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1. Introduction

Proprotein convertase subtilisin kexin type 9 (PCSK9) influences lipid metabolism by accelerating the degradation of low-density lipoprotein (LDL) receptor (LDLR) (1). The clearance of human LDL-cholesterol (LDL-C) depends on the circulation of LDLR in the liver (2). PCSK9, synthesised in hepatocytes, is subsequently secreted and binds to specific LDLRs on hepatocytes, thus forming a trimolecular complex with LDLR and LDL. The PCSK9-LDLR-LDL complexes prevent the recycling of LDLR to the surface of hepatocytes, resulting in elevated plasma levels of LDL-C (3,4). LDL-C is a crucial factor in atherosclerosis and cardiovascular events. In the subendothelial space, LDL-C undergoes oxidation and aggregation, and its small and dense metabolites enter the arterial wall, leading to the progression of atherogenesis and major adverse cardiovascular events (MACEs) (5).

In the early stages, PCSK9 research predominantly centred on abnormal lipid metabolism. In 2000, a locus for autosomal dominant hypercholesterolaemia was found within the PCSK9 gene (human chromosome 1p33-p34.3) (6), later established as the causative gene for familial hypercholesterolaemia in 2003 (7). Subsequently, naturally occurring PCSK9 nonsense mutations were detected and associated with significantly low LDL-C levels and the risk of developing cardiovascular disease shortly thereafter. Currently, over a hundred mutations influencing PCSK9 function have been identified (8). Since the launch of the first PCSK9 inhibitor in 2015, unexpected effects have been progressively unveiled. In 2020, patients undergoing treatment with PCSK9 inhibitor were found to exhibit a reduced platelet reactivity and increased sensitivity to aspirin suppression (9). Later that year, it was first confirmed that short-term treatment with evolocumab significantly ameliorated coronary endothelial dysfunction (10). Mechanistic studies on the impact of PCSK9 on thrombosis and inflammation have also garnered attention during this period (please see section 4 below). Beyond atherosclerotic heart disease, the connection between PCSK9 and heart failure, atrial fibrillation and calcific aortic valve disease has come to light (11). The association between PCSK9 and cardiovascular events appears to transcend lipid levels, yet critical pieces of the puzzle are still missing to explain these findings.

Thrombosis and haemostasis are the leading causes of premature deaths. Apart from increasing plasma levels of LDL-C, PCSK9 regulates the hypercoagulable state and thrombosis processes in disease states. Subsequent studies have underscored this point, indicating that PCSK9 levels in diabetic patients following percutaneous coronary intervention (PCI) were independently associated with MACEs, including thromboembolism risk, but no such association was found in non-diabetic patients (12). The mechanism behind this phenomenon has yet to be elucidated, thereby hindering the tailoring of personalized therapies for patients with concurrent metabolic disorders to achieve further mitigation of residual cardiovascular risk. PCSK9 inhibitors are expected to reach US$ 15,140.3 million by the year 2034, growing at an annual rate of 18.7% (13). The population of patients receiving PCSK9 inhibitors with multiple diagnoses is rapidly increasing. Hence, comprehending the role of PCSK9 in disease states is paramount for enhancing the cost-effectiveness and quality of life for hyperlipidaemia patients globally.

The present review focuses on the effects of changes in PCSK9 levels on thrombosis and haemostasis, and the related modulatory mechanisms of platelet function, coagulation factor activity, inflammatory response and endothelial function in pathological conditions. The aim of the present review was not to comprehensively cover all information regarding the effects of PCSK9 on thrombosis and haemostasis, but rather to focus on disease states, as it was considered that these warrant further extensive research and may have a potential influence on the drug market, regarding the expansion of indications, dose adjustment and drug interactions.

2. PCSK9 structure

PCSK9 is a serine protease consisting of 692 amino acids (14). The structure of PCSK9 reveals three distinct structural domains: The prodomain (aa 32-152), the catalytic domain (aa 153-421) and the C-terminal Cys/His-rich domain (CHRD; aa 453-692) (Fig. 1).

Figure 1 Structure of PCSK9 and the mechanisms of LDLR downregulation. PCSK9, proprotein convertase subtilisin kexin type 9; LDLR, low-density lipoprotein receptor; SREBP, sterol regulatory element-binding protein; HNF1, hepatocyte nuclear factor 1; LDL-C, low-density lipoprotein-cholesterol; Pro, prodomain; Cata., catalytic domain; CHRD, C-terminal Cys/His-rich domain; EGF-A, epidermal growth factor-A.

The prodomain is not only essential for the correct folding PCSK9 in the endoplasmic reticulum, but is also related to the lack of activity against exogenous substrates (15). The C-terminus Gln152 of the prodomain binds to His226, filling the oxyanion hole between Asn317 and Ser386 and shielding the active site cleft, thus inhibiting interaction with other proteins and peptides (16). Consequently, the activity of PCSK9 is manifested through its binding to target substrates and escorting the complexes into intracellular degradation compartments, rather than as a protein hydrolase. The prodomain remains in the catalytic groove after self-cleavage, hindering the binding of PCSK9 to other proteins. Removal of the prodomain increases the affinity of PCSK9 for LDLR by 10-fold (17). The PCSK9 Arg93Cys variant is associated with a reduced risk of premature myocardial infarction (18).

