1985 — 1987 |
Brass, Lawrence F |
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. |
Calcium Homeostasis in Platelets @ University of Pennsylvania
Platelet activation is an ordered sequence of events that begins with the binding of an agonist to its receptor on the platelet surface and concludes with platelet aggregation and secretion. Ca2+ ions play a critical role in these events. Extracellular Ca2+ is necessary for some of the events that take place on the platelet surface, such as fibrinogen binding and, ultimately, platelet aggregation. Intracellular Ca2+ serves as a secondary messenger during platelet activation. The studies that are contained in this proposal focus upon two main areas: the mechanisms of Ca2+ transport that help to maintain Ca2+ homeostasis in platelets and the properties of the Ca2+ binding proteins on the platelet surface, especially glycoproteins IIb and IIIa. Some of the specific issues that will be addressed are: [1]\Identification of the mechanism that mediates Ca2+ efflux across the platelet plasma membrane. [2] Consideration of the role of plasma membrane glycoproteins IIb and IIIa in Ca2+ transport. [3]\Re-examination of the relative contributions of the platelet dense tubular system and mitochondria to Ca2+ homeostasis in resting and stimulated platelets. [4]\Examination of the mechanisms involved in platelet recovery from reversible activation, including the possibility that Na+/Ca2+ exchange is involved. [5] Elucidation of the changes in the IIb/IIIa complex that occur when platelets are activated and the complex is altered to expose the fibrinogen receptor. [6] Identification of Ca2+ binding sites on the cytosolic surface of the platelet plasma membrane that may play a role in platelet activation. [7] Identification of additional Ca2+ binding proteins on the platelet surface.
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1 |
1990 — 1993 |
Brass, Lawrence F |
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. |
Characterization of a Novel G Protein in Human Platelets @ University of Pennsylvania
This proposal will examine the structure and function of a 40 kDa protein in platelets which we believe to be the alpha subunit of a guanine nucleotide-binding regulatory protein or G protein. Based upon our recent studies, this protein has several novel properties. First, it is phosphorylated during platelet activation, apparently by protein kinase C. Second, it is immunologically cross-reactive with G(z-alpha), a putative alpha subunit that has been cloned from two non-platelet cDNA libraries, but not yet isolated. Third, in contrast to the other G proteins which have been a focus for platelet research, it is neither ADP-ribosylated by pertussis toxin nor recognized by antisera that are specific for known pertussis toxin-sensitive G proteins, such as G(i-alpha). Even though G(z- alpha) is not known to be a substrate for protein kinase C, we have provisionally named the platelet protein "G(z-alpha)(plt)" in acknowledgement of the immunologic cross-reactivity of the two proteins. The goal of our studies will be to understand the structure and biology of G(z-alpha)(plt). Specifically, we will: (1) determine the identity of G(z- alpha)(plt) and define its relationship to G(z-alpha), (2) identify the sites at which G(z-alpha)(plt) is phosphorylated, (3) determine the biochemical consequences of phosphorylation and (4) define the role of G(z- alpha)(plt) in platelet function.
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1 |
1993 — 2018 |
Brass, Lawrence F |
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. |
Subcellular Mechanisms of Platelet Activation @ University of Pennsylvania
Platelet activation at sites of vascular injury begins when a locally- generated agonist such as thrombin or collagen interacts with receptors on the platelet surface. This initiates a signal within the platelet that is propagated by a succession of molecules that includes G proteins, phospholipases, various second messengers including Ca++, and a variety of serine/threonine- and tyrosine-specific protein kinases. Ultimately, this results in platelet shape change, the exposure of fibrinogen receptors on the platelet surface, granule secretion and aggregation. Arguably, the most biologically-significant trigger for these events in vivo is thrombin. Recent studies in this laboratory have concentrated on the regulation of Ca++ homeostasis in platelets, the role of IP(3), the structure and function of platelet G proteins, the mechanisms of fibrinogen receptor expression and, most recently, the structure and function of platelet receptors for thrombin. The studies proposed for the next 5 years represent a natural evolution of several of these themes. The major focus will be upon the platelet receptor, including the manner in which it interacts with G proteins and the mechanisms by which its activity is initiated and terminated. By extension these results are applicable to other platelet receptors for biologically significant agonists, and these will also be examined. Our specific goals are to: (1) Define the mechanisms of platelet activation by thrombin. (2) Identify the molecular basis for signal termination and receptor down-regulation following platelet activation by thrombin. (3) Define the role of G proteins in platelet activation, particularly those G proteins that interact with thrombin receptors in platelets and endothelial cells. (4) Apply the approaches used in studying thrombin receptors to other platelet receptors, including the thromboxane A(2) receptor.
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1 |
1994 — 1995 |
Brass, Lawrence F |
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. |
Novel G Protein in Platelets @ University of Pennsylvania
This proposal will examine the structure and function of a 40 kDa protein in platelets which we believe to be the alpha subunit of a guanine nucleotide-binding regulatory protein or G protein. Based upon our recent studies, this protein has several novel properties. First, it is phosphorylated during platelet activation, apparently by protein kinase C. Second, it is immunologically cross-reactive with G(z-alpha), a putative alpha subunit that has been cloned from two non-platelet cDNA libraries, but not yet isolated. Third, in contrast to the other G proteins which have been a focus for platelet research, it is neither ADP-ribosylated by pertussis toxin nor recognized by antisera that are specific for known pertussis toxin-sensitive G proteins, such as G(i-alpha). Even though G(z- alpha) is not known to be a substrate for protein kinase C, we have provisionally named the platelet protein "G(z-alpha)(plt)" in acknowledgement of the immunologic cross-reactivity of the two proteins. The goal of our studies will be to understand the structure and biology of G(z-alpha)(plt). Specifically, we will: (1) determine the identity of G(z- alpha)(plt) and define its relationship to G(z-alpha), (2) identify the sites at which G(z-alpha)(plt) is phosphorylated, (3) determine the biochemical consequences of phosphorylation and (4) define the role of G(z- alpha)(plt) in platelet function.
