Blood Coagulation Factor Xa Interacts with a Linear Sequence of the Kringle 2 Domain of Prothrombin

Abstract

Prothrombin is a vitamin K-dependent plasma protein composed of several functional domains, which is proteolytically activated into thrombin by factor Xa in the presence of factor Va, Ca²⁺, and phospholipids. During the activation, prothrombin is cleaved into three fragments: fragment 1, containing a domain rich in γ-carboxyglutamic acid residues and kringle 1 domain; fragment 2, containing the kringle 2 domain; and a protease catalytic domain, thrombin. Here we studied the interaction site for factor Xa in human prothrombin during the activation. The isolated fragment 2 inhibited the activation of prothrombin by either prothrombinase complex or factor Xa alone in a dose-dependent manner, whereas fragment 1 and diisopropylphosphate (DIP)-thrombin did not. Factor Xa directly bound to fragment 2 immobilized to microwell plates with a Kd of 9.0×10^-8M, but not to fragment 1 or DIP-thrombin. Factor Xa also bound to immobilized prothrombin and prethrombin 1 with K_ds of 2.O×10^-7and 1.5×10^-7M, respectively, suggesting that factor Xa interacts with the kringle 2 domain in these molecules. The binding of factor Xa to immobilized fragment 2 was Ca²⁺-dependent with an optimal concentration at 6mM. In the presence of Ca²⁺, the interaction was enhanced by phospholipids in a concentration-dependent manner. To localize the factor Xa-binding site in the kringle 2 domain, fragment 2 was digested with lysyl endopeptidase and then trypsin after reduction and S-carboxymethylation. The resulting peptides were immobilized to microwell plates and assayed for factor Xa binding ability. The amino acid sequence of the peptide positive in the assay was determined to be residues His²⁰⁵ to Arg²²⁰. Factor Xa bound to a synthetic peptide corresponding to the residues His²⁰⁵ to Arg²²⁰ immobilized to microwell plates. The peptide inhibited factor Xa-catalyzed activation of prothrombin, but a peptide with the reversed sequence of His²⁰⁵ to Arg²²⁰ did not. These findings indicate that factor Xa interacts with at least a linear sequence, His²⁰⁵ to Arg²²⁰, in the kringle 2 domain of prothrombin during its activation into thrombin.