The catalytic domain of PCSK9 (aa 153-421) contains essential active sites of PCSK9 and comes into contact with the epidermal growth factor-A (EGF-A) domain of the LDLR, resulting in a ~1,000 Å2 interface (19). As the complex PCSK9-LDLR transitions from extracellular (high pH) to endosomes/lysosomes (low pH), the conformation of PCSK9 due to the pH changes. The difference between the closed and extended conformations revolves around the residue Ser376, which serves as a hinge connecting LDLR. It has been observed that PCSK9 Asp374 forms an extra salt bridge with the EGF-A His306 side chain at a low pH (20). The binding affinity of PCSK9 for LDLR exhibits an increase, from Kd=750±80 nM at pH 7.5 to Kd=8±1 nM at pH 5.5, thus hindering the recycling of LDLR to the cell surface (20). However, the structure of CD36, an important ligand of PCSK9, does not exhibit the EGF-A-like domain, thus the respective binding sequences have yet to be confirmed (14). Variants in the catalytic domain lead to PCSK9 loss or gain of function (21,22).

The hinge region (aa 422-452) links the CHRD. The absence of the CHRD does not affect the binding of PCSK9 to LDLR at a neutral pH (20). However, there is an interaction between CHRD and ligand-binding domain of LDLR at low pH, assisting in the formation of the complex (23). The CHRD comprises three similar modules (M1: aa 453-529, M2: aa 530-603, and M3: aa 604-692), each forming a six-stranded two-sheet β-sandwich. Asn533 in the M2 module is the only glycosylation site in PCSK9, but it is not necessary for PCSK9 activity (16). The three modules exhibit a structural homology to the homotrimeric form of resistin (24). Resistin is an adipocyte-specific hormone associated with hypercoagulative, hypofibrinolytic activities and insulin resistance (25,26).

The PCSK9 proximal promoter encompasses a functional sterol regulatory element (SRE) that is modulated by intracellular cholesterol levels. SRE binding protein (SREBP) is connected with the SRE of the PCSK9 and LDLR genes, regulating PCSK9 synthesis at the transcriptional level. Following protein cleavage in the Golgi apparatus, mature SREBP-2 enters the nucleus and interacts with the promoters of PCSK9 and LDLR, leading to the increased transcription and translation of both (17,27). The experimental overexpression of SREB-1c stimulates the expression of PCSK9 (28). Another regulator of PCSK9 expression (but not LDLR) is hepatocyte nuclear factor 1 (HNF1). The deficiency or inhibition of HNF1 leads to reduced transcription levels of PCSK9. There is synergy between HNF1 and SRE. Mutations in HNF1 significantly diminish the transactivation activity of SREBP2 on the PCSK9 promote (29). Statins not only deplete intracellular cholesterol, leading to SREBP-2 expression (30,31), but also stimulate hepatic HNF1 expression, thus activating PCSK9 transcription (32).

3. Pleiotropic effects of PCSK9 on cardiovascular disease

Based on the results of several landmark trials of PCSK9 inhibitors (33-35), drugs targeting PCSK9 have swiftly entered the market to improve clinical outcomes by preventing myocardial infarction and coronary revascularization (Table I) (36,37). The enthusiasm for developing PCSK9 inhibitors is still rapidly increasing. A small binding protein of the PCSK9 gene, lerodalcibep, has been shown to lead to a 65% reduction in LDL-C levels in subjects with heterozygous familial hypercholesterolemia at weeks 22 and 24 of phase III clinical trials (38). The oral formulation MK-0616 (an administered macrocyclic peptide), has progressed through phase I clinical trials (39).

Table I US Food and Drug Administration (FDA)-approved PCSK9 inhibitors.

Table I

US Food and Drug Administration (FDA)-approved PCSK9 inhibitors.

Year	Agent	Mechanism of action	Indication	Mechanism	Specific populations	(Refs.)
2015	Alirocumab	Block interaction with LDLR	· Reduce risk of MI, stroke, coronary revascularization: adults with established cardiovascular disease · Reduce LDL-C: Adults with primary hyperlipidemia including HeFH · Reduce LDL-C: Patients (adult, alirocumab; >10 years, evolovumab) with HoFH · Reduce LDL-C: HeFH (>8 years, alirocumab; >10 years, evolovumab)	· MI, stroke, coronary revascularization: Accumulation and transformation of lipids, inflammatory cells, and necrotic cell debris in the intimal space underneath a monolayer of endothelial cells in the vessel wall, leading to clinical complications · Hyperlipidemia: Characterized by elevated cholesterol and triglycerides. Chronic elevated lipid levels promote the development of atherosclerosis. Oxidative stress, mitochondrial dysfunction and apoptosis exerts negative impact on myocardium	· Renal Impairment: No dose adjustment · Hepatic Impairment: · No dose adjustment	(36)
2015	Evolocumab	Block interaction with LDLR				(10)
2021	Inclisiran	Block mRNA translation	Reduce LDL-C: Adults with primary lipidemia including HeFH	· HoFH and HeFH: A autosomal co-dominant hereditary disorder (LDLR, APOB, PCSK9). Characterized by elevated LDL-C, with a 3-13-fold increased risk of atherosclerotic cardiovascular disease		(37)

[i] PCSK9, proprotein convertase subtilisin kexin type 9; LDLR, low-density lipoprotein receptor; MI, myocardial infarction; LDL-C, low-density lipoprotein-cholesterol; HeFH, heterozygous familial hypercholesterolemia; HoFH, homozygous familial hypercholesterolemia; APOB, apolipoprotein B.

