Date on Master's Thesis/Doctoral Dissertation


Document Type

Doctoral Dissertation

Degree Name

Ph. D.



Committee Chair

Maurer, Muriel C.

Author's Keywords

Ligand binding; Blood coagulation; Thrombin; Factor XIII; Fibrinogen


Prothrombin; Ligand binding (Biochemistry); Blood--Coagulation


In blood coagulation, the serine protease thrombin is a multifaceted enzyme that interacts with multiple proteins. Thrombin utilizes two anion binding exosites (ABE-I and II) to supplement binding to fibrinogen and to the platelet receptor GpIba, two proteins instrumental in clot formation. Approximately 7% to 15% of the fibrinogen ? chain exists as the highly anionic ?' variant ( 410 PEHPAETEY S DSLY S PEDDL 427) , which has been shown to bind ABE-II. GpIba possesses a similar anionic stretch of residues ( 269 DEGDTDLY S DY S Y S PEEDTEG 286 ); however, the exact destination of thrombin binding is more ambiguous. 1D and 2D solution NMR have been employed to characterize the structural features of the bound ?' and GpIba peptides. The results indicate that the ?' residues A 414 -L 427 make significant contact with the enzyme, a turn is present between residues Y S 422 -D 425 , and there is a hydrophobic cluster involving Y S 418 -Y S 422 . For the GpIba peptide, NMR results suggest the peptide exists in an extended conformation with residues D 274 -E 285 contributing to binding. Hydrogen deuterium exchange (HDX) coupled with MALDI-TOF MS indicate that at a 20:1 peptide-enzyme ratio both peptides are interacting with residues within ABE-II fragments. Both peptides affect the dynamics of HDX for regions not associated with ABE-II including ABE-I, the autolysis loop, and the A-chain. Curiously, at 40:1 GpIba peptide to thrombin, the GpIba peptide appears to interact with ABE-I, with unique allosteric consequences. Thrombin also activates FXIII, and the resultant transglutaminase covalently crosslinks fibrin through the formation of isopeptide bonds. Studies were performed to examine the effects of activation and inhibition on the conformational dynamics of FXIII using MALDI-TOF MS. A peptide-based FXIIIa inhibitor has been developed with the glutamine isostere 6-diazo-5-oxo-norleucine (DON). The K9 DON peptide blocks chemical modification of C 409 within the catalytic core and promotes significant HDX protection for the ß barrel 1 fragment 526-546. These results suggest that inhibition of FXIIIa leads to local and long-range effects on protein dynamics. Finally, thrombin activation of FXIII appears to influence regions near the activation peptide and Ca 2+ -activated FXIII presents more evidence for the existence of additional Ca 2+ binding sites. Ultimately, these studies further detail the dynamic nature of thrombin and FXIII.