Abstract
Aims: In-stent restenosis (ISR) is a significant limitation of coronary stent implantation, but the exact mechanism of ISR remains unclear. Patients after percutaneous coronary intervention (PCI) are in a hypercoagulable state; however, there is less information on its association with chronic coronary artery disease (CAD) in patients with ISR after PCI. We aimed to clarify whether or not CAD patients with ISR after PCI are in a hypercoagulable state and whether or not PS exposure on extracellular vesicles (EVs), blood cells (BCs), and endothelial cells (ECs) is involved in the hypercoagulable state.
Methods: Phosphatidylserine (PS) exposure to EVs, BCs, and ECs was analyzed using flow cytometry. Procoagulant activity (PCA) was analyzed by clotting time (CT), purified clotting complex assays, and fibrin production assays.
Results: Compared with pre-PCI or controls, levels of exposed PS on EVs, BCs, and ECs were significantly increased from 1 day, peaked at 3 months, and gradually decreased within 1 year in CAD patients after PCI, especially in CAD patients with ISR after PCI. Furthermore, their increased levels significantly decrease CT and enhance intrinsic/extrinsic FXa, thrombin, and fibrin generation. PCA was weakened by approximately 80% when lactadherin was used.
Conclusions: Our results revealed that CAD patients after PCI, especially those patients with ISR after PCI, are associated with a hypercoagulable state in which PS exposure on EVs, BCs, and ECs plays a more important role than tissue factors. Therefore, blocking PS exposure to EVs, BCs, and ECs may provide a new target for preventing ISR in these patients.
Jihe Li, Wei Xia, and Yibing Shao are joint senior authors.
Introduction
Since the introduction of percutaneous coronary intervention (PCI) in cardiovascular medicine, it has become an important treatment option for stable angina, acute coronary syndromes, and coronary multivessel diseases1). However, with the use of stents in more complex lesions and clinically demanding situations, in-stent restenosis (ISR) has become the most important limiting factor for percutaneous coronary stenting2). There are numerous mechanisms for the pathogenesis of ISR, including biological or patient-related factors, anatomical factors, surgical factors, and stenting factors, and the prevalence of ISR is higher within one year of stent implantation3-5). It is well known that patients are in a hypercoagulable state after PCI6), but the relationship between this hypercoagulable state and chronic coronary artery disease (CAD) in patients with ISR after PCI has been poorly investigated. Furthermore, the efficacy of dual antiplatelet therapy (DAPT) after PCI in preventing ISR remains unclear7). Therefore, there is an urgent need to clarify the coagulation mechanism of patients with ISR after PCI in order to provide new ideas for the prevention of ISR.
Mechanistic studies on ISR have shown that both blood cells (BCs) and endothelial cells (ECs) are activated in these patients and participate in this process8). Our previous study also showed that non-ST-elevated myocardial infarction (NSTEMI) patients in a hypercoagulable state after PCI are associated with the activation of BCs as well as extracellular vesicles (EVs)9), a type of nanoparticle released by stimulated or apoptotic cells following plasma membrane remodeling that contains proteins and nucleic acids, thereby interacting with and modifying local and distant cellular targets.
Traditionally, EVs have been classified into four categories based on their size and biogenesis: exosomes, microvesicles, apoptotic bodies, and large oncosomes10-11). They can be released by all cardiac, endothelial, and blood cell types and play a procoagulant role in patients with certain cardiovascular diseases12). Previous studies have shown that EV levels are elevated in patients after PCI and associated with the induction of thrombin generation13). Therefore, we wondered whether CAD patients with ISR after PCI are associated with this hypercoagulable state and explored the mechanisms underlying hypercoagulability formation in these patients.
Phosphatidylserine (PS) is an anionic phospholipid that produces thrombin by providing a catalytic surface for the FXa and prothrombinase complexes14). PS is normally sequestered to the inner plane of the bilayer membrane and is only externalized when the cell is activated or apoptotic and when ECs are shed15). Lactadherin, a more sensitive PS probe than annexin V, is homologous to the PS-binding structural domains of clotting factors V and VIII and is proportional to PS levels16). It is used to detect PS exposure and acts as an anticoagulant by competing with the coagulation protein assembly on PS externalization. Tissue factor (TF) may also be an important source of EVs, and its presence significantly increases their procoagulant activity (PCA)17-18). However, changes in the levels of EVs of cellular or TF origin in ISR patients and whether or not PS or TF have an effect on coagulation activation remain to be determined.
Given the above, we explored the PCA of PS-exposed (PS＋) EVs, BCs, and ECs in CAD patients with ISR after PCI and used lactadherin to perform an inhibition assay, thus clarifying the contribution of PS or TF to coagulation activation in these patients.
Aim
In this study, we investigated whether or not CAD patients with ISR after PCI were in a hypercoagulable state and clarified the PCA of PS＋ EVs, BCs, and ECs. We used lactadherin to measure the origin and dynamics of EVs as well as to analyze the extent of PS exposure on BCs and ECs and their changes in CAD patients with ISR within one year after PCI. We further defined the clotting time (CT), intrinsic Xa, extrinsic Xa, thrombin, and fibrin formation in PS＋ EVs, BCs, and ECs and elucidated the dynamic changes within one year in these patients.