In addition to reducing cardiovascular events by decreasing LDL-C in a dose-dependent manner, PCSK9 inhibition exerts a cardiovascular protective effect beyond lipid management. For decades, statins were known as first-line lipid-lowering agents and for the protection of cardiovascular health in hyperlipidaemia. However, the benefits of statins in patients with resistance or intolerance are limited (40). Researchers have observed the plaque stabilization and regression of atherosclerosis using CT angiography and intravascular ultrasound in patients treated with PCSK9 inhibitors (41,42). With the advent of inclisiran, a siRNA PCSK9 inhibitor, appearing on the market, the rate of major adverse cardiovascular events is expected to decrease by as much as 24-30% (43). The effects of PCSK9 on oxidative stress, endothelial injury, inflammation and immunity have been widely described (44-46). The improvement in MACEs due to PCSK9 inhibition may be attributed to various reasons; primarily of interest are the potential direct and indirect roles of PCSK9 in thrombosis and haemostasis.

A high level of PCSK9 has been found to increase platelet reactivity and significantly increase the risk of ischaemic events (47). The results of the Further Cardiovascular Outcomes Research with PCSK9 Inhibition in Subjects with Elevated Risk (FOURIER) study (34) demonstrated that the incidence of venous thromboembolism (VTE) was decreased in patients treated with PCSK9 inhibitors, which cannot be completely explained by lipid-lowering (48,49). In another study, several platelet activation markers were observed to be significantly reduced after 12 months of PCSK9 monoclonal antibody therapy in patients with hypercholesterolaemia (9), indicating a potential impact of PCSK9 inhibition on platelet function. Additionally, circulating platelets store PCSK9 and release it upon activation, which may promote platelet aggregation and thrombus formation (50).

Abnormal PCSK9 levels are present in several disease states. Patients on maintenance dialysis and following successful renal transplantation have been shown to have higher serum levels of PCSK9 than subjects without chronic kidney disease (51). Patients with liver cirrhosis exhibit lower serum PCSK9 levels, while these levels are increased in patients with hepatitis C virus (HCV)-induced liver cirrhosis (52). PCSK9 levels increase with the severity of breast cancer (53). These specific pathological conditions may lead to changes in thrombosis or haemostasis. Combined with the regulatory role of PCSK9 in thrombosis and the difference in the level of the disease states, thrombosis in metabolism-related diseases may also be regulated by PCSK9, and its regulatory pathway may differ from that in the physiological state.

4. Mechanisms of PCSK9 in thrombosis and haemostasis

Platelet reactivity, coagulation factor activity and endothelial function are crucial factors affecting thrombosis and haemostasis. Inflammation has been found to contribute to thrombosis mechanisms over the past decade (54). Several studies have proven that PCSK9 has an essential effect on these processes, which may provide a new direction for studying the regulatory mechanisms of thrombosis or bleeding in particular disease states.

PCSK9 and platelets

PCSK9-/- mice have been shown to exhibit slower and less stable thrombus formation compared to PCSK9+/+ mice in a model of ferric chloride-induced carotid thrombosis, resulting in incomplete artery occlusion (55). Platelet function is linked to the lipid profile, as cholesterol acquired by platelets modifies the membrane, rendering platelets hypersensitive (46). Previous research has demonstrated that patients with hypercholesterolaemia exhibit elevated membrane cholesterol levels, and treatment with rosuvastatin normalizes the membrane composition, including membrane cholesterol, tissue factor (TF) and TF-dependent procoagulant activity (56). A similar effect between cholesterol and PCSK9 inhibitors appears to be plausible. PCSK9 inhibitors reduce the risk of developing peripheral artery disease following acute coronary syndrome by reducing plasma lipoprotein A [Lp(a)] levels (57,58). However, the exact mechanisms underlying this association are not yet fully understood. Lp(a) possesses anti-fibrinolytic and pro-inflammatory properties, potentially enhancing the occurrence of both venous and arterial thrombosis (59). In addition, Lp(a) contains a Ca2+-independent phospholipase A2, which serves as a platelet-activating factor and contributes to increased atherogenicity by combining with oxidised phospholipids (60). Apolipoprotein (Apo)B100 is an essential marker for liver cells to recognize LDL-C, while ApoB100 danger-associated signal 1 (ApoBDS-1) is a peptide fragment within ApoB100 (61). Assinger et al (61) discovered that ApoBDS-1 activated platelets via p38-mitogen-activated protein kinase (MAPK) and other signalling pathways, contributing to boosting platelet-leukocyte proinflammatory responses. A recent study confirmed that ApoB levels <35 mg/dl reduced the residual risk following acute coronary syndrome, with a greater absolute reduction in MACEs observed in patients with higher baseline levels when treated with alirocumab (62).

