Ichiro Tanaka

Role of factor VIII C2 domain in factor VIII binding to factor Xa

Factor VIII (FVIII) is activated by proteolytic cleavages with thrombin and factor Xa (FXa) in the intrinsic blood coagulation pathway. The anti-C2 monoclonal antibody ESH8, which recognizes residues 2248–2285 and does not inhibit FVIII binding to von Willebrand factor or phospholipid, inhibited FVIII activation by FXa in a clotting assay. Furthermore, analysis by SDS-polyacrylamide gel electrophoresis showed that ESH8 inhibited FXa cleavage in the presence or absence of phospholipid. The light chain (LCh) fragments (both 80 and 72 kDa) and the recombinant C2 domain dose-dependently bound to immobilized anhydro-FXa, a catalytically inactive derivative of FXa in which dehydroalanine replaces the active-site serine. The affinity (K d ) values for the 80- and 72-kDa LCh fragments and the C2 domain were 55, 51, and 560 nm, respectively. The heavy chain of FVIII did not bind to anhydro-FXa. Similarly, competitive assays using overlapping synthetic peptides corresponding to ESH8 epitopes (residues 2248–2285) demonstrated that a peptide designated EP-2 (residues 2253–2270; TSMYVKEFLISSSQDGHQ) inhibited the binding of the C2 domain or the 72-kDa LCh to anhydro-FXa by more than 95 and 84%, respectively. Our results provide the first evidence for a direct role of the C2 domain in the association between FVIII and FXa.

Factor VIII C2 domain contains the thrombin-binding site responsible for thrombin-catalyzed cleavage at Arg1689

Thrombin-catalyzed factor VIII activation is an essential positive feedback mechanism regulating intrinsic blood coagulation. A factor VIII human antibody, A-FF, with C2 epitope, exclusively inhibited factor VIII activation and cleavage at Arg1689 by thrombin. The results suggested that A-FF prevented the interaction of thrombin with factor VIII and that the C2 domain was involved in the interaction with thrombin. We performed direct binding assays using anhydro-thrombin, a catalytically inactive derivative of thrombin in which the active-site serine is converted to dehydroalanine. Intact factor VIII, 80-kDa light chain, 72-kDa light chain, and heavy chain fragments bound dose-dependently to anhydro-thrombin, and the K d values were 48, 150, 106, and 180 nm, respectively. The C2 and A2 domains also dose-dependently bound to anhydro-thrombin, and theK d values were 440 and 488 nm, respectively. The A1 domain did not bind to anhydro-thrombin. A-FF completely inhibited C2 domain binding to anhydro-thrombin (IC50, 18 nm), whereas it did not inhibit A2 domain binding. Furthermore, C2-specific affinity purified F(ab)′2 of A-FF, and the recombinant C2 domain inhibited thrombin cleavage at Arg1689. Our results indicate that the C2 domain contains the thrombin-binding site responsible for the cleavage at Arg1689.

Common inhibitory effects of human anti‐C2 domain inhibitor alloantibodies on factor VIII binding to von Willebrand factor

Summary. Factor VIII (FVIII) Inhibitor alloantibodies obtained from seven severe haemophilia A patients were examined for their binding regions and their effects on FVHI binding to von Willebrand factor (vWF). Immunoblotting analysis with a panel of recombinant fragments demonstrated that the binding regions of antibodies in cases 1‐5 were contained in the C2 domain of the light chain. Antibodies from cases 1 and 2, which recognized an epitope within residues 2248‐2312, completely inhibited FVIII/ vWF binding in an FXISA (IC50: 5‐0 and 9‐0μg/ml, respectively). Antibodies from case 3 recognizing 2170‐2312 and case 5 recognizing 2170‐2327 also inhibited FVIII/vWF binding (IC50:110 and 400μg/mI, respectively). Case 4 antibodies recognizing 2218‐2307 showed barely detectable inhibition and cases 6 and 7 antibodies recognizing the 44 kD heavy chain, did not inhibit. Our results demonstrate that all anti‐C2 alloantibodies with epitopes that extend to the residue 2312 inhibit vWF binding and that an overlap of the inhibitor epitope with residues 2308‐2312 is critical for maximal inhibition of vWF binding. Prevention of FVIII/vWF binding appears to be a common property of anti‐C2 domain inhibitor alloantibodies.

