Sanford Shattil - US grants

The surface membrane of the platelet is the site at which agonists stimulate aggregation and secretion, and it is the surface upon which thrombin generation is facilitated. Platelets are activated upon the binding of agonists to specific surface receptors. The long term goal of this proposal is to better characterize the structural changes within the plasma membrane that occur subsequent to the binding of an agonist and that culminate in the platelet's hemostatic responses. Three general approaches will be taken. First, because the platelet receptor for epinephrine has already been well characterized pharmacologically, the structural features of this Alpha2-adrenergic receptor will be explored. Specifically, the receptor will be solubilized and an attempt will be made to identify the native receptor using polyacrylamide gel electrophoresis under non-denaturing conditions. In addition, the receptor will be purified by affinity chromatography using both and Alpha2-adrenergic antagonist and anti-Alpha2-receptor monoclonal antibodies as the affinity reagents. The receptor will also be reconstituted into artificial membranes in order to examine the influence of specific ions, membrane lipids and proteins on receptor function. Second, because platelets contain saturable and high affinity binding sites for Ca++ on their surface, the effect of receptor stimulation on the binding of Ca++ to specific proteins on the surface membrane will be examined. These membrane Ca++ binding proteins will be identified using 45Ca++ as well as the lanthanides, gadolinium and terbium, which are known for their affinity for Ca++ binding sites. Third, the relationship between the binding of Ca++ to the surface membrane and specific Ca++ dependent membrane changes, such as phospholipid hydrolysis and protein phosphorylation will be examined. These changes may occur subsequent to epinephrine receptor stimulation and membrane Ca++ association and appear to be involved in platelet activation. The above studies will be carried out using normal platelets as well as platelets with known membrane lipid and protein structural defects. As a result, we hope to clarify not only the role of membrane-bound Ca++ in receptor-mediated platelet activation, but also the pathophysiology of some congenital and acquired disorders of platelet function.

During hemostasis, the platelet membrane becomes a conduit for the flow of information into and out of the cell. The long-term goal of this project is to test whether platelet adhesion, aggregation and procoagulant activity are regulated by specific interactions of plasma membrane constituents with intracellular regulatory molecules on the one hand and extracellular ligands on the other. The four specific aims will focus primarily on integrin GP IIb-IIIa, a receptor for RGD- containing adhesive proteins. First, the intracellular processes that regulate binding of fibrinogen to GP IIb-IIIa will be examined. To identify the signaling pathways involved, antibodies specific for and peptides derived from potential mediators, including GTP-binding proteins and protein kinases, will be introduced into permeabilized platelets, and their effects on fibrinogen receptor function measured by flow cytometry. A series of chemical cross-linking and immuno- precipitation strategies will also be employed to identify specific regulatory molecules that interact directly with the cytoplasmic tails of GP IIb or IIIa. Second, the process of inward signaling across GP IIb-IIIa will be characterized. To initiate fibrinogen binding without agonists, platelets will either be incubated with antibodies that directly induce ligand binding or allowed to adhere to immobilized fibrinogen. Then changes in cytoplasmic free Ca++, protein tyrosine and serine/threonine phosphorylation, F-actin assembly, and granule secretion will be monitored. In parallel studies, fluorescence resonance energy transfer techniques will be used to examine whether clustering of receptors is required for these fibrinogen-dependent platelet responses. Third, an antigen-antibody interaction will be studies as a model for integrin-fibrinogen interactions. PAC1 is an antibody specific for activated GP IIb-IIIa and its antibody-combining site mimics GP IIb-IIIa recognition sites in fibrinogen. To establish the molecular basis for this mimicry, recombinant Fab and Fv fragments of PAC1 will be expressed in mammalian and insect cell expression systems. Site-directed mutations will be introduced in the hyper- variable regions of PAC1 and their effects on antibody affinity and specificity determined. Fourth, a flow cytometric assay will be used to study clinical abnormalities of platelet membrane activation in whole blood. Platelets from individuals with reduced platelet aggregation responses will be studied to identify defects either in the structure or activation of GP IIb-IIIa or in signaling reactions distal to GP IIb-IIIa. An assay will also be developed to quantitate occupancy of GP IIb-IIIa by potential therapeutic agents, such as peptides (disintegrins), peptidomimetics and antibody fragments. Finally, we will test the hypothesis that the procoagulant activity of platelets contributes to thrombosis in two acquired disorders that affect the platelet membrane, paroxysmal nocturnal hemoglobinuria and the anti-phospholipid syndrome. Accordingly, platelets from bleeding time wounds in these patients will be studied for over-expression of membrane binding sites for factors Va and VIIIa. Overall, these studies should lead to a more detailed understanding of the platelet membrane events required for normal hemostasis and the role of membrane activation in hemorrhagic and thrombotic disorders.

