Long range communication between exosites 1 and 2 modulates thrombin function.
Although exosites 1 and 2 regulate thrombin activity by binding substrates and cofactors and by allosterically modulating the active site, it is unclear whether there is direct allosteric linkage between the two exosites. To begin to address this, we first titrated a thrombin variant fluorescently labeled at exosite 1 with exosite 2 ligands, HD22 (a DNA aptamer), gamma'-peptide (an analog of the COOH terminus of the gamma'-chain of fibrinogen) or heparin. Concentration-dependent and saturable changes in fluorescence were elicited, supporting inter-exosite linkage. To explore the functional consequences of this phenomenon, we evaluated the capacity of exosite 2 ligands to inhibit thrombin binding to gamma(A)/gamma(A)-fibrin, an interaction mediated solely by exosite 1. When gamma(A)/gamma(A)-fibrinogen was clotted with thrombin in the presence of HD22, gamma'-peptide, or prothrombin fragment 2 there was a dose-dependent and saturable decrease in thrombin binding to the resultant fibrin clots. Furthermore, HD22 reduced the affinity of thrombin for gamma(A)/gamma(A)-fibrin 6-fold and accelerated the dissociation of thrombin from preformed gamma(A)/gamma(A)-fibrin clots. Similar responses were obtained when surface plasmon resonance was used to monitor the interaction of thrombin with gamma(A)/gamma(A)-fibrinogen or fibrin. There is bidirectional communication between the exosites, because exosite 1 ligands, HD1 (a DNA aptamer) or hirudin-(54-65) (an analog of the COOH terminus of hirudin), inhibited the exosite 2-mediated interaction of thrombin with immobilized gamma'-peptide. These findings provide evidence for long range allosteric linkage between exosites 1 and 2 on thrombin, revealing further complexity to the mechanisms of thrombin regulation.
Petrera
Nicolas S
NS
Department of Medicine, McMaster University, and Henderson Research Center, Hamilton, Ontario L8V 1C3, Canada.
Stafford
Alan R
AR
Leslie
Beverly A
BA
Kretz
Colin A
CA
Fredenburgh
James C
JC
Weitz
Jeffrey I
JI
D000494:Allosteric Regulation / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D005337:Fibrin / Q000737:chemistry* / Q000494:pharmacology; D005340:Fibrinogen / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D020349:Surface Plasmon Resonance; D013917:Thrombin / Q000737:chemistry* / Q000494:pharmacology
D010455:Peptides; D005337:Fibrin; D005340:Fibrinogen; D013917:Thrombin