1985 |
Parise, Leslie V. |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Reconstruction of the Platelet Fibrinogen Receptor @ J. David Gladstone Institutes |
0.903 |
1987 — 1988 |
Parise, Leslie V. |
R29Activity Code Description: Undocumented code - click on the grant title for more information. |
Ligand Binding Domains On Platelet Glycoprotein Iib-Iiia @ J. David Gladstone Institutes |
0.903 |
1988 |
Parise, Leslie V. |
R29Activity Code Description: Undocumented code - click on the grant title for more information. |
Ligand Binding Domains On Platelet Glycoprotein Iib-Iia @ University of North Carolina Chapel Hill
This proposal is a request for five years funding for a FIRST award, designed to identify and characterize ligand binding domains on the platelet membrane glycoprotein (GP) IIb-IIIa complex. Previous work demonstrated that GP IIb-IIIa is sufficient to account for the fibrinogen and fibronectin receptor on platelets. However, the number and location of fibrinogen and fibronectin binding sites on GP IIb-IIIa are unknown. Preliminary results suggest that GP IIb and GP IIIa each have fibrinogen binding domains. The specific aims of this proposal are threefold. First, fibrinogen and fibronectin binding domains will be identified by testing the ability of fragments of cleaved GP IIb and GP IIIa to inhibit 125I-GP IIb-IIIa binding to immobilized ligands. This approach will be supplemented with photoaffinity labeling of GP IIb-IIIa. Fragments of GP IIb and GP IIIa containing ligand binding domains will be isolated and characterized with regard to sites recognized on fibrinogen and fibronectin and will serve as antigens in monoclonal antibody production. Second, ligands and monoclonal antibodies will be used to locate ligand binding regions on GP IIb-IIIa by rotary shadowing electron microscopy. Third, monoclonal antibodies will be used to determine if the identified ligand binding domains function in whole platelets to bind ligands and mediate aggregation. In addition, monoclonal antibodies and 125I surface labeling will be used to determine if the conversion of GP IIb-IIIa to a competent receptor on activated platelets is due to an increased exposure of the identified ligand binding domains. Completion of these studies will contribute to the long- term goal of relating GP IIb-IIIa structure to function. Such knowledge will increase our understanding of the molecular mechanisms of hemostasis and thrombosis.
|
1 |
1989 — 1991 |
Parise, Leslie V. |
R29Activity Code Description: Undocumented code - click on the grant title for more information. |
Ligand Binding Domains On Platelet Glycoprotein @ University of North Carolina Chapel Hill
This proposal is a request for five years funding for a FIRST award, designed to identify and characterize ligand binding domains on the platelet membrane glycoprotein (GP) IIb-IIIa complex. Previous work demonstrated that GP IIb-IIIa is sufficient to account for the fibrinogen and fibronectin receptor on platelets. However, the number and location of fibrinogen and fibronectin binding sites on GP IIb-IIIa are unknown. Preliminary results suggest that GP IIb and GP IIIa each have fibrinogen binding domains. The specific aims of this proposal are threefold. First, fibrinogen and fibronectin binding domains will be identified by testing the ability of fragments of cleaved GP IIb and GP IIIa to inhibit 125I-GP IIb-IIIa binding to immobilized ligands. This approach will be supplemented with photoaffinity labeling of GP IIb-IIIa. Fragments of GP IIb and GP IIIa containing ligand binding domains will be isolated and characterized with regard to sites recognized on fibrinogen and fibronectin and will serve as antigens in monoclonal antibody production. Second, ligands and monoclonal antibodies will be used to locate ligand binding regions on GP IIb-IIIa by rotary shadowing electron microscopy. Third, monoclonal antibodies will be used to determine if the identified ligand binding domains function in whole platelets to bind ligands and mediate aggregation. In addition, monoclonal antibodies and 125I surface labeling will be used to determine if the conversion of GP IIb-IIIa to a competent receptor on activated platelets is due to an increased exposure of the identified ligand binding domains. Completion of these studies will contribute to the long- term goal of relating GP IIb-IIIa structure to function. Such knowledge will increase our understanding of the molecular mechanisms of hemostasis and thrombosis.
