FUNCTIONAL PROTEIN TYROSINE PHOSPHATASES IN PLATELETS ______
A Dissertation
Submitted to
The Temple University Graduate Board ______
In Partial Fulfillment
Of the Requirements for the Degree
DOCTOR OF PHILOSOPHY ______
by
Vaishali Vijay Inamdar
December 2017
Dissertation Examining Committee: Dr. Satya P Kunapuli, Physiology Dr. Rosario Scalia, Physiology Dr. Laurie Kilpatrick, Physiology Dr. Xiongwen Chen, Physiology Dr. Ulhas Naik, Professor, Thomas Jefferson University
ABSTRACT
FUNCTIONAL PHOSPHATASES IN PLATELETS
Platelets are small anucleate cells in blood that are derived from megakaryocytes and their primary function is to prevent bleeding. Upon vascular injury, the sub-endothelial collagen gets exposed to which platelets bind and aggregate eventually forming a platelet plug. There are several receptors on platelet surface that can be divided into two broad categories; the immune-receptor tyrosine-based activation motif (ITAM) and the
G protein-coupled receptors (GPCRs). The role of several protein tyrosine kinases
(PTKs) downstream of ITAM and GPCRs has been extensively studied. However, the role of protein tyrosine phosphatases (PTPs) have been under-investigated in platelets.
PTPs are important for dephosphorylating and activating or inactivating the protein.
Proteomics studies show presence of 10 receptor like and 10 cytoplasmic phosphatases in platelets. To date, only five non-transmembrane PTPs (NTPTPs), PTP-1B, Shp1,
Shp2, LMW-PTP, MEG2-PTP and a one receptor- like PTP (RPTP), CD148, have been identified in platelets. Therefore, there is a need to explore the role of other PTPs in platelet activation. The main objective of this thesis was to determine the role of two
PTPs – CD45 and PTPN7 in platelets.
We first studied the role of CD45 in platelets. CD45 is a receptor protein tyrosine phosphatase present on the surface of all hematopoietic cells except for erythrocytes and platelets. Proteomics studies, however, have demonstrated the presence of a CD45 c- terminal catalytic peptide in platelets. Therefore, we investigated the functional role of
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this truncated isoform of CD45 in platelets, which contains the c-terminal catalytic domain but lacks the extracellular region. We used an antibody specific to the c-terminus of CD45 to confirm the presence of a truncated CD45 isoform in platelets. We also examined ex vivo and in vivo platelet function using CD45 KO mice. Aggregation and secretion mediated by the glycoprotein VI (GPVI) receptor was impaired in CD45 KO platelets. Consequently, CD45 KO mice had impaired hemostasis indicated by increased tail bleeding times. Also, using a model of pulmonary embolism we showed that CD45
KO mice had defective in vivo thrombus formation. Next, we investigated whether or not the truncated isoform of CD45 had a role in GPVI signaling. The full-length isoform of
CD45 is known to regulate Src family kinase (SFK) activation in lymphocytes. We find a similar role for the truncated isoform of CD45 in platelets. SFK activation was impaired downstream of the GPVI receptor in the CD45 KO murine platelets.
Consequently, Syk, and PLC2 activations were also impaired in CD45 KO murine platelets. Thus, we concluded that the truncated CD45 isoform regulates GPVI-mediated signaling and platelet functional responses by regulating SFK activation.
Next, we investigated the role of another phosphatase PTPN7 in platelets. Thromboxane is a short-lived lipid mediator and acts as a local mediator to recruit more platelets to site of vascular injury and dysregulation of thromboxane generation can lead to several clinical complications like stroke, myocardial infarction etc. Extracellular signal- regulated protein kinase 1/2 (Erk1/2) plays an important role in promoting thromboxane generation in platelets. However, currently we have a poor understanding of how ERK is regulated in platelets. In lymphocytes ERK phosphorylation is regulated by a non- iii
transmembrane protein tyrosine phosphatase PTPN7. Thus, we hypothesized that PTPN7 is present in platelets and it negatively regulates ERK activation and consequently thromboxane generation. We tested this hypothesis using a PTPN7 knockout mouse model. Using PTPN7 specific antibody for western blot analysis we demonstrated that
PTPN7 is present in human as well as murine platelets. PTPN7 KO murine platelets show increased platelet functional responses such as aggregation, dense granule secretion and thromboxane generation upon stimulation with both GPCR and GPVI agonists. In presence of COX inhibitor indomethacin, the PTPN7 KO murine platelets aggregate and secrete to the same extent as the WT. This suggests that elevated thromboxane A2 levels are responsible for potentiation of platelet functional responses.
ERK phosphorylation is also potentiated in PTPN7 KO and the activity of signaling molecules upstream of ERK such as MEK and PKC which are upstream of is unaffected.
This shows that specificity of the PTPN7 towards ERK in platelets. Lastly, using the pulmonary embolism model we demonstrate a role for PTPN7 in thrombosis. Thus, we concluded that the PTPN7 regulates ERK activation and thromboxane generation in platelets.
In conclusion, we demonstrated that truncated CD45 is a positive regulator of GPVI- mediated SFK activation and platelet functional responses and PTPN7 negatively regulates ERK phosphorylation and thromboxane generation in platelets.
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DEDICATION
This dissertation is dedicated to my nephew Samved.
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ACKNOWLEDGEMENT
I would like to first of thank my mentor, Dr. Kunapuli, for giving me the opportunity to be a part of his lab and engage in platelet research for the last five years. Dr. Kunapuli is an excellent mentor and his teachings are invaluable. He has taught me how to systematically approach a problem, think critically and work efficiently and therefore has been a catalyst for my scientific career.
I would like to acknowledge my committee members: Dr. Rosario Scalia, Dr. Laurie
Kilpatrick, and Dr. Xiongwen Chen. I greatly appreciate all of their support, comments, and feedback during committee meetings. I would like to thank Dr. Laurie Kilpatrick for the guidance she provided while writing my outside research proposal for Ph.D. candidacy exam. I thank Dr. Ulhas Naik for his valuable inputs about my projects during
Gordon research conference.
I would like to thank Carol Dangelmaier for teaching me all the techniques in the lab and also helping me systematically troubleshoot. She has played a pivotal role in my understanding of techniques. I thank John Kostyak for helping me with all my projects and giving valuable advice and pep talks.
All the current and former members of Dr. Kunapuli’s laboratory have been very supportive. I was fortunate to share my journey in Kunapuli lab with Rachit Badolia and
Akruti Patel, they have truly made the Ph.D. journey more enjoyable. I would like to thank my seniors Dheeraj Bhavanasi and Bhanukanth Manne for their help with vi
understanding the field. I would also like to thank Priyanka and Andrew for the initial support. I would like to thank Anushka Dixit who has been with me through thick and thin. My parents Vijay and Smita, sister Pallavi and brother in law Sumeet, have been extremely supportive, incessantly encouraging and constantly inspiring me to give my best. I can’t thank them enough; I am here because of them. I would also like to mention my 6-month-old nephew Samved, who is my lucky charm. Last but not the least, I would be indebted forever to all the mice that I have sacrificed during my projects, if it was not for them I would have never been able to conduct my research and write this thesis.
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TABLE OF CONTENTS
ABSTRACT ...... ii DEDICATION...... v ACKNOWLEDGEMENT ...... vi LIST OF FIGURES ...... x
CHAPTER 1 ...... 1 1 INTRODUCTION...... 1 1.1 Platelet Physiology ...... 1 1.1.1 Introduction to platelets...... 1 1.1.2 Platelet Genesis ...... 1 1.1.3 Platelet Anatomy ...... 2 1.1.4 Platelet function in Hemostasis ...... 5 1.1.5 Other functions of platelets...... 9 1.2 Platelet Receptors...... 11 1.3 Protein Tyrosine Kinases and Protein Tyrosine Phosphatases ...... 18 1.4 Protein Tyrosine Phosphatases ...... 18 1.4.1 Classification of PTPs ...... 18 1.4.2 Mechanism of action of PTPs ...... 21 1.4.3 Regulation of PTPs ...... 23 1.5 Protein Tyrosine Phosphatases in platelets ...... 28 1.5.1 PTP-1B ...... 30 1.5.2 Shp1 and Shp2 ...... 30 1.5.3 LMW-PTP...... 32 1.5.4 MEG-2 ...... 32 1.5.5 CD148 ...... 33 1.6 Potential phosphatases in platelets ...... 34 1.6.1 CD45 ...... 34 1.6.2 PTPN7 ...... 37
CHAPTER 2 ...... 38 2 MATERIALS AND METHODS ...... 38
CHAPTER 3 ...... 44 3 IMPAIRED GLYCOPROTEIN VI-MEDIATED SIGNALING AND PLATELET FUNCTIONAL RESPONSES IN CD45 KNOCKOUT MICE ...... 44 Introduction ...... 44 Results ...... 46 Discussion...... 64
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CHAPTER 4 ...... 69 4 PTPN7 IS A NEGATIVE REGULATOR OF ERK ACTIVATION AND THROMBOXANE GENERATION IN PLATELETS...... 69 Introduction ...... 69 Results ...... 71 Discussion...... 85
CHAPTER 5 ...... 88 5 GENERAL DISCUSSION/ FUTURE DIRECTIONS ...... 88
REFERENCES ...... 98
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LIST OF FIGURES
Figure 1.1: Platelet structure...... 4 Figure 1.2: Steps in platelet activation...... 8 Figure 1.3: Major platelet receptors and their agonists ...... 16 Figure 1.4: Outline of proximal signaling events regulating platelet functional responses ...... 17 Figure 1.5: Classification of PTPs...... 20 Figure 1.6: The reaction mechanism catalyzed by the PTPs...... 22 Figure 1.7: Regulation of PTPs at different levels...... 26 Figure 1.8: Dimerization of PTPs...... 27 Figure 1.9: Protein Tyrosine Phosphatases in platelets and megakaryocytes...... 29 Figure 1.10: Structure of CD45 ...... 36 Figure 3.1: Presence of CD45 in platelets...... 49 Figure 3.2 Checking the specificity of CD45 c-terminal domain antibody...... 50 Figure 3.3: Platelet aggregation and dense granule in WT and CD45 KO murine platelets upon stimulation with CRP...... 53 Figure 3.4: Platelet aggregation and dense granule in WT and CD45 KO murine platelets upon stimulation with Collagen...... 54 Figure 3.5: Platelet aggregation and dense granule in WT and CD45 KO murine platelets upon stimulation with AYPGKF...... 55 Figure 3.6: -granule secretion, IIb3 integrin activation and GPVI surface expression in WT and CD45 KO platelets...... 56 Figure 3.7: In vivo platelet functional responses in WT and CD45 KO mice...... 58 Figure 3.8: Syk and PLC2 activation in CD45 KO and WT murine platelets upon stimulation with CRP 2g/ml...... 61 Figure 3.9: Syk and PLC2 activation in CD45 KO and WT murine platelets upon stimulation with CRP 10g/ml...... 62 Figure 3.10: SFK activation in CD45 KO and WT murine platelets upon stimulation with CRP...... 63 Figure 4.1: Expression of PTPN7 in human and murine platelets...... 73 Figure 4.2: Platelet aggregation and secretion in WT and PTPN7 KO murine platelets upon stimulation with AYPGKF...... 74 Figure 4.3: Platelet aggregation and secretion in WT and PTPN7 KO murine platelets upon stimulation with 2-MesADP...... 75 Figure 4.4: Platelet aggregation and secretion in WT and PTPN7 KO murine platelets upon stimulation with CRP...... 76 Figure 4.5: Aggregation and secretion in presence of indomethacin in WT and PTPN7 KO platelets...... 78 Figure 4.6: Thromboxane generation in WT and PTPN7 KO murine platelets...... 79 Figure 4.7: ERK phosphorylation in WT and PTPN7 KO murine platelets...... 81 Figure 4.8: p38 and MEK phosphorylation in WT and PTPN7 KO murine platelets...... 82 Figure 4.9: In vivo platelet functional responses in WT and PTPN7 KO mice...... 84
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CHAPTER 1
1 INTRODUCTION
1.1 Platelet Physiology
1.1.1 Introduction to platelets.
Platelets are main cellular components of hemostasis. They are discoid in shape and circulate at a relatively high concentration of 1.5-4 x 108/ml. They are in a quiescent state in circulation and get activated only in an event of a vascular injury. They have a small size and shape. Upon vascular injury, the sub-endothelial collagen gets exposed in the blood vessels. Platelets have receptors to recognize collagen that leads to primary activation of platelets. Primary platelet activation leads to the release of secondary mediators and there is also generation of thrombin which lead to further activation of platelets (1). Several diseased conditions may result in pathologic platelet activation which can lead to thrombotic disorders such as myocardial infarction and stroke.
Therefore, anti-platelet agents (e.g. aspirin and clopidogrel) are widely prescribed for individuals who have a high risk of developing thrombotic disorders. However, the major side effect of these drugs is bleeding. Thus, the search for ideal drug targets to treat platelet disorders is still on going.
1.1.2 Platelet Genesis
Platelets are derived from megakaryocytes. Megakaryocytes are highly specialized cells whose sole purpose is to generate platelets. Megakaryocytes are derived from pluripotent 1
stems cells and they undergo a process named as endomitosis. After endomitosis the megakaryocytes undergo cytoplasmic expansion to accumulate cytoplasmic proteins and granules that are essential for platelet formation. Formation of demarcation membrane system (DMS) is a characteristic of the cytoplasmic expansion stage. The cytoplasmic expansion is followed by massive re-organization of megakaryocyte cytoplasm into beaded cytoplasmic extensions known as pro-platelets. The pro-platelets then lead to platelet formation. Therefore megakaryocytes can be viewed as factories that fragment, releasing 2000-3000 platelets into the blood stream (2).
1.1.3 Platelet Anatomy
Platelets are anucleate cells, which have very limited de novo protein synthesis capacity.
