Pro-inflammatory activation of vascular endothelium leading to increased surface expression of adhesion molecules and neutrophil (PMN) sequestration and subsequent activation is paramount in the development of acute lung (ALI) and organ injury in injured patients. We hypothesize that α-enolase, which accumulates in injured patients primes PMNs and causes pro-inflammatory activation of endothelial cells leading to PMN-mediated cytotoxicity. Methods Proteomic analyses of field plasma samples from injured vs. healthy patients was used for protein identification. Human pulmonary microvascular endothelial cells (HMVECs) were incubated with α-enolase or thrombin, and ICAM-1 surface expression was measured by flow cytometry. A two-event in vitro model of PMN cytotoxicity HMVECs activated with α-enolase, thrombin, or buffer was used as targets for lysophosphatidylcholine-primed or buffer-treated PMNs. The PMN priming activity of α-enolase was completed, and lysates from both PMNs and HMVECs were immunoblotted for protease activated receptor-1 (PAR-1) and PAR-2 and co-precipitation of α-enolase with PAR-2 and plasminogen/plasmin. Results α-enolase increased 10.8-fold in injured patients (p<0.05). Thrombin and α-enolase significantly increased ICAM-1 surface expression on HMVECs, which was inhibited by anti-proteases, induced PMN adherence, and served as the first event in the two-event model of PMN cytotoxicity. α-enolase co-precipitated with PAR-2 and plasminogen/plasmin on HMVECs and PMNs and induced PMN priming, which was inhibited by tranexamic acid, and enzymatic activity was not required. We conclude that α-enolase increases post-injury and may activate pulmonary endothelial cells and prime PMNs through plasmin activity and PAR-2 activation. Such pro-inflammatory endothelial activation may predispose to PMN-mediated organ injury.
1119 Thrombin is a multifunctional serine protease involved in hemostasis, fibrinolysis, and pro-inflammatory activation of innate immunity through stimulation of protease-activated receptors (PARs). Thrombin is considered the prototypical protease to activate PARs through cleavage of the N-terminal exodomain of the receptor, thereby unmasking a tethered ligand which activates it. Neutrophils (PMNs) are reported to express PAR-2; however, little is known about the changes in PMN physiology following protease activation of PAR-2. In addition, proteomic analyses (two dimensional gel electrophoresis and mass spectroscopy (MALDI-TOF)) of injured patients (blunt trauma) who developed acute lung injury (ALI) and multiple organ failure (MOF) have identified a number of serine proteases which accumulate post-injury. Moreover, these identical proteases were also found via proteomics in stored, but not fresh, packed red blood cells, which are used to resuscitate these injured patients. We hypothesize that thrombin primes PMNs through activation of PAR-2. Methods: PMNs were isolated from whole blood drawn from healthy human donors using dextran sedimentation, ficoll-hypaque gradient centrifugation and hypotonic lysis of red blood cells. The proteins from whole cell lysates were separated by SDS-polyacrylamide electrophoresis, transferred to nitrocellulose and immunoblotted with a monoclonal antibody to PAR-2. The PMNs were also incubated for 3–30 minutes at 37°C with thrombin [1-10 U/ml] followed by fMLP [1 μM] activation of the NADPH oxidase. Oxidase activity was measured by the superoxide dismutase-inhibitable reduction of cytochrome c at 550 nm. Results: PMNs express PAR-2 as detected by western blotting of whole cell lysates. Thrombin [1 U/ml] for 30 min, significantly primed the fMLP-activated respiratory burst: buffer control: 1.6 +/− 0.5 vs. 1 U/ml thrombin: 2.2 +/− 0.3* (*p<.05, n=5). Higher concentrations of thrombin or shorter incubation times did not induce PMN priming. Furthermore, pre-incubation with leupeptin [50 μM] and 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) [250 μM], two effective protease inhibitors, abrogated the thrombin-induced PMN priming by 100±18%. We conclude that thrombin primes the PMN respiratory burst, which is dependent upon its protease activity. Because PMNs are the effector cell in ALI/MOF and priming agents have been linked to the development of clinical ALI and MOF, proteases that accumulate in the patient plasma post-injury or are transfused with stored PRBCs may be etiologic in post-injury ALI/MOF. Disclosures: No relevant conflicts of interest to declare.
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