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The metalloprotease, NN-PF3 from Naja naja venom inhibits platelet aggregation primarily by affecting α2β1 integrin

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Abstract

NN-PF3 is a non-toxic, anticoagulant, high-molecular-mass (67.81 kDa) metalloprotease from Indian cobra (Naja naja) venom. In the present study, NN-PF3 was investigated for the mechanism of inhibition of collagen-induced aggregation of human platelets. The complete inhibition of collagen-induced aggregation and partial inhibition of ADP- and epinephrine-induced aggregation has the respective IC50 of 75 ± 5, 185 ± 10, and 232 ± 12 nM, whereas no inhibition of thrombin-, arachidonic acid-, and ristocetin-induced aggregation of platelets was observed in platelet-rich plasma. Further, native NN-PF3 and EDTA-inactivated NN-PF3 inhibited collagen-induced aggregation of washed platelets with respective IC50 of 75 ± 4 and 180 ± 6 nM. The higher inhibitory effect of native NN-PF3 compared with EDTA-inactivated NN-PF3 suggests the enzymatic and non-enzymatic mechanism of inhibition. NN-PF3 pretreatment affected the collagen binding but not the fibrinogen, and fibronectin binding of washed platelets in adhesion assay suggested that the collagen receptors are affected. Western blot study using anti-integrin α2β1 mAb 6F1 suggested that NN-PF3 binds to integrin α2β1 in a primary structure-dependent manner only and is not cleaved. There was a drastic reduction in the intensity of several intracellular signaling phosphotyrosine protein bands when monoclonal anti-phosphotyrosine antibody was used, suggesting that the major activation pathway of platelets get affected, which occurs through glycoprotein VI. NN-PF3 did not bind to collagen as revealed by Western blot using anti-collagen mAb. Furthermore, neither the proteolytic cleavage of fibrinogen nor its degradation products by NN-PF3 contributed for the collagen-induced platelet aggregation inhibition.

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Acknowledgement

The authors thank the Department of Science and Technology, New Delhi, India (SR/SO/BB-56/2004 dated 01-06-2006) for financial assistance. We also thank Prof. Barry S. Coller, Professor and Physician-in-Chief, Rockefeller University, New York, USA for generously providing anti-integrin α2β1 mAb 6F1.

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Authors and Affiliations

  1. Department of Biochemistry, Government College for Women, Mandya, 571401, Karnataka, India

    M. S. Kumar

  2. Department of Studies in Biochemistry, University of Mysore, Manasagangotri, Mysore, 560006, Karnataka, India

    K. S. Girish, B. S. Vishwanath & K. Kemparaju

  3. Department of Studies in Biochemistry, University of Mysore, Manasagangotri, Mysore, 570 006, India

    K. Kemparaju

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  1. M. S. Kumar
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  2. K. S. Girish
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  4. K. Kemparaju
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Correspondence to K. Kemparaju.

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Kumar, M.S., Girish, K.S., Vishwanath, B.S. et al. The metalloprotease, NN-PF3 from Naja naja venom inhibits platelet aggregation primarily by affecting α2β1 integrin. Ann Hematol 90, 569–577 (2011). https://doi.org/10.1007/s00277-010-1103-1

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  • DOI: https://doi.org/10.1007/s00277-010-1103-1

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