Novel Regulators of Gpvi-Mediated Platelet Activation

NOVEL REGULATORS OF GPVI-MEDIATED PLATELET ACTIVATION.

A Dissertation Submitted to the Temple University Graduate Board

In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY

By Akruti Patel December 2017

Dissertation Examining Committee:

Dr. Satya Kunapuli, Advisory Chair, Physiology Dr. Rosario Scalia, Physiology Dr. Laurie Kilpatrick, Physiology Dr. Abdelkarim Sabri, Physiology Dr. Ulhas Naik, Physiology, External Member, Thomas Jefferson University ABSTRACT

NOVEL REGULATORS OF GPVI-MEDIATED PLATELET ACTIVATION

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

ii 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

iii

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.

DEDICATION This dissertation is dedicated to my family and friends.

ACKNOWLEDGEMENTS

I would like to thank my mentor Dr. Satya Kunapuli for providing me the opportunity to gain both theoretical and practical knowledge about the field of thrombosis and hemostasis. He fostered an intellectual environment that helped me grow as a scientist and create a framework to which I will build upon in my career. I truly appreciate the time, knowledge, and effort that he contributed to ensuring the success of my thesis.

I would also like to thank Dr. Rosario Scalia, Dr. Abdelkarim Sabri and Dr. Laurie

Kilpatrick for accepting to be my thesis review committee members. I greatly appreciate the time, effort, and the constructive criticism during the committee meetings. I would also like to thank Dr. Ulhas Naik for accepting to be my external committee member and providing insights into my projects at various conferences.

I am deeply indebted to Dr. John Kostyak and Carol Dangelmaier who taught me the practical skills necessary to be efficient in the laboratory. I would like to thank Dr. John

Kostyak for his constant support, encouragement, and constructively mediating differences of opinions when necessary. I would also like to thank Carol Dangelmaier for her continuous support and help in the laboratory. She not only taught me new techniques as well as enhance them.

I would also like to thank my colleagues Dr. Mario Rico, Dr. Elisabetta Liverani, Dr.

Soochong Kim, Monica Wright, Dr. Rachit Badolia, Vaishali Inamdar, and Jeremy

Wurtzel for their moral support especially when my experiments were not going as predicted.

Finally, I would like to thank my parents, siblings, and friends who were during this

vi endeavor a constant motivation and support.

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TABLE OF CONTENTS ABSTRACT ...... II DEDICATION ...... V ACKNOWLEDGEMENTS ...... VI LIST OF TABLES ...... X LIST OF FIGURES ...... XI

CHAPTER 1 ...... 1 1 INTRODUCTION ...... 1 1.1 Overview of Platelets...... 1 1.2 Platelet production...... 1 1.3 Anatomy of Platelets...... 2 1.4 Platelet function ...... 6 1.5 Platelet receptors ...... 11 1.6 Immunoglobulin superfamily-GPVI...... 17 1.7 Potential regulators of GPVI signaling...... 27

CHAPTER 2 ...... 29

2 MATERIALS AND METHODS ...... 29

CHAPTER 3 ...... 38

3 ELMO1 NEGATIVELY REGULATES GLYCOPROTEIN VI-MEDIATED SIGNALING BY RHOG IN PLATELETS ...... 38 3.1 Introduction ...... 38 3.2 Results ...... 40 3.3 Discussion ...... 61

CHAPTER 4 ...... 67

4 PROTEIN KINASE CY155F KNOCK-IN MICE REVEAL POSITIVE REGULATORY ROLE OF Y155 IN GPVI-MEDIATED PLATELET ACTIVATION . 67 4.1 Introduction ...... 67 4.2 Results ...... 69 4.3 Discussion ...... 85

CHAPTER 5 ...... 87

5 GENERAL DISCUSSION/FUTURE DIRECTIONS ...... 87

viii

REFERENCES ...... 95 APPENDIX ...... 111

LIST OF TABLES

Table 1: Summary of PH domain containing proteins from proteomics data...... 41

Table 2: Hematologic analysis of WT and ELMO1-/- mice...... 56

Table 3: Hematologic parameters in WT and PKCY155F mice...... 73

LIST OF FIGURES

Figure 1: Platelet anatomy ...... 5

Figure 2: Overview of steps in platelet activation ...... 9

Figure 3: Major platelet receptors and their corresponding agonists ...... 15

Figure 4: Platelet signaling by major receptors ...... 16

Figure 5: Pictorial depiction of GPVI receptor ...... 19

Figure 6: GPVI signaling cascade...... 20

Figure 7: PKC isozymes categorized based on their lipid and cofactor requirement ...... 25

Figure 8: ELMO1 interacts with PIP3 in platelets...... 42

Figure 9: Platelet responses are unaltered in ELMO1-/- platelets downstream of PAR4 pathway...... 44

Figure 10: Enhanced aggregation and granular secretion in response to CRP in ELMO1-/- platelets...... 45

Figure 11: Enhanced aggregation and granular secretion in response to collagen in ELMO1-/- platelets...... 46

Figure 12: Enhanced -granule secretion and integrin IIb3 activation following GPVI stimulation in ELMO1-/- platelets...... 47

Figure 13: Enhanced thromboxane generation in response to GPVI agonists in ELMO1-/- platelets...... 49

Figure 14: ELMO1 regulates thrombus formation in vitro...... 51

Figure 15: ELMO1 regulates thrombus formation in vivo...... 53

Figure 16: ELMO1 negatively regulates thrombus formation following FeCl3-injury on carotid artery...... 54

Figure 17: ELMO1 regulates hemostasis in vivo...... 55

Figure 18: ELMO1 regulates Syk phosphorylation in the GPVI pathway...... 58

Figure 19: ELMO1 regulates RhoG activity in the GPVI pathway...... 60

Figure 20: Model of ELMO1 mediated GPVI signaling in platelets...... 66

Figure 21: PKCY155 phosphorylation is specific to GPVI pathway...... 70

Figure 22: Generation of PKCY155F knock-in model...... 72

Figure 23: Aggregation and dense granule secretion is unaltered in response to AYPGKF in PKCY155F platelets...... 75

Figure 24: Absence of PKCY155 phosphorylation in platelets leads to diminished aggregation and granule secretion following GPVI stimulation...... 76

Figure 25: Absence of PKCY155 phosphorylation in platelets leads to diminished aggregation and granule secretion following CRP stimulation...... 77

Figure 26: Diminished -granule secretion and integrin IIb3 activation following GPVI stimulation in PKCY155F platelets...... 78

Figure 27: PKCY155F regulates thrombus formation in vitro...... 80

Figure 28: PKCY155 regulates thrombus formation in vivo...... 82

Figure 29: PKCY155F regulates Syk in the GPVI pathway...... 84

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CHAPTER 1

1 INTRODUCTION

1.1 Overview of Platelets.

Platelets are anucleate cells that are found in the circulation and are derived from the cytoplasm of megakaryocytes in the bone marrow. The lifespan of platelets is 7-9 days

(1) and they are 2.0 – 5.0 m (2) in diameter. The platelet count within the circulation ranges between 150-450 x 109 per liter. They are discoid shape when quiescent and undergo shape change upon activation. Platelets are primarily known for their crucial role in thrombosis and hemostasis. Under physiological conditions, platelets patrol the healthy vasculature in a quiescent state. Upon vascular injury, the interaction of platelets with the exposed sub-endothelial collagen leads to aggregation and thrombus formation as a physiological response to limit bleeding and tissue injury. However, in a diseased vessel, unwarranted platelet activation can lead to fatal thrombotic conditions such as ischemic heart disease and stroke. Current antiplatelet therapies such as Aspirin and Clopidogrel are effective treatments for cardiovascular diseases; however, a side effect of these medicines is bleeding (3). Therefore, understanding the biology of platelets may aid in the future design of targeted therapies that limits bleeding risks.

1.2 Platelet production.

Megakaryocytes are precursor cells for platelet production, and each megakaryocyte can release up to 5000 platelets into the circulation (4). Mature megakaryocytes are 50-100

m in diameter and are derived from hematopoietic stem cells in the bone marrow. The

1 process of platelet production is termed thrombopoiesis, and one of the key drivers of this process is thrombopoietin. Currently, the mechanism behind the creation of platelets from megakaryocytes is not entirely understood. Briefly, megakaryocytes undergo a process called endomitosis whereby these cells replicate their DNA in the absence of cell division up to 128N (5). The purpose of this process is to increase the protein and lipid content that eventually leads to the development of invaginated membrane system, a reservoir for cytoplasmic extensions called proplatelets (5). The megakaryocyte then migrates to a vascular sinusoid in the bone marrow where it extends the proplatelets and releases the proplatelets into the vascular sinusoid (5). These proplatelets later bud off to platelets in the circulation (5-8).

1.3 Anatomy of Platelets.

Platelets are formed from the cytoplasm of megakaryocytes that are released into the circulation. Resting platelets that patrol the circulation are discoid shape (Fig. 1A) where they are smooth and lack protrusions (9, 10). However, they do contain membrane invaginations that are connected to the open canicular system (OCS) that functions to allow specific molecules into the platelets. Upon activation of platelets, they undergo shape change (Fig. 1B) where these membrane invaginations on the platelets are a source of surface membrane for spreading and adhesion to the site of injury thereby increasing the surface area of the platelets (9, 10). The structure of platelets can be organized into four zones (9) as follows:

• Peripheral zone includes the plasma membrane with the surface receptors and the

membrane invaginations that are connected to the OCS (9). These membrane

invaginations allow specific molecules to enter the platelets. The receptors on the

plasma membrane sense and react to the environment within the vasculature and

ensures a signal gets relayed for the necessary platelet function.

• Sol-Gel zone is the matrix of platelet cytoplasm which includes the cytoskeletal

components, such as microtubules and microfilaments (9). These components

maintain the discoid shape of resting platelets and undergo reorganization

following platelet activation leading to shape change.

• Organelle zone consists of cytoplasmic components that excludes cytoskeletal

components of the Sol-Gel zones. The cytoplasmic components include the

mitochondria and granules (9). Platelets contain three major granules as follows:

alpha (dense (), and lysosomes. These granules are secreted upon platelet

activation at the site of injury. -granules are larger in size than dense granules

and contains various components that are important for adhesion, pro-coagulation,

angiogenesis, and more. Adhesion molecules such as P-selectin in the alpha

granules are only exposed on the surface upon platelets activation and support

binding of leukocytes. Dense granules, named due to its electron-dense core on

electron microscopy, contain components such as adenosine triphosphate (ATP),

adenosine diphosphate (ADP), calcium, polyphosphate, and serotonin (9). In

human platelets, the large amount of calcium in the dense granules can complex

with pyrophosphate and serotonin which determines the opacity of these granule

in electron microscopy (11, 12). ADP is an important secondary mediator that

upon release recruits more platelets to the site of injury and enhances platelet

activation.

• Membranous zone consists of components such as Golgi complexes, OCS, and

dense tubular system (DTS) (9). OCS is an invaginated membrane reserve

connected to the plasma membrane in the cytoplasm of platelets. It is the source

of the membrane that increases the surface area of platelets during shape change

following platelet activation. The DTS is a derivative of the smooth endoplasmic

reticulum from megakaryocytes that sequesters Ca2+ from the cytoplasm in resting

platelets (9). However, upon platelet activation, phospholipase C (PLC)-mediated

generation of inositol 1,4,5-triphosphate (IP3) leads to release of calcium from

DTS, in turn, increasing the cytoplasmic Ca2+ concentration (13). Furthermore,

when the Ca2+ concentration in the DTS is depleted, stromal interaction molecule

1 (STIM) on the DTS undergoes a conformational change enabling it to interact

with calcium release-activated calcium channel protein 1 (ORAI) on the plasma

membrane. The STIM-ORAI interaction facilitates the entry of extracellular Ca2+

into the cytoplasm of platelets further raising the cytoplasmic Ca2+ concentration

in platelets.

Figure 1: Platelet anatomy (10). (A.) Resting platelets that are smooth and lack protrusions. (B.) Activated platelets that contain protrusions and are bridged together by

IIb3 (GPIIb/IIIa) binding to fibrinogen. Platelet activation leads to shape change, granular secretion, thromboxane generation, and integrin IIb3-mediated platelet aggregation.

1.4 Platelet function

1.4.1 Role of platelets in hemostasis

Platelets are crucial mediators of hemostasis and thrombosis. Under physiological conditions, platelets are maintained in a quiescent state within the circulation. The intact endothelial lining within the circulation acts as a physical barrier and produces the following: nitric oxide, ecto-ADPase (CD39) and prostacyclin (PGI2) to prevent platelet activation. Upon vascular injury, platelets adhere, activate and form thrombus on the exposed sub-endothelial matrix containing von Willebrand factor (VWF) and collagen to arrest bleeding. The stages of platelet plug formation is a dynamic process involving a multitude of signaling by various platelets receptors and are characterized as the following overlapping steps: initiation, perpetuation, and stabilization (14). These events in platelet activation and thrombus formation are summarized as follows (Fig. 2):

1. Initiation. The disruption of endothelial lining upon vascular injury leads to

tethering, adherence, and activation of platelets to the sub-endothelial collagen

forming platelet monolayer (14). Platelets contain four important receptors that

directly (21 and GPVI) bind to collagen or indirectly (GPIb-IX-V and IIb3)

bind to collagen via von Willebrand factor (VWF) (13). Under high shear flow

rates such as in arteries, platelets tether and roll on the immobilized von

Willebrand factor (VWF)/collagen complexes in the sub-endothelium via

glycoprotein GPIb-IX-V (15, 16). However, this interaction is reversible therefore

firm adhesion of platelets to the sub-endothelium requires the activation of

platelets. A crucial receptor that initiates platelet signaling and activation is

glycoprotein VI (GPVI) upon interaction with the sub-endothelial collagen (17,

18). The activation of platelets subsequently leads to inside-out signaling

mediated activation of integrin 21 that further stabilizes platelet adhesion to the

sub-endothelium (16).

Following GPVI activation, platelets undergo shape change which is driven by

actin cytoskeletal rearrangement. The process of shape change involves filopodia

formation followed by lamellipodia formation. This increases the surface area of

platelets by spreading on the damaged surface further facilitating adhesion to the

site of injury (19).

2. Extension. Secondary mediators: secreted ADP and thromboxane (TxA2)

generation, enhance platelet activation and recruit additional platelets to the site of

injury following shape change eventually leading to integrin IIbmediated

platelet aggregation and thrombus formation (14). Furthermore, locally generated

thrombin also enhances platelet activation and the fibrin mesh produced by the

coagulation cascade further stabilizes the thrombus. Resting platelets contain

ADP in the dense granules and is secreted upon platelet activation along with the

contents of the other granules in platelets:  and lysosomes. granules contain a

vast array of molecules including adhesion molecule, P-selectin that is surface

exposed upon platelet activation. Platelet aggregation is initiated by inside-out

signaling mediated conformational change and activation of integrin

IIbthereby enabling the interaction with fibrinogen that acts as a bridge

between platelets.

3. Stabilization. The thrombus is further stabilized by a process termed clot

retraction which decreases the volume of the clot thereby preventing premature

7 disaggregation and increasing the local concentration of agonists at the site of injury (14). Clot retraction is a result of force that is generated upon contraction of actin cytoskeleton due to outside-in signaling initiated upon the interaction of fibrinogen with activated integrin IIb

Figure 2: Overview of steps in platelet activation (14). Under physiological conditions, platelets are maintained in a quiescent state by nitric oxide, ecto-ADPase (CD39) and prostacyclin (PGI2) produced by endothelial cells. Upon vascular injury, platelets tether, roll, adhere, and spread on the exposed sub-endothelium forming a monolayer of activated platelets. This subsequently leads to recruitment of additional platelets to the site of injury by secondary mediators: ADP and TxA2. Furthermore, these secondary mediators along with locally generated thrombin enhance platelet activation eventually leading to platelet aggregation and thrombus formation. The growth and stabilization of the thrombus are mediated by a process called clot retraction.

1.4.2 Beyond hemostasis

While platelets are primarily known for their role in hemostasis, they also contribute to other physiological conditions such as angiogenesis, wound repair, and blood/lymph separation (20). Platelets are also known to contribute to diseases such as cancer, sepsis, atherosclerosis, obesity and more (21). Platelets patrol and react to changes in the environment within the vasculature to prevent blood loss. However, dysregulated platelet activation in a diseased vessel can result in pathological conditions such as thrombosis or bleeding.

a. Atherosclerosis. Atherosclerosis is a pathological condition that involves

narrowing of the arterial vessel wall due to plaque formation. The progression of

atherosclerosis involves various facets including endothelial dysfunctions,

inflammation, and plaque formation (22). Platelets encounter a plethora of

agonists within the microenvironment of a dysfunctional endothelium including

VWF, collagen, and thrombin, which leads to adhesion and platelet activation (22,

23). The secreted granules upon platelet activation further enhances inflammation,

and recruit’s leukocytes to the microenvironment contributing to the progression

of this disease (22). Platelets can also form aggregates with the circulating

leukocytes. Eventually, plaque rupture can lead to thrombosis, which can lead to

fatal conditions such as stroke or heart attack (22).

b. Angiogenesis. Angiogenesis is a physiological process that leads to the formation

of a new blood vessel from pre-existing vessels. This process occurs due to the

activation, migration, and differentiation of endothelial cells which is regulated by

pro-angiogenic molecules such as vascular endothelial growth factor (VEGF).