Methods
Patients
The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Qingdao Municipal Hospital. Written informed consent was obtained from all of the patients. Patients with CAD who were diagnosed with coronary stenosis by coronary angiography and had their first stent implanted at the Heart Centre of Qingdao Municipal Hospital between February 2021 and May 2023 were consecutively selected for this study. The target patients were limited to those 18-75 years old with no plan for revascularization within 1 year and who had completed the 1-year postoperative follow-up. ISR was clinically defined as recurrence of angina or objective evidence of myocardial ischemia. Angiography showed 50% diameter stenosis within the stented segment, including the stent and its edges (within 5 mm)19). During follow-up, patients with recurrent angina underwent immediate coronary angiography, and the extent of ISR was assessed using intravascular ultrasound (IVUS). CAD patients with ISR were to be included in the ISR group based on the angiographic and IVUS results, with the remaining asymptomatic patients undergoing coronary angiography at the end of one year of follow-up to determine whether or not ISR had occurred. The degree of ISR was assessed by IVUS.
Based on the results of coronary angiography and IVUS, we continued to follow patients with ＜75% lumen stenosis in the stent and to intervene in patients with ≥ 75% lumen stenosis in the stent. Patients who received intervention had follow-up terminated and withdrew from the study.
A total of 119 CAD patients, including 93 without ISR and 26 with ISR after PCI, met the inclusion and exclusion criteria and completed the 1-year follow-up after surgery. Fifty healthy individuals with normal physical examination results and no coronary artery pathology were recruited during the same period as the healthy control group. A flow chart of the control group and CAD patients with and without ISR after PCI during the 1-year follow-up period is shown in Supplementary Fig.1. The inclusion and exclusion criteria are described in detail in the Supplementary Materials section.
Reagents
Human umbilical vein endothelial cells (HUVECs) and EC medium were obtained from ScienCell (San Diego, CA, USA). The names and manufacturers of the remaining reagents are described in detail in the Supplementary Materials section.
Sample Preparation
Blood samples were collected from healthy controls and patients with or without ISR at the following time points: 24 h before PCI and 1 day, 1 month, 3 months, 6 months, and 12 months after PCI. Venous blood was collected in the morning with 21-gauge needles from patients who had fasted overnight, and the blood was preserved in tubes containing 3.2% sodium citrate. Isolation of platelet-rich plasma (PRP), platelet-free plasma (PFP), EV-depleted plasma (EDP), erythrocytes, and leukocytes is detailed in the supplemental material.
Flow Cytometric Analyses of PS Exposure on Total EVs, BCs, and ECs
Lactadherin binding was used to quantify PS exposure on total EVs, blood cells, and ECs using flow cytometry. See the Supplementary Materials section for further details.
Determination of the Levels of White Blood Cells (WBCs), Neutrophils, Red Blood Cells (RBCs), Platelets, D-dimer, and TAT
The WBC, neutrophil, RBC, platelet, D-dimer, TAT, and other laboratory values of the study participants were tested in the central laboratory of Qingdao Municipal Hospital.
Measurement of Coagulation Time, Intrinsic/ Extrinsic FXa and Prothrombinase Formation and Inhibition Assays
The coagulation times of total EVs, BCs, and ECs were evaluated by a one-stage recalcification time assay using a KC4A-coagulometer. The formation and inhibition assays of intrinsic/extrinsic FXa and pro-thrombinase in the presence of total EVs, BCs, and ECs are described in detail in the Supplementary Materials section.
Fibrin Formation and Inhibition Assays of Total EVs, BCs, and ECs
Fibrin formation was evaluated based on turbidity, and the process is described in detail in the Supplementary Materials section.
Statistical Analyses
Statistical analyses were performed using the SPSS v16.0 software program (SPSS Software Products, Chicago, IL, USA). Data are expressed as the mean±standard deviation (SD) and were compared using a one-way analysis of variance or Student’s t-test. The least significant difference (LSD) method was used for post-hoc tests after an analysis of variance (ANOVA). Ordered variables were assessed using the Kruskal-Wallis test. Spearman’s rank correlation was used to explore the relationships between specific continuous variables. P＜0.05 was considered to indicate statistical significance.
Results
Characteristics of Participants
The clinical features and laboratory findings of patients with CAD with (n = 26) or without (n = 93) ISR after PCI and healthy controls (n = 50) are presented in Table 1. Compared to controls, patients with and without ISR after PCI had higher values for the age, body mass index (BMI), WBCs, neutrophils, RBCs, platelets, D-dimer, fibrinogen, fasting glucose, creatinine, uric acid, triglyceride, total cholesterol, and low-density lipoprotein cholesterol; higher proportions of current smoking or drinking; lower high-density lipoprotein levels; and a shorter prothrombin time and activated partial thromboplastin time (Table 1, all P＜0.05). However, the proportions of smoking, hypertension, diabetes mellitus, and stroke as well as values for WBCs, neutrophils, RBCs, platelets, D-dimer, fibrinogen, creatinine, triglyceride, total cholesterol, high-density lipoprotein, and low-density lipoprotein cholesterol were all higher in patients with ISR after PCI than in those without ISR after PCI. Furthermore, CAD patients with ISR after PCI had a larger number of stents, longer stent lengths, and smaller stent diameters than those without ISR after PCI (Table 1, all P＜0.05). The remaining indicators were not significantly different between controls and patients with or without ISR after PCI.
