The Dual Regulatory Role of Amino Acids Leu480 and Gln481 of Prothrombin

Prothrombin (FII) is activated to α-thrombin (IIa) by prothrombinase. Prothrombinase is composed of a catalytic subunit, factor Xa (fXa), and a regulatory subunit, factor Va (fVa), assembled on a membrane surface in the presence of divalent metal ions. We constructed, expressed, and purified several...

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Bibliographic Details
Published in:The Journal of biological chemistry 2016-01, Vol.291 (4), p.1565-1581
Main Authors: Wiencek, Joesph R., Hirbawi, Jamila, Yee, Vivien C., Kalafatis, Michael
Format: Article
Language:English
Subjects:
activation
Amino Acid Motifs
Amino Acid Sequence
blood
coagulation factor
factor v
Factor Va - genetics
Factor Va - metabolism
factor Xa
Factor Xa - genetics
Factor Xa - metabolism
Glutamine - genetics
Glutamine - metabolism
Humans
Leucine - genetics
Leucine - metabolism
Molecular Sequence Data
mutation
Protein C - genetics
Protein C - metabolism
Protein Processing, Post-Translational
Protein Structure and Folding
prothrombin
Prothrombin - chemistry
Prothrombin - genetics
Prothrombin - metabolism
prothrombinase
thrombin
Thromboplastin - genetics
Thromboplastin - metabolism
thrombosis
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Summary:Prothrombin (FII) is activated to α-thrombin (IIa) by prothrombinase. Prothrombinase is composed of a catalytic subunit, factor Xa (fXa), and a regulatory subunit, factor Va (fVa), assembled on a membrane surface in the presence of divalent metal ions. We constructed, expressed, and purified several mutated recombinant FII (rFII) molecules within the previously determined fVa-dependent binding site for fXa (amino acid region 473–487 of FII). rFII molecules bearing overlapping deletions within this significant region first established the minimal stretch of amino acids required for the fVa-dependent recognition exosite for fXa in prothrombinase within the amino acid sequence Ser478–Val479–Leu480–Gln481–Val482. Single, double, and triple point mutations within this stretch of rFII allowed for the identification of Leu480 and Gln481 as the two essential amino acids responsible for the enhanced activation of FII by prothrombinase. Unanticipated results demonstrated that although recombinant wild type α-thrombin and rIIaS478A were able to induce clotting and activate factor V and factor VIII with rates similar to the plasma-derived molecule, rIIaSLQ→AAA with mutations S478A/L480A/Q481A was deficient in clotting activity and unable to efficiently activate the pro-cofactors. This molecule was also impaired in protein C activation. Similar results were obtained with rIIaΔSLQ (where rIIaΔSLQ is recombinant human α-thrombin with amino acids Ser478/Leu480/Gln481 deleted). These data provide new evidence demonstrating that amino acid sequence Leu480–Gln481: 1) is crucial for proper recognition of the fVa-dependent site(s) for fXa within prothrombinase on FII, required for efficient initial cleavage of FII at Arg320; and 2) is compulsory for appropriate tethering of fV, fVIII, and protein C required for their timely activation by IIa.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M115.691956