Aspirin resistance in adult patients after Fontan surgery Lidia Tomkiewicz-Pajak, Tomasz Wojcik, Stefan Chłopicki, Maria Olszowska, Jacek Pajak, Jakub Podolec, Barbara Sitek, Piotr Musiałek, Paweł Rubis, Monika Komar, Piotr Podolec PII: DOI: Reference:
S0167-5273(14)02402-4 doi: 10.1016/j.ijcard.2014.11.219 IJCA 19376
To appear in:
International Journal of Cardiology
Received date: Revised date: Accepted date:
21 September 2014 15 November 2014 26 November 2014
Please cite this article as: Tomkiewicz-Pajak Lidia, Wojcik Tomasz, Chlopicki Stefan, Olszowska Maria, Pajak Jacek, Podolec Jakub, Sitek Barbara, Musialek Piotr, Rubis Pawel, Komar Monika, Podolec Piotr, Aspirin resistance in adult patients after Fontan surgery, International Journal of Cardiology (2014), doi: 10.1016/j.ijcard.2014.11.219
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.
ACCEPTED MANUSCRIPT
1
Original article
T
Aspirin resistance in adult patients after Fontan surgery
IP
Lidia Tomkiewicz-Pajak1, Tomasz Wojcik2, Stefan Chłopicki2,3, Maria Olszowska1,
SC R
Jacek Pajak3 , Jakub Podolec4, Barbara Sitek2, Piotr Musiałek1 , Paweł Rubis1, Monika
1
NU
Komar1, Piotr Podolec1
Department of Cardiac and Cardiovascular Diseases, Institute of Cardiology, Jagiellonian
MA
University Medical College and John Paul II Hospital, Krakow, Poland; 2Jagiellonian Centre for Experimental Therapeutics (JCET), Bobrzynskiego 14, Krakow, Poland; 3Department of
D
Experimental Pharmacology, Chair of Pharmacology, Jagiellonian University, Medical
TE
College, Grzegorzecka 16, Krakow, Poland; 4Department of Pediatric Cardiology, Silesian
CE P
Pediatric Medical Center, Katowice, Poland; 5Department of Interventional Cardiology, Institute of Cardiology, Jagiellonian University Medical College and John Paul II Hospital,
AC
Krakow, Poland
Address correspondence to: Lidia Tomkiewicz-Pajak, MD, PhD Institute of Cardiology, Jagiellonian University Medical College 80 Pradnicka St., 31-202 Cracow, Poland phone: +48-126142287 fax: +48-12 423 43 76 e-mail: [email protected]
2
ACCEPTED MANUSCRIPT
T
There are not conflicts of interest.
SC R
inflammation, endothelial function, aspirin resistance
List of abbreviations:
NU
FC – Fontan circulation G-CSF- granulocyte colony-stimulating factor ,
MA
MCP-1- monocyte chemotactic protein-1,
MIP-1 β - macrophage inflammatory protein-1β,
D
sVCAM-1- vascular cell adhesion protein 1,
TE
SVEF – single ventricle ejection fraction, TAT – thrombin antithrombin
CE P
TNFα-Tumor necrosis factor-alfa, TXB2 - thromboxane B2
vWF- von Willebrand Factor,
AC
IP
Key words: Fontan surgery, platelet reactivity, thromboxane B2 generation test,
ACCEPTED MANUSCRIPT
3
Structured abstract Background: Thrombotic complications are common in adult patients who have had a
T
Fontan operation early in life for treatment of congenital heart disease.
IP
Objective: To characterize platelet function and responsiveness to aspirin in relation to
SC R
thrombogenesis, systemic inflammation, and markers of endothelial function in adults with Fontan circulation (FC).
NU
Methods: Thirty-four FC patients (age 18-40 years; 62% taking aspirin chronically and 38% not taking aspirin) and 32 age- and sex-matched healthy controls were studied. Platelet
MA
function was evaluated by measurement of basal concentrations of thromboxane B2 (TXB2) and sCD40L and ex-vivo generation of TXB2 and sCD40L. Plasma concentrations of
D
thrombin-antithrombin, endothelin-1, vWF, IL-6, IL-8, MCP-1, MIP-1β, TNFα, sVCAM-1,
TE
and syndecan-1 also were measured.