Compared to other LDL-C-lowering drugs, where a reduction of 1 mmol/l is associated with a relative risk (RR) of 0.77 [95% confidence interval (CI), 0.75-0.79] for major vascular events, the estimated RR for PCSK9 inhibitors is lower at 0.49 (95% CI, 0.34-0.71) (63). This suggests that although the LDL-C-lowering effect is undoubtedly a key contributor to the haemostasis and thrombosis benefits of PCSK9 inhibition, PCSK9 has also been found to stimulate platelet activation beyond lipids (Fig. 2). First, scavenger receptor CD36, a major platelet glycoprotein, plays a crucial role in dyslipidaemia and the prothrombotic phenotype (64). PCSK9 in the plasma directly combines with CD36 on platelets, thereby enhancing Src kinase phosphorylation. This activation triggers NAD phosphate oxidase-2 (NOX-2), leading to the increased generation of reactive oxygen species (ROS) and the subsequent activation of MAPK-ERK5, ultimately promoting platelet hyperactivity (65,66). Moreover, NOX-2 induces the generation of 8-ISO-prostaglandin F2α, which forms platelet aggregation via GP-IIb/IIIa activation (67). Second, PCSK9 binding to CD36 also activates the JNK and p38 MAPK pathways. The activation of p38 MAPK stimulates cytosolic phospholipase A2, which releases arachidonic acid from phospholipid membranes. Arachidonic acid is then converted into thromboxane A2 (TXA2) through the cyclooxygenase (COX)-1 and Tx synthase pathways (66). TXA2 acts synergistically with platelet agonists to enhance platelet aggregation by activating GP-IIb/IIIa receptors. TXA2 is rapidly metabolised to TXB2, which can be detected in urine as 11-dehydro-TxB2 by liver metabolism, indicating platelet activation in patients (68). Third, positive feedback interplay was found between PCSK9 expression and oxidised LDL (oxLDL) generation (69). Elevated levels of oxLDL combined with CD36 and lectin-like oxLDL receptor 1 (LOX-1) follow a pattern similar to that of PCSK9, leading to platelet aggregation and secretion (70). A recent study demonstrated that inclisiran inhibited human umbilical vein endothelial cell death induced by oxLDL, suggesting a potential role in the treatment of atherosclerosis (71). Finally, the aforementioned mechanism enhances integrin αIIbβ3 activation, P-selectin release, agonist-induced platelet aggregation and clot retraction (65). The PCSK9 regulation of platelets may partly influence the haemostasis process in liver disease.

Figure 2 Possible mechanisms of PCSK9 in thrombosis and haemostasis. PCSK9, proprotein convertase subtilisin kexin type 9; LDL, low-density lipoprotein; oxLDL, oxidized low-density lipoprotein; Apo, apolipoprotein; LOX-1, lectin-like oxLDL receptor 1; Lp(a), lipoprotein A; ROS, reactive oxygen species; NOX-2, NAD phosphate oxidase-2; cPLA2, cytosolic phospholipase A2; AA, arachidonic acid; TXA2, thromboxane A2; TXB2, thromboxane B2; PAI-1, plasminogen activator inhibitor type 1; tPA, tissue-type plasminogen activator; uPA, urokinase-type plasminogen activator; LDLR, low-density lipoprotein receptor; SRA, scavenger receptor class A; TLR4, toll-like receptor 4; COX-2, cyclooxygenase 2; LRP, LDLR-related protein; TF, tissue factor; PF4, platelet factor 4.

PCSK9 and coagulation factors

Another mechanism by which the haemostasis process is impacted by PCSK9 involves blood coagulation factors. Coagulation factor VIII (FVIII) is unequivocally a vital participant in the intrinsic pathway, where the lack of FVIII may cause haemophilia (72). Together with LDLR, LDLR-related protein (LRP) modulates FVIII levels (73). Although there are still no experimental results in support of this premise, it is possible that PCSK9 indirectly increases the circulation of FVIII, given its considerable effect on LDLR reduction (73). Additionally, PCSK9 augments plasma FVIII levels in vivo by decreasing LRP-1 expression (74). LRP-1 is also associated with TF levels on the cell surface. TF downregulation occurs through rapid internalization when the factor VII (FVII)/TF complex bridges LRP-1 (75). Furthermore, PCSK9 induces the de novo synthesis of TF, attributed to the TF/FVIII interaction via the Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB) signalling pathway (76), thereby initiating the extrinsic pathway, where abnormal TF levels may contribute to coagulation initiation.

The increased expression of PCSK9 has been reported to contribute to lower a plasma prothrombin time (PT) and to also be associated with a higher risk of MACEs (77). It is known that PT is crucial for exposing the activation of the extrinsic pathway; thus, a low PT suggests that TF and FVII could be in abnormal states (77). It is possible that PCSK9 could promote the formation of the TF-FVII complex.

PCSK9 affects fibrinolysis through plasminogen activator inhibitor-1 (PAI-1). Levine et al (78) performed a series of experiments to support this view. RNA sequencing revealed that TM5614, a PAI-1 inhibitor, led to an 86% gene transcript downregulation of PCSK9 in mice. Similar results were also found in vivo in mice with hyperlipidaemia. Furthermore, the authors of that study observed that patients with heart failure treated with PCSK9 monoclonal antibodies exhibited a substantial increase in plasma PCSK9 and a reduction in plasma PAI-1 levels (78).

In addition, fibrinogen is also known as coagulation factor I. Candidate gene analysis detected significant associations between total fibrinogen and polymorphisms of PCSK9 and LDLR genes (79). PCSK9 levels were found to be associated with fibrinogen in patients with isolated hyperlipidaemia and stable coronary atherosclerosis (80,81). However, studies on familial hypercholesterolaemia found that PCSK9 inhibitors did not affect fibrinogen and D-dimer levels (82). Conflicting evidence suggests that the association between PCSK9 and fibrinogen should be further investigated for detailed analysis.

PCSK9 and Inflammation

Inflammation plays a cruical role in atherosclerotic plaque and thrombosis. As a result, persistent inflammation is strongly linked to higher risk of ischaemic events (83). Anti-inflammatory therapy can significantly reduce the risk of recurrent cardiovascular events without affecting lipid levels in patients with previous myocardial infarction (84). The PCSK9 pathway promotes the inflammatory response and plays a role in in the thrombosis and atherosclerosis processes (85,86).

