The effect of corn trypsin inhibitor and inhibiting antibodies for FXIa and FXIIa on coagulation of plasma and whole blood: comment

Abstract

We read with interest the article by Hansson et al. 1.Hansson K.M. Nielsen S. Elg M. Deinum J. The effect of corn trypsin inhibitor and inhibiting antibodies for FXIa and FXIIa on coagulation of plasma and whole blood.J Thromb Haemost. 2014; 12: 1678-86Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar reporting that corn trypsin inhibitor (CTI), in addition to being an efficient inhibitor of factor XIIa proteolytic activity, also partially inhibits FXIa amidolytic activity at the concentrations used in many laboratories worldwide. On the basis of their data, the authors recommend using CTI for the prevention of contact pathway‐initiated thrombin generation at concentrations not exceeding 20 μg mL−1. We have been using CTI for the prevention of the contact pathway in tissue factor (TF)‐triggered thrombin generation and clot formation for almost two decades 2.Rand M.D. Lock J.B. van‘t Veer C. Gaffney D.P. Mann K.G. Blood clotting in minimally altered whole blood.Blood. 1996; 88: 3432-45Crossref PubMed Google Scholar, 3.Mann K.G. Whelihan M.F. Butenas S. Orfeo T. Citrate anticoagulation and the dynamics of thrombin generation.J Thromb Haemost. 2007; 5: 2055-61Crossref PubMed Scopus (111) Google Scholar. The concentration of CTI in all of our experiments has been 100 μg mL−1 (8.0 μm), which is, by coincidence, almost identical to the Ki of 8.1 μm for FXIa reported by Hansson et al. 1.Hansson K.M. Nielsen S. Elg M. Deinum J. The effect of corn trypsin inhibitor and inhibiting antibodies for FXIa and FXIIa on coagulation of plasma and whole blood.J Thromb Haemost. 2014; 12: 1678-86Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar. Our choice of this CTI concentration was based on the observation 2.Rand M.D. Lock J.B. van‘t Veer C. Gaffney D.P. Mann K.G. Blood clotting in minimally altered whole blood.Blood. 1996; 88: 3432-45Crossref PubMed Google Scholar that, at lower concentrations, particularly at 20 μg mL−1, CTI inhibition of contact pathway‐related plasma clotting is quite limited. A similar conclusion could be drawn from the data published by Hansson et al. (see Fig. 1 in 1.Hansson K.M. Nielsen S. Elg M. Deinum J. The effect of corn trypsin inhibitor and inhibiting antibodies for FXIa and FXIIa on coagulation of plasma and whole blood.J Thromb Haemost. 2014; 12: 1678-86Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar): their data suggest that, at recommended CTI concentrations (below 20 μg mL−1), efficient inhibition of the contact pathway is impossible, especially if the goal of an experiment is to analyze processes occurring in blood or plasma triggered with low TF concentrations 4.van Veen J.J. Gatt A. Cooper P.C. Kitchen S. Bowyer A.E. Makris M. Corn trypsin inhibitor in fluorogenic thrombin‐generation measurements is only necessary at low tissue factor concentrations and influences the relationship between factor VIII coagulant activity and thrombogram parameters.Blood Coagul Fibrinolysis. 2008; 19: 183-9Crossref PubMed Scopus (84) Google Scholar, 5.Spronk H.M. Dielis A.W. Panova‐Noeva M. van Oerle R. Govers‐Riemslag J.W. Hamulyak K. Falanga A. Cate H.T. Monitoring thrombin generation: is addition of corn trypsin inhibitor needed?.Thromb Haemost. 2009; 101: 1156-62Crossref PubMed Scopus (81) Google Scholar, 6.Chatterjee M.S. Denney W.S. Jing H. Diamond S.L. Systems biology of coagulation initiation: kinetics of thrombin generation in resting and activated human blood.PLoS Comput Biol. 2010; 6Crossref Scopus (116) Google Scholar. It is even more important to efficiently inhibit contact pathway initiation when endogenous FXIa present in plasma of cardiovascular patients and of those with inflammation 7.Butenas S. Undas A. Gissel M.T. Szuldrzynski K. Zmudka K. Mann K.G. Factor XIa and tissue factor activity in patients with coronary artery disease.Thromb Haemost. 2008; 99: 142-9Crossref PubMed Scopus (69) Google Scholar, 8.Undas A. Owszarek D. Gissel M. Salapa K. Mann K.G. Butenas S. Activated factor XI and tissue factor in inflammatory bowel disease.Inflamm Bowel Dis. 2010; 16: 1447-8Crossref PubMed Scopus (14) Google Scholar is quantified. Inefficient inhibition of FXIIa would lead to the activation of FXI during a thrombin generation assay in the absence of an exogenous trigger. Conversely, inhibition of endogenous FXIa by CTI would result in an underestimation of the concentration of FXIa in a patient's plasma. To address these issues, we analyzed the effect of CTI on clotting of a congenital FXII‐deficient plasma (George King Bio‐Medical, Overland Park, KS, USA) triggered with purified human FXIa. CTI (in‐house preparation 9.Cawthern K.M. van ‘t Veer C. Lock J.B. DiLorenzo M.I. Branda R.F. Mann K.G. Blood coagulation in hemophilia A and hemophilia C.Blood. 