DESCRIPTION (provided by applicant): allb-beta3 and aVbeta3 integrins mediate adhesion of platelets and other cells to matrix ligands at sites of vascular injury. Ligand binding is regulated by activating or "inside-out" signals that modulate integrin affinity through conformational changes and integrin avidity through microclustering. Recently, integrin conformational changes have been characterized structurally and talin has been identified as a key regulator of these changes by binding to the beta3 cytoplasmic tail. Here, three major unresolved questions will be asked concerning the molecular basis of inside-out signaling. First, what is the relationship between talin and two other proteins, Rap1b and ADAP, recently implicated as positive regulators of beta3 integrin affinity? The role of Rap1b or ADAP in talin-dependent beta3 integrin activation will be assessed in primary mouse megakaryocytes, platelets and an improved CHO cell model system in which beta3 integrins are sensitive to inside-out signals. Over-expression and knockdown approaches will be used to investigate the inter-dependency of these mediators and their relative effects on beta3 integrin affinity and microclustering. Then, Rap1b and ADAP effectors that regulate beta3 integrins will be identified and their actions characterized. Second, are there regulators of beta3 integrin affinity still to be discovered? Here, the effects on allb-beta3 affinity of PTB domain- containing proteins other than talin will be determined, focusing particular attention on moesin and Dok-2, platelet proteins implicated in cytoskeletal regulation. In a complementary approach, affinity regulation of beta3 integrins will be characterized in zebrafish to evaluate mechanistic similarities and differences with the regulatory process in mammals. Third, what are the mechanisms and consequences of beta3 integrin activation in neoplastic cells? Over-expression and knockdown approaches will investigate whether Rap1b or talin are required for constitutive aVbeta3 activation in certain tumor cells and whether this integrin activation promotes a metastatic phenotype. The potential for oncogenic activation of allb-beta3 in chronic myeloproliferative diseases will be evaluated in mouse megakaryocytes and platelets by over-expressing constitutively-active JAK2 (V617F), a common mutation in these bleeding- and thrombosis-prone diseases. Together, these studies will provide insights into how beta3 integrin activation is regulated, how the activation mechanism is co-opted in clinical disorders, and how it may be harnessed to develop better anti-platelet drugs.

Integrin activation is tightly regulated by intracellular signals, and integrins localize signaling molecules to sites of cell adhesion. These relationships are highly relevant to blood and vascular cells, where integrins regulate growth, differentiation, survival and function. The purpose of this Program Project is to characterize fundamental mechanisms of integrin signaling in blood and vascular cells, with the common goal of identifying general rules of integrin signaling. Project 1 will test the hypothesis that integrin alphaIIbbeta3 utilizes a novel pathway involving protein tyrosine kinases and hematopoietic cell- specific adapter molecules to promote cytoskeletal reorganization in adherent platelets and megakaryocytes. Particular emphasis will be placed on identifying protein-protein interactions that initiate and propagate signal relay form alphaIIbbeta3 to actin. Project 2 will identify and characterize signaling pathways that suppress integrin activation. Special emphasis will be placed on proteins identified in genetic screens to influence integrin activation, including Ras GTPases, MAP kinases, and PEA-15, a death effector, domain-containing protein. Project 3 will test the hypothesis that integrins are components of complex intracellular signaling networks whose spatially and temporally regulated activation governs endothelial cell and monocytic migration. It will determine how these networks regulate localized integrin activity, and how integrins and mechanical forces regulate localized activation of Rac, Cdc42, PAK and ERK kinases to control cell migration. Project 4 will examine relationships between Abl tyrosine kinases and integrin signaling by determining how Abl modulates filopodia formation, cell migration and chemotaxis. It will also determine how integrins stimulate the nuclear export of c-Abl and how they override the inhibition of c-Abl activity by F-actin. These projects will be supported by core units that provide recombinant proteins, cell microinjection and imaging capabilities and administrative coordination. The synergy achieved by this Program will lead to a better understanding of integrin signaling, with implications for hemostasis, vascular biology and blood diseases.

