Concentration of prothrombin fragment 1+2 (PF1+2) is ascertained with a sandwich ELISA. PF1+2 in test plasma is captured by antibodies bound to microtitre plate wells, incubated, and unbound material then washed off. A solution of antibody to PF1+2 that is conjugated to an enzyme is added to ‘tag’ onto any captured PF1+2. Unbound conjugate is washed off and a substrate for the enzyme is added, the product of the enzyme-substrate reaction being coloured. Colour intensity is in direct proportion to the degree of conjugate-binding, itself proportional to the amount of PF1+2 capture and thus, concentration.
Assembly of the prothrombinase complex on the activated platelet surface occurs when FXa binds to surface phospholipid via Gla domain-calcium interactions, in association with its co-factor FVa. The substrate, prothrombin, is converted to the serine protease thrombin via cleavage in two locations, Arg271 and Arg320. The first cleavage at Arg271 produces fragment 1+2 and the intermediate prethrombin 2, the fragment 1+2 being released as an activation peptide. Prethrombin 2 is cleaved at Arg320 to yield active thrombin.
Detection of elevated levels of prothrombin fragment 1+2 is indicative of increased thrombin generation and a hypercoagulable state."