The -granules within platelets contain a vast array of pro-angiogenic factors

such as VEGF and anti-angiogenic factors that influence angiogenesis upon

secretion. Although, platelets contain anti-angiogenic factors within the -

granules, the overall impact is to promote blood vessel formation (24, 25).

However, dysregulated angiogenesis leading to excessive vascular growth can

cause disease conditions such as cancer where secreted platelet granules can cause

complications such as tumor growth (20).

1.5 Platelet receptors

Platelets contain a vast array of receptors that contribute to all aspects of platelet function. Some of the major receptors that are important for platelet function (Fig. 2) are organized into the following structural class: integrin, immunoglobulin superfamily and seven transmembrane receptors (G-protein-coupled receptors (GPCR)) (26). The integrin family includes IIb3 and 21, the immunoglobulin superfamily includes GPVI, and the GPCR family includes P2Y1 and P2Y12, protease-activated receptor (PAR), and thromboxane receptor (TP). These receptors are described in detail (Fig. 3 and Fig. 4) below:

1.5.1 Seven transmembrane receptors

GPCR are seven transmembrane receptors coupled to heterotrimeric G protein complex consisting of an alpha (beta (and gamma ( subunits. The signal relayed from the

GPCR depends on the type of G proteins it is coupled with. The Gsubunit of G protein is categorized into the following: Gs, Gq, G12/13 and Gi subfamily. In the basal state, the

Gsubunit of the heterotrimeric G proteins on the GPCR is bound to guanosine diphosphate (GDP). The activation of GPCR leads to a conformational change which then acts as guanine exchange factor (GEF) that exchanges the GDP to guanosine triphosphate (GTP) on G. Consequently, causing the dissociation of G from the G subunits eventually leading to downstream signaling and function. Below are some

GPCR that is activated during vascular injury.

Thrombin receptors

Thrombin, a serine protease, is generated during the coagulation process and it leads to the formation of fibrin mesh that is important in stabilizing thrombus during hemostasis.

It also elicits a potent response on platelets by PARs coupled to G12/13 and Gq heterotrimeric G proteins. Thrombin proteolytically cleaves PAR receptors at the extracellular N-terminus resulting in a newly exposed N-terminus that serves as a tethered ligand to the receptor initiating downstream signaling. The Gq-mediated signaling activates PLC generating secondary messengers: diacylglycerol (DAG) and

IP3. Consequently, triggering IP3-mediated calcium mobilization and PKC activation eventually resulting in platelet aggregation and secretion (13). The G12/13 signaling leads to RhoA mediated p160ROCK activation ultimately resulting in shape change (13).

Human platelets express PAR1 and PAR4 while murine platelets express PAR3 and

PAR4 (27). Murine PAR3 does not signal on its own instead acts as a cofactor to thrombin-mediated PAR4 activation at low concentrations of thrombin (28, 29).

Thromboxane receptor

TxA2 is a short-lived lipid mediator that is generated during platelet activation. It is

12 produced by TxA2 synthase from prostaglandin H2 (PGH2) which is derived from arachidonic acid via cyclooxygenase (Cox-1). Once TxA2 is generated, it diffuses across the plasma membrane to enhance platelet activation and recruit platelets to the site of injury. TxA2 activates thromboxane receptors (TP) on platelets coupled to Gq and G12/13

(13). Like thrombin receptor signaling, the Gq signaling leads to platelet aggregation and secretion via PLC-mediated generation of DAG and PKC. The G12/13 signaling leads to shape change via RhoA-mediated signaling. Aspirin is an irreversible inhibitor of COX1 and is used as a treatment for cardiovascular diseases (13).

ADP receptors

ADP is stored in the dense granules of resting platelets and is secreted upon platelet activation. ADP along with thromboxane are secondary mediators that act in an autocrine and paracrine manner to enhance platelet activation and recruit more platelets to the site of injury, respectively. Human and murine platelets express both P2Y1 and P2Y12 that are activated by ADP (30). P2Y1 is coupled to Gq whereas P2Y12 is coupled to Gi. As described above, Gq-mediated signaling leads to calcium mobilization and protein kinase

C (PKC) activation mediated via PLCβ. However, Gq-mediated signaling by P2Y1 leads to shape change (30) whereas the Gi-mediated signaling by P2Y12 leads to platelet aggregation and secretion via the inhibition of adenyl cyclase and PI3K activation.

Furthermore, P2Y12-mediated aggregation and secretion requires functional P2Y1- mediated Gq signaling (31, 32).

1.5.2 Integrin IIb3

The activation of integrin IIb3 is crucial for platelet aggregation that supports thrombus formation during vascular injury (33). Integrin IIb3 exists in a low affinity state (inactive) in resting platelets and undergoes conformational change to a high affinity state (active) upon platelet activation (13, 33). This conformational change of integrin

IIb3 requires signaling via other receptors such as GPVI on platelets termed inside-out signaling. Once activated, integrin IIb3 bind to its ligand fibrinogen/VWF which acts a bridge between platelets important for aggregation. Additionally, the interaction of activated integrin IIb3 with fibrinogen also initiates outside-in signaling activating Src family kinases (SFK) and spleen tyrosine kinase (Syk) eventually leading to spreading, aggregation, and clot retraction (13, 33).

1.5.3 Immunoglobulin superfamily

GPVI belong to the immunoglobulin superfamily and is discussed in detail below in section 1.6.

Figure 3: Major platelet receptors and their corresponding agonists (26).

Figure 4: Platelet signaling by major receptors (14).

1.6 Immunoglobulin superfamily-GPVI

1.6.1 Structure of GPVI

GPVI belongs to the immunoglobulin (Ig) receptor family that is constituently associated with a homodimer of Fc receptor (FcR) -chain consisting of immunoreceptor tyrosine- based motif (ITAM). The structure of GPVI contains two Ig C2-like domains followed by a mucin-rich stalk, transmembrane domain, and a cytoplasmic tail as depicted in Fig. 5

(33). The mucinlike stalk extends the Ig C2-like domain from the platelet surface consisting of collagen binding sites (34, 35). The transmembrane region contains a conserved arginine residue that forms a salt bridge with conserved aspartic acid in the transmembrane domain of FcR-chain (17). The salt bridge in the transmembrane domain and the sequence in the juxtamembrane region of the cytoplasmic tail of GPVI are important for the constitutive association of FcR-chain with GPVI (36, 37). The proline- rich region in the cytoplasmic tail of GPVI interacts with SH3 domain containing SFK,

Fyn and Lyn, that are constituently associated with GPVI (38, 39). Human and murine

GPVI receptor share 64 % identity by protein sequence (40). The cytoplasmic tail of human and murine GPVI differ by sequence length as human GPVI contains 51 amino acids whereas the murine GPVI receptor contains 27 amino acids (40).

1.6.2 GPVI-mediated signaling

GPVI interacts with sub-endothelial collagen and initiates platelet activation during vascular injury (18, 33). The expression of GPVI in platelets requires FcR-chain expression since FcR-chain null mice do not express GPVI (36, 41). Furthermore, GPVI

17 mediated signaling also requires FcR-chain (37, 41, 42) and SFKs (38, 43). The crosslinking of GPVI upon interaction with collagen leads to tyrosine phosphorylation of the ITAM motif (44, 45) on the FcRγ chain via SFK, Fyn and Lyn (46, 47). Each FcRγ chain contains an ITAM which consists of two YXX(L/I) motif that is separated by 6-12 amino acids. The tyrosine phosphorylation of the ITAM motif on the FcRγ chain leads to recruitment and binding of Syk to this motif via the tandem SH2 domains present on Syk resulting in auto-phosphorylation and phosphorylation of Syk by SFK (45, 48, 49). The activation of Syk initiates formation of linker for activation of T cells protein (LAT) signalosome (Fig. 6) constituting various adaptors and kinases eventually leading to platelet activation (33). The focus of this thesis is on PI3K (50) and PLC2 (51) that are activated downstream of Syk in GPVI signaling.

Figure 5: Pictorial depiction of GPVI receptor (33).

Figure 6: GPVI signaling cascade (33).

1.6.3 Regulators of GPVI signaling and their associated function

1.6.3.1 Phosphoinositide 3-kinase (PI3K)

PI3K are kinases that phosphorylate the hydroxyl group in the 3’-position on the inositol ring of phosphatidylinositol. These kinases are subdivided into three classes (class I, class

II, and class III) based on the primary structure and substrate specificity (52). The focus of this thesis is on class I PI3K. However, class II PI3K specifically C2 does regulate

GPVI-mediated platelets responses however this is due to its function in regulating the platelet membrane structure (53, 54). Recently, class III PI3K (VPS34) was characterized in platelets by two separate groups however the role of VPS34 is unclear in GPVI- mediated platelet due to contradictory results between the groups (55, 56).

Class I PI3K

Class I PI3K catalyze the production of phosphatidylinositol 3, 4, 5 triphosphate

(PI(3,4,5)P3) from phosphatidylinositol 4, 5 biphosphate (PI(4,5)P3) which recruits pleckstrin homology (PH) domain containing proteins such as bruton’s tyrosine kinase

(Btk) to the membrane (52). These kinases are activated by GPCR and tyrosine kinase receptors. These kinases are heterodimers containing a regulatory and a p110 catalytic subunit. There are four isoforms of catalytic subunit p110 (PIK3CA), p110 (PIK3CB), p110 (PIK3CG) and p110 (PIK3CD). Class I PI3K are further subdivided into two classes: class IA and class IB (52). Class IA consists of p85 regulatory subunit and p110

(, , and ) catalytic subunit. There are five variants of the regulatory subunit p85

(p85, p55, p50, p85, & p85). Class Ib consists of p101 regulatory subunit and p110 catalytic subunit. All isoforms of PI3K are expressed in platelets (57). Class I

PI3K are activated downstream of various receptor in platelets however below are class I

PI3K isoforms that are involved in GPVI-mediated platelet activation.

a. PI3KThis isoform is thought to be predominant form that regulates GPVI-

mediated platelet signaling. The use of kinase-dead mice and pharmacological

inhibitors have shown that PI3Kis important for positive regulation GPVI-

mediated aggregation and secretion. At the molecular level, Akt, PLC2, and

Rap1b phosphorylation are diminished upon inhibition of PI3K downstream of

GPVI (58, 59). This isoform does not have a role in hemostasis (59) and in a

pulmonary thromboembolism model. However, PI3Kregulates thrombus

formation and stability following arterial injury in vivo (60, 61).

b. PI3K. This isoform also positively regulates GPVI-mediated aggregation and

secretion however not to the same extent as PI3K Compared to PI3K

PI3K is not important for thrombus formation (61).

1.6.3.2 Protein Kinase C

Overview of PKC

Protein kinase C (PKC) family are part of the protein kinase A, G, & C (AGC) group of serine/threonine kinases that regulate their function by phosphorylating the hydroxyl group on their substrates (62). PKCs consist of 10 isozyme that are categorized into three

22 classes based on their lipid and cofactor requirement (Fig. 7) as follows: Conventional,

Novel and Atypical. Conventional PKCs (PKC, PKCI, PKCII, and PKC) depend on calcium and diacylglycerol (DAG), novel PKCs (PKC, PKC, PKC, and PKC) depend on DAG and atypical PKCs (PKC and PKC/) which depend on neither. PKCs are maintained in an inactive confirmation by the association of its pseudosubstrate sequence to the substrate-binding pocket. The activity and the function of PKC is regulated by allosteric binding of cofactors and phospholipids releasing the pseudosubstrate sequence from the substrate binding pocket, a series of serine/threonine phosphorylation’s which renders it catalytically competent, and the specific localization of PKC upon association with scaffold proteins such as receptor for activated C-kinase

(RACK) (62, 63). The activity of some PKC isozymes such as PKC can be further regulated by tyrosine phosphorylations. Of the above-mentioned PKC isozymes, human and murine platelets express the following: PKCPKC, PKC,

PKCPKCPKCand PKC. PKCis possibly expressed at low levels in human platelets.

Structure of PKC

PKCs consist of N-terminal regulatory domain followed by hinge region, and a C- terminal catalytic domain (Fig. 7). These motifs are described in detail below:

• Regulatory domain: This domain consists of the auto-inhibitory pseudosubstrate

sequence, and membrane targeting domains (C1 and C2) (62, 71). The

pseudosubstrate sequence maintains the PKCs in an inactive state by interacting

with the substrate binding pocket in the catalytic motif. Upon allosteric interaction

of cofactors with their respective domains, pseudosubstrate sequence is released

from the substrate pocket (62). The DAG binds to the C1 domain which consists

of tandem repeats termed C1a and C1b which are present only in classical and

novel PKCs. Classical PKCs contain C2 domain that binds to Ca2+ while novel

PKCs contains atypical C2 domain that cannot bind to Ca2+. Atypical PKCs do

not contain C2 domain and contains an atypical C1 domain that does not bind to

DAG.

• Catalytic domain: The catalytic motif consists of C3 and C4 domains important

for the activity of the enzyme and substrate binding (62). The C4 domains

consists of the activation motif followed by turn motif and hydrophobic motif

which undergoes series of ordered serine/threonine phosphorylation at these

motifs that keeps PKCs enzymatically competent. This is restricted to classical

and the novel PKC since atypical PKCs only get phosphorylated at the activation

and the turn motif (62).

Figure 7: PKC isozymes categorized based on their lipid and cofactor requirement (71).

PKC isozymes that regulate GPVI-mediated platelet activation

PKC

PKC has been previously shown to positively regulate granule secretion and integrin

IIb3 activation downstream of GPVI-signaling, with the use of pharmacological inhibitors and gene knockout studies (72). Furthermore, PKCwas also found to regulate thrombus formation however these platelet responses is potentially result of fewer dense granules in PKC-/- platelets (72, 73). Therefore, PKCdoes regulate alpha granule secretion but the role of PKCin dense granule secretion and integrin IIb3 activation downstream of GPVI signaling remains to be understood.

PKC

The inhibition of PKCusing pharmacological inhibitors have implicated PKCas a positive regulator of GPVI-mediated dense granule secretion and aggregation in platelets

(72, 74). The absence of PKChas showed the importance of PKCin thrombus formation in vitro (75). PKCis thought to play redundant role as PKC downstream of

GPVI signaling. However, interestingly, PKCwas found to negatively regulate Syk tyrosine phosphorylation downstream of GPVI signaling only in human platelets though the mechanism of this regulation remains to be understood (74).

PKC

Our group has previously shown that PKCis activated downstream of GPVI receptors in human platelets indicated by T538 phosphorylation, a marker of PKCactivation (69).

With the use of pharmacological inhibitor and gene knock-out model, PKCpositively regulates granular (dense and alpha) secretion, thromboxane generation, and integrin-

3 activation downstream of GPVI pathway (69). PKCpotentially regulates granular secretion and thromboxane generation by Syntaxin 4 and Erk (extracellular signal- regulated kinase) respectively. Consistently, PKCalso positively regulates thrombus formation (69) and hemostasis (70).

PKC

Our group has previously shown that PKCis activated downstream of GPVI signaling

(65). PKC was also found to negatively regulate GPVI mediated dense granule secretion, thromboxane generation, and aggregation by pharmacological inhibitors and gene-knockout models (65, 66, 76). PKCmediated platelet aggregation is possibly regulated by interaction and inhibition of VASP-mediated filopodia formation (76).

PKC potentially regulates dense granule secretion upon interaction with SHIP-1 and

Lyn. Furthermore, PKCwas also found to regulate thrombus formation in an in vitro flow over collagen model (73). However, in an in vivo ferric chloride injury model,

PKCwas not found to regulate thrombus formation (66).

1.7 Potential regulators of GPVI signaling.

The Role of PIP3 interacting proteins in GPVI-mediated platelet activation (chapter 3)

PI3K is an important signaling molecule that is activated downstream of GPVI. The activation of PI3K leads to generation of PIP3 from PIP2 which in turn recruits PH

27 domain containing proteins to the membrane. Btk and Ras GTPase-activating protein 3

(RASA3) are well known PIP3 interacting proteins in platelets and are important regulators of GPVI-mediated platelet activation (77, 78). However, whether there are other PIP3 interacting proteins that may regulate GPVI-mediated platelet activation is not understood. Therefore, we plan on identifying novel proteins that interact with PIP3 using a proteomic approach (details in chapter 3).

The role tyrosine phosphorylation on PKC in platelets (chapter 4)

PKC has been previously shown to differentially regulate dense granule secretion downstream of GPVI and PAR signaling (65, 66). However, how PKC regulates this function is not understood. Tyrosine phosphorylation on PKChave been previously shown to be specific to agonist stimulation and can regulate the activity of

PKCTherefore, we speculate that the differential tyrosine phosphorylation on

PKCmay aid in understanding differential function of PKCin platelets (details in chapter-4)

CHAPTER 2

2 MATERIALS AND METHODS Materials.

All materials were obtained from ThermoFisher Scientific unless noted. PI(3,4,5)P3 PIP

BeadsTM (P-B345a) were obtained from Echelon (Salt Lake City, UT). Acetylsalicylic acid (ASA), apyrase (Type V), Thrombin, MRS2179 (M3808), Heparin, Epinephrine,

Indomethacin (I7378) and anti-ELMO1 antibody (E2533) were obtained from Sigma (St.

