Fujio Sekiya

Magnesium(II) Is a Crucial Constituent of the Blood Coagulation Cascade POTENTIATION OF COAGULANT ACTIVITIES OF FACTOR IX BY Mg IONS

We recently showed that not only Ca ions but also Mg ions play a crucial role in stabilizing the native conformation of coagulation factor IX. We here report that Mg ions at physiological concentrations greatly augment the biological activities of factor IX. In clotting assays with dialyzed plasma, addition of Mg ions enhanced the apparent coagulant activity of factor IXa, while that of factor Xa was scarcely affected. Activation of factor X by factor IXa in the presence of factor VIIIa, phospholipids, and Ca ions was accelerated by Mg ions. It appeared that the cation increased the affinity between factor IXa and factor VIIIa, thereby increasing the apparent catalytic efficacy of the enzyme. We also evaluated the effect of Mg ions in the coagulation pathway initiated by tissue factor and found that activation of factor IX by factor VIIa•tissue factor was accelerated by the cation. Consequently, clotting of normal plasma induced by factor VIIa•tissue factor was shortened by the cation, while no such effect was observed in plasma deficient in factor IX or VIII. These results indicate that the previously unrecognized plasma component, Mg ions, plays crucial roles in blood coagulation and, moreover, that contributions of factors IX and VIII in the coagulation cascade have been seriously underestimated in previous investigations.

12S-Hydroxyeicosatetraenoic acid plays a central role in the regulation of platelet activation

When platelets are activated by the recognition of exposed collagen fibers, they start synthesizing two major arachidonic acid metabolites, i.e. thromboxane A2 and 12S-hydroxyeicosatetraenoic acid (12-HETE) via cyclooxygenase and 12-lipoxygenase pathways, respectively. Although the physiological role of the former is well established, that of the latter has not been fully elucidated. Recently, we have revealed that 12-HETE interferes with collagen-induced platelet aggregation [Sekiya, F. et al. (1990) Biochim. Biophys. Acta 1044, 165-168]. In the present paper, we show that this substance enhances thrombin-induced aggregation of bovine platelets, in sharp contrast with the case of collagen. Additionally, 12-HETE is able to prevent the prostaglandin E1-induced elevation of platelet cAMP level and counteracts its inhibitory effect on platelet aggregations. With these observations, we propose a novel self-regulatory mechanism of platelets where 12-HETE plays a key role; it switches sensitivity of platelets from the primary agonist (collagen) to the secondary one (thrombin), and cancels the inhibitory effect of cAMP elevators.

Prothrombin and factor X activator activities in the venoms of Viperidae snakes

A Ca(2+)-dependent prothrombin activator, carinactivase-1 (CA-1), was previously found in the venom of Echis carinatus leucogaster. In the present study, the activities of CA-1-like enzymes were screened in the venoms of various Viperidae snakes. The addition of 1 mM Ca2+ ions to the venoms of only Echis snakes in Viperidae produced considerably high prothrombin activator activity, indicating that only the Echis snake venoms contain not only the Ca(2+)-independent prothrombin activator, ecarin, but also Ca(2+)-dependent activator(s). CA-1-like activators and ecarin in the venom of each Echis snake were efficiently separated by Blue Sepharose column chromatography. The venoms of the various Viperidae snakes were also examined for factor X activator activity. The venoms of genera Daboia, Vipera, Cerastes, Echis, Calloselasma and Bothrops contained factor X activator activity in the presence of Ca2+ ions. Cerastes cerastes and Calloselasma rhodostoma venoms also had Ca(2+)-independent factor X activator activity.

Feedback regulation of platelet function by 12 S-hydroxyeicosatetraenoic acid: Inhibition of arachidonic acid liberation from phospholipids

We have proposed a mechanism that platelet aggregation is regulated by its 12-lipoxygenase product, 12S-hydroxyeicosatetraenoic acid (12-HETE) (Sekiya, F., Takagi, J. and Saito, Y. (1989) Thrombos. Res. 56, 407-415). Inhibition of endogenous 12-HETE production by 15-HETE, a specific inhibitor of 12-lipoxygenase, accelerated aggregation of bovine platelets in response to collagen and arachidonic acid liberation from phospholipids was enhanced. Exogenously added 12-HETE suppressed collagen-induced liberation of arachidonic acid and the aggregation was also inhibited. On the other hand, 12-HETE did not interfere with thromboxane synthesis from free arachidonic acid in a cell-free system. These observations suggest that 12-HETE exerts a negative feedback to prevent excess aggregation through interference with arachidonic acid liberation from membrane phospholipids.

Venom from southern copperhead snake (Agkistrodon contortrix contortrix). II. A unique phospholipase A2 that induces platelet aggregation.

