Contact factor proteases and the complexes formed with alpha 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 Protac. This spontaneous activity makes a background substraction 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 epsilon-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 alpha- and beta-FXIIa. Immunoabsorption with alpha 2-macroglobulin (alpha 2M) antibodies removed 60% of the alpha 2M 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 alpha 2M (chromogenic and ELISA) and plasma kallikrein (S-2302), but FXII (measured with a substrate insensitive to kallikrein) eluted separately.(ABSTRACT TRUNCATED AT 250 WORDS)
D000577:Amides / Q000737:chemistry; D003080:Cold Temperature; D003276:Contraceptives, Oral / Q000494:pharmacology*; D003951:Diagnostic Errors; D007692:Diatomaceous Earth; D004359:Drug Therapy, Combination; D015945:Factor XIa / Q000737:chemistry*; D005260:Female; D006801:Humans; D007616:Kaolin; D008297:Male; D009842:Oligopeptides; D011233:Precipitin Tests; D011480:Protease Inhibitors; D011486:Protein C / Q000032:analysis*; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D000511:alpha-Macroglobulins / Q000737:chemistry*
D000577:Amides; D003276:Contraceptives, Oral; D009842:Oligopeptides; D011480:Protease Inhibitors; D011486:Protein C; D000511:alpha-Macroglobulins; D007616:Kaolin; D007692:Diatomaceous Earth; C026148:prolyl-phenylalanyl-arginine-4-nitroanilide; C040190:S 2366; D015945:Factor XIa; D011761:Pyrrolidonecarboxylic Acid