PB0597 - Specificity of contact pathway inhibitors with regard to other coagulation and fibrinolysis proteases.

Background: There has been much recent interest in studying the role of contact activation in hemostasis and thrombosis. As a component of these studies, we use various inhibitors including Kunitz (soybean trypsin inhibitor (SBTI), corn trypsin inhibitor (CTI)), peptide (ecallantide), and small molecule (avoralstat) inhibitors of kallikrein, factor XIIa, and factor XIa. In these studies, it may be informative to use these inhibitors at concentrations that are significantly outside of their therapeutic range.

Aims: We investigated the off target effects of some contact factor inhibitors across a very large range of concentrations by studying inhibition of multiple coagulation and fibrinolytic enzymes.

Methods: Ecallantide was produced by the Protein Production Core at the University of North Carolina. Avoralstat was purchased from MedChemExpress. CTI was purchased from Prolytix. SBTI was purchased from Sigma-Aldrich. Inhibitor and enzyme were mixed and substrate appropriate for the enzyme was added. Substrate cleavage was proportional to amount of enzyme that was not bound to the inhibitor.

Results: CTI was very specific for factor XIIa with little inhibition of other factors (Figure). SBTI, ecallantide, and avoralstat were, as expected, very potent inhibitors of kallikrein. Avoralstat, at concentrations well above therapeutic levels, had weak inhibitory effect on many other coagulation enzymes. Ecallantide inhibited factor XIa well and plasmin weakly but otherwise had minimal effects on the other coagulation factors. SBTI, generally used as an inhibitor of kallikrein, was also a good inhibitor of plasmin, but exhibited modest inhibition of factor Xa and weak inhibition of factor XIa.

Conclusion(s): Appropriate concentrations of contact factor inhibitors can be used for in vitro research studies to selectively dissect the role of kallikrein, factor XIIa, and factor XIa on models of hemostasis and thrombosis.