Previous experiments by our laboratory examining the signaling events linking PAR-1 to activation of iPLA2 have demonstrated that a member of the novel protein kinase C (PKC) isoenzyme family (calcium-independent, phorbol 12-myristate, 13-acetate dependent) mediates iPLA2 activation following thrombin stimulation of the isolated membrane fraction. to thrombin or tryptase Obtusifolin activation demonstrated an increase in iPLA2 activity and arachidonic acid release. Additionally, stimulated HCAEC demonstrated increased platelet-activating factor (PAF) production and cell surface P-selectin expression, resulting in increased adhesion of neutrophils to HCAEC monolayers. Pretreatment with bromoenol lactone to inhibit iPLA2, blocked membrane phospholipid-derived metabolite production, increased cell surface P-selectin expression and neutrophil adherence. Conclusions The comparable biochemical and cellular responses in HCAEC exposed to thrombin or tryptase activation suggest that the cleavage of two individual PAR serve to extend the range of proteases to which the cells respond rather than resulting in individual intracellular events. This suggests that in conditions such as thrombosis and atherosclerosis that multiple mechanisms can activate the inflammatory response. strong class=”kwd-title” Keywords: thrombin, tryptase, inflammation, endothelium, protease activated recepotors, atherosclerosis The PAR represent a family of G-protein coupled receptors that are activated by proteolytic cleavage of their N-terminus [observe 1 for evaluate]. Recent evidence suggests that conversation of proteases with PAR have far-reaching implications in diversified cellular responses, particularly in inflammation and host defense [2,3]. PAR couple to multiple intracellular signaling pathways that are related to growth and inflammation including activation of phospholipases and MAP kinases [1]. PAR may play important roles in both the acute anti-inflammatory and chronic inflammatory behavior of both endothelial and epithelial cells that form the defensive barriers of the body [2]. We have previously exhibited that thrombin (activates PAR-1) and tryptase (activates PAR-2) activation of endothelial cells results in activation of phospholipase A2 (PLA2) [4,5] These data agree with a previously published study that suggest that the presence of multiple PARs around the endothelial cell surface serve to extend the number of proteases to which the cells respond rather then being coupled to different intracellular responses [6]. Myocardial infarction and the development of thrombotic coronary artery occlusion are associated with the presence of the serine proteases thrombin and tryptase. Thrombin generated at sites of vascular injury is the most potent activator of blood platelets [7,8] and its action on inflammatory cells has been well characterized, providing as a chemotactic agent for monocytes [9] and a mitogenic for both lymphocytes [10] and vascular easy muscle mass cells [11,12]. Thrombin activation of the vascular endothelium occurring in response to vascular injury or wounding can be beneficial in the repair process, but has the potential to mediate a prolonged inflammatory response and proliferative cellular events in the blood vessel wall, such as those that occur in atherosclerosis and restenosis [13]. Similarly, increased numbers of degranulated mast cells have been found in the adventitia of infarct-related coronary arteries [14] and the mediators released from these granules, including tryptase, are mitogens and Obtusifolin co-mitogens for human fibroblasts, stimulating collagen synthesis [15]. Though these studies demonstrate the presence of either thrombin or tryptase associated with atherosclerosis, a defined role has yet to be established for these proteases. Materials and Methods Reagents Human tryptase (200 g/mL recombinant skin tryptase with 0.5 mg/mL heparin) was purchased from Promega Corporation, Madison, WI. BEL was obtained from Cayman Chemical, Ann Arbor, MI. Goat anti-P-selectin antibody and horse raddish peroxidase-conjugated rabbit anti-goat antibody were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. [3H] arachidonic acid and [3H] acetic acid were obtained from Perkin Elmer Rabbit Polyclonal to DDX3Y Life Sciences, Boston, MA. AACOCF3 was purchased from Calbiochem, La Jolla, CA. PX-18 was a gift from Richard Berney (Richard Berney Associates, LLC), Bethesda, MD. All other reagents were purchased from Sigma Chemical, St. Louis, MO. Culture of Endothelial Cells HCAEC were obtained from Cambrex (Walkersville, MD). Cells were grown to confluence, as determined by visual examination utilizing an inverted light microscope. Cells were cultured in EGM-2MV medium from Cambrex (Walkersville, MD) and incubated at 37C, 95% O2/5% CO2. To passage cells, the Sub-culture Reagent Pack (Cambrex, Walkersville, MD) was used. Approximately 3105 of cells in 2 mL of EGM-2MV medium were placed in each well of a 6 well plate. Unless otherwise stated, Obtusifolin cells from passages 3C4 were used for experiments. Thrombin or Tryptase Obtusifolin Stimulation Thrombin or tryptase were diluted with medium (for assay of iPLA2 activity, arachidonic acid release, resistance measurements, and neutrophil adhesion), or Hanks balanced salts solution (for assay of PAF production and P-selectin surface expression) to the working concentration. Thrombin.
