Modulation of scavenger receptor lectin-like oxidised low-density lipoprotein receptor -1 and plasmin inhibitor activities using Affimer technology, improves the hypofibrinolytic environment in high vascular-risk individuals

Abstract

Atherothrombotic disease is a major cause of morbidity and mortality worldwide, with thrombus formation representing the final step in the pathophysiology, leading to end organ damage. Oxidised low-density lipoprotein (oxLDL) and its receptor Lectin-like oxidised low-density lipoprotein (LOX-1) play key roles in the pathogenesis of atherosclerosis and may have a role in thrombus formation but this remains controversial. In contrast, the antifibrinolytic protein plasmin inhibitor (PI) is well known to stabilise the fibrin network, which forms the backbone of the blood clot, through inhibition of the fibrinolytic process, protecting against excessive bleeding after vessel injury. However, there is a fine balance as excessive inhibition of fibrinolysis is associated with increased vascular thrombosis and adverse clinical outcomes. My hypothesis is that oxLDL affects fibrin clot formation and/or lysis and therefore represents a new therapeutic target to reduce thrombosis risk. I also hypothesise that PI activity can be modulated to reduce thrombosis risk and improve fibrinolysis in high- vascular risk conditions. Therefore, the aims of my work are to i) study the effects of oxLDL, with and without LOX-1, on clot structure/lysis to understand whether this pathway represents a new therapeutic target for reducing thrombosis risk, ii) use a new technology, employing small proteins called Affimers, to modulate PI activity and enhance the fibrinolytic process iii) probe into the mechanisms for Affimer-induced modulation of PI activity (if any).LDL was purified from plasma samples and oxidised using validated in vitro techniques. The effects of oxLDL on clot structure and lysis were studied using a turbidimetric assay in the absence and presence of sLOX-1. My results show that oxLDL, with and without sLOX-1, has no effects on clot structure/lysis using turbidimetric assays, indicating this is not a viable pathway for modulation of thrombosis risk. In the second part of my work, I show that the Affimer technology can be successfully used to modulate PI activity. One PI-specific Affimer shortened lysis of clots made from plasma samples and whole blood from healthy controls and also had similar effects across high vascular risk individuals, including patients with cardiovascular disease (CVD) and type 1 and type 2 diabetes (T1DM, T2DM). The mechanism of action of the PI-specific Affimer appears to be related to interference with plasmin generation with less of an effect on plasmin activity. In conclusion, my thesis demonstrates that oxLDL does not represent a credible target for reducing thrombosis risk. On the other hand, the affimer technology can be successfully employed to alter clot lysis by targeting the key anti-fibrinolytic protein, PI.