Identification of fibrinogen fragment D as site of F5 interaction. (A) Affimer F5 was incubated with fibrinogen before plasmin digest, and pulldown of F5-fibrinogen degradation products with His-tag isolating beads. Proteins marked with an asterisk (*) were identified by mass spectrometry (LC-MS/MS). The position of Affimer F5 on the gel is marked. Gel image is representative of 3 independent experiments. (B-C) Autodock 4.2 prediction of Affimer F5 binding to the γ chain of the D fragment of fibrinogen. Fibrinogen fragment D is shown as a space-filling model, with the α chain in green, β chain in turquoise, and γ chain in yellow. The tPA/plasminogen binding site α148-160 and the tPA binding site γ312-324 are highlighted in red. (B) In the lowest-energy pose, the variable region loops of F5 (F5 depicted with magenta ribbons) are interacting with the fibrinogen γ chain near to the γ312-324 tPA binding site. (C) The most populated clusters of predicted docking poses also placed Affimer F5 (orange ribbons) to a similar area of fibrinogen fragment D. (D) Amino acid sequence of fibrinogen γ chain, with those amino acids within 4 Å of Affimer F5′s variable region loops marked. Fibrinogen residues close to Affimer loops in the lowest-energy pose are highlighted yellow, and those in the most populated pose are underlined blue.