Cardiac hypertrophy is a common pathophysiological component of cardiac remodeling in many kinds of cardiovascular diseases, such as valvular heart disease, hypertension, and hypertrophic cardiomyopathy (Pillai et al., 2015). This process is usually considered to be a compensatory response of the heart to an increased hemodynamic load, which could cause contractile depression, ventricular dilatation, interstitial cardiac fibrosis, and eventually heart failure (HF) (Ooi et al., 2015). Studies have shown that platelet activation played a critical role in several cardiovascular diseases, such as HF (Schafer et al., 2003), coronary artery disease (George et al., 2016), and atrial fibrillation (Lim et al., 2014). Platelet activation is followed by platelet adhesion that is characterized by expression of variety of glycoproteins on platelet surface. In platelet-related thrombogenesis, platelets adhere to injured endothelium undergoing a conformational transition followed by activation and degranulation. This activation results in the combination of fibrinogen with the platelet surface receptors and consequently leading to thrombus formation (Santhakumar et al., 2015), thereby leading to coronary artery disease and HF.

Si-Miao-Yong-An decoction (SMYAD) is a traditional Chinese medicine formula, which consists of Lonicerae Japonicae Flos (Jinyinhua), Scrophulariae Radix (Xuanshen), Angelica Sinensis Radix (Danggui), and Glycyrrhizae Radix et Rhizoma (Gancao). SMYAD was reported to reduce the atherosclerosis plaque area, promote the recruitment of vasa vasorum pericytes, and stabilize atherosclerosis vulnerable plaques in ApoE^-/- mice (Qi et al., 2019). SMYAD has also been verified to ameliorate the stability of atherosclerotic plaque by lowering blood lipid in rabbit model (Peng et al., 2012). The formula was also reported to have anti-inflammatory and anti-oxidation properties (Wang and Tian, 2014). We previously demonstrated that SMYAD promoted isoprenaline-induced HF through antioxidant effects (Ren et al., 2019). However, the role of SMYAD in pressure overload-induced cardiac hypertrophy has yet not been explored.

In this study, we found that SMYAD attenuated pressure overload-induced cardiac dysfunction through inhibiting platelet aggregation and activation, suggesting SMYAD as a promising therapeutic agent for adverse cardiac remodeling.

HF represents a heterogeneous condition characterized by vulnerabilities in the blood, vasculature, and impaired flow dynamics that predispose to both arterial and venous thrombosis. Despite the fact that treatment with angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, beta adrenergic antagonists, and mineralocorticoid receptor antagonists in HF patients has improved long-term outcomes, morbidity or mortality remain high in patients with acute decompensated HF (Zannad et al., 2013). Furthermore, there has been little progress in preventing the adverse cardiac remodeling that initiates HF.

Immune activation, inflammation, oxidative stress, alterations in mitochondrial bioenergetics, and autophagy have been postulated as important pathophysiological events in HF (Ayoub et al., 2017). It is commonly recognized that platelet activation plays a main role in cardiovascular disease. There is evidence that platelet activation and hypercoagulability present in HF, and stroke were reduced by warfarin therapy in the HF patient (Gurbel and Tantry, 2014). Platelets normally maintained a quiescent state and were sensitive to homeostasis changes resulting from endothelial cell damage, stress conditions, and hemodynamic abnormality (Hansen et al., 2015). A variety of adhesion molecules were prestored inside platelet granules and expressed on the platelet surface responding to activation. β-TG and platelet factor 4 represented specific platelet proteins of alpha-granules, which could be secreted into surrounding medium during cell activation. It was demonstrated that β-TG and P-selectin were significantly associated with the occurrence of atrial fibrillation and consequent stroke (Weymann et al., 2017). Thus, platelet activation contributes to the progress of cardiovascular disease.

