Development of a Method to Measure Plasma Levels of Activated Protein C in Complex with Protein C Inhibitor

Abstract: Acute myocardial infarction (MI) and deep venous thrombosis (DVT) are conditions caused by thrombus formation with activation of the coagulation system. The plasma concentration of the complex between activated protein C (APC) and protein C inhibitor (PCI), APC–PCI complex is increased in such states. We have devised an immunofluorometric assay employing a catcher antibody (M36) that recognizes a neoepitope in APC–PCI complexes and cleaved PCI. The M36 Mab has a particularly strong binding affinity ( KD = 5 x 10-11M) for the complexed/cleaved PCI, which in combination with the neoepitoe specificity are the major differences compared to previous methods for APC–PCI measurements. The analytical detection limit of the method (0.06 µg/L, +3SD) is sufficient to allow accurate measurements of normal and low plasma concentrations of the APC–PCI complex. The mean in healthy individuals was 0.30 µg/L (0.17–0.54, ±2SD). In patients undergoing hip-arthroplasty, the APC–PCI level was maximal four hours postoperatively and returned to normal within 24 hours postoperatively. Two case-control studies were conducted. In the first, patients presenting at the Emergency Department with suspicion of DVT were included. Based on phlebography results, 123 patients were included. The APC–PCI levels were significantly higher in the 40 DVT patients than in the 83 controls, who had negative phlebography. Receiver operating characteristics (ROC) curves were calculated and demonstrated a discriminatory capacity most comparable to D-dimer, measured in the same patients. Compared to D-dimer, the APC–PCI complex is a more well-defined analyte. In the second study, plasma samples were collected from 74 patients admitted to the Coronary Care Unit for chest pain in combination with ECG changes. MI is preceded by activation of the coagulation system and thrombus formation initiated by a rupture in an atherosclerotic plaque. APC–PCI levels correlated closely with maximal CKMB levels, reflecting MI size, whereas there was no correlation between APC–PCI and CKMB or troponin I on arrival to hospital. Our studies indicate that the APC–PCI complex measured with the new method could be used to indicate patients at risk of developing venous thrombosis, and, the APC–PCI level is an early marker of activation of the coagulation system preceding MI.