PLATELET-ACTIVATING FACTOR IN THROMBOTIC PLATELET-LEUKOCYTE INTERACTIONS

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The phospholipid Platelet-activating factor (PAF) is perhaps the most powerful pro-inflammatory and prothromobtic lipid mediator yet defined. All cellular components of the acute inflammatory system express the single known receptor for PAF. PAF is synthesized by, and retained on the surface of, activated endothelial ceils where it stimulates tethered leukocytes. PAF accumulation normally is tightly controlled, but PAF has a role in thrombosis, sepsis and reperfusion damage. There are deficits in our knowledge of how, when, and where PAF receptor ligands are formed, how PAF is presented to cells of the innate immune system and how PAF stimulated cells interact with other cells. We do not know whether PAF receptor ligands are formed during stent placement, whether PAF stimulated leukocytes provide platelets with substrate for thromboxane production even when platelet cyclooxygenase has been inhibited by aspirin, and we do not know whether PAF production by leukocytes stimulated with endotoxin is mimicked by endogenous agonists that include anti-phospholipid antibodies. We propose to identify the pathways leading to activation of the rate-limiting PAF synthetic activity, determine whether individuals vary in their ability to make PAF, take advantage of new information and techniques to purify the enzyme responsible for PAF synthesis for eventual rational inhibitor design, and identify and modulate mechanisms that cause microparticle formation and PAF release, and to determine whether these parameters correlate with thrombosis. We will define the pro-thrombotic lipids released during stent placement, we will investigate alternate routes to generate thromboxane Aa, and determine whether this correlates with susceptibility to slow reflow after stent placement, and we will test alternate, endogenous ligands of TLR4 as leukocyte agonists. We have four aims: Aim 1. Mechanistically define adhesion-dependent PAF synthesis in PMN. Aim 2. Purify and molecularly characterize the leukocyte PAF acetyltransferase synthetic enzyme. Aim 3. Identify factors that extend the effect of PAF through microparticle release. Aim 4. Define novel routes to leukocyte activation and inflammatory mediator production