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1 |
1994 — 2021 |
Brass, Lawrence F |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Hematology Clinical Research Training Program @ University of Pennsylvania
DESCRIPTION (provided by applicant): The Hematology Research Training Program at the University of Pennsylvania was established in 1978 and currently supports 8 postdoctoral trainees per year. Since its inception 24 years ago, the goal of our program has been to identify individuals interested in the broadly-defined discipline of hematology, and to help them prepare for academic careers as independent investigators using their scientific training in basic, translational and patient-oriented research. Originally the candidates were drawn almost exclusively from physicians enrolled in the clinical hematology-oncology training programs in the Departments of Medicine and Pediatrics, but as specified at the time of the last renewal, we have deliberately sought non-physician postdoctoral trainees in order to provide a mix of backgrounds and interests. During the first 24 years of 'funding, 87 postdoctoral trainees and one pre-doctoral trainee have been supported: 63 M.D., 12 M.D.-Ph.D. and 12 Ph.D. Twenty five of the trainees are women and 63 are men. Of the 77 trainees who have completed training, 47 (61%) hold full-time appointments at an academic institution and are engaged in either laboratory or clinical research. An additional 7 (9%) are engaged primarily in clinical practice, but have university appointments and participate in clinical research projects and teaching part-time. Thirty four of our former trainees who have completed training have extramural research funding. Thirty nine hold faculty appointments at 24 institutions, including Penn, Harvard and the NIH. Although historically this program has supported postdoctoral trainees almost exclusively, we now seek added support in order to appoint up to 4 predoctoral trainees per year. These additional trainees will be drawn primarily from physician-scientist trainees in Penn's MD-PhD program - a program that is also directed by Lawrence Brass, the principal investigator on this grant. The addition of predoctoral students to our program reflects our belief in the long term value of attracting talented students in hematology-related research at an earlier point in their training. A detailed plan for identifying appropriate pre- and postdoctoral candidates, providing them with the research and laboratory skills needed for an independent career, and mentoring them throughout the process is included in the application.
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1 |
1996 — 2007 |
Brass, Lawrence F |
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. |
Subcellular Mechanisms of Platelet Function @ University of Pennsylvania
Platelet activation typically begins with the exposure of the subendothelial collagen matrix and continues as additional platelets are recruited into the growing platelet mass by soluble agordsts. The signal transduction pathways that underlie these events have been the subject of intense scrutiny, with particular emphasis on pathways leading to integrin activation. Building upon observations made during the last funding cycle, the proposed studies will test the hypothesis that intracellular signaling continues even after platelet aggregation has occurred, helping to perpetuate the growth and stability of the platelet plug. Our primary focus will be upon Eph kinases, a family of receptor tyrosine kinases whose tigands, the ephrins, are also cell surface molecules. Interactions between Eph kinases on one cel! and ephfins on another can produce signaling in both. Although Eph kinases and ephrins are best known for their role during development, we have found that human platelets express at least two Eph kinases (EpEA4 and EphB1) and an ephrin (ephrin BI) that is a ligand for both. We have also shown that signaling through Eph kinases and ephrins promotes platelet adhesion, and that blockade of Eph/ephrin interactions will cause platelets to disaggregate. Our investigations of mechanism show that EphA4 forms signaling complexes during platelet aggregation that include the kinases, Fyn and Lyn, and the cell adhesion molecule, L1. Consistent with their proposed role, the binding of ephrins to Eph kinases would not be expected to occur until after platelet aggregation has begun. Drawing on these observations, we now propose the following four specific aims. Aim #1 will focus on the composition of the signaling complexes that form as a consequence of Eph/ephrin interactions during platelet aggregation, and examine the role of Eph and epbdn phosphorylation in this process. Aim #2 will focus on the signaling pathways activated by Eptdephrin interactions in platelets, starting with two that we have already identified: activation of the Ras family member, Rap 1B, and phosphorylation of the cytoplasmic domain of the beta chain of alphalIb/beta3. Aim #3 will test the proposed role of Eph kinases and ephrins in vivo using mouse models. Aim #4 will examine the hypothesis that interactions between EphA4 and L1 affect the ability of L1 to support platelet aggregation by interacting in trans with L1 and beta3 integrins on the surface of adjacent platelets. Taken together, the proposed studies should provide new insights into the late events of platelet activation, an area of investigation that is relatively unexplored.
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1 |
1996 — 2000 |
Brass, Lawrence F |
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. |
Novel G Protein in Human Platelets @ University of Pennsylvania
DESCRIPTION: (Adapted from Applicant's Abstract) Dr. Brass proposes to examine intracellular signaling events that are responsible for platelet activation and megakaryocyte growth. Specifically, this proposal focuses on the role of the heterotrimeric G protein, Gz. Gz is of interest due to its restricted tissue distribution (platelets, megakaryocytes, nerve cells, but not megakaryocytic cell lines). Unlike other G proteins, Gz alpha becomes phosphorylated by PKC during platelet activation, on ser27. The goals of the present proposal are first, to identify the roles of Gz in platelets and megakaryocytes by developing a transgenic mouse model in which Gz alpha expression is knocked out in mice or repressed in megakaryocytes. Second the role of Gz in coupling cell surface receptors to intracellular effectors will be probed with a library of Gz alpha-directed antibodies and by examining the GTP:GDP exchange to detect receptor coupling to Gz. Finally, Dr. Brass proposes to define factors responsible for the selective expression of Gz alpha by studying the promoter region of this gene. It is suggested that these aims will help to define the role of Gz in platelet activation and understand factors responsible for megakaryocyte-selective gene regulation.