|
1 |
1990 — 1996 |
Parise, Leslie V. |
M01Activity Code Description: An award made to an institution solely for the support of a General Clinical Research Center where scientists conduct studies on a wide range of human diseases using the full spectrum of the biomedical sciences. Costs underwritten by these grants include those for renovation, for operational expenses such as staff salaries, equipment, and supplies, and for hospitalization. A General Clinical Research Center is a discrete unit of research beds separated from the general care wards. R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Sickle Cell Adhesion to the Endothelium @ University of North Carolina Chapel Hill
This proposal is designed to define the molecular basis of sickle cell adhesion to the endothelium. Vasoocclusion is believed cause the painful, reoccurring crisis suffered by sickle cell patients. Recent evidence suggests that this vasoocclusion may be precipitated by sickle cell adhesion to the endothelium. The molecules involved on the sickle cell surface, the endothelial cell surface, and any intermediary ligands have not been clearly identified. Our working hypothesis is that sickle cells and possible normal reticulocytes contain a receptor or receptors that bind to a ligand such as unusually large von Willebrand factor (ULvWF), which in turn binds to an endothelial cell receptor(s), precipitating the vasoocclusive crises in sickle cell disease. The specific aims of this grant are fourfold. First, we propose to identify and characterize the receptor(s) on the sickle cell surface that mediate adhesion to endothelial cells. This will involve a variety of approaches such as flow cytometry and sorting, immunoelectron microscopy, monoclonal antibody production, and adhesion assays. Second, we will identify and characterize the receptor(s) on the endothelial cell surface that mediate adhesion to sickle cells, using techniques similar to those listed above, and by testing specific receptor-deficient mutants in an endothelial-like cell line. Third, we will evaluate the role that ULvWF and other potential ligands play in the adhesion of sickle cells to endothelial cells. Finally, we will examine the contribution of activated platelets and platelet secretory products to the adhesion of sickle cells to endothelial cells. Completion of these studies should result in a greater understanding of the molecular events that contribute to the damaging, painful events in sickle cell disease and should provide a rationale for future treatment of this disease.
|
1 |
1992 — 2002 |
Parise, Leslie V |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Activation of the Platelet Fibrinogen Receptor @ University of North Carolina Chapel Hill
fibrinogen receptors; integrins; protein structure function; platelet activation; vitronectin; receptor binding; ligands; lipids; conformation; thrombosis; fibrinogen; hemostasis; protein reconstitution; glycoproteins; tissue mosaicism; leukocytes; tissue /cell culture; flow cytometry; human tissue; transfection;
|
1 |
1999 — 2002 |
Parise, Leslie V. |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Cork--a Novel, Putative Alpha-2 Integrin Kinase @ University of North Carolina Chapel Hill
The alpha2gbeta1 integrin is a collagen receptor involved in platelet activation, smooth muscle cell and T lymphocyte migration, endothelial attachment and other events in the vasculature. We previously demonstrated that collagen-induced platelet aggregation depends upon occupancy of this integrin (along with an unknown co-receptor, possible glycoprotein VI). We also found that collagen activates, in an alpha2beta1-dependent manner, the non-receptor tyrosine kinase Syk, followed by phospholipase Cgamma2, events that appear necessary for collagen-induced aggregation. How the alpha2beta1 integrin activates platelets or induces these signal transduction events is unknown. Much evidence in the integrin field suggests that the short cytoplasmic domains of integrins play critical roles in transmitting signals into cells, potentially due to regulatory molecular that bind to these domains. To identify potential cytoplasmic domain regulatory proteins, we screened a library in the yeast two-hybrid system, using the lapha2 integrin cytoplasmic domain as bait. We have identified a partial cDNA sequence that encodes what appears to be an extremely interesting protein. The mRNA for this protein is large (9.5kb), widely distributed, and appears to be expressed in platelets, smooth muscle cells and other vascular and non-vascular cells. Moreover, nucleotide sequence analysis indicates a region of this protein that is highly homologous to numerous serine/threonine and dual specificity kinases, suggesting that the protein is a kinase. A separate smaller region is homologous to RasGAP, a Ras GTPase activating protein, and indeed this protein binds to Ras in the yeast two- hybrid system, but not to other control proteins, suggesting that his protein might also play a role in regulating Ras or Ras-related proteins. We propose to first complete the sequencing of the cDNA encoding this protein, and second to further characterize the protein with regard to its structure, cell and tissue distribution, and function. Characterizing of this novel protein may help us to better understand alpha2beta1-mediated in platelets and other vascular cells.