1998; 91: 4581-92Crossref PubMed Google Scholar) and FXIa (Haematologic Technologies, Essex Junction, VT, USA) were added at varying concentrations to FXII‐deficient plasma, and this was followed by the addition of either an activated partial thromboplastin time (APTT) reagent (Trinity Biotech PLC Bray, Wicklow, Ireland) or 20 μm synthetic phospholipid vesicles 9.Cawthern K.M. van ‘t Veer C. Lock J.B. DiLorenzo M.I. Branda R.F. Mann K.G. Blood coagulation in hemophilia A and hemophilia C.Blood. 1998; 91: 4581-92Crossref PubMed Google Scholar and recalcification. Concentrations of CTI varied between 25 μg mL−1 and 300 μg mL−1, and concentrations of FXIa varied between 50 pm and 500 pm. Clotting times were measured in the ST‐8 apparatus (Diagnostica Stago, Parsippany, NJ, USA). Neither with the APTT reagent nor with synthetic phospholipids did the clotting times of FXII‐deficient plasma become prolonged at any combination of FXIa and CTI concentrations, indicating the lack of detectable inhibition of FXIa activity by CTI. The most likely cause of the discrepancy between the data published by Hannson et al. 1.Hansson K.M. Nielsen S. Elg M. Deinum J. The effect of corn trypsin inhibitor and inhibiting antibodies for FXIa and FXIIa on coagulation of plasma and whole blood.J Thromb Haemost. 2014; 12: 1678-86Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar and our results is the environment of FXIa in plasma. In the purified system, CTI has no competitors for the active site of FXIa (with the exception of a synthetic substrate, which is an essential component of the assay), whereas in plasma there are several known natural substrates for FXIa, such as FIX 10.Fujikawa K. Legaz M.E. Kato H. Davie E.W. The mechanism of activation of bovine factor IX (Christmas factor) by bovine factor XIa (activated plasma thromboplastin antecedent).Biochemistry. 1974; 13: 4508-16Crossref PubMed Scopus (129) Google Scholar, 11.Naito K. Fujikawa K. Activation of human blood coagulation factor XI independent of factor XII. Factor XI is activated by thrombin and factor XIa in the presence of negatively charged surfaces.J Biol Chem. 1991; 266: 7353-8Abstract Full Text PDF PubMed Google Scholar, 12.Whelihan M.F. Orfeo T. Gissel M.T. Mann K.G. Coagulation procofactor activation by factor XIa.J Thromb Haemost. 2010; 8: 1532-9Crossref PubMed Scopus (47) Google Scholar, FXI 11.Naito K. Fujikawa K. Activation of human blood coagulation factor XI independent of factor XII. Factor XI is activated by thrombin and factor XIa in the presence of negatively charged surfaces.J Biol Chem. 1991; 266: 7353-8Abstract Full Text PDF PubMed Google Scholar, FV, and FVIII 10.Fujikawa K. Legaz M.E. Kato H. Davie E.W. The mechanism of activation of bovine factor IX (Christmas factor) by bovine factor XIa (activated plasma thromboplastin antecedent).Biochemistry. 1974; 13: 4508-16Crossref PubMed Scopus (129) Google Scholar, and a number of serine protease inhibitors 13.Wuillemin W.A. Minnema M. Meijers J.C. Roem D. Eerenberg A.J. Nuijens J.H. ten Cate H. Hack C.E. Inactivation of factor XIa in human plasma assessed by measuring factor XIa–protease inhibitor complexes: major role for C1‐inhibitor.Blood. 1995; 85: 1517-26Crossref PubMed Google Scholar. All of these substrates and inhibitors are competitors of CTI for the active site of FXIa, and, because of the relatively low affinity of CTI for the active site of FXIa, they will minimize CTI binding and its influence on FXIa activity in plasma. In conclusion, our data indicate that CTI, at concentrations as high as 300 μg mL−1, does not inhibit FXIa activity in plasma or, presumably, in blood. S. Butenas and K. G. Mann designed the experiments and analyzed the data. S. Butenas contributed to the data collection and wrote the manuscript. K. Mann is consultant for Baxter and is the Chairman of the Board of Haematologic Technologies Inc. K. Mann reports receiving grants from NHLBI and DoD during the conduct of the study, as well as grants from NHLBI, DoD and Haematologic Technologies Inc. outside the submitted work. In addition, K. Mann has a patent for the use of CTI as an anticoagulant. K. Mann is a major shareholder of Haematologic Technologies, Inc. S. Butenas has no Conflicts of interest. This work was supported by NIH grant RFA‐HL‐13‐025. We would like to thank S. Prior for her technical assistance.NIHRFA‐HL‐13‐025