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Interaction of LSPR with thrombin as an Inhibitor of Blood Clotting. Brandon T. Brown and Toni A. Trumbo Department of Chemistry and Biochemistry, Bloomsburg University of P ennsylvania, Bloomsburg PA 17815. Blood Clotting. Thrombin Catalytic Site. Vitamin K Antagonists. - PowerPoint PPT Presentation
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Brandon T. Brown and Toni A. TrumboDepartment of Chemistry and Biochemistry, Bloomsburg University of
Pennsylvania, Bloomsburg PA 17815
INTERACTION OF LSPR WITH THROMBIN AS AN INHIBITOR OF BLOOD CLOTTING
BLOOD CLOTTING
THROMBIN CATALYTIC SITE
VITAMIN K ANTAGONISTS
• Warfarin= most commonly used
• “Gold Standard”
• Oral available
• One dose daily
• Short comings
• Self-testing
• Risk of hemorrhage
• Interactions
DIRECT THROMBIN INHIBITORS
• Dabigatran
• Oral available
• Less testing
• Fewer life-threatening bleeds
• Two doses daily
• Risks
• Harder to reverse or completely irreversible
PEPTIDE TABLE
Peptide Sequences of InterestFibrinogen Aα (7-20) DFLAEGGGVRGPRV
Fibrinopeptide A ADSGEGDFLAEGGGVRGPRV
Fibrinogen Bβ (5-18) DNEEGFFSARGHRP
Fibrinopeptide B QGVNDNEEGFFSARGHRP
Factor V (699-713) “B1” NRLAAALGIRSFRN
Factor V (1008-1022) “B2” HTHHAPLSPRTFHP
Factor V (1535-1549) “B3” PDNIAAWYLRSNNG
Factor XIII AP (28-41) TVELQGVVPRGVNL
Thrombin Receptor PAR 1 (32-45) KATNATLDPRSFLL
LSPR• Inhibit thrombin directly
• Reversible
• Orally available
CLOT ASSAY
• 5 µM Fibrinogen was added to Assay buffer
• Equilibrated to Room Temperature for 5 minutes
• 2.2 nM Thrombin was added, the solution mixed
• The solution was inserted into the instrument which then measured the absorbance
• 7200 sec
• 325 nm
REPRESENTATIVE UNINHIBITED SPECTRUM
Hill EquationF=(a(x^b))/((c^b)+(x^b))
AVERAGE SPECTRUM AND ERROR BARS
5 µM LSPR
COMPARATIVE PLOT
COEFFICIENTSClot Max T 1/2 Binding coefficient
Uninhibited 2416493.1026 1.1194 609836547.8615
1 µM LSPR 15655.7115 5.1651 _______
2 µM LSPR 38863.6399 7.7950 _______
5 µM LSPR 98706.3974 17.4201 _______
CONCLUSION
• LSPR does inhibit clot formation
• Maximal inhibition found
FUTURE WORK
• HPLC
• Minimum dosage
ACKNOWLEDGEMENTS• Elizabeth Ola
• Jackie North
• Department of Chemistry and Biochemistry
REFERENCES