SMYAD is a famous traditional Chinese medicine formula that was mainly applied to treat thromboangiitis obliterans in China for centuries and presented satisfactory curative effect. The main function of SMYAD is activation of blood circulation to dissipate blood stasis and promote vital energy regulation. Clinically, SMYAD was reported to improve myocardial infarction and HF (Zhou et al., 2012). Recently, SMYAD has been verified to improve liver function and reduce fat accumulation in hyperlipidemia rat by accelerated transformation of cholesterol into bile acids (Liu et al., 2017b). It has not been reported whether SMYAD had effect on cardiac dysfunction induced by TAC. Thus, our study is aimed to investigate the effects and mechanisms of SMYAD on cardiac hypertrophy.

We demonstrated that SMYAD improved systolic function in HF mice, as well as modestly reduced LVID and LV Vol. However, the molecular mechanism by which SMYAD mediates its anti-hypertrophic effects remains unclear, and the signaling pathways that interact to drive hypertrophy are very complicated. To understand the molecular mechanisms responsible for SMYAD’s effects in HF, we investigated platelet, which has been shown to be activated in TAC-treated mice to induce cardiomyocyte hypertrophy and collagen synthesis (Wu et al., 2017a). We observed increased activation of cardiac CD41/61 and P-selectin in TAC-treated mice, as well as the reverse effect of SMYAD. P-selectin (CD62P) is a member of the selectin family of cell adhesion molecules and is stored on the membrane of platelet α-granules and endothelial Weibel–Palade bodies (Qi et al., 2015). It was the main platelet activation marker appearing on the surface of platelet membrane (Xie et al., 2014). It was reported that the expression of platelet P-selectin was increased in hyperlipidemic patients after ischemic stroke (Pawelczyk et al., 2015). In our study, SMYAD reduced the activation of platelet by downregulating P-selectin. P-selectin is responsible for the adhesion of certain leukocytes and platelets to the endothelium when thrombosis develops and inflammation is caused. It rapidly translocated to the cell surfaces of platelets and endothelial cells upon activation (Qi et al., 2015). Platelets can induce cell survival and growth by secreting several sorts of growth factors, including vascular endothelial growth factors, fibroblast growth factors, and platelet-derived growth factors. The plasma concentration of soluble P-selectin is now recognized as a predictor of adverse cardiovascular events (Ishida et al., 2014).

CD41 and CD61 are the main receptors mediating platelet aggregation, and CD61 is the most abundant receptor expressed on the platelet surface. Increased expression of CD61 on platelet surfaces results in enhanced fibrinogen binding and, subsequently, platelets cross-linking and thrombogenesis. SMYAD is suggested to inhibit platelet aggregation by CD41/61 expression and the elevation of vWF. CD41/61 and P-selectin significantly increased in atrial fibrillation patient with hypercoagulable state (Zhang et al., 2018). Besides, the prothrombotic state was characterized by the expression of CD41/61 and P-selectin mediating binding of vWF and fibrin interactions that led to different abnormalities in platelet function (Karmakar et al., 2015). In our study, inhibition of platelet activation by SMYAD reduced the infiltration of inflammatory cell into the heart.

Anticoagulants (e.g., warfarin) and antiplatelet agents (e.g. aspirin) are the principle antithrombotic agents. Many HF patients with sinus rhythm took aspirin because coronary artery disease was the leading cause of HF (Shantsila and Lip, 2016). The prescription of antiplatelet agents, such as clopidogrel and aspirin, has indeed become a stronghold of atherosclerotic cardiovascular disease and heart attack therapy. The therapeutic efficiency of clopidogrel is manipulated by the actions of hepatic cytochrome P450 (CYP) enzymes and influenced by individual genetic variations. The metabolism of clopidogrel into its active metabolites is affected by individual polymorphisms of CYP enzymes (Jarrar et al., 2016). Patients with high on-treatment platelet reactivity upon clopidogrel were at increased risk for thrombotic events, particularly for stent thrombosis and myocardial infarction, but cardiovascular mortality was also elevated in patients undergoing percutaneous coronary intervention (Gross et al., 2016). It was reported that aspirin use after discharge for HF hospitalization was associated with reduced risk of death, all-cause readmission, and HF readmission (Kim et al., 2016). However, data on aspirin in HF were inconclusive because of bleeding complications. There was evidence that a lower risk for death or stroke was associated with warfarin therapy in the HF patient (Hart et al., 2007). Warfarin (vitamin K antagonist) blocks multiple steps of coagulation by reducing the synthesis of vitamin K-dependent coagulation factors. As warfarin was metabolized by the CYP isoenzymes, the pharmacokinetics and pharmacodynamics of warfarin were influenced by interactions with other drugs that were metabolized by the same CYP isoenzymes (Gurbel and Tantry, 2014). Thus, it is of great urgency to find anti-platelet drugs with fewer adverse reaction.