Platelets derive their organelles, proteins and low levels of mRNA from the parental megakaryocyte. The platelets maintain the resting discoid shape due to the actin cytoskeleton and ring of microtubules along the plasma membrane of platelets. The open canilicular system (OCS) is a large network of invaginations in the plasma membrane which greatly enhances the surface area of the platelet membrane thereby increasing the sites for release of granular content. The OCS contains the dense tubular system, which is a Ca+2 store and the site for cyclooxygenase 1 (COX-1), critical for the production of thromboxane A2 (TxA2) which is a positive feedback platelet agonist. Apart from OCS, the plasma membrane expresses various platelet receptors such as glycoprotein receptors and G-protein coupled receptors (Figure 1.1).
2
The platelet cytoplasm contains several granules such as, -granules and dense granules, which secrete their contents upon activation. The -granules contain a wide variety of proteins such as chemokines, proteins that are involved in aggregation and growth factors. Fibrinogen and von Willebrand Factor (vWF) present in -granules plays a critical role in aggregation and thrombus formation. Chemokines such as platelet factor 4
(PF4) and SDF-1 attract leukocytes to the sites of vascular damage; and growth factors including PDGF, EGF and VEGF, are involved in the wound repair and angiogenesis.
The -granules also express a number of membrane proteins on their surface such as P- selectin. Second major type of granules are the dense granules and they contain the major feedback mediators, adenosine diphosphate (ADP) and adenosine triphosphate (ATP), as well as Ca+2, serotonin, and polyphosphates (Figure 1.1) (3, 4).
3
Figure 1.1: Platelet structure. a) Discoid shaped resting platelet b) Activated platelets interacting to form platelet aggregates via the interaction between GPIB/IIIa and fibrinogen (5).
4
1.1.4 Platelet function in Hemostasis
In normal conditions, platelets exist in a quiescent state and platelets are not able to bind to the endothelium. Endothelium releases PGI2 (prostacyclin) and nitric oxide, which inhibits platelet activation via the Gs signaling pathway (6). Endothelium also expresses the ectonucleoside triphosphate diphosphohydrolase (CD39/ENTPD1) that hydrolyzes
ATP and ADP to AMP, which prevents platelet activation (7). However, damage to the endothelium leads to the exposure of sub-endothelial matrix proteins such as vWF and collagen. Platelets recognize these sub-endothelial matrix proteins via their surface receptors. Binding of the receptor to the ligands results in a cellular signaling which activates the platelets. The role of platelets in the thrombus formation in hemostasis or under pathological thrombosis can be described in several stages (outlined in Figure 1.2):
(a) Tethering: In the arteries blood flows at a high shear rate. Under these conditions
platelets are unable to bind or tether to the damaged endothelium. However, the
exposed collagen gets coated with vWF and platelets adhere to the vWF via the
GP1b-IX-V complex on platelet surface. This leads to capture (or tethering) of
platelets however this interaction is relatively weak and platelets need a stronger
adhesion in the arterial blood flow. Under low shear stress (venous flow) the
platelets can bind to the collagen via the collagen binding integrin 21. However,
this interaction has a slow rate of association.
(b) Activation and stable adhesion: Platelets tethering to vWF brings the GPVI
receptor on platelet surface in close proximity to exposed collagen. Once the
GPVI receptor binds to collagen, there is GPVI-mediated signaling that leads to
platelet activation and also the integrins such as IIb3 and 21 on platelet 5
surface are transformed into an active conformation. This in turn leads to stable
adhesion through interactions with vWF and collagen, respectively. The GP1b-
IX-V complex also generates intracellular signals however they are incapable of
mediating rapid platelet activation.
(c) Spreading: Filopodia and lamellipodia are formed upon platelet activation.
Lamellopodia and filopodia both increase the surface area and therefore the area
in contact with the exposed sub-endothelial matrix is more. This leads to more
stable attachment (8).
(d) Secretion: Platelets release the contents of the - and dense-granules upon
activation. Platelets also generate thromboxane A2 (TXA2) which is also released
upon platelet activation. ADP is secreted from dense granules and TXA2 both
feedback on platelets to further activate the platelets. -granule contents such as
Fibrinogen and vWF, further support platelet aggregation by formation of a stable
hemostatic plug (9).
(e) Aggregation and thrombus growth: More platelets from the circulation are
recruited to the growing thrombus by a combination of membrane tethers and
binding to vWF (10). These additional platelets are activated by ADP and TxA2.
Fibrinogen from plasma membrane and secreted from -granules helps in cross-
linking of activated platelets (aggregation) via integrin IIb3. Activated platelets
also provide a pro-coagulant surface for the generation of thrombin through the
coagulation cascade. Thrombin plays two important roles. First, it activates
platelets and second it mediates the conversion of fibrinogen to fibrin which is
6
crosslinked to get a fibrin mesh. Platelets are crosslinked in this fibrin network
and form a plug that occludes the wound and prevents further blood loss.
(f) Clot retraction: The thrombus is further stabilized by clot retraction. Clot
retraction is mediated by the contraction of the actin cytoskeleton when IIb3
integrin binds fibrin/fibrinogen. This results in a platelet contractile force on the
fibrin network, which leads the clot to compact on itself and hence reduce its total
volume. This mechanism is known as clot retraction and its main physiological
function is to clear the obstructed vessel for renewed blood flow.
7
Figure 1.2: Steps in platelet activation. A schematic representation of the various stages of platelet activation including: tethering, integrin activation and stable adhesion, spreading and secretion, secretion and thrombus stability and clot retraction (11).
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1.1.5 Other functions of platelets.
Platelets are involved in several other processes apart from hemostasis. Platelet act as immune cells and are involved in fighting microbial infections. They also play a negative role in cancer by promoting tumor angiogenesis and metastasis (12-14). One more important function of platelets is to maintain vascular integrity and blood-lymph separation (15, 16). Platelets are not designed to differentiate between a disrupted vessel wall due to vascular injury or disruption of the vessel wall due to atherosclerotic disruption. Thus, platelet activation in such abnormal conditions can lead to dysregulated thrombus formation, further leading to blockage of blood vessels and ischemia.
Thrombotic diseases such as heart attack and ischemic stroke, are major causes of mortality in the modern world and therefore studying the platelets in this context is important.
a) Inflammation: Atherosclerosis is an example of a chronic inflammatory process.
During atherosclerosis, there is accumulation of lipids and lymphocytes within the intima of the large arteries. The fatty streak lesions are susceptible to rupture and plaque disruption can lead to platelet adhesion and aggregation. Platelets also play an important role in promoting the recruitment of inflammatory cells towards the lesion sites.
Activated platelets release several of inflammatory mediators which can lead to more influx of inflammatory cells. Platelets release cytokines such as CD40L and interlukin-1 which further inflames the endothelium and leads to the expression of adhesion molecules like ICAM-1, VCAM and p-selectin on the endothelial surface. Change in the endothelial morphology recruits more leukocytes like monocytes and neutrophils which 9
bind to adhesion molecules and transmigrate through the endothelial monolayer into the intima. This transmigration of monocytes plays a central role in atherosclerotic lesion formation (17).
b) Antimicrobial host defense: Platelets are known to function in innate host defense.
Platelets attack the microbial infections in the following in two different ways. (i) Release of anti-microbial proteins: platelet - granules contains one class of molecules know as platelet microbicidal proteins (PMPs) and kinocidins that exert direct antimicrobial effects and also potentiate the antimicrobial mechanisms of leukocytes. (ii) Recruitment to the sites of infection: Platelets express a number of chemotactic receptors such as
CXCR, CCR, N-f-MLF which recognize different components of the microbe. The chemoattractants released from the microbes, guide platelets toward sites of microbial infection. Platelets then interact with microorganisms leading to either opsonization, or antibody-dependent cell cytotoxicity (ADCC) of the microbe (18). Many bacterial infections can lead to severe conditions such as sepsis. Platelets play an important role in sepsis. During sepsis platelets interact with neutrophils to promote the formation of neutrophil extracellular traps (NETs) which play an important role to combat infections
(19). However uncontrolled release of NETs can lead to exacerbation of sepsis. The present antiplatelet therapies have the potential to modulate the interactions of platelets with other immune components and thereby reducing the secretion of inflammatory proteins into the milieu.
10
d) Angiogenesis: Angiogenesis is a process of new blood vessel growth from pre- existing vessels. Platelet secretory granules contain various proteins that regulate angiogenesis. For example platelets trigger and regulate the angiogenic response by secreting factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), Fibroblast growth factors (FGF), Epidermal growth factor (EGF)
(20). Platelets also have angiostatic factors however the overall effect of platelets is stimulatory or angiogenic on blood vessel development.
1.2 Platelet Receptors
Platelets express several receptors on their surface. The receptors can be divided into two broad groups based on their signaling pathways: 1) Immunoglobulin-superfamily receptors, which activate platelets via tyrosine kinase pathway. This class includes the
GPVI/ FcRγ, integrin αIIbβ3, FcγRIIA and CLEC-2. 2) The second class of surface receptors is the G-protein-coupled receptors (GPCRs), e.g. Protease activated receptors
(PARs), P2Y receptors and thromboxane prostanoid receptor (TP) (21-23) (Figure 1.3 and 1.4).
(A) Tyrosine kinase-linked receptors
There are two main sub-classes of the tyrosine kinase-linked receptors: (i) ITAM
(immunoreceptor tyrosine-based activation motif)-containing receptors. This class includes the Fc receptor-γ chain (FcRγ) which is associated with the GPVI receptor and
FcγRIIA. ITAMs have the classic YXX(L/I)X6-12 YXX(L/I) with two tyrosine residues
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in its cytoplasmic domain. (ii) hemITAM-containing receptor, e.g. CLEC-2, which has only one tyrosine residue (YXX(L/I)) in its cytoplasmic domain (24, 25)
(1) Collagen receptor-GPVI
Collagen is a sub-endothelial matrix protein, which activates platelets via GPVI/ FcRγ receptor. Upon stimulation of the GPVI receptor by collagen, there is a signaling cascade that results in platelet activation. SFKs downstream of the GPVI receptor get activated upon GPVI receptor stimulation, and they phosphorylate the ITAMs on the two tyrosine residues. Syk gets recruited to the phosphorylated ITAMs and then gets auto- phosphorylated and activated. Further steps lead to phosphorylation and activation of
PLCγ2. PLCγ2 hydrolyze the membrane phospholipids to generate secondary inositol-
1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG), which in turn mediate a cellular increase in both Ca+2 and protein kinase C (PKC) activity. IP3 mediates Ca+2 release from intracellular stores. This Ca+2 release stimulates extracellular Ca+2 entry through the
CRAC (Ca+2 -release-activated Ca+2) channel Orai-1. Also, DAG and Ca+2 together control CALDEG-GEF1, a Rap1b GTP-exchange factor. Rap1b plays a significant role in integrin αIIbβ3 activation (26). PKCs that are activated have been shown to regulate secretion, i.e., the release of both dense and α-granules in mouse platelets (27).
(2) Fibrinogen receptor-integrin IIb3
αIIbβ3 is a major integrin receptor on platelets. αIIbβ3 mediates platelet aggregation by cross-linking platelets through soluble fibrinogen, vWF and other adhesion proteins, including fibronectin and vitronectin. In resting platelets αIIbβ3 exists in a ‘low-affinity’ 12
conformation and the inside-out signaling leads to a conformational change to a ‘high- affinity’ state. In high-affinity conformation, αIIbβ3 binds to fibrinogen, and triggers a signaling cascade called as outside-in signaling that leads to filopodia and lamellipodia formation, secretion, and clot retraction. Glanzmann’s thrombasthenia is a condition in which either subunit of the integrin patients is absent. Platelets from these patients show defective platelet aggregation, which indicates that αIIbβ3, is essential for aggregation.
Outside-in signaling through the integrin is similar to GPVI signaling and hence leads to further PLCγ2 activation though SFK and Syk activation, reinforcing platelet activation, and activation of myosin light chain (MLC) which plays a role in shape change, actin stress fiber formation and clot retraction.
(B) G protein-coupled receptors
GPCRs play a central role in regulating platelet activation and inhibition. Some of the most important platelet activation receptors are the thrombin protease-activated receptors
(PAR)-1 and PAR-4; the ADP receptors P2Y1 and P2Y12; and the thromboxane (TxA2) receptor, thromboxane prostanoid (TP).
(1) Thrombin receptors- Protease activated receptors (PARs)
Thrombin (Factor IIa) is generated via the coagulation cascade from prothrombin.
Thrombin cleaves fibrinogen (Factor Ia) to form fibrin, which gets crosslinked to form a mesh-like network in which blood cells and platelets get trapped forming a thrombus.
Thrombin can also activate the PAR receptors on platelets, by cleaving the N-terminus of the PAR receptors, exposing a tethered ligand. Human platelets express two isoforms of
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PAR receptor- PAR-1 and PAR-4 whereas murine platelets have PAR-4 receptor but they lack the PAR-1 receptor instead they have PAR-3. The PAR-3 receptor does not directly signal but facilitates the interaction between thrombin and PAR-4 (28, 29). Both PAR-1 and PAR-4 on human platelets are associated with Gq and G12/13. Gq signaling downstream of PAR-1 and PAR-4 activation by thrombin leads to the activation of PLCβ followed by generation of IP3 and DAG, which in turn activate PKC and Ca+2 mobilization respectively. G12/13 leads to activation of Rho kinases via the activation of p115Rho, a GTP exchange factor. Rho kinases then regulate myosin light chain phosphatase, thereby playing a role in shape change and stress fiber formation.
(2) ADP receptors- P2Y1 and P2Y12
P2Y1 and P2Y12 receptors are activated by ADP released from dense granules upon platelet activation. P2Y1 is coupled to Gαq while P2Y12 is coupled to Gαi classes of G proteins. Gq activates PLCβ, which is followed by generation of IP3 and DAG, which in
+2 turn activate PKC and Ca mobilization respectively. Activation of P2Y12 coupled Gi acts in two different pathways. The first pathway inhibits the formation of cAMP through inhibition of adenylyl cyclase and the second pathway activates PI3-kinase. PI3-Kinase phosphorylates inositol-bisphosphate in the membrane to give inositol 3,4,5-triphosphate to provide binding sites for the pleckstrin-homology domains of proteins such as Akt. Co- activation of P2Y1 and P2Y12 is necessary for normal ADP-induced platelet activation as inhibition of either receptor results in a marked decrease in platelet aggregation. Platelets also express an ATP receptor, P2X1, which is a Ca+2 ion channel. The physiological significance of P2X1 is uncertain due to its low expression level.
