A monoclonal antibody against activated protein C allows rapid detection of activated protein C in plasma and reveals a calcium ion dependent epitope involved in factor Va inactivation

Activated protein C (APC) serves as an 'on demand' anticoagulant. Defects in the APC anticoagulant pathway are underlying risk factors for the development of venous and arterial thrombosis. APC has recently been shown to significantly reduce mortality in patients with severe sepsis, presumably by virtue of its ability to down-regulate coagulation as well as inflammation. Our objective was to develop an assay that, for the first time, permits rapid detection of plasma APC. This assay will expedite studies of APC in a variety of vascular disease states including sepsis, severe atherosclerosis, diabetes, and vasculitis. By generating a highly APC-specific monoclonal antibody (HAPC 1555), we have developed an assay that, for the first time, allows rapid detection of plasma APC. The Kd measured for the interaction between APC and HAPC 1555 based on BIAcore studies and binding to immobilized HAPC on microtiter plates is 6.2 +/- 0.9 and 8.8 +/- 1.0 nmol L(-1), respectively. The interaction between HAPC 1555 and APC is Ca2+-dependent, with a Ca2+ concentration of 313 +/- 48 micro mol L(-1) required for half maximal binding. HAPC 1555 interferes with APC-mediated inactivation of factor (F)Va in the presence and absence of phospholipids, suggesting that HAPC 1555 binds to the FVa binding domain of APC. When HAPC 1555 was used in an APC enzyme capture assay, therapeutic APC levels could be measured in 1.5 h, and physiologic levels of APC could be detected between 3 and 19 h. APC levels were also shown to vary markedly in patients with severe sepsis. The rapidity of our APC assay makes APC detection in patients practical clinically. This assay will expedite studies of APC in a variety of vascular disease states including sepsis, severe atherosclerosis, diabetes, and vasculitis.