Thrombin hydrolysis of human osteopontin is dependent on thrombin anion-binding exosites

Abstract

The cytokine osteopontin (OPN) can be hydrolyzed by thrombin exposing a cryptic α4β1/α9β 1 integrin-binding motif (SVVYGLR), thereby acting as a potent cytokine for cells bearing these activated integrins. We show that purified milk OPN is a substrate for thrombin with a kcat/Km value of 1.14 × 105 M-1s-1. Thrombin cleavage of OPN was inhibited by unsulfated hirugen (IC50 = 1.2 ± 0.2 μM), unfractionated heparin (IC50 = 56.6 ± 8.4 μg/ml) and low molecular weight (5 kDa) heparin (IC50 = 31.0 ± 7.9 μg/ml), indicating the involvement of both anion-binding exosite I (ABE-I) and anion-binding exosite II (ABE-II). Using a thrombin mutant library, we mapped residues important for recognition and cleavage of OPN within ABE-I and ABE-II. A peptide (OPN-(162-197)) was designed spanning the OPN thrombin cleavage site and a hirudin-like C-terminal tail domain. Thrombin cleaved OPN-(162-197) with a specificity constant of kcat/Km = 1.64 × 104 M-1s-1. Representative ABE-I mutants (K65A, H66A, R68A, Y71A, and R73A) showed greatly impaired cleavage, whereas the ABE-II mutants were unaffected, suggesting that ABE-I interacts principally with the hirudin-like OPN domain C-terminal and contiguous to the thrombin cleavage site. Debye-Hückel slopes for milk OPN (-4.1 ± 1.0) and OPN-(162-197) (-2.4 ± 0.2) suggest that electrostatic interactions play an important role in thrombin recognition and cleavage of OPN. Thus, OPN is a bona fide substrate for thrombin, and generation of thrombin-cleaved OPN with enhanced pro-inflammatory properties provides another molecular link between coagulation and inflammation.