Louis, MO). CRP-XL was purchased from Dr. Richard Farndale at the University of

Cambridge. AYPGKF was obtained from GenScript (Piscataway, NJ). Type I collagen

(385) and CHRONO-LUME™ (395) were obtained from CHRONO-LOG Corporation

(Havertown, PA, USA). Anti-Syk-01 (sc-51703), anti-PLC2 (sc-5283) and anti-

PKC(Tyr155, sc-23770; Tyr311, sc-18364; Tyr332, sc-18365; Tyr525, sc-18368-R; &

Tyr565, sc-18372-R) were obtained from Santa Cruz Biotechnology (Dallas, TX).

Antibodies against phosphorylated Syk (Tyr525/6, 2711S), PLC2 (Tyr759, 3874S),

PLC2 (Tyr1217, 3871S), Akt (S473, D9E) and -actin (13E5) were purchased from Cell

Signaling Technology (Danvers, MA). Anti-Akt (TA504230) was obtained from Origene

(Rockville, MD). Anti-PKC was obtained from BD Biosciences (San Jose,

CA). Infrared dye-labeled goat anti-mouse (926-68020) and anti-rabbit antibodies (926-

32211) were purchased from LI-COR (Lincoln, NE). Anti-RhoG antibody (04-486) was purchased from Millipore (Temecula, CA). Anti FITC-GPVI (M011-1), Anti FITC-P- selectin (D200) and Anti PE-JON/A (D200) was purchased from Emfret (Würzburg,

Germany). Prostaglandin E1 (BML-PG006-0010) was purchased from Enzo (New York,

NY). ARC-69311MX was a gift from The Medicines Co. (Parsippany, NJ).

Preparation of human platelets.

Blood was collected from informed healthy donors into one-sixth volume of ACD (85 mM sodium citrate, 71.4 mM citric acid and 111 mM dextrose). The protocol approval was obtained from the institutional review board of Temple University and informed consent was provided prior to blood donation, in accordance with the Declaration of

Helsinki. Platelet rich plasma (PRP) was obtained from whole blood by centrifugation at

230 x g for 20 minutes at room temperature. PRP was incubated with 1 mM Aspirin for

30 minutes at 37 °C and centrifuged at 980 x g for 10 minutes at room temperature to isolate platelets. The platelet pellet was re-suspended in Tyrode’s buffer (137 mM sodium chloride, 2.7 mM potassium chloride, 2 mM magnesium chloride, 0.42 mM sodium phosphate monobasic, 10 mM HEPES and 0.1 % dextrose adjusted to pH 7.4) containing 0.2 U/mL of apyrase. The platelet count was adjusted to 2 x 108 cells/mL unless otherwise stated. For the PIP3 pull-down assay, platelets were washed with PIPES buffer (137 mM sodium chloride, 2.7 mM potassium chloride, 2 mM magnesium chloride, 0.42 mM sodium phosphate monobasic, 0.1% dextrose, 10 mM PIPES adjusted to pH 6.5) containing 0.2 U/mL of apyrase, 500 M EGTA, and 10 nM Prostaglandin E1

(PGE1) prior to being re-suspended in Tyrode’s buffer. The platelet count was adjusted to

9 2 x 10 cells/mL for the PIP3 pull-down assay.

Genotyping of PKCY155F mice.

The PRKCDY115F mice were identified based on PCR product size difference as the

PRKCDY115F mice contain LoxP site within intron-6 using gtF (forward 5’-

GATGCTGTATAGGGGCAGGA-3’) and gtR (reverse 5’-

GCAGGTGGTGAGTGTTCCTT-3’) oligonucleotides (Fig. 13B). Additionally, oligonucleotides for sequencing (forward 5’-AGCTCTTTTGCCAGGTCAGA-3’ and reverse 5’-TGCATTTGTAGCCTTGCTTG-3’) and oligonucleotides that recognize the mutated allele (forward 5’-ATTTCCTGGAGGATGGGGGTA-3’ and reverse 5’-

CGATAAACTGTGGTTCTTGATGA-3’) were used to confirm Y155F mutation.

Preparation of murine platelets.

All mice were maintained and housed in a specific pathogen-free facility, and animal procedures were carried out in accordance with the institutional guidelines after the

Temple University Institutional Animal Care and Use Committee approved the study protocol. ELMO1-deficient mice (ELMO1-/-) were obtained from Kodi Ravichandran at the University of Virginia School of Medicine (80). The background of ELMO1-/- mice is C57BL/6J (80) and the background of PKCY155F mice is mixed (C57BL/6J and

129/sv). The appropriate age-matched wildtype littermates were used as controls for both the ELMO1-/- and PKCY155F mice. Whole blood count was measured using the

Hemavet (Drew Scientific; Miami Lakes, FL). Whole blood was collected from mice, 10-

12 weeks old, via cardiac puncture into one-tenth volume of 3.8 % sodium citrate. Blood was dispensed into 1.5 mL microcentrifuge tube containing 400 L of 3.8% sodium citrate and PRP was obtained by centrifugation of whole blood at 100 x g for 10 minutes at room temperature. The PRP was transferred to a clean tube and 400 L of 3.8 % of

31 sodium citrate was added again to whole blood. PRP was again obtained by centrifugation at 100 x g for 10 minutes and combined. PGE1 (1 M) was added to the

PRP and platelets were isolated by centrifugation at 400 x g for 10 minutes. The platelet pellet was re-suspended in Tyrode’s buffer containing 0.2 U/mL of apyrase and the platelet count was adjusted to 1.0 - 1.5 x 108 cells/mL. Platelets were allowed to rest at room temperature prior to use for experiments.

Sample preparation.

Reactions were stopped with one-tenth volume of 6.6 N perchloric acid to precipitate proteins unless otherwise stated. The samples were centrifuged at 15,000 x g for 5 minutes at 4 °C, washed with water, and the proteins were solubilized in 1x sample buffer

(0.1 M Tris-base, 1 % glycerol, 2% sodium dodecyl sulfate (SDS) and 100 mM

Dithiothreitol (DTT)). The samples were boiled at 95 °C for 10 minutes. For the PIP3 pull-down assay, resting human platelets were lysed using equal volume of 2x cold NP-

40 buffer (150 mM sodium chloride, 20 mM HEPES, 2 mM EGTA, and 0.5 % Nonidet p-40 adjusted to pH 7.4) containing Halt Protease and Phosphatase cocktail solution

(Pierce, Rockford, IL) and were centrifuged at 12,000 x g for 10 minutes at 4 °C to pellet cell debris.

PIP3 Pull-down assay.

PIP3 pull-down assay was performed as per manufacturer’s instructions. Briefly, cleared

32 lysates were incubated with 100 L of PI(3,4,5)P3 PIPTM beads or control beads overnight at 4 °C. Beads were washed three times with 1x cold NP-40 buffer, eluted with

2x SDS-sample buffer (Boston BioProducts), and boiled at 95 °C for 10 minutes.

In-gel trypsin digestion and LC-MS analysis.

Samples from the PIP3 pull-down were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by liquid chromatography-mass spectroscopy (LC-MS) as previously described (81).

Immunoblotting.

Human and murine platelet lysate were subjected to SDS-PAGE (8% acrylamide) and were electro-transferred onto nitrocellulose membrane. Membranes were blocked with

Odyssey blocking buffer (LI-COR) for 1 hour at room temperature and incubated with the indicated primary antibodies overnight at 4 °C. Membranes were washed 3 x 10 minutes with Tris-buffered saline containing 0.1% Tween-20 (TBST) and probed with infrared-labelled secondary antibodies or alkaline phosphatase (AP)-conjugated secondary antibodies for 1 hour at room temperature. The blots were washed 3 x 10 minutes with TBST and analyzed using Odyssey imaging system (LI-COR).

Platelet aggregation and dense granule secretion.

Platelet aggregation and ATP secretion were simultaneously detected using a lumi- aggregometer (CHRONO-LOG Corporation) at 37 °C while stirring. ATP secretion was detected using CHRONO-LUME (CHRONO-LOG Corporation), a luciferin/luciferase reagent.

Flow cytometry

All flow cytometry experiments were performed under non-stirring conditions with platelet density of 1 x 106 cells/mL. Washed murine platelets were re-suspended in

Tyrode’s buffer containing 1 mM CaCl2 and were activated with various concentrations of CRP for 10 minutes at 37 °C in a 50 L reaction volume prior to fixing along with 5

L of P-selectin or JON/A antibody to detect surface P-selectin exposure or Integrin

IIb3 activation respectively. GPVI surface expression in resting platelets was detected using 5 L of FITC-GPVI antibody to the 50 L reaction without the agonist. Platelets were fixed with 1% paraformaldehyde and detected using FACSCalibur flow cytometer

(BD Biosciences). A total of 30,000 events were acquired per sample and the appropriate isotype control was used as a negative control.

Thromboxane generation.

Thromboxane B2 (TXB2) generation was quantified using a kit from Enzo as per manufacturer’s instructions. Briefly, murine platelets (1.5 x 108 cells/mL) in Tyrode’s buffer (pH 7.4) were stimulated with varying concentration of CRP or collagen for 4

34 minutes and the reaction was stopped via flash-freezing. The samples were stored at -80

°C until analysis. The samples were centrifuged at 15,000 x g for 10 minutes at 4 °C and the supernatant was diluted 100-fold with the provided assay buffer. The level of thromboxane B2 (TXB2), a stable product of thromboxane A2 (TXA2), was detected using a competitive enzyme immunoassay kit generation by Enzo.

Flow over collagen

Flow over collagen assay was performed as previously described (82). Briefly, whole blood was isolated from age-matched mice (10-12 weeks) by cardiac puncture, anticoagulated using 400 M PPACK (Enzo, BML-PI117-0005) and 5U/mL Heparin, and perfused over a collagen-coated dish using a chamber from Glycotech

(Gaithersburgh, MD) under arterial (1000 s-1) and venous (200 s-1) shear rates. Thrombus formation was observed using Nikon Eclipse TE300 inverted microscope (objective

200x) and analyzed using ImageJ (NIH) analysis.

Pulmonary thromboembolism

Pulmonary thromboembolism model was performed as previously described (83) with the following changes: age-matched mice (age: 10-12 weeks) were weighed and anesthetized with Ketamine/xylazine. Mice were then injected via the retro-orbital sinus with 400

μg/kg collagen and 60 μg/kg epinephrine or phosphate buffered saline as a control and the time to cessation of respiration was recorded. Upon respiratory arrest, the heart was

35 perfused with Chicago sky blue dye to confirm pulmonary emboli.

Ferric chloride (FeCl3) injury model

The left carotid artery was injured using FeCl3 as previously described (84). Briefly, 10-

12 weeks old mice were anesthetized and the carotid artery was exposed to filter paper (1 x 1 mm) saturated with 7.5% or 5% FeCl3 for 90 seconds and the flow rate was recorded using Transonic T402 flow meter (Ithaca, NY). The time to occlusion was defined as the time required for the flow rate to reach 0 mL/min. The operator was blinded to mouse genotype while performing all experiments.

Tail bleeding.

Four-week old mice were anesthetized and the distal 3 mm of the tail was cut, and immediately immersed in 37 °C saline. The time for bleeding to halt was recorded.

Bleeding was manually stopped when mice blead for more than 10 minutes.

Expression of ELMO1-GST and the measurement of RhoG activation.

The expression of bacterially expressed ELMO1-GST and the measurement of RhoG activity was determined as previously described (85).

Statistics

All statistical analysis was performed using KaliedaGraph and Microsoft Excel. Data was represented as mean ± SE of at least three independent experiments. All statistics were analyzed using a Student’s t-test. p<0.05 was considered statistically significant.

CHAPTER 3

3 ELMO1 NEGATIVELY REGULATES GLYCOPROTEIN VI- MEDIATED SIGNALING BY RHOG IN PLATELETS

3.1 Introduction

Class I PI3K is an important signaling molecule that is activated downstream of GPVI- mediated platelet activation. The inhibition of class I PI3K with pharmacological and genetic approaches leads to diminished platelet functional responses such as platelet aggregation, granular secretion, and thrombus formation (57). In platelets, the activation of class I PI3K is not restricted to the GPVI pathway but is also activated downstream of

P2Y12 (57) and IIb3 receptors (57). PI3K phosphorylates the 3-hydroxyl (OH) group of the inositol ring of phosphatidylinositol leading to the generation of

Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) from Phosphatidylinositol 4,5- bisphosphate (PIP2) in the plasma membrane (57). PIP3 formation in turn leads to the recruitment of pleckstrin homology (PH) domain-containing proteins to the plasma membrane (88) such as Bruton’s tyrosine kinase (Btk) (89-91) and Ras GTPase- activating protein 3 (RASA3) (92).

ELMO (engulfment and cell motility) is a scaffold protein with no catalytic activity (93).

There are three mammalian and murine isoforms of ELMO proteins, ELMO 1-3. These proteins contain series of five armadillo repeats at the N-terminus, a PH domain followed by a proline-rich region at the C-terminus (93). ELMO1 and ELMO2 interact with SH3- domain containing DOCKA/DOCKB family consisting of DOCK1-5 isoforms via its

38 proline-rich region (93, 94). ELMO1 and ELMO2 regulate actin cytoskeletal rearrangement via Rac1 upon interaction with dedicator of cytokinesis (DOCK), a guanine nucleotide exchange factor (93).

Active RhoG, a small GTPase, is another ELMO1 interacting protein that interacts with

ELMO1 via the armadillo repeats. RhoG regulates the actin cytoskeleton via the ELMO,

DOCK, and Rac1 axis and is involved in cell migration (95-97), engulfment of apoptotic cells (93), and neurite outgrowth (98). However, in platelets, the interaction of active

RhoG with ELMO1 and DOCK180 does not appear to regulate the actin cytoskeleton via

Rac1 following GPVI-mediated platelet activation indicting that RhoG’s function is different in platelets than in nucleated cells (99). ELMO1 expression has been established in human platelets (99) but the function of ELMO1 is not yet known.

In the present study, we screened for proteins that interacted with PIP3 in platelets using a proteomic approach and found ELMO1. We utilized ELMO1-/- mice to characterize the functional role of ELMO1 in platelets. We provide evidence that absence of ELMO1 leads to enhanced GPVI-mediated platelet aggregation, secretion, and thromboxane generation. Consistently, whole blood from ELMO1-/- exhibit enhanced thrombus formation in an in vitro flow over collagen model under arterial and venous shear conditions. Additionally, we demonstrate that ELMO1 regulates hemostasis and thrombotic functions in vivo. Finally, we show that ELMO1 negatively regulates Syk activation in platelets downstream of GPVI signaling pathway through binding to RhoG.

3.2 Results

ELMO1 interacts with PIP3 in platelets.

Class I PI3K is an important signaling molecule that is activated downstream of various receptors in platelets (57). The inhibition of class I PI3K using pharmacological and genetic approaches leads to diminished platelet activation and thrombus formation (57).

The activation of PI3K leads to the generation of PIP3 and, in turn, the recruitment of PH domain-containing proteins to the plasma membrane (57). In platelets, RASA3 and Btk, are known to interact with PIP3 (100) and are important mediators of platelet activation

(78, 101). However, whether there are other proteins that can interact with PIP3 in platelets is not known. Therefore, we used a proteomic approach to screen for novel proteins that interact with PIP3 in resting human platelets by performing a pull-down assay using PIP3 beads. We used control beads to detect non-specific interactions.

Proteins bound to PIP3 or control beads were separated by SDS-PAGE and identified by

LC-MS. When compared to control beads, 24 proteins out of 154 proteins bound specifically with PIP3 (Appendix). Within these 24 proteins, we found 5 that are known to contain PH domains (Table 1). It is well known that Ras GTPase-activating protein 3

(RASA3) (92), Btk (90, 91), Cytohesin-2 (102, 103), and Dual adapter for phosphotyrosine and 3-phosphotyrosine and 3-phosphoinositide (DAPP1) (104, 105) can interact with PIP3 in nucleated cells. However, whether ELMO1 can interact with PIP3 in platelets has not been previously established. Therefore, we confirmed that ELMO1 interacts with PIP3 beads in platelets using Btk as a positive control by Western blot analysis (Fig. 8A). This indicates that endogenous ELMO1 specifically interacts with

PIP3 in platelets.

Accession Protein Score Coverage # of Peptides Q14644 Ras GTPase-activating protein 3 2029.17 27.22 24 Q06187 Tyrosine-protein kinase BTK 473.89 25.49 14 Q99418 Cytohesin-2 120.69 11.50 4 Q9UN19 Dual adapter for phosphotyrosine and 49.84 10.36 2 3-phosphotyrosine and 3- phosphoinositide Q92556 Engulfment and cell motility protein 1 41.93 1.51 1 Table 1: Summary of PH domain containing proteins from proteomics data.

