To determine whether PDI and ERp57 share substrate specificity, we performed kinetic trapping experiments in platelet-rich plasma with ERp57 using ERp57-CCCC, ERp57-CACC, ERp57-CCCA, ERp57-CACA, and ERp57-AAAA. vascular injury may serve as a regulatory switch that allows activation of proteins, among them vitronectin, critical for thrombus formation. Thrombus formation is usually a host defence mechanism that protects the high-pressure mammalian circulatory system from significant loss of pressure following vascular injury. Limited proteolysis, cell activation and receptor conformational transitions are important mechanisms for rapidly transforming a quiescent system into an active system, as characterized by the activation of the blood coagulation cascade and fibrin generation, endothelial cell activation and degranulation and platelet thrombus formation1, but we have recently decided that protein disulfide isomerase (PDI) and several additional thiol isomerases may play an important role 7-Amino-4-methylcoumarin in maintaining the patency of the circulatory system. PDI and other thiol isomerases are required for thrombus formation and their absence from blood in the intact circulatory system is usually a mechanism to maintain patency in the absence of a breach. PDI is usually secreted from endothelial cells and bound platelets following vessel wall injury during thrombus formation2,3,4,5. From animal studies, PDI is required for both fibrin generation and platelet thrombus formation2,3. Similarly, ERp57 and ERp5, both users of the thiol isomerase family, also play a critical role in thrombus formation6,7,8,9. However, the molecular and cellular basis by which these thiol isomerases participate in the haemostatic process is usually unknown. PDI, a prototype of a family of thiol 7-Amino-4-methylcoumarin isomerases with common thioredoxin-like domains and active sites characterized by the sequence CXXC (ref. 10), is an oxidoreductase involved in the formation of protein disulfide bonds during protein synthesis. PDI has two active thioredoxin domains, and and and domains sharing 50% amino acid identity and the CGHC motif21. To determine whether PDI and ERp57 share substrate specificity, we performed kinetic trapping experiments in platelet-rich plasma with ERp57 using ERp57-CCCC, 7-Amino-4-methylcoumarin ERp57-CACC, ERp57-CCCA, ERp57-CACA, and ERp57-AAAA. Analysis of the experiment by SDSCPAGE and Western blot showed trapping variants of ERp57 produce a fingerprint of mixed disulfide complexes unique from PDI, suggesting unique units of substrates (Fig. 2c). Immunoblot for the presence of vitronectin among the ERp57 mixed disulfide complexes show that none of the ERp57 trapping variants produce a disulfide-linked complex containing vitronectin. This demonstration of substrate specificity distinguishes PDI Rabbit Polyclonal to HOXA6 and ERp57. To further characterize the reductase reaction between PDI and plasma vitronectin, we decided the number of disulfide bonds in vitronectin that are reduced by PDI. Since purified plasma vitronectin assumes different conformations depending on the method of purification (that is, 7-Amino-4-methylcoumarin multimer formation with urea treatment and conformational changes after heparin-affinity purification22,23), we performed the PDI-vitronectin reaction using vitronectin in new platelet-poor plasma. Since vitronectin has two free thiols, the plasma was initially treated with NEM and extra NEM removed by dialysis, then reacted with either PDICCCCC or PDICAAAA. The reduction of disulfide bonds and subsequent alkylation of the new thiols in vitronectin were visualised by immunoblotting and monitoring gel mobility shifts associated with the incorporation of PEG5000-maleimide (Fig. 3). The time-dependent (Fig. 3a) and dose-dependent (Fig. 3b) reduction of plasma vitronectin by PDICCCCC but not PDICAAAA led to the appearance of vitronectin species of increased molecular excess weight at 89,900. This molecular excess weight increase corresponds to the incorporation of 3.60.06 moles of the PEG5000-maleimide per mole of vitronectin. No incorporation of PEG5000-maleimide was observed when ERp57 was added to plasma. Similar results, with the.