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(3) Thromboxane A2 receptor-TP
TXA2 is a short-lived lipid mediator synthesized by activated platelets, which serve to recruit more platelets to the site of injury and also amplify the initial activation signals.
TXA2 is generated from prostaglandin H2 (PGH2) by thromboxane-A synthase. TXA2 is lipid soluble and crosses the plasma membrane, exiting the platelet and binding to the TP receptor on the surface of same platelet or other platelets in the vicinity in an autocrine or paracrine manner respectively. The TP receptor couples to Gq and G12/13, therefore, signal in the same manner as the thrombin receptors. Patients deficient in TXA2 production, which display a mild bleeding disorder this suggests the importance of the positive feedback by TXA2. Thus, TXA2 and ADP work synergistically to amplify platelet activation signals.
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Figure 1.3: Major platelet receptors and their agonists (30).
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Figure 1.4: Outline of proximal signaling events regulating platelet functional responses (31).
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1.3 Protein Tyrosine Kinases and Protein Tyrosine Phosphatases
Downstream of the platelet receptors there are several signaling molecules, which help to convey the signal for platelet activation. Protein tyrosine phosphorylation is one of the critical cell signaling mechanism, which helps to relay the signal in platelets. Regulation of the protein tyrosine phosphorylation is a combined activity of the protein-tyrosine kinases (PTKs) and protein- tyrosine phosphatases (PTPs). If the delicate balance of tyrosine phosphorylation within the cell is disrupted, it can lead to the onset of several human diseases, such as cancer (32-34). Therefore, studying the role of these phosphatases has significant implications for the development of novel therapies to treat disease.
The role of PTK's has been explored to a greater extent as compared to PTPs (35, 36).
Also, earlier, PTPs were thought to be constitutively active and dephosphorylated every substrate they encountered. However, in recent years, it was demonstrated that PTPs have specific substrates. (37-41)
1.4 Protein Tyrosine Phosphatases
1.4.1 Classification of PTPs
All PTPs have a signature motif, HCX5R, which is present in the PTP catalytic domain, where the cysteine is essential for catalysis and acts as a nucleophile (42). 107 genes in the human genome encode for PTPs (35). There are 105 Mouse orthologs for these genes.
Out of the 107 PTPs, only 81 of the PTPs are active protein phosphatases with the ability
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to dephosphorylate phosphotyrosines. Classification of PTPs into four groups (Classes I –
IV) is based on the amino acid sequences of their catalytic domains and their substrate specificity (Figure 1.5) (35). The first class of phosphatases is the class I cysteine-based
PTPs. There are two broad sub-categories of class I cysteine-based PTPs, the ‘classical’
PTPs, which are tyrosine-specific, and the ‘dual-specific’ PTPs which have a broad substrate specificity. The classical PTPs are further subdivided into the 21 receptor-like
PTPs (RPTPs), and the 17 non-transmembrane (NTPTPs); and the dual-specific PTPs are divided into seven subgroups based on substrate specificity (Figure 1.5). There is only one class II cysteine-based PTP known as the Low molecular weight PTP (LMW- PTP).
The Class III cysteine-based PTP family contains three members, which comprise of three cell cycle regulators CDC25A, CDC25B, and CDC25C. The last types of phosphatases are the Asp-based phosphates, which are Eya and have been shown to have
Tyr/Ser phosphatase activity.
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Figure 1.5: Classification of PTPs. Class I Cys-based PTPs (green), class II Cys-based PTPs (yellow), class III Cys-based
PTPs (blue), and Asp-based PTPs (pink). The substrate specificity of each class of PTP is listed (grey) (35).
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1.4.2 Mechanism of action of PTPs
The phosphate is removed from the substrate by phosphates in a two-step reaction. In the first step of the reaction, the active site cysteine carries out a nucleophilic attack on the phosphorus atom of the substrate. During this time, there is cleaving of the ester bond and the proton from the well-positioned general acid (aspartic acid) is donated to the leaving group oxygen. Therefore, after the first substitution the phosphate group is gets covalently attached to the cysteine through a thioester linkage.
The second step of the reaction is hydrolysis of the phosphate group from the cysteine- phosphate linkage. The aspartate molecule that donates its proton in the first phase of the reaction abstracts one proton from water. Thus, the water molecule acts as a nucleophile for the phosphate. This results in efficient hydrolysis of the phosphate (43) (Figure 1.6)
21
Figure 1.6: The reaction mechanism catalyzed by the PTPs. The above figure shows the two step hydrolysis of the phosphate molecule (43).
22
1.4.3 Regulation of PTPs
Cells express various PTKs and PTPs and tyrosine phosphorylation can occur if PTPs are tightly regulated. Following are the mechanisms of regulation for the PTPs.
(A) Expression of protein tyrosine phosphatase:
Differential expression of PTPs is an important regulator of PTP function. There is a ubiquitous expression of certain PTPs whereas some PTPs are restricted to a cell type.
For example, there is a ubiquitous expression of PTP1B and Shp2 whereas PTPN7 and
CD45 are limited to the hematopoietic cells (44). There is upregulation of certain PTPs when cell density increases such as including PTP-LAR (45) and RPTP (46). Also, the time of expression of the PTPs may vary within the cells. For example, at the beginning and end of smooth muscle cell proliferation in restenosis, there is a dynamic expression pattern for PTPs (47) . (outlined in figure 1.7)
(B) Subcellular localization.
The correct location of PTP within the cell is necessary for the appropriate function of
PTP. Distribution of the PTPs in cells ranges from the plasma membrane to cytoplasm.
Out of the 38 classical PTPs, 21 PTPs are receptor type, and 17 are cytoplasmic. CD148 and CD45 are the examples of receptor-type PTP's whereas PTPN7, PTP1B, and Shp1 are cytoplasmic PTPs (48). Although Shp1 and PTP1B are cytoplasmic phosphatases, they are recruited to regulate the phosphorylation of the surface receptors. Some studies have suggested the presence phosphatases in the endoplasmic reticulum. PTP1B is the 23
best-studied example phosphates localized in the endoplasmic reticulum (49). Another interesting example of cellular targeting is the localization of PTP-MEG2 to secretory vesicles (50).
(C) Post-translational modification: phosphorylation.
Phosphorylation of PTPs is one of the mechanisms by which PTPs are regulated however there is limited knowledge on how phosphorylation regulates PTPs. Serine and Tyrosine residues of RPTPα, CD45, PTP-1B, PTP-PEST, Shp1, and Shp2 are phosphorylated.
Phosphorylation of RPTPα at Tyr-789 and Tyr-810 have entirely different functions.
Phosphorylation at Tyr 810 increases the catalytic activity of the PTP whereas phosphorylation of Tyr-789 is required for mediating activation of Src and also recruitment of Grb-2, which antagonizes Src activation (51, 52). The cytoplasmic phosphatase, PTPN7, is phosphorylated at Ser-23 by PKA (53). The phosphorylation of
Ser-23 results in dissociation of PTPN7 from its substrate which allows the substrate to get phosphorylated. Phosphorylation of PTPN7 at ser-225 by PKC theta results in lipid raft targeting of PTPN7 (54).
(D) Post-translational modification: oxidation.
It is well known that reactive oxygen species (ROS) reversibly inhibit PTPs by transiently oxidizing the active site cysteine (55, 56). The primary sources of ROS appear to be the mitochondria or NADPH oxidases. PTP oxidation results in the formation of a covalent bond between the sulfur of cysteine and the nitrogen of serine which makes the catalytic cysteine unavailable for its function (57-59). Two commonly used general PTP
24
inhibitors, vanadate, and pervanadate, work by oxidizing the active site cysteine of all classical PTPs (60). Vanadate is a reversible inhibitor of PTPs while pervanadate is irreversible (60).
(E) Ligands.
The extracellular regions of RPTPs are large and highly glycosylated and may regulate
PTP function. There is insufficient knowledge about the ligands for RPTPs. RPTPζ is one of the best-characterized RPTPs that is regulated by ligand binding. Binding of growth factor pleiotrophin (PTN) to RPTPζ leads to inhibition of the phosphatase. Following binding of the PTN to RPTPζ, there is an increase in phosphorylation of RPTPζ substrates (61).
(F) Dimerisation.
Transmembrane proteins are regulated by a mechanism known as dimerization.
Dimerization leads to inactivation of Several RPTPs. The ‘inhibitory wedge’ model proposes that an helix-turn-helix wedge domain in the membrane-proximal region of one
RPTP obstructs the active site of the partner RPTP (62). CD45 is an example of RPTP that gets inactivated by dimerization. Another example of homo-dimerization is RPTP.
Fluorescence resonance energy transfer between chimeras of RPTP proteins fused to derivatives of green fluorescent protein show that RPTP homodimerizes in living cells, as demonstrated. Co-immunoprecipitation experiments have been used to show dimerization of full-length RPTP (63), CD45 (64) and RPTP (65). (Figure 1.8)
25
Figure 1.7: Regulation of PTPs at different levels. (top to bottom) PTPs are differentially expressed in specific organs, tissues or cells.
Within cells, PTPs are directed to specific subcellular locations. At the molecular level,
PTPs are regulated by post-translational modifications (66).
26
Figure 1.8: Dimerization of PTPs.
27
1.5 Protein Tyrosine Phosphatases in platelets
Although PTPs play a major role in cellular function, the role of PTPs in regulating platelet functional responses is an understudied area. The proteomic studies have shown the presence of 10 receptor type, and ten cytoplasmic tyrosine phosphatases in platelets
(Figure 1.9). To date, out of the classical PTPs, one RPTP (CD148) and five NTPTPs
(PTP-1B, Shp1, Shp2, MEG-2, and LMW-PTP) were conclusively demonstrated to be expressed in platelets using antibody-based techniques (Figure 1.9).
Phosphatases have remained understudied in platelets partly due to unavailability of specific phosphatase inhibitors. However, genetically modified mouse models for phosphatases are now used to understand the function of different phosphatases in platelets and thrombosis. PTP-1B was the first PTP identified in platelets and is the most well studied. Far less is known about the other four NTPTPs in platelets. Also, the function of only one RPTP, CD148 has been demonstrated in platelets. Below is a summary of the role of PTPs identified in platelets to date.
28
Figure 1.9: Protein Tyrosine Phosphatases in platelets and megakaryocytes. List of PTPs expressed in platelets and megakaryocytes using mass spectrometry (48).
29
1.5.1 PTP-1B
PTP-1B is abundant in platelets and plays a major role in regulating outside-in integrin signaling downstream of αIIbβ3 (67). PTP-1B is cleaved by calpain-mediated cleavage within the C-terminal tail of PTP-1B which results in the release of PTP-1B from the cytosolic surface of the ER. This release leads to increases in catalytic activity of PTP-
1B. Shattil et al. (68-70) demonstrated the molecular mechanism on how PTP-1B initiates outside-in αIIbβ3 signaling. They showed that PTP-1B plays a specialized role in regulating outside-in integrin αIIbβ3 signaling using PTP-1B knockout mice. They demonstrated that PTP-1B plays a major role in displacing Csk from a complex with the
β3 subunit and Src, followed by dephosphorylation of Src thereby leading to its activation. Response to fibrinogen is impaired in PTP-1B KO murine platelets, and consequently, the thrombus formation is reduced in PTP-1B knockout mice as demonstrated by in vivo thrombosis model (68). However, PTP-1B-KO mice have only a minor role in the hemostatic function of platelets. It is interesting to note that PTP-1B- deficient platelets responds normally to other platelet agonists, including the GPVI agonist convulxin, ADP, and thrombin (68).
1.5.2 Shp1 and Shp2
Shp1 and Shp2 are classical PTPs which have a similar structure. Both Shp1 and Shp2 have tandem SH2 domains at their N-terminal and single PTP domain in their C-terminal.
There is differential expression of Shp1 and Shp2. Shp1 is expressed in hematopoietic and epithelial cells whereas there is a ubiquitous expression of Shp2. Shp1 and Shp2 also
30
have differential functions, Shp1 negatively regulates immune receptor signaling, on the other hand, Shp2 acts as a positive regulator of growth factor and cytokine receptor signaling (44).
Shp1 and Shp2 are associated with ITIM containing receptors such as PECAM. PECAM receptors inhibit collagen and thrombin-induced platelet activation. Thus, the main function of Shp1 and Shp2 was also proposed in inhibition of platelet activation (65, 71-
75). However, several studies have suggested a complex role for Shp1 and Shp2 in platelets.
There is a diverse role for Shp1 in platelets. Studies by Brass et al. (76) demonstrate that
Shp1 negatively regulates G protein-coupled receptor signaling in platelets. In their studies, Brass et al. used a non-selective Shp1/2 inhibitor NSC-87877 to show the importance of Shp1 in the release of regulators of G protein signaling (RGS)10 and
RGS18 from the scaffold protein spinophilin. A separate study by Tadokoro et al. (77) has shown a positive role for Shp1 in outside-in αIIbβ3 signaling. This study suggests that Shp1 dephosphorylates and releases the cytoskeletal protein α-actinin from αIIbβ3.
Shp1-deficient platelets show a defect in spreading on fibrinogen although expression of
αIIbβ3 levels is comparable to the control (78). This confirms a positive role for Shp1 in platelets. Furthermore, Shp-1 deficient platelets also show impaired platelet aggregation and secretion when stimulated with CRP. However, there is a reduction of GPVI expression on the Shp1 deficient platelet surface, suggesting that the impaired platelet aggregation and secretion is due to reduced GPVI surface expression.
31
Moraes et al. (79) proposed that Shp2 negatively regulate GPVI signaling. According to their model, Shp2 sequesters PI3-kinase away from a LAT-Gab-1 complex found in lipid rafts, by binding to PECAM-1 present in non-lipid rafts (79). However, in contrast to the above proposal, Shp2-deficient platelets show normal platelet activation when stimulated with CRP. Studies in Shp2 conditional KO show that Shp2 defcient platelets are hyper- responsive to fibrinogen and anti-CLEC-2 antibody.