CE P
Results: Platelet numbers were significantly lower in FC patients than in controls, but the patients had significantly higher platelet activity, as evidenced by higher TXB2 and sCD40L concentrations and higher ex vivo generation of TXB2. Chronic aspirin treatment had no
AC
effect on plasma concentrations of TXB2 and sCD40L in FC, but in 52% of aspirin-treated FC subjects, TXB2 concentrations remained elevated at 60 min of TXB2 generation, indicating aspirin resistance. In addition, FC patients had increased levels of thrombin-antithrombin, endothelin-1, vWF, IL-8, MCP-1, MIP-1β, TNFα, sVCAM-1, and syndecan-1 but not of IL-6. Conclusion: Adults with FC had lower platelet numbers but increased platelet activity, increased thrombogenesis, systemic inflammation, and endothelial dysfunction. A significant proportion of aspirin-treated FC adults had aspirin resistance, which may be at least in part responsible for their increased incidence of thrombotic complications.
ACCEPTED MANUSCRIPT
4
Introduction
T
The Fontan operation is performed for treatment of various complex congenital heart
IP
malformations. The purpose of the operation, in which the systemic venous return is
SC R
connected to the pulmonary arteries without the interposition of a pumping chamber (1), is to separate the pulmonary and systemic circulation and achieve normal or near normal levels of arterial oxygen saturation.
NU
Long-term follow-up of patients with Fontan circulation (FC) has revealed increased rates of late complications, including thrombosis. The reported incidence of this life-threatening
MA
complication in FC patients ranges from 17% to 33% (1, 2). The pro-thrombotic risk factors include previous thrombosis, type of arteriopulmonary connection, presence of
TE
D
thrombogenic foreign material, arrhythmias, slow venous flow, elevated central venous pressure, ventricular dysfunction, liver injury, effusion protein-losing enteropathy, and
CE P
prolonged immobilization (3).
The mechanisms leading to thromboembolism in patients with FC remain to be elucidated.
AC
Abnormalities in any of the regulatory mechanisms in hemostasis - plasma coagulation/fibrinolytic proteins, platelets, and endothelial function - might contribute to increased thrombotic risk (4). Indeed, changes in plasma activity of coagulation factors and inhibitors, endothelial dysfunction, and enhanced platelet activation have been reported in children after Fontan surgery (5-7). However, only a few studies of hemostatic abnormalities in adult patients, late after Fontan surgery (8-10), have been reported. Also, optimal antithrombotic treatment for adult FC patients is controversial (3); some authors have reported that Fontan patients taking either aspirin or warfarin had lower thromboembolic rates than those not on antithrombotic therapy, but others have found no benefit of the therapy (3, 8, 9).
ACCEPTED MANUSCRIPT
5
Increased platelet activation has been described in some patients with FC (4, 6). Accordingly, long-term antiplatelet therapy for prevention of thrombosis after Fontan operations is a
T
reasonable consideration, even though thrombosis is a common feature (2-4). However, it is
IP
not known whether patients after Fontan operation respond to aspirin and whether aspirin
SC R
resistance is prevalent in them (11-13). In reported studies, aspirin resistance has been defined by the presence of incomplete inhibition of ex vivo platelet aggregation induced by
NU
arachidonic acid, suboptimal inhibition of platelets in various other ex vivo tests (14-16), or increased thromboxane B2 (TXB2) metabolites in urine (13). Recently, Dobaczewski, et al.
MA
(17) suggested that in healthy and streptomycin-induced diabetic rats, measurement of dynamic generation of TXB2 is a more sensitive determination of aspirin resistance than is
D
measurement of plasma concentration of TXB2 and urine concentration of 11-dehydro-
TE
TXB2..
