Masahiro Takeyama

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.  Background: Precise assessment of clotting function is essential for monitoring of hemostatic treatment for hemophilias A and B.Materials and methods: Clot 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: Overall 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 (0–0.1 IU dL−1) of both factors.Conclusions: These 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.

The Factor VIIIa C2 Domain (Residues 2228–2240) Interacts with the Factor IXa Gla Domain in the Factor Xase Complex

Factor VIIIa functions as a cofactor for factor IXa in the phospholipid surface-dependent activation of factor X. Both the C2 domain of factor VIIIa and the Gla domain of factor IXa are involved in phospholipid binding and are required for the activation of factor X. In this study, we have examined the close relationship between these domains in the factor Xase complex. Enzyme-linked immunosorbent assay-based and surface plasmon resonance-based assays in the absence of phospholipid showed that Glu-Gly-Arg active site-modified factor IXa bound to immobilized recombinant C2 domain (rC2) dose-dependently (Kd = 108 nm). This binding ability was optimal under physiological conditions. A monoclonal antibody against the Gla domain of factor IXa inhibited binding by ∼95%, and Gla domainless factor IXa failed to bind to rC2. The addition of monoclonal antibody or rC2 with factor VIIIa inhibited factor IXa-catalyzed factor X activation in the absence of phospholipid. Inhibition was not evident, however, in similar experiments in the absence of factor VIIIa, indicating that the C2 domain interacted with the Gla domain of factor IXa. A fragment designated C2-(2182–2259), derived from V8 protease-cleaved rC2, bound to Glu-Gly-Arg active site-modified factor IXa. Competitive assays, using overlapping synthetic peptides encompassing residues 2182–2259, demonstrated that peptide 2228–2240 significantly inhibited both this binding and factor Xa generation, independently of phospholipid. Our results indicated that residues 2228–2240 in the factor VIIIa C2 domain constitutes an interactive site for the Gla domain of factor IXa. The findings provide the first evidence for an essential role for this interaction in factor Xase assembly.

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

Relationship between the binding sites for von Willebrand factor, phospholipid, and human factor VIII C2 inhibitor alloantibodies within the factor VIII C2 domain.

Some factor VIII (FVIII) inhibitor alloantibodies block FVIII binding to von Willebrand factor (VWF) and phospholipid (PL) and recognize a C2 domain epitope that overlaps both binding sites. We previously showed that FVIII peptide 2315-2330 neutralized FVIII inhibitors and that Cys2326 and Glu2327 contributed to the maximum neutralizing effect. In the present study, we investigated the relationship between the essential binding sites for VWF, PL, and anti-C2 inhibitors by means of competitive-inhibition assays with overlapping synthetic peptides that span the C terminus of the C2 domain (residues 2288-2332). We identified 2 peptides (residues 2303–2317 and 2315–2330) that specifically blocked FVIII binding to VWF or PL by approximately 80% (50%-inhibitory concentration [IC50], 9.0 μM) and 95% (IC50, 0.12 ?M), respectively. To examine in detail the residues responsible for PL binding, we prepared mutants of peptide 2315-2330 in which we sequentially substituted each residue with Gly. Two residues, Ile2317 and Met2321, were shown to be essential for PL binding. Their substitution with Gly reduced the inhibitory effect by >90%.The data suggest that the binding sites for VWF, PL, and anti-C2 inhibitors in the C2 domain are in very close proximity but are not identical.

Heparin-induced inhibitory effects of a prothrombin complex concentrate on global tests of haemostasis

Prothrombin complex concentrates (PCC) have been used as bypassing agents for the treatment of haemophilia A patients with inhibitor as well as for replacement therapy in congenital and acquired deficiencies of vitamin-K-dependent clotting factors. The efficacy of PCC is variable, however, especially during long-term and high-dose use, and all currently available products of this nature contain heparin. We have examined the haemostatic properties of PCC using reconstituted whole blood made by mixing coagulation-factor-deficient plasma and washed blood cells. In rotation thromboelastometry (ROTEM), the recommended therapeutic dose of Proplex ST corrected the abnormal patterns. At higher concentrations, however, the ROTEM patterns regressed. In addition, specific assays of coagulation factors appeared unreliable in the presence of 2.5 U/ml Proplex ST; the abnormalities were corrected when protamine sulfate was added. The findings suggest that the presence of heparin in PCC might have a greater effect on global haemostasis. Careful attention to the anticoagulant effect as well as thrombogenicity of PCC is required. Monitoring therapy using such as ROTEM analysis could be highly informative.