The measurement of low levels of factor VIII or factor IX in hemophilia A and hemophilia B plasma by clot waveform analysis and thrombin generation assay

Summary.u2002 Background:u2002Precise assessment of clotting function is essential for monitoring of hemostatic treatment for hemophilias A and B.Materials and methods:u2002Clot waveform analysis and thrombin generation assays were performed on factor (F) VIII‐ and FIX‐deficient plasmas, which had been reconstituted with known amounts of recombinant FVIII (rFVIII) and affinity‐purified FIX respectively. Clot waveforms were assessed qualitatively and quantitatively by measuring the parameters clotting time, maximum coagulation velocity (Min1), and maximum coagulation acceleration (Min2). The thrombin generation assay was also assessed qualitatively and measurements made of time to peak and peak height.Results:u2002Overall results obtained with both assays showed good correlation for both clotting factors confirming that the changes in clotting waveform reflected changes in thrombin generation. Both assays demonstrated a predictable dose response to the addition of FVIII or IX. However, clot waveform analysis was more sensitive than the thrombin generation assay, particularly in detecting very low levels (0–0.1u2003IUu2003dL−1) of both factors.Conclusions:u2002These data suggest that the application of clot waveform analysis to the routine management of the hemophiliacs could increase our understanding of the clinical significance of low levels of FVIII and FIX that cannot be measured by assays in current use. This may be particularly useful in the management of hemophiliacs with inhibitors or undergoing gene therapy.

Unresponsiveness to factor VIII inhibitor bypassing agents during haemostatic treatment for life-threatening massive bleeding in a patient with haemophilia A and a high responding inhibitor.

Summary.u2002 u2002We report a case of haemophilia A with a high responding inhibitor of factor VIII (FVIII) who had a serious retroperitoneal haematoma caused by penetration of a duodenal ulcer. Inhibitor‐bypassing therapy was commenced immediately on admission. On the 17th day of treatment with activated prothrombin complex concentrate (APCC; FEIBA®), re‐bleeding occurred and thrombelastography (TEG) demonstrated resistance to therapy. Treatment was changed to recombinant activated factor VII (rFVIIa; NovoSeven®) and resulted in clinical improvement together with an improvement in TEG parameters. On the 10th day of continuous infusion with NovoSeven®, however, TEG again showed resistance to therapy. FEIBA® infusions were re‐introduced and TEG results remained satisfactory for 7u2003days. On day 34, however, further retroperitoneal bleeding was evident and a decline in the haemostatic efficiency of FEIBA® was recorded by TEG. NovoSeven® was again successfully administered for 7u2003days. There were no laboratory findings to indicate disseminated intravascular coagulation (DIC), hypercoagulability or abnormal fibrinolysis. The plasma‐based clotting tests did not show any additional prolongation on the occasions when the TEG demonstrated unresponsiveness to FEIBA® or NovoSeven®. These findings suggested that some component of whole blood, other than plasma might have governed the TEG data. The long‐term use of APCC such as FEIBA® or rFVIIa, requires careful monitoring in terms of FVIII inhibitor bypassing activity as well as the tendency to DIC.

Mechanisms of plasmin-catalyzed inactivation of factor VIII: a crucial role for proteolytic cleavage at Arg336 responsible for plasmin-catalyzed factor VIII inactivation.

