Prothrombin residues 473-487 contribute to factor Va binding in the prothrombinase complex.
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 PT473-487 and Fl-fVa. These functional and spectroscopic data suggest that fII residues 473-487 provide fVa-binding sites and mediate interactions between fVa and fII in the prothrombinase complex.
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
Subramanian
S
Yegneswaran
Rolf M
RM
Mesters
José A
JA
Fernández
John H
JH
Griffin
D000818:Animals; D016880:Anisotropy; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D001777:Blood Coagulation; D015153:Blotting, Western; D002918:Chymotrypsin / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004792:Enzyme Precursors / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D015943:Factor Va / Q000737:chemistry*; D015951:Factor Xa / Q000737:chemistry; D005340:Fibrinogen / Q000737:chemistry; D019793:Fluorescein / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D010314:Partial Thromboplastin Time; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011516:Prothrombin / Q000737:chemistry* / Q000235:genetics*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013917:Thrombin / Q000737:chemistry; D013925:Thromboplastin / Q000378:metabolism*; D014757:Viper Venoms / Q000494:pharmacology
D004792:Enzyme Precursors; D010455:Peptides; D014757:Viper Venoms; D015943:Factor Va; D011516:Prothrombin; D005340:Fibrinogen; D013925:Thromboplastin; D001120:Arginine; D010450:Endopeptidases; D002918:Chymotrypsin; D013917:Thrombin; C026812:meizothrombin; D015951:Factor Xa; C020097:ecarin; D019793:Fluorescein