PCSK9 is associated with key inflammatory markers, such as white blood cells, fibrinogen and high-sensitivity C-reactive protein (45). However, the underlying mechanisms remain unclear. The increased levels of adhesion molecules and cytokines (e.g., PF4) expressed by endothelial cells under atherosclerosis lead to the attachment of monocytes and lymphocytes at the intima and further stimulate monocytes to differentiate into macrophages (87,88). PF4 is a protein synthesised from platelet particles. Recent research has found that PF4 interacts with von Willebrand factor (VWF) to form immune complexes. The PF4-VWF complex stimulates platelet activation and inflammation, and promotes the formation of immune-associated thrombosis by suppressing a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 in a concentration-dependent manner (89). A cohort study supported the assumption that after 12 months of PCSK9 inhibition therapy, the plasma level of PF4 decreased, which may play a role in cardiovascular event reduction (9). VWF participates in the coagulation process as a carrier and protector of FVIII. Since plasma FVIII levels are increased by PCSK9 (74), it can be hypothesized that low PCSK9 levels may further become a barrier to thrombosis. In summary, the PCSK9-FVIII-PF4 pathway in thrombosis is a direction worthy of further research. This may be helpful to explain VTE reduction in patients with PCSK9 inhibitors. Yurtseven et al (90) observed a significant increase in Ly6C (hi) monocytes, which are the inflammatory monocytes, following treatment with PCSK9, followed by an enhancement of their recruitment to the arterial wall. Furthermore, oxLDL and PCSK9 potently bind to LDLR, CD36, LOX-1, TLR4 and scavenger receptor class A on macrophages, thereby stimulating the NF-κB, MAPK and COX-2 pathways. This induction leads to an increased oxLDL uptake by macrophages, as well as to the increased expression of cytokines and mRNA of adhesion molecules, including interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-α, chemokine C-X-C motif ligand 2 and monocyte chemotactic protein 1 (MCP1) (70,91,92). In a previous study, compared with the atherosclerotic plaques of untreated ApoE-null mice, mice treated with PCSK9 small hairpin RNA were found to have less macrophage infiltration. The mice in which PCSK9 was suppressed had lower levels of vascular inflammatory regulators, such as TNF-α, IL-1β, MCP1, TLR4 and NF-κB (93). Macrophages turn into foam cells (94). Foam cells, which participate in atherosclerotic plaque rupture, mediate the sustained inflammatory response, resulting in vascular remodelling. After the plaque is disrupted, tissue factor, collagen and other plaque components are exposed, which leads to platelet activation and aggregation, and thrombus formation (95). Clinical research has confirmed that circulating PCSK9 levels are significantly associated with an increased risk of developing myocardial infarction (96).

Neutrophil extracellular traps (NETs) composed of DNA and histones released by neutrophils play a crucial role in trapping bacteria and driving bactericidal activity. However, NETs also play a role in the initiation, formation and propagation of thrombosis. Lipopolysaccharide activates platelets and neutrophils via TLR4, inducing the release of PF4, the expression of P-selectin and NETosis. The formation of NETs results in the release of TF via autophagy and the activation of FVII (97). An analysis of the inferior vena cava conducted in mice suggested that in comparison with PCSK9-deficient mice, wild-type mice displayed an inferior vena cava thrombus with greater weight, length, myeloid cell recruitment and a greater number of NETs (98). Therefore, PCSK9 promotes thrombosis by affecting leukocyte mobilisation and NET formation. These mechanisms may be of particular importance for patients with diabetes.

PCSK9 and endothelial dysfunction

Vascular endothelial injury is closely associated with thrombosis. Elevated levels of cholesterol expose the vascular walls to oxidative stress, leading to endothelial dysfunction (99). Inflammation induced by endothelial dysfunction abnormally induces platelet attachment to endothelial cells in the arterial walls (100), increasing the cardiovascular risk. Even with short-term treatment, the PCSK9 inhibitors, evolocumab and alirocumab, have been found to improve endothelial function (101,102). Damage to the vascular wall by PCSK9 can occur via a variety of mechanisms.

First, a study on a cohort of 26 patients with cardiovascular disease found that reducing PCSK9 levels significantly increased circulating endothelial progenitor cells, suggesting the promotion of vascular repair (103). Second, the expression of PCSK9 in smooth muscle cells (SMCs) and endothelial cells was found to be highest at proper endothelial shear stress (3-6 dynes/cm2), and the levels of ROS followed the same pattern as PCSK9 (104). Haemodynamic factors stimulate local lipid accumulation and oxidative stress, accelerating vascular remodelling (105). It has been shown that anti-PCSK9 therapy with alirocumab ameliorates oxidative stress in rats with biliary cirrhosis, without affecting the plasma levels of oxLDL (106). Third, the Bcl-2 protein family and caspases play an essential role in apoptosis. Breaking the balance between the apoptosis inhibitor, B-cell lymphoma-2 (Bcl-2), and the apoptosis inducer, Bcl2-associated X (Bax), can activate caspase-9 and caspase-3 sequentially by regulating the release of cytochrome c from the mitochondria to induce cell apoptosis. Studies using human umbilical vein-derived endothelial cells have demonstrated that Bax phosphorylation under oxLDL pressure decreases the Bcl-2/Bax ratio and activates the apoptotic process (107). Following the silencing of PCSK9 with specific small hairpin RNA, the Bcl-2/Bax ratio increases, and the phosphorylation levels of the p38 and JNK signalling pathways are significantly decreased. The programmed death process of endothelial cells is inhibited (108). In addition, PCSK9 affects the metabolism of SMC. It promotes age-related atherosclerosis by downregulating the expression of ApoER2 on SMCs (109). The dysfunction of vascular endothelial cells may be related to abnormal plasma and local PCSK9 levels in patients with and without renal disease undergoing dialysis.

