Background: Previous studies have demonstrated that platelet activation occurs in peripheral blood of patients with rheumatic mitral stenosis (MS). However, in patients with MS, the plasma level of soluble P-selectin (a marker of platelet activation) remains unsettled.
Methods and results: A total of 20 patients with symptomatic MS undergoing percutaneous transluminal mitral valvuloplasty (PTMV) were studied (group 1; 16 patients in permanent atrial fibrillation, and 4 patients in sinus rhythm). The plasma levels of soluble P-selectin in the femoral vein and artery, and right and left atria before PTMV and those in the peripheral venous blood at the 1-week and 4-week follow-ups after PTMV were determined by solid-phase, sandwich, enzyme-linked immunosorbent assay. The mitral valve area was calculated by means of the Doppler pressure half-time method. In addition, we measured plasma concentrations of soluble P-selectin in the peripheral venous blood samples obtained from 22 control patients (including 14 healthy volunteers in sinus rhythm [group 2] and 8 patients in permanent lone atrial fibrillation [group 31). The plasma levels of soluble P-selectin were significantly elevated in group 1 patients (49.78 [+ or -] 37.72 ng/mL) [mean [+ or -] SD] compared with group 2 (25.52 + or -] 15.38 ng/mL) and group 3 patients (32.17 [+ or -] 14.18 ng/mL) [p < 0.005]. In group 1 patients, the plasma levels of soluble P-selectin in the left atrium did not significantly differ from those in the right atrium, femoral vein, or femoral artery (p = 0.05). The area of mitral valve increased significantly after PTMV (1.06 [+ or -] 0.17 [cm.sup.2] vs 1.48 [+ or -] 0.32 [cm.sup.2], p < 0.0001). The mean left atrial pressure fell significantly and immediately after PTMV (23.0 [+ or -] 5.1 mm Hg vs 17.6 [+ or -] 5.9 mm Hg, p < 0.0001). The peripheral venous plasma levels of soluble P-selectin obtained before PTMV did not significantly fall after PTMV (before, 49.8 [+ or -] 37.7 ng/mL; 10 min after, 39.8 [+ or -] 19.1 ng/mL; 1 week after, 46.1 [+ or -] 20.8 ng/mL; and 4 weeks after, 41.2 [+ or -] 15.9 ng/mL; p = 0.145).
Conclusions: The venous plasma levels of soluble P-selectin in patients with moderate-to-severe MS were significantly higher than those in healthy volunteers or patients with lone atrial fibrillation. In addition, in patients with MS, there was no difference in the plasma levels of soluble P-selectin between the left and right atrial blood and between peripheral and atrial blood. Moreover, there was no change in soluble P-selectin levels as a result of PTMV.
Key words: mitral stenosis; P-selectin
Abbreviations: MS = mitral stenosis; PTMV = percutaneous transluminal mitral valvuloplasty
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P-selectin (a biologically relevant molecule that is released to the surface of platelet from [alpha] granules on activation) (1-3) is thread constitutively in a preformed state in the Weibel-Palade bodies of endothelial cells and in the [alpha] granules of platelets. This stored P-selectin is mobilized to the cell surface within minutes in response to a variety of inflammatory or thrombogenic agents, and is involved in the adhesion of myeloid cells to activated endothelium and in the adhesion of platelets to monocytes and neutrophils.
Previous studies (4,5) have demonstrated that platelet activation occurs in peripheral blood of patients with rheumatic mitral stenosis (MS). We have demonstrated that in patients with moderate-to-severe rheumatic MS, increased regional left atrial platelet P-selectin expression has a significantly direct relationship with the severity of MS. (6) In addition, the increased regional left atrial platelet P-selectin expression is not reflected in peripheral venous blood samples. In that study, (6) platelet activity was determined by measuring the fractions of platelets expressing P-selectin by flow cytometry, reflecting changes in individiual platelets and allowing us to see changes in individual platelets long before they can be detected in the plasma. However, Li-Saw-Hee et al (7) and Goldsmith et al (8) reported that plasma levels of soluble P-selectin (a marker of platelet activation) in patients with MS were lower than those in normal control subjects. Accordingly, we undertook the present study to test the hypothesis that plasma levels of soluble P-selectin were increased in patients with rheumatic MS, and there was no difference in the plasma levels of soluble P-selectin between the left and right atrial blood and between peripheral and atrial blood.