Plasmin not only functions as a key enzyme in the fibrinolytic system but also directly inactivates factor VIII and other clotting factors such as factor V. However, the mechanisms of plasmin-catalyzed factor VIII inactivation are poorly understood. In this study, levels of factor VIII activity increased ∼2-fold within 3 min in the presence of plasmin, and subsequently decreased to undetectable levels within 45 min. This time-dependent reaction was not affected by von Willebrand factor and phospholipid. The rate constant of plasmin-catalyzed factor VIIIa inactivation was ∼12- and ∼3.7-fold greater than those mediated by factor Xa and activated protein C, respectively. SDS-PAGE analysis showed that plasmin cleaved the heavy chain of factor VIII into two terminal products, A137–336 and A2 subunits, by limited proteolysis at Lys36, Arg336, Arg372, and Arg740. The 80-kDa light chain was converted into a 67-kDa subunit by cleavage at Arg1689 and Arg1721, identical to the pattern induced by factor Xa. Plasmin-catalyzed cleavage at Arg336 proceeded faster than that at Arg372, in contrast to proteolysis by factor Xa. Furthermore, breakdown was faster than that in the presence of activated protein C, consistent with rapid inactivation of factor VIII. The cleavages at Arg336 and Lys36 occurred rapidly in the presence of A2 and A3-C1-C2 subunits, respectively. These results strongly indicated that cleavage at Arg336 was a central mechanism of plasmin-catalyzed factor VIII inactivation. Furthermore, the cleavages at Arg336 and Lys36 appeared to be selectively regulated by the A2 and A3-C1-C2 domains, respectively, interacting with plasmin.

Immunological characterization of factor VIII autoantibodies in patients with acquired hemophilia A in the presence or absence of underlying disease

The development of a factor VIII autoantibody results in a severe hemorrhagic diathesis known as acquired hemophilia A. Underlying pathologies, such as autoimmune disease or chronic inflammatory disease, are observed in about half of the patients. We have investigated a total of 16 cases with acquired hemophilia A and divided the patients into two groups according to the presence or absence of other clinical conditions. Group A comprised nine cases with no detectable associated pathology. Group B consisted of seven cases with other clinical diagnoses. Significant levels of factor VIII activity (FVIII:C) and factor VIII antigen (FVIII:Ag) were detected in Group A and the pattern of FVIII:C inactivation was characteristic of Type 2 inhibitors. In contrast, no FVIII:C was detected in Group B and, in five of seven cases, the inhibitory pattern was Type 1. IgG(4) antibody subclass specificity was dominant in both groups. IgG1 antibody reactivity was higher in Group B than in Group A. Our results suggested a close relationship between the presence of underlying disease and immunological and coagulation characteristics in acquired hemophilia A.

MEASUREMENT OF ANTI-FACTOR IX IGG SUBCLASSES IN HAEMOPHILIA B PATIENTS WHO DEVELOPED INHIBITORS WITH EPISODES OF ALLERGIC REACTIONS TO FACTOR IX CONCENTRATES

We have established a simple enzyme-linked immunosorbent assay (ELISA) for the detection of anti-factor IX IgG subclasses in haemophilia B patients with inhibitors. The assay was performed using immobilized purified factor IX. Specific IgG subclasses were detected by peroxidase-conjugated anti-human IgG1,2,3 and 4. Ten plasma samples from 6 haemophilia B patients with inhibitors ranging from 1.0 to 253 Bethesda Units/ml were analyzed. All samples were positive for IgG4. Six out of 10 samples were positive only for IgG4. Three samples were positive for IgG2. Five of the 6 patients had previously had allergic reactions to factor IX concentrates. Three patients had allergic episodes within the past month. Three samples from these latter patients taken on the day when the allergy had occurred showed positive also for IgG1. In later samples, however, taken at 4 days and 4 weeks respectively from two of these same patients. IgG1 was not detected. In two of the five patients in whom allergic reactions had occurred more than one month previously IgG1 was not detected. The results suggested that allergic reactions in patients with haemophilia B treated with factor IX concentrates were associated with the development of the specific IgG1 subclass of antibody to factor IX.

22q13 Microduplication in two patients with common clinical manifestations: a recognizable syndrome?

