Orthner C L, Kolen B, Drohan W N
Plasma Derivatives Laboratory, American Red Cross, Rockville, MD.
Thromb Haemost. 1993 May 3;69(5):441-7.
Activated protein C (APC) is a serine protease which plays an important role as a naturally occurring antithrombotic enzyme. APC, which is formed by thrombin-catalyzed limited proteolysis of the zymogen protein C, functions as an anticoagulant by proteolytic inactivation of the coagulation cofactors VIIIa and Va: APC is inhibited by several members of the serpin family as well a by alpha 2-macroglobulin. APC is being developed as a therapeutic for the prevention and treatment of thrombosis. We have developed an assay to quantify circulating levels of enzymatically active APC during its administration to patients, in healthy individuals, and in various disease states. This assay utilizes an EDTA-dependent anti-protein C monoclonal antibody (Mab) 7D7B10 to capture both APC and protein C from plasma, prepared from blood collected in an anticoagulant supplemented with the reversible inhibitor p-aminobenzamidine. Mab 7D7B10-derivatized agarose beads are added to the wells of a 96-well filtration plate, equilibrated with Tris-buffered saline, and incubated for 10 min with 200 microliters of plasma. After washing, APC and protein C are eluted from the immunosorbent beads with a calcium-containing buffer into the wells of a 96-well microtiter plate containing antithrombin III (ATIII) and heparin. The amidolytic activity of APC is then measured on a kinetic plate reader following the addition of L-pyroglutamyl-L-prolyl-L-arginine-p-nitroanilide (S-2366) substrate. The rate of substrate hydrolysis was proportional to APC concentration over a 200-fold concentration range (5.0 to 1,000 ng/ml) when measured continuously over a 15 to 30 min time period.(ABSTRACT TRUNCATED AT 250 WORDS)
活化蛋白C（APC）是一种丝氨酸蛋白酶，作为一种天然存在的抗血栓酶发挥着重要作用。APC由凝血酶催化酶原蛋白C进行有限的蛋白水解形成，通过对凝血辅因子VIIIa和Va进行蛋白水解失活来发挥抗凝作用：APC受到丝氨酸蛋白酶抑制剂家族的几个成员以及α2-巨球蛋白的抑制。APC正在被开发用于预防和治疗血栓形成。我们已经开发出一种检测方法，用于在给患者、健康个体以及各种疾病状态下的人群施用APC期间，定量循环中具有酶活性的APC水平。该检测方法利用一种依赖乙二胺四乙酸（EDTA）的抗蛋白C单克隆抗体（Mab）7D7B10从血浆中捕获APC和蛋白C，血浆由采集于添加了可逆抑制剂对氨基苯甲脒的抗凝剂中的血液制备而成。将Mab 7D7B10衍生化的琼脂糖珠加入到96孔过滤板的孔中，用Tris缓冲盐水平衡，然后与200微升血浆孵育10分钟。洗涤后，APC和蛋白C用含钙缓冲液从免疫吸附珠上洗脱到含有抗凝血酶III（ATIII）和肝素的96孔微量滴定板的孔中。然后在加入L-焦谷氨酰-L-脯氨酰-L-精氨酸-对硝基苯胺（S-2366）底物后，在动力学酶标仪上测量APC的酰胺水解活性。在15至30分钟的时间段内连续测量时，底物水解速率在200倍的浓度范围内（5.0至1000纳克/毫升）与APC浓度成正比。（摘要截短于250字）