Figure 8: ELMO1 interacts with PIP3 in platelets. (A.) Resting human platelets (1 x

109 cells/mL) were lysed with NP-40 lysis buffer and incubated with PI(3,4,5)P3 PIPTM beads or control beads overnight at 4 C. Pulldowns were analyzed by Western blot and probed with ELMO1 or Btk antibody. Blots are a representative of at least 3 independent experiments. (B.) The proteins from resting washed human, wildtype (WT) littermate controls, and ELMO1-/- murine platelets were precipitated and were analyzed by Western blot. The blots were probed for ELMO1 and -actin as a loading control. Blot is a representative of at least 3 independent experiments. MW, molecular weight marker.

ELMO1 negatively regulates platelet aggregation and granule secretion downstream of GPVI.

The function of ELMO1 is not known in platelets. Therefore, we utilized ELMO1

(ELMO1-/-) deficient mice (80) to study the function of ELMO1 in platelets. As previously reported, ELMO1 is present in human platelets (99) and we also found

ELMO1 present in murine platelets (Fig. 8B). Furthermore, the absence of ELMO1 was confirmed in ELMO1-/- platelets (Fig. 8B).

To investigate the function of ELMO1 in platelets, we performed ex vivo platelet aggregation and dense granule secretion studies from ELMO1-/- and WT littermate control platelets. In response to AYPGKF (PAR4 agonist), platelet aggregation and dense granule secretion were unaltered in platelets isolated from ELMO1-/- mice compared to

WT littermate control platelets (Fig. 9). However, CRP (GPVI agonist)-induced platelet aggregation and dense granule secretion were enhanced in ELMO1-/- platelets in a concentration-dependent manner (Fig. 10). Similarly, platelet aggregation and dense granule secretion in response to the physiological GPVI agonist, collagen, were also potentiated in ELMO1-/- platelets (Fig. 11). This indicates that ELMO1 negatively regulates platelet aggregation and dense granule secretion specifically downstream of

GPVI pathway and not the PAR4 pathway. Consistently, alpha-granule secretion and integrin IIb3 activation was also enhanced downstream of GPVI pathway in the

ELMO1-/- compared to the WT littermate control (Fig. 12) indicating ELMO1’s negative regulatory role in platelets.

Figure 9: Platelet responses are unaltered in ELMO1-/- platelets downstream of

PAR4 pathway. Representative aggregation tracings (A) and dense granule secretion (B) of washed platelets from ELMO1-/- or wildtype littermate (WT) stimulated with indicated concentration of AYPGKF for 4 minutes under stirring conditions at 37 °C. Platelet aggregation and dense granule secretion was detected by lumi-aggregometry.

Quantification of extent of aggregation (C.) and dense granule secretion (D.) of at least three independent experiments from panel A. & B. Data is represented as mean ± SE and was analyzed using Student’s t-test (p<0.05). A.U is arbitrary units.

Figure 10: Enhanced aggregation and granular secretion in response to CRP in

ELMO1-/- platelets. (A.) Representative aggregation and (B.) dense granule secretion tracings of washed platelets from ELMO1-/- or wildtype littermate (WT) control activated with the indicated concentration of CRP for 4 minutes. Platelet aggregation and dense granule secretion was detected by lumi-aggregometry under stirring conditions at 37 °C.

Quantification of (C.) extent of aggregation and (D.) dense granule secretion of at least three independent experiments from panel A. and B. Data is represented as mean ± SE and was analyzed by Student’s t-test. (* p < 0.05). A.U is arbitrary units.

Figure 11: Enhanced aggregation and granular secretion in response to collagen in

ELMO1-/- platelets. (A.) Representative aggregation and (B.) dense granule secretion tracings of washed platelets from ELMO1-/- or wildtype littermate (WT) control activated with the indicated concentration of collagen for 4 minutes. Platelet aggregation and dense granule secretion was detected by lumi-aggregometry under stirring conditions at 37 °C.

Figure 12: Enhanced -granule secretion and integrin IIb3 activation following

GPVI stimulation in ELMO1-/- platelets. Flow cytometry analysis of (A.) P-selectin exposure and (B.) integrin IIb3 activation in ELMO1-/- or wildtype (WT) littermate control stimulated with the indicated concentration of CRP for 10 minutes at 37 °C. P- selectin exposure and integrin IIb3 activation was detected using P-selectin-FITC and

JON/A-PE antibodies respectively. The data is represented as fold increase over basal ±

SE and was analyzed by Student’s t-test. (* p < 0.05).

Enhanced GPVI-mediated thromboxane generation in the ELMO1-/- platelets.

We investigated the contribution of ELMO1 in thromboxane generation downstream of the GPVI pathway. In response to CRP, thromboxane generation was enhanced in platelets from ELMO1-/- mice compared to the WT mice, in a concentration-dependent manner (Fig. 13A). Similarly, thromboxane generation was also enhanced in the ELMO1-

/- platelets in response to the physiological GPVI agonist, collagen (Fig. 13A). To investigate whether the enhanced thromboxane generation contributes to the enhanced

GPVI-mediated platelet responses observed in the ELMO1-/- platelets, we performed ex vivo platelet aggregation and dense granule studies from ELMO1-/- and WT littermate control platelets in the presence of 10 M Indomethacin. As expected, platelet aggregation and dense granule secretion is decreased in the Indomethacin-treated WT platelets compared to the WT platelets in Fig. 10 that is not treated with indomethacin.

However, platelet aggregation and dense granule secretion were enhanced in the ELMO1-

/- platelets pre-treated with Indomethacin compared to the WT littermate controls pre- treated with Indomethacin in response to GPVI agonist, CRP (Fig. 13B & C). This indicates that ELMO1 negatively regulates GPVI-mediated platelet functional responses even in the absence of generated thromboxane.

Figure 13: Enhanced thromboxane generation in response to GPVI agonists in

ELMO1-/- platelets. (A.) Washed murine platelets were stimulated with the indicated concentrations of CRP or collagen for 4 minutes and the reaction was stopped via flash- freezing. ThromboxaneB2 generation was determined using an ELISA kit as per manufacturer’s instructions. The data is represented as mean ± SE of at least three independent experiments and was analyzed by Student’s t-test. (* p <0.05). (B.)

Representative aggregation tracings and (C.) dense granule secretion of washed platelets from ELMO1-/- or wildtype (WT) littermate controls pre-incubated with Indomethacin 10

M for 5 minutes and activated with CRP 5 g/mL for 4 minutes. Aggregation and dense granule secretion was detected by using lumi-aggregometry under stirring conditions at

37 °C.

ELMO1 regulates thrombus formation in vitro.

Our previous data suggests that ELMO1 specifically regulates GPVI-mediated platelet activation. Therefore, we investigated the role of ELMO1 in thrombus formation in an in vitro flow over collagen model under arterial (1000 s-1) and venous (200 s-1) shear conditions. Whole blood from ELMO1-/- mice exhibited enhanced thrombus formation compared to the WT littermate control in both arterial and venous conditions (Fig. 14).

This is consistent with the enhanced dense granule secretion and thromboxane generation observed with ex vivo ELMO1-/- murine platelets in response to GPVI agonists.

Figure 14: ELMO1 regulates thrombus formation in vitro. (A.) Representative light microscopic image of thrombus formation on collagen coated surface and (B) percent thrombus area. Whole blood from wildtype (WT) littermate controls and ELMO1-/- perfused over collagen (50 g/mL) coated surface at arterial shear rate 1000 s-1 or venous shear rate 200 s-1 for 4 minutes. The direction of flow was right to left. (B.) The images were analyzed using ImageJ and the data is represented as percent thrombus area ± SE.

The data was analyzed by Student’s t-test (* p <0.05). WT and ELMO1-/- count = 3.

ELMO1 regulates both thrombosis and hemostasis in vivo.

To determine if ELMO1 plays a similar role in vivo, we utilized a pulmonary thromboembolism model via administration of collagen and epinephrine into the retro- orbital sinus in the WT littermate and ELMO1-/- mice. ELMO1-/- mice exhibited shorter survival rate compared to the WT littermate control (Fig. 15). We also evaluated the functional implication of the absence of ELMO1 on arterial thrombus formation upon

-/- FeCl3 injury. ELMO1 mice exhibited shorter times to occlusion compared to the WT littermate controls (Fig. 16). This indicates that ELMO1 negatively regulates GPVI- mediated thrombus formation in vivo.

To investigate whether ELMO1 has any hemostatic function, we performed a tail bleeding assay using ELMO1-/- and WT littermate mice. ELMO1-/- mice have shorter bleeding times compared to the WT littermate control indicating that the importance of

ELMO1 in hemostasis (Fig. 17). The hematological parameters, including platelet count and mean platelet volume, were unaltered in ELMO1-/- mice compared to the WT littermate control (Table 2). This indicates importance of ELMO1 in both hemostasis and thrombosis despite unaltered platelet count and mean platelet volume.

Figure 15: ELMO1 regulates thrombus formation in vivo. Survival curve of wildtype

(WT) and ELMO1-/- mice in a pulmonary thromboembolism model. Time to cessation of respiration was recorded after retro-orbital administration of 400 μg/kg collagen and 60

μg/kg epinephrine or phosphate buffered saline (PBS). WT count = 7. ELMO1-/- count =

6. The background of WT and ELMO1-/- mice is C57BL/6J.

Figure 16: ELMO1 negatively regulates thrombus formation following FeCl3-injury on carotid artery. (Ai.) Representative carotid artery blood flow tracings and (Aii.) time to occlusion from WT and ELMO1-/- mice. Carotid artery was isolated from the indicated mice and was exposed to 7.5% FeCl3-injury for 90 seconds. Data is represented as mean

± SE. WT count = 12. ELMO1-/- count = 14. Statistical analysis performed was Student’s t-test. (* p <0.05). The background of WT and ELMO1-/- mice is C57BL/6J.

Figure 17: ELMO1 regulates hemostasis in vivo. Tail Bleeding times of WT and

ELMO1-/-. The distal 3 mm of the tail was cut and the time it took for bleeding to stop was recorded. Each symbol represents one animal. WT count = 38. ELMO1-/- count = 18.

Statistical analysis performed was Student’s t-test. (* p <0.05). The background of WT and ELMO1-/- mice is C57BL/6J.

WT ELMO1-/-

White blood cells (x 109 cells/L) 3.39 ± 0.51 3.89 ± 0.39

Neutrophils (x 109 cells/L) 0.31 ± 0.03 0.45 ± 0.12

Lymphocytes (x 109 cells/L) 2.87 ± 0.51 3.22 ± 0.26

Red blood cells (x 109 cells/L) 7.89 ± 0.16 8.04 ± 0.11

Platelets (x 109 cells/L) 623.6 ± 24.6 714.1 ± 160.5

Mean platelet volume (fl) 4.08 ± 0.09 4.06 ± 0.09

Table 2: Hematologic analysis of WT and ELMO1-/- mice. Data is represented as mean ± standard error. (n = 5; unpaired Student’s t-test). The background of WT and

ELMO1-/- mice is C57BL/6J.

ELMO1 negatively regulates Syk activation in the GPVI signaling pathway.

The activation of Syk (45, 48, 49) upon activation of GPVI/FcRγ complex leads to downstream signaling events eventually leading to activation of PI3K (50) and PLC2

(51). Thus, we evaluated these signaling molecules to understand the role of ELMO1 in the GPVI signaling pathway. The phosphorylation of Syk and PLC2 are enhanced as early as 30 seconds in ELMO1-/- platelets compared to the WT littermate control platelets in response to CRP (Fig. 18A). Furthermore, Akt phosphorylation, a measure of PI3K activation, is also enhanced in the ELMO1-/- platelets downstream of GPVI signaling

(Fig. 18B). This indicates that ELMO1 plays an important role in regulating Syk downstream of GPVI signaling pathway. However, the enhanced signaling events downstream of the GPVI receptor in the ELMO1-/- platelets could be due to altered GPVI expression. Therefore, we evaluated the surface expression of GPVI in the ELMO1-/- and

WT littermate platelets by flow cytometry. The surface expression of GPVI was unaltered in ELMO1-/- platelets compared to the WT littermate control (Fig. 18C). This indicates that the ELMO1 plays an important role in negatively regulating Syk in the

GPVI signaling pathway even though the GPVI surface expression is unaltered.

Figure 18: ELMO1 regulates Syk phosphorylation in the GPVI pathway. (A. & B.)

ELMO1-/- and WT platelets were activated with CRP 1.25 g/mL at the indicated time points under stirring conditions at 37 °C at the indicated time points, proteins were precipitated and analyzed by Western blot. Blots were probed with the indicated antibodies. (C.) Surface expression of GPVI in washed murine platelets from WT and

ELMO1-/- assessed by flow cytometry. Black dashed and gray solid line is WT and

ELMO1-/- platelets with isotype control respectively. Black solid line is WT platelets and the gray filled histogram is ELMO1-/- platelets with FITC-GPVI antibody.

ELMO1 regulates RhoG activity in the GPVI signaling pathway.

We and others have previously shown that the absence of RhoG leads to decreased

GPVI-mediated platelet activation and thrombus formation (85, 99). RhoG is activated as early as 30 seconds in response to CRP and it regulates GPVI-mediated platelet activation by regulating Syk (85). Active RhoG was also found to interact with ELMO1 in platelets (99) however, the functional implication of this interaction has not been described in platelets. Therefore, we utilized full length bacterially expressed and purified

ELMO1-GST to determine the activity of RhoG in platelets from ELMO1-/- and WT littermate controls in response to CRP. RhoG activity was enhanced in the ELMO1-/- platelets in response to CRP compared to the WT littermate control (Fig. 19). This indicates that ELMO1 regulates RhoG activity by acting as a brake in GPVI-mediated platelet activation.

Figure 19: ELMO1 regulates RhoG activity in the GPVI pathway. RhoG activity in

ELMO1-/- and WT murine platelets in response to CRP. Washed murine platelets were activated with CRP 10 g/mL for 1 minute and lysed with NP-40 lysis buffer. Active

RhoG was pull-down using GST-ELMO1 fusion protein and RhoG activity was detected by Western blot.

3.3 Discussion

The generation of PIP3 upon class I PI3K activation acts as a central hub to recruit downstream effectors that are involved in a vast array of platelet functions such as platelet aggregation and thrombus formation (57). Effectors such as Btk and RASA3 are known to be important mediators of platelet activation (78, 89). However, there may be other proteins that can interact with PIP3 in platelets. Therefore, we performed a proteomic screen using PIP3 beads as bait to identify novel proteins that interact with

PIP3 in platelets. Our data confirms previous study using PIP3 beads to identify novel

PIP3 interacting proteins in platelets (106). We chose to focus on proteins that contain a

PH domain and found the following; RASA3, Btk, Cytohesin-2, DAPP1, and ELMO1.

Among these proteins, we chose to study ELMO1 since its function is unknown in platelets.

With the use of ELMO1-/- mice, we provide evidence that ELMO1 is important for platelet function and thrombosis. ELMO1-/- platelets exhibit enhanced platelet aggregation, granule secretion, and thromboxane generation specifically downstream of

GPVI pathway despite normal surface expression of GPVI. Consequently, whole blood from ELMO1-/- mice exhibit enhanced thrombus formation in an in vitro flow over collagen model under arterial and venous shear conditions. Consistently, ELMO1-/- mice exhibit decreased survival in an in vivo pulmonary thromboembolism model and have a

-/- shorter time to occlusion in the FeCl3-injury model. ELMO1 mice also exhibit shorter bleeding times compared to the wildtype littermate controls indicating its importance in thrombosis and hemostasis in vivo. However, ELMO1-/- mice are global knockout and

ELMO1 is ubiquitously expressed therefore we cannot rule out the contribution of other cell types in thrombus formation in vivo. Therefore, a platelet specific conditional

ELMO1-/- mice would be required to determine ELMO1’s role in thrombus formation devoid of contribution of other cells types in thrombus formation.

It is known that some PH domain containing proteins interact with PIP3 in nucleated cells

(88) and ELMO1-3 contain PH domain at the c-terminus (93). Previous in vitro studies indicate that the PH domain of ELMO1 does not interact with PIP3 along with other phosphoinositides (107). However, in this study, we provide evidence that endogenous

ELMO1 can interact with PIP3 in platelets. One possibility could be that ELMO1 can directly interact with PIP3 via its PH domain or indirectly by associating with other proteins such as DOCK in an endogenous system. DOCK proteins contain DOCK homology region (DHR)-1 domain that is well known to interact with PIP3 in nucleated cells (108-110), and ELMO1 can interact with DOCKA (DOCK180, 2, and 5) and

DOCKB subfamilies (DOCK 3 and 4). The proteomic screen in this study also reveals

DOCK5 (Appendix) as a PIP3 interacting protein in platelets therefore there is possibility that ELMO1 interacts with PIP3 indirectly via DOCK5. Future studies need to be conducted to determine whether ELMO1 directly or indirectly interacts with PIP3 in platelets.