Table 1.Clinical features and laboratory findings of the study participants
|
Controls (n = 50)
|
Non ISR (n = 93)
|
ISR (n = 26)
|
| Gender (M/F) |
27/23 |
51/42 |
15/11 |
| Age (years) |
43.25±3.67 |
55.63±8.58＃
|
56.12±8.57＃
|
| BMI (kg/m²)
|
22.53±2.97 |
25.73±4.13＃
|
24.65±3.56＃
|
| Current smoking (yes/no) |
9/41 |
19/74＃
|
6/12＃
|
| Current alcohol (yes/no) |
17/33 |
42/51＃
|
12/15＃
|
| Hypertension (yes/no) |
— |
56/37 |
20/6＊
|
| Diabetes mellitus (yes/no) |
— |
32/61 |
11/15＊
|
| Atrial fibrillation (yes/no) |
— |
15/78 |
4/19 |
| Stroke (yes/no) |
— |
9/82 |
6/20＊
|
| WBC (109/L)
|
4.55±0.68 |
6.48±1.27＃
|
7.16±1.36＃＊
|
| Neutrophils (109/L)
|
3.25±0.55 |
3.55±0.56＃
|
3.96±0.73＃＊
|
| RBC (1012/L)
|
5.15±0.38 |
5.65±0.55＃
|
5.96±0.56＃＊
|
| Platelets (109/L)
|
235.13±25.53 |
257.68±27.55＃
|
268.35±27.53＃＊
|
| PT (s) |
12.97±1.35 |
11.51±1.22＃
|
11.11±1.34＃＊
|
| APTT (s) |
32.16±3.56 |
27.42±3.42＃
|
25.55±3.46＃＊
|
| D-dimer (mg/L) |
0.33±0.11 |
0.76±0.15＃
|
0.92±0.19＃＊
|
| Fibrinogen (g/L) |
2.51±0.38 |
3.38±0.45＃＃
|
3.89±0.59＃＃＊
|
| Fasting glucose (mmol/L) |
4.53±1.39 |
6.17±1.85＃＃
|
5.97±1.76＃＃
|
| Cr (μmol/L) |
55.37±9.57 |
82.44±13.39＃＃
|
83.61±15.29＃＃
|
| Uricacid |
351.32±39.56 |
416.55±49.33＃＃
|
415.34±55.27＃＃
|
| TG (mmol/L) |
1.36±0.25 |
1.59±0.31＃
|
1.83±0.42＃＊
|
| TC (mmol/L) |
4.05±0.57 |
5.19±0.78＃
|
5.67±0.76＃＊
|
| HDL-C (mmol/L) |
1.21±0.18 |
1.03±0.19＃
|
0.95±0.26＃＊
|
| LDL-C (mmol/L) |
2.64±0.40 |
3.22±0.61＃
|
3.49±0.68＃＊
|
| Number of stent |
— |
1.83±0.75 |
2.67±1.17＊
|
| Total length of stent (mm) |
— |
46.35±22.16 |
55.76±33.45＊
|
| Stent diameter (mm) |
— |
3.32±0.45 |
2.83±0.39＊
|
Data are presented as means±SD or number (%). BMI: body mass index; WBC: white blood cell; RBC: red blood cell; PT: prothrombin time; APTT: activated partial thromboplastin time; Cr: creatinine; TG: triglyceride; TC: total cholesterol; HDL-C: high-density lipoprotein; LDL-C: low-density lipoprotein cholesterol.
＃P＜0.05, ＃＃P＜0.01 Vs Controls; ＊P＜0.05 Vs Non ISR. P＜0.05 was considered statistically significant.
Flow cytometry was used to measure EV levels in healthy controls and CAD patients with or without ISR within 12 months after PCI. Compared with pre-PCI patients and controls, the levels of total PS＋ total EVs/PEVs/ErEVs/LEVs/TF＋EVs/EEVs were significantly elevated in patients after PCI, especially in CAD patients with ISR after PCI, with a gradually increasing trend from 1 day, which peaked at 3 months and decreased gradually until 12 months. Total PS＋ EV levels were also positively correlated with hypercoagulation markers (D-dimer, TAT) at 3rd month after PCI (Fig.1A-F and Table 2, all P＜0.05). Flow cytometry analyses of PS exposure on RBC, PLT, and WBC counts in controls and CAD patients with ISR at the third month after PCI are presented in Supplementary Fig.2.
