long term serial changes in platelet activation indices following sirolimus eluting and bare metal stents implantation in patients with stable coronary artery disease

Long term serial changes in platelet activation indices following sirolimus eluting andbare metal stents implantation in patients with stable coronary artery disease Maria Marketou, Geor

Long term serial changes in platelet activation indices following sirolimus eluting and

bare metal stents implantation in patients with stable coronary artery disease

Maria Marketou, George E Kochiadakis, Aikaterini Giaouzaki, Katerini Sfiridaki,

Stelios Petousis, Fragiskos Maragoudakis, Konstantinos Roufas, Despoina Vougia,

John Logakis, Gregory Chlouverakis, Panos E Vardas

DOI: 10.1016/j.hjc.2017.01.009

Reference: HJC 118

To appear in: Hellenic Journal of Cardiology

Please cite this article as: Marketou M, Kochiadakis GE, Giaouzaki A, Sfiridaki K, Petousis S,

Maragoudakis F, Roufas K, Vougia D, Logakis J, Chlouverakis G, Vardas PE, Long term serial changes

in platelet activation indices following sirolimus eluting and bare metal stents implantation in patients with

stable coronary artery disease, Hellenic Journal of Cardiology (2017), doi: 10.1016/j.hjc.2017.01.009.

This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo

copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

M AN

IP T

Long term serial changes in platelet activation indices following sirolimus eluting and bare

metal stents implantation in patients with stable coronary artery disease

Maria Marketou,1 George E Kochiadakis, 1 Aikaterini Giaouzaki, 1 Katerini Sfiridaki,2 Stelios Petousis, 1 Fragiskos Maragoudakis, 1 Konstantinos Roufas, 1 Despoina Vougia, 1 John Logakis,1

Gregory Chlouverakis,3 Panos E Vardas. 1

1

: Cardiology Dept, Heraklion University Hospital, Crete, Greece

2

: Regional Blood Bank Centre, Venizelion Hospital, Crete, Greece

;3: Division of Biostatisctics, School of Medicine, University of Crete

Address for correspondence:

Maria Marketou

Cardiology Dept., Heraklion University Hospital

P.O Box 1352,

71110, Heraklion, Crete, Greece

Tel: +30 2810 392422 Fax: +30 2810 542055 or +30 2810 542111

E-mail: maryemarke@yahoo.gr

M AN

IP T

Abstract

Background: Platelet activation is crucial in the development of stent thrombosis following

percutaneous coronary intervention (PCI) We carried out a long-term assessment of multiple factors implicated in the thrombotic process and markers of platelet activation, after implantation

of sirolimus-eluting stents (SES) in patients with stable coronary artery disease (CAD), and we compared the results with those after bare-metal stent (BMS) implantation

Methods: Forty-seven consecutive patients, aged <70 years, with severe stenosis (>70%

narrowing of the lumen) of one major epicardial coronary artery and stable CAD, underwent successful elective PCI and were randomly allocated to SES (n=25) or BMS (n=22) Venous blood was obtained 24 hours before, and 24 hours, 48 hours, 1 month, and 6 months after PCI, for the measurement of plasma sP-selectin, von Willebrand Factor (vWF), fibrinogen, d-dimer, sCD40, factorVIII, b-thromboglobulin (b-TG) and platelet factor 4 (PF-4)

Results: There were no significant differences between the two groups in the levels of fibrinogen

and d-dimers in peripheral blood However, we observed a significant time effect (p<0.001) and a stent-effect (p<0.015) on vWF levels, and a significant time effect (p = 0.012) on factor VIII, sP-selectin (p=0.04), b-TG (p<0.001), and PF4 (p=0.016) A trend towards a statistically significant stent effect on sCD40 was also revealed (p=0.06)

Conclusions: SES and BMS do not show significant differences in relation to markers of platelet

activation and coagulation in patients with stable CAD Although some markers showed an increase after stent implantation, they returned to their initial levels 6 months later

M AN

IP T

Although drug-eluting stents (DES) are associated with significantly lower rates of in-stent stenosis than are bare-metal stents (BMS), early and late stent thrombosis is still an issue.1,2 Stent thrombosis is a rare, but potentially fatal complication of the percutaneous treatment of coronary artery disease (CAD) In patients undergoing coronary stenting, antithrombotic drugs are needed to prevent intraluminal thrombus formation Although these patients routinely receive dual antiplatelet treatment to reduce the risk of stent thrombosis, the later remains a dramatic complication following stent implantation, while it is estimated that the rate of acute and subacute thrombosis after percutaneous coronary intervention (PCI) with stent implantation may reach 16% in high-risk patients.3

One of the most important and most frequently used drugs in DES is sirolimus Notably, it has been reported that sirolimus significantly potentiates agonist-induced platelet aggregation in a time- and dose-dependent manner.4 In addition, DES are associated with delayed

endothelization, a condition that predisposes to thrombosis, and for this reason a longer period of antiplatelet therapy is mandated compared to BMS However, the mechanisms involved are not entirely clear, and the influence of these two types of coronary stent on platelet activation and the blood’s hemostatic system has not been fully clarified or understood