Interestingly, we did not find ELMO2 and ELMO3 in our proteomic screen. However, by western blot analysis we did find ELMO2, not ELMO3, in human and murine platelets

(data not shown) which suggests that ELMO2 may not be interacting with PIP3 in platelets. Furthermore, ELMO2 is upregulated (data not shown) in platelets from

ELMO1-/- mice. However, ELMO2 does not seem to be compensating for the function of

ELMO1 since the platelet functional responses are intact. Whereas, the function of macrophages and fibroblasts isolated from ELMO1-/- mice were unaltered due to upregulation of ELMO2, suggesting a compensatory role for ELMO2 in these cells (80).

In this study, we provide evidence that Syk phosphorylation and the downstream signaling of Syk such as PLC2, and Akt phosphorylation, a measure of PI3K activation, are enhanced in the absence of ELMO1 in GPVI signaling. Therefore, we speculate that the potential ELMO1/ PIP3 interaction does not contribute to the GPVI-mediated platelet functional responses observed in the ELMO1-/- mice. Previous studies have shown that class I PI3K (86, 101) are activated downstream of Syk in the GPVI signaling pathway indicating that any alteration of Syk would lead to alteration of PI3K. Hence, we propose that the enhanced GPVI-mediated platelet function observed in ELMO1-/- platelets is due to altered Syk activation not PI3K.

However, how ELMO1 regulates Syk downstream of GPVI pathway is not understood. A potential mechanism on understanding how ELMO1 may regulate Syk activation could be RhoG. We and others have previously shown that RhoG, using genetic approaches, positively regulates GPVI-mediated platelet activation (85, 99). Furthermore, we reported that the absence of RhoG leads to diminished Syk activation (85) indicating that any

63 alteration of RhoG activity would lead to alteration of Syk activity downstream of GPVI signaling. Goggs et al, have also shown that ELMO1 specifically interacts with active

RhoG in platelets (99) however the functional implication of this interaction is not understood. Therefore, we utilized recombinant ELMO1 as a bait as a mode to detect active RhoG in the WT vs ELMO1-/- platelets downstream of GPVI signaling. Our data provides evidence that RhoG activity is enhanced in the ELMO1-/- murine platelets compared to WT platelets upon stimulation with CRP indicating that ELMO1’s ability to regulate Syk may be due to the interaction of active RhoG. Therefore, we propose that upon binding to RhoG, ELMO1 acts a brake in regulating RhoG activity downstream of

GPVI pathway.

Even though we observed enhanced RhoG activity in ELMO1-/- platelets using recombinant ELMO1 as a bait, this may be due to the absence of competition by endogenous ELMO1/active RhoG in ELMO1-/- platelets. Since the bait is added in excess to outcompete the endogenous ELMO1’s ability to bind to active RhoG, we speculate the competition of endogenous ELMO1 binding to active RhoG in the WT platelets should be minimal. Currently, the only mode to measure RhoG activity is the use of recombinant

ELMO1 as a bait.

The ability of ELMO1 to act as brake on RhoG activity relies on RhoG activation to occur first because ELMO1 specifically interacts with active RhoG (99) indicating RhoG is upstream of ELMO1 in platelets. This is consistent with previous studies in nucleated

64 cells where RhoG is known to be upstream of ELMO1 (98). Therefore, we propose the following model (outlined in Fig. 20) regarding the regulation of ELMO1 in platelets.

Under physiological conditions, RhoG is maintained inactive. Upon vascular injury, the activation of GPVI/FcR complex leads to activation of RhoG (85, 99). The activation of

RhoG leads to activation of Syk in turn leading to activation of downstream signaling molecules such as PI3K and PLC2 eventually leading to platelet activation (85).

Concomitantly to RhoG activation, ELMO1 interacts with active RhoG (99) which then acts as a brake to ensure platelets remain quiescent.

In conclusion, ELMO1 negatively regulates GPVI-mediated platelet activation and thrombus formation through binding to RhoG.

Figure 20: Model of ELMO1 mediated GPVI signaling in platelets.

CHAPTER 4

4 PROTEIN KINASE CY155F KNOCK-IN MICE REVEAL POSITIVE REGULATORY ROLE OF Y155 IN GPVI- MEDIATED PLATELET ACTIVATION

4.1 Introduction

Protein kinase C’s (PKC) are serine/threonine kinases that belong to the extended AGC family (protein kinases A, G & C) comprising of 10 isozymes (62). PKC isoforms are categorized into three classes based on their lipid and cofactor requirements; conventional (PKC, PKCI, PKCII, and PKC) depend on calcium and diacylglycerol

(DAG), novel (PKC, PKC, PKC, and PKC) depend on DAG and atypical (PKC and PKC/) which depend on neither. PKC isoforms contain an n-terminal regulatory motif, a hinge region, and a c-terminal catalytic motif. The activity of PKC is regulated by allosteric binding of cofactors and phospholipids, a series of serine/threonine phosphorylation’s which renders it catalytically competent, and the specific localization of PKC upon association with scaffold proteins such as receptor for activated C-kinase

(RACK) (62, 63). The activity of some PKC isozymes such as PKCcan be further regulated by tyrosine phosphorylation (79). Unlike the serine/threonine residues on PKC isozymes, the tyrosine residues on PKC are not conserved among the PKC isozymes

(79).

PKCcontains several tyrosine residues that have been shown to be phosphorylated and regulate its activity (79). In nucleated cells, specific tyrosine phosphorylation of PKC

67 upon different stimuli have been shown to play a role in PKC’s ability to be involved in various functions (79). Our group has previously shown that PKC negatively and positively regulates dense granule secretion in platelets downstream of GPVI and protease-activated receptor (PAR) pathways, respectively (65, 66). Furthermore, we and others have shown that PKCis phosphorylated on Y311 (human Y313) and Y565

(human Y567) downstream of GPVI and PAR pathways (112, 113). We hypothesize that the differential regulation of platelet activation by PKC may be due to differential tyrosine phosphorylation on PKC by different agonists.

In this study, we identified that of several tyrosine residues on PKC,Y155 is selectively phosphorylated by GPVI agonists. Therefore, we generated PKCY155F knock-in mice to characterize the function of Y155 phosphorylation in platelets. We provide evidence that platelet functional responses are diminished in PKCY155F platelets with GPVI agonists, but unaltered with PAR activation both ex vivo and in vivo. Finally, we show that PKCY155 regulates primary signaling downstream of GPVI via Syk.

4.2 Results

Phosphorylation on PKCY155 is specific to the GPVI pathway.

PKC is involved in a vast array of cellular functions and tyrosine phosphorylation on

PKC has been previously reported to modulate its activity depending on the cell type and stimulus (79). In platelets, PKC positively and negatively regulates dense granule secretion and thromboxane generation downstream of PAR and GPVI signaling pathways, respectively (65, 66). PKChas several tyrosine residues, in both human and mice, that could be phosphorylated downstream of platelet agonists (shown in Fig. 21A).

We hypothesized that PKC’s ability to differentially regulate platelet function may be due to differential tyrosine phosphorylation on PKC. We evaluated the tyrosine phosphorylation of PKC in human platelets in response to convulxin, a GPVI agonist, and thrombin, a PAR agonist by Western blot analysis, using phospho-specific antibodies. As previously reported by our group and others (65, 113), human PKCis phosphorylated on tyrosine residues 313 and 567 (Fig. 21B) in response to both convulxin and thrombin in human platelets respectively. We also observed that PKC is phosphorylated on Y332 and Y525 (Fig. 21B) by both convulxin and thrombin.

Interestingly, PKCY155 phosphorylation occurred only in response to the GPVI agonist, convulxin (Fig. 21B). We further evaluated the kinetics of PKCY155 phosphorylation in response to convulxin and found that PKCY155 is phosphorylated as early as 15 seconds in human platelets (Fig. 21C). These data indicate that PKCis phosphorylated on Y155 and is specific to the GPVI pathway in human platelets.

Figure 21: PKCY155 phosphorylation is specific to GPVI pathway. (A.) Pictorial representation of the murine tyrosine phosphorylation sites on PKC. Human (H) tyrosine phosphorylation sites on PKCare indicated above the murine tyrosine phosphorylation sites. (B.) Human platelets (2 x 108 cells/mL) were stimulated with the indicated agonists for 1 minute under stirring conditions at 37 °C, proteins were precipitated, and analyzed by Western blot analysis. (C.) Human platelets (2 x 108 cells/mL) were stimulated with 100 ng/mL of convulxin (CVX) at the indicated time points, proteins were precipitated, and analyzed by Western blot analysis. (B & C) Blots are representative of 3 independent experiments.

Generation and characterization of PKCY155F knock-in mice.

PKCY155 is located on the C1 domain of the regulatory motif and is restricted to PKC since it is not conserved within other PKC isozymes. PKCtyrosine 155 is also conserved in mice, therefore we generated PKC knock-in mice where PKCtyrosine

155 was mutated to phenylalanine, using the strategy outlined in Fig. 16A. Murine

PKCis a 674-amino acid protein consisting of 18 exons and tyrosine 155 is located on exon-5 of PKC. The Y155F mutation was introduced into exon-5 of PRKCD locus in the 129Sv/C57BL6j mice embryonic stem cells by homologous recombination (Fig.

22A). The clones positive for Y155F mutation were confirmed by dual selection using

G418 and Gancyclovir along with PCR and sequencing. The resulting chimeras were bred with transgenic mice expressing Cre recombinase to remove the PGKneo cassette.

The PKCY155F mice were identified by PCR containing a copy of LoxP in intron-6 which is 271 bp product compared to 181 bp wildtype littermate control (Fig. 22B). The mutation in the PCR product was confirmed by DNA sequence analysis (not shown). We further confirmed Y155F mutation by another PCR using oligonucleotides that recognize the mutant allele (Fig. 22C). PKCY155F mice are viable and follow predicted

Mendelian ratios. The blood cell profile is shown in Table 3. PKCY155F mice have a statistically significant increase in neutrophil count, and a slight but significant decrease in platelet count compared to WT control mice. All other parameters are unaltered compared to WT controls. However, the WT hematological parameters in Table 3 compared to the hematological parameters of WT in Table 2 (chapter-3) are significantly different and we speculate that these differences may potentially due to the different background of these mice.

Figure 22: Generation of PKCY155F knock-in model. (A.) Schematic representation of targeted generation of PKCY155F knock-in mice. Exons 2-9 are represented as grey vertical lines. PKCtyrosine 155 is located on Exon 5 and is indicated with an asterisk.

Identification of wildtype and PKCY155F knock-in mice using (B.) gtF/gtR primer pair

(indicated in panel A) and (C.) primers that specifically recognize PKCY155F site by

PCR.

Parameter WT PKCY155F

WBC (106/mL) 7.98 ± 0.21 10.73 ± 1.35

NE (106/mL) 0.91 ± 0.12 1.74 ± 0.25*

LY (106/mL) 6.56 ± 0.17 8.67 ± 1.14

Plt (106/mL) 776 ± 8.67 674 ± 40.43*

MPV (fL) 4.26 ± 0.04 4.24 ± 0.05

Table 3: Hematologic parameters in WT and PKCY155F mice. Whole blood was collected from WT and PKCY155F mice and analyzed using a Hemavet hematology analyzer. Abbreviations: WBC, white blood cells; NE, neutrophils; LY, lymphocytes;

Plt, platelets; MPV, mean platelet volume. Data is represented as mean ± SE and analyzed by Student’s t-test. * p < 0.05, n = 5. The background of the WT littermate control and PKCY155F is 129Sv/C57BL6j.

PKCY155 positively regulates GPVI-mediated platelet activation.

In order to investigate the function of PKCY155 in platelets, we performed ex vivo aggregation and dense granule secretion studies using platelets from PKCY155F and

WT littermate control mice. In response to a PAR4 agonist (AYPGKF), platelet aggregation and dense granule secretion were unaltered in PKCY155F mice compared to the wildtype littermate controls (Fig. 23). However, PKCY155F platelets exhibited decreased platelet aggregation and dense granule secretion in a concentration-dependent manner in response to the physiological GPVI agonist, collagen (Fig. 24). Similarly, platelet aggregation and dense granule secretion were also diminished in PKCY155F platelets with the GPVI agonist, CRP (Fig. 25). Both -granule release, as measured by

P-selectin exposure, and llb3 activity were reduced at all concentrations of CRP in platelets from PKCY155F mice compared to platelets from WT control mice (Fig. 26).

These data indicate that PKCY155 positively regulates GPVI-mediated platelet aggregation and dense granule secretion.

Figure 23: Aggregation and dense granule secretion is unaltered in response to

AYPGKF in PKCY155F platelets. (A.) Representative aggregation (C.) dense granule secretion tracings of washed platelets from PKCY155F (grey) and wildtype littermate control (WT, Black) activated with indicated concentration of AYPGKF for 4 minutes.

Platelet aggregation and dense granule secretion was performed under stirring conditions at 37 °C and detected by lumi-aggregometry. Quantification of (B.) aggregation and (D.) dense granule secretion of at least 3 independent experiments from panel A. and panel C. respectively. The quantification of aggregation and dense granule secretion is represented as mean ± SE and was analyzed by Student’s t-test.

Figure 24: Absence of PKCY155 phosphorylation in platelets leads to diminished aggregation and granule secretion following GPVI stimulation. (A.) Representative aggregation and (B.) dense granule secretion tracings of washed platelets from

PKCY155F or wildtype littermate controls (WT) treated with indicated concentrations of collagen for 4 minutes under stirring conditions at 37 °C. Platelet aggregation and dense granule secretion was detected by lumi-aggregometry. (C.) Quantification of extent of aggregation and (D.) dense granule secretion of panel A. and B. respectively and are represented as mean ± SE. The data was analyzed by Student’s t-test (*p<0.05).

Figure 25: Absence of PKCY155 phosphorylation in platelets leads to diminished aggregation and granule secretion following CRP stimulation. (A.) Representative aggregation and (B.) dense granule secretion tracings of washed platelets from

PKCY155F or wildtype littermate controls (WT) treated with indicated concentrations of CRP for 4 minutes under stirring conditions at 37 °C. Platelet aggregation and dense granule secretion was detected by lumi-aggregometry. (C.) Quantification of extent of aggregation and (D.) dense granule secretion of panel A. and B. respectively and are represented as mean ± SE. The data was analyzed by Student’s t-test (*p<0.05).

Figure 26: Diminished -granule secretion and integrin IIb3 activation following

GPVI stimulation in PKCY155F platelets. (A.) Surface P-selectin exposure and (B.) integrin IIb3 activation was detected by Flow Cytometry using P-selectin and JON/A antibodies respectively. Washed murine platelets were incubated with indicated concentration of CRP for 10 minutes at 37°C and reaction was stopped with formaldehyde. The data is represented as fold increase over basal and analyzed using

Student’s t-test (*p<0.05).

PKCY155 regulates thrombus formation in vitro.

Our previous data suggest that the function of PKCY155 is specific to the GPVI pathway. Therefore, we investigated the role of PKCY155 in thrombus formation in an in vitro flow over collagen assay under arterial shear conditions (1000 s-1). Whole blood isolated from PKCY155F mice exhibited decreased thrombus formation compared to that observed with whole blood isolated from WT littermate control mice (Fig. 27). This is consistent with the phenotype observed with ex vivo PKCY155F platelets in response to GPVI agonists.

Figure 27: PKCY155F regulates thrombus formation in vitro. (A.) Representative light microscopic image of thrombus formation on collagen coated surface. Whole blood from wildtype littermate (WT) and PKCY155F perfused over collagen (50 g/mL) coated surface at arterial shear rate 1000 s-1 for 4 minutes. The direction of flow is right to left. (B.) Quantification of thrombus area from panel A of at least three independent experiments by using ImageJ. The data is represented as percent thrombus area and analyzed by Student’s t-test (* p < 0.05).

PKCY155 regulates thrombus formation in vivo.

To determine if PKCY155 phosphorylation plays a similar role in vivo, we evaluated the role of PKCY155 in thrombus formation using a model of pulmonary thromboembolism. PKCY155F mice exhibited longer survival compared to the WT littermate control mice (Fig. 28A). We further evaluated the functional implication of

PKCY155 on arterial thrombus formation upon 5% FeCl3 injury. PKCY155F mice had longer time to occlusion compared to the WT littermate controls (Fig. 28B). This indicates that PKCY155 phosphorylation positively regulates GPVI-mediated thrombus formation in vivo.

However, we do observe differences in survival times and time to occlusion in these WT mice following pulmonary thromboembolism model and FeCl3 injury respectively, compared to the WT from Fig. 15 & 16. The WT littermate control in Chapter-3 are

C57BL/6J whereas the WT littermate control used in this chapter are mixed (C57BL/6J and 129/sv). Therefore, we speculate that the differences observed in the survival times and time to occlusion following pulmonary thromboembolism model and FeCl3 injury respectively, may potentially due to the different backgrounds of these mice.