1.5.3 LMW-PTP
LMW-PTP is a small (18 kDa) PTP that consists of a single PTP domain containing the active-site signature motif HCX5R and no other known domains or motifs (35). It is only class II NTPTP based on its unique structural features. Its physiological function remains less understood however some studies have implicated its role in regulating cell adhesion and spreading (80, 81). Mancini et al. (81) demonstrate that human platelets express high levels of LMW-PTP (~0.05% of total protein) and that it dephosphorylates the ITAM- containing Fc RIIA receptor in vitro and in cell lines.
1.5.4 MEG-2
MEG-2 is an NPTP consisting of N-terminal lipid-binding domain. MEG-2 is located on the internal membranes of secretory vesicles and granules in neutrophils and lymphocytes. MEG-2 modulates the murine development and platelet and lymphocyte activation through secretory vesicle function (82). Wang et al (82)demonstrated that 32
platelets from MEG-2-deficient mice did not aggregate when stimulated with a high dose of thrombin compared to wild-type platelets (82). In contrast, ADP-induced aggregation was only slightly impaired in the MEG-2 deficient platelets. The author suggests a link with the defective release of α-granules however, the exact mechanism remains unclear.
1.5.5 CD148
CD148 is the first receptor-like phosphatase identified in platelets is CD148 (also referred to as DEP-1, PTPRJ, and RPTPη) (83). Senis et al. (83) used CD148 KO mice to study the function of CD148 in platelets. CD148 deficient platelets show impaired GPVI mediated aggregation. However, CD148 KO mice have approximately 50% reduction in
GPVI surface expression which partially contributes to impaired GPVI mediated aggregation. However, in vitro studies using recombinant CD148 show that CD148 can dephosphorylate the SFK which suggests that SFK is a substrate for CD148. Elevated
SFK phosphorylation levels at the negative tyrosine residue in CD148 KO murine platelets strengthens the observations made in the in vitro studies. Outside- in signaling is also impaired in CD148KO platelets. However, CD148 show comparable aggregation and secretion pattern when stimulated with GPCR agonists. CD148 KO mice show impaired thrombosis and hemostasis. The above results collectively suggest that CD148 plays a role of a positive regulator of SFK signaling in platelets (48).
33
1.6 Potential phosphatases in platelets
Since the role of PTPs have been explored to a lesser extent in platelets, there is need to characterize the role of few more phosphatases in platelets. Therefore, we investigated the role of one receptor like and cytoplasmic phosphatase, CD45 and PTPN7 respectively in platelets. Following section is a brief overview of the function of CD45 and PTPN7 in hematopoietic cells
1.6.1 CD45
CD45 is a protein tyrosine phosphatase known to regulate SFKs in lymphocytes (84).
CD45 is a large molecule (180-220 kDa) with an extracellular, transmembrane, and catalytic cytoplasmic domain (Figure 1.10). The extracellular domain consists of three different regions 1) A, B, and C regions which are encoded by exons 4,5, and 6, respectively, and can be alternatively spliced to give various isoforms, 2) a cysteine rich domain and 3) a fibronectin domain (85). All CD45 isoforms have the same cytoplasmic region, which contains domain 1 and 2. Domain 1 has phosphatase activity whereas the function of domain 2 is not known function of domain 2 is not known (86).
CD45 function has been described in both lymphoid and myeloid lineage cells. In lymphoid lineage cells like T-cells, B-cells and natural killer cells, CD45 is a positive regulator of proximal signaling events (87). Studies in CD45 KO mouse models have revealed a significant role for CD45 in T-cell development (88, 89). Deficiency of CD45 leads to a severe defect in thymocyte development, which results in only 5-10% of the normal number of mature T-cells exiting into the periphery (88). Also, CD45 KO B-cells
34
exhibit subtle developmental defects and the amount and proportion of B-cell subsets are significantly altered (90). In natural killer cells, CD45 is necessary for ITAM- specific functions such as degranulation, cytokine production and expansion during viral infection
(91). For myeloid lineage cells, like neutrophils, CD45 is known to play a role in chemotaxis. Enhanced migration is observed in CD45 KO neutrophils since they are found to be hyper-responsive to chemokines such as CCL3 and CXC1 (92).
Given the critical role of CD45 in regulating the activation of SFKs in cells of hematopoetic lineage, it would be interesting to evaluate the role of CD45 in platelets.
35
Figure 1.10: Structure of CD45 A Representative figure showing the structure of CD45 in lymphocytes adapted from
(93). CD45 consists of extracellular, transmembrane and intracellular regions. The extracellular region of CD45 consists of alternatively spliced region liked to O-glycans, cysteine rich region and fibronectin domains associated with N-glycans. The intracellular region contains the D1 and D2 domain. The D1 domain is the phosphatase domain that has the catalytic activity. 36
1.6.2 PTPN7
Protein tyrosine phosphatase PTPN7 is a cytoplasmic protein tyrosine phosphatase, alternatively called as hematopoietic protein tyrosine phosphatase (HePTP). PTPN7 is a
38kDa protein consisting of a C-terminal catalytic domain and a short N-terminal extension, which contains the Kinase Interaction Motif (KIM, residues 15-31) (Figure
1.11). Both lymphoid and myeloid lineage cells express PTPN7. In T-cells, PTPN7 dephosphorylates ERK on the positive regulatory p-tyrosine residue. PTPN7 KO mice were used to characterize the role of PTPN7 in T cell signaling. T-cells show enhanced
ERK activation in PTPN7 KO mice indicating that PTPN7 is a negative regulator of TCR induced T-cell activation. Similarly, ERK is hyper-phosphorylated in K562 myelogenous leukaemia cells undergoing megakaryocytic differentiation when subjected to inhibition by antisense for PTPN7. In B-cells, PTPN7 negatively regulates p38 MAPK activation.
Based on these previous pieces of evidence, we evaluated the role of PTPN7 as a negative regulator of MAPK activation in platelets. Mass spectrometry has demonstrated the presence of PTPN7 in platelets, however; the presence of PTPN7 has never been evaluated using biochemical techniques. Therefore, it will be interesting to evaluate the presence of PTPN7 in platelets and study the role it plays in platelet signaling.
37
CHAPTER 2
2 MATERIALS AND METHODS
Material – Apyrase (grade VII), Indomethacin, MRS 2179, 2-MeSADP was obtained from Sigma-Aldrich (St Louis, MO). AR-C69931MX (Cangalore) was a gift from gift from the Medicines Company, Parsippany NJ). Hexapeptides, AYPGKF was custom synthesized at Invitrogen (Carlsbad, CA). Collagen-related peptide (CRP) was purchased from Dr. Richard Farndale (University of Cambridge, UK). Antibody for phospho- tyrosine Src-Family Kinase Y416 (catalog# 6943P), pSyk Y525/526 (catalog# 2711), pSyk Y352 (catalog#2701), pPLC2 Y759 (catalog#3874), pPLC2 Y1217
(catalog#3871), -Actin 13E5 (catalog # 4970S) pMEK1/2 S217/221 (41G9) antibody
(catalog# 9154T), P-S PKC substrate antibody (catalog# 2261S), and p p38 MAPK
(T180/Y182) (D3F9) antibody (catalog # 4511T) were purchased from Cell Signaling
Technology (Danvers, MA). Antibodies - CD45 H-230 (catalog# sc-25590), total Syk
(Syk-01; catalog#51703), Total PLC2 (B-10; catalog#sc-5283), and PTPN7 (HePTP) antibody (calatlog # sc-21008) were from Santa Cruz Biotechnology (Santa Cruz, CA).
Antibody for phospho-tyrosine ERK1/2 (catalog# M9692) and Total ERK (catalog#
M5670) were purchased from Sigma. Luciferin-luciferase reagent was purchased from
Chrono-Log (Havertown, PA). The anti-mouse JON/A-PE, CD62-FITC, and GPVI-FITC antibodies were obtained from Emfret Analytics (Wuerzburg, Germany). Anti-Human
CD45RO PE antibody was from eBiosciences (reference # 12-0457-42) and C-terminal domain specific CD45 recombinant protein were purchased from Enzo life sciences
(catalog#BML-SE135-0020). Ferric Chloride anhydrous (catalog# 153499) was
38
purchased from MP biomedicals; PTPN7 knockout mice were obtained from Riken
Research institute, Japan. CD45 knockout mice were obtained from Dr.Tak Mak from
University Health Network in Toronto, Canada. They were bred in the central animal facility of Temple University Medical School. All other reagents were of reagent grade, and deionized water was used throughout.
Isolation of murine platelets – Mouse blood was collected as previously described (94).
Blood was drawn via cardiac puncture into one-tenth volume of 3.8% sodium citrate.
Blood was spun at 100g for 10 min, and the platelet rich plasma (PRP) was separated.
The remainder of blood was mixed with 400 l of 3.8% sodium citrate and spun for another 10 min at 100 g. Resulting PRPs were combined, 1 prostaglandin E1 was added, and samples were centrifuged for 10 min at 400 g. Platelet-poor plasma was removed and the platelet pellet was resuspended in Tyrode’s buffer (138 mM NaCl, 2.7 mM KCl, 2 mM MgCl2, 0.42 mM NaH2PO4, 5 mM glucose, 10 mM HEPES, and 0.2 units/ml apyrase, pH 7.4). Platelet counts were determined using a Hemavet 950FS blood cell counter (Drew Scientific Inc., Dallas, TX). Platelet counts were adjusted to 1.5 x 108 platelets/ml.
Preparation of human platelets– Blood was collected from informed healthy volunteers in to one-sixth volume of acid/citrate/dextrose (2.5 g sodium citrate, 2 g glucose, and 1.5 g citric acid in 100 ml de-ionized water). Blood was centrifuged at 250g for 20 minutes to obtain platelet rich plasma (PRP). Platelets were isolated from plasma by centrifugation at 980 g for 10 minutes and resuspended in Tyrode’s buffer pH 7.4 as described above. 39
The platelet count was adjusted to 5x109 cells/ml. Approval was obtained from the institutional review board of Temple University for these studies. Informed consent was provided prior to blood donation, in accordance with the Declaration of Helsinki
Preparation of human platelet lysate, membrane fraction and cytoplasmic fraction - 500
l of Tyrode’s buffer containing 2X Halt Protease and Phosphatase cocktail solution
(Pierce, Rockford, IL) was added to 500 l of platelets. Platelets were lysed by 4 freeze/thaw cycles and centrifuged at 1500 g for 10 minutes at 4 °C to remove cells which are not lysed. 300 μl the lysed cells was mixed with 100 μl of 1% triton (whole cell lysate). Remaining part of the lysed cell suspension was ultra-centrifuged at 10,000g for
30 minutes at 4 °C. The supernatant was separated (cytoplasmic fraction). The pellet, containing the membrane and cytoskeleton was resuspended in 100 μl of 1% TritonX-100 and spun at 12,000g for 10 minutes at 4 °C to separate the soluble membrane fraction from the insoluble cytoskeleton. Protein estimation was performed using the Pierce BCA
Protein assay kit (Thermo Scientific, Rockford, IL). The protein concentration was adjusted to 1 μg/μl using 1X sample buffer containing 1 mM DTT. Five or 3 μg of protein was loaded on SDS-PAGE gel for separation and subjected to western blotting.
Preparation of megakaryocyte lysate – Bone marrow femurs and tibiae of WT mice was flushed into IMDM. Megakaryocytes were separated from mononuclear cells using a discontinuous BSA gradient (0%, 1.5%, 3%), washed in PBS, and lysed using an NP-40 lysis buffer (1% NP- 40, 150 mM NaCl, 50 mM Tris-HCl, and 1:100 Halt® (Thermo
40
Fisher) protease and phosphatase cocktail. Samples were then boiled in 2X Laemmeli buffer for 5 minutes prior to resolving via SDS-PAGE.
Aggregometry – Aggregation of 0.25 ml washed platelets was analyzed using a lumiaggregometer (Chrono-log Corp. Havertown, PA). Aggregation was measured using light transmission under stirring conditions (900 rpm) at 37 °C for the indicated time.
Western Blotting – Platelets were stimulated with agonists or vehicle control for the appropriate time under stirring conditions at 37 °C. The reaction was terminated by addition of 1/10 volume 6.6 N HClO4 and placed at 4 °C. The samples were centrifuged at
10,000 × g for 5 minutes and the protein precipitate was washed with 0.5 ml of deionized water. The samples were again centrifuged at 13,000 × g for 5 minutes, and the protein pellets were solubilized in sample buffer containing 0.1 M Tris, 2% SDS, 1% (v/v) glycerol, 0.1% Bromophenol blue and 100 mM Dithiothreitol (DTT). The samples were incubated at 95 °C for 10 minutes prior to loading onto the gel. Proteins were resolved by
SDS-PAGE and transferred to nitrocellulose membrane (Whatman Protran, Pittsburgh,
PA). Membranes were blocked with Odyssey blocking buffer for 1 hour at room temperature, incubated overnight at 4 °C with the desired primary antibody and then washed four times with 1X Tris-Buffered Saline and Tween 20 (TBST). Membranes were then incubated with appropriate secondary infrared dye-labeled antibody (1:10,000) for 60 minutes at room temperature and washed four times with TBST. Membranes were imaged using a LI-COR Odyssey infrared imaging system.
41
Flow Cytometry – All data was collected using a Calibur flow cytometer (Becton
Dickinson, San Jose, CA). Washed platelets were analyzed before and after activation with CRP and AYPGKF, and surface expression of P-selectin and JON/A binding was examined. Platelets were incubated with P-selectin-FITC or JON/A-PE antibodies and stimulated with the appropriate agonist at 37 °C under non-stirring conditions. A set of unstimulated platelets was incubated with GPVI-FITC antibody. After 30 minutes of stimulation, the platelets were fixed using 1% formalin. Light-scatter and fluorescence data from 10,000 platelet events were collected with all detectors in logarithmic mode.
Data was analyzed using Flow Jo software.
