Interaction of LSPR with thrombin as an Inhibitor of Blood Clotting

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