MATERIALS AND METHODS
Study Population
Twenty patients who had symptomatic, moderate-to-severe rheumatic MS (mitral valve area, 1.06 [+ or -] 0.17 [cm.sup.2]; range, 0.6 to 1.29 [cm.sup.2]) without significant mitral, tricuspid, or aortic regurgitation, and left atrial thrombus and had undergone percutaneous transluminal mitral valvuloplasty (PTMV) were studied (group 1). There were '2 men and 18 women (age range, 39 to 72 years; mean age, 55 [+ or -] 12 years). Sixteen patients had permanent atrial fibrillation, and 4 patients were in sinus rhythm. Six patients had a history of cerebral thromboembolism. Ten patients were in New York Heart Association functional class III, and B) patients were in New York Heart Association functional class II. No patients had a history of malignancy, inflammatory disease, collage vascular disease, renal or liver disease, diabetes mellitus. hypertension, hyperlipidemia, infectious disease, deep venous thrombosis, pulmonary embolism, or recent surgery.
Peripheral venous plasma levels of soluble P-selectin were also measured in 22 control patients. The group of control patients included 14 healthy volunteers in sinus rhythm (group 2) and 8 patients in permanent lone atrial fibrillation without systemic disease or structural heart disease (group 3). In group 3, two patients had a history of systemic arterial thromboembolism. None of the control patients had a history of active malignancy, inflammatory disease, renal or liver disease, diabetes mellitus, hypertension, hyperlipidemia, deep venous thrombosis, pulmonary embolism, or recent surgery.
Informed consent was obtained from all study subjects. The study protocol was approved by the Institutional Review Committee on Human Research in our institution.
Doppler Echocardiography and Medications
In patients with rheumatic MS, transthoracic echocardiographic examinations were performed on the day' of PTMV and before the valvuloplasty procedure with a 2.5-MHz transducer attached to a commercially available Doppler echocardiography machine (Sonos 5500; Hewlett-Packard; Palo Alto, CA) to assess left and right atrial dimensions, and mitral valve area. M-mode measurements were performed according to the recommendation of the American Society of Echocardiography. Left and right atrial areas were planimetered in the four-chamber view, and maximum areas were measured (at the end of the T wave on the ECG) and averaged more than five beats. The mitral valve area was calculated by means of the Doppler pressure half-time method. The absence of left atrial cavity or appendage thrombus was confirmed by transesophageal echocardiography.
In group 1 patients, digoxin, [beta]-blockade, and calcium-channel blockade were discontinued for at least 5 half-life before study, and therapy with diuretic agents was discontinued on the day of PTMV. Warfarin was discontinued for at least 3 days before PTMV and was administered on the second day after PTMV. Heparin, 5,000 U, was administered into the left atrium after transseptal puncture in each patient. In group 3 patients, therapy with aspirin was discontinued for at least 7 days, digoxin and calcium-channel blockade were discontinued for at least 5 half-life before study, and warfarin was discontinued for tit least 3 days before the study.
Valvuloplasty Procedure
PTMV was performed by the transseptal approach with the use of an Inoue balloon catheter (Toray Medical Corporation; Tokyo, Japan). Details of the procedure have been described previously. (9) Measurements of left atrial pressure and transmitral pressure gradient were performed immediately before and after valvuloplasty.
Blood Sample Collection and Measurement of Plasma Soluble P-Selectin Concentrations
Blood samples were obtained in the fasting, nonsedative state at 9 to 10 AM in the control and study groups to exclude the possible influence of circadian variations. (10) In group 1 patients, blood was obtained from the femoral vein :and artery through introducer sheaths immediately alter puncture with the patients in the supine position for at least 20 min. Bight atrial blood was obtained through balloon catheter, and left atrial blood was obtained immediately after transseptal puncture before heparin administration. Another set of blood samples from the femoral vein, femoral artery, and right and left atria were obtained at 10 min alter optimal PTMV. At the 1-week and 4-week follow ups after PTMV, peripheral venous blood was obtained under minimal tourniquet pressure from the antecubital vein using a sterile 22-gauge needle syringe in a single attempt with the study patients in the supine position for at least '20 min. Five milliliters of blood were drawn into an evacuated tube containing [K.sub.3] ethylenediamine tetra-acetic acid (Vacutainer; Becton Dickinson; Franklin Lakes, NJ). In groups 2 and 3, blood was also obtained from the antecubital vein. Blood samples with gross hemolysis were discarded. Mixtures of blood and [K.sub.3] ethylenediamine tetra-acetic acid were immediately centrifuged (model 5400; Kubota Corporation; Tokyo, Japan) at 3,000 revolutions per minute for 10 min at room temperature. The plasma was immediately separated and frozen at - 80[degrees]C until the assay. Blood samples were also obtained for whole blond counts, and biochemical and electrolytes measurements by standard laboratory methods.
