Platelets are small anucleate cells in blood that are derived from megakaryocytes and their primary function is to prevent bleeding. Upon vascular injury, the sub-endothelial collagen gets exposed to which platelets bind and aggregate eventually forming a platelet plug. There are several receptors on platelet surface that can be divided into two broad categories; the immune-receptor tyrosine-based activation motif (ITAM) and the G protein-coupled receptors (GPCRs). The role of several protein tyrosine kinases (PTKs) downstream of ITAM and GPCRs has been extensively studied. However, the role of protein tyrosine phosphatases (PTPs) have been under-investigated in platelets. PTPs are important for dephosphorylating and activating or inactivating the protein. Proteomics studies show presence of 10 receptor like and 10 cytoplasmic phosphatases in platelets. To date, only five non-transmembrane PTPs (NTPTPs), PTP-1B, Shp1, Shp2, LMW-PTP, MEG2-PTP and a one receptor- like PTP (RPTP), CD148, / Biomedical Sciences

Platelets are anucleate cells that are crucial mediators of hemostasis and thrombosis. Under physiological conditions, platelets are maintained in a quiescent state within the vasculature. Upon vascular injury, an essential receptor that initiates platelet activation upon interaction with sub-endothelial collagen is Glycoprotein VI (GPVI). The activation of platelets leads to platelet shape change, granular secretion, thromboxane A2 (TXA2) synthesis, and integrin IIb3-mediated platelet aggregation and thrombus formation. In the past, a lot of effort has been placed in understanding GPVI and its signaling in platelets, however, much is still unknown. Therefore, the focus of this thesis is to identify novel regulators of GPVI-mediated platelet signaling. Phosphoinositide 3-kinase (PI3K) is an important signaling molecule that is activated downstream of various receptors including GPVI upon platelet activation. PI3K activation leads to the generation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) subsequently leading to the recruitment of pleckstrin homology (PH) domain-containing proteins to the plasma membrane. We performed a proteomic screen to identify proteins that interacted with PIP3 using PIP3 beads, and among these proteins, we found engulfment and cell motility-1 (ELMO1). ELMO1 is a scaffold protein with no catalytic activity that regulates the actin cytoskeleton during cell motility and cell spreading in nucleated cells. ELMO1 is expressed in platelets and interacts with active RhoG. However, the function of ELMO1 is not known. Therefore, we utilized ELMO1-/- mice to investigate the role of ELMO1 in platelets. Aggregation, granular secretion, and thromboxane generation was enhanced in ELMO1-/- platelets in response to glycoprotein VI (GPVI) agonists, collagen-related peptide (CRP) and collagen, but unaltered using protease-activated receptor 4 (PAR4) agonist (AYPGKF). This suggests that ELMO1 plays a specific role downstream of GPVI despite normal surface expression level of GPVI. Furthermore, whole blood from ELMO1-/- mice, perfused over collagen, under arterial shear conditions, exhibited enhanced thrombus formation compared to blood from WT littermate controls. In an in vivo pulmonary thromboembolism model, ELMO1-/- mice showed reduced survival compared to WT littermate control. ELMO1-/- mice also showed shorter time to occlusion using the ferric-chloride injury model and reduced tail bleeding times compared to WT littermate control. This indicates that ELMO1 plays an essential role in hemostasis and thrombosis in vivo. At the molecular level, RhoG activity was enhanced in ELMO1-/- murine platelets compared to the WT littermate control in response to CRP. Together, these data suggest that ELMO1 negatively regulates GPVI-mediated thrombus formation via RhoG. Protein kinase C delta (PKC) is a serine/threonine kinase that positively and negatively regulate dense granule secretion downstream of PAR and GPVI receptors, respectively. However, the mechanism of such differential regulation is not known. We hypothesize that this differential regulation occurs via the phosphorylation of specific tyrosine sites on PKC downstream of GPVI and PARs. We observed that many of the tyrosine residues in PKC were phosphorylated in response to both GPVI and PAR activation. Interestingly, PKCY155 phosphorylation only occurred following GPVI stimulation. Hence, we generated PKCY155F KI mice to characterize the function of PKCY155 phosphorylation in platelets. Aggregation and dense granule secretion were unaffected in PKCY155F platelets upon stimulation with a PAR agonist. However, these platelet functional responses were decreased upon stimulation of PKCY155F platelets with GPVI agonists, compared to WT littermates, despite normal surface GPVI expression. Whole blood from PKCY155F mice perfused over collagen under arterial shear conditions showed decreased thrombus formation. Similarly, we observed that PKCY155F mice survive longer than controls using a pulmonary thromboembolism model. PKCY155F mice also exhibited longer time to occlusion using the ferric-chloride injury model. At the molecular level, Syk and PLC2 phosphorylation was decreased in the PKCY155F platelets following GPVI stimulation. In conclusion, PKCY155 phosphorylation positively regulates GPVI-mediated platelet activation. Together, the studies proposed in this thesis provide insights into regulation of GPVI-mediated platelet function by ELMO1 and PKCY155. ELMO1 negatively regulates GPVI-mediated platelet activation via RhoG and may provide a suitable target for antihemorrhagic therapy. While PKCY155, being a positive regulator of GPVI-mediated platelet activation, could be a potential drug target for anti-thrombotic therapy. / Biomedical Sciences

