Prothrombin residues 473-487 contribute to factor Va binding in the prothrombinase complex

The Journal of Biological Chemistry
Subramanian YegneswaranJohn H Griffin

Abstract

To identify sequences in prothrombin (fII) involved in prothrombinase complex (fXa.fVa.fII.phospholipids) assembly, synthetic peptides based on fII sequences were prepared and screened for their ability to inhibit factor Xa (fXa)-induced clotting of normal plasma. The fII peptide (PT473-487, homologous to chymotrypsin residues 149D-163) potently inhibited plasma clotting assays and prothrombinase activity, with 50% inhibition of 12 and 10 microm peptide, respectively. Prothrombinase inhibition by PT473-487 was factor Va (fVa)-dependent and sequence-specific, because the peptide did not inhibit fII activation in the absence of fVa, and a scrambled sequence peptide, PT473-487SCR, was not inhibitory. Peptide PT473-487 did not inhibit the amidolytic activities of fXa and thrombin, suggesting that the peptide did not alter the integrity of their active sites. To determine whether PT473-487 interacted directly with fVa, fluorescein-labeled fVa (Fl-fVa) was prepared. When PT473-487 was titrated into samples containing phospholipid-bound Fl-fVa, the peptide increased fluorescein anisotropy (EC(50) at 3 microm peptide), whereas the control peptide PT473-487SCR did not alter the anisotropy, suggesting a direct binding interaction between...Continue Reading

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Citations

Jul 11, 2006·Proceedings. Biological Sciences·Thomas Cavalier-Smith
May 14, 2014·Proceedings of the National Academy of Sciences of the United States of America·Nicola PozziEnrico Di Cera
Aug 1, 2007·Journal of Thrombosis and Haemostasis : JTH·P E BockI M A Verhamme
Jan 27, 2006·Proteins·Ludovic AutinBruno O Villoutreix
May 30, 2014·Blood·Lacramioara IvanciuRodney M Camire
Jun 8, 2007·The Journal of Biological Chemistry·Andrew J GaleJohn H Griffin
Jan 17, 2008·The Journal of Biological Chemistry·Sinh TranBjörn Dahlbäck
Jul 25, 2007·The Journal of Biological Chemistry·Subramanian YegneswaranJohn H Griffin
Jun 19, 2013·The Journal of Biological Chemistry·Nicola PozziEnrico Di Cera
Oct 21, 2014·Expert Review of Proteomics·Nicola Pozzi, Enrico Di Cera
Jan 15, 2016·The Journal of Biological Chemistry·Nicola PozziEnrico Di Cera
Apr 18, 2019·Scientific Reports·Laura AcquasalienteEnrico Di Cera
Nov 26, 2015·The Journal of Biological Chemistry·Joesph R WiencekMichael Kalafatis

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