INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-5 IS CLEAVED BY PHYSIOLOGICAL CONCENTRATIONS OF THROMBIN

Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) is cleave d by a serine protease that is secreted by fibroblasts and porcine smo oth muscle cells (pSMC) in culture. To investigate whether other serin e proteases could cleave this substrate at physiologically relevant co ncentrations, we determined the proteolytic effects of thrombin on IGF BP-5. Human alpha-thrombin (0.0008 NIH U/ml) cleaved IGFBP-5 into 24-, 23-, and 20-kDa non-IGF-I-binding fragments. Cleavage occurred at a p hysiologically relevant thrombin concentration. The effect was specifi c for IGFBP-5, as other forms of IGFBPs, e.g. IGFBP-1, IGFBP-2, and IG FBP-4 were not cleaved by thrombin. Although IGFBP-3 was cleaved by th rombin, this effect required a 50-fold greater thrombin concentration. [S-35]Methionine labeling followed by immunoprecipitation confirmed t hat IGFBP-5 that was constitutively synthesized by pSMC cultures was a lso degraded by thrombin into 24-, 23-, and 20-kDa fragments. The bind ing of IGF-I to IGFBP-5 partially inhibited IGFBP-5 degradation by thr ombin, and an IGF analog that does not bind to IGFBP-5 had no effect. Thrombin did not account for the serine protease activity that had bee n shown previously to be present in pSMC-conditioned medium. This was proven by showing that 1) no immunoreactive thrombin could be detected in the pSMC-conditioned medium; 2) the IGFBP-5 fragments that were ge nerated by thrombin showed three cleavage sites (Arg(192)-Ala(193), Ar g(156)-Ile(157), and Lys(120)-His(121)), whereas the serine protease i n conditioned medium cleaves IGFBP-5 at a different site; and 3) hirud in had no effect on IGFBP-5 cleavage by the protease in pSMC medium; h owever, it inhibited IGFBP-5 degradation by thrombin. To determine the physiological significance of IGFBP-5 cleavage, the effect of an IGFB P-5 mutant that is resistant to cleavage by the pSMC protease and has been shown to inhibit IGF-I actions in pSMC was determined. This mutan t inhibited IGF-I-stimulated DNA synthesis, but if thrombin was added simultaneously, IGF-I was fully active. In summary, physiological conc entrations of thrombin degrade IGFBP-5. Degradation can be blocked by hirudin and is partially inhibited by IGF-I binding. Generation of act ive thrombin in vessel walls may be a physiologically relevant mechani sm for controlling IGF-I bioactivity.