|
1 |
2000 — 2007 |
Parise, Leslie V. |
M01Activity Code Description: An award made to an institution solely for the support of a General Clinical Research Center where scientists conduct studies on a wide range of human diseases using the full spectrum of the biomedical sciences. Costs underwritten by these grants include those for renovation, for operational expenses such as staff salaries, equipment, and supplies, and for hospitalization. A General Clinical Research Center is a discrete unit of research beds separated from the general care wards. |
Red Blood Cell Adhesion to the Endothelium @ University of North Carolina Chapel Hill |
1 |
2002 — 2005 |
Parise, Leslie V. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Signaling Pathways Activating Adhesion in Sickle Cells @ University of North Carolina Chapel Hill
DESCRIPTION (provided by applicant): Agonist-induced signaling is a primary mechanism regulating adhesiveness of platelets, leukocytes and other cells. However, RBCs are generally considered to be inert to agonist-stimulated enhancement of adhesion; thus signaling in sickle (SS) RBCs by any agonist to directly activate SS RBC adhesiveness is relatively unexplored. Thrombospondin (TSP) is an adhesive protein that is abnormally elevated in the plasma of sickle cell patients. While TSP has known agonist properties towards other cells, it has been proposed in sickle cell disease to function purely as an adhesive molecule, by bridging adherent SS RBCs to the endothelium and subendothelial matrix. Here we establish that SS RBCs, in contrast to normal (AA) RBCs, respond to agonist stimulation by TSP to become significantly more adhesive. We also identify a site within TSP, which when immobilized can support SS RBC adhesion, or when soluble, can activate SS RBC adhesion. We further establish a previously unappreciated role for integrin-associated protein (TAP or CD47) on SS RBCs as both a basal adhesion receptor for immobilized TSP and a signal-transducing receptor in response to soluble TSP, and detect a potential physical difference in TAP on SS RBC that may contribute to the higher adhesion of SS versus AA RBCs. Evidence is also provided for a unique synergy between TAP-mediated signaling and shear stress-induced signaling, involving activation of large G-proteins and tyrosine kinases, to ultimately activate an a4B1 integrin-dependent increase in SS RBC adhesion. We therefore propose to: 1) identify proximal events in the activation of a4B1-mediated SS RBC adhesion, 2) define the basis for the apparent physical difference in sickle cell TAP and ask whether that difference contributes to the enhanced basal and stimulated sickle cell adhesion, 3) delineate the specific signaling pathway(s) induced by stimulation of lAP that results in increased SS RBC adhesiveness, 4) determine the role and mechanism of TAP-mediated SS RBC activation in SS RBC adhesion to endothelial cells, and 5) test the hypothesis that TAP contributes to SS RBC adhesion and pathology in vivo in studies of human sickle cell flow through a rat cremaster muscle model system. These data therefore provide new fundamental models of sickle cell adhesion and identify multiple potential therapeutic targets for down-regulating sickle cell adhesiveness and potentially preventing vaso-occlusive crises.
|
1 |
2004 — 2007 |
Parise, Leslie V. |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Structure and Function of Vascular Integrins @ University of North Carolina Chapel Hill
DESCRIPTION (provided by applicant): Interactions between vascular cells and matrix proteins are mediated by integrins. These reactions are essential for vascular homeostasis and play an important role in thrombosis and hemostasis. Key integrins in the vasculature include glycoproteins llb/llla (allbb3) on the platelet surface and the fibronectin receptor (FnR, a5b1) and vitronectine receptor (avb3 and avb5) on the surface of endothelial and other cells. Studies performed as part of the Program Project and in other laboratories have shown that the interaction between integrin receptors and their ligands not only define a mechanism for cell adhesion and aggregation, but also initiates a variety of cellular signals that are essential for diverse cellular functions such as motility, production of cytokines, and cell division. These signals include activation of kinases, rearrangement of the cellular cytoskeleton and induction of genes. The overall focus of this Program Project continues to be on the identification and characterization of pathways through which these integrin-mediated signals are transduced. The function of CIB, a calcium and integrin binding protein, in integrin activation will be determined. The role of the low molecular weight G protein, rap1b, in integrin activation will be examined using a knockout mouse. The role of VASP in actin polymerization and activation of integrins will be determined. The cooperative effects of integrin mediated cell anchorage and mitgoens on signal generation and regulation of cell cycle traverse will be explored. These studies should continue to clarify the roles of integrins as signal transduction molecules and the unique effects of these proteins in vascular biology.