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1 |
1999 — 2003 |
Brass, Lawrence F |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Hematology Clinical Research Training Grant @ University of Pennsylvania
The Hematology Research Training Program at the University of Pennsylvania was established in 1978 and is currently in its 19th year of funding, the last four of which have been under the current principal investigator, Lawrence Brass. Throughout that time, the goal has been to help top candidates with an interest in the broadly-defined disciplines of hematology and, more recently, vascular biology to prepare themselves for academic careers in which their scientific training in laboratories could be applied to either laboratory or clinical research. During the first 19 years of funding, 70 postdoctoral trainees and one pre-doctoral trainee have been supported for work in 33 different laboratories: 57 M.D., 9 M.D.-Ph.D. and 4 Ph.D. Twenty of the trainees were women, 51 were men. Of the 64 trainees who have completed training to date, 41 (64%) hold full-time appointments at an academic institution and are engaged in either laboratory (22%) or clinical (42%) research. An additional 7 (11%) are engaged primarily in clinical practice, but have university appointments and participate in clinical research projects and teaching part-time. The remaining 25% are either not working for a variety of reasons (5%), employed in industry (3%) or engaged in full time private practice (17%). Approximately half of our former trainees have extramural research funding with direct costs in the current year exceeding $7 million. Graduates of this program hold appointments at 21 institutions around the United States, including the University of Pennsylvania, Harvard and the NIH. Among the program graduates there are currently 3 full professors, 11 associated professors, 20 assistant professors and 4 instructors. This proposal provides follow-up information on all of the trainees since the inception of the grant and describes how the program has evolved during the past two decades. Based on the success of the training program to date, the availability of additional strong candidates, and the growth of the faculty and programs in hematology and vascular biology, this proposal also makes the case for an increase in funding from the current 5 M.D., M.D.-Ph.D. and Ph.D. postdoctoral positions per year to 8 per year.
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1 |
1999 — 2021 |
Brass, Lawrence F |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Medical Scientist Training Program @ University of Pennsylvania |
1 |
2000 |
Brass, Lawrence F |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Gordon Conference On Hemostasis 2000 @ Gordon Research Conferences
This application is for partial support of the biannual Gordon Research Conference on Hemostasis to be held at Plymouth State College in Plymouth, New Hampshire July 9-14, 2000. This international conference has been in existence since 1973 and has a long-standing tradition of providing a forum for the presentation and discussion of cutting edge research in the field of hemostasis. It also has a longstanding tradition of providing a setting in which scientists from academia and industry can meet, discuss their mutual interests and help to foster the development of promising graduate students and postdoctoral researchers. Topics for the program were selected with considerable input from the wider community of hemostasis investigators, including those who attended previous meetings. Based on that input, special emphasis will be placed on megakaryocyte and platelet biology, predisposing factors in the development of arterial and venous thrombosis, and the translation of recent basic science discoveries to the early stages of clinical development. Scientists from the United States and from all parts of the world are expected to participate. Total attendance will be limited to approximately 135, allowing the conference to retain the informality and close interchange that are hallmarks of the Gordon Conference series. Many of the speakers have been or will be invited in advance. Others will be chosen from late-breaking developments and from the abstracts that are submitted for presentation as posters. Nine lecture sessions and two poster events are planned and will cover: 1. Megakaryocytes: regulation of platelet formation 2. Platelet signaling receptors and their effectors 3. Regulation of the platelet cytoskeleton and secretion 4. Mechanisms of integrin activation 5. Genetic approaches to hyperactive hemostasis and its consequences 6. Hemostatic mechanisms in development and the central nervous system 7. Proteases and other coagulant proteins 8. Slugging it out at the vessel wall 9. Therapeutic applications of hemostasis research: moving toward clinical development
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0.901 |
2002 — 2005 |
Brass, Lawrence F |
P50Activity 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 grants differ from program project grants 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. |
Regulation of G Protein Signaling in Platlets @ University of Pennsylvania
Human platelets are rapidly activated at sites of vascular injury by agonists such as collagen, thrombin and AP. With the notable exception of collagen, most agonists activate platelets via cell surface receptors coupled to heterotrimeric G proteins. Collectively, G proteins and G protein coupled receptors provide a sensitive mechanism that allows rapid platelet activation. However, this sensitivity also increases the risk that inappropriate platelet activation will, if unchecked, cause tissue ischemia and infarction. This is particularly true when progressive diseases such as atherosclerosis narrow the vascular lumen. Since passage through the circulatory system will predictably expose platelets to conditions that could cause unwanted activation, it is reasonable to propose that mechanisms exist to place limits on signaling through G proteins. It is our hypothesis that in platelets such regulatory mechanisms are directed towards both receptors and G proteins, but those working at the level of the G proteins are particularly important and work in concert with endothelial but those working at the level of the G proteins Are particularly important and work in concert with endothelial PGI2, NO and CD39 to prevent inappropriate platelet activation. The goal of the proposed studies is to test this hypothesis and to extend current information about the regulation of G protein-dependent signaling in platelets. Three related issues will be addressed using human platelets and platelets from genetically-engineered mice. First, there are at least 1o different G proteins in platelets. To what extend does each make a unique contribution to signaling and to what extent are they redundant? Second, will persistent activation of G proteins in platelet hyperactivity ex vivo and will it contribute to the development of thrombosis and the progression of atherosclerosis in vivo. Third, what are the roles of RGS proteins in limiting G protein signaling in platelets and do they provide a means of preventing unwarranted platelet activation? We will address these questions primarily using transgenic engineered for both a gain and loss of function of platelet G protein signaling. The results of these studies should new information about normal platelet biology, identify potential targets for therapeutic intervention, and create model systems in which the consequences of dysregulated platelet activation can be studied and understood.
