Affimer proteins as a tool to modulate fibrinolysis, stabilize the blood clot, and reduce bleeding complications

Katherine J. Kearney, Nikoletta Pechlivani, Rhodri King, Christian Tiede, Fladia Phoenix, Ramsah Cheah, Fraser L. Macrae, Katie J. Simmons, Iain W. Manfield, Kerrie A. Smith, Benjamin E. J. Spurgeon, Khalid M. Naseem, Robert A. S. Ariëns, Michael J. McPherson, Darren C. Tomlinson, Ramzi A. Ajjan

Research output: Contribution to journalArticlepeer-review

16 Citations (Scopus)

Abstract

Bleeding complications secondary to surgery, trauma, or coagulation disorders are important causes of morbidity and mortality. Although fibrin sealants are considered to minimize blood loss, this is not widely adopted because of its high cost and/or risk for infection. We present a novel methodology employing nonantibody fibrinogen-binding proteins, termed Affimers, to stabilize fibrin networks with the potential to control excessive bleeding. Two fibrinogen-specific Affimer proteins, F5 and G2, were identified and characterized for their effects on clot structure/fibrinolysis, using turbidimetric and permeation analyses and confocal and electron microscopy. Binding studies and molecular modeling identified interaction sites, whereas plasmin generation assays determined effects on plasminogen activation. In human plasma, F5 and G2 prolonged clot lysis time from 9.8 6 1.1 minutes in the absence of Affimers to 172.6 6 7.4 and more than 180 minutes (P < .0001), respectively, and from 7.6 6 0.2 to 28.7 6 5.8 (P < .05) and 149.3 6 9.7 (P < .0001) minutes in clots made from purified fibrinogen. Prolongation in fibrinolysis was consistent across plasma samples from healthy control patients and individuals at high bleeding risk. F5 and G2 had a differential effect on clot structure and G2 profoundly altered fibrin fiber arrangement, whereas F5 maintained physiological clot structure. Affimer F5 reduced fibrin-dependent plasmin generation and was predicted to bind fibrinogen D fragment close to tissue plasminogen activator (tPA; residues g312-324) and plasminogen (a148-160) binding sites, thus interfering with tPA–plasminogen interaction and representing 1 potential mechanism for modulation of fibrinolysis. Our Affimer proteins provide a novel methodology for stabilizing fibrin networks with potential future clinical implications to reduce bleeding risk.

Original languageEnglish
Pages (from-to)1233-1244
Number of pages12
JournalBlood
Volume133
Issue number11
DOIs
Publication statusPublished - 14 Mar 2019
Externally publishedYes

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