Thromboxane generation assay – 0.25 ml washed platelets were stimulated with agonist for 3.5 minutes. The reaction was stopped by immediately freezing the samples on dry ice and methanol. The samples were thawed and centrifuged at 10,000 g for 10 minutes to pellet the cells. The supernatant was diluted 1:500 using water. The diluted samples were used to evaluate thromboxane generation using TXB2 EIA kit from enzo life sciences
(catalog # ADI-901-002)
Pulmonary Thromboembolism – Mice were weighed, anesthetized and injected via retro- orbital sinus with 400 g/kg of collagen and 60 mg/kg epinephrine or PBS (control). The time to cessation of respiration was recorded. Chicago sky blue dye perfused through the right ventricle of the heart. Lungs of the mice were examined to verify that pulmonary embolism occurred as the dye is excluded from the lungs if the circulation is obstructed due to thrombosis.
42
Bleeding Time – Mice (4-5 weeks old) were anesthetized using isofluorane. Four mm of the tail was excised and immediately immersed in 0.9% isotonic saline at 37 °C. The bleeding time was defined as the time required for blood flow into the saline to stop.
Ferric Chloride injury of the carotid artery model: Ferric Chloride-induced Carotid Artery
Injury—Adult mice (6 – 8 weeks old) were anesthetized using a controlled flow of isoflurane. Both WT mice and PTPN7 KO mice were utilized in this experiment. The carotid artery on the left-hand side was surgically exposed, and the artery was placed on a miniature Doppler flow probe (model 0.7PSB, Transonic Systems (Ithaca, NY)). Two drops of Saline were put on the Doppler for monitoring and recording the baseline blood flow using a Transonic model T402 flow meter. Blood flow was recorded for 2 minutes.
In the meantime, Whatman no. 1 filter paper (1 X 1 mm) was saturated with 7.5% FeCl3.
Then we stopped recording the blood flow by hitting the zero button on the transonic apparatus. After two minutes of recording, zero blood flow, the filter paper saturated filter paper was applied to the adventitial surface of the carotid artery, immediately proximal to the flow probe. After 1.5 mins, the filter paper was removed; the artery was washed with distilled water to remove surrounding excess FeCl3. Saline solution was placed in the wound, and carotid blood flow was monitored.
Statistical Analysis –Student t-test was used. Statistically significant data bearing P value
< 0.05 are annotated by an asterisk. Data are expressed as mean ± SEM.
43
CHAPTER 3
3 IMPAIRED GLYCOPROTEIN VI-MEDIATED SIGNALING AND
PLATELET FUNCTIONAL RESPONSES IN CD45 KNOCKOUT
MICE
Introduction
Platelets are the primary mediators of hemostasis and thrombosis and their activation is tightly regulated under normal physiological conditions. Upon vascular injury, circulating platelets bind to the exposed sub-endothelial collagen via 21 and signal through the
GPVI/FcR–chain complex. This interaction results in the primary activation of platelets and subsequent release of the secondary mediators ADP and thromboxane (TXA2). These mediators, along with generated thrombin, further activate platelets via G protein-coupled receptor (GPCR) signaling (23).
GPVI receptors lack intrinsic receptor tyrosine kinase activity; instead, they rely on SFKs to phosphorylate the immunoreceptor tyrosine-based activation motif (ITAM) on the
FcR chain to initiate GPVI signaling. Therefore, SFKs are indispensable for GPVI- mediated platelet activation (95). Human platelets have seven SFK family members - Src,
Yes, Lyn, Hck, Fyn, Fgr, and Lck, whereas mouse platelets have four Src, Lyn, Fyn, and
Fgr (95). SFKs have highly conserved tyrosine residues that regulate their activation. For example, SFKs have a positive regulatory site (Y416), which is present in the activation loop between the N-terminal and C-terminal domains. The negative regulatory site
(Y527) is present in the c-terminal tail (96, 97). When phosphorylated, the negative 44
regulatory site keeps the SFKs in an inactive conformation and dephosphorylation of this residue primes SFKs for activation (98). Primed SFKs then undergo auto- phosphorylation at the Y416 leading to their full activation (96). Although the role of
SFKs is well established in platelets, we have very little understanding of their regulation by protein tyrosine phosphatases (PTP).
PTPs are important regulators of signal transduction (99). One of the most well studied
PTPs in platelets is PTP-1B, which is an essential positive regulator of Src downstream of IIb3 activation (67, 100, 101). Another receptor tyrosine phosphatase known to regulate SFKs is CD148. CD148 deficient platelets exhibit impaired GPVI mediated platelet activation. However, CD148 knockout mice have a 42% reduction in GPVI platelet surface expression and thus exhibit defective platelet function (102). Thus, the mechanism behind SFK regulation in platelets remains unclear.
CD45 is a protein tyrosine phosphatase known to regulate SFKs in lymphocytes (84).
CD45 is a large molecule (180-220 kDa) with an extracellular, transmembrane, and catalytic cytoplasmic domain (Figure 1.9). The extracellular domain consists of three different regions 1) A, B, and C regions which are encoded by exons 4,5, and 6, respectively, and can be alternatively spliced to give various isoforms, 2) a cysteine rich domain and 3) a fibronectin domain (85). All CD45 isoforms have the same cytoplasmic region, which contains domain 1 and 2. Domain 1 has phosphatase activity whereas the function of domain 2 is not known (86). In lymphocytes CD45 regulates SFK activation by dephosphorylating the negative regulatory tyrosine domain (103). Given the critical 45
role of CD45 in regulating the activation of SFKs, it would be interesting to evaluate the role of CD45 in platelets.
Data published by Karisch et al. (104) in 2011 suggests that CD45 may be expressed in platelets. Using mass spectrometry, they show the presence of a c-terminal peptide of
CD45. Conversely, in 1988, Leon et al (105) have established that CD45 is not expressed on the surface of platelets by using flow cytometry. Based on these studies, we hypothesized that platelets express a truncated isoform of CD45, which has the catalytic cytoplasmic domain but lacks the extracellular domain. In this manuscript, we demonstrate that platelets express a truncated CD45 isoform of approximately 65 kDa.
We also show that deficiency of CD45 leads to impaired GPVI-mediated signaling and subsequent platelet function.
Results
Investigating the presence of a truncated isoform of CD45 in platelets:
First, we verified the absence of CD45 from the platelet surface using an antibody specific for the extracellular region of CD45 by flow cytometry (data not shown). Next we investigated the presence CD45 in platelets by using an established antibody specific for the c-terminal domain of CD45 (106) via western blotting. We observed that a protein of approximately 65 kDa was recognized in WT murine platelets but not in CD45 KO murine platelets (Figure 3.1A). Based on the bioinformatics tools; Uniprot and ExPASy, the molecular weight of the CD45 c-terminal domain 1 and 2 is approximately 65-68
46
kDa. Thus, we believe that the observed protein is a truncated form of CD45, consisting of only a part of the c-terminal domain.
Flow cytometry studies have shown the presence of CD45 on the megakaryocyte surface
(107). Using western blotting we determined that full-length CD45 is present in WT but not in CD45 KO megakaryocytes, confirming CD45 deletion in the megakaryocyte lineage (Figure 3.1B). Additionally, we investigated the presence of CD45 in human platelets. We observed that, similar to murine platelets, human platelets also express a
65 kDa protein (Figure 3.1C). In order to further evaluate the location of CD45 in human platelets, we separated the membrane and cytoplasmic fractions. We observed the 65 kDa band to be specifically present in the cytoplasmic fraction (Figure 3.1C).
To validate the specificity of the CD45 antibody towards the c-terminal domain, we used a CD45 c-terminal domain recombinant protein. The molecular weight of the CD45 c- terminal domain recombinant protein is 80 kDa. The CD45 c-terminal domain recombinant protein contains amino acids from 584-1281 that encompasses a part of the transmembrane region and the entire c-terminal region. Therefore, the CD45 c-terminal domain recombinant protein has additional amino acids as compared to the CD45 truncated isoform expressed in platelets. The CD45 recombinant protein, WT platelet lysates, CD45 KO murine platelet lysates, and human platelet lysates were subjected to western blotting and probed with either the CD45 antibody or the CD45 antibody blocked with CD45 recombinant protein. We observed that the CD45 antibody recognizes an 80 kDa recombinant protein and a 65 kDa protein in both WT murine and human platelet
47
lysates (Figure 3.2 A-C). However, blocking the CD45 antibody with the CD45 recombinant protein prevents the recognition of the 80 kDa recombinant protein and a 65 kDa protein in both WT murine platelet lysates and human platelet lysates (Figure 3.1D-
F). These data suggest that the CD45 antibody is specific for the c-terminal domain of
CD45.
48
Figure 3.1: Presence of CD45 in platelets. A) Murine platelet lysate from WT and CD45 KO B) Murine megakaryocyte lysate from
WT and CD45 KO mice and C) human platelet – whole cell lysate, membrane and cytoplasmic lysates (5 g) were subjected to western blot analysis using a c-terminal domain specific antibody and -actin was probed as a loading control.
49
Figure 3.2 Checking the specificity of CD45 c-terminal domain antibody. A) CD45 c-terminal domain recombinant protein (30 and 60 ng) B) WT and CD45 KO murine platelet lysates and C) human platelet lysate (3 g) was subjected to western blotting and probed with either CD45 antibody or CD45 antibody pre-incubated with
CD45 c-terminal domain recombinant protein.
50
Evaluation of ex vivo platelet functional responses in CD45 KO and WT mice:
We evaluated whether or not the truncated CD45 isoform has a role in platelet functional responses. Platelets have two important classes of receptors – ITAM based receptors, such as GPVI receptors, and GPCRS, such as protease activated receptors (PARs) (23).
We stimulated WT and CD45 KO platelets with the GPVI agonists such as CRP and collagen, as well as the PAR-4 agonist AYPGKF. CD45 KO murine platelets exhibited impaired platelet aggregation and dense granule secretion when stimulated with 2 g/ml of CRP compared to WT platelets. Upon stimulation with 10 g/ml CRP the differences with respect to aggregation became less apparent, however dense granule secretion was significantly impaired in the CD45 KO murine platelets (Figure 3.3 A-C). Similar results were obtained using 0.5 g/ml and 1 g/ml of the physiological GPVI agonist collagen
(Figure 3.4 A-C). When WT and CD45 KO platelets were stimulated with 100 M or 200
M AYPGKF there was no difference in aggregation and dense granule secretion (Figure
3.5 A-C). These results suggest a functional role for CD45 downstream of only GPVI- mediated platelet activation.
Likewise, we investigated alpha granule release and activation of IIb3 integrin in WT and CD45 KO mouse platelets using flow cytometry. CD45 KO murine platelets showed a significant decrease in the surface expression of P-selectin (a marker for -granule release) as compared to WT murine platelets when stimulated with CRP (2 g/ml and 10
g/ml) (Figure 3.6 A). Furthermore, we investigated the activation of IIb3 using JON/A
51
antibody, which binds only to active IIb3. We observed decreased levels of JON/A binding in CD45 KO murine platelets stimulated with CRP (2 g/ml and 10 g/ml) as compared to WT (Figure 3.6 B). We did not observe any differences in alpha granule secretion or activation ofIIb3 between WT and CD45 KO platelets when stimulated with AYPGKF (data not shown).
It was previously revealed that the absence of CD148 phosphatase results in a 42% reduction of GPVI surface expression on the platelet surface (102). A decrease in the surface expression of GPVI would explain why the authors saw impaired GPVI-mediated platelet functional responses. Hence, we evaluated the GPVI surface expression on CD45
KO and WT platelets using flow cytometry. We observed that the CD45 KO and WT mouse platelets had similar GPVI surface expression (Figure 3.6 C). This indicates that
CD45 does not regulate GPVI surface expression in platelets and cannot explain the reduction of CRP-induced platelet functional responses in the CD45 KO murine platelets.
52
Figure 3.3: Platelet aggregation and dense granule in WT and CD45 KO murine platelets upon stimulation with CRP.
Washed murine platelets (1.5 X 108) were stimulated with low and high concentrations of
GPVI and GPCR agonists under stirring conditions; (A) CRP (2 g/ml and 10 g/ml); (B and C) Quantitation of extent of aggregation and ATP secretion of (A), * p <0.05
53
Figure 3.4: Platelet aggregation and dense granule in WT and CD45 KO murine platelets upon stimulation with Collagen.
Washed murine platelets (1.5 X 108) were stimulated with low and high concentrations of
GPVI and GPCR agonists under stirring conditions; (A); Collagen (0.5 g/ml and 1
g/ml); (B and C) Quantitation of extent of aggregation and ATP secretion of (A), * p
<0.05.
54
Figure 3.5: Platelet aggregation and dense granule in WT and CD45 KO murine platelets upon stimulation with AYPGKF.
Washed murine platelets (1.5 X 108) were stimulated with low and high concentrations of
GPVI and GPCR agonists under stirring conditions; (A) AYPGKF (100 M and 200
M). (B and C) Quantitation of extent of aggregation and ATP secretion of (A), * p
<0.05. 55
Figure 3.6: -granule secretion, IIb3 integrin activation and GPVI surface expression in WT and CD45 KO platelets.
A, B) The difference in mean fluorescence intensity (MFI) of CD62 and JON/A binding between knockout and wild type murine platelets was calculated. WT was considered as
100% and values for KO were calculated accordingly, * p <0.05. C) Washed WT and
CD45 KO murine resting platelets were incubated with a FITC-labeled anti-mouse GPVI antibody. The gray and black histograms represent the WT and CD45 KO surface expression, respectively, whereas the light gray histogram represents the IgG control. 56
Evaluation of in vivo platelet functional responses in CD45 KO and WT mice:
As CD45 KO platelets exhibited impaired GPVI-mediated ex vivo platelet functional responses, we investigated the physiological implications of CD45 deficiency. Initially, we measured tail-bleeding time, which reflects primary hemostatic function. We observed prolonged tail bleeding times in CD45 KO mice compared to WT mice (Figure
3.7 A). These data are indicative of impaired hemostatic function in CD45 KO mice.
We also used a model of pulmonary thromboembolism to evaluate the ability of platelets to form a thrombus in vivo (108-110). We injected 400 g/kg of collagen and 60 mg/kg of epinephrine into the retro-orbital space of WT and CD45 knockout mice. This leads to activation of platelets in circulation and formation of emboli, which travel to the lungs and block blood flow thereby resulting in cessation of respiration. We observed that the time to cessation of respiration was prolonged in CD45 KO as compared to WT, which is indicative of a defect in thrombus formation (Figure 3.7 B). We confirmed that mortality was due to pulmonary embolism by injecting Chicago sky blue dye in the right ventricle of the heart. This dye is excluded from the lungs if the circulation is obstructed due to thrombosis (data not shown). Thus, we concluded that CD45 KO mice have impaired thrombosis and hemostasis.