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1 |
2002 — 2005 |
Brass, Lawrence F |
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. |
Characterization of a Novel G Protein in Platelets @ University of Pennsylvania
DESCRIPTION (provided by applicant): Human platelets are activated at sites of vascular injury by the combined effects of multiple agonists, most of which activate platelets via G protein coupled receptors. The G proteins that are normally expressed in platelets include three ubiquitous members of the G1 family (Gi1, Gi2 and Gi3) and one (Gz), that is expressed most prominently in platelets and brain. Our previous goals for this project included identifying unique roles for Gz in platelet activation, identifying receptors and effectors that interact with Gz, identifying sites of phosphorylation in the a subunit of Gz, and analyzing the promoter region of the gene encoding Gzalpha. Substantial progress was made towards all of these goals, culminating with the development of Gzalpha knockout mice and the demonstration that Gz is required for the potentiation of platelet aggregation and the suppression of cAMP formation by physiological concentrations of epinephrine. In addition, we have recently observed that Gz and other Gi family members in platelets are needed for maximal activation of the low molecular weight GTP-binding protein, Rap1B, and obtained data implicating Rap1B in platelet aggregation. Based on these results, the studies in this proposal will continue our efforts to understand the role of all four G family members in platelet activation. It is our hypothesis that the biochemical properties that distinguish the various Gi family members determine how they are regulated, which receptors and effectors they interact with, and, ultimately, the efficiency with which different agonists cause platelet activation. The proposed studies are divided into four specific aims. The first will address the differences in receptor and effector coupling among the Gi family members and the role of Gi-derived Gbetagamma. The second aim will examine the regulatory impact of the phosphorylation of Gzalpha by protein kinase C and the Rac1/cdc42-activated kinase, PAK, during platelet activation. The third aim will extend our studies on the regulation of Rap1B in platelets by Gi family members and the fourth will examine the regulation of cAMP formation in circulating platelets. All four of these aims will combine biochemical and pharmacologic approaches with studies on genetically engineered mice lacking selected receptors and G proteins, allowing us to better understand platelet activation in vivo.
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1 |
2006 |
Brass, Lawrence F |
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.) |
Proteomic Studies Normal and Abnormal Platelet Function @ University of Pennsylvania
[unreadable] DESCRIPTION (provided by applicant): [unreadable] Many useful insights into normal and pathologic platelet function have come through studies that focused on single molecules. There are, however, many unanswered questions that do not lend themselves to a "one molecule at a time" approach. These questions would benefit from the application of technologies that can identify complex mixtures of proteins in a quantitative manner using amounts of blood that can reasonably be obtained from single individuals or small numbers of inbred mice. Some of those questions will be addressed in the studies that comprise this proposal. The goal of the first specific aim is to define the components of the platelet sheddome - the set of proteins that are proteolytically shed from the platelet surface when platelets are activated. Protein shedding from platelets releases bioactive molecules into the surrounding plasma. Shedding can also alter the function of essential cell surface receptors, thereby modulating thrombus growth and the interaction of platelets with other types of cells. The goal of the second aim is to understand how the loss of critical signaling and adhesion molecules, either by mutation or by the long term administration of therapeutic antagonists, can have unanticipated effects on the expression of other proteins. Such compensatory changes are of interest because of the insights that they can provide into the normal events of platelet activation, and because of their potential relevance to differences among individuals that may affect susceptibility to disease and responses to anti-platelet agents. The third specific aim will address the molecular basis for the decrease in platelet function and survival that occurs when human platelets are stored prior to transfusion. We will test the hypothesis that these changes are due in part to the shedding of critical membrane proteins. The fourth specific aim will test the hypothesis that an unbiased screen of the platelet proteome can help to establish a molecular diagnosis when other methods have failed to do so. Studies will be performed on platelets from two cohorts of patients with clinical evidence for platelet dysfunction, but no established molecular diagnosis. Each of the proposed studies rests on the combined experience in clinical hematology, transfusion medicine, platelet activation and proteomic technologies represented by the PI, co-PI and their collaborators. The studies are designed using methods that we have recently applied to the identification of platelet membrane proteins. This includes novel methods that permit quantitation of individual molecules so that changes in levels of expression can be measured. [unreadable] [unreadable] [unreadable]
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1 |
2006 — 2008 |
Brass, Lawrence F |
R33Activity Code Description: The R33 award is to provide a second phase for the support for innovative exploratory and development research activities initiated under the R21 mechanism. Although only R21 awardees are generally eligible to apply for R33 support, specific program initiatives may establish eligibility criteria under which applications could be accepted from applicants demonstrating progress equivalent to that expected under R33. |
Blood Systems Biology @ University of Pennsylvania
