Evaluation of PCSK9 Enzyme in Non-Cholesterol Biological Pathways: In Vitro Demonstration of Direct Platelet-Related Effects of PCSK9 Enzyme
试验速览
- 阶段
- 不适用
- 状态
- 尚未招募
- 入组人数
- 80
- 主要终点
- Difference in ADP-induced platelet aggregation (%) with and without PCSK9 enzyme
研究概览
简要总结
Proprotein convertase subtilisin/kexin type 9 (PCSK9) has evolved as a pivotal enzyme in lipid metabolism and therapeutic target for hypercholesterolemia and atherosclerotic cardiovascular disease (CVD). Furthermore, research from in-vitro and in-vivo animal models suggest that the inhibition of PCSK9 expression may suppress platelet activation, and systemic inflammation thereby reducing thrombotic risk. Taken together, these data support the lower rates of myocardial infarction, stroke, and coronary revascularization in subjects with increased CVD risk in clinical trials with alirocumab (Praluent) and evolocumab (Repatha) as compared to placebo. The main aim of this single center, exploratory in-vitro evaluation is to investigate the direct role of PCSK9 on platelet activation and aggregation using blood collected from a diverse population of antiplatelet naïve healthy volunteers (n=40) and subjects with CVD risk factors (n=40) between the 18-50 years of age. All procedures including consenting, collection of clinical data, and lab processing will occur at the Sinai Center for Thrombosis Research. The results of this study will provide a better understanding of cardioprotective effects of PCSK9 inhibition beyond lipid lowering.
详细描述
The pivotal role of platelets during arterial ischemic events, such as myocardial infarction (MI), stroke, and cardiovascular (CV) death, and the significant role of potent antiplatelet therapy in reducing these events have been well defined. An independent association between plasma PCSK9 levels, elevated platelet reactivity, and ischemic outcomes in patients with acute coronary syndromes (ACS) has been demonstrated. The association of plasma PCSK9 levels with coronary artery disease (CAD) risk, independent of low-density lipoprotein cholesterol (LDL-C) levels, has been suggested. Based on a meta-analysis of trials of medium-high risk cardiovascular disease (CVD) patients, the investigators have demonstrated a significant mortality and MI reduction with PCSK9 antibodies and suggested a relevant ant ischemic/antiplatelet role of PCSK9 antibodies. These observations support the hypothesis that the anti-ischemic effects of PCSK9 inhibitor therapy are related to its pleiotropic effects on platelets.
In vitro, animal, and human studies have suggested the direct effects of PCSK9 and PCSK9 inhibitors on platelets. PCSK9 indirectly activates platelets through elevated LDL-C and oxLDL levels. oxLDL can activate platelets by binding to the scavenger receptors CD36 and LOX-1. In addition, a direct relationship between PCSK9 and platelet activation through the scavenger receptors CD36 and Lectin-like oxidized LDL receptor (LOX)-1 has been suggested. Incubation of human recombinant PCSK9 protein with platelet-rich plasma collected from healthy subjects significantly enhanced activated GPIIb/IIIa receptor expression and platelet aggregation induced by suboptimal concentrations of epinephrine. In in vitro experiments, human recombinant PCSK9 exposure enhanced LOX-1 expression in human vascular endothelial cells and smooth muscle cells. Similarly, LOX-1 KO mice showed lower PCSK9 levels and PCSK9-KO mice showed much lower LOX-1 levels, indicating a reciprocal relationship between PCSK9 and LOX-1. A similar reciprocal relationship between PCSK9 levels and LOX-1 and CD36 expression has not been demonstrated in human platelets.
In vitro immunoblotting experiments demonstrated the presence of PCSK9 in platelets obtained from patients with CAD, and flow cytometry experiments demonstrated that collagen-related peptide (CP) significantly enhanced the surface expression of PCSK9 on platelets. Furthermore, PCSK9 antibodies significantly inhibited ADP- and CP-induced platelet aggregation and platelet-dependent thrombus formation on the immobilized collagen surface under high shear rate (1000s-1). Binding of PCSK9 to CD36 triggers platelet activation through activation of nicotinamide adenine dinucleotide phosphate oxidase (Nox)2, release of reactive oxygen species (ROS), and subsequent activation of GPIIb/IIIa receptor and p-selectin release. In addition, Nox2 induces the generation of 8-Iso-prostaglandin F2α and thromboxane (Tx) A2 through the activation f cytosolic phospholipase A2 and finally results in the activation of the GP-IIb/IIIa receptor and p-selectin release. The relationship between plasma PCSK9, urinary 11-dehydro-TxB2, and CV events in patients with atrial fibrillation has been demonstrated. Furthermore, washed platelets resuspended in plasma from patients with familial hypercholesterolemia after PCSK9 inhibitor treatment induced lower platelet aggregation and soluble Nox2-derived peptide (sNox2-dp) release than those obtained using plasma before PCSK9 inhibitor treatment in an in vitro study. This reduction was reversed by the addition of oxLDL. Furthermore, oxLDL-induced platelet aggregation was reduced by inhibiting CD36, LOX-1, and Nox2. In the same study, 6-month treatment with PCSK9 inhibitors was shown to reduce serum LDL-C, oxLDL, serum TxB2, sNox2-dp, and PCSK9 levels.