57
Figure 3.7: In vivo platelet functional responses in WT and CD45 KO mice. A) Bleeding time was measured in WT and CD45 KO mice following excision of the distal 4 mm of the tail. B) WT and CD45 KO mice were anesthetized and injected either with PBS or appropriate amounts of collagen and epinephrine and time to cessation of respiration was recorded, * p <0.05.
58
Evaluation of GPVI signaling in CD45 KO and WT murine platelets:
Since CD45 KO murine platelets were defective in GPVI-mediated platelet functional responses, we evaluated the signaling downstream of the GPVI receptor. First, we investigated the phosphorylation status of two proximal signaling molecules in the GPVI pathway- Syk and PLC2. When CD45 KO murine platelets were stimulated with 2
g/ml CRP (Figure 3.8) or 10 g/ml CRP (Figure 3.9) for 10 and 30 seconds we saw a significant decrease in the phosphorylation of Syk (Y352, Y525/526) and PLC2 (Y759,
Y1217) compared to WT control platelets. SFKs are upstream of Syk and are indispensable for GPVI receptor activation (95). CD45 is known to regulate SFK activation in lymphocytes by dephosphorylating the negative regulatory tyrosine (103).
Thus, we evaluated whether or not the truncated isoform of CD45 regulates SFK phosphorylation in platelets. However, SFKs are also important for signaling downstream of GPCRs (95, 111). GPCRs (P2Y12, P2Y1, and TP receptor) are stimulated via secreted
TXA2 and ADP, which act in an autocrine and paracrine fashion leading to further activation of platelets (11)
To evaluate SFK phosphorylation downstream of only the GPVI receptor, we pre- incubated the platelets with the feedback inhibitors indomethacin, which inhibits TXA2 generation, and the ADP receptor antagonists, MRS-2179 and ARC-69931. This was followed by stimulation of the platelets with 10 g/ml of CRP. Using the feedback inhibitors enabled us to eliminate any contribution by TXA2 and ADP to SFK phosphorylation. Under these conditions, we observed that CD45 KO murine platelets have reduced CRP-induced SFK Y416 phosphorylation compared to WT murine platelets
59
(Figure 3.10). Therefore, we concluded that the truncated isoform of CD45 regulates SFK phosphorylation in platelets.
60
Figure 3.8: Syk and PLC2 activation in CD45 KO and WT murine platelets upon stimulation with CRP 2g/ml
A) Murine platelets from WT and CD45 KO murine platelet lysates were stimulated with
CRP (2 g/ml) for 10 and 30 seconds subjected to western blot analysis and probed with pSyk Y525/526, pSyk Y352, pPLC2 Y759, and pPLC2 Y1217 antibodies. The same blot was probed with total Syk and total PLC2 antibodies as protein loading control in each lane. B, C, D, E) Quantitation for phosphorylation of Syk and PLC2, *p <0.05
61
Figure 3.9: Syk and PLC2 activation in CD45 KO and WT murine platelets upon stimulation with CRP 10g/ml
A) Murine platelets from WT and CD45 KO murine platelets were stimulated with CRP
(10 g/ml) for 10 and 30 seconds. Platelet lysates subjected to western blot analysis and probed with pSyk Y525/526, pSyk Y352, pPLC2 Y759, and pPLC2 Y1217 antibodies.
The same blot was probed with total Syk and total PLC2 antibodies as protein loading control in each lane. B, C, D, E) Quantitation for phosphorylation of Syk and PLC2, *p
<0.05 62
Figure 3.10: SFK activation in CD45 KO and WT murine platelets upon stimulation with CRP.
A) Platelets from WT and CD45 KO mice were incubated with secondary signaling inhibitors - indomethacin 10 M, MRS-2179 100 M, ARC-69931 100 nM for 5 mins and then stimulated with GPVI agonist CRP (10 g/ml) for 10 and 30 seconds and reaction was stopped using the 1/10 volume 6.6 N HClO4. These lysates were subjected to western blot analysis using pSFK Y416 antibody. The same blot was probed with total
PLC2 antibody as protein loading control in each lane. B) Quantitation for phosphorylation of SFK, *p <0.05.
63
Discussion
The extracellular domain of CD45 consists of three different regions (A, B and C), which are coded by three different exons and can be alternatively spliced to give various isoforms. The largest isoform of CD45 is RABC (240 kDa), which includes all three exons encoding the A, B, and C regions, while the smallest isoform is RO (180 kDa), which lacks those three exons (85). Expression of different CD45 isoforms is cell-type specific and depends on differentiation and the activation state of the cell (112). The majority of developing thymocytes and activated T-cells express the low molecular weight isoform (180 kDa), whereas mature T-cells express multiple isoforms. B-cells express only the high molecular weight isoform (113). However, it is important to note that all isoforms of CD45 have a common c-terminal cytoplasmic domain, which has catalytic activity. Apart from these most commonly known isoforms in lymphocytes, human Langerhans cells express an unusual isoform that is similar to the RO (180 kDa) epitope but lacks the oligosaccharide with terminal sialic acid moiety (114). Also, a report from Kirchberger et al (115) demonstrated the presence of another uncommon
CD45 isoform in monocytes and neutrophil granulocytes. They found that upon stimulation of phagocytes, a 95 kDa fraction of CD45 consisting of the cytoplasmic tail is cleaved by proteases. This 95 kDa fraction of CD45 aligns with the c-terminal region of
CD45 and is cytoplasmic (115). It is clear from above examples that numerous isoforms of CD45 exist in different cells.
Our study shows that platelets express a truncated isoform of CD45 of approximately 65 kDa, and consists of only a part of the c-terminal region. It is important to note that the c- 64
terminal region of CD45 contains a catalytic domain which is indispensable for its function (112). Since, the extracellular domain of CD45 in lymphocytes does not contribute to the catalytic activity of CD45 (84); it is likely that the truncated isoform of
CD45 functions even in the absence of the extracellular domain. Furthermore, the data sheet of the recombinant CD45 c-terminal domain protein used in figure 2 suggests that it is a catalytically active protein. This further supports our above hypothesis.
We also investigated the presence of CD45 in megakaryocytes. Flow cytometry studies have shown that megakaryocytes express a full-length isoform of CD45 (107). We have verified this via western blotting. We observed that the CD45 antibody recognized a 180 kDa protein in WT but not in the CD45 KO. Also, the 65 kDa protein observed in platelets was not present in the megakaryocytic protein lysates. Therefore, we ruled out the possibility that the truncated CD45 is an alternatively spliced variant. We speculate that the truncated CD45 isoform in platelets is generated by proteolytic cleavage when platelets are formed from megakaryocytes. Currently, we do not know the exact residue where full length CD45 is cleaved and this needs to be investigated in the future. Our data also suggests that human platelets express a 65kDa of CD45, which is present in the cytosol. A 100 kDa band is recognized by the antibody only in the membrane fraction and not in other lanes. We predict that the 100kDa band is a non-specific band and is recognized due to the enrichment of membrane bound proteins in the membrane fraction.
The whole cell lysate would contain both membrane and cytosolic proteins, which would serve to dilute the membrane fraction. Therefore, we do not see a prominent 100 kDa band in the whole cell lysate.
65
CD45 plays a very crucial role in all hematopoietic cells studied (87-92), however, the function of CD45 in platelets had never been evaluated. In this manuscript, we demonstrate that the truncated isoform of CD45 is a positive regulator of SFK activation downstream of the GPVI receptor. Consequently, the absence of CD45 specifically impairs GPVI-mediated platelet signaling and platelet functional responses. However,
CD45 deficiency does not completely abolish the platelet signaling or functional response, which suggests that other phosphatases might play a role in GPVI signaling.
We speculate that another phosphatase; CD148 plays a role in regulating SFK activation along with CD45. Although the CD148 KO studies suggest that the impaired GPVI responses are due to the decreased GPVI surface expression, the in vitro studies using recombinant CD148 have shown that CD148 recombinant protein directly dephosphorylates the c-terminal inhibitory and activation loop sites of SFKs (102).
Therefore, CD45 and CD148 both may play a role in regulating the SFK activation in platelets.
Stimulation of CD45 KO murine platelets with lower concentrations of GPVI agonist
CRP results in impaired platelet aggregation and secretion in CD45 KO murine platelets.
With an increase in the CRP concentration, the WT and CD45 KO platelets show the same extent of aggregation. However, the dense granule secretion is impaired at both low and high concentrations of CRP. This discrepancy in aggregation and secretion at higher concentration of CRP is explained by the fact that aggregation saturates at a relatively low concentration of
66
We also evaluated the alpha granule secretion and IIb3 integrin activation in the CD45
KO platelets stimulated with CRP. CD45 KO platelets show a defect in alpha granule secretion and IIb3 integrin activation at both low and high concentrations of CRP. It is interesting that at higher concentrations of CRP, although IIb3 integrin activation is impaired the CD45 KO murine platelets aggregate fully. However, we can explain this observation using the example of patients who are heterozygous for Glanzmann's thrombasthenia. These patients have a 50% reduction in IIb3 integrin activation; however, there are no demonstrable defects in aggregation (116, 117). This demonstrates that 50% IIb3 integrin activation is sufficient for full aggregation.
Because of the defects noted in the response of CD45 KO platelets to the GPVI agonist
CRP, we investigated GPVI signaling in CD45 KO murine platelets. SFKs are essential for signaling downstream of GPVI receptors. SFK activation is dependent on dephosphorylation of the negative regulatory site Y527 that leads to a partially active conformation of SFKs. SFKs then undergoes auto-phosphorylation at the Y416 residue to attain fully active conformation. CD45 KO murine platelets stimulated with GPVI agonist in presence of feedback inhibitors show reduced SFK Y416 phosphorylation as compared to the WT platelets. This indicates that absence of CD45 results in impaired
SFK activation downstream of the GPVI receptor. We speculate that the truncated CD45 dephosphorylates the negative regulatory tyrosine residue and activates SFKs. Therefore, in the absence of CD45; there is no dephosphorylation of the negative regulatory tyrosine residue and thus no further activation of SFKs. 67
Syk and PLC2 phosphorylation is dependent on SFK activation. Activated SFKs phosphorylate the tyrosine residues on the ITAM domain of the FcRγ chain, thereby creating docking sites for Syk recruitment and activation (118). Syk undergoes auto- phosphorylation on tyrosine residues including Y352, and Y525/526. Activation of Syk is followed by downstream signaling which eventually leads to phosphorylation of PLC2
(119). Therefore, our observation that SFK Y416 phosphorylation is reduced in CD45
KO mice compared to WT following stimulation with a GPVI agonist is supported by reduced Syk and PLC2 phosphorylation also observed in CD45 KO platelets.
In conclusion, we show that CD45 is expressed in platelets as a truncated isoform that regulates GPVI–mediated platelet functional responses through SFK activation.
68
CHAPTER 4
4 PTPN7 IS A NEGATIVE REGULATOR OF ERK ACTIVATION
AND THROMBOXANE GENERATION IN PLATELETS
Introduction
Platelets are primary mediators of hemostasis and thrombosis, and their activation is tightly regulated under normal physiological conditions. Upon vascular injury, circulating platelets bind to exposed sub-endothelial collagen, which leads to initial activation of platelets. Activated platelets secrete their granular contents such as ADP and generate
Thromboxane (TXA2). Both ADP and TXA2 are positive feedback activators of platelets
(23).
TXA2 is a lipid mediator which feedbacks on the platelets to amplify the initial signal and helps in stable thrombus formation (120, 121). Patients having the deficiency of TXA2 have a mild bleeding disorder (122). On the contrary, high levels of TXA2 can lead to clinical conditions such as myocardial infarction (123) and stroke (124). The above examples reiterate the importance of generated TXA2 in maintaining normal hemostasis.
Therefore, it is essential to understand the regulation of TXA2 generation in platelets.
Several steps are involved in generation of TXA2 in platelets. ERK and p38 are crucial
MAPK enzyme that is involved in TXA2 generation in platelets. Studies have shown that extracellular signal-regulated protein kinase 1/2 (ERK 1/2) play a major role in TXA2 generation downstream of the protease activated receptors (PARs) (125, 126) and ADP
69
receptors such as P2Y1 and P2Y12 receptors (127, 128). Activation of ERK1/2 leads to activation of an enzyme cytoplasmic phospholipase A2 (cPLA2). ERK phosphorylates cPLA2 on serine residue 505 that is required for activation of cPLA2 (129, 130).
Activated cPLA2 is involved in hydrolysis of the membrane phospholipids to release
Arachidonic Acid (AA). Furthermore, enzymes such as the prostaglandin G/H synthase and TXA2 synthase convert the free AA to TXA2. Given the importance of ERK in TXA2 generation in platelets it is crucial to understand the mechanisms of ERK regulation in platelets.
Protein tyrosine phosphatase PTPN7 is a cytoplasmic protein tyrosine phosphatase, originally cloned from human T-lymphocytes (131, 132), and is alternatively called as hematopoietic protein tyrosine phosphatase (HePTP). PTPN7 is a 38kDa protein consisting of a C-terminal catalytic domain and a short N-terminal extension, which contains the Kinase Interaction Motif (KIM). PTPN7 is expressed in cells of hematopoietic lineage such as neutrophils, megakaryoctyes, erythrocytes and lymphocytes (133). Mass spectrometry has demonstrated the presence of PTPN7 in platelets (76, 104), however; the presence of PTPN7 has never been evaluated using biochemical techniques. In T-cells, PTPN7 dephosphorylates ERK on the positive regulatory p-tyrosine residue and thereby negatively regulates T-cell activation (133,
134). Therefore, it will be interesting to evaluate the role of PTPN7 as a regulator of ERK and thromboxane generation in platelets.
70
In this study, we evaluated the presence of PTPN7 in human and murine platelets.
