The Structural Integrity of Anion Binding Exosite I of Thrombin Is Required and Sufficient for Timely Cleavage and Activation of Factor V and Factor VIII

α-Thrombin has two separate electropositive binding exosites (anion binding exosite I, ABE-I and anion binding exosite II, ABE-II) that are involved in substrate tethering necessary for efficient catalysis. α-Thrombin catalyzes the activation of factor V and factor VIII following discrete proteolytic cleavages. Requirement for both anion binding exosites of the enzyme has been suggested for the activation of both procofactors by α-thrombin. We have used plasma-derived α-thrombin, β-thrombin (a thrombin molecule that has only ABE-II available), and a recombinant prothrombin molecule rMZ-II (R155A/R284A/R271A) that can only be cleaved at Arg320 (resulting in an enzymatically active molecule that has only ABE-I exposed, rMZ-IIa) to ascertain the role of each exosite for procofactor activation. We have also employed a synthetic sulfated pentapeptide ( DY(SO3−)DY(SO3−)Q, designated D5Q1,2) as an exosite-directed inhibitor of thrombin. The clotting time obtained with β-thrombin was increased by ∼8-fold, whereas rMZ-IIa was 4-fold less efficient in promoting clotting than α-thrombin under similar experimental conditions. α-Thrombin readily activated factor V following cleavages at Arg709, Arg1018, and Arg1545 and factor VIII following proteolysis at Arg372, Arg740, and Arg1689. Cleavage of both procofactors byα-thrombin was significantly inhibited by D5Q1,2. In contrast, β-thrombin was unable to cleave factor V at Arg1545 and factor VIII at both Arg372 and Arg1689. The former is required for light chain formation and expression of optimum factor Va cofactor activity, whereas the latter two cleavages are a prerequisite for expression of factor VIIIa cofactor activity. β-Thrombin was found to cleave factor V at Arg709 and factor VIII at Arg740, albeit less efficiently than α-thrombin. The sulfated pentapeptide inhibited moderately both cleavages by β-thrombin. Under similar experimental conditions, membrane-bound rMZ-IIa cleaved and activated both procofactor molecules. Activation of the two procofactors by membrane-bound rMZ-IIa was severely impaired by D5Q1,2. Overall the data demonstrate that ABE-I alone of α-thrombin can account for the interaction of both procofactors with α-thrombin resulting in their timely and efficient activation. Because formation of meizothrombin precedes that of α-thrombin, our findings also imply that meizothrombin may be the physiological activator of both procofactors in vivo in the presence of a procoagulant membrane surface during