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?