A platelet aggregation factor was purified from the venom of southern copperhead snake (Agkistrodon contortrix contortrix) by DEAE-cellulose ion-exchange chromatography, precipitation with ammonium sulfate, affinity chromatography using bovine serum albumin as ligand, and gel filtration on Cellulofine GCL-2000. It had molecular weights of 11,000 and 14,000, as determined by gel filtration chromatography and sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE), respectively. It consists of a single polypeptide, and was identified as a phospholipase A2. It was quite resistant to heat and various denaturing reagents including urea and SDS. It lost both phospholipase A2 activity and platelet aggregating activity upon modification of histidine residue(s) with p-bromophenacyl bromide. Its specificity towards the beta-position of phospholipid in esterolytic reaction was confirmed by gas-liquid chromatography using a pure synthetic phosphatidylcholine. Platelet aggregation by this phospholipase A2 was completely inhibited by prostacyclin, but was little inhibited by aspirin which indicates almost no direct participation of released arachidonic acid in the aggregation mechanism.

Localization of the specific binding site for magnesium(II) ions in factor IX

We demonstrated recently that coagulation factor IX has a specific binding site(s) for Mg2+ ions, independent of the Ca2+‐binding sites, and that binding of Mg2+ ions is very important for expression of the functional conformation of this protein. We report here the localization of this Mg2+‐specific binding site. We prepared three Gla‐containing fragments of bovine factor IX, namely GlaEGFNC (residues 1–144+286–296), GlaEGFN (1–83) and the Gla domain peptide (1–46). Fragments GlaEGFNC and GlaEGFN retained the ability to undergo a conformational change upon binding of Mg2+ ions in the presence of excess Ca2+ ions. This change could be detected by a conformation‐specific antibody. Furthermore, the Gla domain peptide was capable of binding Mg2+ ions, as determined by the metal ion‐induced quenching of the intrinsic fluorescence. It appears that the Mg2+‐specific binding site of factor IX is located in the N‐terminal Gla domain.

Elucidation of a role of plasma albumin during collagen-induced aggregation of bovine platelets.

Bovine platelets of which phospholipids were labeled with [14C] arachidonate were stimulated by collagen. Omitting albumin in suspending medium suppressed aggregation and arachidonate liberation from phospholipids. Analysis of [14C] arachidonate metabolites released into medium revealed that release of 12S-hydroxyeicosatetraenoic acid (12-HETE) from stimulated platelets was dependent on the existence of albumin, while thromboxane B2 release was not. Moreover, exogenously added 12-HETE inhibited collagen-induced aggregation. These observations suggest that albumin potentiates aggregation by preventing intracellular accumulation of 12-HETE, and strengthen our previous finding that albumin in plasma is very essential for collagen-induced aggregation of platelets (Sekiya, F., Takagi, J., Kasahara, K., Inada, Y. and Saito, Y. (1988) Thrombos. Res. 50, 837-846.).

Structure and Functions of Coagulation Factor IX/Factor X-Binding Protein Isolated from the Venom of Trimeresurus flavoviridis

Snake venoms contain various anticoagulants that affect blood coagulation system (1, 2). The anticoagulant is classified into two categories: anticoagulant enzymes and non-enzymatic anticoagulants. The examples of the former category are phospholipases, fibrinogenolytic enzymes, protein C activators, and proteolytic enzymes that convert coagulation factors into degraded inactive forms. An interesting example of the non-enzymatic anticoagulants is an inhibitor of the activation of prothrombin. The inhibitors in this category have been first found in the venom of Agkistrodon acutus (3) and Trimeresurus gramineus (4). The anticoagulant protein isolated from A. acutus inhibits the participation of factor Xa in the prothrombinase complex formation (5, 6). In order to understand the anticoagulant mechanism we have isolated and characterized another anticoagulant protein with Mr 27,000 from the venom of the habu snake Trimeresurus flavoviridis (7). This anticoagulant protein forms a complex with either coagulation factor IX or factor X with a stoichiometry of 1 to 1 in a calcium-dependent fashion, and thereby blocks the amplification of the coagulation cascade (7). Thus, we named it IX/X-bp (factor IX/factor X-binding protein). This chapter describes the structural and functional properties of IX/X-bp from the venom of the habu snake (T. flavoviridis).

Plasma albumin is essential for collagen-induced platelet aggregation

We found that platelets must have albumin on the surface to respond to collagen and aggregate. Albumin, however, was not absolutely necessary for ADP-, platelet activating factor-, serotonin- or thrombin-induced aggregation, while fibrinogen was required for ADP- or serotonin-induced aggregation. Immunofluorescent microscopy revealed that albumin was retained on gel-filtrated platelets but not on washed platelets. Albumin was not required for platelet adhesion to immobilized collagen. Without albumin thromboxane formation upon collagen-stimulation was diminished. These data suggest that albumin is essential in some step(s) that results in production of thromboxane A2.

Cooperativity between platelet-activating factor and collagen in platelet aggregation

Cell lysate obtained from cultured vascular endothelial cells contained a substance which induced platelet aggregation. This substance was identified as a phospholipid, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor; PAF), by thin-layer chromatography, phospholipase A2 digestion, inhibition by a specific antagonist, CV-3988, and agonist-specific refractory state. It was further found that PAF and collagen together induced extensive aggregation of platelets even with the concentrations by which each agonist alone could not induce aggregation of platelets at all.