Functional Mapping and Protein Engineering of Thrombin

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DESCRIPTION (provided by applicant): Thrombin cleavage of osteopontin (OPN), an RGD-containing proinflammatory cytokine, greatly augments its cell interactive properties by the exposure of a new alpha4beta1and alpha9beta1 integrin binding site SVVYGLR at its C-terminus. Both OPN and alpha4beta1 integrin are important in experimental autoimmune encephalitis (EAE), a mouse model of multiple sclerosis. We hypothesize that thrombin-cleavage of OPN, with its resultant enhanced interaction with alpha4beta1 inteqrin, is important in the pathogenesis of EAE. We showed that thrombin cleavage of recombinant OPN markedly increased the binding of Jurkat cells (which express alpha4beta1), and activated thrombin-activatable fibrinolysis inhibitor (TAFla) abolished this enhanced cell binding by cleaving the C-terminal arginine. TAFI is a latent plasma carboxypeptidase, which is activated by the thrombin-thrombomodulin (TM) complex on endothelial cells. We showed that TAFla is more effective than plasma carboxypeptidase N in inactivating bradykinin (BK) and activated complement C5a. We hypothesize that TAFla is an anti-inflammatory molecule, which, together with aPC, counteract the proinflammatory effects of thrombin. E229K thrombin, an engineered human thrombin with minimal procoagulant functions, activated PC and TAFI in mice, and blocked BK-induced hypotension. Aim #1 Structure-function analysis of the thrombin-OPN interaction and TAFla inactivation of the SVVYGLR site. We shall characterize thrombin cleavage of soluble vs immobilized OPN and phosphorylated vs non-phosphorylated OPN. We will determine the structural requirements on thrombin for OPN cleavage, and characterize the efficiency of TAFla cleavage of SVVYGLR and other relevant substrates. Aim #2 Cell biology of thrombin cleavage of OPN and its modulation by TAFla We hypothesize that the engagement of the RGD and the SVVYGLR sites on the thrombin-cleaved OPN fragment to RGD-dependent and RGD-independent integrins respectively will lead to different cellular effects. We have generated various GST-OPN fusion proteins that represent the two binding sites either singly or in combination. We will study their effects on Jurkat cells in terms of haptotaxis, metalloprotease secretion, cytokine release, and gene transcription profiles. We will test whether SDF-1alpha TGF-beta1, and the thrombin receptor activation peptide activate alpha4beta1on Jurkat cells. We will test whether thrombin-cleaved OPN induces a prothrombotic phenotype in endothelial cells. Aim #3 The in vivo role of TAFla in mouse inflammation model To test whether TAFla is important in modulating the proinflammatory effect of thrombin-cleaved OPN, we will compare the EAE phenotype in WT and TAFI-null mice and test the OPN-fusion proteins in an OPN-induced peritonitis model. We will confirm that E229K thrombin blockage of BK-induced hypotension is mediated by TAFI activation. We will test the role of TAFla inactivation of C5a in a neutrophil alveolitis model.