Measurement of protein C

Protein C, also known as autoprothrombin IIA and blood coagulation factor XIV,[1]:6822[3] is a zymogenic (inactive) protein, the activated form of which plays an important role in regulating blood clotting, inflammation, cell death, and maintaining the permeability of blood vessel walls in humans and other animals. Activated protein C (APC) performs these operations primarily by proteolytically inactivating proteins Factor Va and Factor VIIIa. APC is classified as a serine protease as it contains a residue of serine in its active site.[4]:35 In humans, protein C is encoded by the PROC gene, which is found on chromosome 2.

Principles
Protein C may be measured by:
a) Immunological by means of an ELISA assay Remember this measures only the amount of Protein C present and NOT its functional activity.

b) A clot-based functional APTT assay - the time to clot formation after addition of a Protein C activator is determined and from this the amount of Protein C present can be determined.

c) A Chromogenic assay - Protein C is activated using (commonly) Protac™, an extract of the venom of Akistrodon contortrix contortrix and the concentration of Protein C is determined from the rate of colour change in the test sample due to cleavage of a chromogenic substrate.

d) A thrombin-generation-based test has also been shown to detect Protein C deficiency.

Method

Commercial kits are readily available for Protein Assays and should use a reference standard calibrated against the current International Standard for Protein C.

1. ELISA Assays: Most ELISA assays use either monoclonal or polyclonal antibodies against Protein C. 

2. Functional, Clotting-based Protein C Assays: These are based on either the PT or the APTT, although the APTT is more commonly used. Patient platelet poor plasma is incubated at 37°C with phospholipid, a contact activator (e.g. Kaolin) and a Protein C activator (e.g. Protac). After incubation (typically 1-4 minutes) calcium is added to initiate clotting. The time taken to form a clot is timed. From this the Protein C level is determined from a reference curve. 

The clotting time of the APTT [or PT] will be influenced by the amount of Va or VIIIa present in the reaction mixture and in turn this will be influenced by the activity of the Activated Protein C [APC]. APC is generated from the conversion of Protein C to activated Protein C by Protac. So if there is a reduction in circulating Protein C levels, then less APC will be generated, less Va and VIIIa will be inactivated and the clotting times will be shorter.

http://www.practical-haemostasis.com/Thrombophilia%20Tests/pc_assays.html