[unreadable] DESCRIPTION (provided by applicant): The University of Pennsylvania, in response to RFA-HL-06-004, has assembled an interdisciplinary team of faculty from the School of Engineering and Applied Sciences and the School of Medicine with expertise in experimental and computational hemodynamics, bond mechanics and biorheology, transport physics, platelet biology, coagulation and protease biochemistry, continuum/stochastic simulation, inverse problems, and knockout mice for thrombosis research. The Cluster Team will deploy integrative and hierarchical computational models and experimental studies to predict spatial-temporal processes in mouse and human blood under hemodynamic conditions. Specific Aims are defined for 3 Cluster projects: Specific Aim 1 (Project I: D. A. Hammer, Collaborating PI) will focus on platelet hydrodynamics and receptor bonding and signaling (GPIb/vWF and GPVI/collagen) with outside-in/inside-out signaling leading to alpha2beta1 and alphallb-betaS activation. Platelet Adhesive Dynamics simulation of platelet capture, rolling, activation, arrest, and embolism as a function of fluid shear rate will be compared to experiment using parallel-plate flow chambers. Specific Aim 2 (Project II: S. L. Diamond, Lead PI) will focus on simulation and experiment of platelet deposition on a reactive surface in the presence of coagulation under flow conditions. Kinetic Monte Carlo/Continuum simulation of agonist activation, platelet deposition/fragmentation, granule release, and thrombin generation will be compared to experiments run in well plates, cone-and-plate viscometer, and parallel-plate flow cells. Specific Aim 3 (Project III: L. F. Brass, Collaborating PI) will focus on thrombin receptor function and platelet- platelet interactions within formed aggregates relating to signaling, clot stability, and retraction. Both human blood and normal and knockout mouse blood will be used for in situ detection of platelet function in formed thrombi and testing of intracellular signaling models for platelets under realistic hemodynamic conditions. Lay Statement: Blood is ideal for Systems Biology research since it is easily obtained from donors or patients, amenable to high throughput liquid handling experiments, and clinically relevant. Better elucidation and quantitative simulation of blood reactions and platelet signaling pathways under hemodynamic conditions are directed at clinical needs in thrombosis risk assessment, anti-coagulation therapy, platelet targeted therapies, and stroke research. [unreadable] [unreadable] [unreadable]
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1 |
2006 — 2010 |
Brass, Lawrence |
P50Activity 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 grants differ from program project grants 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. |
The Role of Sema4d/Cd100 and Its Receptors in Platelet Biology and Thrombosis @ University of Pennsylvania
Project 5: In addition to adhering to each other and to the vessel wall, platelets contribute to thrombotic[unreadable] events by releasing bioactive molecules such as ADP, TxA2 and CD40L. In studies that form the basis for[unreadable] this proposal, human and mouse platelets were found to express on their surface the class IV semaphorin,[unreadable] sema4D or CD 100, a protein best known for its role in B-cell/T-cell interactions. It was also found that[unreadable] activated platelets shed the exodomain of sema4D and the "sheddase" was identified as the TNFa cleaving[unreadable] enzyme, ADAM17. Based on these observations and preliminary studies on the effects of soluble sema4D on[unreadable] platelets, we have developed the following hypotheses: 1) sema4D, either as a soluble molecule or surfacebound,[unreadable] contributes to platelet activation by binding to receptors expressed on nearby platelets, 2) plateletderived[unreadable] sema4D can also affect cells other than platelets within the circulation and the vessel wall, and 3)[unreadable] plasma levels of soluble sema4D will increase when pathological platelet activation occurs. To test these[unreadable] hypotheses, Aim #1 will examine the role of sema4D in platelet activation. Aim #2 will investigate the[unreadable] regulated shedding of the sema4D extracellular domain. Aim #3 will examine the role of CD72 and plexin-[unreadable] Bl as candidate receptors for platelet-derived sema4D in platelets, monocytes and endothelial cells, and Aim[unreadable] #4 will ask whether platelet activation in vivo causes a measurable increase in plasma sema4D levels that[unreadable] correlates with the extent of platelet activation. Aims #1-3 will take advantage of existing mouse lines[unreadable] lacking sema4D, CD72 or ADAM17. Aim #4 will make use of samples from two clinical trials in which[unreadable] platelet activation is expected. The first trial includes the 1,000 patients undergoing cardiopulmonary bypass[unreadable] in the prospective heparin-induced thrombocytopenia (HIT) trial that is part of Project #1. All of these[unreadable] individuals should have transient platelet activation while on bypass. Those that develop HIT will have[unreadable] persistent platelet activation. The second trial includes 4,000 patients with well-characterized atherosclerotic[unreadable] cardiovascular disease, a setting where platelet activation is predicted to occur, but be less pronounced.[unreadable] Collectively, these aims will address the basic biology of platelet sema4D and its receptors, explore the[unreadable] consequences of sema4D release when platelets are activated, and begin to assess the role of soluble sema4D[unreadable] as a contributor to thrombotic events in vivo.
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0.915 |
2007 |
Brass, Lawrence F |
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.) |
Proteomic Studies of Normal and Abnormal Platelet Function @ University of Pennsylvania
[unreadable] DESCRIPTION (provided by applicant): [unreadable] Many useful insights into normal and pathologic platelet function have come through studies that focused on single molecules. There are, however, many unanswered questions that do not lend themselves to a "one molecule at a time" approach. These questions would benefit from the application of technologies that can identify complex mixtures of proteins in a quantitative manner using amounts of blood that can reasonably be obtained from single individuals or small numbers of inbred mice. Some of those questions will be addressed in the studies that comprise this proposal. The goal of the first specific aim is to define the components of the platelet sheddome - the set of proteins that are proteolytically shed from the platelet surface when platelets are activated. Protein shedding from platelets releases bioactive molecules into the surrounding plasma. Shedding can also alter the function of essential cell surface receptors, thereby modulating thrombus growth and the interaction of platelets with other types of cells. The goal of the second aim is to understand how the loss of critical signaling and adhesion molecules, either by mutation or by the long term administration of therapeutic antagonists, can have unanticipated effects on the expression of other proteins. Such compensatory changes are of interest because of the insights that they can provide into the normal events of platelet activation, and because of their potential relevance to differences among individuals that may affect susceptibility to disease and responses to anti-platelet agents. The third specific aim will address the molecular basis for the decrease in platelet function and survival that occurs when human platelets are stored prior to transfusion. We will test the hypothesis that these changes are due in part to the shedding of critical membrane proteins. The fourth specific aim will test the hypothesis that an unbiased screen of the platelet proteome can help to establish a molecular diagnosis when other methods have failed to do so. Studies will be performed on platelets from two cohorts of patients with clinical evidence for platelet dysfunction, but no established molecular diagnosis. Each of the proposed studies rests on the combined experience in clinical hematology, transfusion medicine, platelet activation and proteomic technologies represented by the PI, co-PI and their collaborators. The studies are designed using methods that we have recently applied to the identification of platelet membrane proteins. This includes novel methods that permit quantitation of individual molecules so that changes in levels of expression can be measured. [unreadable] [unreadable] [unreadable]