Persistent hemodynamic overload will result in pathological cardiac hypertrophy, which is a leading predictor for the development of HF. In the present study, we used a TAC mice model to mimic pressure overload-induced stress. We demonstrated that SMYAD improved pressure overload-induced cardiac dysfunction and remodeling, and promoted histological changes in heart tissues. We also found that SMYAD decreased protein expressions of platelet aggregation markers (CD41, CD61) and platelet activation marker (P-selectin) in pressure overload-induced failing hearts. We have previously isolated and identified some chemical constituents, but the relationship between pharmacological effects and chemical constituents needs to be further studied.

The healthy endothelium in large vessels has multiple mechanisms that inhibit the adhesion or activation of platelets, either directly or by actively degrading platelet agonists. One of the earliest events in atherosclerosis is the loss of normal endothelial functions, including disruption of anti-platelet mechanisms. Endothelial cells respond to injury by releasing numerous factors, including vWF. vWF is an established marker of endothelial activation, and patients with elevated plasma levels of the ultra large vWF molecules are at high risk for future cardiovascular events (Warlo et al., 2017). vWF is a glycoprotein involved in both platelet activation and aggregation through its binding sites for GpIb and GpIIb/IIIa, respectively. High circulating levels of unusually large vWF multimers have strong procoagulant activity and facilitate platelet adhesion and aggregation by interacting with platelets after an acute event superimposed on coronary artery disease (Akyol et al., 2016). There was increasing evidence that platelet–endothelial interactions also contributed to early atherosclerotic plaque initiation and growth (Wu et al., 2017b). Platelets may also play a key role in neutrophil extracellular traps (Carestia et al., 2016).Through these interactions, platelet-derived factors can contribute to the pro-inflammatory and mitogenic status of resident mural cells. There was evidence that transendothelial migration of platelet monocyte complexes may result in dissociation and surface deposition of platelets (van Gils et al., 2008). In the next study, we will further investigate the interaction between platelet, endothelial cells, and neutrophil.

In this study, we characterized the property of SMYAD against cardiac hypertrophy. SMYAD inhibited pressure overload-induced platelet activation, cardiac hypertrophy, and dysfunction. It inhibited TAC-induced hypertrophy, myocardial necrosis, platelet aggregation, and platelet activation. CD41/CD61 served as a mediator of the platelet aggregation effect, while P-selectin/β-TG mediated the platelet activation effect. This study provided evidence that SMYAD may be a promising therapeutic agent for adverse cardiac remodeling.

All datasets generated for this study are included in the manuscript and/or the Supplementary files.

All the animal experiments were performed in accordance with the “Guide for the Care and Use of Laboratory Animals” by the National Institutes of Health. All studies were conducted in line with the approval of the Animal Care Committee of Beijing University of Chinese Medicine.

CS, SG, WW, and SL conceived and designed the experiments. CS, QW, HZ, WJ, and HL carried out the experiments, analyzed the data, and drafted the manuscript. BL, WW, XY, LL, and XC participated in its design and prepared the paper. SL, SG, and QW revised the manuscript. All authors read and approved the final manuscript.

This study was funded by the National Nature Science Foundation of China (grant NO. 81874387, 81774101, and 81703942) and the Fundamental Research Funds for the Central Universities (2019-JYB-TD-002 and 2019-JYB-XS-012).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.