Table 2.Correlations between PS
＋ EVs/ErEVs/PEVs/LEVs/EEVs and hypercoagulability markers at 3 months after the PCI
|
Non ISR |
ISR |
| Variable |
D-dimer |
TAT |
D-dimer |
TAT |
| Total PS＋ EVs(/μL)
|
r = 0.612＊
|
r = 0.621＊
|
r = 0.744＊＊
|
r = 0.745＊＊
|
| LAC＋ RBC (%)
|
r = 0.535＊
|
r = 0.532＊
|
r = 0.673＊＊
|
r = 0.668＊＊
|
| LAC＋ PLT (%)
|
r = 0.621＊＊
|
r = 0.634＊＊
|
r = 0.742＊＊
|
r = 0.746＊＊
|
| LAC＋ WBC (%)
|
r = 0.611＊＊
|
r = 0.603＊＊
|
r = 0.719＊＊
|
r = 0.726＊＊
|
| LAC＋ ECs (%)
|
r = 0.501＊
|
r = 0.512＊
|
r = 0.657＊
|
r = 0.671＊
|
PCI: percutaneous coronary intervention; EV: Extracellular vesicle; ISR: LAC: lactadherin; TAT: thrombin–antithrombin complex.
＊P＜0.05, ＊＊P＜0.01. P＜0.05 was considered statistically significant.
Previous studies have shown that EVs can be derived from RBCs, platelets, WBCs and ECs20-21). Flow cytometry was used to measure the extent of PS exposure in BCs and ECs. Compared with pre-PCI or controls, CAD patients without ISR after PCI, especially CAD patients with ISR after PCI, had increased percentages of PS＋ RBCs, platelets, WBCs, and ECs from 1 day post-procedure, peaking at 3 months, and decreasing gradually until the 12th month (Fig.2A-D, all P＜0.05). Relationships between PS＋ BCs, PS＋ ECs, and hypercoagulation markers (D-dimer, TAT) were analyzed at 3 months when PS＋ BC and PS＋ EC levels were the highest. Interestingly, we found that the hypercoagulation markers (D-dimer, TAT) followed the same trend as the percentage of PS＋ RBCs, platelets, WBCs, and ECs and were positively correlated at 3 months after PCI (Fig.2E-F and Table 2, all P＜0.05).
PCA of EVs/RBCs/Platelets/WBCs and ECs in CAD Patients with or without ISR after PCI
A recalcification clotting time assay was used to assess CT. Compared with pre-PCI or controls, total PS＋ EVs/RBC/platelets/WBCs and ECs in CAD patients after PCI, especially in CAD patients with ISR after PCI, showed a gradually shortened CT from 1 day to 3 months, a minimum at 3 months, and a gradual extension from 3 months to 12 months (Fig.3A-E, all P＜0.05). To explore whether the PCA of EVs was associated with PS or TF, we performed inhibition assays. The results showed that lactadherin effectively prolonged the CT of total PS＋ EVs/RBCs/platelets/WBCs and ECs in CAD patients with or without ISR at 3 months after PCI, whereas the anti-TF antibody did not (Fig.3F, all P＜0.05), indicating that PS played an important role in the PCA of EVs.
We further explored PCA using intrinsic factor Xa, prothrombinase, and extrinsic factor Xa assays. Changes in intrinsic factor Xa, prothrombinase activity, and extrinsic factor Xa in BCs, ECs, and EVs followed the same trends as the changes in PS exposure in patients with or without ISR after PCI. The production of procoagulant enzyme complexes in both groups was higher than that in the healthy controls or pre-PCI, especially in the ISR group (Fig.4A and Fig.5A-D, all P＜0.05). Inhibition assays were also performed at 3 months, which was the longest time of PCA. Lactadherin significantly weakened the procoagulant enzyme complex activities of PS＋ EVs, BCs, and ECs, whereas the anti-TF antibody did not (Fig.4B and Fig.5E, all P＜0.05).
Fibrin deposition is an important step in neointimal formation, which is one of the main processes of ISR22). Thus, we determined fibrin formation using turbidity measurement assays. Compared with pre-PCI or controls, fibrin formation of total PS＋ EVs/RBC/platelets/WBCs and ECs was elevated in CAD patients after PCI, especially in CAD patients with ISR after PCI, with a gradually increasing trend from 1 day, peaking at 3 months and decreasing gradually until 12 months (Fig.6A-E, all P＜0.05). Lactadherin markedly inhibited fibrin formation, whereas anti-TF did not (Fig.6F, all P＜0.05).
PS Exposure on EVs, BCs and ECs can Predict Future Restenosis
We analyzed the predictive value of PS exposure on EVs, BCs, and ECs for ISR occurrence in patients with CAD 1 day after PCI using ROC curves. The results showed that the areas under the curve for PS exposure on EVs, RBC, PLT, WBC, and ECs were 0.861, 0.998, 0,837, 0.795, and 0.836, respectively (Supplementary Fig.3). We further confirmed that all of these indicators can predict future restenosis using Kaplan-Meyer analyses (Supplementary Fig.4, all P＜0.001).