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1 |
2008 — 2012 |
Brass, Lawrence F |
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. |
Subcellular Mechanisms Pf Platelet Activation @ University of Pennsylvania
2H-1,3,2-Oxazaphosphorin-2-amine, N,N-bis(2-chloroethyl)tetrahydro-, 2-oxide; 2H-1,3,2-oxazaphosphorin-2-amine, N,N-is(2-chloroethyl)tetrahydro-,2-oxide; Adhesion Molecule; B2 antigen, Xenopus; Binding; Binding (Molecular Function); Bizzozero's corpuscle/cell; Bleeding; Blood Platelets; Blood Vessels; Body Tissues; CD100 antigen; CD72; CD72 gene; CTX; CYCLO-cell; Carloxan; Cek5 Ligand; Cek5 RPTK Ligand; Cell Adhesion Molecules; Cell Communication and Signaling; Cell Signaling; Cell surface; Ciclofosfamida; Ciclofosfamide; Cicloxal; Clafen; Claphene; Cycloblastin; Cycloblastine; Cyclophospham; Cyclophosphamide; Cyclophosphamidum; Cyclophosphan; Cyclophosphane; Cyclophosphanum; Cyclostin; Cyclostine; Cytophosphan; Cytophosphane; Cytoplasmic Domain; Cytoplasmic Tail; Cytoxan; Data; Deetjeen's body; Deposit; Deposition; EC 2.7; EFNB1 Gene Product; EPH Tyrosine Kinase 2; EPHT2 Protein; EPLG1; Eck Ligand; Eck RPTK Ligand; Elk-L Protein; Endothelium Secreted Protein B61; Endoxan; Endoxana; Enduxan; Ensure; Eph Family Receptor Interacting Protein B1; EphB1 Protein; Ephrin Receptor EphB1; Ephrin Type-B Receptor 1; Ephrin-A1; Ephrin-B1; Epl1 Protein; Event; Extracellular Matrix, Integrins; Family; Family member; Fosfaseron; Funding; Generalized Growth; Genes; Genoxal; Genuxal; Goals; Growth; Hayem's elementary corpuscle; Hemorrhage; Hemostatic Agents; Hemostatics; In Vitro; Injury; Integrins; Intracellular Communication and Signaling; Kinases; Knockout Mice; LERK-1 Protein; LERK-2 Protein; LYB2; Ledoxina; Ligand Binding; Ligands; Localized; Mammals, Mice; Marrow platelet; Mice; Mice, Knock-out; Mice, Knockout; Microscopy; Mitoxan; Modeling; Molecular; Molecular Interaction; Murine; Mus; Neosar; Neuronally Expressed EPH-Related Tyrosine Kinase; Null Mouse; Pathologic; Phosphotransferases; Platelet Activation; Platelet aggregation; Platelets; Play; Process; Procytox; Proteins; Receptor Protein; Receptor, EphB1; Recruitment Activity; Reticuloendothelial System, Platelets; Role; Scaffolding Protein; Sendoxan; Signal Transduction; Signal Transduction Systems; Signaling; Surface; Syklofosfamid; Testing; Thrombocytes; Thrombus; Time; Tissue Growth; Tissues; Transphosphorylases; Tyrosine-Protein Kinase Receptor EPH-2; Work; Zytoxan; base; biological signal transduction; blood loss; cell adhesion protein; comparative; digital; elk Ligand; gene product; in vivo; injury response; instrument; novel; ontogeny; plexin; prevent; preventing; protein protein interaction; receptor; recruit; response to injury; restraint; scaffold; scaffolding; sema4D; semaphorin 4D; semaphorin CD100; social role; thrombocyte/platelet; vascular
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2010 — 2013 |
Brass, Lawrence F |
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. |
Regulation of the Early Events of Platelet Activation @ University of Pennsylvania
DESCRIPTION (provided by applicant): Regulation of the early events of platelet activation Lawrence F. Brass, MD PhD Platelets are essential for normal hemostasis, but can also contribute to thrombosis and to the evolution and consequences of common diseases of the vessel wall including arteriosclerosis. Although a great deal is known about platelet activation, much less is known about the events within platelets that modulate responsiveness, limiting platelet activation when it is not needed and ensuring that the response to injury halts bleeding without causing vascular occlusion. The focus of this proposal is on the events of platelet activation that occur immediately downstream of agonists such as thrombin, ADP and TxA2, all of whose receptors are coupled to heterotrimeric G proteins. Our hypothesis is that dysregulation of G protein dependent events is prothrombotic and potentially contributes to vascular disease progression. This hypothesis will be tested in studies with human platelets and selected mouse models. In our preliminary studies, we have identified a previously-undescribed regulatory complex in resting platelets in which at least two RGS (regulators of G protein signaling) proteins and the tyrosine phosphatase, SHP-1, are bound to the 130 kDa scaffold protein, spinophilin (SPL), which in resting platelets is tyrosine phosphorylated. Platelet activation causes SHP-1- dependent dephosphorylation of spinophilin and agonist-selective dissociation of the SPL/RGS/SHP-1 complex. These events can be recapitulated in transfected CHO cells. Based on these observations, we propose that spinophilin first sequesters RGS proteins, allowing signaling to begin, and then releases them in order to limit signaling magnitude and duration. Support for this model is drawn from our studies on SPL(-/-) mice and mice expressing Gi2a(G184S), a mutation that renders the a subunit of the G protein, Gi2, resistant to inactivation by RGS proteins. Those studies show that 1) loss of spinophilin impairs platelet responses to agonists, 2) this defect is limited to agonists that can cause dissociation of the SPL/RGS/SHP-1 complex, and 3) blocking RGS-dependent negative feedback on Gi2 produces, as the model would predict, a gain of platelet function in vitro and in vivo. The proposed studies are divided into three specific aims. Aim 1 will test our hypothesis that RGS proteins help to regulate platelet responsiveness by limiting the duration of G protein signaling during platelet activation. Aim 2 will test our hypothesis that the decay of the SPL/RGS/SHP-1 complex provides a timed brake on G protein signaling and identify the mechanisms involved. Finally, Aim 3 will focus on the role of RGS proteins and the SPL/RGS/SHP-1 complex in regulating the conversion of adherent platelets to a fully activated state and in avoiding the adverse consequences of a chronic increase in platelet reactivity.
