Mutant N143P reveals how Na+ activates thrombin.

The molecular mechanism of thrombin activation by Na(+) remains elusive. Its kinetic formulation requires extension of the classical Botts-Morales theory for the action of a modifier on an enzyme to correctly account for the contribution of the E*, E, and E:Na(+) forms. The extended scheme establishes that analysis of k(cat) unequivocally identifies allosteric transduction of Na(+) binding into enhanced catalytic activity.

Thrombin mutant W215A/E217A acts as a platelet GPIb antagonist.

Objective: Thrombin containing the mutations Trp215Ala and Glu217Ala (WE) selectively activates protein C and has potent antithrombotic effects in primates. The aim of this study was to delineate the molecular mechanism of direct WE-platelet interactions under static and shear conditions.

Methods And Results: Purified platelets under static conditions bound and spread on immobilized wild-type but not WE thrombin.

Mechanism of Na(+) binding to thrombin resolved by ultra-rapid kinetics.

The interaction of Na(+) and K(+) with proteins is at the basis of numerous processes of biological importance. However, measurement of the kinetic components of the interaction has eluded experimentalists for decades because the rate constants are too fast to resolve with conventional stopped-flow methods. Using a continuous-flow apparatus with a dead time of 50 micro s we have been able to resolve the kinetic rate constants and entire mechanism of Na(+) binding to thrombin, an interaction that is at the basis of the procoagulant and prothrombotic roles of the enzyme in the blood.

Important role of the cys-191 cys-220 disulfide bond in thrombin function and allostery.

Little is known on the role of disulfide bonds in the catalytic domain of serine proteases. The Cys-191-Cys-220 disulfide bond is located between the 190 strand leading to the oxyanion hole and the 220-loop that contributes to the architecture of the primary specificity pocket and the Na+ binding site in allosteric proteases. Removal of this bond in thrombin produces an approximately 100-fold loss of activity toward several chromogenic and natural substrates carrying Arg or Lys at P1.

Crystal structures of murine thrombin in complex with the extracellular fragments of murine protease-activated receptors PAR3 and PAR4.

It has been proposed that the cleaved form of protease-activated receptor 3 (PAR3) acts as a cofactor for thrombin cleavage and activation of PAR4 on murine platelets, but the molecular basis of this physiologically important effect remains elusive. X-ray crystal structures of murine thrombin bound to extracellular fragments of the murine receptors PAR3 ((38)SFNGGPQNTFEEFPLSDIE(56)) and PAR4 ((51)KSSDKPNPR downward arrow GYPGKFCANDSDTLELPASSQA(81), downward arrow = site of cleavage) have been solved at 2.0 and 3.

Structural basis of Na+ activation mimicry in murine thrombin.

Unlike human thrombin, murine thrombin lacks Na+ activation due to the charge reversal substitution D222K in the Na+ binding loop. However, the enzyme is functionally stabilized in a Na+-bound form and is highly active toward physiologic substrates. The structural basis of this peculiar property is unknown.

Thrombin allostery.

Thrombin is a Na(+)-activated, allosteric serine protease that plays multiple functional roles in blood pathophysiology. Binding of Na(+) is the major driving force behind the procoagulant, prothrombotic and signaling functions of the enzyme. This review summarizes our current understanding of the molecular basis of thrombin allostery with special emphasis on the kinetic aspects of Na(+) activation.