Schematic diagram of plasmin cleavage and inactivation of human F.IX.

Human F.IX is composed of a light chain (residues 1-145), an activation peptide (residues 146-180), and a heavy chain (residues 181-415). The light chain comprises a domain containing 10-12 γ-carboxyglutamic acid (GLA) residues, 2 human epidermal growth factor (EGF)-like domains, and a linker (L) region.25 The activation peptide (AP) is cleaved and removed upon F.IX activation by F.VIIa/tissue factor or F.XIa. The heavy chain contains the catalytic domain (CD), which is made up of the active site formed by the triad of residues: Asp270, His221, and Ser365. The results of the NH₂-terminal sequencing indicate that plasmin inactivates the coagulant activity of F.IX after hydrolysis at Lys43, Arg145, Arg180, Lys316, and Arg318. The results of reducing and nonreducing SDS-PAGE, NH₂-terminal sequencing, and F.IX aPTT coagulant activity assays are collectively consistent only with plasmin hydrolysis following 2 pathways, which are indicated (A and B), as are the cleavage sites and the apparent molecular masses of the cleavage products. By this scheme, no single molecule is cleaved at both Arg145 and Arg180 and nowhere else. Thus, although plasmin can catalyze the activation cleavages individually, plasmin does not generate F.IXa.