Calpains are cysteine proteases implicated in a wide variety of cellular processes such as cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. We propose to test the hypothesis that calpain-1 regulates platelet signaling by modulating the activity of PTP1B under the following specific aims: (1) Regulation of PTP1B by calpain-1. We will determine the status of PTP1B cleavage in the wild type and calpain-1 null mouse platelets upon activation using domain-specific antibodies and immune complex phosphatase assays. We will measure the degradation of PTP1B and specific activity of degraded fragments and investigate the effect of PTP1B on c-Src activation in platelets. These studies will help to clarify the role of calpain-1 in the regulation of PTP1B and provide an explanation for the increased tyrosine phosphatase activity in calpain-I null mouse platelets. (2) Mechanism of calpain-1 dependent beta-3 integrin interactions with the platelet cytoskeleton. We plan to determine the identity of PTP1B substrates in platelets by gel electrophoresis and mass spectrometry. Using beta-3 integrin cytoplasmic domain constructs carrying conservative and radical replacement of tyrosines at 747 and 759, we will determine their phosphorylation stoichiometry, susceptibility to dephosphorylation by PTP1B, and quantify their interactions with Shc, myosin, and newly identified PTP1B substrates. These experiments, assisted by the BIAcore technology, will help to clarify the downstream effectors of PTP1B that are critical for outside-in signaling in platelets. (3) Role of calpain-1 signaling in platelet spreading. Using calpain-1 null mice with pure C57BL/6J genetic background, we will investigate the mechanism of enhanced platelet spreading by identifying the potential PTP1B-independent substrates of calpain-1 in mouse platelets. We will also test whether the calpain-1 null mice with the pure C57BL/6J genetic background display abnormalities in the platelet secretion and in vivo thrombosis status. Together, these studies will help to clarify the role of calpain-1 in integrating common and divergent aspects of platelet signaling, reveal novel aspects of PTP1B regulation, and provide a better understanding of the mechanism of platelet aggregation and clot retraction pathways. Ultimately, these studies may lead to the development of new therapeutic strategies against thrombosis.

Public Health Relevance

Calpains are cysteine proteases implicated in a wide variety of cellular processes such as cell proliferation, apoptosis, cell motility, and hemostasis. The proposed studies will clarify the role of calpain-1 in platelet signaling and provide a better understanding of the mechanism of platelet aggregation and clot retraction pathways. Ultimately, these studies may lead to the development of new therapeutic strategies against thrombosis.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL089517-04
Application #
8204714
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Sarkar, Rita
Project Start
2009-01-21
Project End
2014-12-31
Budget Start
2012-01-01
Budget End
2014-12-31
Support Year
4
Fiscal Year
2012
Total Cost
$412,500
Indirect Cost
$162,500
Name
Tufts University
Department
Physiology
Type
Schools of Medicine
DUNS #
039318308
City
Boston
State
MA
Country
United States
Zip Code
02111
Howatt, Deborah A; Balakrishnan, Anju; Moorleghen, Jessica J et al. (2016) Leukocyte Calpain Deficiency Reduces Angiotensin II-Induced Inflammation and Atherosclerosis But Not Abdominal Aortic Aneurysms in Mice. Arterioscler Thromb Vasc Biol 36:835-45
Nwankwo, Jennifer O; Lei, Jianxun; Xu, Jian et al. (2016) Genetic inactivation of calpain-1 attenuates pain sensitivity in a humanized mouse model of sickle cell disease. Haematologica 101:e397-e400
Srinivasan, Subhashini; Schiemer, James; Zhang, Xiaowei et al. (2015) Gα13 Switch Region 2 Binds to the Talin Head Domain and Activates αIIbβ3 Integrin in Human Platelets. J Biol Chem 290:25129-39
Wu, Zhengli; Chen, Xiaochun; Liu, Fang et al. (2014) Calpain-1 contributes to IgE-mediated mast cell activation. J Immunol 192:5130-9
De Franceschi, Lucia; Franco, Robert S; Bertoldi, Mariarita et al. (2013) Pharmacological inhibition of calpain-1 prevents red cell dehydration and reduces Gardos channel activity in a mouse model of sickle cell disease. FASEB J 27:750-9
Kemp, C M; Oliver, W T; Wheeler, T L et al. (2013) The effects of Capn1 gene inactivation on skeletal muscle growth, development, and atrophy, and the compensatory role of other proteolytic systems. J Anim Sci 91:3155-67
Subramanian, Venkateswaran; Moorleghen, Jessica J; Balakrishnan, Anju et al. (2013) Calpain-2 compensation promotes angiotensin II-induced ascending and abdominal aortic aneurysms in calpain-1 deficient mice. PLoS One 8:e72214
Li, Xuerong; Marinkovic, Marina; Russo, Crystal et al. (2012) Identification of a specific region of Plasmodium falciparum EBL-1 that binds to host receptor glycophorin B and inhibits merozoite invasion in human red blood cells. Mol Biochem Parasitol 183:23-31
Wieschhaus, Adam; Khan, Anwar; Zaidi, Asma et al. (2012) Calpain-1 knockout reveals broad effects on erythrocyte deformability and physiology. Biochem J 448:141-52
Wieschhaus, Adam J; Le Breton, Guy C; Chishti, Athar H (2012) Headpiece domain of dematin regulates calcium mobilization and signaling in platelets. J Biol Chem 287:41218-31

Showing the most recent 10 out of 13 publications