inhibitory effects of mns and bda-410 on human platelet aggregation greg czaplewski research...
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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, 2009MNS : 3,4-Methylenedioxy-β-nitrostyreneBDA-410 : (2S)-N-{(1)-l-[(S)-hydroxy(3-oxo-2-phenyl-1-cyclopropen-1-yl)methyl]-2 methylpropyl}-2-benzenesulfony-lamino-4-methylpentanamide
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
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
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
http://www.integrilin.com/popups/platelet2.html
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
MNS and BDA-410 inhibitors• MNS:
– 3,4-Methylenedioxy-β-nitrostyrene (C10H9NO4, 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 (C26H32N2O5S, 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.
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)
Thrombin 0.15 U/ml
5 10 15-100
-80
-60
-40
-20
0Vehicle (1% DMSO)MNS 10uMBDA-410 100uMBDA 100uM, MNS 10uM
Time (min)
% A
gg
reg
atio
n
Inhibition of Aggregation using Thrombin as the Agonist
Thrombin 0.15 U/ml
0
20
40
60
80
100
1 min 2 min 3 min 4 min 5 min
% A
ggre
gatio
n
Vehicle (<1% DMSO)
MNS 10uM
BDA 100uM
MNS 10uM + BDA 100uM
Repeated measures ANOVA test: p=0.0791 (n=3)
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)
TRAP-4 200 uM
5 10 15-100
-80
-60
-40
-20
0Vehicle (1% DMSO)MNS 10uMBDA-410 100uMBDA 100uM, MNS 10uM
Time (min)
% A
gg
reg
atio
n
Inhibition of aggregation using TRAP-4 as the agonist
TRAP-4 100-200 µM
0
20
40
60
80
100
1 min 2 min 3 min 4 min 5 min
% A
ggre
gatio
n
Vehicle (<1% DMSO)
MNS 10uM
BDA 100uM
MNS 10uM + BDA 100uM
Repeated measures ANOVA test: p=0.145 (n=4)
Future Direction
• Use other agonists and more trials to continue to establish the relationship between the two systems. E.g. collagen, ADP, U46619.
-Parallel- -Series- -Independent-
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
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?