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2010 |
Brass, Lawrence F |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
Confocal Upgrade For Intravital Microscopy Following Vascular Injury @ University of Pennsylvania
DESCRIPTION (provided by applicant): Confocal upgrade for intravital microscopy following vascular injury 6. Project summary The goal of this proposal is to modernize an existing shared instrument that has been used for the past 5 years to observe the hemostatic response to vascular injury in genetically engineered mice. Hemostasis refers to the rapid accumulation of circulating platelets and fibrin at a site of injury, thereby limiting blood loss. The unintended consequence of having a rapid response hemostatic mechanism is that inappropriate formation of platelet/fibrin clots is a primary contributor to heart attacks and strokes, particular in the setting of atherosclerosis. Although much has been learned about these events in vitro, it has only recently been possible to study them in vivo. Basic and clinical research in hemostasis and thrombosis research is a major focus at Penn Medicine and Children's Hospital of Philadelphia (CHOP). Five years ago, we assembled a (then) state of the art instrument that has enabled us to use digital Intravital fluorescent microscopy to observe events within the microcirculation. The original instrument has been put to good use in studies that range from testing new ideas about platelet activation to efforts to finding novel ways to cure hemophilia. It has been shared by members of the immediate community as well as by visiting investigators. When originally assembled, there was only one comparable instrument in the United States and even now there are few others. However, the original instrument included confocal capabilities whose usefulness proved to be limited by the relatively long times (>45 sec) required to capture an entire set of images. Better, faster confocal heads are now available and can be retrofitted to our existing microscope. The requested funds will be used to upgrade the confocal head, lasers and camera, and replace the wavelength changer with one that is much faster. The net effect will be to allow us to observe events in greater detail and with a greater sense of spatial relationships, bringing us back to the level of state of the art technology needed to answer biologically-important questions. We know that the new system will work as expected, because a similar instrument was recently completed in the Furie lab at Harvard. Although our specific biological questions differ, their experience is informative about the capabilities of the system. This proposal includes examples of the NIH-funded research that will benefit from the new instrument, as well as a management plan for its continued use. Economic impact: Penn Medicine and CHOP contribute substantially to the local economy. In 2008, they created and supported more than 54,000 jobs and $11 billion in regional economic activity. The current proposal will help us to maintain our edge in competing for grant support and create or retain at least 4 jobs at Penn plus more at Intelligent Imaging Innovations, our partner in developing and maintaining the instrument. 1
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2014 — 2016 |
Brass, Lawrence F |
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. |
Spatial Determinants of Thrombus Growth and Stability in Vivo @ University of Pennsylvania
Project 3 - Summary Vascular injury and vessel wall diseases serve as triggers for the accumulation of platelets and fibrin, sometimes with disastrous consequences. Platelet activation mechanisms are commonly studied in vitro using tools that work best at the level of individual platelets or small groups of platelets. Here we have made a paradigm shift, viewing both hemostatic and pathologic thrombus formation as the product of large platelet populations and combining observational, experimental and computational approaches to understand the relationships among those populations. Our overall goal is to obtain new insights into platelet activation as it occurs in vivo and identify better ways to limit thrombosis without overly impairing hemostasis. Our premise is that as platelets begin to accumulate at a site of injury, they alter their local microenvironment in ways that affect subsequent events. We and others have shown previously that there are regional differences in the extent of platelet activation within hemostatic thrombi, resulting in a core of fully-activated, closely-packed platelets overlaid by a shell of less-activated platelets. Here we hope to understand how these regional differences arise and influence subsequent thrombus growth and stability. In Aim #1 we will apply novel technologies to determine how regional differences in platelet packing density, intrathrombus solute transport and agonist distribution arise and interact with the platelet signaling network. In Aim #2, we will collaborate with fellow PPG members, Sriram Krishnaswamy and Rodney Camire, to test the hypothesis that thrombus architecture, as well as the location of procoagulant membranes, dictate the distribution of thrombin and fibrin during the hemostatic response. Finally, in Aim #3 we will combine observational and experimental data from Aims #1 and #2 with computational methods to test hypotheses about thrombus formation that cannot be addressed by experimental approaches alone, including the hypothesis that the formation of a core-and-shell architecture is a biological mechanism evolved to limit thrombus growth and prevent vascular occlusion. All three aims are built upon considerable preliminary data and take advantage of new and existing methods to study thrombus formation. They will also take advantage of the collective expertise of a team of investigators with strong backgrounds in platelet biology, mouse models of hemostasis, methods development, and the use of computational and applied engineering approaches to answer biological questions.
