FACTOR IXA INHIBITION BY PROTEASE NEXIN-2 AMYLOID BETA-PROTEIN PRECURSOR ON PHOSPHOLIPID-VESICLES AND CELL-MEMBRANES

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-bind ing inhibitor of enzymes, factors XIa and IXa (FIXa), suggesting a rol e for this protein in hemostasis. Since coagulant reactions are modula ted on biologic surfaces, we investigated how 25:75 (mol/mol) phosphat idylserine/phosphatidylcholine vesicles (PSPC), thrombin-activated pla telets, or umbilical vein endothelial cells influence inactivation of FIXa by PN-2/A beta PP. The K-m of human or porcine FIXa activation of human factor X in the presence of PSPC, activated platelets, or endot helial cells in the absence or presence of thrombin-activated factor V III (FVIIIa) was similar, (0.05-0.39 mu M). The presence of FVIIIa inc reased the catalytic efficiency (k(cat)/K-m ratio) of human and porcin e factor IXa's activation of factor X 4952-406-fold, respectively. In the presence of PSPC, the K-i of human and porcine FIXa inhibition by PN-2/A beta PP was K-i = 1.9 x 10(-9) M and 5.8 x 10(-9) M, respective ly. After the addition of FVIIIa to the reaction, the K-i for both hum an and porcine FIXa inhibition by PN-2/A beta PP on PSPC increased 13- and 4-fold to K-i = 2.5 x 10(-8) M and 2.4 x 10(-8) M, respectively. These K-i for inhibition of human FIXa on phospholipid vesicles by PN- 2/A beta PP were similar when factor X activation was measured by chro mogenic or activation peptide release assays. FVIIIa reduced the inhib ition of FIXa by PN-2/A beta PP only in the presence of PSPC. Inhibiti on of human and porcine FIXa on PSPC by the isolated KPI domain of PN- 2/A beta PP was not influenced by FVIIIa. Activated human platelets pr ovide further protection of human or porcine FIXa from inhibition by P N-2/A beta PP over that seen with PSPC and FVIIIa or endothelial cells . PN-2/A beta PP is an inhibitor of FIXa in the presence of the assemb led tenase complex.