Annexin Lectins: Ca2+-Dependent Heparin-Binding Activity, Phosphatidylserine-Binding Activity, and Anticoagulant Activity.

Among numerous heparin-binding proteins identified in animal tissues and body fluids, annexins are unique because their activies depend on their Ca2+ binding. Annexins are known to have other Ca2+-dependent activities. For example, they bind to phosphatidylserine in the plasma membrane, and some of them exhibit potent anticoagulant activity. This chapter describes three protocols that measure the Ca2+-dependent activities using recombinant annexins: solid-phase heparin-binding assay using bovine serum albumin-conjugated heparin, solid-phase phosphatidylserine-binding assay, and plasma coagulation inhibition assay.

Annexin A4 inhibits sulfatide-induced activation of coagulation factor XII.

BACKGROUND: Factor XII (FXII) is a plasma serine protease that initiates the intrinsic pathway of blood coagulation upon contact with anionic substances, such as the sulfated glycolipid sulfatide. Annexins (ANXs) have been implicated in the regulation of the blood coagulation reaction by binding to anionic surfaces composed of phospholipids and sulfated glycoconjugates, but their physiological importance is only partially understood. OBJECTIVE: To test the hypothesis that ANXs are involved in suppressing the intrinsic pathway initiated by sulfatide, we examined the effect of eight recombinant ANX proteins on the intrinsic coagulation reaction and their sulfatide binding activities. METHODS: Recombinant ANXs were prepared in Escherichia coli expression systems and their anticoagulant effects on the intrinsic pathway initiated by sulfatide were examined using plasma clotting assay and chromogenic assay. ANXA4 active sites were identified by alanine scanning and fold deletion in the core domain. RESULTS AND CONCLUSIONS: We found that ANXA3, ANXA4, and ANXA5 strongly inhibited sulfatide-induced plasma coagulation. Wild-type and mutated ANXA4 were used to clarify the molecular mechanism involved in inhibition. ANXA4 inhibited sulfatide-induced auto-activation of FXII to FXIIa and the conversion of its natural substrate FXI to FXIa but showed no effect on the protease activity of FXIIa or FXIa. Alanine scanning showed that substitution of the Ca2+ -binding amino acid residue in the fourth fold of the core domain of ANXA4 reduced anticoagulant activity, and deletion of the entire fourth fold of the core domain resulted in complete loss of anticoagulant activity.