Atherogenic apoC3 may not be as bad as we thought

Hyperlipidemia and thrombosis both play major roles in driving cardiovascular disease, the leading cause of mortality and disability worldwide [1]. One pathophysiological feature associated with hyperlipidemia is platelet hyperreactivity, which contributes to adverse thrombotic events. Platelet hyperreactivity is increasingly recognized as being associated with high levels of atherogenic lipoprotein-associated cholesterol [2, 3]. However, the precise mechanism behind this association remains unknown.

Apolipoprotein C-III (apoC3) is a 79-amino acid, exchangeable apolipoprotein primarily synthesized in hepatocytes and enterocytes [4, 5] and transported in plasma on lipoproteins. When newly secreted triglyceride-rich lipoproteins (TRLs) enter circulation, they acquire apoC3 as part of their surface apolipoprotein complement. During intravascular lipolysis, lipoprotein lipase hydrolyzes core triglycerides, resulting in progressive particle shrinkage and surface remodeling. As TRLs decrease in size, excess surface exchangeable apolipoproteins, including apoC3, are transferred to high-density lipoproteins (HDLs), which function as surface acceptors [6]. Conversely, newly secreted TRLs initially contain a limited complement of exchangeable apolipoproteins and subsequently acquire them, including apoC3, from circulating HDL [6]. The C-terminal region (residues 41–78) of apoC3 contains a class-A amphipathic helix enriched in hydrophobic and aromatic residues, which constitute a major portion of the nonpolar surface and mediate insertion into lipid interfaces [7, 8].

Genetic studies established apoC3 as a contributor to plasma triglyceride levels, and loss-of-function variants in the APOC3 gene are associated with lower plasma triglyceride levels and reduced risk of atherosclerotic cardiovascular disease [9–11]. Although low-density lipoprotein (LDL) remains the primary therapeutic target for reducing risk of cardiovascular disease, accumulating evidence indicates that triglycerides and TRLs also contribute independently to cardiovascular risk. Large-size epidemiological studies have demonstrated that elevated apoC3 levels correlate with increased cardiovascular risk, independent of low-density lipoprotein (LDL) cholesterol [12–14]. Functionally, apoC3 promotes hypertriglyceridemia by inhibiting lipoprotein lipase (LPL)-mediated hydrolysis of TRLs [7, 15, 16]. ApoC3 also interferes with the interactions between TRLs with heparan sulfate proteoglycans and members of the LDL receptor family, thereby reducing the hepatic uptake of TRLs [17, 18]. Approximate 60% of apoC3 resides on HDL in plasma from healthy individuals [19]. Importantly, apoC3 affects HDL functions. HDL particles containing apoC3 display reduced anti-inflammatory and endothelial-protective properties compared with HDL lacking apoC3 [20, 21]. HDL subspecies enriched in apoC3 are associated with increased cardiovascular risk, whereas HDL particles without apoC3 maintain inverse associations with coronary heart disease [22, 23].

Given the role of apoC3 at the intersection of triglyceride metabolism and cardiovascular risk, it has become a therapeutic target to treat patients with severe hypertriglyceridemia and familial chylomicronemia syndromes (FCS). In patients with FCS, GalNac-conjugated antisense oligonucleotides (ASO) therapy targeting APOC3 mRNA significantly lowered apoC3 and triglycerides and was associated with fewer acute pancreatitis events compared with placebo, supporting a clinically meaningful benefit in the high-risk population [24]. In adults with moderate hypertriglyceridemia, ASO targeting apoC3 also showed a placebo-adjusted reduction in fasting triglycerides of approximately 58–61%, demonstrating triglyceride-lowering in a broader population [25]. Collectively, these trials support apoC3 as a validated therapeutic target for reducing TRLs, with current efforts on confirming pancreatitis and cardiovascular outcomes while maintaining long-term safety. However, in clinical trials of first-generation ASO targeting apoC3, thrombocytopenia was recognized as an adverse effect, with platelet recovery after withdrawing from the therapy [26]. The second-generation GalNAc-conjugated ASO, designed to enhance hepatocyte specificity, demonstrated an improved safety profile; nevertheless, a modest reduction in platelet counts was still reported [27]. It remains unclear why the ASO targeting apoC3 affects platelet count, but this study may provide some insight into apoC3 effects on platelet function.

