Protease nexin-2/amyloid β protein precursor (PN-2/AβPP) is an abundant, secreted platelet protein which is a potent inhibitor of coagulation Factor XIa. We examined other potential anticoagulant activities of PN-2/AβPP. Purified Kunitz protease inhibitor domain of PN-2/AβPP and PN-2/AβPP itself were found to prolong the coagulation time of plasma and pure Factor IXa. The Kunitz protease inhibitor domain also inhibited the ability of Factor IXa to activate Factor X. PN-2/AβPP inhibited Factor IXa with a K(i) of 7.9 to 3.9 x 10-11 M in the absence and presence of heparin, respectively. When the second-order rate constant of PN-2/AβPP's inhibition of Factor IXa (2.7 x 108 M-1min-1) was compared to that of antithrombin III (3.8 x 106 M- 1min-1), PN-2/AβPP was at least a 71-fold more potent inhibitor of Factor IXa than antithrombin III. PN-2/AβPP formed a complex with Factor IXa as detected by gel filtration and ELISA. The finding that PN-2/AβPP is a potent inhibitor of Factor IXa could help to explain the spontaneous intracerebral hemorrhages seen in patients with hereditary cerebral hemorrhage with amyloidosis Dutch-type where there is an extensive accumulation of PN- 2/AβPP in their cerebral blood vessels.