[unreadable] DESCRIPTION (provided by applicant): Following vascular injury, adhesive ligands such as fibrinogen and von Willebrand factor engage integrin allbbeta3 to effect platelet aggregation and spreading during hemostasis and thrombosis. These responses are triggered by ligand-mediated allbbeta3 clustering, which initiates "outside-in" signals to reorganize the actin cytoskeleton. Recent work from this laboratory has established that outside-in signaling in platelets requires Src family tyrosine kinases (SFKs), c-Src in particular, which bind to beta3 and are activated by allbbeta3 clustering in a manner dependent on PTP-1B, a protein tyrosine kinase. Here, three major unresolved questions will be asked concerning the molecular basis of outside-in signaling in platelets and its biological consequences. First, do direct interactions between integrins and SFKs represent a general mechanism for spatio-temporal initiation of outside-in signaling in platelets? Since platelets contain five different integrins and at least six different SFKs, this possibility will be evaluated by co-immunoprecipitation techniques, by bimolecular fluorescence complementation imaging in live cells, and by localization of Src activation in live cells using a FRET-based reporter. In addition, integrin/SFK interactions will be assessed in Drosophila cells to determine the extent to which direct activation of SFKs by integrins is an evolutionary conserved process. Second, how does PTP-1B activate c-Src downstream of integrins? The mechanism by which PTP-1B is recruited to the allbbeta3/c-Src complex, and possibly to other integrin/SFK complexes, will be evaluated in platelets and model cell systems, focusing on the possible role of adapter proteins. In addition, the effect of integrin clustering on PTP-1B catalytic activity will be determined. Third, does selective disruption of outside- in signaling affect thrombus formation in vivo? Here arterial thrombosis will be studied in novel gene-targeted mice predicted to have selective defects in the interaction of allbbeta with c-Src or other SFKs, or defects in downstream events required for actin reorganization. Altogether, these studies will provide molecular insights into how outside-in integrin signaling is initiated and establish the extent to which this process regulates platelet function in vivo. Thus, this line of investigation may lead to identification of new anti-thrombotic drug targets and serve as a paradigm for integrin signaling in other blood cells. [unreadable] [unreadable] [unreadable]

Objective: The purpose of the Administrative Core Unit is to provide overall direction and oversight of the Program, promote interaction among the investigators, and provide effective budget management and administrative assistance. Description: The offices of Dr. Shattil and Tawana Young are located immediately adjacent to the laboratories of Dr. Shattil and Ginsberg. The program will require extensive interactions between the participants, as well as access to the central UCSD electronic mail system, computing facilities and fiscal management software. The Administrative Core Unit will coordinate the monitoring of research within and between projects, optimize utilization of the other core units, interact with departmental and UCSD fund managers, schedule "work-in-progress" research meetings and meetings of the Advisory Boards, and prepare reports to the NIH.

Vascular cells, including platelets, express numerous integrins, and bidirectional signaling appears to be a general function of most of them. While studies of B3 integrins allbB3 and aVB3 have contributed to our current understanding of integrin signaling, important questions remain. Specifically, which intracellular proteins interact with integrin B cytoplasmic tails and how do they transmit signals to and from integrins? The goal of this project is to test two hypotheses relevant to these unresolved questions using advanced experimental approaches. The first hypothesis is that inside-out regulation of allbps affinity is controlled by the coordinated recruitment of proteins such as talin and kindlin-3 to B3. Binary and ternary interactions among these proteins will be examined in living cells, including murine platelets, using bimolecular fluorescence complementation, FRET, and in situ proximity ligation. Studies will address the degree to which talin and kindlin-3 recruitment are dependent on Rap1 GTPase, whether kindlin-3 promotes talin recruitment or vice-versa, and whether adhesive ligand binding to allbp3 is sufficient to promote recruitment of either of these proteins to the B3 tail. The second hypothesis is that interactions of the aV integrin B cytoplasmic domain with talin, kindlins and Src family kinases (SFKs), either alone or in combination, dictate the outcome of aV-mediated processes in vivo. Our preliminary studies with zebrafish embryos using morpholino oligonucleotides to knockdown aV, and aV mRNA to rescue knockdown phenotypes, reveal that gastrulation events required for left-right body axis specification are dependent on aV, as are certain neurological and vascular developmental events also reported in aV knockout mice. Therefore, additional knockdown and rescue experiments will be carried out to identify the relevant zebrafish integrin aV B subunit that regulates specification of laterality. To determine whether integrin interactions with talin, kindlins or SFKs are involved, rescue experiments will be conducted with mutant p subunits that are predicted and demonstrated to selectively or collectively disrupt interactions with these proteins. The proposed studies should clarify basic and conserved mechanisms of allb and aV integrin signaling and inform followup studies in gene-targeted mice, with implications for human platelet and vascular biology.