Discussion
In this study, we identified four important findings. First, compared with pre-PCI or controls, EVs derived from BCs, ECs, and TFs in CAD patients after PCI, especially in CAD patients with ISR after PCI, were significantly elevated within 12 months. Second, compared to pre-PCI or controls, the levels of PS＋ RBCs, platelets, WBCs, and ECs were significantly higher in CAD patients after PCI, especially those with ISR after PCI, and their levels were strongly correlated with hypercoagulability markers. Third, in patients with CAD after PCI, especially in patients with ISR after PCI, the exposed PS on EVs, BCs, and ECs significantly shortened CT and increased both intrinsic/extrinsic Xase and fibrin generation. Fourth, lactadherin, a PS inhibitor, significantly attenuated PCA of EVs, BCs, and ECs, whereas anti-TF did not. In summary, we confirmed that CAD patients with ISR after PCI are in a hypercoagulable state, which can be partially attributed to elevated levels of PS exposure in EVs, BCs, and ECs.
Previous studies have shown that one of the causes of ISR is the alteration of the local hemodynamic environment induced by stent implantation, which negatively affects the physiological behavior of biomolecules (e.g. ECs)23). Studies of the pathological mechanism of ISR have shown that its etiology includes neointimal hyperplasia, elastic recoil, and negative arterial remodeling, with neointimal hyperplasia predominating. Its pathological mechanism is that intracoronary stent implantation induces local barotrauma with endothelial denudation and subsequent activation of the inflammatory response. Specifically, stent implantation results in endothelial damage and activation of vascular smooth muscle cells (VSMCs), with subsequent aggregation of platelets and fibrin at the site of injury. The activated platelets express P-selectin, which leads to the binding of WBCs to platelet plaques and their rolling along them. Activated WBCs can in turn bind tightly to ECs via adhesion molecules3-5, 8). Conversely, the relevant ECs and VSMCs release monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-18, etc., which contributes to the aggregation of inflammatory cells, such as monocytes and neutrophils, and triggers a series of inflammatory xenobiotics responses24-25). It has also been shown that RBCs, which make up the largest volume of blood at approximately 45%, remain in the stent implantation area for a long time and are prone to form granular stagnation zones in the arterial wall, where their erosive and depositional behaviors may damage vascular ECs and interfere with their physiological functions23). Our findings on patients’ baseline data are in agreement with the above studies; compared to controls or pre-PCI patients, CAD patients after PCI, especially those with ISR after PCI, had higher levels of WBCs, RBCs, and platelets.
Our study demonstrated that levels of PS＋ total EVs/PEVs/ErEVs/LEVs/TF＋ EVs/EEVs in CAD patients without ISR after PCI, especially in CAD patients with ISR, were elevated from 1 day to 12 months and peaked at 3 months. We believe that this was related to postoperative EC damage and BC activation. Damage to ECs after stent implantation leads to a series of inflammatory responses8, 24-25) associated with EC and BC activation, apoptosis, and release of EVs10), which consequently leads to increased EV levels. Relevant studies have shown that the VCAM-1 expression on coronary ECs in patients with myocardial infarction correlates with the severity of inflammation26) and that inflammation persists for 90 days after the procedure27), which is consistent with the trend of changes in the above indices. Zhou et al.’s study on EV levels after PCI showed that platelets, leukocytes, and total EVs continued to rise up to 2 days after PCI, which is consistent with our findings.
Our previous studies have shown that patients with NSTEMI after PCI are in a hypercoagulable state, and PS exposure on EVs and BCs promotes this hypercoagulable state9). However, whether or not CAD with ISR after PCI is associated with this hypercoagulable state and the specific mechanisms of this hypercoagulable state have not been clearly defined. We measured the degree of PS exposure on EVs, BCs, and ECs with lactadherin and showed that PS＋ EVs, RBC, platelets, WBCs, and ECs in CAD patients without ISR, especially those with ISR, increased from 1 day, peaked at 3 months, and then gradually decreased until 12 months but remained higher than values in pre-PCI patients and controls. We speculate that this is related to EC damage, recovery, and changes in the levels of inflammatory factors. EC damage after stent implantation leads to an inflammatory response. ECs are damaged and release inflammatory mediators (MCP-1, IL-18, etc.), which induce the activation of leukocytes and platelets and aggregation at the damaged endothelium8, 24-25). Consequently the levels of EVs derived from BCs, ECs, and PS exposed to these substances were also elevated. Previous studies have shown that the prevalence is higher within 1 year of stent implantation, and that the persistence of local inflammation for 90 days after PCI is associated with a high risk of ISR27-28). After 3 months, however, with the reversal of EC damage and the reduction in the levels of associated inflammatory factors, these markers gradually declined but remained higher than those in the pre-PCI or control groups. In addition, we further determined that CAD patients with ISR after PCI had significantly higher hypercoagulation markers (D-dimer and TAT), which followed the same trend as the percentage of PS＋ RBCs, platelets, WBCs, and ECs, and were positively correlated at 3 months after PCI. Thus, we confirmed that CAD patients with ISR after PCI were in a hypercoagulable state and were significantly correlated with BCs and ECs activation.
