The Impact of Convertase Subtilisin/Kexin Type 9 Monoclonal Antibodies with and without Apheresis on Platelet Aggregation in Familial Hypercholesterolemia

There is no doubt that enhanced low-density lipoprotein cholesterol (LDL-C) level is one of the main risk factors for atherosclerosis leading to coronary artery disease. Indeed, patients suffering from familial hypercholesterolemia (FH) die from cardiovascular disease prematurely. FH is an inherited disease causing elevated levels of LDL-C. A causal relationship between elevated LDL-C and hyperaggregability is known as well and it can be assumed that increased platelet function depends on the higher amount of circulating lipoproteins with atherogenic apolipoprotein B 100 (ApoB100) on their surface [1]. FH is mainly caused by one of four basic mutations. A mutation in the gene for the LDL receptor (LDL-R) is detected most often (90% of cases). This mutation prevents LDL-C from binding to their receptor because there are few or no LDL-R. Other common mutations include ApoB100, proprotein convertase subtilisin/kexin type 9 (PCSK9) or LDL-R adaptor protein (LDL-RAP1). ApoB100 is needed for interaction of LDL-C with the LDL receptor. PCSK9ab is responsible for LDL-R degradation and its overexpression is hence associated with more extensive degradation of LDL-R [2]. LDL-RAP1 collaborates with LDL-R and enables endocytosis of LDL-C after binding to that receptor and hence mutation of the LDL-RAP1 gene (autosomal recessive disease) leads to FH. Regardless, in all cases, LDL-C levels are markedly elevated in comparison with healthy population [3‐5].

Current treatment possibilities are based on these pathophysiological issues. Statins are used as in other hypercholesterolemic patients, but their impact on the level of LDL-C is low in this case. For a long time, repeated lipid apheresis was the only reasonable choice for decreasing LDL-C levels in FH. Antisense nucleotide mipomersen against mRNA for ApoB100, approved in 2013, was one of the earliest targeted drugs for FH but its effect is burdened by potential hepatotoxicity. Finally, the development of PCSK9 monoclonal antibodies (PCSK9ab) brought a marked improvement in FH treatment. Most recently, a small interfering RNA against PCSK9 inclisiran was registered by both FDA and EMA [6]. As the drugs against PCSK9ab are relatively new, there is very limited information on their impact on platelet aggregation in contrast to many publications reporting increased platelet aggregation in FH and the positive effect of LDL-apheresis [7‐9]. The last options for lipid-lowering therapy are (1) lomitapide as adjuvant therapy, for those patients who have not achieved LDL-C goals with apheresis and any other lipid modifying agent combination [10], or (2) the newest drug evinacumab approved in 2021 [11]. They might be less suitable due to possible occurrence of specific side effects, which encompass liver toxicity in the former and a decrease in HDL cholesterol in the latter case [12, 13]. The effect of inclisiran, lomitapide and evinacumab on LDL-C in homozygous FH is comparable and achieves a 50% reduction [10, 14].

Platelet aggregation is physiologically an essential part of haemostasis which is strictly regulated by a number of biological factors. It is known that pathological conditions such as atherosclerosis or inflammation lead to hyperactive platelets which are linked to an elevated risk of cardiovascular disease [15]. High levels of oxidized LDL-C trigger platelet aggregation after binding to their specific receptors on thrombocyte surface, mainly scavenger receptor CD36. This cascade is associated with activation of cytosolic phospholipase A₂ which releases arachidonic acid (AA). AA is then enzymatically transformed to thromboxane A₂ by cyclooxygenase 1 and thromboxane synthase. The process is associated with enhanced cytosolic Ca²⁺ content and inhibition of nitric oxide release. Nitric oxide has important antiplatelet effects and hence it is not surprising that platelet aggregation is enhanced in FH [1, 7, 15‐18].

In this study, we aimed (1) to examine the effect of treatment by monoclonal antibodies against PCSK9 on different aggregatory cascades using 7 platelet aggregation triggers as well as on the antiplatelet potency of three clinically used drugs in FH patients, and (2) to compare platelet responses to these triggers and antiplatelet drugs between FH patients and age-matched generally healthy controls.

In this study, we focused on the analysis of platelet function in patients suffering from familial hypercholesterolemia treated with lipid apheresis, but also with a relatively novel therapeutic option based on PCSK9ab. In addition, the impact of these treatments on platelet aggregation was compared with healthy volunteers. The main findings identified in this study were (i) PCSK9ab significantly decreased platelet aggregation and the effect was not inferior to apheresis, (ii) apheresis significantly decreased platelet response to collagen and ristocetin triggered aggregation, whereas PCSK9ab without apheresis achieved the same effect as apheresis with the exception of ristocetin, (iii) our FH treated patients had lower platelet reactivity to most inducers compared to age-matched healthy controls and (iv) low blood lipid level reduces the sensitivity to platelet inducers and thus increases the effectiveness of antiplatelet drugs, which was observed for ASA and vorapaxar.