CE P
In the present work we aimed to characterize platelet responsiveness to aspirin in patients with FC on the basis of dynamic generation of TXB2 and sCD40L in ex vivo assay as well as
AC
to characterize thrombogenesis, inflammation, and endothelial function.
Material and methods
Study participants We studied 21 men (62%) and 13 women (48%) who had received a Fontan operation and 32 healthy control subjects matched for age, sex, and basal metabolic index. Main exclusion criteria were: age below 18 years, neoplastic diseases, infection, inflammation, major trauma within the three months preceding the study, pregnancy, alcohol abuse and diabetes mellitus. Healthy subjects were not receiving any medication.
ACCEPTED MANUSCRIPT
6
Study protocol
T
Patients’ previous interventions, complications, medical therapy, and clinical, demographic,
IP
and anatomic data were collected at the time of the examination from the medical records.
SC R
Cardiac echocardiography was performed in all patients, and single ventricle ejection function (SVEF) was assessed. Arterial blood oxygen saturation (SaO2) was measured by means of
NU
transcutaneous pulse oximetry with subjects breathing room air. Twenty one (62%) patients were taking aspirin; 13 (38%) were receiving either warfarin (10) or enoxaparin (3) (Figure
MA
1).
Warfarin was replaced with enoxaparin for five days before the study. Aspirin had been taken
D
in doses 75 mg/day for at least seven day before the study. To minimize the influence of
TE
concomitant medications on platelet function, all of them were withheld for 24 hours prior to
CE P
the study.
The University Ethical Committee approved the study, and all the subjects provided written,
AC
informed consent.
Laboratory investigations Blood samples were collected from the antecubital vein after overnight fasting for at least 12 hours. White blood cells (WBC), platelet count, red blood cells (RBC), hematocrit (HCT), hemoglobin (HGB), total protein, alanine aminotransferase (ALT), gamma glutamyl transpeptidase (GGTP), cystatin C, creatinine, C-reactive protein (CRP), serum bilirubin, international normalized ratio (INR), N-terminal pro B-type natriuretic peptide (NT-proBNP) were assayed with routine laboratory techniques. Plasma thrombin-antithrombin concentration was measured with ELISA (Siemens).
Platelet and endothelial cell activation
7
ACCEPTED MANUSCRIPT
Plasma samples were analyzed with human soluble vascular cell adhesion protein-1 (sVCAM1) and Endothelin-1 Quantikine ELISA kits (R&D Systems), according to manufacturer’s
T
instructions. Syndecan-1 was detected with a Human Syndecan-1 DuoSet ELISA
IP
Development kit (R&D Systems). Plates were coated with 0.8 g/ml capture antibody in
SC R
phosphate-buffered saline solution and incubated overnight at room temperature, then washed three times with 400 ml of wash buffer and blocked by 1-h incubation with 300 l of
NU
reagent diluent. Plates were washed as in the previous step, 100 l of samples and standards per well were added, and the plates were incubated for 2 h at room temperature and washed as
MA
in the previous step. Next, 100 l of 400 ng/ml of detection antibody per well were added, and the plates were incubated 2 h at room temperature and washed. One hundred microliters of
D
the working dilution of streptavidin-horse radish peroxidase were added to each well, and the
TE
plates were incubated for 20 min in the dark at room temperature and washed as above. Next,
CE P
100 l per well of 2N H2SO4 were added to stop the reaction. Optical density of each well was read immediately with a microplate reader set to 450 nm. The wavelength reaction was
AC
set to 570 nm. Plasma von Willebrand Factor (vWF) antigen was also measured (Diagnostica Stago, Asnieres, France).
