Factor Va increases the affinity of factor Xa for prothrombin: a binding study using a novel photoactivable thiol-specific fluorescent probe.
The multiprotein complex of factor Xa, factor Va, and prothrombin efficiently generates the blood-clotting agent, thrombin. Here, the formation of the factor Xa*prothrombin complex and the effects of factor Va on this complex were examined using a photoactivable thiol-specific fluorescent probe (LWB), which was synthesized and incorporated into the active site of factor Xa. The use of fluorescent LWB illustrated that factor Xa has an increased affinity for prothrombin in the presence of factor Va. Further exposure of these components to UV light resulted in a specific photocrosslinking of LWB-factor Xa to prothrombin, suggesting a physical association between these proteins. These data demonstrate that LWB can successfully function both as a spectroscopic probe and as a photocrosslinking reagent for studying protein-protein interactions.
Yegneswaran
Subramanian
S
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.
Fernández
José A
JA
Griffin
John H
JH
Dawson
Philip E
PE
D003432:Cross-Linking Reagents / Q000528:radiation effects; D015943:Factor Va / Q000494:pharmacology*; D015951:Factor Xa / Q000378:metabolism*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000528:radiation effects; D006801:Humans; D008722:Methods; D019920:Photoaffinity Labels / Q000528:radiation effects; D011485:Protein Binding / Q000187:drug effects; D011516:Prothrombin / Q000378:metabolism*; D013438:Sulfhydryl Compounds; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D019920:Photoaffinity Labels; D013438:Sulfhydryl Compounds; D015943:Factor Va; D011516:Prothrombin; D015951:Factor Xa