Low Molecular Weight Heparin Modulates the Binding and Reactivity of Factor Xa towards Basic Pancreatic Trypsin Inhibitor: A Surface Plasmon Resonance and Kinetics Study.
Authors: Neuenschwander Pierre F. , Baker-Deadmond Kimberly J. , Jones Angela D. 
Published: October 2019

Abstract
Previous studies by us have shown that the blood coagulation factor IXa is modulated by low molecular weight heparin (LMWH), resulting in greater reactivity of this protease with basic pancreatic trypsin inhibitor (BPTI). Since thrombin, factor Xa and factor VIIa share a high degree of homology with factor IXa and all are capable of binding heparin, we examined the potential ability of LMWH to modulate the reactivity of these coagulation proteases towards BPTI. Recombinant BPTI containing a His6-tag on the amino terminal end was constructed, expressed in bacteria and purified by nickel-chelating affinity chromatography (HisTrap). His6-BPTI had identical inhibitory activity as wild-type BPTI towards both trypsin and factor IXa. Surface plasmon resonance (Biacore 3000) was used to examine the binding and reacitvity of His6-BPTI to various coagulation proteases under different conditions. Initial biacore studies (using factor IXa) suggested that the carboxymethyl dextran moeity of CM5 biacore chips can paritally mimic heparin binding to the coagulation proteases, resulting in all sensed interactions being largely a reflection of the heparin-bound enzyme conformer. Thus, a hydrophobic HPA chip that lacks the carboxymethyl dextran matrix was used to prepare self-assembling monolayer surfaces of 100 mol% 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 90 mol% DOPC and 10 mol% of the nickel-chelating phospholipid 1,2 dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} (DOGS-NTA). His6-BPTI was efficiently, specifically and reproducibly captured only onto the surface containing DOGS-NTA. This binding was also dependent on the presence of nickel, and the generated BPTI-phospholipid surface was found to bind trypsin in a BPTI-dependent fashion. Efficient regeneration of the surface was accomplished between cycles by sequential injections of 10 mM EDTA and 10 mM NaOH (to completely remove the enzyme-BPTI complexes) followed by re-loading of the surface with nickel and re-capture of BPTI. Subsequent injections of thrombin, factor IXa, factor Xa, factor VIIa (both in the absence and presence of saturating levels of soluble tissue factor) over the BPTI-phospholipid surface was examined in the absence and presence of 10 μM of the LMWH enoxaparin (15 oligosaccharide units). As previously observed, the binding of factor IXa to BPTI was greatly enhanced by the presence of enoxaparin. Interestingly, the binding of factor Xa to the BPTI surface was also substantially enhanced by enoxaparin, while that of thrombin was only moderately (though consistently) enhanced and that of factor VIIa remained unaffected. Solution-based inhibition studies with factor Xa and BPTI or the isolated second Kunitz-type inhibitor of tissue factor pathway inhibitor (TFPI-K2) confirmed a 2- to 3-fold enhancing effect of enoxaparin on factor Xa reacitivity towards BPTI, with no effect apparent towards TFPI-K2. The enhancing effect of LMWH towards BPTI was also observed with high levels of fondaparinux (100 μM). Although the high levels of fondaparinux required are likely not clinically relevant and presumably reflect the poorer binding of fondaparinux (5 oligosaccharide units) to factor Xa compared with enoxaparin, these results underscore the ability of LMWH to modulate factor Xa reactivity upon occupation of the heparin-binding exosite and may have implications for the rational drug design of specific factor Xa inhibitors.