The soluble P-selectin concentrations of human plasma samples were quantified with the use of a commercially available, solid-phase, sandwich enzyme-linked immunosorbent assay kit (R&D Systems; Minneapolis, MN). The samples were processed according to the instructions of the manufacturer. The samples, which included standards of known recombinant human soluble P-selectin concentrations in buffer and reconstituted extracts of the quality control and test samples and an enzyme (horseradish peroxidase)-labeled second antibody, were sequentially added to a 96-well microplate precoated with a monoclonal antibody to human soluble P-selectin. After 1 h of incubation at room temperature and removal of unbound materials, the amount of enzyme-conjugated tracer bound to the wells was detected through reaction with a substrate (tetramethylbenzidine) specific for the enzyme. The reaction product was measured by using a microplate reader (MRX; Dynex Technologies; Chantilly, VA) and reading the absorbance at 450 nm with a correction wavelength of 630 mm. A standard curve was determined with the use of the mean absorbance values of the included P-selectin standards, and the soluble P-selectin concentrations in all unknown plasma samples was then calculated with linear regression. All standards and samples were tested in duplicate. The assay was sensitive to detect < 0.5 ng/mL of P-selectin, according to the manufacturer of the assay kits. The mean interassay coefficients of variance of 7 plasma samples was 10.9%, and the mean intra-assay coefficients of variance (if 40 plasma samples was 4.2%.
Statistical Analysis
Continuous variables were described as the mean [+ or -] SD. Categorical variables were compared using the Fisher exact test (two tailed). Continuous variables within the same group were compared using paired t test. Log transformation of continuous variables was used to improve the normality for statistical analysis. Continuous variables among the three groups were compared using the one-way analysis of variance. A Scheffe test was used for post hoc comparisons. Analysis of covariance was used to determine the significance of dependent variable among multiple independent variables. The plasma levels of soluble P-selectin of the four different sampling sites were compared using the repeated-measures analysis of variance. The correlation between the plasma level of soluble P-selectin and mitral valve area, the mean left atrial pressure, or left atrial dimension were performed with the Pearson correlation. Statistical analysis was performed using a statistical software program (SAS for Windows. version 8.02; SAS Institute; Cary, NC); p < 0.05 was considered statistically significant.
RESULTS
Comparison of Baseline Characteristics and Peripheral Venous Plasma P-Selectin Concentrations Among the Three Groups
The baseline characteristics for each group are summarized in Table 1. There were no statistically significant differences among the three groups in terms of blood cell counts and biochemistry data. There were no differences among the three groups in terms of the use of [beta]-blockade (group 1, 15.0% vs group 2, 0.0% vs group 3, 0.0%), calcium-channel blockade (group 1, 25.0% vs group 2, 0.0% vs group 3, 12.5%), amiodarone (group 1, 5.0% vs group 2, 0.0% vs group 3, 0.0%), propafenone (group 1, 5.0% vs group 2, 0.0% vs group 3, 12.5%), and aspirin (group 1, 0.0% vs group 2, 0.0% vs group 3, 12.5%). As a result of atrial fibrillation, the use of warfarin therapy in patients from group 1 (95.0%) and group 3 (87.5%) was more frequent than that in group 2 patients (0.0%). The use of digoxin in patients from group 1 (70.0%) was more frequent titan that in group 2 (0.0%) and group 3 patients (25.0%). The duration of atrial fibrillation of group 1 patients did not differ from that of group 3 patients. Group 3 patients were significantly older than group 2 patients. There were fewer male patients in group 1 than in groups 2 and 3. By analysis of covariance, the peripheral venous plasma levels of soluble P-selectin were significantly higher in group 1 patients than in group 2 or 3 patients (p < 0.005) [Table 1]. The peripheral venous plasma levels of soluble P-selectin among group "2 subjects did not differ from those among group 3 patients.
