Insight into Factor IXa Selectivity and Reactivity Using Kunitz-Type Inhibitors: Importance of Factor IXa Residue K98.
Authors: Neuenschwander Pierre F. , Williamson Stephen R. , Nalian Armen 
Published: October 2019

Previous studies by us have shown that blood coagulation factor IXa is relatively resistant to inhibition by the Kunitz-type inhibitor bovine pancreatic trypsin inhibitor (BPTI; aprotinin), but that this resistance can be partially alleviated by the presence of low molecular weight heparin. In order to gain insight into potential mechansims of factor IXa selectivity and its modulation by heparin we undertook an examination of the reactivity of factor IXa with several other Kunitz-type inhibitors: the Kunitz-type inhibitor domain of protease nexin-2 (PN2KPI) and the first two Kunitz-type inhibitor domains of tissue factor pathway inhibitor (TFPI K1 and TFPI K2). As expected, factor IXa exhibited remarkable specificity towards these highly-homologous inhibitors and expressed the following order of reactivity: PN2KPI > TFPI K1/K2 > BPTI. Surprisingly, the enhancing effect of heparin (enoxaparin) was limited to factor IXa reactivity with BPTI and was not observed with PN2KPI or TFPI inhibitory domains. This effect of heparin was not due to a simple template or bridging mechanism between factor IXa and BPTI since progressively smaller oligosaccharides (H14, H10, H6) retained function. We performed molecular modeling and molecular dynamic simulation studies that suggested that BPTI residue R39 may sterically clash with the 99 loop region of factor IXa (specifically residue K98). In contrast, the corresponding position in PN2KPI (G39) does not appear to clash with factor IXa residue K98 in modeling studies. In addition, the 99 loop region of factor IXa is in close proximity to the heparin binding site. Thus, based on these observations we hypothesized that factor IXa residue K98 could be restricting occupation of the factor IXa active site, and that this steric hindrance could be partly alleviated by heparin binding. We therefore examined a mutant of factor IXa in which residue K98 was mutated to A. The fIXK98A mutant was expressed as a zymogen in human 293 cells and purified by a combination of ion exchange and heparin affinity chromatography. The fIXK98A mutant exhibited identical procoagulant activity compared to wild-type factor IXa in a standard clotting assay. In addition, upon activation with RVV-X the fIXaK98A enzyme showed similar activity as wild-type factor IXa towards the small peptide substrate CBS 31.39. In sharp contrast, however, examination of the inhibition of fIXaK98A by BPTI showed a dramatic enhancement in inhibition compared to the wild-type enzyme: Ki = 20 μM in the absence of heparin and Ki = 10 μM in the presence of heparin for fIXaK98A; and Ki > 500 μM in the absence of heparin and Ki = 40 μM in the presence of heparin for wild-type factor IXa. We conclude from this that factor IX residue K98 limits access of specific molecules into the active site of factor IXa and protects it from inhibition by BPTI. It would seem that some or all of this interference is alleviated upon heparin binding to factor IXa. The smaller effect of heparin on the mutant enzyme (2-fold) compared to the wild-type enzyme (>10-fold) further supports the supposition that heparin binding to factor IXa may in part be facilitating movement of the 99 loop of factor IXa to enhance access to the active site. This may have implications in the selectivity and reactivity of factor IXa with other inhibitors as well as certain substrates and provides some important insight into factor IXa function.