Cytokines/chemokines analysis Bio-Plex Pro human cytokine, chemokine, and grow factors magnetic beads kit (Bio-Rad) were used, in accordance with the manufacturer’s instruction. Data were acquired on a validated and calibrated Bio-Plex 200 system (Bio-Rad) and analyzed with Bio-Plex Manager 6.0 software (Bio-Rad) with a detection target of 50 beads per region, low RP1 target for CAL2 calibration, and recommended doublet discriminator gates of 5000–25,000. We also measured interleukin 6 (IL6), interleukin 8 (IL8), granulocyte colony-stimulating factor (GCSF), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1β
ACCEPTED MANUSCRIPT
8
(MIP-1β). Tumor necrosis factor-alfa (TNFα) was measured with an ELISA method (R&D Systems).
T
Dynamic generation of TXB2 and sCD40L
IP
Blood was collected into vials (Monovette®) containing heparin as an anti-coagulant agent.
SC R
Briefly, an aliquot of whole blood was supplemented with 500 μM aspirin immediately upon withdrawal, then centrifuged (2320×g, 12 min), and the separated effluent was frozen (−20
NU
°C) until assayed for TXB2 or sCD40L. Another portion of the sample was diluted 5 times with buffered saline. The specimens were incubated for 1 h at 37oC with continual stirring.
MA
At 0, 30, and 60 min of incubation, 200 μl of sample were taken and supplemented with 500 μM aspirin and processed before further analyses in the same way as described above. The
TE
analyses (stored at -20 °C).
D
rest of the collected blood was centrifuged (1000 g, 15 min) to separate plasma for cytokine
CE P
TXB2 concentrations were measured by use of a previously validated enzyme-linked immunoassay method (EISA-kit by Enzo Life Sciences, sensitivity 10,54 pg/mL), and sCD40L concentrations were measured by use of R&D Systems ELISA kit (sensitivity 10.1
AC
pg/mL), both according to the manufacturers’ instructions. Obtained concentrations were normalized for each individual’s platelet count. Statistical analysis Continuous data were expressed as median (interquartile range) and categorical variables as frequencies and percentages. Categorical values were analyzed by use of the Chi2 test. The standard Student’s t-test was used for comparison of data having no departures from normality (according to D’Agostino and Person omnibus test), and the nonparametric Mann– Whitney U-test was used for the remaining variables. Correlations between the individual measurements were calculated by use of the Pearson or Spearman rank correlation, as
9
ACCEPTED MANUSCRIPT
appropriate. A p-value
S0167-5273(14)02402-4 doi: 10.1016/j.ijcard.2014.11.219 IJCA 19376
To appear in:
International Journal of Cardiology
Received date: Revised date: Accepted date:
21 September 2014 15 November 2014 26 November 2014
Please cite this article as: Tomkiewicz-Pajak Lidia, Wojcik Tomasz, Chlopicki Stefan, Olszowska Maria, Pajak Jacek, Podolec Jakub, Sitek Barbara, Musialek Piotr, Rubis Pawel, Komar Monika, Podolec Piotr, Aspirin resistance in adult patients after Fontan surgery, International Journal of Cardiology (2014), doi: 10.1016/j.ijcard.2014.11.219
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.
ACCEPTED MANUSCRIPT
1
Original article
T
Aspirin resistance in adult patients after Fontan surgery
IP
Lidia Tomkiewicz-Pajak1, Tomasz Wojcik2, Stefan Chłopicki2,3, Maria Olszowska1,
SC R
Jacek Pajak3 , Jakub Podolec4, Barbara Sitek2, Piotr Musiałek1 , Paweł Rubis1, Monika
1
NU
Komar1, Piotr Podolec1
Department of Cardiac and Cardiovascular Diseases, Institute of Cardiology, Jagiellonian
MA
University Medical College and John Paul II Hospital, Krakow, Poland; 2Jagiellonian Centre for Experimental Therapeutics (JCET), Bobrzynskiego 14, Krakow, Poland; 3Department of
D
Experimental Pharmacology, Chair of Pharmacology, Jagiellonian University, Medical
TE
College, Grzegorzecka 16, Krakow, Poland; 4Department of Pediatric Cardiology, Silesian
CE P
Pediatric Medical Center, Katowice, Poland; 5Department of Interventional Cardiology, Institute of Cardiology, Jagiellonian University Medical College and John Paul II Hospital,
AC
Krakow, Poland
Address correspondence to: Lidia Tomkiewicz-Pajak, MD, PhD Institute of Cardiology, Jagiellonian University Medical College 80 Pradnicka St., 31-202 Cracow, Poland phone: +48-126142287 fax: +48-12 423 43 76 e-mail: [email protected]
2
ACCEPTED MANUSCRIPT
T
There are not conflicts of interest.