5. Roles of PCSK9 in thrombosis and haemostasis under disease states

Some pathological states may alter the PCSK9 levels, activity and downstream pathways (Fig. 3). Moreover, this may lead to abnormal LDL-C levels and an increased risk of bleeding and thrombosis.

Figure 3 PCSK9 is associated with a variety of disease states with special metabolic characteristics. PCSK9, proprotein convertase subtilisin kexin type 9; LRP1, LDLR-related protein; tPA, tissue-type plasminogen activator; RAAS, renin-angiotensin-aldosterone system; PAI-1, plasminogen activator inhibitor type 1; SMC, smooth muscle cell; GDM, gestational diabetes mellitus; LDLR, low-density lipoprotein receptor; TF, tissue factor; HCV, hepatitis C virus; TLR, Toll-like receptor; TAT, thrombin-antithrombin complex; GVD, graft vascular disease.

PCSK9 and liver disease

The liver is the predominant synthesis site for multiple proteins involved in coagulation and liver diseases may lead to coagulation disorders presenting as either the impairment or promotion of haemostasis (110). Bleeding was historically considered to be a frequent and fatal complication of liver disease; however, a significant rate of VTE was also observed in patients with chronic liver disease (CLD) (111). The effects of liver disease on haemostasis involve a several mechanisms, and some of the haemostatic changes counteract each other. In patients with liver dysfunction, the reduction of fibrinogen and coagulation factors, such as FII, FV, FVII, FIX, FX and FXI increase the risk of bleeding, while elevated FVIII levels, decreased levels of protein C/S and antithrombin, together with VWF compensation, promote clotting (112). Mild to moderate thrombocytopenia and platelet function defects have been described in patients with CLD, and this can be more severe in those with cirrhosis and splenomegaly (113). However, enhanced in vivo platelet activation in patients with liver cirrhosis may contribute to the thrombosis of large vessels (114). There are also abnormalities in the fibrinolytic system in the condition of liver disease, the most prominent of which is the increased level of tissue-type plasminogen activator (tPA) due to reduced hepatic clearance. This may explain the high rate of hyperfibrinolysis (30 to 50%) that occurs in patients with end-stage liver disease (115). However, a causal role of hyperfibrinolysis in bleeding is uncertain due to concomitant changes in haemostasis.

PCSK9 is synthesized mainly in the liver. Impaired hepatic function in patients with liver cirrhosis affects the synthesis and secretion of PCSK9. Feder et al (116) reported that patients with liver cirrhosis had lower plasma levels of PCSK9 compared to non-cirrhotic patients. Moreover, the association between serum cholesterol and PCSK9 levels is lacking, which may be due to the overriding effect of PCSK9 on cholesterol metabolism. Lower PCSK9 levels in patients with liver disease are associated with reduced levels of pro-inflammatory factors, suggesting a reduction in the occurrence of atherosclerotic events (117). In a previous study, serum PCSK9 levels and the international normalized ratio were shown to be negatively associated with each other, suggesting a higher risk of bleeding (118). In that study, low systemic levels of PCSK9 were associated with decreased albumin levels and an increased mortality rate in patients suffering from end-stage liver disease.

PCSK9 mediates the degradation of LDLR homologous and non-homologous receptors, including CD36 (119) and LRP1 (120), to regulate inflammation and vascular function. CD36 is a multifunctional signalling molecule inducing inflammation through JNK activation (121). Since CD36 is a key receptor for platelet activation, decreased levels of PCSK9 in patients with liver disease may inhibit CD36 degradation, thereby enhancing the stimulation of the CD36 pathway and promoting platelet aggregation (122). In addition, low levels of PCSK9 have a potential effect on fibrinolysis. tPA is the major intravascular activator of fibrinolysis and is cleared by LRP1 (115). Low PCSK9 levels in liver disease may decrease LRP1 degradation, thereby promoting LRP1-mediated tPA clearance and alleviating hyperfibrinolysis to a certain extent (120).

PCKS9 and chronic kidney disease

In a previous prospective study involving 5,138 individuals with chronic kidney disease, elevated levels of PCSK9 were shown to be associated with an increased risk of cardiovascular disease (123). Despite an increase in PCSK9 levels in cases of nephrotic syndrome, no association was found with the estimated glomerular filtration rate (124). While some studies have reported an association between PCSK9 and proteinuria, others have revealed opposite results (124,125). PCSK9 is expressed in the kidneys to a lesser extent. The collecting duct is a major source of increased PCSK9 expression in the kidneys, which may be a new driver of hyperlipidaemia in nephrotic syndrome (126).

Chronic kidney disease affects the haemostatic system due to complex alterations in platelets, vessel walls, microparticles and even drugs (127). The changes in PCSK9 levels among patients with nephropathy add a further layer of complexity to haemostasis. The renin-angiotensin-aldosterone system (RAAS) is abnormally active in patients with renal failure, and is associated with increased levels of PAI-1 (127). Given the high level of PCSK9, the interaction between PCSK9 and PAI-1 may promote hyperfibrinolysis (as aforementioned in the sub-section entitled 'PCSK9 and coagulation factors'). Intracellular PCSK9 promotes arterial medial calcification. SMCs overexpressing PCSK9 release an abundance of extracellular vesicles (EVs) that are rich in Ca2+ and alkaline phosphatase (128). EVs increase pro-calcific markers, which results in renal impairment by regulating the calcium-phosphorus balance, and eventually promoting calcification of SMCs (129). Arterial medial calcifications promote the progression of atherosclerotic lesions and affect platelet-vessel wall interactions (130).

