleeding under high shear conditions

Red blood cells (RBCs) contribute to hemostasis under blood‐flow, and anemia might contribute to a hemorrhagic diathesis. The majority of current laboratory techniques to assess hemostasis do not consider the effects of RBCs. An assay to determine the role of RBCs in hemostasis could be beneficial for clinical management.

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.

Highly elevated plasma level of von Willebrand factor accelerates the formation of platelet thrombus under high shear stress in plasma with deficient ADAMTS13 activity

Upshaw-Schulman syndrome (USS) is a thrombo-hemorrhagic disease caused by congenital deficiency of ADAMTS13 due to ADAMTS13 gene mutations. USS is characterized by repeated episodes of thrombocytopenia and microangiopathic hemolytic anemia that respond dramatically to infusions of fresh frozen plasma. There are two phenotypic expressions of USS: one is the early-onset type and the other, the late-onset type, is asymptomatic during childhood with the first bout of thrombotic thrombocytopenic purpura (TTP) developing after adolescence or during adulthood. We found that gravida with the latter phenotype developed thrombocytopenia and hemolytic anemia during the second or third trimesters, often followed by thrombotic microangiopathies (TMAs). These phenomena suggest that elevated plasma von Willebrand Factor (VWF) might be crucial because plasma levels of VWF antigen usually increase by 200-500% during this period of gestation. Here, we performed platelet function assays using a mixture of anti-coagulated blood from normal volunteers, human VWF, anti-ADAMTS13 monoclonal antibody A10, and purified plasma-derived ADAMTS13 to investigate the effect of plasma VWF levels on platelet thrombus formation in the context of deficient ADAMTS13. In vitro studies showed that mural thrombus formation and platelet aggregation under high shear stress were markedly augmented by increasing the amounts of exogenously added VWF when ADAMTS13 activity was deficient, as may be the case in the in vivo circulation of gravida with USS. These results suggest that highly elevated plasma VWF might accelerate platelet thrombus formation not only in the circulation but also on the surface of vascular endothelial cells in the setting of ADAMTS13 deficiency in USS.

The utility of VWF multimer analysis in response to the desmopressin administration for the diagnosis of severe type 1 von Willebrand disease

Within 8 months of commencing Wilate ITI, the recovery levels at two consecutive follow-up appointments were found to be greater than 66% of predicted with an estimated terminal half-life of 6 hours. After 17 months of daily Wilate at 100 U kg , and 10 months at daily doses of 50 U kg , inhibitor titers have remained below detectable limits, and the patient has not experienced any treatment-related adverse effects or any major or minor bleeding episodes. Wilate ITI has therefore been declared a success in this patient. He remains on daily Wilate prophylaxis to this date. In conclusion, Wilate ITI was found to be a safe and effective treatment approach for this high-risk, adult patient with severe HA complicated by longstanding FVIII inhibiting alloantibody. Eradication of the inhibitor was achieved with Wilate ITI despite the presence of several risk factors associated with poor ITI outcome, i.e. previous ITI failures, onset of ITI after 6 years of age, and ITI initiation more than 12 months from inhibitor detection. The patient has not experienced further breakthrough bleeding or reappearance of the inhibitor despite discontinuation of prophylactic bypassing therapy and reduction in the Wilate dose. We conclude that VWF containing products, such as Wilate may be considered as an ITI alternative for adults with HA complicated by inhibitor.

Successful haemostatic management of replacement of the ascending aorta for type A acute aortic dissection in a patient with mild haemophilia B.

The mortality from cardiovascular disease, particularly coronary artery disease, is reported to be lower in haemophilia patients than in age-matched male population [1]. While, comparison between haemophilia patients and healthy controls suggested only minimally reduced intima-media onto the femoral artery in individuals with bleeding tendency and hypocoagulability caused by the disorder has a limited effect on atherogenesis [2]. In recent decades, the treatment for haemophilia patients has been developing, consequently their life expectancy is close to that of normal male population. This change would attribute to an increased risk of developing age-related disorders such as acquired heart diseases. Only few hospitals can be performed the cardiac surgery for haemophilia patients, however [3]. We had a case with replacement of the ascending aorta for acute aortic dissection classified as Stanford type A for a 65 year-old haemophilia B patient admitted in our hospital, and now present the successful haemostatic management for the monitoring of factor IX activity (FIX:C) during the support of extracorporeal circulation with heparinization. The patient was previously diagnosed as mild haemophilia B, because FIX:C and FIX antigen were 13 and 37 IU dL , respectively. On August in 2014, he had a temporary faintness and was admitted to our hospital. Ultrasound and chest CT findings revealed the acute aortic dissection with Stanford type A (Fig.1), and he had to undergo an urgent surgery with arch replacement. The FIX:C level measured at our laboratory was 14 IU dL 1 prior to surgery. The prothrombin time was 11.3 s (reference 10.0–15.0 s), activated partial thromboplastin time was 33.9 s (reference 22.0–32.0 s), haemoglobin was 14.5 g dL . Since the FIX:C level was required to maintain ~100 IU dL 1 on the open cardiac surgery, he received 6000 IU of recombinant factor IX (rFIX) (BeneFIX ; Pfizer, peak FIX:C 83 IU dL ) at initial bolus infusion, showing the 70% recovery rate of expected value, although his FIX inhibitor was not detected (<0.3 BU mL ). He received additional 3000 IU of rFIX, followed by continuous infusion of rFIX at a rate of 6.7 IU kg 1 h 1 during the operation. A left radial artery catheter and a pulmonary artery catheter via right internal jugular vein were placed for continuous haemodynamic monitoring. He was anticoagulated with 10 000 U of heparin (~150 IU kg ) for extracorporeal circulation, resulting in significant prolongation (>400 s) of activated clotting time. Meanwhile, the presence of heparin at high level prevented to measure the FIX:C in onestage clotting assay. To solve this, we attempted to evaluate the FIX:C in his plasma by counteracting the heparin due to addition of heparinase (Hep-tem ; Pentapharm GmbH, Munich, Germany) into blood samples (v/v = 1:20) during the bypass. Just after the beginning of cardiopulmonary bypass, his FIX:C was 4.0 IU dL 1 in the absence of heparinase, while was 41 IU dL 1 in its presence, suggestive of the usefulness of heparinase for measurement of coagulation factor activity when heparinazation. Also, this result indicated the more requisition of rFIX than was anticipated, may be due to the hemodilution, and the successful assessment of FIX:C in this methodology. He had an additional bolus infusion of 5000 U rFIX, followed by continuous infusion with up to 13 IU kg 1 h . The replacement of the ascending aorta graft and a pacing wire were placed, and the atrial pacing was required to separation from cardiopulmonary bypass. When reversal of anticoagulation with protamine was achieved, FIX:C was 126 and 123 IU dL 1 in the absence or presence of heparinase, respectively. From this data, we decreased the rate of continuous infusion of rFIX to 6.7 IU kg 1 h . The operation was uneventfully successful. Total time using cardiopulmonary bypass for the operation was 2 h 58 min and the bleeding volume was 1320 mL. Correspondence: Keiji Nogami, MD, PhD, Department of Pediatrics, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan. Tel.: +81 744 29 8881; fax: +81 744 24 9222; e-mail: roc-noga@naramed-u.ac.jp