PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9 PREFERENTIALLY ASSOCIATES WITH A SPECIFIC LDL SUBFRACTION: A DETAILED CHARACTERIZATION AND STUDY OF THE EFFECTS OF ANTI-PCSK9 MABS TREATMENT

BACKGROUND AND AIM: Proprotein Convertase Subtilysin/Kexin Type 9 is a key regulator of LDL-C levels. Nevertheless, its physiological modulation is not fully understood. It is unclear whether PCSK9 has biological effects other than degrading the LDLR; consensus is lacking also on how PCSK9 is transported in the bloodstream, and whether this influences PCSK9 function. There are several conflicting data about PCSK9’s possible association with different lipoprotein subtypes. The biological function of the lipoprotein-bound PCSK9, also, is still a matter of debate. Due to its role in modulating plasma cholesterol levels, several approaches have been developed to modulate PCSK9 activity. Anti-PCSK9 mAbs represent the most advanced clinical available strategy for PCSK9 pharmacological inhibition, followed by the siRNA approach which seems promising. In this context, our aims were: 1) to establish a valid experimental procedure for the study of the possible association between PCSK9 and plasma lipoproteins. This would help us reach a clear idea about PCSK9’s lipoprotein compartmentation. 2) To characterize the lipoprotein fraction that binds PCSK9; 3) to address whether anti-PCSK9 mAbs therapy interferes with the PCSK9-LPs association, with resulting biological consequences. The capacity of PCSK9 to bind different lipoproteins raises the possibility that changes in the lipoprotein compartmentalization could regulate PCSK9 activity, and mAbs possible effects on such interaction may have biological consequences thus affecting their own pharmacological action. The study of such regulatory mechanisms could represent a potential avenue for developing new cholesterol-lowering drugs. METHODS: To reach our first aim, we compared several well-established methods in the lipoprotein isolation technique to clarify whether PCSK9 associates differently with circulating lipoproteins. The lipoprotein fractions collected were quantified for their cholesterol, triglycerides, PCSK9, apoB, apo AI and Lp(a) content using clinical grade reactives or ELISA. They were further characterized through immunoblotting, agarose gel electrophoresis, transmission electron microscopy (TEM), lipidomic and mass spectrometry. Following this, we analyzed PCSK9’s lipoprotein distribution among OptiPrep UC-isolated lipoproteins, obtained from anti-PCSK9 mAbs treated subjects before therapy (T0), and 1 (T1), 3 (T3) and 6 (T6) months after the beginning of treatment. The collected lipoprotein fractions were further characterized as described above. RESULTS AND CONCLUSION: Based on our results, the PCSK9-LDL association exists and is sensitive to high salt concentrations. OptiPrep UC and FPLC are both suitable methods for the study of this association. We believe that PCSK9 circulates mostly as a free type of protein, and partially (approx. 10-20% of total recovered PCSK9) associated through its active form with a light LDL subfraction resembling an IDL. We believe that this association originates in the circulation, as we did not find PCSK9 in the VLDL-containing fractions. PCSK9 did not associate with Lp(a) or HDL. Anti-PCSK9 mAbs administration induced a large decrease in LDL-C levels, - from 140,3±64,8 mg/dL to 51,5±35,6 mg/dL before and after treatment, respectively - parallel with a drastic increase in PCSK9 plasma levels - from 409,4±123,3 ng/mL to 3802,3±1001,0 ng/mL (n=22); despite this, they did not affect the PCSK9-LDL association but instead induce a more than 10-fold increase in the absolute quantity of LDL-bound PCSK9. Interestingly, the amount of PCSK9 recovered in the LDL fractions after therapy was about 242,3±164,8 ng/mL, like that of plasma of untreated subjects; this suggested that the LDL-bound PCSK9 may be inactive. Preliminary data obtained from Inclisiran-treated subjects suggest that the PCSK9- LDL association is maintained also when reducing both LDL and PCSK9 levels, therefore reinforcing the idea that the PCSK9-LDL association might be biologically meaningful.