Zymogen PCSK9 (73 kDa) undergoes processing in the endoplasmic reticulum and Golgi body and is secreted as mature PCSK9 (63 kDa) (131). Dialysis may influence PCSK9 kinetics, although available literature on this topic is limited. A prevoius small cohort study indicated increased PCSK9 levels in patients receiving peritoneal dialysis as compared to those on haemodialysis and in control subjects (124). Notably, abnormal platelet activation occurs in patients treated with peritoneal dialysis (127). Therefore, the potential benefits of anti-PCSK9 therapy in these patients are worthy of investigation.

PCSK9 and diabetes mellitus

Cardiovascular disease tends to develop in patients with either type 1 (T1DM) or type 2 (T2DM) diabetes mellitus. The lipid profile of patients with well-controlled T1DM is similar to that of the general population (132). However, patients with T2DM frequently exhibit elevated levels of triglycerides and non-high-density lipoprotein-cholesterol, even with good glycaemic control (133). Patients with T2DM are considered to be in a hypercoagulable state induced by insulin resistance and endothelial dysfunction, which causes microvascular and macrovascular complications by augmenting atherosclerosis (134). Due to the increased risk of MACEs, clinical guidelines the stricter lipid targets for individuals with diabetes (135). Hyperglycaemia leads to platelet hyperactivity through various mechanisms, including the glycation of platelet surface proteins, the expression of GPⅡb and P-selectin, and PKC activation (136). Moreover, T2DM is associated with chronic inflammatory disorders (137). Neutrophils isolated from diabetic individuals are more prone to forming NETs, and the interaction between NETs and macrophages contributes to persistent macrophage inflammation (138). Hypofibrinolysis is also widely observed in T2DM, partly due to the elevated concentration and activity of PAI-1 (139). Platelet hyperactivity, fibrinolytic inhibition, oxidative stress and inflammation further promote thrombosis (136).

Circulating levels of PCSK9 are significantly higher in pregnant women with gestational diabetes mellitus (GDM) compared to those with a normal glucose tolerance. PCSK9 level is positively associated with fasting plasma glucose, glycosylated haemoglobin, total cholesterol, and LDL-C levels in patients with GDM (140). PCSK9 deficiency leads to an increased expression of LDLR in pancreatic β-cells (141). PCSK9 knockdown also promotes intracellular cholesterol accumulation, islet function and insulin secretion (141,142). Specifically, PCSK9 interacts with human pancreatic β-cells through LDLR and an intracellular signalling mechanism that regulates the expression of CD36, anti-programmed death 1 and HLA-ABC receptors (141). It remains unclear whether the use of PCSK9 inhibitors increases the risk of developing diabetes (143,144). Considering the inconsistent results, further investigations are required to elucidate the association between PCSK9 levels and diabetes.

PCSK9 monoclonal antibody therapy is safe and effective for patients with diabetes (145). Plasma PCSK9 levels appear to affect inflammation and coagulation in patients with diabetes mellitus. A previous observational study reported that patients with diabetes and PCSK9 levels of ≤43.5 ng/ml had the highest survival from MACEs (12). High PCSK9 levels were found to be associated with MACEs in diabetic patients undergoing primary PCI, possibly due to their strong association with inflammation and platelet activation markers, such as high-sensitivity C-reactive protein, P-selectin and CD40L (12). In diabetic patients treated with ticagrelor, PCSK9 levels were positively associated with maximal platelet aggregation and maximum amplitude of ADP-induced platelet-fibrin clots, while no association was found in non-diabetic patients (12). In addition, PCSK9 levels have been shown to be independently associated with the platelet count in patients with T2DM (146).

It appears that patients with DM experience additional anticoagulation benefits from PCSK9 inhibitors. On the one hand, diabetes is associated with chronic low-grade inflammation, suggesting that the inhibition of PCSK9 in diabetic patients may yield greater cardiovascular benefits through its anti-inflammatory effects than in non-diabetic patients. On the other hand, PCSK9 may also interact with fibrinolytic processes. Patients with T2DM exhibit impaired fibrinolysis due to the downregulation of insulin receptor substrate 1 in endothelial cells. The level of PAI-1 is increased by 25-280% compared to that in non-diabetic patients (139). Since PCSK9 levels positively correlate with PAI-1 levels (78), diabetic patients are more likely to have high PCSK9 levels and hypercoagulable states. Although there is a lack of evidence on the effect of PCSK9 levels on the fibrinolytic state, the role of PCSK9 in the regulation of the fibrinolytic process should be further explored.

PCSK9 and cancer

Thromboembolism is the second leading cause of mortality among patients with cancer undergoing outpatient chemotherapy, immediately following the progression of cancer (147). To the best of our knowledge, to date, no large-scale clinical studies have evaluated the effects of anti-PCSK9 therapy on thrombosis in patients with cancer, although it is likely a promising research area. It is well-known that hypercoagulability, endothelial injury and abnormal haemodynamics contribute to thrombosis, and cancer-associated thrombosis (CAT) presents peculiarities in pathophysiological mechanisms. First, host and malignant cells abnormally synthesize excessive TF and TF-positive EVs, which initiate the extrinsic pathway (148). TF-positive EVs generated from endothelial cells, monocytes and macrophages play a role in thrombosis, while platelet-derived TF and TF-positive EVs are controversial. The exposed phosphatidylserine on TF-positive EVs appears to promote the binding of coagulation factors, rather than TF. NETs serve as a scaffold for various procoagulants and act as mediators of CAT (149). It has been shown that 4T1-derived exosome range EVs induce NET formation in the neutrophils of granulocyte colony-stimulating factor-treated mice (150). Human studies have shown that blood and tissue samples from patients with gastric cancer were more likely to form NETs, promoting the conversion of thrombin and fibrin to develop thrombosis compared with healthy individuals (151). Moreover, other substances produced by cancer cells also exacerbate the hypercoagulable state, such as carcinoma mucins, distinct cancer procoagulant factors and PAI-1 (152). Second, surgical procedures, catheters and chemotherapy often lead to endothelial injury (153). Third, oncologic pain causes immobility, which aggravates circulatory stasis (154).