SC R
inflammation, endothelial function, aspirin resistance
List of abbreviations:
NU
FC – Fontan circulation G-CSF- granulocyte colony-stimulating factor ,
MA
MCP-1- monocyte chemotactic protein-1,
MIP-1 β - macrophage inflammatory protein-1β,
D
sVCAM-1- vascular cell adhesion protein 1,
TE
SVEF – single ventricle ejection fraction, TAT – thrombin antithrombin
CE P
TNFα-Tumor necrosis factor-alfa, TXB2 - thromboxane B2
vWF- von Willebrand Factor,
AC
IP
Key words: Fontan surgery, platelet reactivity, thromboxane B2 generation test,
ACCEPTED MANUSCRIPT
3
Structured abstract Background: Thrombotic complications are common in adult patients who have had a
T
Fontan operation early in life for treatment of congenital heart disease.
IP
Objective: To characterize platelet function and responsiveness to aspirin in relation to
SC R
thrombogenesis, systemic inflammation, and markers of endothelial function in adults with Fontan circulation (FC).
NU
Methods: Thirty-four FC patients (age 18-40 years; 62% taking aspirin chronically and 38% not taking aspirin) and 32 age- and sex-matched healthy controls were studied. Platelet
MA
function was evaluated by measurement of basal concentrations of thromboxane B2 (TXB2) and sCD40L and ex-vivo generation of TXB2 and sCD40L. Plasma concentrations of
D
thrombin-antithrombin, endothelin-1, vWF, IL-6, IL-8, MCP-1, MIP-1β, TNFα, sVCAM-1,
TE
and syndecan-1 also were measured.
CE P
Results: Platelet numbers were significantly lower in FC patients than in controls, but the patients had significantly higher platelet activity, as evidenced by higher TXB2 and sCD40L concentrations and higher ex vivo generation of TXB2. Chronic aspirin treatment had no
AC
effect on plasma concentrations of TXB2 and sCD40L in FC, but in 52% of aspirin-treated FC subjects, TXB2 concentrations remained elevated at 60 min of TXB2 generation, indicating aspirin resistance. In addition, FC patients had increased levels of thrombin-antithrombin, endothelin-1, vWF, IL-8, MCP-1, MIP-1β, TNFα, sVCAM-1, and syndecan-1 but not of IL-6. Conclusion: Adults with FC had lower platelet numbers but increased platelet activity, increased thrombogenesis, systemic inflammation, and endothelial dysfunction. A significant proportion of aspirin-treated FC adults had aspirin resistance, which may be at least in part responsible for their increased incidence of thrombotic complications.
ACCEPTED MANUSCRIPT
4
Introduction
T
The Fontan operation is performed for treatment of various complex congenital heart
IP
malformations. The purpose of the operation, in which the systemic venous return is
SC R
connected to the pulmonary arteries without the interposition of a pumping chamber (1), is to separate the pulmonary and systemic circulation and achieve normal or near normal levels of arterial oxygen saturation.
NU
Long-term follow-up of patients with Fontan circulation (FC) has revealed increased rates of late complications, including thrombosis. The reported incidence of this life-threatening
MA
complication in FC patients ranges from 17% to 33% (1, 2). The pro-thrombotic risk factors include previous thrombosis, type of arteriopulmonary connection, presence of
TE
D
thrombogenic foreign material, arrhythmias, slow venous flow, elevated central venous pressure, ventricular dysfunction, liver injury, effusion protein-losing enteropathy, and
CE P
prolonged immobilization (3).
