Involvement of Thrombin Anion-binding Exosites 1 and 2 in the Activation of Factor V and Factor VIII

Esmon, Charles T.; Lollar, Pete

doi:10.1074/jbc.271.23.13882

Cited by 112 publications

(166 citation statements)

References 64 publications

Supporting

Mentioning

157

Contrasting

Unclassified

Order By: Relevance

“…This latter observation was based upon results showing that mutations at Arg-740, impairing this cleavage, significantly reduced cleavage rates at the two other P1 sites. Thrombin-catalyzed activation of factor VIII is dependent upon interactions involving the anion binding exosites of the proteinase (11,12). Exosite binding is believed to determine substrate affinity, whereas subsequent active site docking primarily affects V max (13).…”

mentioning

confidence: 99%

Cleavage at Arg-1689 Influences Heavy Chain Cleavages during Thrombin-catalyzed Activation of Factor VIII

Newell

Fay

2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

The procofactor, factor VIII, is activated by thrombin or factor Xa-catalyzed cleavage at three P1 residues: Arg-372, Arg-740, and Arg-1689. The catalytic efficiency for thrombin cleavage at Arg-740 is greater than at either Arg-1689 or Arg-372 and influences reaction rates at these sites. Because cleavage at Arg-372 appears rate-limiting and dependent upon initial cleavage at Arg-740, we investigated whether cleavage at Arg-1689 influences catalysis at this step. Recombinant B-domainless factor VIII mutants, R1689H and R1689Q were prepared and stably expressed to slow and eliminate cleavage, respectively. Specific activity values for the His and Gln mutations were ϳ50 and ϳ10%, respectively, that of wild type. Thrombin activation of the R1689H variant showed an ϳ340-fold reduction in the rate of Arg-1689 cleavage, whereas the R1689Q variant was resistant to thrombin cleavage at this site. Examination of heavy chain cleavages showed ϳ4-and 11-fold reductions in A2 subunit generation and ϳ3-and 7-fold reductions in A1 subunit generation for the R1689H and R1689Q mutants, respectively. These results suggest a linkage between light chain cleavage and cleavages in heavy chain. Results obtained evaluating proteolysis of the factor VIII mutants by factor Xa revealed modest rate reductions (<5-fold) in generating A2 and A1 subunits and in cleaving light chain at Arg-1721 from either variant, suggesting little dependence upon prior cleavage at residue 1689 as compared with thrombin. Overall, these results are consistent with a competition between heavy and light chains for thrombin exosite binding and subsequent proteolysis with binding of the former chain preferred.Factor VIII, a plasma protein missing or defective in individuals with hemophilia A, is synthesized as an ϳ300-kDa single chain polypeptide corresponding to 2332 amino acids. Within the protein are six domains based on internal homologies and ordered as NH 2 -A1-A2-B-A3-C1-C2-COOH (1, 2). Bordering the A domains are short segments containing high concentrations of acidic residues that follow the A1 and A2 domains and precede the A3 domain and are designated a1 (residues 337-372), a2 (residues 711-740), and a3 (1649 -1689). Factor VIII is processed by cleavage at the B-A3 junction to generate a divalent metal ion-dependent heterodimeric protein composed of a heavy chain (A1-a1-A2-a2-B domains) and a light chain (a3-A3-C1-C2 domains) (3).The activated form of factor VIII, factor VIIIa, functions as a cofactor for factor IXa, increasing its catalytic efficiency by several orders of magnitude in the phospholipid-and Ca 2ϩ -dependent conversion of factor X to factor Xa (4). The factor VIII procofactor is converted to factor VIIIa through limited proteolysis catalyzed by thrombin or factor Xa (5, 6). Thrombin is believed to act as the physiological activator of factor VIII, as association of factor VIII with von Willebrand factor impairs the capacity for the membrane-dependent factor Xa to efficiently activate the procofactor (5, 7). Activation of factor VIII occu...

show abstract

mentioning

confidence: 99%

Cleavage at Arg-1689 Influences Heavy Chain Cleavages during Thrombin-catalyzed Activation of Factor VIII

Newell

Fay

2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…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). Interestingly, proexosite I of prothrombin, which is present at a low affinity state on the molecule, and its affinity for its ligands increases by ϳ100-fold following activation and formation of thrombin (33,39), was found to be directly involved in the productive interaction with factor Va within prothrombinase (32,33).…”

mentioning

confidence: 99%

Structural Requirements for Expression of Factor Va Activity

Kalafatis

Beck

Mann

2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

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....

show abstract

“…The critical roles of exosites I and II in regulation of macromolecular interactions of thrombin are well established (12,13), including the role of exosite I in fibrinogen substrate recognition (14,15), and both exosites in thrombomodulin-regulated protein C activation (16 -18), factor V (19 -22), and factor VIII (19) activation. In addition, exosite II binds heparin (23), regulating inactivation of thrombin by antithrombin (24), and Pro fragment 2 (F2, 25).…”

mentioning

confidence: 99%

Effects of Activation Peptide Bond Cleavage and Fragment 2 Interactions on the Pathway of Exosite I Expression during Activation of Human Prethrombin 1 to Thrombin

Anderson¹,

Nesset

Bock

2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

Involvement of Thrombin Anion-binding Exosites 1 and 2 in the Activation of Factor V and Factor VIII

Cited by 112 publications

References 64 publications

Cleavage at Arg-1689 Influences Heavy Chain Cleavages during Thrombin-catalyzed Activation of Factor VIII

Cleavage at Arg-1689 Influences Heavy Chain Cleavages during Thrombin-catalyzed Activation of Factor VIII

Structural Requirements for Expression of Factor Va Activity

Effects of Activation Peptide Bond Cleavage and Fragment 2 Interactions on the Pathway of Exosite I Expression during Activation of Human Prethrombin 1 to Thrombin

Contact Info

Product

Resources

About