PCSK9 and other disease states

Changes in immune-related factors can lead to alterations in PCSK9 levels and functions, which can also affect clotting functions. Herein, the changes in the levels of PCSK9 in infection and transplantation are described. Research on the marine fish, Epinephelus coioides, has demonstrated that the upregulation of PCSK9 expression increased the activities of NF-κB promoter, Singapore grouper iridovirus-induced apoptosis, and pro-inflammatory factors (155). These findings highlight the role of PCSK9 in modulating innate immunity in pathogen infections. Moreover, PCSK9 reduces the infectivity of HCV by acting on LDLR, very LDLR, scavenger receptor class B type 1 and CD81 receptors on hepatocytes (156). PCSK9 expression is upregulated during sepsis, increasing cholesterol levels and promoting cholesterol accumulation in immune cells (157). These factors improve TLR function and promote inflammation (157,158). Thrombin-antithrombin complex levels have been found to be elevated in PCSK9-overexpressing mice, which indicates that PCSK9 exacerbates the hypercoagulation state in early sepsis (159). Therefore, although PCSK9 promotes pathogen clearance, it is possible that PCSK9 also exacerbates the procoagulant state of the body. A number of organ transplant patients suffer from graft vascular disease (GVD). Previous studies have reported that PCSK9 levels are upregulated in wild-type GVD mice, while PCSK9 knockout mice have a reduced vascular stenosis, intimal hyperplasia area and collagen deposition (160). Furthermore, the lack of PCSK9 probably inhibits the inflammasome through the nod-like receptor protein 3 pathway and suppresses the migration and proliferation of vascular SMCs (161). Previous studies on patients who have undergone a heart transplant suggested that PCSK9 inhibitors can stabilise coronary intimal hyperplasia (161). Additionally, donor-specific antibodies do not increase after 1 month of treatment (162). PCSK9 is a potential marker of ischemia/reperfusion injury during visceral vascular surgery or myocardial infarction (163). Ischemia/reperfusion triggers the upregulation of PCSK9 and increased the levels of autophagy regulatory proteins light chain 3 and beclin-1, at the same time, activating the Bcl-2/adenovirus E1B 19-kDa interacting protein pathway aggravated ischemia/reperfusion injury (164).

6. Conclusion and future prospects

Based on their superior safety profile, current PCSK9 inhibitors have been approved for use in the majority of patients with hyperlipidaemia, including those with liver disease, chronic kidney disease, diabetes and cancer. Nevertheless, both clinical evidence and in vitro data suggest that during pathological conditions, the concentration and effects of PCSK9 could differ from those in general patients under pathological conditions. The effect of PCSK9 on thrombosis and haemostasis could also be intricate. Generally, PCSK9 levels are elevated in several metabolic states, primarily inciting thrombophilia through platelet activation and inflammation pathways. Nonetheless, in certain cases, reduced levels of PCSK9 may alleviate hyperfibrinolysis by mediating the PAI-1 pathway.

To date, numerous key questions remain to be addressed. The PAI-1/PCSK9 pathways regulate the fibrinolysis process; however, the specific mechanism that dominates fibrinolysis in patients with liver damage remains to be determined. It also remains unclear whether the concurrent inhibition of the RAAS and PCSK9 inhibitors may be beneficial in patients with chronic kidney disease. Future research is thus required to elucidate the following: i) The mechanisms through which different disease states alter PCSK9 function and its impact on the clotting process beyond serum concentrations; ii) the specific roles of PCSK9 in local coagulation within particular tissues; and iii) provide further evidence of the extent of the inhibitory effects of PCSK9 on haemostasis and thrombosis, and determine the associated clinical outcomes. Therefore, the use of anti-PCSK9 therapy in specific patients may alter the course of haemostasis and thrombosis, presenting a double-edged sword. On the one hand, the incidence of bleeding events may be increased, while on the other hand, patients with thrombotic tendencies may benefit. At the same time, drug interactions can be reshuffled. It is expected that more high-quality evidence will emerge, as it may change medication regimens for certain patients.

Availability of data and materials

Not applicable.

Authors' contributions

SC, GM and QX were involved in the investigative aspects of the review, such as the literature search for related studies. SC, GM and XC were involved in visualization (preparation of the figures). SC and GM were involved in the writing of the original draft of the manuscript. XC, QX and YC were involved in the writing, reviewing and editing of the manuscript. GM, QX and YC were involved in the conceptualization and supervision of the study, and in funding acquisition. All authors have read and agreed to the published version of the manuscript. Data authentication is not applicable.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

The authors would like to thank Professor Yan Zhang (Department of Cardiology, Peking University First Hospital) for his professional guidance of this review during the revision process.

Funding

The present study was supported by the National Key Research and Development Program of China (grant nos. 2021YFC2500500 and 2021YFC2500503), the National High Level Hospital Clinical Research Funding (Scientific Research Fund of Peking University First Hospital (grant no. 2022SF15), and the National Natural Science Foundation of China General Program (grant nos. 82073935 and 82373950).

References