Protein S down-regulates factor Xase activity independent of activated protein C: specific binding of factor VIII(a) to protein S inhibits interactions with factor IXa.

Protein S functions as an activated protein C (APC)‐independent anticoagulant in the inhibition of intrinsic factor X activation, although the precise mechanisms remain to be fully investigated. In the present study, protein S diminished factor VIIIa/factor IXa‐dependent factor X activation, independent of APC, in a functional Xa generation assay. The presence of protein S resulted in an c. 17‐fold increase in Km for factor IXa with factor VIIIa in the factor Xase complex, but an c. twofold decrease in Km for factor X. Surface plasmon resonance‐based assays showed that factor VIII, particularly the A2 and A3 domains, bound to immobilized protein S (Kd; c. 10 nmol/l). Competition binding assays using Glu‐Gly‐Arg‐active‐site modified factor IXa showed that factor IXa inhibited the reaction between protein S and both the A2 and A3 domains. Furthermore, Sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed that the cleavage rate of factor VIIIa at Arg336 by factor IXa was c. 1·8‐fold lower in the presence of protein S than in its absence. These data indicate that protein S not only down‐regulates factor VIIIa activity as a cofactor of APC, but also directly impairs the assembly of the factor Xase complex, independent of APC, in a competitive interaction between factor IXa and factor VIIIa.

Identification of a protein S-interactive site within the A2 domain of the factor VIII heavy chain

We have recently demonstrated that protein S impairs the intrinsic tenase complex, independent of activated protein C, in competitive interactions between the A2 and A3 domains of factor VIIIa and factor IXa. In the present study, we have identified a protein S-interactive site in the A2 domain of factor VIIIa. Anti-A2 monoclonal antibody recognising a factor IXa-functional region (residues 484-509) on A2, and synthetic peptide inhibited the A2 binding to protein S by approximately 60% and approximately 70%, respectively, in solid-phase binding assays. The 484-509 peptide directly bound to protein S dose-dependently. Covalent cross-linking was observed between the 484-509 peptide and protein S following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide). The cross-linked adduct was consistent with 1:1 stoichiometry of reactants. Cross-linking formation was blocked by addition of the 484-497 peptide, but not by the 498-509 peptide. Furthermore, N-terminal sequence analysis of the 484-509 peptide-protein S adduct showed that three sequential residues (S488, R489, and R490) in A2 were not identified, suggesting that these residues participate in cross-link formation. Mutant A2 molecules where these residues were converted to alanine were evaluated for the binding of protein S. The S488A, R489A, and R490A mutants demonstrated approximately four-fold lower affinity than wild-type A2. These results indicate that the 484-509 region in the A2 domain of factor VIIIa, in particular sequential residues at positions 488-490, contributes to a unique protein S-interactive site.

Factor VIII Light Chain Contains a Binding Site for Factor X That Contributes to the Catalytic Efficiency of Factor Xase

