Protein C, a 62,000-dalton glycoprotein with approximately 28% carbohydrates, is synthetised in the liver. Activated protein C (APC) is a key anticoagulant enzyme in the down-regulation of coagulation. Protein C consists of two polypeptide chains with a heavy chain linked by a disulfide bond to a light chain. The heavy chain contains the serine active site and the activation peptide and it also contains proposed binding sites for factors Va and VIIIa. The light chain contains a region of g-carboxyglutamic acid residues (calcium ion and phospholipid binding region) and the epidermal growth factor region (proposed protein S-binding region). The average concentration of protein C in human plasma is 4 mg/mL and its half-life in plasma is 7 to 9 hours. One Unit of Protein C corresponds to the amount of Protein C contained in one mL of freshly pooled normal plasma. In order to facilitate comparison of results from analysis of protein C in plasma, an international standard has been prepared and with the assigned potency expressed in international units (IU). Thus 1 IU corresponds to about 4 mg of functional protein C.
A freeze-dried human plasma (denoted 86/622) has been established by the WHO Expert Committee on Biological Standardization as the 1st International Standard for protein C in plasma, which is available from NIBSC.1 The assigned functional activity of this plasma is 0.82 IU/ampoule.
Protein C in plasma may be activated by the thrombin/thrombomodulin complex or, more conveniently, by a specific venom enzyme from the snake Agkistrodon controtrix contortrix (Southern Copperhead Snake).2
The most suitable chromogenic substrate for the assay of APC described so far is chromogenic substrate S-2366.3-5 The Chromogenix protein c reagent (Art. No 82 20 98), which contains a purified preparation of the snake venom enzyme allows activation of protein C without interference from other coagulation factors. The amount of activated protein C is determined by the rate of hydrolysis of the chromogenic substrate S-2366. The activity of human APC, derived from the rate of hydrolysis of chromogenic substrate S-2366 in a purified system under properly standardized conditions (0.1 mol/L Tris-HCl pH 8.3, 0.26 mol/L CaCl, 4 mmol/L CaCl2 and with 0.2% BSA as bulking agent) is about 37 nkat / mg of active enzyme.
The corresponding activity obtained in a plasma containing system after snake venom activation, using Coamatic Protein C, is about 25 nkat / mg of active enzyme.
- Hubbard AR. Standardization of protein C in plasma: Establishment of an International Standard. Thromb Haemost 59, 464-467 (1988).
- Stocker K, Fischer H, Mejer J, Brogli M, Svendsen L. Protein C activators in snake venoms. Behring Inst Mitt 79, 37-47 (1986).
- Exner T, Vaasjoki R. Characterisation and some properties of the protein C activator from Agkistrodon Contortrix venom. Thromb Haemost 59, 40-44 (1988).
- Odegaard OR, Try K, Andersson TR. Protein C: an automated activity assay. Haemostasis 17, 109-113 (1987).
- McCall F, Conkie JA, Walker ID, Davidson JF. Measurement of protein C in plasma – a fully automated assay. Thromb Res 45, 681-685 (1987).