These preliminary observations suggest that:
- CV benefits associated with PCSK9 inhibitor therapy may be in part related to its pleiotropic effects on platelets.
- In vitro, animal, and clinical studies suggested direct effects of PCSK9 on platelets.
- PCSK9 influence platelet function through CD36/LOX-1 receptor mediated Nox2 activity.
研究设计
- 研究类型
- Observational
- 观察模型
- Cohort
- 时间视角
- Prospective
入排标准
- 年龄范围
- 18 Years 至 50 Years（Adult）
- 性别
- All
- 接受健康志愿者
- 是
入选标准
- •Healthy subjects (n=40): 18-50 years old male and female subjects without any conditions or taking any agents or food supplements that may influence platelet function as determined by the study principal investigator.
- •Subjects with CV risk factors (n=40): 18-50 years old male and female subjects with at least three of the following CV risk factors:
- •History of hypercholesterolemia, hypertension, overweight, current smoker, prediabetes or diabetes who are only on metformin and insulin, and family history of coronary artery, peripheral artery, or cerebrovascular disease.
- •Overweight is defined as subjects with BMI ≥25 kg/m2 (https://www.heart.org/en/healthy-living/healthy-eating/losing-weight/bmi-in-adults).
- •Diabetes is defined as haemoglobin (Hb)A1c ≥6.5%, prediabetes =5.7%-6.4%) (https://diabetes.org/about-diabetes/diagnosis)
排除标准
- •Subjects with Diabetes mellitus, who are on GLP-1 agonists or SGLT2 inhibitors.
- •Subjects on high dose statin therapy or PCSK9 therapy.
- •Prior coronary of cerebrovascular event requiring intervention.
- •Use of any antiplatelet or anticoagulant therapy within 14 days of enrollment.
- •Subjects with a history of chronic kidney disease, liver disease, pancreatitis, HIV, hepatitis, and active cancer, active infection, or any other medical condition or concomitant medications as determined by the investigator that may affect study results.
研究组 & 干预措施
Healthy
Participants (approximately 40) 18-50 years old male and female without any major medical conditions or taking any agents or food supplements that may influence platelet function as determined by the study principal investigator.
干预措施: PCSK9 Antibody, PCSK9 Enzyme (Drug)
Cardiovascular Risk Factor
Participants (approximately 40) 18-50 years old male and female with at least three of the following risk factors: history of hypercholesterolemia, hypertension, overweight, current smoker, prediabetes or diabetes who are only on metformin and insulin, and a family history of coronary artery, peripheral artery, or cerebrovascular disease.
干预措施: PCSK9 Antibody, PCSK9 Enzyme (Drug)
结局指标
主要结局
Difference in ADP-induced platelet aggregation (%) with and without PCSK9 enzyme
时间窗: Throughout study completion, an average of about 1 year
Difference in ADP-induced platelet aggregation (%) with and without addition of PCSK9 enzyme in blood collected from healthy volunteers and subjects with cardiovascular risk factors.
Difference in collagen related peptide -induced platelet aggregation (%) with and without PCSK9 enzyme
时间窗: Throughout study completion, an average of about 1 year
Difference in collagen related peptide-induced platelet aggregation (%) with and without PCSK9 enzyme in blood collected from healthy volunteers and subjects with cardiovascular risk factors.
次要结局
- Difference in ADP-induced platelet aggregation (%) with PCSK9 enzyme alone and with addition of PCSK9 antibody(Throughout study completion, an average of about 1 year)
- Difference in collagen related peptide-induced platelet aggregation (%) with PCSK9 enzyme alone and with addition of PCSK9 antibody(Throughout study completion, an average of about 1 year)
研究者
Paul A. Gurbel
Director of Cardiovascular Research
LifeBridge Health