Previous work in the field has demonstrated that apoC3 acts as an inhibitor of platelet activation through the α_IIbβ₃ receptor [29]. In this issue of the Journal, Schrottmaier et al. [28] further investigated the impact of apoC3 on platelet function and thrombosis. To evaluate the effects on platelet secretion, the authors measured surface exposure of CD62P and CD40L as α-granule markers and CD63 as a dense and lysosomal granule marker. All these three markers showed reduced surface exposure, indicating that apoC3 inhibits platelet activation and granule release. To further characterize apoC3-platelet interactions, the RGDS (Arg-Gly-Asp-Ser) peptide, which competes with the RGD_-binding domain in α_IIbβ₃ and α₅β₁ integrins, was incubated with platelets and restored platelet function that was inhibited by apoC3, suggesting α_IIbβ₃ or α₅β₁ may mediate apoC3-platelet interactions. In addition, the authors investigated the impact of apoC3 on intracellular signaling pathways. VASP (Vasodilator-stimulated phosphoprotein) phosphorylation was mildly increased, confirming that apoC3 interacts with and inhibits the α_IIbβ₃ receptor. To evaluate the in vivo function of apoC3, a FeCl₃ thrombosis mouse model was used, where apoC3-primed platelets were infused into mice, and thrombus formation in the mesenteric arterioles was significantly delayed, and thrombi were less stable. Ultimately, this study uncovers that apoC3 outside of its pro-atherogenic function is multifaceted and can act as a direct inhibitor of platelet activation and thrombus formation.

The central findings of this study should be considered as a starting point for uncovering the effects of apoc3 on platelet function. Some limitations should be considered when interpreting these results. A key point to address first is the use of apoC3 that was purified from delipidated VLDL. Lipid-free apoC3 represents a small portion of apoC3 present in circulation; therefore, lipid-bound apoC3 functional impacts on platelet function may differ from what the authors have reported [30, 31]. In addition, it should be noted that previous studies suggest that apoC3 is associated with not only VLDL, but chylomicron, LDL, and HDL as well [32, 33].

ApoC3 and its different proteoforms have various functions when present on different lipoproteins, and this could be further influenced by oxidative stress [32–34]. The authors [28] touched on this aspect by examining the influence of lipid-free apoC3 with either oxLDL or VLDL on platelet aggregation, where they observed a reduction in platelet aggregation. However, other studies have shown that platelets are hyperreactive when treated with oxLDL or in hyperlipidemic conditions [35–37]. Whether different proteoforms of apoC3 contributed to these discrepancies requires further investigation. The authors [28] performed preliminary studies aimed at determining which platelet receptors interact with apoC3, and their data suggested interactions with the integrin receptors, α_IIbβ₃ or α5β₁, and potentially the scavenger receptors. Future studies using monoclonal antibodies or mouse knockout models can provide deeper insight into which platelet receptors lipid-free apoC3 interact with. In addition, it would be invaluable to use similar methods to define lipid-bound apoC3 interactions with the α_IIbβ₃ receptor and functional consequences on platelets. Lipid-free apoC3 was shown to reduce platelet secretion by reducing CD62P levels; however, platelet-monocyte aggregation was unaffected, suggesting that lipid-free apoC3 affects monocyte function as well and would be of great interest to study in the future, because monocytes are key drivers of atherosclerosis [38]. The impact of lipid-free apoC3 in the FeCl₃ thrombosis model was an elegant experiment that, if expanded, could offer more insight into the mechanistic role of lipid-free apoC3. Addressing these points in future work could yield insights that are highly valuable to the broader research community.

Although apoC3 has been primarily studied in the context of lipid metabolism and atherogenesis, emerging work suggests potential roles in platelet function and thrombosis. ApoC3 is well established as a regulator of triglyceride metabolism, and elevated levels are associated with an increased risk of atherosclerotic cardiovascular disease [39]. ApoC3 deficiency, on the other hand, is cardioprotective. Mouse and rabbit models of apoC3 deficiency exhibited lower triglyceride levels and faster clearance of triglyceride-rich lipoproteins, and were protected from the development of atherosclerotic plaque lesions [16, 40–42]. Studies investigating the effects of apoC3-lowering drugs in models of hypercholesterolemia have shown that it is only an effective treatment when lipid lowering is achieved, and it results in an attenuation in atherosclerotic plaque size and improved plaque stability [43, 44]. These studies show the impact of apoC3 on atherogenesis but have not investigated its functional effects on specific cell types, such as platelets, which play a critical role in the development of atherosclerotic plaques.

Only limited studies have examined apoC3 function in platelets, highlighting the importance of the current investigation into apoC3’s functional impact on platelet function and thrombosis. A previous study suggests that apoC3 is an inhibitor of platelet function through the α_IIbβ₃ receptor using isolated apoC3 from human serum, likely in form of lipid-bound apoC3 [29]. When platelets were incubated with apoC3-depleted plasma, platelet aggregation was increased. The combined results from both studies showed that increasing levels of apoC3 inhibit platelet aggregation regardless of whether it’s lipid-bound or not. More studies are needed to define the potential impact of apoC3-lowering drugs on platelet function, and risks of thrombocytopenia and thrombosis.

In conclusion, Schrottmaier et al. [28] illuminate an unexpected role for lipid-free apoC3 in platelet function. Their study suggests that outside of its atherogenic function, apoC3 can act as a direct inhibitor of platelet activation and thrombus formation, as shown in the Figure. These results set the stage for future studies to clarify the mechanistic pathways involved in lipid-free apoC3-platelet interactions. Overall, these findings provide a valuable foundation for ongoing work on how apolipoproteins influence platelet functions.

Figure:

Apoc3 exerts dual effects by inhibiting platelet activation and clot formation while elevating the risk of thrombotic cardiovascular disease.