POTENTIAL GENE VARIANTS INFLUENCING FVIII LEVELS: THE ROLE OF LDL RECEPTOR ON FVIII CLEARANCE.

Background Genetic variants in the LDL receptor (LDLR) gene have been associated with higher LDL cholesterol levels, premature atherosclerotic cardiovascular disease (ASCVD) and increased cardiovascular risk. However, the increased arterial thrombotic risk observed in LDLR carriers is partly independent from the lipoprotein levels and could be related to the LDLR modulation of FVIII levels. Indeed, a role of LDLR in FVIII clearance has been shown and LDLR variants have been associated with higher FVIII levels. To date, it is not clear which are the LDLR variants involved in FVIII clearance and the underlying mechanism. With this background and gap of knowledge, the present study aims to: - identify the LDLR variants associated with high FVIII coagulant activity (FVIII:C≥150 IU/dL) in a cohort of DVT (Deep Veen Thrombosis) patients and healthy control subjects; - functionally and biochemically characterize the LDLR variants (identified in this study and described in literature) associated with high FVIII levels in order to understand their ability to modulate the FVIII clearance, in terms of FVIII binding and/or uptake. Methods We selected 596 Italian subjects (298 DVT patients and 298 controls) enrolled and sequenced in the frame of the DVT-Milan study. FVIII:C was measured by one-stage clotting assay. A multiplexed next generation sequencing was performed. In order to select the variants to be characterized in vitro, we analyzed the association between LDLR variants and high FVIII:C (FVIII:C≥150 IU/dL). For common and low-frequency LDLR variants a logistic regression model was performed. Rare LDLR variants have been analyzed using cumulative association tests. Wild type (WT) and selected mutant LDLR vectors were transiently transfected in LDLR-deficient Chinese hamster ovary (CHOldlA7) cells with chemical methods. The LDLR expression in total cell lysates was assessed by western blotting (WB). Immunofluorescence (IF) was performed to evaluate total, surface and intracellular expression of LDLR proteins. Results In our cohort of DVT patients and healthy control subjects we identified the missense LDLR variant rs45508991 associated with high FVIII levels (OR=5.47, p=0.06). We selected 4 LDLR variants to be characterized in vitro: the variant rs45508991 and 3 synonymous variants (rs688, rs2228671 and rs5925), previously reported to be associated with higher FVIII levels or increased risk of ischemic stroke and/or coronary artery disease. Among the 4 LDLR variants expressed in vitro, we observed, by both WB and IF, a significant reduced expression of total LDLR protein carrying the rs45508991 variant, compared to the WT LDLR. Moreover, by IF, we observed that the surface LDLR expression was significantly reduced in cells expressing the variant rs45508991. Conclusions and perspectives Our hypothesis is that a reduced expression of LDLR, total and on cell surface, caused by the identified variant rs45508991, could impair FVIII binding and clearance, leading to an increase in FVIII levels. In order to test this hypothesis, we will perform the fluorescence-activated cell sorting (FACS) with the goal to confirm IF findings concerning the cell surface LDLR expression in all samples and to evaluate the LDLR-mediated binding of FVIII.