Furthermore, we used PTPN7 knockout mice to investigate the role of PTPN7 in platelet activation. In this study, we show that PTPN7 is a negative regulator of ERK and thereby
TXA2 generation and platelet functional responses.
Results
Evaluating the presence of PTPN7 in murine and human platelets:
PTPN7 is present as a 38kDa cytoplasmic protein in lymphocytes (131, 132, 135).
However, its presence in platelets has been shown using proteomic approaches (76, 104) but never been evaluated using never been evaluated using biochemical techniques. To assess the presence of PTPN7 in platelets, we used an antibody which recognizes the N- terminus of PTPN7. We observed that PTPN7 is present as a 38kDa protein in WT murine platelets and human platelets. Thus, using western blotting we conclude that
PTPN7 is expressed in platelets. (Figure 4.1).
Studying ex vivo platelet functional responses in PTPN7 KO murine platelets:
As we demonstrated the presence of PTPN7 in platelets, we evaluated the role of PTPN7 in platelet functional responses including aggregation and secretion using PTPN7 KO mice. Platelets have two important classes of receptors – ITAM based receptors, such as
GPVI receptors, and GPCRS, such as protease activated receptors (PARs) and ADP receptors (23). Upon stimulation of WT and PTPN7 KO murine platelets with lower concentrations of the PAR agonist AYPGKF, the ADP receptor agonist 2-MesADP and
GPVI receptor agonist CRP, the extent of aggregation and the amount of ATP secretion
71
was significantly greater in the PTPN7 KO murine platelets as compared to the WT controls (Figure 4.2, 4.3 and 4.4). PTPN7 KO murine platelets that were stimulated with higher concentrations of AYPGKF, 2-MesADP and CRP show higher dense granule secretion as compared to the WT although the extent of aggregation is similar between
WT and PTPN7 KO (Figure 4.2, 4.3 and 4.4). These data suggest that PTPN7 plays an important role in regulating platelet functional responses such as aggregation and secretion in platelets.
72
Figure 4.1: Expression of PTPN7 in human and murine platelets.
10l of murine platelet lysates of WT and PTPN7 KO (count adjusted to 1.5X108) control samples were loaded in the lanes 1 and 2 .5l of human platelet lysate (count adjusted to 2X108) was loaded in lane 3. The blot was probed with PTPN7 antibody
(1:500) and the same blot was reprobed with-Actin antibody (1:2000).
73
Figure 4.2: Platelet aggregation and secretion in WT and PTPN7 KO murine platelets upon stimulation with AYPGKF.
Washed murine platelets (count adjusted to 1.5 X 108) were stimulated with low and high concentrations of GPCR and GPVI agonists under stirring conditions; (A) AYPGKF (75
M and 200 M); (B and C) Quantitation of extent of aggregation and ATP secretion of
(A). * p <0.05.
74
Figure 4.3: Platelet aggregation and secretion in WT and PTPN7 KO murine platelets upon stimulation with 2-MesADP.
Washed murine platelets (count adjusted to 1.5 X 108) were stimulated with low and high concentrations of GPCR and GPVI agonists under stirring conditions; (A); 2-MeSADP (3 nM andnM); (B and C) Quantitation of extent of aggregation and ATP secretion of
(A), * p <0.05.
75
Figure 4.4: Platelet aggregation and secretion in WT and PTPN7 KO murine platelets upon stimulation with CRP.
Washed murine platelets (count adjusted to 1.5 X 108) were stimulated with low and high concentrations of GPCR and GPVI agonists under stirring conditions; (A) CRP (1 g/ml and 5 g/ml). (B and C) Quantitation of extent of aggregation and ATP secretion of (G),
* p <0.05.
76
Evaluating TXA2 generation in PTPN7 KO murine platelets:
As the aggregation and secretion responses were potentiated in PTPN7 KO mice we evaluated whether the potentiation was due to enhanced TXA2 generation. For this purpose, we pre-treated the WT and PTPN7 KO murine platelets with 10 μM of indomethacin, a cyclooxygenase inhibitor, which inhibits TXA2 generation and followed by stimulation with AYPGKF and CRP. We observed that the extent of aggregation and the amount of dense granule secretion in PTPN7 KO was the same as the WT when the platelets are pretreated with indomethacin (Figure 4.5). This result suggests that the potentiation of aggregation and secretion is due to enhanced TXA2 generation.
In order to confirm whether the enhanced platelet aggregation and secretion in PTPN7
KO murine platelets were due to enhanced TXA2 generation, both WT and PTPN7 KO murine platelets were stimulated with agonist and the TXA2 levels were measured using
TXB2 assay, a competitive immunoassay. PTPN7 KO murine platelets showed a significant increase in TXA2 levels as compared to the WT samples when stimulated with all agonists (Figure 4.6). Therefore, we conclude that TXA2 generation is potentiated in
PTPN7 KO platelets.
77
Figure 4.5: Aggregation and secretion in presence of indomethacin in WT and
PTPN7 KO platelets.
Washed murine platelets (count adjusted to 1.5 X 108) were pretreated with 10 M of indomethacin and stimulated with low and high concentrations of GPCR and GPVI agonists under stirring conditions; (A) AYPGKF (75 M and 200 M); (B) CRP (1 g/ml and 5 g/ml).
78
Figure 4.6: Thromboxane generation in WT and PTPN7 KO murine platelets. WT and PTPN7 KO murine platelets (count adjusted to 1.5 X 108) were stimulated with
AYPGKF (75 M and 200 M), MeSADP (3nM andnM) and CRP (1 g/ml and 5
g/ml) for 3.5 min at 37 °C under the stirring conditions. Reactions were terminated and the generated TXB2 levels were measured as described in experimental procedures. Data are represented as percent maximal TXB2 generated in the WT controls. Each bar is the average of three experiments ±SEM from 3 days. Asterisk denotes P < 0.05.
79
Evaluating MAPK phosphorylation in WT and PTPN7 murine platelets.
ERK plays an important role in TXA2 generation in platelets (125, 127, 128, 136) and since TXA2 levels are potentiated in PTPN7 KO murine platelets, we investigated the role of PTPN7 in regulation of ERK. ERK was found to be hyper-phosphorylated in PTPN7
KO murine platelets that upon AYPGKF stimulation that were pre-treated with or without indomethacin (Figure 4.7 A). PTPN7 KO platelets stimulated with CRP show hyper-phosphorylation of ERK. However, in presence of indomethacin, both WT and
PTPN7 KO stimulated with 5 μg/ml of CRP show very negligible ERK phosphorylation
(Figure 4.7 B).
In B-lymphocytes PTPN7 also regulates p38 MAPK (135). Since platelets express both
ERK and p38 we evaluated the role of PTPN7 in p38 regulation. We observed that p38
MAPK is phosphorylated to the same extent in both WT and PTPN7 KO murine platelets
(Figure 4.8 A). Since MEK is upstream of ERK in PAR signaling (137, 138), we also evaluated the MEK activation specific MEK phosphorylation. We observe that activity of
MEK is the same in PAR-activated WT and PTPN7 KO platelets (Figure 4.8 A). Thus, we conclude that PTPN7 selectively regulates ERK in platelets without regulating its upstream kinase MEK.
80
Figure 4.7: ERK phosphorylation in WT and PTPN7 KO murine platelets. Murine platelets from WT and PTPN7 KO murine platelets (count adjusted to 1.5 X 108) were treated with or without 10 M of indomethacin and stimulated with (A) AYPGKF
(200 M); (B) CRP (5 g/ml). Platelet lysates were collected for western blot analysis and probed with pERK antibody. The same blot was probed with total ERK antibody as protein loading control in each lane.
81
Figure 4.8: p38 and MEK phosphorylation in WT and PTPN7 KO murine platelets. A) Murine platelets from WT and PTPN7 KO murine platelets (count adjusted to 1.5 X
108) stimulated with AYPGKF (200 M), CRP (5 g/ml). Platelet lysates were collected for western blot analysis and probed with phosphorylated p38 MAPK antibody. The same blot was probed with total p38 MAPK antibody as protein loading control in each lane.
B) Murine platelets from WT and PTPN7 KO were stimulated with AYPGKF (200 M).
Platelet lysates were collected for western blot analysis and probed with pMEK antibody.
The same blot was probed with total PLC2 antibody as protein loading control in each lane.
82
Evaluating in vivo platelet functional responses in WT and PTPN7 KO mice
As we observed potentiation of platelet functional responses with GPCR and GPVI agonists in in PTPN7 KO murine platelets, we evaluated the implications of PTPN7 deficiency in vivo. We evaluated the hemostatic function by measuring the bleeding times for the WT and PTPN7 KO. Bleeding times was measured as the time to cessation of blood flow after cutting 3mm of mouse tail. We observed that there are no significant differences in the average bleeding times of WT and PTPN7 KO (Figure 4.9 A). Next, we evaluated the in vivo thrombosis using ferric chloride-induced carotid artery injury model. We observed that the average time to occlusion in both WT mice and KO mice is around 10 minutes and not statistically different from one another (Figure 4.9 B and C).
Next, we evaluated in vivo thrombosis using pulmonary embolism model. In this model collagen and epinephrine are injected in the retro-orbital space that leads embolizing thrombi in the lungs, consequently death of the mouse due to the cessation of respiration.
We observed that time to cessation of respiration was significantly lower in the PTPN7
KO as compared to the WT (Figure 4.9 D). Thus, we conclude that platelet responses are potentiated in vivo.
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Figure 4.9: In vivo platelet functional responses in WT and PTPN7 KO mice. A) Bleeding time was measured in WT and CD45 KO mice following excision of the distal 4 mm of the tail. B) WT and PTPN7 KO mice were subjected to FeCl3 injury model as described in the methods section and time to occlusion was measured. C)
Representative tracing showing time to occlusion in WT and PTPN7 KO mice. D) WT and CD45 KO mice were anesthetized and injected either with PBS or appropriate amounts of collagen and epinephrine and time to cessation of respiration was recorded, * p <0.05.
84
Discussion
PTPN7 KO mice were previously used to characterize the role of PTPN7 in T cell signaling (133). T-cells show enhanced ERK activation in PTPN7 KO mice indicating that PTPN7 is a negative regulator of TCR induced T-cell activation. Similarly, ERK is hyperphosphorylated in K562 myelogenous leukemia cells undergoing megakaryocytic differentiation when subjected to inhibition by antisense for PTPN7 (139). In B-cells,
PTPN7 negatively regulates p38 MAPK activation (135). Based on these previous pieces of evidence, we evaluated the role of PTPN7 as a regulator of MAPK activation in platelets using PTPN7 KO mice.
Platelets express several MAPKs out of which ERK1/2 and p38MAPK(140, 141) have been studied exclusively. ERK inhibition by U0126, a MEK inhibitor, leads to inhibition of cPLA2 activation and TXA2 generation downstream of PAR (125) and ADP receptors
(127). Thereby, proving that ERK1/2 is required for cPLA2 activation and consequently
TXA2 production. ERK is hyperphosphorylated downstream of PAR receptors in PTPN7
KO murine platelets. As a result, there is enhanced TXA2 generation, in the PTPN7 KO murine platelets. When the TXA2 production is blocked by indomethacin, the extent of
PAR receptor-mediated aggregation and secretion in PTPN7 KO mice is similar to the
WT. However, under these conditions, ERK remains hyper phosphorylated downstream of the PAR receptors. This data suggests that potentiation of ERK phosphorylation in
PTPN7 KO murine platelets results in an enhanced TXA2 generation however the enhanced TXA2 levels are not responsible for increased ERK phosphorylation. This data also suggests that PTPN7 acts upstream of ERK and TXA2 production. To confirm 85
whether or not PTPN7 is specific for ERK, we evaluated the activity of MEK and PKC, which are also upstream of ERK in PAR signaling (137, 138). We find that activity of both PKC and MEK is unaffected in PTPN7 KO mice downstream of the PAR receptors.
Thus, confirming that PTPN7 specifically regulates ERK downstream of the Gq pathway.
ERK is also hyper-phosphorylated downstream of GPVI receptor in PTPN7 KO murine platelets. However, previous studies show that ERK phosphorylation downstream of
GPVI receptor is solely dependent on the feedback signaling by TXA2 and ADP (142).
Roger et al. (142) show that in the presence of indomethacin, collagen-stimulated platelets to have negligible ERK phosphorylation. Similarly, our studies show that in the presence of indomethacin, ERK phosphorylation is abolished in both WT and PTPN7 KO platelets stimulated with CRP. Thus, our data suggest that potentiation of ERK phosphorylation in PTPN7 KO murine platelets downstream of the GPVI receptor is solely due to enhanced TXA2 generation in PTPN7 KO platelets. Similarly, the GPVI- mediated aggregation and secretion responses are normalized in indomethacin-treated
PTPN7 KO murine platelets. Therefore, we can conclude that potentiation of aggregation and secretion in PTPN7 KO platelets is due to enhanced secondary stimulation.
Apart from ERK platelets also express p38 MAPK which get activated downstream of thrombin (126, 141), and collagen (143, 144). Several studies have suggested that p38
MAPK is essential for cPLA2 activation and TXA2 generation. Our studies demonstrate p38 MAPK phosphorylation is comparable in WT and PTPN7 KO platelets upon PAR
86
and GPVI receptor stimulation. This data suggests that PTPN7 specifically regulates only
ERK activation in platelets.
We evaluated the physiological implications of PTPN7 deficiency in mice. FeCl3- induced injury of the carotid artery model is frequently used to assess the in vivo thrombosis model. The in vivo thrombosis in WT and PTPN7 KO is comparable when mice were subjected to FeCl3-induced injury of the carotid artery model. Eckly et al.
(145) have demonstrated that FeCl3 can also lead to tissue factor-mediated injury to the blood vessel, which may result in thrombin generation. Thus, the authors have suggested the use the model cautiously for studying the thrombus formation in vivo. Therefore, we used pulmonary embolism model, which involves direct activation of platelets in vivo by injecting collagen and epinephrine in the mice, to evaluate in vivo thrombosis. Our results indicate that PTPN7 KO mice were more resistant to pulmonary thromboembolism than controls. Intriguingly, PTPN7 KO mice showed normal tail bleeding times. These findings suggest that PTPN7 plays a significant role in in vivo thrombosis but is dispensable for hemostasis.