|
1 |
2004 — 2005 |
Parise, Leslie V. |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Activation of the Platelet Fivrogen Receptor @ University of North Carolina Chapel Hill |
1 |
2004 — 2008 |
Parise, Leslie V. |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Collagen Receptor Signaling in Platelets @ University of North Carolina Chapel Hill
Platelets become exposed to collagen during rupture of the atherosclerotic plaque and during normal vascular injury. The exposed collagen serves not only as a direct platelet agonist but also provides an adhesive surface to platelets, thus contributing to thrombosis. Two of the major collagen receptors on platelets are glycoprotein (GP) VI and the Alpha-2 Beta-1 integrin. GPVI and Alpha-2 Beta-1 are both required for full collagen mediated platelet adhesion and activation, likely involving a GPVI-mediated activation of Alpha?2 Beta-1 as demonstrated by our lab and others. However, signals leading to Alpha-2 Beta-1 activation by GPVI or other agonist receptors are not well understood. Related to this, the small GTPases Rap1 and R-Ras have each been proposed as positive regulators of integrin activation whereas Ras has been proposed as a negative regulator. We recently demonstrated that GPVI signaling in platelets leads to Rap1 activation in a manner dependent upon secreted ADP activation of the P2Y12 receptor as well as a P2Y12-independent pathway. We also provide preliminary evidence using a transducible primary mouse megakaryocyte system, that Rap1 may promote Alpha-2 Beta-1 activation. In separate studies we found that R-Ras promotes several Alpha-2 Beta-1 mediated events, and that Ras is present in platelets and activated by agonist stimulation. However, the interrelationship of Rap1 to R-Ras or Ras is not understood. In the present proposal, we aim to further define the communication between GPVI and Alpha-2 Beta-1 by first, clarifying the roles of Rap1 and R-Ras in GPVI-induced Alpha-2 Beta-1 activation, second, mapping upstream pathways leading to Rap1 activation with regard to the potential role of R-Ras and other molecules in this event, third, mapping signaling pathways downstream of activated Rap1 leading to Alpha-2 Beta-1 integrin activation, and finally, determining the role of Ras in regulating Alpha-2 Beta-1 integrin activation. Results from these studies will provide fundamental information on how these important collagen receptors communicate with one another via these small G-proteins in platelets and megakaryocytes.
|
1 |
2004 — 2005 |
Parise, Leslie V. |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core B-- Structure/ Proteomics Core @ University of North Carolina Chapel Hill |
1 |
2006 |
Parise, Leslie V. |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Target Discovery Platelets in Situ Proteome Reactivity @ University of North Carolina Chapel Hill
[unreadable] DESCRIPTION (provided by applicant): [unreadable] Circulating platelets generate intracellular signals when exposed to extracellular matrix proteins and soluble agonists, resulting in rapid activation of adhesion receptors such as integrin alphallb-beta3, platelet aggregation, secretion of granular contents and increased procoagulant activity. These changes contribute not only to normal hemostasis but also to thrombotic events such as myocardial infarction and stroke. Platelets are anucleate and therefore not amenable to direct molecular approaches, thus limiting our understanding of the specific components of these activation-dependent signaling pathways. The Parise laboratory has become proficient in the use of knockdown and overexpression approaches in megakaryocytes, which are platelet precursors, to dissect signaling pathways relevant to platelet function. Here we propose to join forces with Dr. Benjamin Cravatt of The Scripps Research Institute, who has developed a novel natural products inspired spiroepoxide probe library that has been used previously to identify enzymes of importance in transformed cells. Probes from this library bind covalently to target enzymes in cell-based screens, thus affecting specific cellular readouts. By use of new technology termed in situ proteome profiling, covalently bound probes are then labeled with a biotin and fluorescent tag by use of "click chemistry", thus facilitating rapid identification of targets via mass spectrometry. However, this library has never been applied to platelets. Therefore, we propose to 1) identify molecular targets necessary for essential platelet functions by screening human platelets with the spiroepoxide probe library and performing in situ proteome reactivity profiling, and 2) validate molecular targets in megakaryocytes and platelets by use of independent counter screens involving overexpression and knock down, as well as pharmacological inhibition and enzyme activity assays, when possible, of candidate targets. Platelet-based screening and proteome reactivity profiling with this library, together with the ability to modulate protein expression levels in megakaryocytes provides a powerful combination of approaches in the platelet field for rapidly identifying and verifying new therapeutic targets for modulating platelet function. [unreadable] [unreadable]
