Jun Mizuguchi

Hemextin AB complex, a unique anticoagulant protein complex from Hemachatus haemachatus (African Ringhals cobra) venom that inhibits clot initiation and factor VIIa activity.

During injury or trauma, blood coagulation is initiated by the interaction of factor VIIa (FVIIa) in the blood with freshly exposed tissue factor (TF) to form the TF·FVIIa complex. However, unwanted clot formation can lead to death and debilitation due to vascular occlusion, and hence, anticoagulants are important for the treatment of thromboembolic disorders. Here, we report the isolation and characterization of two synergistically acting anticoagulant proteins, hemextins A and B, from the venom of Hemachatus haemachatus (African Ringhals cobra). N-terminal sequences and CD spectra of the native proteins indicate that these proteins belong to the three-finger toxin family. Hemextin A (but not hemextin B) exhibits mild anticoagulant activity. However, hemextin B forms a complex (hemextin AB complex) with hemextin A and synergistically enhances its anticoagulant potency. Prothrombin time assay showed that these two proteins form a 1:1 complex. Complex formation was supported by size-exclusion chromatography. Using a “dissection approach,” we determined that hemextin A and the hemextin AB complex prolong clotting by inhibiting TF·FVIIa activity. The site of anticoagulant effects was supported by their inhibitory effect on the reconstituted TF·FVIIa complex. Furthermore, we demonstrated their specificity of inhibition by studying their effects on 12 serine proteases; the hemextin AB complex potently inhibited the amidolytic activity of FVIIa in the presence and absence of soluble TF. Kinetic studies showed that the hemextin AB complex is a noncompetitive inhibitor of soluble TF·FVIIa amidolytic activity, with a Ki of 50 nm. Isothermal titration calorimetric studies showed that the hemextin AB complex binds directly to FVIIa with a binding constant of 1.62 × 105 m–1. The hemextin AB complex is the first reported natural inhibitor of FVIIa that does not require a scaffold to mediate its inhibitory activity. Molecular interactions of the hemextin AB complex with FVIIa/TF·FVIIa will provide a new paradigm in the search for anticoagulants that inhibit the initiation of blood coagulation.

Crystal structure of thrombin in complex with s-variegin: insights of a novel mechanism of inhibition and design of tunable thrombin inhibitors

The inhibition of thrombin is one of the important treatments of pathological blood clot formation. Variegin, isolated from the tropical bont tick, is a novel molecule exhibiting a unique ‘two-modes’ inhibitory property on thrombin active site (competitive before cleavage, noncompetitive after cleavage). For the better understanding of its function, we have determined the crystal structure of the human α-thrombin:synthetic-variegin complex at 2.4 Å resolution. The structure reveals a new mechanism of thrombin inhibition by disrupting the charge relay system. Based on the structure, we have designed 17 variegin variants, differing in potency, kinetics and mechanism of inhibition. The most active variant is about 70 times more potent than the FDA-approved peptidic thrombin inhibitor, hirulog-1/bivalirudin. In vivo antithrombotic effects of the variegin variants correlate well with their in vitro affinities for thrombin. Our results encourage that variegin and the variants show strong potential for the development of tunable anticoagulants.

The 99 and 170 Loop-modified Factor VIIa Mutants Show Enhanced Catalytic Activity without Tissue Factor

To elucidate the functions of the surface loops of VIIa, we prepared two mutants, VII-30 and VII-39. The VII-30 mutant had all of the residues in the 99 loop replaced with those of trypsin. In the VII-39 mutant, both the 99 and 170 loops were replaced with those of trypsin. Thek cat/K m value for hydrolysis of the chromogenic peptidyl substrate S-2288 by VIIa-30 (103 mm − 1s− 1) was 3-fold higher than that of wild-type VIIa (30.3 mm − 1 s− 1) in the presence of soluble tissue factor (sTF). This enhancement was due to a decrease in the K m value but not to an increase in the k cat value. On the other hand, the k cat/K m value for S-2288 hydrolysis by VIIa-39 (17.9 mm − 1 s− 1) was 18-fold higher than that of wild-type (1.0 mm − 1 s− 1) in the absence of sTF, and the value was almost the same as that of wild-type measured in the presence of sTF. This enhancement was due to not only a decrease in the K m value but also to an increase in the k cat value. These results were in good agreement with their susceptibilities to a subsite 1-directed serine protease inhibitor. In our previous paper (Soejima, K., Mizuguchi, J., Yuguchi, M., Nakagaki, T., Higashi, S., and Iwanaga, S. (2001) J. Biol. Chem. 276, 17229–17235), the replacement of the 170 loop of VIIa with that of trypsin induced a 10-fold enhancement of the k cat value for S-2288 hydrolysis as compared with that of wild-type VIIa in the absence of sTF. These results suggested that the 99 and the 170 loop structures of VIIa independently affect the K m andk cat values, respectively. Furthermore, we studied the effect of mutations on proteolytic activity towardS-alkylated lysozyme as a macromolecular substrate and the activation of natural macromolecular substrate factor X.

