Interference of C-reactive protein with clotting times.

Abstract

C-reactive protein (CRP) is an acute phase protein with a known affinity for phospholipids (PLs). Phosphatidylcholine (PC) is the principal CRP ligand, but CRP also interferes with other PL, e.g., phosphatidylethanolamine (PE) [1 – 3] . Recently, we illustrated that CRP interferes with activated partial thromboplastin time (aPTT), prolonging clotting times (CT) proportionally with CRP concentration [4] and depending on the type of aPTT reagent [4, 5] . PLs act as catalytic surfaces in clotting assays such as aPTT, hence this phenomenon is most likely PL-dependent, with both concentration and type of PL taking part. To unravel the mechanism, we studied the influence of CRP on different aPTT reagents. To work in PL-controlled conditions, we performed thrombin generation (TG) measurements by calibrated automated thrombinography (CAT ® ; Thrombinoscope, Maastricht, The Netherlands) [6] . We compared the results with the effect on thrombin time, a PL-independent clotting time (CT). Moreover, we compared the effect of CRP with that of annexin V (AnV), a potent endothelial surface anticoagulant based on its ability to displace PL-dependent coagulation factors by binding to negatively charged PL, including phosphatidylserine (PS) and PE [1, 7] . We attempted to visualize the binding of CRP to the PL surface by flow cytometry. Finally, we measured the PC content and the surface tension of the reagents because differences in interference of CRP may be determined by the accessibility of PL. Platelet-poor normal pooled plasma (NPP) was spiked with CRP or AnV and tested in duplicate on STAR-Evolution (Diagnostica Stago, Asni e res, France) with different aPTT reagents and thrombin time. The reagents are listed in Table 1 in Supplemental Material. Only limited information on the type and concentration of PL in these reagents was provided by the manufacturer. We used purified CRP prepared as described previously [8] to obtain a dilution series with a final CRP concentration of 0, 25, 50, 100, 150, 200, and 300 mg/L. The final concentration range of AnV (Sigma-Aldrich, St. Louis, MO, USA) (0 – 0.09 – 0.18 – 0.36 – 0.72 – 1.14 μ M) was based on current literature [7] . TG was triggered in platelet-poor plasma by tissue factor (TF, 5 pM) or silica (a 1/400 diluted aPTT reagent, PTT-A; Diagnostica Stago) and PL (4 μ M). The CAT was performed as reported previously [6, 9] . Under these conditions, TG is PL-controlled, allowing to perform the test with different PLs separately: PE, PC, PS in purified linear structure (Sigma-Aldrich) and hexagonal PL (PE) of Staclot-LA (Diagnostica Stago) (HPE). Mixtures of PC and PS were used in a 70/30, 50/50, 30/70, 0/100, and 100/0 proportion. The concentration of HPE in Staclot-LA reagent was not revealed by the manufacturer and was therefore used in different dilutions (1/10, 1/50, and 1/100). To evaluate the prolongation of the lag time (LT) and the reduction of the peak height (PH), TG parameters were coupled in a PH/LT ratio [9] . Microparticles (MPs) were prepared by activating platelets with calcium ionophore (Sigma-Aldrich). Flow cytometric analysis of MP on FACSCanto II (Becton Dickinson, Erembodegem, Belgium) was performed with AnV *Corresponding author: Katrien M.J. Devreese, Coagulation Laboratory, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, De Pintelaan 185 (2P8), 9000 Ghent, Belgium, Phone: + 32 9 332 65 67, Fax: + 32 9 332 49 85, E-mail: katrien.devreese@uzgent.be Charlotte J. Verfaillie: Coagulation Laboratory, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Gent, Belgium Frank De Bisschop: Department of Inorganic and Physical Chemistry, Ghent University, Gent, Belgium Joris R. Delanghe: Chemistry Laboratory, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Gent, Belgium