Studies have shown that PS exposure of BCs, ECs, and EVs leads to enhanced PCA in a variety of coagulation-related disorders, including cardiovascular disease, stroke, and nephrotic syndrome9, 17, 18). However, whether or not PS exposure on BCs, ECs, and EVs enhances PCA in CAD patients with ISR after PCI has not yet been studied. To clarify whether or not PS exposure led to the development of a hypercoagulable state in CAD patients with ISR, fibrin production, and inhibition tests were performed. Our results showed that PS exposure of EVs, BCs, and ECs significantly shortened the CT and increased intrinsic/extrinsic FXa and fibrin production. Tissue factor (TF) is the main initiator of cascade coagulation and is involved in a variety of noncoagulant activities29). Therefore, with anti-TF antibodies, inflammation-induced coagulation may be completely inhibited when TF is blocked. To exclude the role of TF, we performed inhibition assays using an anti-TF antibody and lactadherin. The results showed that lactadherin, a more sensitive PS probe than Annexin V, that acts as an anticoagulant by competing with the coagulin assembly for PS externalization, blocked nearly 80% of PCA, whereas the effect of the anti-TF antibody was relatively weak. This may be related to the fact that plasma TF is cryptic16). Previous studies have shown that fibronectin is deposited in ISR lesions, which supports our conclusions30).
Our study demonstrated that CAD patients with ISR after PCI had a larger number of stents, longer stent lengths, and smaller stent diameters than those without ISR after PCI. Furthermore, studies have shown that in addition to the above biological factors of ISR pathogenesis, stent design, stent length, stent overlap, number of stents, stent implantation location, and stent shape all have a significant impact on the development and severity of ISR3-5, 31). Bénard et al. found that stents further contribute to neointimal hyperplasia by influencing hemodynamics32). These studies support our findings. We believe that a greater number of stents, longer stent lengths, and narrower stent diameters would have a greater impact on hemodynamics, which would exacerbate the activation and apoptosis of relevant BCs and vascular ECs, increase levels of PS＋ BCs and ECs, and ultimately lead to the onset and progression of ISR33).
There are several limitations associated with the present study that should be recognized. Patients who did not agree to enroll in this study and those who underwent intracoronary intervention because of ISR or developed acute myocardial infarction during follow-up were excluded. These points have several potential implications. In addition, our study was a single-center study, which may have led to bias and merits further validation in a large-sample, multicenter, population-based cohort study.
Conclusions
Our findings suggest that CAD patients after PCI, especially CAD patients with ISR, are in a hypercoagulable state, which is associated with PS exposure on BCs, ECs, and EVs. Furthermore, lactadherin, a sensitive anticoagulant that competes with coagulation protein assembly on PS externalization, was able to significantly inhibit this hypercoagulable state. Therefore, competitive binding agents of PS coagulation proteins may be novel agents to weaken the hypercoagulable state in patients with ISR, thus reducing the development of ISR in patients with CAD after PCI.
Author Contributions
D.T. and L.K. contributed to the data collection, data analysis, manuscript drafting, Figure creation, critical revision, and final approval of the knowledge content. F.X. contributed to blood sample collection. W.W., B.S., G.L., C.Z., Y.L., and H.W. provided excellent technical assistance. J.L., Y.S., and W.X. contributed to critical revision and final approval of the knowledge content.
Funding
This study was funded by 2023-WJZD158 of the Qingdao Municipal Health Commission and Technological Benefits to the Public by Qingdao Municipal.
Acknowledgements
We thank F.X. for blood sample collection and D.T., B.S., G.L., C.Z., Y.L., and H. W. for excellent technical assistance.
Conflict of Interest
The authors declare no conflicts of interest.
Supplementary Methods
Patients
The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Qingdao Municipal Hospital. All patients signed an informed consent form. Patients with chronic coronary artery disease (CAD) who were diagnosed with coronary stenosis by coronary angiography and had their first stent implanted at the Heart Centre of Qingdao Municipal Hospital from February 2021 to May 2023 were consecutively selected for this study. These patients were limited to 18-75 years of age and had no plan for revascularization within 1 year, and completed 1-year postoperative follow-up. In-stent restenosis (ISR) was clinically defined as angina recurrence and or objective evidence of myocardial ischaemia. Angiography showed the presence of a 50% diameter stenosis within the stented segment, including the stent and its edges (within 5 mm)1). During follow-up, patients with recurrent angina underwent immediate coronary angiography and the extent of ISR was assessed by intravascular ultrasound (IVUS). CAD patients with ISR will be included in the ISR group, and the remaining asymptomatic patients during the follow-up period will have coronary angiography at the end of 1 year of follow-up to determine whether ISR occurred. Based on the results of coronary angiography and intravascular ultrasound, we continued to follow up patients with the degree of lumen stenosis in the stent was less than 75%, and for patients with the degree of lumen stenosis in the stent was greater than 75%, internal coronary intervention was performed. Patients who have received the intervention will terminate the follow-up and withdraw from our study. A total of 119 CAD patients, including 93 patients without ISR and 26 CAD patients with ISR after percutaneous coronary intervention (PCI), met the inclusion and exclusion criteria, and completed 1-year follow-up after operation. Fifty healthy individuals with normal physical examination and no coronary artery pathology were recruited during the same period as a healthy control group. Flow chart of control group, CAD patients with or without ISR after PCI during 12 months followed up is shown in Supplementary Fig.1.
