NITROPHORIN-2 - A NOVEL MIXED-TYPE REVERSIBLE SPECIFIC INHIBITOR OF THE INTRINSIC FACTOR-X ACTIVATING COMPLEX

Nitrophorin-2 (NP-2), isolated from salivary glands of the blood-sucki ng insect Rhodnius prolixus, has been shown to be a specific inhibitor of the intrinsic factor X-(FX)-activating complex. The inhibitory eff ect of NP-2 is most potent in the presence of both FVIIIa and phosphol ipids (artificial phospholipid vesicles or activated human platelets). Detailed kinetic analyses of the inhibitory mechanism of NP-2 demonst rated a decrease in both V-max and K-m of activated FIX-(FIXa)-catalyz ed FX activation in the presence of FVIIIa and phospholipid vesicles, characteristic of a hyperbolic mixed-type reversible inhibitor. NP-2 e xhibits a higher binding affinity for the enzyme-substrate complex, i. e., FIXa/FVIIIa/Ca2+/phospholipids/FX complex (K-i' = 6.2 nM) than for the enzyme complex, i.e., FIXa/FVIIIa/Ca2+/phospholipids (K-i = 16.5 nM). The same inhibitory kinetic mechanism is valid in platelet-mediat ed FIXa-catalyzed FX activation (K-i' = 5.9 nM and K-i = 12.6 nM, resp ectively). The fact that NP-2 increases the concentrations (EC50) of F IXa, FVIIIa, and phospholipid vesicles required for half-maximal rates of FX activation suggests that NP-2 interferes with the functioning o f all three major components of the intrinsic FX-activating complex. N P-2 was found to inhibit FX activation when either phospholipids or FV IIIa are present, but not in the absence of both factors. Taken togeth er, we conclude that NP-2 is a unique, potent, and highly specific inh ibitor of the intrinsic FX-activating complex that inhibits FIXa bound either to the phospholipid or activated platelet surface or to the co factor FVIIIa by interfering with the assembly of FX-activating comple x on these surfaces.