1. Inhibitory Effects of MNS and BDA-410 on Human Platelet Aggregation Greg Czaplewski Research Advisor: Dr. Athar Chishti Graduate Assistant: Adam Wieschhaus Department of Pharmacology July 30, 2009 MNS : 3,4-Methylenedioxy-β-nitrostyrene BDA-410 : (2S)-N-{(1)-l-[(S)-hydroxy(3-oxo-2-phenyl-1-cyclopropen-1-yl)methyl]-2 methylpropyl}-2-benzenesulfony-lamino-4-methylpentanamide

2. Motivation Acute myocardial infarction and ischemic stroke are the first and third leading causes of death in the U.S.* Current treatments (Plavix, Aspirin) have harmful side effects: –Minor/Major bleeding (3.7-5.1%)** –Gastrointestinal Hemorrhage (2.0-2.7%)** Interaction between Syk/Src tyrosine kinase and cysteine protease systems unknown *Heron MP, Hoyert DL, Murphy SL, Xu J, Kochanek KD, Tejada-Vera B. Deaths: Final Data for 2006. National vital statistics reports; 2009 Apr; Vol 57 No 14. Hyattsville, MD: National Center for Health Statistics. **http://products.sanofi-aventis.us/plavix/plavix.html

3. Objective Develop effective protocol for platelet isolation Establish aggregation curves using Thrombin and TRAP-4 agonists Establish inhibition curves using MNS, BDA-410, and combination Analyze curves to obtain relationship between the two systems

4. Coagulation Cascade Chain of Events Tear in endothelial cell lining –Exposes collagen and other factors that activate platelets –Platelet-platelet interactions result in aggregation, forming a plug –Plug adheres to damaged area Problem when thrombus blocks >50% of blood flow –Interruption in blood flow causes infarction –Embolus occludes another blood vessel

5. http://www.integrilin.com/popups/platelet2.html

6. First Step: Isolation Protocol Gel-Filtered Platelet Isolation Platelet-rich-plasma (PRP) acquired from human donors Filter paper and Sepharose 2B gel layered in column Small size of platelets causes fast descent through gel Low volume (<3-4 ml) platelet collection ideal to avoid presence of fibrinogen

7. MNS and BDA-410 inhibitors MNS: –3,4-Methylenedioxy-β-nitrostyrene (C 10 H 9 NO 4, MW 207.05 Da) –Inhibits Src and Syk family tyrosine kinases –Was shown in 2007 to inhibit thrombin- or collagen-induced human platelet aggregation, ATP secretion, GPIIb/IIIa activation and protein tyrosine phosphorylation. [1] BDA-410 –(2S)-N-{(1)-l-[(S)-hydroxy(3-oxo-2-phenyl-1-cyclopropen-1-yl)methyl]-2 methylpropyl}-2-benzenesulfony-lamino-4-methylpentanamide (C 26 H 32 N 2 O 5 S, MW 484.61 Da) –inhibits cysteine proteases (primarily calpain I and II) but has not been tested on platelet aggregation. –Was shown in 2007 to inhibit P. falciparum cysteine proteases in blocking Malaria parasite growth. [2] [1] Wei-Ya Wang, Pei-Wen Hsieh, Yang-Chang Wu, Chin-Chung Wu. Biochemical Pharmacology 74: 601– 611, 2007. [2] Xuerong Li, Huiqing Chen, Jong-Jin Jeong, Athar H. Chishti. Molecular & Biochemical Parasitology 155:26–32, 2007.

8. Inhibition of Aggregation using Thrombin as the Agonist Thrombin, the most potent platelet agonist, is a serine protease that catalyzes reactions in the coagulation cascade. Has three known receptors on the surface of platelets. (PAR 1, 3, 4)

9. Inhibition of Aggregation using Thrombin as the Agonist Repeated measures ANOVA test: p=0.0791 (n=3)

10. Inhibition of aggregation using TRAP-4 as the agonist Thrombin receptor-activating peptides (TRAPs) are synthetic peptides –Have been shown to mimic the effects of Thrombin –Target specific Thrombin receptors (In this case, PAR-4)

11. Inhibition of aggregation using TRAP-4 as the agonist Repeated measures ANOVA test: p=0.145 (n=4)

12. Future Direction Use other agonists and more trials to continue to establish the relationship between the two systems. E.g. collagen, ADP, U46619. Platelet Excitation Cysteine Protease System Tyrosine Kinase System Platelet Excitation Cysteine Protease System Tyrosine Kinase System Platelet Excitation Tyrosine Kinase System Cysteine Protease System -Parallel--Series--Independent-

13. Summary Need to reduce platelet aggregation to prevent acute myocardial infarction and ischemic stroke Developed platelet isolation protocol Obtained inhibition curves for MNS, BDA-410, and combination Results suggest combination therapy is a potential modality to reduce platelet aggregation and thrombosis in vivo

14. Acknowledgements We are appreciative and thankful for the financial support of this project by the National Science Foundation and the Department of Defense Grant EEC-NSF # 0755115. Questions?