BLOOD-COAGULATION FACTOR XA INTERACTS WITH A LINEAR SEQUENCE OF THE KRINGLE-2 DOMAIN OF PROTHROMBIN

Prothrombin is a vitamin K-dependent plasma protein composed of severa l functional domains, which is proteolytically activated into thrombin by factor Xa in the presence of factor Va, Ca2+, and phospholipids. D uring the activation, prothrombin is cleaved into three fragments: fra gment 1, containing a domain rich in gamma-carboxyglutamic acid residu es and kringle 1 domain; fragment 2, containing the kringle 2 domain; and a protease catalytic domain, thrombin. Here we studied the interac tion site for factor Xa in human prothrombin during the activation. Th e isolated fragment 2 inhibited the activation of prothrombin by eithe r 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 K-d of 9.0 x 10(-8) M, but not to fragment 1 or DIP-thrombin. Factor Xa also bound to immobilized prothrombin and prethrombin 1 with K(d)s of 2.0 X 10(-7) and 1.5 X 10(-7) M, respectively, suggesting th at factor Xa interacts with the kringle 2 domain in these molecules. T he binding of factor Xa to immobilized fragment 2 was Ca2+-dependent w ith an optimal concentration at 6 mM. In the presence of Ca2+, the int eraction was enhanced by phospholipids in a concentration-dependent ma nner. To localize the factor Xa-binding site in the kringle 2 domain, fragment 2 was digested with lysyl endopeptidase and then trypsin afte r reduction and S-carboxymethylation. The resulting peptides were immo bilized to microwell plates and assayed for factor Xa binding ability. The amino acid sequence of the peptide positive in the assay was dete rmined to be residues His(205) to Arg(220). Factor Xa bound to a synth etic peptide corresponding to the residues His(205) to Arg(220) immobi lized to microwell plates. The peptide inhibited factor Xa-catalyzed a ctivation of prothrombin, but a peptide with the reversed sequence of His(205) to Arg(220) did not. These findings indicate that factor Xa i nteracts with at least a linear sequence, His(205) to Arg(220), in the kringle 2 domain of prothrombin during its activation into thrombin.