In conclusion, although currently we lack understanding of how PTPN7 is regulated in platelets, through this study we have established that PTPN7 negatively regulates ERK activation and thereby TXA2 generation in platelets.
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CHAPTER 5
5 GENERAL DISCUSSION/ FUTURE DIRECTIONS
Understanding mechanisms of platelet activation and signaling components that regulate platelet activation are necessary because dysfunction of platelets signaling can lead to life-threatening conditions. Therefore, a better understanding of the signaling leading to platelet activation is critical to identify molecular targets for developing therapeutic interventions. A classic example of an anti-platelet drug is aspirin. Aspirin inhibits thromboxane generation by targeting the cyclooxygenase enzyme. Aspirin is given to patients suffering from myocardial infarction.(104) However, since we are aware of the role of thromboxane in platelets, we can predict that aspirin may lead to a risk of bleeding in these patients (146). The above example of aspirin, further emphasizes the importance of understanding mechanisms of platelet signaling.
Signals are transmitted in platelets via phosphorylation of proteins by PTK's. As discussed in the introduction, the phosphorylation of these proteins is regulated by PTP's.
Studying the role of PTP's will not only allow us to gain insight into the regulation of platelet activation but also evaluate their potential as a target for platelet therapies.
In our studies, we have shown a role for two PTP's- CD45 and PTPN7 in platelets. In
Chapter 3, we show a functional role for truncated CD45 in platelets using CD45 knockout mice. The most significant finding of this study is the presence of a novel truncated CD45 isoform, potentially consisting c-terminal domain 1 and 2, in platelets.
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Our study shows also emphasizes the importance of CD45 in regulating GPVI mediated signaling and platelet functional responses. Also, CD45 plays a critical role in regulating hemostasis and thrombosis in vivo. This data collectively suggests that CD45 is a positive regulator of GPVI mediated platelet function and hemostasis.
Additionally, in Chapter 4, we show for the first time that PTPN7 is present in both human and murine platelets. Using PTPN7 KO mice, we established that PTPN7 is a negative regulator of ERK in platelets. Thus, in PTPN7 KO murine platelets we observe potentiation in thromboxane levels and consequently elevated platelet functional responses. Our results demonstrate that PTPN7 negatively regulates in vivo thrombosis but not hemostasis. Thus, our finding suggests that PTPN7 acts as a break for platelet activation.
Although in the above studies we have gathered an understanding of the function of
CD45 and PTPN7 in platelets, we still need to address several questions. For example, it will be interesting to evaluate the role of truncated CD45 and PTPN7 in human platelets.
Following is a discussion of several concerns that need to be addressed.
1) Study the function of CD45 in human platelets:
Our research has demonstrated the presence of the truncated CD45 in the cytoplasmic fraction of human platelet lysate. We postulate that the truncated isoform of CD45 acts as a negative regulator of GPVI signaling in human platelets. To study the role of truncated
CD45 in human platelets, we will use a CD45 inhibitor. Previously, a monoclonal antibody against the RB isoform of CD45 has been used to inhibit the alloreactivity of
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human CD4+ lymphocytes in vitro (147). Since the monoclonal antibody is targeted toward the extracellular domain, it will not be advisable to use this antibody as an inhibitor for platelets. Instead, we will opt for an inhibitor that is specific to the catalytic domain. However, the problem associated with the use of analogs targeted towards the catalytic domain is non-specificity because many phosphatases share substrates and the structure of the catalytic pocket (148). Perron et al. (149) have identified a small molecule inhibitor- compound 211 which binds to an allosteric pocket, near the linker or
D1–D2 interface that is unique to the CD45 intracellular domain. Compound 211 was shown to be specific for CD45 and did not affect several related PTPs (149). Therefore, using compound 211 will be ideal for our studies. Firstly, we will evaluate the specificity of compound 211 for CD45 in platelets. One way to address is the using compound 211 in CD45 KO murine platelets. If the inhibitor is specific, CD45 KO platelets treated with and without inhibitor should show the same extent of inhibition of platelet functional responses. After proving the specificity of the inhibitor, we will use the inhibitor to further will evaluate ex vivo platelet functional responses in human platelets such as platelet aggregation, dense and alpha granule secretion and IIb3 activation.
Furthermore, we will also evaluate the GPVI signaling in human platelets in presence and absence of the inhibitor.
2) Assessing the stage of platelet development at which CD45 gets truncated.
Our results suggest that megakaryocytes have a full-length isoform of CD45 whereas platelets express a truncated isoform. Therefore, we hypothesize that CD45 gets truncated at the pro-platelet stage. We will use confocal microscopy to evaluate our hypothesis. We 90
will culture megakaryocytes from WT mice and CD45 KO mice (negative control). After maturation of megakaryocytes, mature megakaryocytes will be transferred to fibrinogen- coated plates, which will allow pro-platelet formation. The cells will then be fixed and permeablized to take up the CD45 c-terminal domain antibody. After incubation with the secondary fluorescent antibody, the results will be viewed using confocal microscopy.
We anticipate that we will observe the full-length isoform in megakaryocytes and a cytoplasmic, truncated isoform of CD45 in pro-platelets and platelets.
3) Evaluating the mechanism by which CD45 is cleaved and investigating the region where CD45 is cleaved.
(i) We observed that the CD45 antibody recognized a 180 kDa protein in WT but not in the CD45 KO. Also, the 65 kDa protein found in platelets was not present in the megakaryocytic protein lysates. Therefore, we ruled out the possibility that the truncated
CD45 is an alternatively spliced variant. We speculate that the truncated CD45 isoform in platelets is generated by proteolytic cleavage when platelets are formed from megakaryocytes. To identify the protease involved in truncating CD45, we will treat the megakaryocytes with different protease inhibitors such as serine protease inhibitor, metalloprotease inhibitor, a calpain inhibitor, a proteasome inhibitor, cysteine protease inhibitor and aspartyl protease inhibitor. Following the treatment, we will stimulate the megakaryocytes to form pro-platelets and evaluate the results using confocal microscopy.
The conditions under truncation of CD45 isoform is not observed in pro-platelets will the potential mechanism of proteolytic cleavage.
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(ii)The whole of the cytoplasmic domain of CD45 is approximately 95kDa in size. The truncated isoform of CD45 in platelets is approximately 65kDa suggesting that CD45 is cleaved within the cytoplasmic domain. To identify the exact location where CD45 gets cleaved, we will use mass spectrometry. We will subject human, WT and CD45 KO platelets to SDS-PAGE and the bands between 50-75 kDa will be analyzed using mass spectrometry analysis. The peptide sequences will be aligned with the full-length CD45 peptide sequence to identify the position where CD45 is cleaved.
4) The mechanism by which PTPN7 is regulated in platelets.
Studies in lymphocytes have suggested that interaction between PTPN7 and ERK is regulated by phosphorylation of a serine residue. Tryptic-peptide mapping was used to show that PTPN7 gets phosphorylated at Ser-23 apart from Ser-72 and Thr-45 (53, 150).
Ser-23 is present in the KIM, and therefore phosphorylation at Ser-23 leads to dissociation of PTPN7 from ERK. Saxena et al. (53) show that in T-cells, cAMP signaling leads to activation of PKA which further phosphorylates PTPN7 on Ser-23. A similar mechanism exists in B-cells, where phosphorylation PTPN7 via the
Gs/cAMP/PKA pathway leads to increase of free p38 MAPK (135). Currently, we don't know if a similar mechanism exists in platelets. Activation of Gs/cAMP/PKA pathway in platelets leads to inhibition of platelet activation, and ERK is not activated downstream of this pathway. Therefore, it will be interesting to evaluate the mechanism by which
PTPN7 gets phosphorylated downstream of the Gq-mediated signaling. We speculate that serine/threonine kinases such as PKC's, which share same consensus sequence with PKA,
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may play a significant role in phosphorylating PTPN7. As of now, we are not able to test this hypothesis since an antibody that binds to phosphorylated Ser-23 of PTPN7 is not available commercially. To address this question, firstly, we will generate an antibody that specifically recognizes phosphorylation on Ser-23 of PTPN7. We predict that PTPN7 will be phosphorylated at Ser-23 when platelets are stimulated. To test the hypothesis whether or not PKC's are involved in this phosphorylation, we will inhibit the PKC activity using PKC inhibitors. If our hypothesis is correct, then we will observe reduced phosphorylation on the Ser-23 in samples treated with PKC inhibitor as compared to the untreated samples. We can further test if similar results are obtained by using KO mice for several PKC isoforms.
5) The role of PTPN7 in human platelets.
Small molecule modulators of PTPN7 interact with unique amino acid residues in the
PTPN7 substrate-binding loop and WPD-loop, thereby making this compound specific for PTPN7 (151). PTPN7 inhibitor specifically augments ERK1/2 and p38 activation in human T cells. Whereas activation of MEK was unchanged, proving the specificity of the antibody (151). We will investigate the role of PTPN7 in human platelets using the above-mentioned inhibitor. We will evaluate the ex vivo platelet functional responses in human platelets such as platelet aggregation, dense and alpha granule secretion.
Furthermore, we will also evaluate the ERK phosphorylation in human platelets in presence and absence of the inhibitor.
6) Study different PTPs in platelets
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We are only just beginning to understand the role of PTPs in platelets. Using proteomics approach ten receptor type and ten non-receptor type phosphatases have been identified.
However, till date function of one receptor type tyrosine kinase (CD148) and 3 Non- receptor type phosphatases (PTP1B, SHP-1, and SHP-2) have been studied in platelets.
Our studies on CD45 and PTPN7 have further added to the existing knowledge base.
However, the role of several other phosphatases needs to be investigated. We propose to study one more receptor type PTP, PTPRO in platelets. We have chosen to study this molecule due to commercial availability of a PTPRO KO mouse model.
PTPRO family is a group of highly conserved receptor-type protein tyrosine phosphatases. They have a single catalytic domain, a transmembrane domain and an extracellular domain of variable length (152). Alternative splicing of the exons encoding the extracellular domain give rise to two main isoforms of PTPRO – GLEPP-1(PTPRO) a full-length isoform, which has an extended extracellular domain with 8 fibronectin motifs and PTPROt, which has only 8 amino acids in the extracellular region (152). PTPRO is expressed mainly in epithelial, brain and kidney cells whereas PTPROt, previously identified as a lymphoid PTP, is expressed in B-lymphoid tissues, macrophages and osteoclasts (152-156). PTPROt has a critical role in B-cell proximal signaling, as SYK has been identified as its major substrate. Chen et al have shown the functional consequences of overexpression of PTPROt on overall B-cell signaling. They found that overexpression of PTPROt leads to decreased SYK phosphorylation, BCR signaling and cellular proliferation (157). In addition to SYK, PTPROt also dephosphorylates ZAP70 and Src Family Kinases like Lyn/Lck. PTPROt dephosphorylates the activating tyrosine
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(Y394) on Lyn/Lck, which is indicated hypo-phosphorylation of Lyn/Lck in presence of overexpressed PTPROt. Thus suggesting that PTPROt prevents activation of Lyn/Lck which further suppresses SYK/ZAP70 activation (158).PTPRO knockout mouse was generated by Wharram et al by disrupting the exon coding for NH2-terminal region by gene targeting in embryonic stem cells (159).These mice have been used to study the role of PTPRO in kidney or hepatic cells (160), however no studies have been performed in hematopoietic cells using PTPRO KO mice.
We hypothesize that PTPRO plays a significant role in regulating SFK and Syk phosphorylation in platelets. Firstly, we will establish the presence of PTPRO in platelets using biochemical techniques. Next, investigate the role of PTPRO in platelet activation by characterizing the ex vivo platelet functional responses in WT and PTPRO KO mice.
If observe a difference in platelet functional responses between WT and PTPRO KO mice we will investigate the molecules in platelet signaling that PTPRO could regulate.
Finally, we will evaluate the physiological implications of PTPRO deficiency in mice by measuring tail bleeding times and in vivo thrombosis models using both a FeCl3 injury model and pulmonary embolism.
Significance:
Given the role of Protein Tyrosine Phosphatases in the regulation of platelet activation, they make lucrative drug targets. However, PTPs have always been considered un- druggable targets due to the traditional approach of targeting the active site, which is similar in all PTPs. More recently, efforts are being made to target the structural features surrounding the active site, which is unique to the PTP.
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CD45 is a positive regulator of GPVI- mediated platelet activation and can be a good target for an anti-thrombotic therapy. An allosteric inhibitor of CD45 - compound 211, has been characterized and in vitro, experiments have suggested that the compound 211 prevents T-cell receptor-mediated activation of Lck, Zap-70, and mitogen-activated protein kinase, and interleukin-2 production. Furthermore, in a delayed-type hypersensitivity reaction in vivo, compound 211 abolishes inflammation (149). Therefore, it will be interesting to see its use as an anti-thrombotic agent.
PTPN7 is a negative regulator of GPCR - mediated ERK activation thus it will be interesting to evaluate its potential as an anti-bleeding target. PTPN7 gene is located on the long arm of chromosome 1, and this gene locus is highly susceptible to genetic mutations (132). PTPN7 is often is dysregulated in the pre-leukemic disorder myelodysplastic syndrome and myelogenous leukemia. PTPN7 currently under evaluation as a drug target for leukemia and can also be evaluated as an anti-bleeding target.
A thorough understanding of the role of PTPs in platelets can also help to predict the effect of already existing drugs on hemostatic functions. For example, inhibitors used to treat PTPN7 related leukemia will also have a profound effect on platelet activation. We can speculate that PTPN7 inhibitors would lead to hyper platelet activation, which would lead to thrombosis in these patients.
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It will be interesting to evaluate the potential of these new drug targets in preventing thrombosis or bleeding disorders as opposed to the currently existing therapies. Current antiplatelet therapies have several short comings such as increased risk of bleeding or resistance. Therefore, it will be necessary to see if targeting CD45 and PTPN7 can eliminate these potential risks.
In conclusion understanding the role of above phosphatases in platelet activation will be helpful for the development of new generation of antiplatelet therapies and also understand effect of several existing therapies on platelet activation.
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