Platelet activation is crucial in the development of stent thrombosis following PCI In this study we assessed the behavior of platelet activation markers and other factors that are implicated in the thrombotic process after DES implantation in patients with stable CAD and we compared the results with those following BMS implantation The DES used were sirolimus-eluting stents (SES), and levels of sCD40, fibrinogen, factor VIII, von Willebrand factor (vWF),

M AN

IP T

d-dimer, sP-selectin, platelet factor 4 (PF-4), and beta-thromboglobulin (b-TG) were used as indices of thrombogenesis in peripheral blood

Methods

We enrolled 47 consecutive patients aged <70 years, with severe stenosis (>70% narrowing of the lumen) of one major epicardial coronary artery and stable CAD, who underwent successful elective PCI and were randomly allocated to SES (n=25) or BMS (n=22) Patients with any of the following were excluded from the study: in-stent restenosis, disease of the left main coronary artery, chronic total occlusion (>3 months), bifurcation stenting, adjacent stented segments >3

cm, periprocedural complications, prior PCI or bypass surgery; ejection fraction <55%; allergy to aspirin, heparin, or clopidogrel; significant valvular disease; pregnancy, myocarditis, history or signs of neoplastic or hematological disease; heart, renal or hepatic failure; history of any inflammatory disease during the last 6 months; and those who were heavy smokers Other exclusion criteria were a personal or family history of bleeding disorders or thrombotic events,

hematocrit levels <35% or >50%, and platelet counts <150,000/µL or >500,000/ µL

Cardiovascular medications were not discontinued for the study and treatment remained unchanged during the study period All patients were on dual antiplatelet therapy with aspirin

100 mg and clopidogrel 75 mg Clopidogrel loading (300 mg) was administered a day before coronary intervention while the patients were on aspirin for at least 7 days before the procedure

Blood samples were obtained 24 hours before PCI, at 24 hours and 48 hours after, and on the first visits 1 month and 6 months after the coronary intervention Study subjects were asked to refrain from eating food and drinking alcohol or coffee for 12 hours before every blood sampling All studies were performed between 9:00 and 11:00 a.m and all participants were rested for >30 min

M AN

IP T

in a supine position A medical history was obtained and a full clinical examination was performed on each visit

All participants gave written informed consent to their participation in the study The hospital’s ethics committee approved the study protocol

Biochemical Assays

Venous blood was obtained atraumatically 24 hours before, and 24 hours, 48 hours, 1 month, and

6 months after PCI, for the measurement of plasma sP-selectin, vWF, fibrinogen, d-dimer, sCD40, factor VIII, b-TG and PF- 4 Blood samples were immediately centrifuged at 3000 rpm for 20 minutes at 4°C, and the serum and plasma were separated and stored at -80°C until analyzed

sP-Selectin, sCD40, b-TG and PF- 4 were measured by enzyme-linked immunosorbent assay (ELISA, R&D Systems, Abingdon, United Kingdom) using commercial reagents, and results were reported in nanograms per milliliter The quantitative assay of vWF was based on the immunoturbidimetric determination of vWF antigen (vWF Ag, Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany) Fibrinogen was measured by modification of Clauss method (Multifibren U, Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany) Results were reported in grams per liter D-dimer levels were evaluated using a fully automated quantitative dimer assay, the particle-enhanced immunoturbidimetric assay Innovance D-DIMER (Siemens Medical Solutions, Marburg, Germany)

Statistical analysis

Summary descriptive data are presented as mean ± SD or frequency (%), as appropriate Repeated measures ANOVA was used to assess the time course (the within factor with 5 levels)

M AN

IP T

of various parameters that are implicated in the thrombotic process and to compare the drug-eluting and bare-stent groups (the between factor with 2 levels) Post-hoc Bonferroni-adjusted tests were employed in case of overall significant findings to pinpoint differences All statistical tests were performed at the two sided 5% level of significance, using the IBM-SPSS 21 statistical software package

Results

Demographic and clinical data, angiographic findings, and procedural variables were similar in the two groups (Table 1) Figure 1 shows the serial changes of the measured parameters in the two groups, SES and BMS, between the study time points before and after stenting All baseline measurements were comparable between the two groups

Our analysis did not reveal any significant differences between the two stent groups in the levels

of fibrinogen and d-dimers in peripheral blood However, repeated measures ANOVA showed a significant time effect (p<0.001) and a stent-effect (p<0.015) on vWF levels, and a significant time effect (p=0.012) on factor VIII, sP-Selectin (p=0.04), b-TG (p<0.001), and PF-4 (p=0.016)

A trend towards a statistically significant stent effect on sCD40 was also revealed (p=0.06) (Figure 1) Our analysis did not have sufficient statistical power to detect time-stent interactions between groups

The results of the post-hoc tests showed that vWF levels in the SES group were higher at 24 h and