We report here on two unrelated patients (Patients 1 and 2) with a cryptic microduplication involving a 22q13 segment. Both patients manifested infantile hypotonia, developmental delay, and growth deficiency. In addition, an abnormal signal intensity area was detected in the frontal white matter of Patient 2 by brain MRI. Whole‐genome microarray comparative genomic hybridization for Patient 1 and fluorescence in situ hybridization analysis with 22q‐subtelomeric probes performed in both patients showed a submicroscopic 22q13 duplication that involved the SHANK3 gene. The duplication in Patient 1 was de novo type, while that in Patient 2 was derived from a familial 17;22 translocation. The presence of common clinical manifestations in the two patients with the common duplicated region led to a conclusion that 22q terminal duplication is a recognizable clinical entity, that is, the 22q13 microduplication syndrome.

Higher recovery of factor VIII (FVIII) with intermediate FVIII/von Willebrand factor concentrate than with recombinant FVIII in a haemophilia A patient with an inhibitor.

The first line of therapy for acute bleeding in patients with low-responding factor VIII (FVIII) inhibitors is FVIII concentrates (1). Most inhibitors, recognizing the FVIII light chain, inhibit von Willebrand factor (VWF) and phospholipid binding to FVIII (2,3), and appears to be less active in vitro against plasmaderived FVIII concentrates containing VWF (pdFVIII/VWF) than VWF-free FVIII concentrates (4–7). These findings suggest that pdFVIII/VWF might be therapeutically more effective than recombinant FVIII (rFVIII) in patients with FVIII inhibitors. However, no clinical studies supporting this concept have been reported. In the present study, we have compared the recovery of FVIII activity (FVIII:C) after treatment with pdFVIII/VWF and rFVIII for massive intramuscular bleeding that occurred during regular infusion of FVIII for immune tolerance induction (ITI) therapy in a young male haemophilia A patient with an inhibitor. Immune tolerance induction therapy was commenced in our patient at the age of 9 years (18 October 1999) with the administration of 100 U kg of rFVIII (Recombinate; Baxter Healthcare Corp., Westlake Village, CA, USA) daily for 3 weeks, followed by infusions three to four times a week. Inhibitor levels fluctuated for 3 years after ITI therapy were initiated (maximum inhibitor level, 152.0 BU mL) and regular infusions of FVIII were continued. The number of bleeding episodes appeared to decline, and since January 2003, the inhibitor level has been kept constant within a low range from 0.9 to 2.1 BU mL. He was admitted into our hospital with severe pain in his right buttock and walking difficulties on 6 January 2004. He had suffered from painful swelling in his right buttock 2 weeks before admission without improvement in spite of daily infusions of FVIII (100 U kg). A subcutaneous haematoma (7 · 8 cm) was evident on the right buttock, with heat sensation, and impaired flexion and extension of the right hip joint. Computer tomography scanning demonstrated a massive intramuscular haematoma, measuring 10 cm in diameter, in the right gluteus maximus and gluteus medius muscles. On admission, 12 h after infusion of 4000 U (87 IU kg) of rFVIII, the FVIII inhibitor titre was 1.7 BU mL. As the bleeding manifestations had not responded to the infusion of rFVIII, 4000 U of activated prothrombin complex concentrate (Feiba Immuno; Baxter Healthcare Corp.) were administered. Nevertheless, the swelling and pain in the right buttock increased. Subsequently, replacement therapy with the same dose of rFVIII (4000 IU) was administered and continued (Fig. 1). Clinical symptoms gradually improved and the patient was discharged on 30 January. Regular prophylaxis in this patient is now maintained using FVIII/VWF concentrate. The inhibitor titre in this patient remained constant, within the range of 1.5–2.0 BU mL, throughout the present series of investigations. Therefore, it was possible to compare the recovery Correspondence: Midori Shima MD, Department of Pediatrics, Nara Medical University, 840 Shijo-cho, Kashihara city, Nara 634-8522, Japan. Tel.: +81 744 29 8881; fax: +81 744 24 9222; e-mail: mshima@naramed-u.ac.jp