The use of antiplasmin-specific Affimer as a tool to modulate fibrin clot properties and thrombosis risk

Abstract

Introduction: Vascular thrombosis represents one of the most common causes of mortality in the western world. The obstructive thrombus is composed of a mesh of fibrin fibres with blood cells trapped in these networks. Fibrin clot lysis, that limits thrombus formation and helps to prevent extensive vascular occlusion, is controlled by a number of factors including incorporation of anti-fibrinolytic proteins into the clot, most importantly plasmin inhibitor (PI). Aim: The aim of the study is to identify the role of artificial binding proteins (also termed Affimers) in modifying the effects of PI on fibrin clot lysis. Method: A large library of Affimers was screened for PI binding using a phage display system. High affinity PI-binding Affimers were screened for modulation of fibrin clot lysis. Validated turbidimetric assays were employed to assess the role of PI-binding Affimers on fibrin clot structure/lysis, using both purified and plasma systems. In order to investigate mechanistic pathways, a number of techniques were employed including confocal and electron microscopy, immunoblotting, pull-down assays, mutagenesis work, mass spectometry and molecular modelling. Result: Following three rounds of phage panning of two independent screening tests, a total of 167 high affinity PI-binding Affimers were isolated. Out of these 167 Affimers 24 had different sequences and these were subcloned and expressed E. coli. One Affimer (A68) consistently inhibited the prolongation of PI-induced clot lysis in a purified system and also reduced plasma clot lysis from 650±23 to 420±21 sec (p<0.001). The effects of the Affimer were maintained in whole blood systems and a number of different clinical conditions indicating consistency of action. Mechanistic work indicates that Loop 1 of Affimer A68 is responsible for PI inhibition with possibly multiple areas on the protein responsible for interaction with the Affimer.

Conclusion: The work so far provides proof of concept that PI-binding Affimers represent a viable tool to modulate fibrin clot lysis and may help to identify novel therapeutic targets. Future animal work is warranted to understand whether Affimer A68 reduces thrombosis in vivo, with the potential to offer a new therapeutic agent to decrease the risk of vascular occlusion.