Thrombin hydrolysis of human osteopontin is dependent on thrombin anion-binding exosites.
The cytokine osteopontin (OPN) can be hydrolyzed by thrombin exposing a cryptic alpha(4)beta(1)/alpha(9)beta(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 k(cat)/K(m) value of 1.14 x 10(5) m(-1) s(-1). Thrombin cleavage of OPN was inhibited by unsulfated hirugen (IC(50) = 1.2 +/- 0.2 microm), unfractionated heparin (IC(50) = 56.6 +/- 8.4 microg/ml) and low molecular weight (5 kDa) heparin (IC(50) = 31.0 +/- 7.9 microg/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 k(cat)/K(m) = 1.64 x 10(4) m(-1) s(-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.
Myles
Timothy
T
Division of Hematology, Stanford University School of Medicine, Stanford, CA 94305, USA. timothy.myles@maxygen.com
Leung
Lawrence L K
LL
D020816:Amino Acid Motifs; D000838:Anions; D001665:Binding Sites; D001777:Blood Coagulation; D006801:Humans; D006868:Hydrolysis; D007249:Inflammation; D020128:Inhibitory Concentration 50; D016023:Integrins / Q000737:chemistry; D007700:Kinetics; D008895:Milk, Human / Q000378:metabolism; D009154:Mutation; D053495:Osteopontin / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013917:Thrombin / Q000737:chemistry*
D000838:Anions; D016023:Integrins; D053495:Osteopontin; D013917:Thrombin