Figure 28: PKCY155 regulates thrombus formation in vivo. (A.) Survival curve of

WT and PKCY155F mice following pulmonary thromboembolism model. Time to cessation of respiration was recorded after retro-orbital administration of Collagen 400

g/kg and epinephrine 60 g/kg or phosphate buffered saline (PBS). WT count = 25 and

PKCY155F count = 22. (B.) Time to vessel occlusion of WT and PKCY155F carotid artery upon FeCl3-injury. Carotid artery was isolated from PKCY155F or WT mice and was exposed to 5% FeCl3 for 90 seconds. Data is represented as mean ± SE and analyzed by Student’s t-test (* p < 0.05). WT and PKCY155F count = 7. The background of the

WT littermate control and PKCY155F is 129Sv/C57BL6j.

PKCY155 regulates GPVI-mediated platelet activation via Syk.

The GPVI/FcR complex is an important receptor that is activated upon vascular injury

(18) leading to the phosphorylation of the ITAM motif (44, 45) on the FcRchain by the constitutively associated SFK, Fyn and Lyn(46, 47), leading to the activation of Syk (45,

48, 49). The activation of Syk leads to downstream signaling events eventually leading to activation of PLC2 (51). Hence, we evaluated the phosphorylation’s of these signaling molecules in order to understand the role of PKCY155 in the GPVI signaling pathway.

Platelets from PKCY155F mice exhibited decreased Syk and PLC2 phosphorylation in response to GPVI agonist as early 10 seconds (Fig. 29A-B). As the decreased signaling observed downstream of GPVI in PKCY155F could be due to altered GPVI receptor number, we evaluated the surface expression of GPVI receptor by flow cytometry in

PKCY155F and the WT littermate control platelets. GPVI surface expression is unaltered in platelets from PKCY155F mice compared to the WT littermate controls

(Fig. 29C). This indicates that the decreased dense granule secretion and platelet aggregation observed in the PKCY155F platelets is due to reduced Syk activation.

Figure 29: PKCY155F regulates Syk in the GPVI pathway. (A. & B.)

Representative Western blot analysis of PKCY155F and WT platelets pre-incubated with Indomethacin 10 M, AR-C69931MX 100 nM, and MRS2179 100 M for 5 minutes and activated with CRP 10 g/mL at indicated time points. Time course was performed under stirring conditions at 37 °C, proteins were precipitated and analyzed by

Western blot. (C.) Surface expression of GPVI in washed murine platelets from WT and

PKCY155F assessed by flow cytometry. Black dashed line and grey solid line is WT and PKCY155F platelets respectively with isotype control. Black solid line is WT platelets with FITC-GPVI antibody and the gray filled histogram is PKCY155F platelets with FITC-GPVI antibody.

4.3 Discussion

We have previously published that dense granule secretion in platelets is differentially regulated by PKC using knockout mice (66). GPVI-mediated dense granule secretion appears to be negatively regulated by the association of SHIP1, Lyn, and PKC. As

SHIP1 associates with phospho-tyrosine residues, and PKC has several tyrosine residues, we postulated that the GPVI pathway and the GPCR pathways differentially phosphorylate tyrosine residues on PKC and that would differentially regulate SHIP1-

Lyn-PKC association and dense granule release (66, 114). We have identified that

PKCY155 phosphorylation occurs only downstream of GPVI pathways but not by

GPCR pathways. Hence, we evaluated the role of Y155 phosphorylation in platelet function using Y155F knock in mice.

PKCY155F platelets exhibited diminished GPVI-mediated dense granule secretion specifically downstream of the GPVI pathway despite normal surface expression of

GPVI. Consequently, PKCY155F mice exhibited diminished thrombus formation under arterial shear conditions using an in vitro flow over collagen model, longer survival in an in vivo pulmonary thromboembolism model, and longer time to occlusion in the ferric- chloride injury model. At the molecular level, PKCY155 regulates GPVI-mediated platelet activation potentially by regulating Syk phosphorylation and activation.

In platelets, Hall et al and our group have previously shown that PKC is tyrosine

85 phosphorylated on 311 (112-114) and 565 (113) upon GPVI and PAR stimulation. Hall et al. has also shown that PKC is not phosphorylated on Y52, Y155, Y332 and Y525 in platelets (113). Conversely, our study provides evidence that Y155, Y332, and Y525 on

PKC are phosphorylated upon GPVI activation. We speculate that one of the reasons for the observed differences could be due to the kinetics of activation. Hall et al (113) did not observe tyrosine phosphorylation of PKCY155 at 3 minutes and we provide evidence that Y155 phosphorylation occurs within 15 seconds with a GPVI agonist and is dramatically decreased within 3 minutes.

PKC negatively regulates GPVI-mediated dense granule secretion in platelets (65, 66) however, in this study, we provide evidence that PKCY155 positively regulates dense granule secretion downstream of the GPVI pathway. We do not know why there is a contrasting phenotype between the PKCknockout vs the PKCY155F knock-in. One explanation could be that the PKC might bind to other signaling molecules, at sites other than Y155, and this possibility occurs in the knock-in mouse but not in the knockout mouse. There is also the possibility that Y155 phosphorylation on PKCacts to sequester a phosphatase that regulates Syk. There are several tyrosine phosphatases in platelets that could regulate the function of Syk, such as TULA-2 (115), PTPRO (116-118), and SHP-1

(119).

In conclusion, PKCY155 phosphorylation is specific to the GPVI pathway in human platelets and it regulates GPVI-mediated thrombus formation possibly through Syk regulation.

CHAPTER 5

5 GENERAL DISCUSSION/FUTURE DIRECTIONS

GPVI is an important receptor that initiates platelet activation upon vascular injury resulting in platelet aggregation and thrombus formation. Within the past four decades, a lot of effort has been dedicated to understanding this receptor and the downstream signaling molecules however there are still gaps in knowledge. Therefore, to better understand the biology of platelets, the focus of this thesis to understand the bridge between the signaling molecules and their biological function downstream of GPVI pathway which may aid in development of future therapeutics.

In chapter 3, we provide evidence of novel proteins that interacted with PIP3 in platelets and among these proteins was ELMO1. We further utilized ELMO1-/- mice to study the function of ELMO1, a previously uncharacterized protein in platelets. In response to

GPVI agonists, we show that aggregation, granular secretion, integrin IIb activation, and thromboxane generation are enhanced in the ELMO1-/- platelets compared to the wildtype littermate control but unaltered with PAR4 agonist (AYPGKF). Furthermore,

ELMO1 negatively regulates aggregation and dense granule secretion devoid of thromboxane indicating its role in primary signaling. Consistently, we show that ELMO1 plays a role in hemostasis and thrombus formation in vivo. ELMO1 negatively regulates

GPVI-mediated platelet function by acting as a brake on RhoG activity.

In chapter 4, we provide evidence of differential tyrosine phosphorylation of PKC downstream of PAR and GPVI pathway. The phosphorylation of PKCY155 is specific to GPVI pathway not PAR pathway. Therefore, we generated PKCY155F knock-in mice to understand the function of PKCY155 in platelets. In response to GPVI agonists, granular secretion and integrin IIb activation is diminished in the absence of

PKCY155 and unaltered with PAR4 agonist. Consistently, PKCY155 regulates thrombus formation both in vitro and in vivo. PKCY155 positively regulates GPVI- mediated platelet activation by regulating Syk activity in primary signaling.

In conclusion, our study provides two novel regulators of GPVI-mediated platelet activation: ELMO1 and PKCY155. ELMO1 negatively regulates GPVI-mediated platelet activation by RhoG. PKCY155 phosphorylation is specific to GPVI pathway and Y155 phosphorylation on PKCpositively regulates GPVI-mediated platelets activation potentially regulating Syk.

The Role of ELMO1 in other major platelet receptors.

This study provides evidence that ELMO1 negatively regulates Syk in GPVI-mediated platelet function upon interaction with RhoG rather than by ELMO1/PIP3 interaction.

Therefore, we speculate that ELMO1’s ability to regulate Syk relies on its ability to bind to active RhoG and this might be restricted to ITAM signaling. Our laboratory and others have shown that RhoG only positively regulates GPVI/FcR (ITAM) signaling and does

88 not play a role downstream of P2Y12 and integrin IIb3 (85, 99). However, there is a possibility that ELMO1 may play a role downstream of P2Y12 and integrin IIb3 independent of RhoG via the ELMO1/PIP3 interaction since PI3K is an important signaling molecule that is activated downstream of both P2Y12 and integrin IIb3 (57).

Role of ELMO1 downstream of P2Y12

P2Y12 is a GPCR that is coupled to Gi and is activated by ADP in platelets. The activation of P2Y12 receptor leads to G-mediated activation of PI3K in turn leading to platelet aggregation and secretion (13). We speculate that ELMO1 may regulate P2Y12-mediated platelet response via its ELMO1/PIP3 interaction. To investigate this function, we plan on observing aggregation and secretion in ELMO1-/- and WT platelets in response to 2-

MesADP (P2Y1 and P2Y12 agonist). Furthermore, we plan on evaluating signaling molecules such as Akt phosphorylation, a measure of PI3K activation, downstream of 2-

MesADP to ensure that any phenotype we observe is not due to altered PI3K activation rather due to ELMO1/PIP3 interaction in ELMO1-/- platelets.

The role of ELMO1 in integrin IIb3-mediated platelet spreading and clot retraction

In nucleated cells, ELMO1 has been previously shown to regulate integrin-mediated spreading and cell migration via PI3K (108). In platelets, a major integrin that is not only involved in platelet aggregation but is important for outside-in signaling mediated clot retraction and spreading is IIb3 (120). PI3K is an important signaling molecule that positively regulates IIb3-mediated clot retraction and platelet spreading (59).

Therefore, we speculate that ELMO1 may regulate integrin-mediated platelet spreading and clot retraction. We plan on evaluating platelet spreading on immobilized fibrinogen

89 and clot retraction in platelets isolated from WT and ELMO1-/-. We further plan on evaluating signaling molecules such as Akt and Rac1 downstream of IIb3 in platelets from WT and ELMO1-/-.

Potential GPVI signaling regulator: DOCK5

A well-known ELMO1 interacting protein is DOCK. These proteins are GEFs for Rho

GTPases and consist of 11 family members which can be classified into following subfamilies: DOCKA (DOCK2, 5 and 180), DOCKB (DOCK3 and DOCK4), DOCKC

(DOCK6, 7 and 8) and DOCKD (DOCK9, 10 and 11. ELMO1 is known to interact with

DOCK proteins in the DOCK A and DOCK B subfamilies as these DOCK proteins contain an SH3 domain in the N-terminus that interacts with the Pro-X-X-Pro region of

ELMO1 in the C-terminus (121). In this study, we provide evidence that DOCK5, a GEF for Rac1 (122), is a potential PIP3 interacting protein in platelets using PIP3 beads however the function of this protein in platelets remains to be understood. We plan on determining the presence of DOCK5 in human and murine platelets by Western blot analysis using DOCK5 antibody. Furthermore, we plan on investigating the function of

DOCK5 by obtaining DOCK5 knockout mice from Dr. Côté’s laboratory at the Montreal

Clinical Research Institute (123). Since DOCK5 interacts with PIP3 in platelets and PI3K positively regulates GPVI-mediated platelet activation, we speculate that DOCK5 may play a role downstream of GPVI pathway. With the use of platelets from DOCK5-/-, we will evaluate ex vivo platelet aggregation, granular secretion (dense and alpha granules), thromboxane generation, integrin IIb3 activation in response to various agonists for

GPVI, PAR, P2Y1 and P2Y12 receptors. Furthermore, we will evaluate the role of

DOCK5 in hemostasis by tail bleeding assay and thrombus formation by the following: ex vivo flow over collagen model, in vivo pulmonary thromboembolism model, and ferric-chloride injury model.

To understand the mechanism how DOCK5 would regulate GPVI-mediated platelet activation, we plan on evaluating signaling molecules such Akt (PI3K activation), PLC2 and Rac1 downstream of GPVI signaling. We plan on detecting Rac1 activity using GST-

PAK-PBD as a bait downstream of GPVI pathway since DOCK5 is a GEF for Rac1 and

Rac1 is known to positively regulates GPVI-mediated platelet activation (124).

The role of SFK in Y155 phosphorylation on PKC

In nucleated cells, PKC is tyrosine phosphorylated by SFK (79). In this study, we provide evidence that PKCY155 positively regulates GPVI-mediated platelet activation.

However, it is not understood what phosphorylates PKCY155 in GPVI-signaling, we speculate SFK may phosphorylate PKCY155 in platelets. To do this, we plan on treating platelets (human and murine) with pharmacological inhibitors PP2 (SFK inhibitor) and

PP3 prior to stimulating with GPVI agonist and detect PKCY155 phosphorylation by

Western blot analysis. Furthermore, to figure out the individual SFK that phosphorylates

PKCY155, we plan on using individual SFK knockout mice and detect PKCY155 phosphorylation in response to GPVI agonist.

The role of PKCin CLEC-2-mediated platelet activation.

C-type lectin-like receptor 2 (CLEC2) consists of a hem-ITAM with a single YXXL/I motif (125). The activation of CLEC-2 receptor leads to activation of Syk which in turn leads to downstream signaling eventually leading to platelet aggregation and secretion

(101). In this study, we provide evidence that PKCY155 positively regulates an ITAM

(GPVI)-mediated platelet activation by regulating Syk. We propose that PKCY155 may positively regulate hem-ITAM-mediated platelet activation by Syk. To investigate the role of PKCY155 in CLEC-2 signaling, we plan on activating wildtype and

PKCY155F platelets with CLEC-2 agonists (CLEC-2 antibody and Rhodocytin).

Furthermore, we plan on evaluating signaling molecules such as Syk, PLC2, and Akt in

WT and PKCY155F platelets in response to CLEC-2 agonists.

Significance

Platelets are not only crucial mediators of hemostasis but are crucial mediators of thrombosis. The activation of platelets upon vascular injury is initiated by GPVI resulting in shape change, granular secretion, thromboxane generation eventually leading to aggregation and thrombus formation. However, dysregulation of this hemostatic function in a diseased vessel can lead to complications such as bleeding or thrombosis. Current anti-platelets therapies, aspirin and clopidogrel, are effective treatments options for cardiovascular diseases. However, there is bleeding risk associated with these therapies.

Therefore, understanding the biology of platelets may aid in development of future targeted therapy that can minimize bleeding risk associated with current therapies. Our study provides insights into two novel regulators of GPVI-mediated platelet activation:

ELMO1 and PKCY155.

Currently, there are no pharmacological inhibitors of ELMO1 however it might be interesting to design inhibitors to ELMO1 as a potential therapy for thrombocytopenic conditions to minimize bleeding risk. In this study, we show that ELMO1 acts as a brake in RhoG activity downstream of GPVI and active RhoG is known to interact with

ELMO1 at the armadillo repeats on ELMO1 (126). Therefore, by designing an inhibitor to the armadillo repeat, ELMO1 can no longer act as a brake on RhoG activity thereby resulting in increase in platelet responses which might be beneficial in thrombocytopenic conditions.

The phosphorylation of PKCY155 positively regulates GPVI-mediated platelet functional responses. Therefore, it would be interesting to design an inhibitor to

PKCY155 site for disease conditions with enhanced platelet responses such as cardiovascular diseases. This can be advantageous since the Y155 phosphorylation on

PKC is specific to the cell type and the agonist that induces this phosphorylation.

Although ELMO1 and PKCY155 could pose as beneficial therapeutic targets, both

ELMO1 and PKC are ubiquitously expressed which could lead to non-specific effects in other cells. Therefore, this study provides insights into the implication of any therapeutics that are designed for either ELMO1 or PKCY155 in platelets. Furthermore, the design of platelet specific targeted inhibition of ELMO1 and PKCY155 may aid in minimizing non-specific effects in other cells.

In conclusion, this study provides insights into regulators of GPVI-mediated platelet activation which may provide necessary basis for design of future therapies.

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APPENDIX

TableS1. Proteins that specifically interacted with PIP3. The table below is sorted such that proteins that interacted specifically with PIP3 are highlighted in gray and not with control beads. The PIP3 interacting proteins are ranked from highest abundance. PIP3=PIP3 beads. Ctrl = control beads.