DESCRIPTION (provided by applicant): Integrins mediate cell adhesion and signaling during physiological responses to vascular injury, as exemplified by the requirement for platelet ¿IIb¿3 in hemostasis. Furthermore, evidence indicates that integrins may function abnormally in pathological circumstances, such as arterial thrombosis and neoplasia. A key regulatory step in integrin function is receptor activation, manifest by conversion from a low- to a high-affinity conformational state, and from an unclustered to a clustered, high-avidity state. Integrin activation is regulated by inside-out signals that are triggered by cellular agonists, although te precise nature of these signals remains incompletely understood. The long-term objectives of this proposal are to 1) fully understand the molecular basis of inside-out ¿IIb¿3 signaling in platelets; and 2) extend these fundamental concepts to integrins in human cancer cells, since these integrins have been implicated in tumor progression, including the metastatic cascade. Two specific aims will address major unresolved questions pertaining to these objectives. Aim 1 will test the hypothesis that a specific molecular adapter protein, ADAP, interacts functionally if not physically with one or more recently identified integrin-proximal regulatory proteins (e.g., talin, kindlin), to activate ¿IIb¿3 in response to platelet agonists. Accordingly, biochemical and advanced imaging techniques will be used to determine whether and how ADAP interacts with such integrin-proximal regulators in platelets, in gene-targeted primary murine megakaryocytes, and in model cells engineered to recapitulate ¿IIb¿3 activation. These experimental systems will also be used to assess the role of ADAP in promoting changes in ¿IIb¿3 clustering as a mechanism of ¿IIb¿3 activation complementary to affinity modulation. Aim 2 will test the hypothesis that activation of ¿1 integrins is required for tumor cell extravasation from blood vessels and for the development of metastatic foci. Preliminary data indicate the presence of activated ¿1 integrins in human cancers. Therefore, human melanoma and breast cancer cell lines and a well-characterized vertebrate experimental metastasis model system will be used to address which ¿1 integrins must become activated to promote metastasis, whether specific integrin-interacting proteins such as talin, kindlin and Src family kinases are involved, and whether deletion of a specific tumor suppressor gene, DLC-1, promotes metastasis by activating ¿1 integrins. The proposed studies will establish common and unique mechanisms of integrin activation in physiological and pathological circumstances, with diagnostic and therapeutic implications for vascular biology and beyond.

