Formation of platelet thrombi is dependent on the agonist-induced inside-out signaling to integrin ?IIb?3 that regulates soluble fibrinogen binding, and outside-in signaling through aIIbb3 that controls the platelet cytoskeletal rearrangement. Inside-out signaling is generated by several agonists that engage the G protein coupled receptors (GPCR). Intrinsic to inside-out and outside-in signaling, is the reversible tyrosine (Tyr and serine/threonine (Ser/Thr) phosphorylation-dependent assembly of multiple effectors. The phosphorylation and the activity of several effectors are regulated by protein kinases and phosphatases. While kinase mediated phosphorylation events during inside-out and outside-in signaling has been intensely investigated, the contribution of the catalytic subunits of Ser/Thr protein phosphatase 1 (PP1c) and protein phosphatase 2A (PP2Ac) is relatively unexplored. In the current funding period, we noticed decreased thrombin-induced inside- out signaling and delayed in vivo thrombus formation in mice lacking the catalytic subunit of protein phosphatase 1 ? (PP1c?). Outside-in signaling was unaffected by the loss of PP1c?, but increased in the absence of PP2Ac. Our overarching hypothesis is that the specific subtypes of Ser/Thr phosphatases orchestrate a spatial regulation of inside-out and outside-in signaling. Our goal is to decipher the molecular details underpinning the functional coupling of PP1c with the G protein signaling and PP2Ac with the integrin signaling, during physiological responses to injury.
Aim 1 will define the role of PP1c and its interacting protein G?1 during inside-out signaling. G?1, a component of the heterotrimeric G proteins that couple to GPCR, interacted with PP1c in resting platelets, while agonist treatment dissociated this complex. Depletion of Gb1 in murine megakaryocytes or blockade of G?? signaling in platelets, decreased thrombin receptor activating peptide induced fibrinogen binding and aggregation. Using platelets from human and mice deficient in PP1c?, PP1ca and G?1, our goal is to test if G?1 targets PP1c to the GPCR complex and positively regulates inside- out signaling.
Aim 2 will define the role of PP2Ac during aIIbb3 mediated outside-in signaling. Src activation is critical for outside-in signaling and we showed that PP2Ac depletion activates Src. CIN85 is an adaptor protein that associated with PP2Ac and outside-in signaling dissociated this complex in platelets. CIN85 depletion reduced Src activation and ?IIb?3 adhesiveness. Using platelets and aIIbb3 model cells, we will test if PP2Ac negatively regulates outside-in signaling via CIN85. The proposed research is innovative because it represents a departure from the kinase-mediated phosphorylation events to a phosphatase mediated dephosporylation events during platelet activation. The proposed research is significant because it will advance our understanding of the molecular mechanisms of platelet activation, and lay the basic groundwork for identifying phosphatase-interacting proteins as the new therapeutic targets for future anti-thrombotic therapy.

Public Health Relevance

Platelet activation events initiate heart attack and stroke. This proposal will examine new concepts of platelet activation, with the expectation that the studies will identify new targets for anti-thrombotic therapy.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
Project #
Application #
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Kindzelski, Andrei L
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Baylor College of Medicine
Internal Medicine/Medicine
Schools of Medicine
United States
Zip Code
Vijayan, K Vinod (2018) Myosin IIa signal von Willebrand factor release. Blood 131:592-593
Pradhan, Subhashree; Khatlani, Tanvir; Nairn, Angus C et al. (2017) The heterotrimeric G protein G?1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein-coupled receptor signaling in platelets. J Biol Chem 292:13133-13142
Dasgupta, Swapan K; Le, Anhquyen; Vijayan, K Vinod et al. (2017) Dasatinib inhibits actin fiber reorganization and promotes endothelial cell permeability through RhoA-ROCK pathway. Cancer Med 6:809-818
Yao, Hui; He, Guangchun; Chen, Chao et al. (2017) PAI1: a novel PP1-interacting protein that mediates human plasma's anti-apoptotic effect in endothelial cells. J Cell Mol Med 21:2068-2076
Da, Q; Shaw, T; Pradhan, S et al. (2017) Disruption of protein complexes containing protein phosphatase 2B and Munc18c reduces the secretion of von Willebrand factor from endothelial cells. J Thromb Haemost 15:1032-1039
Khatlani, Tanvir; Pradhan, Subhashree; Da, Qi et al. (2016) A Novel Interaction of the Catalytic Subunit of Protein Phosphatase 2A with the Adaptor Protein CIN85 Suppresses Phosphatase Activity and Facilitates Platelet Outside-in ?IIb?3 Integrin Signaling. J Biol Chem 291:17360-8
Lam, Fong W; Vijayan, K Vinod; Rumbaut, Rolando E (2015) Platelets and Their Interactions with Other Immune Cells. Compr Physiol 5:1265-80
Vijayan, K Vinod (2015) DGKE disruption ditches complement and drives p38 signaling. Blood 125:898-9
Wang, Rui; Stone, Rebecca L; Kaelber, Jason T et al. (2015) Electron cryotomography reveals ultrastructure alterations in platelets from patients with ovarian cancer. Proc Natl Acad Sci U S A 112:14266-71
Da, Qi; Behymer, Molly; Correa, Juliana I et al. (2014) Platelet adhesion involves a novel interaction between vimentin and von Willebrand factor under high shear stress. Blood 123:2715-21

Showing the most recent 10 out of 22 publications