Contact factor proteases and the complexes formed with ??2-macroglobulin can interfere in protein C assays by cleaving amidolytic substrates

Plasma from women taking combined oral contraceptives and cold-activated plasma contain proteases which cleave chromogenic substrates in protein C assays in the absence of protein C activators such as ProtacTM. This spontaneous activity makes a background subtraction necessary and makes protein C (PC) assays less accurate. We investigated two commonly used substrates < Glu-Pro-Arg-pNA (S-2366) and 2AcOH.H-D-Lys(Cbo)-Pro-Arg-pNA (PC substrate) and found that cold-activated normal and protein C-deficient plasmas gave absorbance values up to 300 times higher than buffer blanks. FXIa cleaves these substrates but activity was not blocked by corn or lima bean trypsin inhibitors, soy bean trypsin inhibitor (SBTI), hirudin or ±-amino-n-caproic acid (EACA). Kaolin activation of normal, FXI, FIX, FVIII, FVII and protein C-deficient, but not of FXII or prekallikrein (PKK)-deficient plasmas led to cleavage of chromogenic substrate for protein C. The protein C substrates were cleaved by purified kallikrein and α- and ß-FXIIa. Immunoabsorption with α2-macroglobulin (α2M) antibodies removed 60% of the α2M and 70% of the activity on PC Substrate. Gel filtration of normal plasma on Sephadex G-150 gave a single peak of protein C activity and antigen in the included volume. After cold activation of the fractions, a second protein C-like peak appeared in the void volume, but with no detectable protein C antigen. This peak coincided with α2M (chromogenic and ELISA) and plasma kallikrein (S-2302), but FXII (measured with a substrate insensitive to kallikrein) eluted separately. Gel filtration of a cold activated plasma gave a similar peak of spontaneous activity on PC Substrate, which was not inhibited by SBTI. We suggest that the protein C substrates may be cleaved by kallikrein and other enzymes complexed to α2M, which retain activity on small substrates. Samples exceptionally susceptible to cold activation should be collected into anticoagulant containing kallikrein inhibitors and methylamine.