Date on Master's Thesis/Doctoral Dissertation

5-2018

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Chemistry

Degree Program

Chemistry, PhD

Committee Chair

Maurer, Muriel

Committee Co-Chair (if applicable)

Wittebort, Richard

Committee Member

Wittebort, Richard

Committee Member

Buchanan, Robert

Committee Member

D'Souza, Stanley

Author's Keywords

blood coagulation; prothrombin; thrombin; exosites; protease activated receptors; NMR

Abstract

The serine protease thrombin plays important roles in coagulation, anticoagulation, and platelet activation. Thrombin is initially expressed as the inactive zymogen prothrombin (ProT). The final cleaved and activated form of thrombin has mature anion binding exosites (ABEs) and several regulatory loops. Engagement with these exosites helps define the fate of thrombin as a procoagulant or an anticoagulant. Researchers previously reported that zymogen ProT may already bind exosite ligands. Little is known about conformational changes associated with ProT maturation, resultant ligand binding affinities, individual ligand-protein contacts, and long-range communication between thrombin exosites. Protease Activated Receptors (PARs) play critical roles in controlling platelet activation. Using solution NMR methods, we demonstrated that PAR3 (44-56) and weaker binding version PAR3G (44-56, P51G) can already bind to immature pro-ABE I. 1D and 2D 1H-15N HSQC titrations revealed that PAR3G 15N-E48 and 15N-D54 both entered into higher affinity, intermediate exchange regimes as ProT was converted into thrombin. The high affinity of PAR3G D54 suggests that the thrombin R77a region is better oriented for binding than thrombin R75. PAR3G 15N-F47 and 15N-L52 experienced significant changes in chemical shift and thus chemical environment upon ABE I maturation. However, the ProT 30s loop made better contacts with PAR3 than the ProT hydrophobic cluster (F34, L65, and I82). The project was extended to PAR1 (49-62). Both PAR1P and weaker version PAR1G (P54G) bound to ProT and proton NMR line broadening increased with thrombin. 1D and 2D 1H-15N HSQC titrations revealed that unlike PAR3G (44-56), PAR1G (49-62) 15N- K51, E53, F55, D58, and E60 exhibited little interactions with ProT. Affinities increased with mature thrombin ABE I. NMR titrations could probe PAR1 (58DEEKN62), a region previously unresolved by X-ray crystallography. Interestingly, the ABE II ligand GpIbα (269-282, 3YP) influenced the NMR binding affinities of PAR1G and PAR3G supporting long-range communication between the ABE II and ABE I exosites. Finally, our studies shifted toward the thrombin active site region. Kinetic assays and 2D tr-NOESY studies provided clues on why Fibrinogen Bβ (5-16) is such a weak thrombin substrate. The Fibrinogen Bβ 10FFSAR14 sequence contributes towards hindering binding (Km) and product turnover (kcat).

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