Takayoshi Hamamoto

Large-scale preparation of human thrombin: polyethylene glycol potentiates the factor Xa-mediated activation of prothrombin.

We investigated the ability of polyethylene glycol 4000 to accelerate thrombin generation in a mixture of prothrombin and factor X at concentrations of 1-30%. In the presence of 5 mM of CaCl2, polyethylene glycol 4000 promoted prothrombin activation at concentrations above 1%. The peak of activation was seen at levels of 14 and 20% of polyethylene glycol 4000. The effect of the polyethylene glycol was remarkably dependent on its molecular weight; molecular weights greater than 2000 were required for accelerating thrombin generation. Under optimal conditions, polyethylene glycol 4000, in the presence of CaCl2, promoted conversion of all of the prothrombin into thrombin and its derivatives. We conclude that polyethylene glycol 4000, at concentrations ranging from 14 to 20%, effectively accelerates thrombin generation in the presence of 5 mM of CaCl2. This new method for preparing thrombin is based on the use of polyethylene glycol 4000 and CaCl2 and is applicable to the manufacture of thrombin.

Full-length human tissue factor pathway inhibitor inhibits human activated protein C in the presence of heparin

Previous studies have demonstrated that tissue factor pathway inhibitor (TFPI) purified from a hepatoma cell line failed to inhibit human activated protein C (APC) or human thrombin. In the present study, we have examined the ability of full-length TFPI and a truncated form of TFPI lacking the third Kunitz-type domain and C-terminal tail (TFPI1-161) to inhibit the amidolytic activity of human APC in the presence and absence of heparin. TFPI readily inhibited APC amidolytic activity only in the presence of heparin, whereas TFPI1-161 failed to inhibit APC amidolytic activity in the presence or absence of heparin. Optimal inhibition of APC by TFPI was observed at 1 U/ml heparin. The results of competition studies between factor Xa and APC for inhibition by TFPI in the presence of heparin suggested that the second Kunitz-type domain in TFPI was responsible for the inhibition of APC.

Stable complex formation between serine protease inhibitor and zymogen: coagulation factor X cleaves the Arg393-Ser394 bond in a reactive centre loop of antithrombin in the presence of heparin.

Antithrombin (AT) inhibits several blood coagulation proteases, including activated factor X (FXa), by forming stable complexes with these proteases. Herein, we demonstrate that AT forms a stable complex with zymogen factor X (FX). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusion chromatography analyses showed that AT and FX formed an SDS-stable complex, which is distinct in apparent molecular mass from an FXa-AT complex, in the presence of heparin. Amino-terminal sequence analysis of the complex following SDS-PAGE under reducing conditions provided clear evidence that AT forms this complex with the heavy chain of FX, because two sequences, HGSPVDI (residues 1-7 of AT) and SVAQATS (residues 1-7 of the heavy chain of FX), were identified. Furthermore, sequence SLNPNRV, which corresponds to residues 394-400 of AT, was identified in the non-reduced FX-AT complex, indicating that FX cleaved the Arg393-Ser394 bond in a reactive centre loop of AT. Unfractionated heparin induced FX-AT complex formation more effectively than low-molecular weight heparin or AT-binding pentasaccharide, and appeared to promote complex formation mainly via a template effect. These data suggest that AT is capable of forming a stable complex with zymogen FX by acting as an inhibitor in the presence of heparin.

Prospective Efficacy and Safety of a Novel Bypassing Agent, FVIIa/FX Mixture (MC710) for Hemophilia Patients with Inhibitors

Hemophilia A and B are hereditary bleeding disorders caused by a deficiency of coagulation factors VIII (FVIII) and IX (FIX), respectively. In substitution therapies using FVIII and FIX concentrates for the management of bleeding, the development of inhibitory antibodies is a serious complication in ~28% and ~7% of hemophilia A and B patients, respectively [1, 2]. Currently, two bypassing agents, plasma-derived activated prothrombin complex concentrates (APCC, FEIBA®) and recombinant activated factor VII (rFVIIa, NovoSeven®), are available for the management of bleeding in hemophilia patients with inhibitors including acquired hemophilia patients. Retrospective studies showed the efficacy of rFVIIa and APCC in 12~36 h in a standard administration regime is assessed to be 66~95% and 39~76%, respectively [3]. A recent comparative study seemed to indicate equivalence between rFVIIa and APCC [4]; however, a considerable number of patients experience treatment failure or insufficient efficacy. In 2002, an anecdotal report suggested that the sequential administration of rFVIIa and prothrombin complex concentrate (PCC) to hemophilia patients with inhibitors in order to obtain a stronger hemostatic effect than with rFVIIa alone [5]. Further, it has been reported that the combination of rFVIIa and APCC appeared to confer beneficial hemostatic synergy in patients refractory to each individual therapy [6-8]. However, repeated infusion of APCC may cause an accumulation of prothrombin and factor X (FX), thereby increasing the risk of thrombosis. Moreover, a lack of suitable laboratory tests for monitoring hemostatic effects is a major concern with current bypassing therapies. As a consequence and in appreciation of the incomplete efficacy and safety of currently available bypassing agents, new drugs are in development, such as rFVIIa analogues featuring higher hemostatic potential [9], glycoPEGylated rFVIIa with a longer half-life than rFVIIa [10], and non-anticoagulant sulphated polysaccharides (AV513) [11]. In order to solve these problems, we searched for alternative factors for enhancing and promoting FVIIa’s hemostatic activity, and found that a combination of plasma-derived FVIIa and FX (FVIIa/FX) may overcome the disadvantages of rFVIIa therapy for hemophilia patients with inhibitors. That combination allows improving APTT remarkably in the