PROJECT SUMMARY Platelet studies have had a profound impact on our understanding of adhesion receptor function as exemplified by the adhesion and signaling roles of integrin ?IIb?3 in hemostasis and thrombosis. Less is known about whether and how ?IIb?3 signaling regulates platelets in their roles as sentinels and effectors of immunity and inflammation. In this regard, we have discovered that ?IIb?3 interacts through its ?IIb cytoplasmic tail with SHARPIN, an obligate component of the linear ubiquitin chain assembly complex (LUBAC). This complex is the only known enzyme responsible for M1 linear ubiquitination of key proteins involved in immune and inflammatory signaling through the NF-?B pathway in leukocytes. Platelets express many proteins of the canonical NF-?B pathway. Therefore, we will now employ complementary approaches, including studies of peripheral blood platelets, megakaryocytes and platelets derived from human induced pluripotent stem cells, and gene-targeted mice to test the following central hypothesis: Through dynamic and mutually exclusive interactions with ?IIb?3 and LUBAC, SHARPIN regulates platelet function in inflammation and immunity as well as in hemostasis and thrombosis. Aim 1 will employ biochemical and advanced imaging techniques to test whether physical and functional linkages exist between ?IIb?3, LUBAC and the NF-?B pathway in human and mouse platelets. Attention will be focused on whether SHARPIN functions to maintain ?IIb?3 in a low-affinity state in resting platelets, but dissociates from ?IIb and assembles the LUBAC complex to facilitate ?IIb?3 and NF-?B activation in response to platelet agonists generated during hemostasis and inflammation. Aim 2 will introduce genetic modifications into human megakaryocytes and platelets derived from induced pluripotent stem cells to model the effects of SHARPIN knockdown or knockout on ?IIb?3 and LUBAC functions in the appropriate primary cells. Using this system, optogenetic techniques new to the platelet field will be used to determine the functional consequences of conditional SHARPIN interactions with ?IIb?3 or with its two LUBAC protein partners. Aim 3 will address whether SHARPIN is required for ?IIb?3 and platelet functions in response to inflammatory stimuli and vascular injury in vivo. To accomplish this, platelet-specific SHARPIN knockout mice will be generated using the Pf4-Cre system and studied in several mouse models of platelet-dependent inflammation, hemostasis and thrombosis. Altogether, these studies will provide new insights into the role of SHARPIN in platelet pathobiology, with mechanistic and potential therapeutic implications beyond hemostasis and for platelets in inflammation and immunity.

PROJECT SUMMARY Integrin ?IIb?3 (GP IIb-IIIa) is the platelet receptor for fibrinogen and is required for platelet aggregation during hemostasis. Fibrinogen binding to platelets is regulated by interactions of specific intracellular proteins, including talin and kindlin-3, with the ?3 cytoplasmic tail. In contrast, proteins that might interact with the ?IIb tail to regulate fibrinogen binding are relatively unexplored. We have found that human and mouse platelets and endothelial cells express the 40 kDa protein, SHARPIN. Studies with human platelets as well as with platelets and megakaryocytes derived from human induced pluripotent stem cells have revealed that SHARPIN can interact directly with either the ?IIb tail or with two other proteins to constitute the linear ubiquitination chain assembly complex (LUBAC). In fact, stimulation of platelets by traditional hemostatic agonists, such as thrombin, or by inflammatory agonists, such as lipopolysaccharide or soluble CD40 ligand (sCD40L), triggers both fibrinogen binding to ?IIb?3 and Met1-linked linear ubiquitination of IKK? (NEMO) to promote NF-kB pathway signaling. SHARPIN knockdown by shRNA in megakaryocytes and platelets results in decreased agonist-induced, linear ubiquitination of NEMO, but increased fibrinogen binding to ?IIb?3, MHC Class I expression, and release of endogenous sCD40L. Here we will test the hypothesis that SHARPIN?s mutually exclusive interactions with integrin ? tails or LUBAC regulate critical platelet and/or endothelial cell responses during hemostasis, thrombosis, inflammation and angiogenesis. Aim 1 will use advanced techniques, including optogenetics, to determine the stoichiometry of SHARPIN and ?IIb?3 in platelets and to test the functional effects of enforcing SHARPIN interactions with either ?IIb or LUBAC. Platelet-specific SHARPIN knockout mice will be generated in order to test the requirement for platelet SHARPIN in hemostasis, thrombosis and inflammation using a range of mouse models. Aim 2 will determine the role of SHARPIN in the adhesive and angiogenic functions of integrin ?V?3 and in NF-kB pathway signaling in endothelial cells. Endothelial cell SHARPIN will be specifically and conditionally knocked out in mice, and lung microvascular endothelial cells from these mice will be evaluated for ?V?3-dependent adhesive responses and for angiogenic sprouting. The effects of deleting endothelial cell SHARPIN in vivo will be determined using established mouse models of developmental and pathological angiogenesis. This project will make heavy use of the Hemostasis, Thrombosis, and Inflammation Models Core and it will collaborate with all other projects in this Program to achieve its aims. Altogether, these studies will provide a comprehensive test of the central hypothesis and establish new mechanistic insights into the regulation of integrin and immune signaling by SHARPIN in vascular cells, with clear implications for hemostasis, thrombosis, inflammation and angiogenesis.