Exclusion criteria for all patients were as follows: 1) Refused to participate in the study, participant in other clinical studies, life expectancy less than 1 year, mental illness, long-term work with radiation exposure, patients with severe renal dysfunction, lack of information during the follow-up period due to death or other reasons, acute coronary syndrome (ACS): including unstable angina, ST-segment elevation myocardial infarction (STEMI) and non-ST-segment elevation myocardial infarction (NSTEMI), patients with previous drug coated balloon intervention, percutaneous transluminal coronary angioplasty, and percutaneous coronary intervention, patients with sudden myocardial infarction or revascularization during the follow-up period; 2) Diseases affecting EVs as well as PS exposure on BCs and ECs: malignant tumors, benign/malignant neuroendocrine tumors, autoimmune diseases, acute and chronic infectious diseases, hypersplenism, myelopathic diseases: e.g., leukemia, myeloma, etc., and patients with long-term use of anti-tuberculosis drugs, chemotherapy drugs, etc.; 3) When the investigator considered that the patient was unable to complete the study or comply with the study.
CAD patients without atrial fibrillation received a combination of aspirin (loading dose 300 mg, 100 mg daily) and clopidogrel (loading dose 300 mg, 75 mg daily) prior to PCI, and CAD patients with atrial fibrillation were treated with warfarin in combination with aspirin (loading dose 300 mg, 100 mg daily) and clopidogrel (loading dose 300 mg, 75 mg daily) prior to PCI. The international normalized ratio (INR) was maintained between 2 and 3 before PCI. It was changed to warfarin combination with aspirin (100 mg daily) three months after PCI. Both treatment programmes lasted 1 year.
Reagents
Human umbilical vein endothelial cells (HUVECs) and endothelial cell medium were from ScienCell (San Diego, CA, USA). Trypsin-EDTA was from Gibco (Grand Island, NY). Purified CD31, CD41a, CD14 were from BD Biosciences (CA, USA). Alexa Fluor 488 or Alexa Fluor 647 conjugated lactadherin were prepared in our laboratory. Human factors Va, VIIa, VIII, X, Xa, IXa, prothrombin, and thrombin were all from Haematologic Technologies (Burlington, VT, USA). Isotype control antibody was from Dako (Carpinteria, CA, USA). Mouse anti-fibrin II chain (clone NYBT2G1) was from Accurate Chemical & Scientific (Westbury, NY, USA). Percoll was from GE Healthcare (Uppsala, Sweden). Tyrode’s buffer containing 1 mM Hepes was prepared in our laboratory and filtered through a 0.22-mm syringe filter from Millipore (UK).
Sample Preparation
We respectively collected blood samples from healthy controls and CAD patients with or without ISR at the following time points: 24 hours before PCI, and 1day, 1 month, 3 months, 6 months, and 12 months after PCI. Venous blood was collected in the morning with the 21-gauge needles from patients that had been fasted overnight, and the blood was then preserved in tubes containing 3.2% sodium citrate. Within 30 min after blood collection, platelet-rich plasma (PRP), erythrocytes and leukocytes from the samples were centrifuged for 15 min at 200 × g, 20℃, with a light brake and were analyzed immediately after isolation. Platelet-free plasma (PFP) from the samples were centrifuged for 20 min at 1,500 × g and then these plasmas were aspirated and re-centrifuged for 30 min at 2,000 × g. PFP aliquots of 0.25 ml were used immediately or quick-frozen in liquid nitrogen, and stored at −80℃ for further use. To isolate EVs, 250µl PFP was centrifuged for 30 min at 20,000 × g, removing 225 mL of EV-depleted plasma (EDP), and resuspending the EV pellet in the remaining 25 mL volume prior to storage at −80℃ for further use.
Flow Cytometric Analysis of PS Exposure on Total EVs, Blood Cells and ECs
Lactadherin binding was used for quantifying PS exposure on total EVs, blood cells and ECs via flow cytometry. The EVs-enriched suspension (5 µl) from each participant was resuspended in 35 µl Tyrode’s buffer, and then were identified as described previously2). EVs were identified via flow cytometry as smaller than 1 µm in size as well as lactadherin＋. The following surface markers were used to determine the cell type of origin for these EVs: CD235a＋ (RBC), CD41a＋ (platelet), CD45＋ (WBC), CD31＋ CD41a− (EC) and CD142＋ (TF). The number of each EV type per µL was calculated using Trucount Tube (with a precise number of fluorescent beads 48678 to determine the number of EVs in a sample) after accumulation of 5,000 gated events. RBCs, PLTs, WBCs and serum-cultured ECs were resuspended at 0.5–1 × 106/mL, and 5 µL AF488-lactadherin was added for 10 min at 25℃ protected from light. Then, 10,000 events per sample were acquired via flow cytometry, with BD FACSDiva used for result analysis.