Factor (F) VIII functions as a cofactor in FXase, markedly accelerating the rate of FIXa-catalyzed activation of FX. Earlier work identified a FX-binding site having μM affinity within the COOH-terminal region of the FVIIIa A1 subunit. In the present study, surface plasmon resonance (SPR), ELISA-based binding assays, and chemical cross-linking were employed to assess an interaction between FX and the FVIII light chain (A3C1C2 domains). SPR and ELISA-based assays showed that FVIII LC bound to immobilized FX (K(d) = 165 and 370 nM, respectively). Furthermore, active site-modified activated protein C (DEGR-APC) effectively competed with FX in binding FVIII LC (apparent K(i) = 82.7 nM). Western blotting revealed that the APC-catalyzed cleavage rate at Arg(336) was inhibited by FX in a concentration-dependent manner. A synthetic peptide comprising FVIII residues 2007-2016 representing a portion of an APC-binding site blocked the interaction of FX and FVIII LC (apparent K(i) = 152 μM) and directly bound to FX (K(d) = 7.7 μM) as judged by SPR and chemical cross-linking. Ala-scanning mutagenesis of this sequence revealed that the A3C1C2 subunit derived from FVIII variants Thr2012Ala and Phe2014Ala showed 1.5- and 1.8-fold increases in K(d) for FX, whereas this value using the A3C1C2 subunit from a Thr2012Ala/Leu2013Ala/Phe2014Ala triple mutant was increased >4-fold. FXase formed using this LC triple mutant demonstrated an ~4-fold increase in the K(m) for FX. These results identify a relatively high affinity and functional FX site within the FVIIIa A3C1C2 subunit and show a contribution of residues Thr2012 and Phe2014 to this interaction.

Identification of a thrombin-interactive site within the FVIII A2 domain that is responsible for the cleavage at Arg372

FVIII is activated by cleavage at Arg372, Arg740, and Arg1689 by thrombin. This study showed that an anti‐A2 monoclonal antibody, with a specific epitope for residues 484–509, and anti‐FVIII inhibitor alloantibodies with similar A2 epitopes, inhibited thrombin‐catalyzed FVIII activation. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis showed that cleavage at Arg372 but not at Arg740 occurred at approximately fourfold decreased rate in the presence of anti‐A2 antibody. Peptide 484–509 also inhibited co‐factor activation, consistent with inhibition of cleavage at Arg372. Direct binding studies using active‐site modified thrombin showed that a 484–509 peptide as well as the anti‐A2 antibodies blocked the A2‐thrombin binding. Furthermore, covalent cross‐linking was observed between the 484–509 peptide and thrombin following reaction with 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide. Mutant A2 molecules in which the clustered basic residues in this sequence were converted to alanine were used to assess the binding reactions in a surface plasmon resonance‐based assay. Mutants R484A, R489A, R490A, H497A and K499A possessed two to fivefold lower affinity than wild‐type A2. These findings demonstrate that clustered basic residues within the 484–509 region of the A2 domain play a part of key role in thrombin‐binding, which is responsible for thrombin‐catalyzed FVIII activation by cleavage at Arg372.

Variable Contributions of Basic Residues Forming an APC Exosite in the Binding and Inactivation of Factor VIIIa

Basic residues contained in the 39-, 60-, and 70-80-loops of activated protein C (APC) comprise an exosite that contributes to the binding and subsequent proteolytic inactivation of factor (F) VIIIa. Surface plasmon resonance (SPR) showed that WT APC bound to FVIII light chain (LC) and the FVIIIa A1/A3C1C2 dimer with equivalent affinity (Kd = 525 and 546 nM, respectively). These affinity values may reflect binding interactions to the acidic residue-rich a1 and a3 segments adjacent to A1 domain in the A1/A3C1C2 and A3 domain in LC, respectively. Results from SPR, using a panel of APC exosite variants where basic residues were mutated, in binding to immobilized FVIIIa A1/A3C1C2 or LC indicated ~4-10-fold increases in the Kd values relative to WT for several of the variants including Lys39Ala, Lys37-Lys38-Lys39/Pro-Gln-Glu, and Arg67Ala. On the other hand, a number of APC variants including Lys38Ala, Lys62Ala, and Lys78Ala showed little if any change in binding affinity to the FVIII substrates. FXa generation assays and Western blotting, used to monitor rates of FVIIIa inactivation and proteolysis at the primary cleavage site in the cofactor (Arg(336)), respectively, showed marked rate reductions relative to WT for the Lys39Ala, Lys37-Lys38-Lys39/Pro-Gln-Glu, Arg67Ala, and Arg74Ala variants. Furthermore, kinetic analysis monitoring FVIIIa inactivation by APC variants at varying FVIIIa substrate concentration showed ~2.6-4.4-fold increases in Km values relative to WT. These results show a variable contribution of basic residues comprising the APC exosite, with significant contributions from Lys39, Arg67, and Arg74 to forming a FVIIIa-interactive site.