|
1 |
2006 — 2007 |
Parise, Leslie V. |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Structure/Proteomics Core @ University of North Carolina Chapel Hill |
1 |
2006 — 2007 |
Parise, Leslie V. |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Activation of the Platelet Fibrogen Receptor @ University of North Carolina Chapel Hill |
1 |
2007 |
Parise, Leslie V. |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Activation of Camp-Mediated Sicke Cell Adhesion
Agonist-induced signaling is a primary mechanism regulating adhesiveness of platelets, leukocytes and other cells. However, RBCs are generally considered to be inert to agonist stimulation; thus signaling in sickle (SS) RBCs by agonists to directly activate SS RBC adhesiveness is relatively unexplored. Because abnormal SS RBC adhesion is believed to contribute to the painful vaso-occlusive crises suffered by sickle cell patients, it is crucial to understand mechanisms that modulate this adhesion. We recently made the important finding that SS RBCs are very responsive to agonist stimulation, resulting in a rapid and pronounced increase in adhesion to multiple vascular proteins. Here we propose to expand upon our findings that agonist-induced cAMP production results in a protein kinase A-dependent activation of SS RBC adhesion to laminin. Thus, epinephrine and several other physiologic agonists rapidly increase SS RBC adhesion in a subset of patients, providing a potential physiologic link between conditions that elevate these agonists, such as stress and pain, with vaso-occlusion. Here we propose to: 1) establish the basis for epinephrine-responsive and non-responsive sickle cell patient RBCs, 2) identify the receptor subtype(s) involved in epinephrine-induced SS RBC adhesion and identify additional naturally occurring agonists and agonist receptors that induce this process, 3) map the AMP-dependent signaling pathway(s) mediating stimulated SS RBC adhesion to laminin, 4) identify additional vascular proteins that support cAMP-stimulated SS RBC adhesion and the receptors/sites mediating these adhesive interactions, and 5) determine the role of cAMP-stimulated SS RBC adhesion in an in vivo system. These studies are likely to lead to new therapeutic targets for the disruption of AMP-mediated adhesive interactions in sickle cell patients.
|
0.97 |
2007 |
Parise, Leslie V. |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Target Discovery in Platelets by in Situ Proteome Reactivity Profiling @ University of North Carolina Chapel Hill
[unreadable] DESCRIPTION (provided by applicant): [unreadable] Circulating platelets generate intracellular signals when exposed to extracellular matrix proteins and soluble agonists, resulting in rapid activation of adhesion receptors such as integrin alphallb-beta3, platelet aggregation, secretion of granular contents and increased procoagulant activity. These changes contribute not only to normal hemostasis but also to thrombotic events such as myocardial infarction and stroke. Platelets are anucleate and therefore not amenable to direct molecular approaches, thus limiting our understanding of the specific components of these activation-dependent signaling pathways. The Parise laboratory has become proficient in the use of knockdown and overexpression approaches in megakaryocytes, which are platelet precursors, to dissect signaling pathways relevant to platelet function. Here we propose to join forces with Dr. Benjamin Cravatt of The Scripps Research Institute, who has developed a novel natural products inspired spiroepoxide probe library that has been used previously to identify enzymes of importance in transformed cells. Probes from this library bind covalently to target enzymes in cell-based screens, thus affecting specific cellular readouts. By use of new technology termed in situ proteome profiling, covalently bound probes are then labeled with a biotin and fluorescent tag by use of "click chemistry", thus facilitating rapid identification of targets via mass spectrometry. However, this library has never been applied to platelets. Therefore, we propose to 1) identify molecular targets necessary for essential platelet functions by screening human platelets with the spiroepoxide probe library and performing in situ proteome reactivity profiling, and 2) validate molecular targets in megakaryocytes and platelets by use of independent counter screens involving overexpression and knock down, as well as pharmacological inhibition and enzyme activity assays, when possible, of candidate targets. Platelet-based screening and proteome reactivity profiling with this library, together with the ability to modulate protein expression levels in megakaryocytes provides a powerful combination of approaches in the platelet field for rapidly identifying and verifying new therapeutic targets for modulating platelet function. [unreadable] [unreadable]