Determination of the Levels of WBCs, Neutrophils, RBCs, Platelets, D-Dimer and TAT
The levels of WBCs, neutrophils, RBCs, platelets, d-dimer, TAT and other laboratory findings of the study participants were tested in the central laboratory of Qingdao Municipal Hospital.
Coagulation Time and Inhibition Assays of Total EVs, Blood Cells and ECs
The coagulation time of total EVs, blood cells and ECs were evaluated by one-stage recalcification time assay in a KC4A-coagulometer as previously described3). We incubated 100 µl of RBC (1 × 108), platelet (1 × 107), WBC (1 × 106), serum-cultured ECs (2 × 105) or EVs-containing suspensions (10 µl of EVs-enriched suspension was resuspended in 90 µl Tyrode’s buffer) with 100 µl of EV-free human plasma from control volunteers at 37℃, respectively. After 180 seconds, 100 µl of warmed 25 mM CaCl2 was added to start the reaction, and the clotting time was recorded. Inhibition assays were performed at the peaked time, these samples were incubated with 128 nM lactadherin or 40 µg/mL anti-TF at 37℃ for 10 min.
Intrinsic/ Extrinsic FXa and Prothrombinase Formation and Inhibition Assays
The formation of intrinsic/extrinsic FXa and pro-thrombinase in the presence of total EVs, blood cells and ECs were performed as previously described4). For the intrinsic FXase formation assays, 1 × 105 RBC, 1 × 104 platelets/WBC, 2 × 105 serum-cultured ECs or 10 µL of EV suspension were incubated with FIXa, FX, FVIII, thrombin and CaCl2 in FXa buffer for 5 min at 25℃, followed by addition of EDTA to stop the reaction. Measurement of extrinsic FXa formation was similar to that for intrinsic FXa except that cells or EVs were incubation with FX, FVIIa and Ca2＋. Each test was performed in triplicate. For prothrombinase measurements, samples were incubated with FXa, FVa, prothrombin, and CaCl2 at 25℃ for 5 min.
Fibrin Formation and Inhibition Assays of total EVs, blood cells and ECs
Fibrin formation was evaluated by turbidity as described5-6). Isolated EVs, RBC, platelets, WBC or serum-cultured ECs were added to recalcified (10 mM, final) EDP (88% EDP, final) in the circumstance of EDP isolated from healthy controls in the presence or absence of lactadherin (128 nM) or anti-TF (40 µg/mL). Fibrin formation was measured by turbidity at 405 nm in a Spectra Max 340PC plate reader. Each test was performed in triplicate. And inhibition assays were also performed at the highest point of 3 months, which EVs, BCs and ECs were added to recalcified (10 mM, final) EDP (88% EDP, final) in the absence /presence of lactadherin or anti-TF.
Statistical Analysis
Statistical analysis was performed using the SPSS v16.0 software. Data are expressed as mean±SD and compared using one way-ANOVA or Student t-test. Ordered variables were assessed via the Kruskal–Wallis test. Spearman’s rank correlation was employed for exploring the relationship between specifc continuous variables. P＜0.05 was considered statistically signifcant.
Supplementary References
1)Kibos A, Campeanu A, Tintoiu I. Pathophysiology of coronary artery in-stent restenosis. Acute Card Care, 2007; 9: 111-119
2)He Z, Si Y, Jiang T, Ma R, Zhang Y, Cao M, Li T, Yao Z, Zhao L, Fang S, Yu B, Dong Z, Thatte HS, Bi Y, Kou J, Yang S, Piao D, Hao L, Zhou J, Shi J. Phosphotidylserine exposure and neutrophil extracellular traps enhance procoagulant activity in patients with inflammatory bowel disease. Thromb Haemost, 2016; 115: 738-751
3)Fu Y, Zhou J, Li H, Cao F, Su Y, Fan S, Li Y, Wang S, Li L, Gilbert GE, Shi J. Daunorubicin induces procoagulant activity of cultured endothelial cells through phosphatidylserine exposure and microparticles release. Thromb Haemost, 2010; 104: 1235-1241
4)Antonelli A, Ferri C, Ferrari SM, Ghiri E, Goglia F, Pampana A, Bruschi F, Fallahi P. Serum levels of proinflammatory cytokines interleukin-1beta, interleukin-6, and tumor necrosis factor alpha in mixed cryoglobulinemia. Arthritis Rheum, 2009; 60: 3841-3847
5)Campbell RA, Overmyer KA, Selzman CH, Sheridan BC, Wolberg AS. Contributions of extravascular and intravascular cells to fibrin network formation, structure, and stability. Blood, 2009; 114: 4886-4896
6)Atkinson BT, Jasuja R, Chen VM, Nandivada P, Furie B, Furie BC. Laser-induced endothelial cell activation supports fibrin formation. Blood, 2010; 116: 4675-4483
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