Protease nexin-2/amyloid beta-protein precursor (PN-2/A beta PP) is a Kunitz-type protease inhibitor which has been shown to be a tight-binding inhibitor of coagulation factors XIa and IXa. Here we show that PN-2/A beta PP and its KPI domain also inhibited isolated factor Xa with a Ki of 10(-8) M. On a solid phase binding assay, PN-2/A beta PP formed a complex with factor Xa. Incubation of molar excess factor Xa to PN-2/A beta PP produced a single cleavage within PN-2/A beta PP's heparin binding domain liberating a 8.2-kDa amino-terminal peptide. PN-2/A beta PP and its KPI domain equally inhibited factor Xa in the prothrombinase complex with a Ki of 1.9 x 10(-8) M and 1.3 x 10(-8) M, respectively. A beta PP695 which does not contain the KPI domain was a substrate of factor Xa but did not inhibit it, indicating the PN-2/A beta PP inhibition of factor Xa was not substrate inhibition. All of the factor Xa inhibition in the prothrombinase complex by PN-2/A beta PP and its KPI domain on the chromogenic assay was accounted for by inhibition of release of prothrombin fragment F1+2 as determined on immunochemical assay. In the prothrombinase complex, PN-2/A beta PP inhibited factor Xa with a kassoc = 1.8 +/- 0.7 x 10(6) M-1 min-1 similar to antithrombin III and heparin inhibition (kassoc of 3.0 +/- 0.2 x 10(6) M-1 min-1). These studies indicated that PN-2/A beta PP in the assembled prothrombinase complex inhibited factor Xa comparable to antithrombin III in the presence of heparin. PN-2/A beta PP's factor Xa inhibitory activity along with its known inhibition of factors XIa and IXa suggest that this protease inhibitor and related proteins could be regulators of hemostatic reactions on membranes of cells in the intravascular compartment.