Medicine (St Vincent's) - Theses
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ItemThe 78kDa glucose regulated protein (GRP78) interacts with thrombomodulin on endothelial surface and demonstrates antithrombotic activity( 2014)Thrombomodulin (TM) is a multi-domain glycoprotein expressed primarily on vascular endothelial cells. Recent research has demonstrated that the N-terminal module, the lectin-like domain (LLD), exhibits anti-inflammatory properties. In this project, a specific interaction between the LLD and the 78kDa glucose regulated protein (GRP78) was identified. This interaction was confirmed by reciprocal immunoprecipitation of both proteins and by colocalization on cell surface. GRP78 is mostly an intracellular protein, best known for its function as a chaperone, guiding the unfolded protein response. Extracellular GRP78 has been demonstrated to inhibit tissue factor (TF)-mediated coagulation. In this project, recombinant GRP78 (full length, various mutants and individual domains) was generated, and its effect on haemostasis was studied in vitro and in vivo. GRP78 prolonged the TF-dependent coagulation in a coagulation assay as well as the biochemical analysis of spectrozyme Xa. Contact-initiated clotting was not altered and there was no effect on thrombin time. Recombinant GRP78 (8 to 10μg/ml) was shown to inhibit platelet aggregation significantly in response to collagen (1U/mL), TRAP (1μM) and ADP (10μM) but not with Ristocetin. Pre-administration of GRP78 (1-5μg/g mouse body weight) demonstrated dose-dependent prolongation of mouse tail bleeding time. Antithrombotic activity of GRP78 was tested in vivo in a model of pulmonary thrombosis, induced by collagen (1.2μg/g) infusion into the jugular vein of anaesthetised mice with monitoring for morbidity and survival at 30 min. GRP78 demonstrated significant dose-dependent (1-8μg/g mouse body weight) improvement in survival from acute pulmonary thrombosis as compared to buffer control.TM is already known to participate in a clinically relevant anticoagulant pathway: the generation of activated protein C (APC) by thrombin binding to the epidermal growth factor-like modules of TM and consequent inhibition of procoagulant proteins Va and VIIIa. Here, another novel mechanism has been identified by which TM can maintain the endogenous antithrombotic phenotype of endothelial cells: the interaction between the LLD and GRP78 recruits the capacity to inhibit TF-mediated coagulation and inhibit platelet aggregation to the site of a developing thrombus. The clinical importance and detailed dissection of mechanisms is ongoing in the laboratory.