Spuriously Low Levels of Protein C with a Protac® Activation Clotting Assay

Protein C (PC) plasma activity is now widely determined in patients who have experienced thrombotic events. Several methods for PC activity measurements have been published and they were compared in a multicenter trial (1). A common feature is the need for an extraction step. More recently an alternative method using direct activation of PC by a snake venom (Protac@) has become commercially available (2, 3), which avoids the tedious extraction step and the thrombin neutraltzatton procedure. Activated PC is then determined either by clotting assay in an aPTT system or with a chromogenic substrate. The main advantages of clotting assays lie in their ability to use the natural substrate (FVIIIa) of PC (and hence to detect some variants with nonnal amidolytic activity) and to be less sensitive to acarboxylated forms of PC (4, 5) In some patients without anticoagulant treatment we have found discrepant results between Protac@ based clotting and chromogenic assays (reagents, Standard Human Plasma and Fibrintimer from Behringwerke, Marburg, FRG), 8S previously reported by Lobermann et al. (3).As shown in the table the values obtained with the chromogenic assay were higher and better correlated with antigen levels measured by electroimmunoassay (Assera Plate Proteine C, Stago, Asnidres, France) than those found with the clotting assay (r 0.975 versus 0.309). However, when the clotting assay was performed at higher plasma dilutions (1120 and Ll40) than that recommended by the manufacturer (1/10) the PC levels were found to increase and the correlation with PC antigen values to improve (r 0.92 at Il40 dilution), even if the correction was not complete. A common feature in this group of patients was a high level of FVIII activity (determined by one-stage clotting assay, reagents from Behringwerke). The apparent low PC levels and their partial correction by higher plasma dilutions could arise from an increase in the total FVIII concentration due to patient plasma FVIII contribution in the assay mixture. The.influence of FVIII on the clotting assay was also tested by measuring PC levels after DDAVP infusion, since its effect on haemostasis is relatively specific for the FVIIUvon Wllebrand complex and t-PA (6). The raise in FVIII (mean three-fold over baseline values) did not change PC levels when measured with the chromogenic assay but gave low values with the clotting one, which could not be totally corrected by increasing dilutions (data not shown). PAI was undetectable in DDAVP plasma (Coa-Set t-PA/PAI, KabiVitrum, Sweden). DDAVP and Normal Human Plasma were also submitted to extraction according to Francis and Patch (7) (except that the MES-citrate-NaCl supernatant was immediately diluted IlI0 in the Veronal buffer supplemented with 0.1 moVl HEPES pH 7 .6), and clotting activity after Protac@ activation was measured. Extraction did abolish the dilution effect, suggesting that some plasma component, probably FVIII, is the cause of the artefact. Thus, the Protac@ clotting method alone cannot be recommended for screening PC deficiency. The partial correction by Table Protein C values Patient FVIII PC Ag PC chrom PC clotting, dilutions utO u20 u40