PIP3 Ctrl_ Σ# Ratio Σ# _Plat Platel ΣCov Pep Accession Description PS elets ets (PIP3/ erage tide Ms A5: B5: Ctrl) s Area Area Q14644 Ras GTPase-activating 27.22 24 15 2.113 0.000 #DIV/ protein 3 OS=Homo 3 E8 E0 0! sapiens GN=RASA3 PE=1 SV=3 - [RASA3_HUMAN] Q06187 Tyrosine-protein kinase 25.49 14 44 7.481 0.000 #DIV/ BTK OS=Homo sapiens E7 E0 0! GN=BTK PE=1 SV=3 - [BTK_HUMAN] P26678 Cardiac phospholamban 21.15 1 2 2.765 0.000 #DIV/ OS=Homo sapiens E7 E0 0! GN=PLN PE=1 SV=1 - [PPLA_HUMAN] P68871 Hemoglobin subunit 15.65 2 4 9.787 0.000 #DIV/ beta OS=Homo sapiens E6 E0 0! GN=HBB PE=1 SV=2 - [HBB_HUMAN] P01857 Ig gamma-1 chain C 14.24 4 5 1.175 0.000 #DIV/ region OS=Homo E7 E0 0! sapiens GN=IGHG1 PE=1 SV=1 - [IGHG1_HUMAN] Q99418 Cytohesin-2 OS=Homo 11.50 4 13 2.342 0.000 #DIV/ sapiens GN=CYTH2 E7 E0 0! PE=1 SV=2 - [CYH2_HUMAN] P69905 Hemoglobin subunit 10.56 1 1 3.270 0.000 #DIV/ alpha OS=Homo sapiens E6 E0 0! GN=HBA1 PE=1 SV=2 - [HBA_HUMAN]

111

Q9UN19 Dual adapter for 10.36 2 2 1.196 0.000 #DIV/ phosphotyrosine and 3- E7 E0 0! phosphotyrosine and 3- phosphoinositide OS=Homo sapiens GN=DAPP1 PE=1 SV=1 - [DAPP1_HUMAN] P68366 Tubulin alpha-4A chain 7.59 3 3 1.353 0.000 #DIV/ OS=Homo sapiens E7 E0 0! GN=TUBA4A PE=1 SV=1 - [TBA4A_HUMAN] P06732 Creatine kinase M-type 7.35 2 4 6.042 0.000 #DIV/ OS=Homo sapiens E6 E0 0! GN=CKM PE=1 SV=2 - [KCRM_HUMAN] P10809 60 kDa heat shock 7.33 2 2 5.300 0.000 #DIV/ protein, mitochondrial E6 E0 0! OS=Homo sapiens GN=HSPD1 PE=1 SV=2 - [CH60_HUMAN] O00159 Unconventional myosin- 5.64 5 5 1.760 0.000 #DIV/ Ic OS=Homo sapiens E7 E0 0! GN=MYO1C PE=1 SV=4 - [MYO1C_HUMAN] Q6UXU1 Putative 4.17 1 1 1.235 0.000 #DIV/ uncharacterized protein E6 E0 0! UNQ6490/PRO21339 OS=Homo sapiens GN=UNQ6490/PRO2133 9 PE=5 SV=1 - [YC002_HUMAN] P05164 Myeloperoxidase 4.16 3 7 1.720 0.000 #DIV/ OS=Homo sapiens E7 E0 0! GN=MPO PE=1 SV=1 - [PERM_HUMAN] O75390 Citrate synthase, 3.43 1 1 6.115 0.000 #DIV/ mitochondrial E6 E0 0! OS=Homo sapiens GN=CS PE=1 SV=2 - [CISY_HUMAN] Q5T750 Skin-specific protein 32 3.20 1 1 1.062 0.000 #DIV/ OS=Homo sapiens E7 E0 0! GN=XP32 PE=1 SV=1 - [XP32_HUMAN]

112

Q6ZU80 Centrosomal protein of 2.10 2 2 2.848 0.000 #DIV/ 128 kDa OS=Homo E7 E0 0! sapiens GN=CEP128 PE=1 SV=2 - [CE128_HUMAN] Q12931 Heat shock protein 75 1.99 1 1 8.896 0.000 #DIV/ kDa, mitochondrial E6 E0 0! OS=Homo sapiens GN=TRAP1 PE=1 SV=3 - [TRAP1_HUMAN] P49748 Very long-chain specific 1.68 1 1 0.000 0.000 #DIV/ acyl-CoA E0 E0 0! dehydrogenase, mitochondrial OS=Homo sapiens GN=ACADVL PE=1 SV=1 - [ACADV_HUMAN] Q92556 Engulfment and cell 1.51 1 1 5.883 0.000 #DIV/ motility protein 1 E5 E0 0! OS=Homo sapiens GN=ELMO1 PE=1 SV=2 - [ELMO1_HUMAN] P08133 Annexin A6 OS=Homo 1.34 1 1 1.707 0.000 #DIV/ sapiens GN=ANXA6 E6 E0 0! PE=1 SV=3 - [ANXA6_HUMAN] Q9Y2L6 FERM domain- 1.16 1 1 8.321 0.000 #DIV/ containing protein 4B E6 E0 0! OS=Homo sapiens GN=FRMD4B PE=1 SV=4 - [FRM4B_HUMAN] Q9H7D0 Dedicator of cytokinesis 1.02 2 4 7.319 0.000 #DIV/ protein 5 OS=Homo E6 E0 0! sapiens GN=DOCK5 PE=1 SV=3 - [DOCK5_HUMAN] O94911 ATP-binding cassette 0.76 1 18 6.421 0.000 #DIV/ sub-family A member 8 E7 E0 0! OS=Homo sapiens GN=ABCA8 PE=1 SV=3 - [ABCA8_HUMAN] P59665 Neutrophil defensin 1 9.57 1 1 0.000 0.000 #DIV/ OS=Homo sapiens E0 E0

113

GN=DEFA1 PE=1 SV=1 0! - [DEF1_HUMAN] P54920 Alpha-soluble NSF 3.73 1 1 0.000 0.000 #DIV/ attachment protein E0 E0 0! OS=Homo sapiens GN=NAPA PE=1 SV=3 - [SNAA_HUMAN] O00124 UBX domain-containing 2.96 1 1 0.000 0.000 #DIV/ protein 8 OS=Homo E0 E0 0! sapiens GN=UBXN8 PE=1 SV=2 - [UBXN8_HUMAN] Q9NXN4 Ganglioside-induced 2.01 1 11 0.000 0.000 #DIV/ differentiation-associated E0 E0 0! protein 2 OS=Homo sapiens GN=GDAP2 PE=2 SV=1 - [GDAP2_HUMAN] Q8NFT8 Delta and Notch-like 1.90 1 1 0.000 0.000 #DIV/ epidermal growth factor- E0 E0 0! related receptor OS=Homo sapiens GN=DNER PE=1 SV=1 - [DNER_HUMAN] P12532 Creatine kinase U-type, 5.52 2 4 4.201 7.110 5.909 mitochondrial OS=Homo E6 E5 E0 sapiens GN=CKMT1A PE=1 SV=1 - [KCRU_HUMAN] Q96Q15 Serine/threonine-protein 0.49 2 10 1.588 3.837 4.140 kinase SMG1 OS=Homo E7 E6 E0 sapiens GN=SMG1 PE=1 SV=3 - [SMG1_HUMAN] P13929 Beta-enolase OS=Homo 10.83 3 5 9.810 2.689 3.649 sapiens GN=ENO3 PE=1 E6 E6 E0 SV=5 - [ENOB_HUMAN] P06576 ATP synthase subunit 28.92 12 67 1.022 2.820 3.623 beta, mitochondrial E8 E7 E0 OS=Homo sapiens GN=ATP5B PE=1 SV=3 - [ATPB_HUMAN] Q9BQE3 Tubulin alpha-1C chain 6.46 2 5 1.142 3.315 3.445 OS=Homo sapiens E7 E6 E0 GN=TUBA1C PE=1

114

SV=1 - [TBA1C_HUMAN] Q6KB66 Keratin, type II 7.30 3 39 3.781 1.331 2.840 cytoskeletal 80 OS=Homo E7 E7 E0 sapiens GN=KRT80 PE=1 SV=2 - [K2C80_HUMAN] P17661 Desmin OS=Homo 9.57 4 10 9.693 3.817 2.539 sapiens GN=DES PE=1 E6 E6 E0 SV=3 - [DESM_HUMAN] P21333 Filamin-A OS=Homo 29.24 54 15 1.621 7.599 2.134 sapiens GN=FLNA PE=1 5 E8 E7 E0 SV=4 - [FLNA_HUMAN] Q9Y490 Talin-1 OS=Homo 9.01 17 33 6.333 3.820 1.658 sapiens GN=TLN1 PE=1 E7 E7 E0 SV=3 - [TLN1_HUMAN] P00505 Aspartate 3.26 1 7 1.338 8.286 1.615 aminotransferase, E7 E6 E0 mitochondrial OS=Homo sapiens GN=GOT2 PE=1 SV=3 - [AATM_HUMAN] P25705 ATP synthase subunit 17.18 8 54 4.108 2.551 1.610 alpha, mitochondrial E7 E7 E0 OS=Homo sapiens GN=ATP5A1 PE=1 SV=1 - [ATPA_HUMAN] P35579 Myosin-9 OS=Homo 44.95 84 42 5.423 3.382 1.604 sapiens GN=MYH9 PE=1 8 E8 E8 E0 SV=4 - [MYH9_HUMAN] Q08554 Desmocollin-1 OS=Homo 3.91 3 17 1.899 1.241 1.531 sapiens GN=DSC1 PE=1 E7 E7 E0 SV=2 - [DSC1_HUMAN] P12259 Coagulation factor V 2.83 5 9 1.929 1.305 1.478 OS=Homo sapiens E7 E7 E0 GN=F5 PE=1 SV=4 - [FA5_HUMAN] Q9Y2K3 Myosin-15 OS=Homo 2.16 4 17 3.009 2.077 1.449 sapiens GN=MYH15 E7 E7 E0 PE=1 SV=5 - [MYH15_HUMAN]

115

P38646 Stress-70 protein, 20.03 10 12 9.070 6.855 1.323 mitochondrial OS=Homo E6 E6 E0 sapiens GN=HSPA9 PE=1 SV=2 - [GRP75_HUMAN] P07996 Thrombospondin-1 12.22 12 27 5.197 3.946 1.317 OS=Homo sapiens E7 E7 E0 GN=THBS1 PE=1 SV=2 - [TSP1_HUMAN] Q5D862 Filaggrin-2 OS=Homo 6.48 6 21 3.006 2.394 1.255 sapiens GN=FLG2 PE=1 E7 E7 E0 SV=1 - [FILA2_HUMAN] P08514 Integrin alpha-IIb 4.33 3 5 6.195 5.286 1.172 OS=Homo sapiens E6 E6 E0 GN=ITGA2B PE=1 SV=3 - [ITA2B_HUMAN] Q86TD4 Sarcalumenin OS=Homo 1.39 1 2 7.642 6.658 1.148 sapiens GN=SRL PE=2 E6 E6 E0 SV=2 - [SRCA_HUMAN] P11142 Heat shock cognate 71 11.46 6 12 1.312 1.219 1.076 kDa protein OS=Homo E7 E7 E0 sapiens GN=HSPA8 PE=1 SV=1 - [HSP7C_HUMAN] P16615 Sarcoplasmic/endoplasmic 5.76 5 7 1.295 1.317 9.830 reticulum calcium ATPase E7 E7 E-1 2 OS=Homo sapiens GN=ATP2A2 PE=1 SV=1 - [AT2A2_HUMAN] P22531 Small proline-rich protein 43.06 2 19 1.036 1.058 9.789 2E OS=Homo sapiens E7 E7 E-1 GN=SPRR2E PE=2 SV=2 - [SPR2E_HUMAN] Q13085 Acetyl-CoA carboxylase 1 5.84 11 18 1.519 1.937 7.845 OS=Homo sapiens E7 E7 E-1 GN=ACACA PE=1 SV=2 - [ACACA_HUMAN] Q5T749 Keratinocyte proline-rich 10.71 5 40 2.801 3.632 7.712 protein OS=Homo sapiens E7 E7 E-1 GN=KPRP PE=1 SV=1 - [KPRP_HUMAN] P28331 NADH-ubiquinone 6.88 4 7 8.649 1.131 7.644 oxidoreductase 75 kDa E6 E7 E-1 subunit, mitochondrial

116

OS=Homo sapiens GN=NDUFS1 PE=1 SV=3 - [NDUS1_HUMAN] P01834 Ig kappa chain C region 33.02 2 6 1.478 1.956 7.555 OS=Homo sapiens E7 E7 E-1 GN=IGKC PE=1 SV=1 - [IGKC_HUMAN] Q04695 Keratin, type I 34.49 18 72 1.192 1.584 7.520 cytoskeletal 17 OS=Homo 4 E9 E9 E-1 sapiens GN=KRT17 PE=1 SV=2 - [K1C17_HUMAN] P63316 Troponin C, slow skeletal 15.53 2 4 1.402 1.868 7.504 and cardiac muscles E7 E7 E-1 OS=Homo sapiens GN=TNNC1 PE=1 SV=1 - [TNNC1_HUMAN] P35609 Alpha-actinin-2 5.59 4 13 8.753 1.170 7.484 OS=Homo sapiens E6 E7 E-1 GN=ACTN2 PE=1 SV=1 - [ACTN2_HUMAN] P0CG48 Polyubiquitin-C 38.10 2 11 1.656 2.233 7.417 OS=Homo sapiens E7 E7 E-1 GN=UBC PE=1 SV=3 - [UBC_HUMAN] Q13201 Multimerin-1 OS=Homo 16.37 17 68 9.090 1.226 7.412 sapiens GN=MMRN1 E7 E8 E-1 PE=1 SV=3 - [MMRN1_HUMAN] P05089 Arginase-1 OS=Homo 18.63 5 15 1.051 1.429 7.356 sapiens GN=ARG1 PE=1 E7 E7 E-1 SV=2 - [ARGI1_HUMAN] P62736 Actin, aortic smooth 35.81 11 21 1.696 2.573 6.590 muscle OS=Homo sapiens 6 E8 E8 E-1 GN=ACTA2 PE=1 SV=1 - [ACTA_HUMAN] P05976 Myosin light chain 1/3, 10.31 2 15 1.351 2.089 6.467 skeletal muscle isoform E7 E7 E-1 OS=Homo sapiens GN=MYL1 PE=1 SV=3 - [MYL1_HUMAN] P11021 78 kDa glucose-regulated 6.27 3 7 7.711 1.200 6.427 protein OS=Homo sapiens E6 E7 E-1 GN=HSPA5 PE=1 SV=2 -

117

[GRP78_HUMAN]

P02675 Fibrinogen beta chain 15.27 5 6 7.606 1.196 6.358 OS=Homo sapiens E6 E7 E-1 GN=FGB PE=1 SV=2 - [FIBB_HUMAN] P10916 Myosin regulatory light 33.13 6 46 6.474 1.036 6.247 chain 2, E7 E8 E-1 ventricular/cardiac muscle isoform OS=Homo sapiens GN=MYL2 PE=1 SV=3 - [MLRV_HUMAN] P02768 Serum albumin 34.65 20 12 7.341 1.212 6.055 OS=Homo sapiens 7 E7 E8 E-1 GN=ALB PE=1 SV=2 - [ALBU_HUMAN] Q9HCC0 Methylcrotonoyl-CoA 7.82 3 5 8.575 1.457 5.886 carboxylase beta chain, E6 E7 E-1 mitochondrial OS=Homo sapiens GN=MCCC2 PE=1 SV=1 - [MCCB_HUMAN] Q8N1N4 Keratin, type II 25.77 13 21 7.515 1.298 5.788 cytoskeletal 78 OS=Homo 1 E7 E8 E-1 sapiens GN=KRT78 PE=2 SV=2 - [K2C78_HUMAN] P25311 Zinc-alpha-2-glycoprotein 7.38 2 17 7.071 1.246 5.677 OS=Homo sapiens E6 E7 E-1 GN=AZGP1 PE=1 SV=2 - [ZA2G_HUMAN] Q9H943 Uncharacterized protein 1.11 1 24 2.034 3.595 5.658 C10orf68 OS=Homo E7 E7 E-1 sapiens GN=C10orf68 PE=2 SV=2 - [CJ068_HUMAN] Q9H4B7 Tubulin beta-1 chain 13.75 5 7 6.496 1.191 5.455 OS=Homo sapiens E6 E7 E-1 GN=TUBB1 PE=1 SV=1 - [TBB1_HUMAN] P60709 Actin, cytoplasmic 1 36.27 12 20 1.488 2.746 5.417 OS=Homo sapiens 9 E8 E8 E-1 GN=ACTB PE=1 SV=1 - [ACTB_HUMAN]

118

P05109 Protein S100-A8 37.63 4 15 4.859 9.066 5.359 OS=Homo sapiens E7 E7 E-1 GN=S100A8 PE=1 SV=1 - [S10A8_HUMAN] P10599 Thioredoxin OS=Homo 12.38 1 9 8.897 1.678 5.301 sapiens GN=TXN PE=1 E6 E7 E-1 SV=3 - [THIO_HUMAN] Q562R1 Beta-actin-like protein 2 11.17 3 90 1.289 2.744 4.696 OS=Homo sapiens E8 E8 E-1 GN=ACTBL2 PE=1 SV=2 - [ACTBL_HUMAN] O43150 Arf-GAP with SH3 0.80 1 18 2.605 5.568 4.679 domain, ANK repeat and 0 E9 E9 E-1 PH domain-containing protein 2 OS=Homo sapiens GN=ASAP2 PE=1 SV=3 - [ASAP2_HUMAN] P02671 Fibrinogen alpha chain 4.62 3 7 1.109 2.400 4.619 OS=Homo sapiens E7 E7 E-1 GN=FGA PE=1 SV=2 - [FIBA_HUMAN] P01040 Cystatin-A OS=Homo 30.61 2 12 1.259 2.737 4.601 sapiens GN=CSTA PE=1 E7 E7 E-1 SV=1 - [CYTA_HUMAN] P04264 Keratin, type II 63.51 41 54 2.804 6.140 4.567 cytoskeletal 1 OS=Homo 53 E9 E9 E-1 sapiens GN=KRT1 PE=1 SV=6 - [K2C1_HUMAN] O95810 Serum deprivation- 7.06 2 3 5.993 1.365 4.391 response protein E6 E7 E-1 OS=Homo sapiens GN=SDPR PE=1 SV=3 - [SDPR_HUMAN] P63104 14-3-3 protein zeta/delta 8.98 2 3 4.615 1.102 4.188 OS=Homo sapiens E6 E7 E-1 GN=YWHAZ PE=1 SV=1 - [1433Z_HUMAN] O94925 Glutaminase kidney 10.61 5 9 1.129 2.765 4.082 isoform, mitochondrial E7 E7 E-1 OS=Homo sapiens GN=GLS PE=1 SV=1 - [GLSK_HUMAN]

