This application is for a Clinical Investigator Developmental Award to an M.D. who is committed to pursuing an academic career in medicine. He will spend 90% of his effort over the next 5 years on the training program described in this application, which emphasizes didactic course work as well as extensive laboratory training. The proposed research will extend the novel observation made in the primary sponsor's laboratory characterizing ICAM-2 on platelets and its interaction with LFA-1 on leukocytes. Based on this study, we hypothesize that sequential adhesion events responsible for mediating leukocyte:endothelium interaction during inflammation may also occur between leukocytes and surface adherent platelets. These would include P-selectin-mediated attachment and rolling, followed by activation-dependent firm adhesion of leukocytes to platelets through integrin receptors and ligands. The objective of the proposal is to identify leukocyte subsets, adhesion receptors, and chemoattractants involved in platelet:leukocyte interactions during hemostasis and inflammation. The consequences of such interactions may contribute to pathological conditions in man such as arteriosclerosis, myocardial ischemia, necrotizing enterocolitis, and complications of cardiopulmonary bypass.
In Aim 1 (a-c), we will use an established in vitro vascular model system to examine the adhesion mechanisms involved in plate-mediated recruitment of leukocytes to thrombogenic surfaces.
In Aim 1 a, we will determine and compare whether leukocyte subsets can adhere to immobilized platelets in flow through P-selectin as previously observed for neutrophils. This will be accomplished by using mAb to P-selectin and confirmed on lipid bilayers in which the purified selectin protein has been incorporated.
In Aim 1 b, we will determine if surface adherent platelets can activate bound leukocytes resulting in up-regulation of leukocyte integrins and thus, adhesion strengthening interactions (""""""""sticking""""""""). If """"""""sticking"""""""" does occur, mAbs and receptor antagonists to platelet-derived chemoattractants such as PAF, PDGF, and NAP-2 will be used to determine the activating agent.
In Aim 1 c, identification of the adhesion receptors and ligands involved in these interactions will be performed using available mAb, receptor antagonists, or by generating mAb to platelet antigens using standard techniques available in the Springer laboratory. Screening will be accomplished by using a novel leukocyte transmigration assay system specifically developed for evaluating platelet:leukocyte interactions. Finally, in Aim 2, we will examine the in vivo behavior of human platelets with respect to P-selectin-mediated interactions with vascular endothelium. This will be performed using intravital microscopy in the mouse cremaster muscle, Peyer's patch, and newly developed peripheral lymph node model. We are confident that the goals of this proposal can be achieved as the Springer and von Andrian laboratories have proven expertise in protein purification, tissue culture, monoclonal antibody production, and intravital microscopy. The strengths of these laboratories in basic science research plus their interests in disease processes involving adhesion molecules make an ideal environment for training a young investigator.
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