|
1 |
2009 |
Parise, Leslie V. |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
2009 Cell Biology of Megakaryocytes and Platelets Gordon Research Conference @ Gordon Research Conferences
DESCRIPTION (provided by applicant): Blood platelets play a critical role in the prevention of excessive blood loss at sites of tissue injury. They are formed from megakaryocytes in the bone marrow and circulate in the bloodstream in a quiescent state, but undergo 'explosive'activation upon damage to the vasculature, supporting formation of a vascular plug. Conditions associated with a reduction in platelet number (thrombocytopenia) or function can lead to excessive bleeding. Platelets play a fundamental role in thrombotic disorders, including stroke and myocardial infarction, and contribute to the metastatic spread of cancer. As such, platelets are a major target for pharmaceutical intervention, both to enhance production and block function. Despite this clinical significance, there are major gaps in our understanding of the molecular events which underlie platelet formation and activation. Research in this area had been hampered by the paucity of megakaryocytes in marrow, and the challenges of growing these cells in vitro with the generation of large numbers of platelets. The availability of genetically-modified mice, the discovery of thrombopoietin and the introduction of powerful technologies such as genomics, proteomics and RNAi has triggered a revolution in terms of molecular progress. However, until the current Gordon Research Conference was convened 4 years ago, there was no focused meeting that brought together scientists working on megakaryocyte and platelet cell biology. This third conference on 'The Cell Biology of Megakaryocytes and Platelets'will bring together academic and pharmaceutical investigators, post-doctoral fellows and graduate students for 5 days of intense discussion of the major scientific discoveries and challenges in the field at the GRC site in Galveston Island, TX March 15-20th, 2009. This application is a request for partial funds to support the attendance of speakers and discussion leaders, and selected postdoctoral fellows and students at this important scientific meeting. This meeting will be held at: Hotel Galvez 2024 Seawall Boulevard Galveston Island, TX 77550
|
0.904 |
2010 — 2013 |
Parise, Leslie V. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cib1 Regulation of Endothelial Function @ Univ of North Carolina Chapel Hill
DESCRIPTION (provided by applicant): This is a revised proposal to examine the role of CIB1, in endothelial function and pathological angiogenesis, or new blood vessel growth. CIB1 is a 22kDa EF- hand containing, Ca2+binding protein, homologous to calmodulin. Several lines of evidence indicate that CIB1 is a key regulator of endothelial cell function. Endothelial cells lacking CIB1 are significantly impaired in their ability to migrate, form tubules and proliferate. In addition, Cib1 knockout mice have impaired pathological and adaptive angiogenesis in response to ischemia. New data also indicate that tumor growth is compromised in these mice, apparently due to a poor angiogenic response. Separate studies have shown that CIB1 binds directly to, and activates, PAK1, a serine/threonine kinase known to regulate endothelial cell migration. New biochemical data show that CIB1 also binds directly to many integrin 1-subunits, several of which are important in endothelial function. Based on these data, we propose to 1) test the hypothesis that CIB1 directly binds to and regulates the function of integrins, especially those relevant to endothelial cells and 2) delineate the mechanisms by which CIB1 regulates pathological and tumor angiogenesis. Here we will determine whether regulation of angiogenesis occurs via a PAK1, integrin and/or another pathway(s). Since pathological angiogenesis contributes to a wide range of diseases involving cancer, atherosclerosis, retinopathies, etc, these studies should allow us to identify fundamental mechanisms regulating endothelial cell function and vascular remodeling.