The mechanisms leading to thromboembolism in patients with FC remain to be elucidated.
AC
Abnormalities in any of the regulatory mechanisms in hemostasis - plasma coagulation/fibrinolytic proteins, platelets, and endothelial function - might contribute to increased thrombotic risk (4). Indeed, changes in plasma activity of coagulation factors and inhibitors, endothelial dysfunction, and enhanced platelet activation have been reported in children after Fontan surgery (5-7). However, only a few studies of hemostatic abnormalities in adult patients, late after Fontan surgery (8-10), have been reported. Also, optimal antithrombotic treatment for adult FC patients is controversial (3); some authors have reported that Fontan patients taking either aspirin or warfarin had lower thromboembolic rates than those not on antithrombotic therapy, but others have found no benefit of the therapy (3, 8, 9).
ACCEPTED MANUSCRIPT
5
Increased platelet activation has been described in some patients with FC (4, 6). Accordingly, long-term antiplatelet therapy for prevention of thrombosis after Fontan operations is a
T
reasonable consideration, even though thrombosis is a common feature (2-4). However, it is
IP
not known whether patients after Fontan operation respond to aspirin and whether aspirin
SC R
resistance is prevalent in them (11-13). In reported studies, aspirin resistance has been defined by the presence of incomplete inhibition of ex vivo platelet aggregation induced by
NU
arachidonic acid, suboptimal inhibition of platelets in various other ex vivo tests (14-16), or increased thromboxane B2 (TXB2) metabolites in urine (13). Recently, Dobaczewski, et al.
MA
(17) suggested that in healthy and streptomycin-induced diabetic rats, measurement of dynamic generation of TXB2 is a more sensitive determination of aspirin resistance than is
D
measurement of plasma concentration of TXB2 and urine concentration of 11-dehydro-
TE
TXB2..
CE P
In the present work we aimed to characterize platelet responsiveness to aspirin in patients with FC on the basis of dynamic generation of TXB2 and sCD40L in ex vivo assay as well as
AC
to characterize thrombogenesis, inflammation, and endothelial function.
Material and methods
Study participants We studied 21 men (62%) and 13 women (48%) who had received a Fontan operation and 32 healthy control subjects matched for age, sex, and basal metabolic index. Main exclusion criteria were: age below 18 years, neoplastic diseases, infection, inflammation, major trauma within the three months preceding the study, pregnancy, alcohol abuse and diabetes mellitus. Healthy subjects were not receiving any medication.
ACCEPTED MANUSCRIPT
6
Study protocol
T
Patients’ previous interventions, complications, medical therapy, and clinical, demographic,
IP
and anatomic data were collected at the time of the examination from the medical records.
SC R
Cardiac echocardiography was performed in all patients, and single ventricle ejection function (SVEF) was assessed. Arterial blood oxygen saturation (SaO2) was measured by means of
NU
transcutaneous pulse oximetry with subjects breathing room air. Twenty one (62%) patients were taking aspirin; 13 (38%) were receiving either warfarin (10) or enoxaparin (3) (Figure
MA
1).
Warfarin was replaced with enoxaparin for five days before the study. Aspirin had been taken
D
in doses 75 mg/day for at least seven day before the study. To minimize the influence of
TE
concomitant medications on platelet function, all of them were withheld for 24 hours prior to
CE P
the study.
The University Ethical Committee approved the study, and all the subjects provided written,
AC
informed consent.
Laboratory investigations Blood samples were collected from the antecubital vein after overnight fasting for at least 12 hours. White blood cells (WBC), platelet count, red blood cells (RBC), hematocrit (HCT), hemoglobin (HGB), total protein, alanine aminotransferase (ALT), gamma glutamyl transpeptidase (GGTP), cystatin C, creatinine, C-reactive protein (CRP), serum bilirubin, international normalized ratio (INR), N-terminal pro B-type natriuretic peptide (NT-proBNP) were assayed with routine laboratory techniques. Plasma thrombin-antithrombin concentration was measured with ELISA (Siemens).
