Activation Of NLRP3. Inflammasome Complex. Regulates The Onset Of Hypoxia. Induced Thrombosis. NEHA GUPTA, Manish Sharma, Tarun Tyagi, Anita Sahu,.
Activation Of NLRP3 Inflammasome Complex Regulates The Onset Of Hypoxia Induced Thrombosis NEHA GUPTA, Manish Sharma, Tarun Tyagi, Anita Sahu, Amit Prabhakar and Mohmmad Zahid Ashraf Defence Institute of Physiology and Allied Sciences, Defence Research and Development Organization (DRDO),Delhi, India Tathagata Chatterjee, Army Hospital (Research & Referral),Delhi, India Abstract 281
Financial Disclosures NONE for all the Authors.
Thrombosis: Hypoxia As A Risk Factor? • Higher incidence of thrombo-embolic disorders on ascension to high altitude (Deep Vein Thrombosis, Pulmonary Embolism, Mesenteric Vein Thrombosis, Cerebral Venous Thrombosis, Portal Vein Thrombosis , arterial thrombosis) Jha et al., High Alt Med Biol 2002;3:21—7; Kotwal et al., Thromb Res. 2007;120(3):391-7; Chohan et al., Def Sci J 1984; 34:361.
• Air travel – another prothrombotic risk (Bendz et al., Lancet
2000;356: 1657-58;
Lapostelle et al., N Engl J Med 2001; 345 (11): 779-783)
Experimental Groups
Hypoxia leads to hyper coagulation ? Role of platelets hyper reactivity is reported (Tyagi et al Blood;123(8):1250-60) BUT Mechanism is poorly understood ?
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1. Cumulative Influence of Systemic Hypoxia on Coagulation & Fibrinolysis A . In situ Inspection of IVC
B . Thrombus Weight
C. Survival Curve
Gross IVC/thrombus
Representative Heat Maps
D. Hyper coagulation: H & E Staining
E: Hemorrhagic lungs
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Systemic Hypoxia Accelerates Coagulation & Fibrinolysis F. Hyper coagulation
G. Hyper Fibrinolysis
Coagulation cascade
2. INVOLVEMENT OF STRONG INFLAMMATION IN HYPOXIA INDUCED THROMBOSIS - Activation Of NLRP3 Inflammasome 2.1 Transcriptome Analyses A. Venn Diagram
NLRP3= NOD (Nucleotide Oligomerization Domain) like receptor family, pyrin domain containing 3 protein
2.2. Cytokine Array Analyses A. Cytokine Array
B. Quantification
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3. NLRP3 Inflammasome in Hypoxia Induced thrombosis 3.1 Immunofluoroscence staining
3.2 ELISA
NLRP3 Inflammasome signaling
3.3 Time dependent mRNA expression of NLRP3 inflammasome component
4. Inflammasome Inhibition Curtails Stasis Induced Thrombosis (Causal role of Inflammation in coagulation ) 4.1 Caspase-1 inhibition (SML0499)
Strategies for Inflammasome inhibition in vivo
4.2 NLRP3 Knockdown (in vivo siRNA)
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4.3 IL-1b Neutralisation (Anti-IL-1b antibody)
Hypoxia Inhibition reduced Inflammasome activation in stasis induced thrombosis
Translational Implication
Conclusion :
Proinflammatory Phenotype Precedes Prothrombotic Phenotype Under Hypoxic Environment
Novelty & Significance: Role of NLRP3 in pathogenesis of Venous Thrombosis was in question (ATVB 2011;31(3):506-12). Present study first time reports the role of NLRP3 in venous thrombosis.
Schematic Illustration
Hypoxia induced Prothrombotic Phenotype is attributed to the platelet hyperreactivity (Blood 2014;20;123(8):125060). Present study reveals NLRP3 inflammasome complex likely to precede platelet aggregation, as an early response to hypoxia. Cross talk between inflammation & Coagulation is still eclipsed by lack of evidences that can distinguish possible cause or consequence relationship. Our experiments pertaining to causal role of early inflammasome in hypoxia induced coagulation is a foot forward in this regard. Inhibition of inflammasome curtailed stasis induced thrombosis demonstrating that venous stasis which is characterized by a hypoxic milieu, is associated with inflammasome activation. Issue of hypoxia induced inflammasome activation encompasses conflicting studies & opinions (NEJM 2011;364(20):1976-7). Present study provides evidences to address such enduring issues.
Acknowledgment •Director, Defence Institute of Physiology & Allied Sciences, Delhi. • This work was funded by Defence Research & Development Organization, Project SL-10/DIP-255