119

P04406 Glyceraldehyde-3- 21.49 6 19 6.968 1.755 3.971 phosphate dehydrogenase E6 E7 E-1 OS=Homo sapiens GN=GAPDH PE=1 SV=3 - [G3P_HUMAN] Q02413 Desmoglein-1 OS=Homo 12.49 10 75 2.145 5.418 3.960 sapiens GN=DSG1 PE=1 E7 E7 E-1 SV=2 - [DSG1_HUMAN] P07339 Cathepsin D OS=Homo 4.37 1 3 1.815 4.933 3.678 sapiens GN=CTSD PE=1 E6 E6 E-1 SV=1 - [CATD_HUMAN] P40926 Malate dehydrogenase, 23.37 6 16 5.072 1.447 3.506 mitochondrial OS=Homo E6 E7 E-1 sapiens GN=MDH2 PE=1 SV=3 - [MDHM_HUMAN] P14923 Junction plakoglobin 19.06 10 28 7.965 2.346 3.395 OS=Homo sapiens E6 E7 E-1 GN=JUP PE=1 SV=3 - [PLAK_HUMAN] P35527 Keratin, type I 78.17 35 23 1.038 3.083 3.367 cytoskeletal 9 OS=Homo 15 E9 E9 E-1 sapiens GN=KRT9 PE=1 SV=3 - [K1C9_HUMAN] P15924 Desmoplakin OS=Homo 8.08 20 49 7.627 2.276 3.352 sapiens GN=DSP PE=1 E6 E7 E-1 SV=3 - [DESP_HUMAN] P81605 Dermcidin OS=Homo 26.36 3 79 3.132 9.817 3.190 sapiens GN=DCD PE=1 E7 E7 E-1 SV=2 - [DCD_HUMAN] Q99798 Aconitate hydratase, 12.82 8 26 1.533 4.853 3.158 mitochondrial OS=Homo E7 E7 E-1 sapiens GN=ACO2 PE=1 SV=2 - [ACON_HUMAN] P61626 Lysozyme C OS=Homo 37.16 4 22 1.163 4.173 2.788 sapiens GN=LYZ PE=1 E7 E7 E-1 SV=1 - [LYSC_HUMAN] Q86YZ3 Hornerin OS=Homo 13.58 10 67 1.067 5.085 2.098 sapiens GN=HRNR PE=1 E7 E7 E-1 SV=2 - [HORN_HUMAN] P08590 Myosin light chain 3 50.77 9 16 8.622 4.263 2.023 OS=Homo sapiens 5 E7 E8 E-1 GN=MYL3 PE=1 SV=3 -

120

[MYL3_HUMAN]

P13533 Myosin-6 OS=Homo 14.18 25 52 1.377 7.026 1.960 sapiens GN=MYH6 PE=1 E7 E7 E-1 SV=5 - [MYH6_HUMAN] P12883 Myosin-7 OS=Homo 13.28 23 51 1.377 7.026 1.960 sapiens GN=MYH7 PE=1 E7 E7 E-1 SV=5 - [MYH7_HUMAN] A6NL28 Putative tropomyosin 11.66 3 29 2.694 1.862 1.447 alpha-3 chain-like protein E8 E9 E-1 OS=Homo sapiens PE=5 SV=2 - [TPM3L_HUMAN] P06753 Tropomyosin alpha-3 17.25 5 34 2.716 1.901 1.429 chain OS=Homo sapiens E8 E9 E-1 GN=TPM3 PE=1 SV=1 - [TPM3_HUMAN] Q9Y623 Myosin-4 OS=Homo 4.95 9 22 7.887 6.148 1.283 sapiens GN=MYH4 PE=1 E6 E7 E-1 SV=2 - [MYH4_HUMAN] P31151 Protein S100-A7 11.88 1 5 5.190 4.861 1.068 OS=Homo sapiens E6 E7 E-1 GN=S100A7 PE=1 SV=4 - [S10A7_HUMAN] Q5SY68 Protein S100-A7-like 2 11.88 1 4 5.190 4.861 1.068 OS=Homo sapiens E6 E7 E-1 GN=S100A7L2 PE=2 SV=1 - [S1A7B_HUMAN] P09493 Tropomyosin alpha-1 29.58 9 75 2.029 1.922 1.056 chain OS=Homo sapiens E8 E9 E-1 GN=TPM1 PE=1 SV=2 - [TPM1_HUMAN] P06702 Protein S100-A9 31.58 4 27 1.007 1.669 6.034 OS=Homo sapiens E7 E8 E-2 GN=S100A9 PE=1 SV=1 - [S10A9_HUMAN] P31025 Lipocalin-1 OS=Homo 17.05 3 22 4.340 8.873 4.891 sapiens GN=LCN1 PE=1 E6 E7 E-2 SV=1 - [LCN1_HUMAN] Q5TH69 Brefeldin A-inhibited 0.55 2 22 2.621 7.284 3.599 guanine nucleotide- E6 E7 E-2 exchange protein 3

121

OS=Homo sapiens GN=ARFGEF3 PE=1 SV=3 - [BIG3_HUMAN] P62805 Histone H4 OS=Homo 19.42 2 2 0.000 5.073 0.000 sapiens GN=HIST1H4A E0 E6 E0 PE=1 SV=2 - [H4_HUMAN] P31944 Caspase-14 OS=Homo 17.77 5 7 0.000 1.076 0.000 sapiens GN=CASP14 E0 E7 E0 PE=1 SV=2 - [CASPE_HUMAN] P07355 Annexin A2 OS=Homo 16.22 5 8 0.000 1.005 0.000 sapiens GN=ANXA2 E0 E7 E0 PE=1 SV=2 - [ANXA2_HUMAN] P60174 Triosephosphate 15.03 3 3 0.000 2.301 0.000 isomerase OS=Homo E0 E7 E0 sapiens GN=TPI1 PE=1 SV=3 - [TPIS_HUMAN] Q01469 Fatty acid-binding protein, 14.81 2 4 0.000 5.653 0.000 epidermal OS=Homo E0 E6 E0 sapiens GN=FABP5 PE=1 SV=3 - [FABP5_HUMAN] O00217 NADH dehydrogenase 13.81 2 3 0.000 1.719 0.000 [ubiquinone] iron-sulfur E0 E8 E0 protein 8, mitochondrial OS=Homo sapiens GN=NDUFS8 PE=1 SV=1 - [NDUS8_HUMAN] P01876 Ig alpha-1 chain C region 12.46 3 4 0.000 1.031 0.000 OS=Homo sapiens E0 E7 E0 GN=IGHA1 PE=1 SV=2 - [IGHA1_HUMAN] P05141 ADP/ATP translocase 2 12.42 4 20 0.000 7.969 0.000 OS=Homo sapiens E0 E7 E0 GN=SLC25A5 PE=1 SV=7 - [ADT2_HUMAN] P27105 Erythrocyte band 7 12.15 2 2 0.000 2.464 0.000 integral membrane protein E0 E6 E0 OS=Homo sapiens GN=STOM PE=1 SV=3 - [STOM_HUMAN] P68431 Histone H3.1 OS=Homo 11.76 2 6 0.000 2.879 0.000 sapiens GN=HIST1H3A E0 E7

122

PE=1 SV=2 - E0 [H31_HUMAN] P12235 ADP/ATP translocase 1 11.74 4 20 0.000 7.641 0.000 OS=Homo sapiens E0 E7 E0 GN=SLC25A4 PE=1 SV=4 - [ADT1_HUMAN] P48047 ATP synthase subunit O, 11.74 2 4 0.000 8.690 0.000 mitochondrial OS=Homo E0 E7 E0 sapiens GN=ATP5O PE=1 SV=1 - [ATPO_HUMAN] P38117 Electron transfer 11.37 3 15 0.000 3.701 0.000 flavoprotein subunit beta E0 E7 E0 OS=Homo sapiens GN=ETFB PE=1 SV=3 - [ETFB_HUMAN] P32119 Peroxiredoxin-2 10.61 2 5 0.000 1.200 0.000 OS=Homo sapiens E0 E7 E0 GN=PRDX2 PE=1 SV=5 - [PRDX2_HUMAN] P29508 Serpin B3 OS=Homo 10.26 3 3 0.000 1.556 0.000 sapiens GN=SERPINB3 E0 E7 E0 PE=1 SV=2 - [SPB3_HUMAN] Q96RQ3 Methylcrotonoyl-CoA 9.66 5 6 0.000 1.368 0.000 carboxylase subunit alpha, E0 E7 E0 mitochondrial OS=Homo sapiens GN=MCCC1 PE=1 SV=3 - [MCCA_HUMAN] P02788 Lactotransferrin 9.15 4 6 0.000 6.303 0.000 OS=Homo sapiens E0 E6 E0 GN=LTF PE=1 SV=6 - [TRFL_HUMAN] Q02978 Mitochondrial 2- 8.92 2 3 0.000 4.588 0.000 oxoglutarate/malate E0 E6 E0 carrier protein OS=Homo sapiens GN=SLC25A11 PE=1 SV=3 - [M2OM_HUMAN] P40925 Malate dehydrogenase, 7.49 2 3 0.000 1.040 0.000 cytoplasmic OS=Homo E0 E7 E0 sapiens GN=MDH1 PE=1 SV=4 - [MDHC_HUMAN]

123

P67936 Tropomyosin alpha-4 7.26 2 6 0.000 1.089 0.000 chain OS=Homo sapiens E0 E8 E0 GN=TPM4 PE=1 SV=3 - [TPM4_HUMAN] P21912 Succinate dehydrogenase 6.79 2 2 0.000 7.310 0.000 [ubiquinone] iron-sulfur E0 E6 E0 subunit, mitochondrial OS=Homo sapiens GN=SDHB PE=1 SV=3 - [DHSB_HUMAN] Q96P63 Serpin B12 OS=Homo 6.67 3 5 0.000 1.051 0.000 sapiens GN=SERPINB12 E0 E7 E0 PE=1 SV=1 - [SPB12_HUMAN] P04220 Ig mu heavy chain disease 6.65 2 2 0.000 7.050 0.000 protein OS=Homo sapiens E0 E6 E0 PE=1 SV=1 - [MUCB_HUMAN] O60361 Putative nucleoside 6.57 1 2 0.000 5.439 0.000 diphosphate kinase E0 E6 E0 OS=Homo sapiens GN=NME2P1 PE=5 SV=1 - [NDK8_HUMAN] O60814 Histone H2B type 1-K 5.56 1 1 0.000 4.355 0.000 OS=Homo sapiens E0 E6 E0 GN=HIST1H2BK PE=1 SV=3 - [H2B1K_HUMAN] P47985 Cytochrome b-c1 complex 5.47 2 3 0.000 4.911 0.000 subunit Rieske, E0 E6 E0 mitochondrial OS=Homo sapiens GN=UQCRFS1 PE=1 SV=2 - [UCRI_HUMAN] P19429 Troponin I, cardiac 5.24 1 5 0.000 5.688 0.000 muscle OS=Homo sapiens E0 E7 E0 GN=TNNI3 PE=1 SV=3 - [TNNI3_HUMAN] Q15040 Josephin-1 OS=Homo 4.95 1 3 0.000 9.654 0.000 sapiens GN=JOSD1 PE=1 E0 E6 E0 SV=1 - [JOS1_HUMAN] P04040 Catalase OS=Homo 4.74 2 2 0.000 2.547 0.000 sapiens GN=CAT PE=1 E0 E6 E0 SV=3 - [CATA_HUMAN]

124

Q13835 Plakophilin-1 OS=Homo 4.55 2 3 0.000 3.885 0.000 sapiens GN=PKP1 PE=1 E0 E6 E0 SV=2 - [PKP1_HUMAN] P35030 Trypsin-3 OS=Homo 4.28 1 4 0.000 1.357 0.000 sapiens GN=PRSS3 PE=1 E0 E8 E0 SV=2 - [TRY3_HUMAN] P35232 Prohibitin OS=Homo 3.68 1 1 0.000 6.011 0.000 sapiens GN=PHB PE=1 E0 E6 E0 SV=1 - [PHB_HUMAN] Q15517 Corneodesmosin 3.40 1 2 0.000 1.508 0.000 OS=Homo sapiens E0 E7 E0 GN=CDSN PE=1 SV=3 - [CDSN_HUMAN] Q00325 Phosphate carrier protein, 3.31 1 4 0.000 3.115 0.000 mitochondrial OS=Homo E0 E7 E0 sapiens GN=SLC25A3 PE=1 SV=2 - [MPCP_HUMAN] P05165 Propionyl-CoA 3.02 2 3 0.000 6.045 0.000 carboxylase alpha chain, E0 E6 E0 mitochondrial OS=Homo sapiens GN=PCCA PE=1 SV=4 - [PCCA_HUMAN] P02679 Fibrinogen gamma chain 2.87 1 1 0.000 1.651 0.000 OS=Homo sapiens E0 E7 E0 GN=FGG PE=1 SV=3 - [FIBG_HUMAN] P05106 Integrin beta-3 OS=Homo 2.54 2 2 0.000 7.053 0.000 sapiens GN=ITGB3 PE=1 E0 E6 E0 SV=2 - [ITB3_HUMAN] P36542 ATP synthase subunit 2.35 1 1 0.000 1.474 0.000 gamma, mitochondrial E0 E7 E0 OS=Homo sapiens GN=ATP5C1 PE=1 SV=1 - [ATPG_HUMAN] P29350 Tyrosine-protein 2.35 1 1 0.000 2.822 0.000 phosphatase non-receptor E0 E6 E0 type 6 OS=Homo sapiens GN=PTPN6 PE=1 SV=1 - [PTN6_HUMAN] P40939 Trifunctional enzyme 2.10 2 3 0.000 2.484 0.000 subunit alpha, E0 E7 E0 mitochondrial OS=Homo sapiens GN=HADHA PE=1 SV=2 -

125

[ECHA_HUMAN]

Q99959 Plakophilin-2 OS=Homo 2.04 2 2 0.000 5.049 0.000 sapiens GN=PKP2 PE=1 E0 E6 E0 SV=2 - [PKP2_HUMAN] P31040 Succinate dehydrogenase 1.96 1 1 0.000 1.868 0.000 [ubiquinone] flavoprotein E0 E7 E0 subunit, mitochondrial OS=Homo sapiens GN=SDHA PE=1 SV=2 - [DHSA_HUMAN] Q86UK7 Zinc finger protein 598 1.88 1 7 0.000 1.263 0.000 OS=Homo sapiens E0 E7 E0 GN=ZNF598 PE=1 SV=1 - [ZN598_HUMAN] Q96QA5 Gasdermin-A OS=Homo 1.80 1 4 0.000 5.062 0.000 sapiens GN=GSDMA E0 E6 E0 PE=2 SV=4 - [GSDMA_HUMAN] Q9UJS0 Calcium-binding 1.78 1 1 0.000 6.661 0.000 mitochondrial carrier E0 E6 E0 protein Aralar2 OS=Homo sapiens GN=SLC25A13 PE=1 SV=2 - [CMC2_HUMAN] Q702N8 Xin actin-binding repeat- 1.63 3 12 0.000 2.053 0.000 containing protein 1 E0 E6 E0 OS=Homo sapiens GN=XIRP1 PE=1 SV=1 - [XIRP1_HUMAN] Q8NG92 Olfactory receptor 13H1 1.62 1 2 0.000 1.299 0.000 OS=Homo sapiens E0 E7 E0 GN=OR13H1 PE=2 SV=1 - [O13H1_HUMAN] Q05823 2-5A-dependent 0.94 1 9 0.000 2.914 0.000 ribonuclease OS=Homo E0 E7 E0 sapiens GN=RNASEL PE=1 SV=2 - [RN5A_HUMAN] Q99973 Telomerase protein 0.76 2 2 0.000 2.889 0.000 component 1 OS=Homo E0 E6 E0 sapiens GN=TEP1 PE=1 SV=2 - [TEP1_HUMAN]

126

Q92888 Rho guanine nucleotide 0.66 1 10 0.000 1.007 0.000 exchange factor 1 E0 E8 E0 OS=Homo sapiens GN=ARHGEF1 PE=1 SV=2 - [ARHG1_HUMAN] Q75N90 Fibrillin-3 OS=Homo 0.43 1 9 0.000 3.851 0.000 sapiens GN=FBN3 PE=2 E0 E7 E0 SV=3 - [FBN3_HUMAN]

127