Free Radical Initiators in Platelets: A Comparison of Hydrogen Peroxide and 2,2’- Azobis (2-amidinopropane) Dihydrochloride.
Poster No.
K. Manasa and R. Vani Jain University Department of Biotechnology, Centre for Post Graduate Studies, Jain University, #18/3, 9th Main, 3rd Block Jayanagar, Bangalore 560011 E-mail:
[email protected] INTRODUCTION
RESULTS & DISCUSSION •Redox changes occur as a
function
of
PROTEIN SULFHYDRYLS
AGGREGATION
normal
platelet activation, but the introduction of additional oxidative stress in certain situations
may
be
prothrombotic. • ROS derived from both platelets
and
other
vascular
sources
alter
platelet
SUPEROXIDE DISMUTASE
TBARS
CONJUGATE DIENES
responses
(Freedman, • H2O2 is a strong physiological oxidant and
SUPEROXIDE GENERATION
2008;
Alexandru et al., 2010) .
induces OS in platelets (Halliwell and Gutteridge,
2000). • It easily penetrates cell membranes, transforms into more active forms and can independently cause or exacerbate platelet aggregation. • H2O2 at a concentration of 200 μM enhances and at 2-20 mM reduces platelet aggregation. • High
concentrations
physiological
levels,
of
H2O2,
seriously
far
from
damage
the
plasma membrane of platelets and associated receptors (Vavaev et al., 2012). • AAPH decomposes to give free carbon-centred radicals, which react with PLATELET MORPHOLOGY
oxygen quite rapidly to yield peroxyl radicals. • AAPH is used to generate free radicals at a controlled and constant rate for specific durations and at specific sites, to study the dynamics of oxidation (Niki, 1990; Yoshida et al., 2004).
R-N=N-R
.
R-N2 R
Cage Products .
N2 + 2R
ROO ̅
HYPOTHESIS & AIM OF THE STUDY Hypothesis: • There may be differential response to these free radical initiators by platelets. Objectives: • To assess the levels of oxidative stress in platelets treated with the free radical initiators - H2O2 and AAPH. • To compare the OS induced by H2O2 and AAPH on platelets.
MATERIALS & METHODS • Blood Sampling (Vani et al., 2012) • Platelet Isolation (Carneiro and Blakely, 2006) • Platelet Morphology (Houwen, 2000; Khachonsaksumet and Riganti, 2002) • Platelet Aggregation (Born and Cross, 1963) • Superoxide Generation (Olas and Wachowicz, 2002) Lipid Peroxidation • Thiobarbituric acid Reactive Substances (TBARS) (Olas et al., 2006) • Conjugate Dienes (Olas and Wachowicz, 2002) Protein Oxidation • Protein Carbonyls (Reznick and Packer, 1994) • Protein Sulfhydryls (Habeeb, 1972) Antioxidant Enzymes • Superoxide Dismutase (SOD) (Misra and Fridovich, 1972) • Catalase (CAT) (Aebi et al., 1984)
A: H2O2 Treated Platelets (2.0 mM, 30 min). B: AAPH Treated Platelets (2.0 mM, 30 min). Circles indicate platelet aggregates. • Aggregation increased in 2.0 mM AAPH but decreased in 2.0 mM H2O2, as H2O2 disrupts the platelet membrane at concentrations > 2.0 mM. • Superoxide generation increased in H2O2 platelets when compared with control, as superoxide overwhelmed platelet SOD; but was insignificant in AAPH platelets. • SOD of H2O2 and AAPH platelets increased in accordance with the concentration of superoxide produced. • TBARS increased in H2O2 platelets in comparison to control due to increased hydroxyl radical production. • AAPH platelets demonstrated increased TBARS at 0.5 mM; but decreased at 1.0 mM and 2.0 mM. • Conjugate dienes increased in H2O2 platelets with respect to control. • AAPH treated platelets demonstrated concentration dependent increase in conjugate dienes. • Decrement in the sulfhydryls of AAPH platelets indicates that the peroxyl radicals have oxidised the protein sulfhydryls to disulfides.
CONCLUSIONS •Both free radical initiators impaired platelets. •H2O2 acted on lipids but did not affect proteins significantly. •AAPH acted on both lipids and proteins (increased conjugate dienes and decreased protein sulfhydryls). •Platelets were more susceptible to AAPH than H2O2. •This study demonstrates the approximate amount of oxidative insult induced through H2O2 and AAPH. •It also gives insight into vulnerability of platelets to different concentrations of these free radical initiators.
REFERENCES 1. 2. 3.
ACKNOWLEDGEMENTS We would like to acknowledge Prof. Leela Iyengar, Ms. Soumya Ravikumar, Mr. Carl Hsieh and Jain University for their support.
4. 5. 6.
Vani R, Koshy AA, Koushik AK, Kaur H, Kumari K, Agrawal M, Priyanka, Ramya, Khathai S, Gowda V and Kumar V. 2012. ‘The efficacy of erythrocytes isolated from blood stored under blood bank conditions’, Transfusion and Apheresis Science. 47: 359-64. Born GVR, Cross MJ. 1963. ‘The aggregation of blood platelets’, Journal of Physiology. 168: 178-195. Reznick AZ and Packer L. 1994. ‘Oxidative damage to proteins: Spectophotometric method of carbonyl assay’, Methods in Enzymology. 233: 357-61. Habeeb AFSA. 1972. ‘Reaction of Protein Sulfhydryl Groups with Ellman’s Reagent’, Methods in Enzymology. 34: 457-64. Misra HP and Fridovich I. 1972. ‘The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase’, Journal of Biological Chemistry. 247:10: 3170-5. Aebi H. 1984. ‘Catalase in vitro’, Methods in Enzymology. Packer L (ed), Acad Press; 105: 121-6.
