VPB0645 - Modulation of plasmin inhibitor activities, using Affimer technology, improves the hypofibrinolytic environment in high vascular risk individuals

Background: Enhanced fibrin clot formation and/or suppression of fibrinolysis are associated with increased risk of vascular occlusion. Plasmin inhibitor (PI), a key anti-fibrinolytic protein, is cross-linked into fibrin networks with higher concentrations of PI documented in fibrin clots and plasma from high vascular risk individuals.

Aims: Use PI-binding proteins, termed Affimers, for improving the hypofibrinolytic environment in high vascular risk individuals, including patients with cardiovascular disease (CVD) and those with type 1 and type 2 diabetes (T1D and T2D).

Methods: PI-specific binders were isolated from a large library of Affimers using a phage display system. Turbidimetric assays and ROTEM were used to assess the effects of a PI-binding Affimers on clot formation and lysis.

Results: Of n=28 of PI binding Affimers, one (A11) reduced clot lysis time from 66.67±5.78 to 28.40±8.70 minutes in clots made from pooled human plasma (p < 0.05), and from 49.40±25.79 to 15.93±7.41 minutes (p < 0.05) in clots made from purified fibrinogen in the presence of PI and factor XIII. The effects of A11 was concentration-dependent with maximum effects reached at 40:1 Affimer:PI molar ratio (107 µg/ml of Affimer in plasma and 319 µg/ml in purified). Moreover, Affimer A11 had consistent effect on clot lysis time in different populations of patients, using tPA at high or low concentrations (83ng/ml or 21ng/ml, respectively). Regardless of tPA concentration, and using individual plasma samples, A11 enhanced clot lysis time by 20-40% in samples from CVD (n=15), T1DM (n=15) and T2DM patients (n=15), as well as healthy controls (n=15). In whole blood ROTEM, A11 also reduced from 25.72±8.89 to 14.75±5.19 minutes (n=6 healthy controls) (p < 0.05) without affecting maximum clot firmness.

Conclusion(s): This work provides proof of concept that PI-binding Affimers can alter protein function and may be a future tool to modulate fibrin clot lysis and reduce vascular thrombotic events in high risk populations.