Fibronectin binds to platelet membrane glycoprotein (GP) IIb-IIIa in Arg-Gly-Asp (RGD)-dependent and -independent manner. We have isolated and characterized the 29-kDa dispase fragment of fibronectin. Binding of 125I-fibronectin to thrombin-stimulated platelets was inhibited by the 29-kDa fragment and the GRGDSPA peptide with IC50 values of 1.5 +/- 0.4 and 8.1 +/- 0.9 microM, respectively. The NH2-terminal sequence of this fragment gave this result: Ala-Val-Thr-Thr-Ile-Pro-Ala-Pro-Thr-Asp. This established the position of this peptide within fibronectin as beginning with the residue tentatively designated 1597. Neither the RGDS sequence nor the RGD-independent binding domain of fibronectin (Bowditch, R. D., Halloran, C. E., Aota, S., Obara, M., Plow, E. F., Yamada, K. M., and Ginsberg, M. H. (1991) J. Biol. Chem. 266, 23323-23328) was contained in this fragment. The 29-kDa fragment inhibited ADP-induced aggregation of platelets and binding of fibrinogen to activated platelets. The fragment bound to immobilized GPIIb-IIIa. The 125I-labeled 29-kDa fragment directly bound to thrombin-stimulated platelets with 98,000 +/- 4,600 molecules/platelet (Kd = 4.6 +/- 0.5 x 10(-6) M). Direct binding was inhibited by the unlabeled 29-kDa fragment but was not blocked by either the GRGDSPA peptide or the monoclonal anti-GPIIb-IIIa antibody. These results indicate that the additional RGD-independent binding domain(s) to GPIIb-IIIa are present on fibronectin. This additional binding domain(s) on fibronectin may prevent thrombus formation by interfering with the interaction of fibrinogen with GPIIb-IIIa.