48 h compared to baseline (p=0.06 and 0.02, respectively) At one and 6 months there was a significant decline from the 48 h levels to levels slightly, but not significantly above baseline In contrast, no significant increase was observed in the BMS group In addition, patients with SES showed a trend towards a statistically significant increase in factor VIII levels at 1 month and

sP-M AN

IP T

selectin levels at 6 months (p=0.08) On the other hand, PF-4 showed a significant increase at one month (p=0.01) only in the BMS group and its levels returned to baseline at 6 months Finally,

b-TG also showed a significant increase at 48 h only in the BMS group, which remained significant

at one month (p<0.01 for both) but returned to baseline at 6 months

Clinical parameters, such as smoking, diabetes mellitus, hypertension, hyperlipidemia, and stent length, did not significantly affect the time course of our parameters for either stent type

Discussion

In this study, we investigated for the first time, and in the long term, changes in multiple markers

of coagulation and platelet activation in the peripheral blood of patients with stable CAD undergoing elective PCI and we compared the effect of SES or BMS implantation We found that the majority of factors we studied did not show any substantial differences in behavior between the two stent groups Our analysis did not reveal any differences in the levels of fibrinogen and d-dimers in peripheral blood between the two groups of stents However, there was a trend towards

an increase in the levels of sCD40, vWF, factor VIII and sP-selectin levels after SES implantation, whereas PF-4 and b-TG levels intermittently showed a higher elevation during the study only in the BMS group

Increased activation of platelets is a consistent finding after coronary stent implantation5 and previous studies have reported changes in the expression of coagulation factors and in platelet activation.6,7 These could be modified by the selection of antithrombotic regimens Similarly, we hypothesized that the drug eluted by SES could influence platelet activation and thrombogenic factors after implantation, behavior that has not been clarified so far Our study is the first to

M AN

IP T

examine so many factors at the same time and over such a long follow up after SES implantation

and to compare the results with those from BMS in patients with stable CAD

Coronary thrombosis has long been recognized as a rare but catastrophic complication after stent implantation Stent thrombosis may lead to an acute myocardial infarction and death, while it has been

reported that 90% of these patients require an emergency reperfusion procedure.8 In patients undergoing

coronary stenting, antithrombotic drugs are needed to prevent intraluminal thrombus formation Although these patients routinely receive dual antiplatelet treatment to reduce the risk of stent thrombosis, it continues to be, albeit in a small number of patients, a dramatic complication following stent implantation The creation of in-stent thrombosis begins initially with platelet adhesion, while at the same time the body’s fibrinolytic and anticoagulant capability is deficient, as a result of factors that regulate the vascular endothelium Following its formation, the thrombus undergoes endothelization and becomes perfused by leukocytes and monocytes, while smooth muscle cells also ultimately migrate to the same site.9 In consequence, the onset, development, and formation of the thrombus within the stent—also causing restenosis—are a complication in which various factors are involved, influencing platelet adhesion, endothelial function, coagulation, and fibrinolysis Various conditions, such as incorrect deployment of the stent or a bad choice of size, a small stent, small vessel, bifurcation of the vessel, implantation of multiple stents, eccentric lesions, acute coronary syndrome, low ejection fraction, and subtherapeutic antiplatelet medication,10-13 are considered to be risk factors for the occurrence of thrombosis However, some patients show none of the above factors

An understanding of the mechanisms and the pathophysiology of stent thrombosis is crucial for its better treatment and prevention There is a debate in the literature about the extent to which SES, which have been proved to reduce in-stent restenosis, tend to increase the incidence of thrombosis Since thrombosis may not appear until late after stent implantation, we decided to conduct this long-term study

M AN

IP T

The thrombogenicity of DES is a matter of controversy and seems to result from a series of complex interactions involving the presence of a thrombogenic surface and the activation of platelets In a large cohort of unselected patients undergoing coronary stenting, there was a significant excess risk of stent thrombosis at 3 years with first-generation DES compared with BMS, driven by an increased risk of stent thrombosis events beyond 1 year Second-generation DES were associated with a similar risk of stent thrombosis compared with BMS.14 Another pooled analysis, including over 5000 patients in trials with drug-eluting stents, showed similar rates of stent thrombosis in patients receiving BMS or DES.15 However, little is known about the pathophysiology or mechanisms underlying this response, and changes in blood coagulation and the fibrinolytic system following stent implantation are of great interest Platelet and thrombin activation are important factors in the development of stent thrombosis.16 Stent thrombosis is a complex phenomenon that has not yet been fully elucidated We investigated whether sirolimus

may enhance thrombogenicity through the activation of platelets and coagulation proteins

Α previous in vitro study showed that there is no substantial difference in the thrombogenicity of

BMS and SES.17 However, that study compared the very early effects of these two types of stent

and conclusions about their long-term effects cannot be drawn We studied the thrombogenic effects of BMS and SES in the long term after implantation Although we found small differences in some factors, they were not sufficient to have a major impact on the clinical view with respect to thrombotic events

We demonstrated that patients with stable CAD undergoing SES implantation have significantly higher levels of vWF in peripheral blood immediately post-procedure, compared with patients receiving BMS The role of vascular endothelium in the pathogenesis of thrombus formation has