Vascular endothelial growth factor (VEGF) is a key factor in endothelial cell biology and blood vessel formation and a candidate therapeutic for the stimulation of angiogenesis-dependent tissue regeneration. The objective of this study was to confer the angiogenic activity of VEGF(121) upon the biomaterial fibrin, a natural substrate for endothelial cell growth and clinically accepted as 'fibrin glue'. To achieve this, we engineered fibrin-based hydrogels that were covalently modified with VEGF(121). Our laboratory has recently developed novel methodology that allows the covalent incorporation of exogenous bioactive peptides by the transglutaminase activity of factor XIIIa into fibrin during coagulation. Here, this ability of factor XIIIa to crosslink additional proteins within fibrin was employed to covalently incorporate VEGF(121). By recombinant DNA methodology, a mutant VEGF(121) variant, alpha(2)-PI(1--8)-VEGF(121), which contains an additional factor XIIIa substrate sequence NQEQVSPL at the aminoterminus, was expressed in E. coli. In soluble form, the mutant protein fully retained its mitogenic activity for endothelial cells. Using (125)I-labeled alpha(2)-PI(1--8)-VEGF(121), its covalent incorporation and the efficiency of incorporation into fibrin was demonstrated and characterized. The immobilized, fibrin-conjugated VEGF(121) protein remained an active and very efficient mitogen for human endothelial cells grown on two-dimensional VEGF(121)-modified fibrin surfaces, and the incorporation of increasing amounts of alpha(2)-PI(1--8)-VEGF(121) resulted in dose-dependent enhancement of endothelial cell growth. The VEGF-modified fibrin matrices can be formed as injectable gels in a single-step reaction under physiological conditions in vivo. When used as a ingrowth matrix, such VEGF incorporating materials could be useful in a variety of clinical situations that require an angiogenic response into an ischemic region or inplant.