|
0.988 |
2015 — 2018 |
Holly, Stephen P Parise, Leslie V. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Platelet Aadacl1 in Thrombosis, Lipidomics and Signaling @ Univ of North Carolina Chapel Hill
? DESCRIPTION (provided by applicant): Activated platelets can induce myocardial infarction and cerebrovascular thrombosis, whereas insufficient platelet activation can result in pathological blood loss. A better understanding of the complex network of events that govern platelet activation could lead to the identification of new therapeutic targets that mitigate thrombosis without significantly increasing bleeding. We previously used a novel chemoproteomic approach to identify arylacetamide deacetylase-like 1 (AADACL1) as a lipid hydrolase in human platelets. We found that AADACL1 is required for agonist-stimulated platelet aggregation and thrombus formation but not platelet adhesion ex vivo. Exciting new data indicate that AADACL1 inhibition significantly prolongs occlusion time in a rat model of thrombosis, suggesting that blocking AADACL1 activity in vivo may effectively reduce thrombosis while preserving other aspects of hemostasis, a hypothesis that we will test in Specific Aim 1. Even though AADACL1 activity contributes to the signaling output of multiple platelet agonists, we have only a cursory understanding of its substrates, products and mechanism of action in platelet aggregation. Obtaining this fundamental knowledge is crucial for evaluating the scope of AADACL1 action in platelets and determining the prospective value of AADACL1 and its substrates, and products as potential regulators of thrombosis. Therefore, in Specific Aim 2 we propose to define the metabolic footprint of AADACL1 in platelets via global and targeted mass spectrometry-based lipidomics. Finally, we determined that AADACL1 is required for optimal activation of Rap1 and PKC, two platelet signaling nodes critical to the platelet activation process in response to multiple agonists. Although the mechanism by which AADACL1 regulates these key signaling nodes is unknown, our intriguing preliminary data suggest that ether lipids metabolized by AADACL1, may function as endogenous regulators of platelet signaling at least in part via direct interaction with the C1 domains of PKC, a hypothesis that will be tested in Specific Aim 3. Completion of these studies will help us dissect an entirely novel lipid signaling pathway in platelets and probe the biological implications of AADACL1 metabolism in thrombosis and hemostasis.
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0.988 |
2019 |
Parise, Leslie V. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Mechanisms of Cib1 Signaling @ Univ of North Carolina Chapel Hill
ABSTRACT CIB1 is an intracellular protein that regulates cell growth, survival and proliferation, most notably under pathologic conditions. It functions by binding directly to effector kinases, integrins and other targets and modulates their activity, thereby serving as a signaling node for key regulatory pathways. Inhibition of CIB1 binding to select partners has excellent therapeutic potential in multiple diseases such as cancer, retinopathies and cardiac hypertrophy. It is therefore critical to understand CIB1 signaling in different cell types. In many breast cancer cell lines but not in normal cells tested to date, CIB1 is required for survival and proliferation via the PI3K/AKT and MEK/ERK pathways. In endothelial cells, CIB1 supports efficient angiogenesis in response to injury via the MEK/ERK pathway. However, how and why CIB1 selectively regulates these pathways under stressed or transformed conditions is poorly understood. We found that CIB1 interacts directly with PAK1 and PDK1, which can feed into these pathways. Moreover, while some data suggest that CIB1 regulates integrin signaling, which can activate the PI3K/AKT and MEK/ERK pathways, it is completely unknown whether or how CIB1 connects integrin signaling to these pathways. Our overall hypothesis is that under select stress or oncogenic conditions in a variety of cell types, CIB1 binds to specific partners to regulate the PI3K/AKT, MEK/ERK and potentially other pathways, to promote cell viability, growth and proliferation. To address this hypothesis, we will use a combination of targeted and unbiased approaches to determine how the CIB1 interaction with PAK1, PDK1 and the ?V integrin support the PI3K/AKT and MEK/ERK pathways. We will complement targeted approaches with unbiased phosphoproteomics and interactomics to delineate on a global scale, how CIB1 signaling affects not only MEK/ERK and PI3K/AKT but other phospho-signaling pathways that may help explain CIB1 biology.
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0.988 |