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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. Ariens, Michael J. McPherson, Darren C. Tomlinson and Ramzi A. Ajjan

Key points

  • We describe a fibrinogen-specific conformational protein (Affimer) that delays fibrinolysis by reducing fibrin-dependent plasmin generation.

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 due to high cost and/or risk of infection. We present a novel methodology employing non-antibody 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, confocal and electron microscopy. Binding studies and molecular modelling 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±1.1 in the absence of Affimers, to 172.6±7.4 and >180 min (P< .0001) respectively, and from 7.6±0.2 to 28.7±5.8 (P< .05) and 149.3±9.7 (P< .0001) min in clots made from purified fibrinogen. Prolongation in fibrinolysis was consistent across plasma samples from healthy controls and individuals at high bleeding risk. F5 and G2 had a differential effect on clot structure, G2 profoundly altered fibrin fiber arrangement while 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 γ312-324) and plasminogen (α148-160) binding sites, thus interfering with tPA-plasminogen interaction and representing one 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.

  • Submitted June 5, 2018.
  • Accepted December 2, 2018.