Thrombin cleaves the C-terminal region of CgA. (A) SDS-PAGE of recombinant CgA (6μM) before and after incubation with thrombin-sepharose (Thr-Seph) or sepharose alone (Seph). Thrombin (60 U, Sigma-Aldrich) was coupled to 200 μL of activated-CH-sepharose (GE Amersham), according to the manu-facturer's instructions. CgA (6μM in PBS) was mixed with the thrombin-CH-sepharose (1:10 suspension) and left to digest for 10 hours at 30°C under gentle agitation. The supernatant was then recovered and stored at −20°C until analysis. (B) Gel filtration chromatography of thrombin-digested CgA. Thrombin-digested CgA was loaded onto a Superdex 75 column and eluted with PBS. Fractions corresponding to the main peaks were collected and pooled (pools 1, 2, and 3). (C) 436/439 and 436/439+FRs-ELISAs of recombinant CgA, thrombin-digested CgA, pools 1, 2, and 3. (D) Molecular weight (Dalton) of fragments present in thrombin-digested CgA as measured by ESI-MS (Obs). The corresponding fragments and their expected molecular weight (Exp) are also shown. (E) Primary sequence of human CgA. Dibasic sites are indicated in bold. Arrows indicate the cleavage sites of thrombin as detected by ESI-MS.