Induction of Acquired Factor IX Inhibitors in Cynomolgus Monkey (Macaca Fascicularis): A New Primate Model of Hemophilia B

Inherited hemophilia dog and other transient hemophilic animal models have been used for evaluation of hemostatic agents for use in treatment of hemophilia. We established the first nonhuman primate hemophilic model by immunizing cynomolgus monkeys with human FIX (hFIX) in adjuvants. FIX activities of all three hFIX-immunized monkeys decreased transiently to less than 10% in accordance with prolongation of activated partial thromboplastin time (APTT). Forty micrograms of human factor VIIa (hFVIIa) per kilogram body weight (that was reported to be clinically effective) was administered to the monkey with the highest inhibitor titer to evaluate its usefulness as a hemophilia inhibitor model. Results of thromboelastography (TEG) after the injection demonstrated that the hemostatic effect of FVIIa in this model would be similar to that in hemophiliacs with inhibitors. The antibodies purified from the monkeys plasma by hFIX-immobilized gel were composed of two types: Ca(2+)-dependent and -independent antibodies, with features of IgG(1) and IgG(4). Both types of antibodies reacted to cynomolgus FIX, and only Ca(2+)-dependent antibodies also expressed inhibitory activity against cynomolgus FIX. Immunoblotting analyses of Ca(2+)-dependent antibodies using hFIX and its derivatives suggested that they recognized the Ca(2+)-dependent conformation related to the gamma-carboxyglutamic acid (Gla) domain. Comparison of FIX cDNA from human, cynomolgus monkey, and other species, and the results of immunization of various animals (goats, beagle dogs, rabbits, and rats) with hFIX in adjuvants strongly suggested that the development of acquired FIX inhibitors in the monkeys might be due to high cross-reactivity of the antibodies to molecular mimic antigens, hFIX, and cynomolgus FIX.

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.

Stepwise Assembly of Fibrinogen Is Assisted by the Endoplasmic Reticulum Lectin-Chaperone System in HepG2 Cells

The endoplasmic reticulum (ER) plays essential roles in protein folding and assembly of secretory proteins. ER-resident molecular chaperones and related enzymes assist in protein maturation by co-operated interactions and modifications. However, the folding/assembly of multimeric proteins is not well understood. Here, we show that the maturation of fibrinogen, a hexameric secretory protein (two trimers from α, β and γ subunits), occurs in a stepwise manner. The αγ complex, a precursor for the trimer, is retained in the ER by lectin-like chaperones, and the β subunit is incorporated into the αγ complex immediately after translation. ERp57, a protein disulfide isomerase homologue, is involved in the hexamer formation from two trimers. Our results indicate that the fibrinogen hexamer is formed sequentially, rather than simultaneously, using kinetic pause by lectin chaperones. This study provides a novel insight into the assembly of most abundant multi-subunit secretory proteins.

Molecular Evolution of Blood Clotting Factors with Special Reference to Fibrinogen and von Willebrand Factor

Comparative studies on “fibrinogen-like domains” and von Willebrand factor (vWF) domains found in various proteins have been performed. The “fibrinogen-like domain” is contained in more than 70 functional proteins, which are involved in innate immunity, angiogenesis, development, and differentiation, such as ficolins, tenascins, angiopoietins, and tachylectins. On the other hand, vWF A and D domains are identified extensively in integrins, extracellular matrix proteins, and secreted mucins. These vWF domains, in addition to “fibrinogen-like domains,” are found throughout the vertebrates and in several invertebrate species, indicating that these domains have deep evolutionary roots associated with vertebrate circulatory systems and their roles in homeostasis. These facts suggest that a mechanism of hemostasis is evolutionarily conserved from arthropods to mammals.