Plasminogen binding to receptors involves both C-terminal lysine- dependent and -independent interactions. The latter are poorly understood. Our earlier work demonstrated a novel Ca (2+) -enhanced bivalent interaction between plasmin-cleaved FXa (FXa33/13) and plasminogen truncated at Lys78 (Lys-Pg). Here we hypothesized that the effects of Ca (2+) may enable dissection of the C-terminal lysine-dependent and -independent interactions. To evaluate the role of the Glu-plasminogen (Glu-Pg) amino acids 1 - 77, binding of FXa33/13 to immobilized Glu-Pg was compared to Lys-Pg by surface plasmon resonance. Under identical conditions, approximately half the amount of FXa33/13 bound to Glu-Pg. The simplest fit of data suggested a 21 plasminogenFXa33/13 stoichiometry for both, which were proportionately enhanced by Ca (2+) . Only Lys-Pg demonstrated significant Ca (2+) -independent binding to FXa33/13. In the presence of Ca (2+) , weak C-terminal lysine-independent binding could be detected, but only for Glu-Pg. The elastase-generated plasminogen fragment encompassing the angiostatin-like kringle domains 1 to 3 (K1 - 3) inhibited binding of FXa33/13 to Lys-Pg, whereas fragments corresponding to kringle 4- and kringle 5-protease domain had no effect. Immobilized K1 - 3 binding to FXa33/13 had both Ca (2+) -dependent and -independent components. The principal K (d) for the interaction was 10-fold higher than Lys-Pg. In the presence of Ca (2+) , eACA inhibited FXa33/13 binding to K1 - 3 by 30%, but eliminated binding in the absence of Ca (2+) . These studies suggest that Ca (2+) -dependent and -independent binding of Lys-Pg to FXa33/13 are C-terminal lysine-dependent. The N-terminal 1 - 77 amino acids of Glu-Pg confer significant C-terminal lysine-independent binding, which may play a role during the initiating stages of plasminogen activation.