Historical emphasis on the left ventricle (LV) has left clinicians with a lack of efficacious right ventricle (RV)-specific therapies, and classical pharmacological interventions for LV failure are often not effective when re-appropriated as interventions for RV failure. Different and still largely unknown molecular mechanisms governing the two ventricles, perhaps related to the distinct embryological origins, might at least in part explain the limited understanding of RV pathophysiology. microRNAs (miRs) are major post-transcriptional regulators and their altered expression has been implicated in several cardiovascular pathologies. We hypothesized that altered miR expression specific to the failing RV may underlie the molecular pathophysiology of progressive mechanical RV dysfunction. We applied an “-omics” approach to a pre-clinical, large animal model of heart failure. Ten dogs were subjected to 4-week tachypacing to induce congestive heart failure (HF) and secondary pulmonary hypertension. Ten non-paced dogs were used as normal controls. Hemodynamic and echocardiographic assessment confirmed development of RV dysfunction and secondary pulmonary hypertension in tachypaced dogs. In HF vs control, RV end-diastolic pressure and mean pulmonary arterial pressure were significantly increased, while tricuspid annular plane systolic excursion, tricuspid annular systolic velocity, and RV fractional area change were significantly decreased. miR microarray and quantitative RT-PCR analyses both showed upregulation of several miRs in HF-RV vs HF-LV and control myocardium. We focused on miR-21, which increased in HF-RV vs control RV (with no change in HF-LV vs control LV) and is known to target phosphatase and tensin homolog (PTEN), a negative regulator of fibroblast proliferation. PTEN also inhibits the phosphorylation/activation of Akt, a positive regulator of fibroblast proliferation. Myocardial PTEN was indeed selectively downregulated in HF-RV and not in HF-LV, consistent with increased fibrosis in HF-RV vs HF-LV. Moreover, Akt phosphorylation was increased by in RV-HF and reduced in HF-LV vs control. Isolated fibroblasts and myocytes from each ventricle were subjected to cyclic stretch and/or aldosterone treatment to mimic mechanical and hormonal stimuli occurring during HF. In RV fibroblasts, miR-21 was increased by both cyclic stretch and by aldosterone, with no significant miR-21 increase in any other cell type. Furthermore, in stretched/treated RV fibroblasts there was a significant downregulation of miR-21 targets PTEN and sprouty homolog 1, as well as a significant upregulation of pro-fibrotic mediator transforming growth factor-β1. These findings suggest that miR-21 upregulation due to RV fibroblast responsiveness to mechanical and hormonal stimuli is a novel determinant of RV fibrosis and functional impairment. This study uncovered a novel, major biological difference between the RV and LV, specifically a peculiar molecular response of RV fibroblasts to mechanical and hormonal stress. It is my hope that my findings will contribute to what will eventually lead to a clear, clinically relevant understanding of the mechanisms underlying RV failure. / Physiology

Ischemic stroke is the third leading cause of death and the leading cause of morbidity in the United States. Cognitive deficits, specifically with respect to learning and memory, are a significant contributor to morbidity in stroke patients. Unfortunately, current treatment options must be administered within a thin therapeutic window of the initial infarct. This requirement results in less than 10% of stroke patients being eligible for treatment. There are currently no treatment options that are effective in the subacute phase of the disease and no treatments that are effective in reversing postischemic learning and memory deficits. We sought to examine the potential efficacy of the anti-inflammatory Cannabinoid-2 Receptor Agonist, O-1966, in attenuating infarct expansion and reversing cognitive deficits in the subacute phase of the disease using a photothrombosis model of stroke. Additionally, we sought to characterize the inflammatory response in photothrombosis. Mice were treated with repeated doses of O-1966 or vehicle and were sacrificed at 24 hours and 7 days to study the acute and subacute phase of the disease respectively. Learning and memory testing, immunohistochemistry, and polymerase chain reaction were used to measure the effect of O-1966 on infarct expansion, inflammatory gene expression, and cognitive function. In addition to PCR, flow cytometry was used to characterize the temporal dynamics of inflammation following photothrombosis. Our studies show that O-1966 is effective in the subacute phase in attenuating infarct expansion and proinflammatory gene expression and reversing learning and memory deficits. / Pharmacology

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