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2017 — 2018 |
Brass, Lawrence F |
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. |
Spatial Determits of Thrombus Growth and Stability in Vivo @ University of Pennsylvania
Project 3 - Summary Vascular injury and vessel wall diseases serve as triggers for the accumulation of platelets and fibrin, sometimes with disastrous consequences. Platelet activation mechanisms are commonly studied in vitro using tools that work best at the level of individual platelets or small groups of platelets. Here we have made a paradigm shift, viewing both hemostatic and pathologic thrombus formation as the product of large platelet populations and combining observational, experimental and computational approaches to understand the relationships among those populations. Our overall goal is to obtain new insights into platelet activation as it occurs in vivo and identify better ways to limit thrombosis without overly impairing hemostasis. Our premise is that as platelets begin to accumulate at a site of injury, they alter their local microenvironment in ways that affect subsequent events. We and others have shown previously that there are regional differences in the extent of platelet activation within hemostatic thrombi, resulting in a core of fully-activated, closely-packed platelets overlaid by a shell of less-activated platelets. Here we hope to understand how these regional differences arise and influence subsequent thrombus growth and stability. In Aim #1 we will apply novel technologies to determine how regional differences in platelet packing density, intrathrombus solute transport and agonist distribution arise and interact with the platelet signaling network. In Aim #2, we will collaborate with fellow PPG members, Sriram Krishnaswamy and Rodney Camire, to test the hypothesis that thrombus architecture, as well as the location of procoagulant membranes, dictate the distribution of thrombin and fibrin during the hemostatic response. Finally, in Aim #3 we will combine observational and experimental data from Aims #1 and #2 with computational methods to test hypotheses about thrombus formation that cannot be addressed by experimental approaches alone, including the hypothesis that the formation of a core-and-shell architecture is a biological mechanism evolved to limit thrombus growth and prevent vascular occlusion. All three aims are built upon considerable preliminary data and take advantage of new and existing methods to study thrombus formation. They will also take advantage of the collective expertise of a team of investigators with strong backgrounds in platelet biology, mouse models of hemostasis, methods development, and the use of computational and applied engineering approaches to answer biological questions.
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2020 |
Brass, Lawrence F |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Enhancing Biomedical Research Training Program Evaluation Capacity @ University of Pennsylvania
Project Summary Evaluating training program efficacy and using that information to improve trainee success has become even more important than ever for biomedical research training programs. In this proposal, the 6 NIGMS-funded T32 training programs for PhD and MD/PhD predoctoral students at the University of Pennsylvania, including the MSTP program directed by the P.I., will work together to put into place an improved infrastructure for program evaluation built around short and long term metrics for success. Our goals are to: 1) improve our ability to assess the impact of courses, workshops and skills training programs, 2) develop sustainable tools for tracking short and long term outcomes, and 3) identify predictors of trainee success during and after completion of the training program. Reaching these goals will improve our ability to answer critical questions, including what has been the impact of each training program? How well do students supported by our NIGMS T32 grants do compared to their colleagues who were not supported in achieving sustainable careers consistent with the goals of each training program? In other words, does being in our training programs make a difference? The P.I. of this proposal has had considerable experience evaluating the outcomes of MD/PhD programs at the national level and in working with others to disseminate the results. Penn's institutional commitment to achieving these goals is strong and will be sustained well beyond the period of the supplement. University resources that will be drawn upon include Penn's Center for Teaching and Learning (CTL), Penn's Office of Institutional Research and Analysis, and the offices of Biomedical Graduate Studies and the Combined Degree and Physician Scholars Programs. 1
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2020 — 2021 |
Brass, Lawrence F |
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. |
A Systems Approach to Hemostasis and Thrombosis @ University of Pennsylvania
Project 3 Abstract Platelet activation is critical for hemostasis and a contributing factor in thrombosis. Although recent studies have highlighted roles for platelets in diverse processes, the rapid accumulation of large numbers of platelets remains the hallmark of hemostasis and arterial thrombosis, and is the major focus of this project. Our recent studies in the mouse microvasculature show that the hemostatic response to small injuries produces a relatively simple structure in which a core of fully-activated platelets is overlaid by a shell of less-activated platelets. Dense packing in the core acts as a molecular trap, establishing an environment in which diffusion replaces convection. This structure allows thrombin and other agonists to form overlapping gradients that produce regional differences in platelet activation and fibrin distribution. Recognizing that transport is regulated by platelet packing density is a paradigm shift, suggesting that platelet procoagulant activity arises from forming a sheltered environment and not just from phospholipid exposure. We believe that this concept is key to understanding the impact of antiplatelet agents and the events of arterial thrombosis. Testing it calls for scaling up to larger injuries in larger vessels, and for extending our analysis from mice to humans and from hemostasis to thrombosis, all with a hybrid experimental and computational approach that integrates with and supports the other projects in this PPG. Aim #1 will examine the spatial and temporal distribution of platelet activation at high resolution, measure transport in the gaps between platelets, and examine the hemostatic response in large arteries and veins. The initial results show a more complex architecture with regions of greater and lesser platelet activation and packing density, and large differences between the luminal and abluminal surfaces. Our subcontract with Brian Storrie at the University of Arkansas will allow 3-dimensional reconstruction of larger hemostatic thrombi at the sub-micron level. In collaboration with Project 4 we will examine the impact of sepsis and systemic inflammation on platelet function in vivo and support studies on the impact of the PF4-directed antibody, KKO. Studies with µ- and m- calpain deficient mice will support work in Project 2, but also be part of understanding the role of clot retraction in limiting transport through larger hemostatic structures. Aim #2 will examine the mechanisms that shape the hemostatic plug, testing the hypothesis that hemostatic structure requires tight regulation of the extent of platelet activation and the delivery of platelet cargoes deep within the hemostatic mass. Studies on NBEAL2-/- (gray platelet syndrome) mice and the ?empty a-granule? mice developed in Project 1 will allow us to examine the role of secretion on hemostatic plug architecture. Aim #3 will determine whether the ordered hemostatic structure that we have observed in mice applies to humans, and how it differs in arterial thrombosis as compared to hemostasis. The human studies will be performed in vivo with Penn trauma surgeon, Carrie Sims, and ex vivo using a novel microfluidics device developed with Dan Huh in Penn?s School of Engineering. Studies of human arterial thrombi will be done in collaboration with Project #2 co-investigator John Weisel.
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