Platelet and endothelial cell activation
7
ACCEPTED MANUSCRIPT
Plasma samples were analyzed with human soluble vascular cell adhesion protein-1 (sVCAM1) and Endothelin-1 Quantikine ELISA kits (R&D Systems), according to manufacturer’s
T
instructions. Syndecan-1 was detected with a Human Syndecan-1 DuoSet ELISA
IP
Development kit (R&D Systems). Plates were coated with 0.8 g/ml capture antibody in
SC R
phosphate-buffered saline solution and incubated overnight at room temperature, then washed three times with 400 ml of wash buffer and blocked by 1-h incubation with 300 l of
NU
reagent diluent. Plates were washed as in the previous step, 100 l of samples and standards per well were added, and the plates were incubated for 2 h at room temperature and washed as
MA
in the previous step. Next, 100 l of 400 ng/ml of detection antibody per well were added, and the plates were incubated 2 h at room temperature and washed. One hundred microliters of
D
the working dilution of streptavidin-horse radish peroxidase were added to each well, and the
TE
plates were incubated for 20 min in the dark at room temperature and washed as above. Next,
CE P
100 l per well of 2N H2SO4 were added to stop the reaction. Optical density of each well was read immediately with a microplate reader set to 450 nm. The wavelength reaction was
AC
set to 570 nm. Plasma von Willebrand Factor (vWF) antigen was also measured (Diagnostica Stago, Asnieres, France).
Cytokines/chemokines analysis Bio-Plex Pro human cytokine, chemokine, and grow factors magnetic beads kit (Bio-Rad) were used, in accordance with the manufacturer’s instruction. Data were acquired on a validated and calibrated Bio-Plex 200 system (Bio-Rad) and analyzed with Bio-Plex Manager 6.0 software (Bio-Rad) with a detection target of 50 beads per region, low RP1 target for CAL2 calibration, and recommended doublet discriminator gates of 5000–25,000. We also measured interleukin 6 (IL6), interleukin 8 (IL8), granulocyte colony-stimulating factor (GCSF), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1β
ACCEPTED MANUSCRIPT
8
(MIP-1β). Tumor necrosis factor-alfa (TNFα) was measured with an ELISA method (R&D Systems).
T
Dynamic generation of TXB2 and sCD40L
IP
Blood was collected into vials (Monovette®) containing heparin as an anti-coagulant agent.
SC R
Briefly, an aliquot of whole blood was supplemented with 500 μM aspirin immediately upon withdrawal, then centrifuged (2320×g, 12 min), and the separated effluent was frozen (−20
NU
°C) until assayed for TXB2 or sCD40L. Another portion of the sample was diluted 5 times with buffered saline. The specimens were incubated for 1 h at 37oC with continual stirring.
MA
At 0, 30, and 60 min of incubation, 200 μl of sample were taken and supplemented with 500 μM aspirin and processed before further analyses in the same way as described above. The
TE
analyses (stored at -20 °C).
D
rest of the collected blood was centrifuged (1000 g, 15 min) to separate plasma for cytokine
CE P
TXB2 concentrations were measured by use of a previously validated enzyme-linked immunoassay method (EISA-kit by Enzo Life Sciences, sensitivity 10,54 pg/mL), and sCD40L concentrations were measured by use of R&D Systems ELISA kit (sensitivity 10.1
AC
pg/mL), both according to the manufacturers’ instructions. Obtained concentrations were normalized for each individual’s platelet count. Statistical analysis Continuous data were expressed as median (interquartile range) and categorical variables as frequencies and percentages. Categorical values were analyzed by use of the Chi2 test. The standard Student’s t-test was used for comparison of data having no departures from normality (according to D’Agostino and Person omnibus test), and the nonparametric Mann– Whitney U-test was used for the remaining variables. Correlations between the individual measurements were calculated by use of the Pearson or Spearman rank correlation, as
9
ACCEPTED MANUSCRIPT
appropriate. A p-value