Atherosclerosis and cardiovascular disease are very tightly related as long-term exposure to high lipid levels causes platelet hyperaggregability. LDL-C and platelet hyperfunction are interrelated because LDL-C is involved in activation and signal transduction in platelets, and therefore lowering blood lipids should reduce platelet activity. The treatment of FH is not simple in particular in relation to patients. There are several options often requiring pharmacological combinations to achieve the goal set by the European Society of Cardiology (ESC) [6, 26]. Statins are the most commonly prescribed drugs in lipid management, but only a small proportion of patients reach the target LDL-C level with maximum tolerated doses [6, 27]. Their effect in FH is rather based on protection against cardiovascular atherosclerotic events [28]. The combination of statins and ezetimibe is more advantageous and results in an about 15% reduction of LDL-C in FH [26, 29]. Moreover, lowering of LDL-C is more challenging in homozygous (HoFH) or severe heterozygous (HeFH) patients, and even the mentioned combination of statins with ezetimibe has often insufficient effect, making the use of a tri-combination necessary [27]. In HeFH patients, the ESC recommended the combination of statin at maximum tolerated doses and the addition of ezetimibe and a PCSK9ab. In HoFH patients, the treatment is enriched with LDL-apheresis [26]. In the present study, all HoFH and most HeFH patients received a tri-combination (statin + ezetimibe + PCSK9ab) and all HoFH patients also received LDL-apheresis (Supplementary information Table S5). In practice, LDL-apheresis is used in patients with HoFH and severe HeFH who respond poorly to pharmacological intervention [6]. Changes in blood LDL-C after lipid apheresis are around 70% [6‐9] which is consistent with our data (Supplementary information Table S1). Our group has recently reported that lipid apheresis was found to reduce not only serum lipids, but biomarkers of inflammation as well [30]. The effects of LDL-apheresis on platelet function vary from study to study. Pares et al. demonstrated that the lowering of LDL-C influenced platelet aggregability triggered by ADP in a short term but the study was performed with just one patient [9]. In contrast to that finding, some other studies did not show the decreased aggregability immediately after apheresis [8, 31]. It is suggested that long-term LDL-apheresis has a more pronounced effect on platelet activity [7], because aggregability is influenced by LDL-C exposure over time. Lowering of LDL-C leads to normalization of platelet function [9]. These findings are consistent with our data. In this study, our patients have been treated for 16.5 years on average. Unfortunately, LDL-apheresis has some limitations such as re-elevation of LDL-C and certain discomfort because it is an invasive method [6]. Furthermore, the availability of this therapy is low in the Czech Republic, because lipid apheresis is carried out in just a few centres [32].

LDL-R is regulated by the serine protease PCSK9 and this is essential for the regulation of blood LDL-C. Excess of PCSK9 in blood leads to LDL-R degradation and elevates blood LDL-C. Moreover, PCSK9 binds to CD36 receptor on platelet surface and activates downstream signal transduction leading to enhanced aggregability [16]. Considering all these aspects, a positive impact of PCSK9ab was expected. Patients included in this study were treated with either alirocumab or evolocumab, both FDA/EMA approved PCSK9ab. These molecules bind specifically to PCSK9 and abolish its interaction with LDL-R [6]. PCSK9 inhibitors reduce LDL-C by 55–60% [33, 34] and, in combination with other hypolipidemics, this could reach a reduction of up to 85% [26]. Torres et al. compared the effect of LDL-apheresis and PCSK9ab, and their combination on LDL-C. The combination of these therapies decreased LDL-C numerically but did not bring a statistically significant improvement [35]. Regardless, PCSK9ab therapy is relatively new, brings a clear profit in terms of cholesterol reduction, but there are almost no data on its effect on platelets in metabolic diseases. In a study by Qi et al. [16], the benefit of evolucumab on aggregation was demonstrated. It was shown that aggregation is correlated with blood PSCK9 and high levels of PCSK9 increase hyperaggregability and evolucumab conversely reduced the potentiating effects of PCSK9 on platelets. However, enhanced expression of the gene for PCSK9 is not ordinary in FH [2]. Importantly, our results showed for the first time that therapy with antibodies against to PCSK9 decreased LDL-C as well as platelet aggregability. In addition, it should be mentioned that the heterozygous form of FH is not rare, its prevalence is estimated to be 1:200, so 40 million people worldwide are potentially at the risk of this disease and consequences. More patients could profit from this therapy because screening, diagnosis and awareness of FH in real population are insufficient [3].

In our study, pharmacotherapy with PCSK9 was not inferior in comparison to treatment with lipid apheresis in relation to platelet reactivity. In the case of TRAP, the pharmacotherapy group had even significantly lower platelet aggregability. This result should be however interpreted with care, as this was likely driven by the fact, that none of the FH patients in the pharmacotherapy group was of HoFH type. On the other hand, it seems that for most HeFH, which is a much more frequent form of FH, pharmacotherapy sufficiently decreases both LDL-levels and platelet hyperactivity. There was only one exception as patients in pharmacotherapy group reacted slightly more to ristocetin-induced aggregation in contrast to the post-apheresis group. The possible explanation can be that apheresis can decrease levels of von Willebrand factor, a pathway stimulated by ristocetin [36].

Current guidelines recommend lowering LDL-C below 2.6 mmol/L for moderate risk or below 1.8 mmol/L for high cardiovascular risk [6, 37]. Three patients out of a total six achieved LDL-C below 1.8 mmol/L in group before apheresis and in the pharmacotherapy group. However, immediately after apheresis, all six patients achieved LDL-C below 1.8 mmol/L. Quite interestingly, the closest relationship between aggregation induced by AA, collagen and ristocetin and metabolic parameters was observed for TG (Supplementary information Figures S5 and S6). Only three patients reached the recommend level of TG (< 1.7 mmol/L [37]) before apheresis with mean 1.4 ± 0.8 mmol/L. The pharmacotherapy group had similar TG with mean 1.5 ± 0.5 mmol/L and likewise three patients had TG below 1.7 mmol/L. TG-rich lipoproteins are also associated with hyperaggregability, although this is a rather less well-known fact compared to LDL-C [38‐40]. Regardless, lowering both LDL-cholesterol and TG can linearly decrease platelet reactivity as we observed in our study.

To our best knowledge, this is the first paper comparing the impact of different therapies in FH patients to their impact on platelet aggregation with many different platelet inducers. Additionally, the effect of these therapies was compared to age-matched generally healthy donors. This study demonstrated the benefit of PCSK9ab treatment in normalizing platelet function.