The S130K fibroblast growth factor-1 mutant induces heparin-independent proliferation and is resistant to thrombin degradation in fibrin glue

Objective: Site-directed mutagenesis is an important technique that can alt er cytokine function, thereby eliciting desired responses. S130K is a mutat ion of fibroblast growth factor-1 (FGF-1), with lysine replacing serine in the heparin-binding site. We measured molecular stability and mitogenic act ivity of FGF-1 and S130K, both in the media and when suspended in fibrin gl ue (FG), on smooth muscle cells (SMCs) and endothelial cells (ECs) to deter mine if the mutation altered the function and potential clinical applicabil ity. Methods: EC and SMC proliferation of soluble FGF-1 or S130K at 0, 0.1, 1, 1 0, or 100 ng/mL, with heparin at 0, 5, 50, or 500 units (U)/mL was measured on growth-arrested cells in serum-free media. EC and SMC proliferation ass ays with cells on FG containing either FGF-1 or S130K at 0, 1, 10, 100, or 1000 ng/mL in combination with heparin at 0, 5, 50 or 500 U/mL were also pe rformed during the exponential growth phase. Molecular degradation by throm bin was measured by sodium dodecylsulfate-polyacrylamide gel electrophoresi s. Results: S130K induces greater EC and SMC proliferation in the absence of h eparin than FGF-1 does (P < .0001 for both the 10 and 100 ng/mL doses). S13 0K is also significantly more potent than FGF-1 in the presence of heparin. Heparin in the media enhances cytokine-induced SMC and EC proliferation at doses of 5 U/mL, but inhibits SMC proliferation at concentrations of 500 U /mL. For the FG data, unlike FGF-1, S130K induces EC and SMC proliferation in the absence of heparin. The addition of 5 U/mL of heparin enhances the p roliferation induced by S130K. For ECs, as the heparin dose increases to 50 U/mL, proliferation decreases, as compared with the 5 U/mL concentration w hen either FGF-1 or S130K in the FG was compared at concentrations of 10, 1 00, and 1000 ng/mL (P < .01). S130K is more potent in FG than is FGF-1 both with and without heparin and exhibits maximal EC and SMC proliferation at 10 ng/mL, whereas FGF-1 activity is maximal at 100 ng/mL. Gel electrophores is demonstrated that S130K was relatively more resistant to thrombin degrad ation than FGF-1. Conclusions: Site-directed mutagenesis changed the potency and the heparin dependency on cellular proliferation of FGF-1 in vitro. These techniques sh ould allow the delivery of mutant growth factors to areas of vascular inter vention to induce specific, desired responses. We believe that these studie s will enhance our knowledge of the function of various regions of the FGF- 1 molecule, allowing us to more precisely design increasingly more useful F GF-1 mutants.