Enzymic properties of proteolytic derivatives of human .alpha.-thrombin

Background: Thrombin utilizes active site and anion-binding exosites (ABE) to fulfill diverse roles. Results: ABE I ligands PAR3(44 -56), PAR1(49 -62), and Hirudin(54 -65) exert different effects on thrombin exosite-active site and exosite-exosite interactions. More consequences occur with added PPACK and ABE II ligands. Conclusion: Thrombin employs ligands to transmit unique events across the enzyme. Significance: Ligand binding may be tailored to therapeutically regulate multifunctional thrombin.

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Ligand Binding to Anion-binding Exosites Regulates Conformational Properties of Thrombin

Malovichko

Sabo²,

Maurer

2013

“…It has been shown that prothrombin and thrombin have two separate electropositive exosites (anion binding exosite I, ABE-I, and anion binding exosite II, ABE-II) that are responsible for the majority of the functions of the molecules (30 -39). Whereas ABE-I has been involved in the binding to thrombomodulin (40), fibrinogen (41), PAR1 (42), the COOH-terminal hirudin peptides (43), and heparin cofactor II (44) among others, ABE-II was found to be involved in the interaction with protease nexin (45) and antithrombin III (44). Data from separate laboratories have demonstrated that both exosites bind factors V and VIII (33,34,35).…”

mentioning

confidence: 99%

Structural Requirements for Expression of Factor Va Activity

Kalafatis

Beck

Mann

2003

Thrombin activated factor Va (factor V IIa , residues 1-709 and 1546 -2196) has an apparent dissociation constant (K d,app ) for factor Xa within prothrombinase of ϳ0.5 nM. A protease (NN) purified from the venom of the snake Naja nigricollis nigricollis, cleaves human factor V at Asp 697 , Asp 1509 , and Asp 1514 to produce a molecule (factor V NN ) that is composed of a M r 100,000 heavy chain (amino acid residues 1-696) and a M r 80,000 light chain (amino acid residues 1509/1514 -2196). Factor V NN , has a K d,app for factor Xa of 4 nM and reduced clotting activity. Cleavage of factor V IIa by NN at Asp 697 results in a cofactor that loses ϳ60 -80% of its clotting activity. An enzyme from Russell's viper venom (RVV) cleaves human factor V at Arg 1018 and Arg 1545 to produce a M r 150,000 heavy chain and M r 74,000 light chain (factor V RVV , residues 1-1018 and 1546 -2196). The RVV species has affinity for factor Xa and clotting activity similar to the thrombin-activated factor Va. Cleavage of factor V NN at Arg 1545 by ␣-thrombin (factor V NN/IIa ) or RVV (factor V NN/RVV ) leads to enhanced affinity of the cofactor for factor Xa (K d,app ϳ 0.5 nM). A synthetic peptide containing the last 13 residues from the heavy chain of factor Va (amino acid sequence 697-709, D13R) was found to be a competitive inhibitor of prothrombinase with respect to prothrombin. The peptide was also found to specifically interact with thrombin-agarose. These data demonstrate that 1) cleavage at Arg 1545 and formation of the light chain of factor V IIa is essential for high affinity binding and function of factor Xa within prothrombinase and 2) a binding site for prothrombin is contributed by amino acid residues 697-709 of the heavy chain of the cofactor.The prothrombinase complex responsible for the generation of ␣-thrombin in the hemostatic process is composed of factor Va and factor Xa associated on a phospholipid membrane in the presence of Ca 2ϩ (1, 2). Although factor Xa alone can convert prothrombin to ␣-thrombin, the prothrombinase complex has a catalytic efficiency five orders of magnitude greater than factor Xa acting alone (3). Plasma factor V circulates as a large single chain protein of M r 330,000 (4 -6). The cDNA sequences for human, murine, porcine, and bovine factor V have been reported previously (7-11). The factor V molecule is composed of triplicated "A" domains, duplicated "C" domains, and a "B" region. Human factor V is cleaved by ␣-thrombin at Arg 709 , Arg 1018 , and Arg 1545 and generates the active cofactor factor Va, which is composed of a heavy chain (A1-A2 domains, M r 105,000, amino acid residues 1-709) non-covalently associated with the light chain (A3-C1-C2 domains, M r 74,000, amino acid residues 1546 -2196). The interaction between the two chains is promoted by divalent cations (12, 13).Activation of factor V by ␣-thrombin is required for the interaction of the cofactor with factor Xa and prothrombin. Factor Va and factor Xa interact stoichiometrically in the absence of phospholipids with a K d of 0....

“…The number of thrombin structures currently deposited in the Protein Data Bank exceeds 150, but not a single one of them was obtained in the absence of Na ϩ , inhibitors, or effector molecules. The presence of inhibitors, either at the active site or exosite I, is made necessary by the tendency of thrombin to cleave itself at Arg-77a in exosite I (13). The cleavage initiates a cascade of autoproteolytic steps that culminates in the excision of the Arg-67-Arg-77a fragment of exosite I and the conversion of the native ␣-thrombin into ␥-thrombin.…”

mentioning

confidence: 99%

Crystal Structure of the Anticoagulant Slow Form of Thrombin

Pineda¹,

Savvides²,

Waksman

et al. 2002

Using the thrombin mutant R77aA devoid of the site of autoproteolytic degradation at exosite I, we have solved for the first time the structure of thrombin free of any inhibitors and effector molecules and stabilized in the Na ؉ -free slow form. The slow form shows subtle differences compared with the currently available structures of the Na ؉ -bound fast form that carry inhibitors at the active site or exosite I. The most notable differences are the displacement of Asp-189 in the S1 specificity pocket, a downward shift of the 190 -193 strand, a rearrangement of the side chain of Glu-192, and a significant shift in the position of the catalytic Ser-195 that is no longer within H-bonding distance from His-57. The structure of the slow form explains the reduced specificity toward synthetic and natural substrates and suggests a molecular basis for its anticoagulant properties.