Comparison of Plasma Levels' of Soluble P-Selectin Among the Four Different Sampling Sites
Analysis of the prevalvuloplasty data of the 20 patients with rheumatic MS revealed that the plasma levels of soluble P-selectin in the left atrium (47.4 [+ or -] 30.1 ng/mL) did not significantly differ from those in the right atrium (39.3 + or -] 26.0 ng/mL), femoral vein (49.8 [+ or -]37.7 ng/mL), or femoral artery (44.2 [+ or -] "29.3 ng/mL) [p = 0.05].
Correlation Between Plasma Levels of Soluble P-Selectin and Hemodynamic and Echocardiographic Variables in Patients With Mitral Stenosis
Correlation analysis demonstrated that there was no significant correlation between the plasma levels of P-selectin in the left atrium and left atrial diameter (p = not significant, r = -0.005), left atrial area (p = not significant, r = -0.037), prevalvuloplasty mean left atrial pressure (p = not significant, r = 0.287), prevalvuloplasty transmitral pressure gradient (p = not significant, r = 0.155), and the severity of MS (p = not significant, r = 0.076).
Peripheral Venous Plasma Levels of Soluble P-Selectin Before and After Mitral Valvuloplasty
There were no significant changes in the left atrial diameter, left ventricular dimension, and ejection fraction after PTMV (Table 2). The area of mitral valve increased significantly after PTMV (p < 0.0001). The left atrial area reduced significantly after PTMV (p < 0.03). The mean left atrial pressure fell significantly and immediately after PTMV (p < 0.0001). The transmitral pressure gradient fell significantly after PTMV (p < 0.0001). The peripheral venous plasma levels of soluble P-selectin obtained before PTMV did not significantly fall after PTMV (before, 49.8 [+ or -] 37.7 ng/mL vs 10 min alter, 39.8 [+ or -] 19.1 ng/mL vs 1 week after, 46.1 [+ or -] 20.8 ng/mL vs 4 weeks after, 41.2 [+ or -] 15.9 ng/mL; p = 0.145).
DISCUSSION
The present study, in which we examined the plasma soluble P-selectin concentrations in atrial and peripheral venous or arterial blood samples of patients with symptomatic rheumatic MS undergoing PTMV, produced two major findings. First, the venous plasma soluble P-selectin levels in patients with MS were significantly higher than those in healthy volunteers or patients with lone atrial fibrillation. Second, in patients with MS, the plasma soluble P-selectin concentrations in the left atrium did not signifcantly differ from those in the right atrium, femoral vein, and femoral artery.
Increased P-Selectin Concentrations in Patients" With Mitral Stenosis
Li-Saw-Hee et [al.sup.7] and Goldsmith et [al.sup.8] reported that plasma levels of soluble P-selectin in patients with MS were significantly lower than those in normal control subjects. (7,8) On the contrary, in the present study we demonstrated that the venous plasma soluble P-selectin levels in patients with MS were significantly higher than those in healthy volunteers. Although the use of warfarin and digoxin therapies in patients with MS was more frequent than that in heal@ volunteers and there were fewer male patients with MS than in healthy volunteers in our study, by analysis of covariance the peripheral venous plasma levels of soluble P-selectin were independently and significantly higher in patients with MS than in healthy volunteers. There were several possible explanations for the discrepancy between our findings and their findings. First, in our study, warfarin was discontinued for at least 3 days; before PTMV, compared with 2 days in their study. There are controversial results regarding the effect of warfarin on platelet activity. (11,12) Second, in our study, blood was drawn into a Vacutainer containing [K.sub.3] ethylenediamine tetra-acetic acid as anticoagulant, compared with sodium citrate as anticoagulant in their study. Platelet activation in response to adenosine diphosphate is higher in blood anticoagulated with citrate compared with ethylenediamine tetra-acetic acid. (13) Our results in the present study were consistent with other and our previous studies. (4-6)
In the present study, we found that the peripheral venous plasma levels of soluble P-selectin obtained before PTMV did not significantly fall after PTMV. There was one possible mechanism that contributed to this observation. P-selectin is found constitutively in a preformed state in the Weibel-Palade bodies of endothelial cells and in the [alpha] granules of platelets. Therefore, increased plasma P-selectin level will be observed in patients with diseases associated with activation of platelets and endothelium. We and others (14,15) have demonstrated that endothelial dysfunction is present in patients with rheumatic MS. In a previous study, (14) we demonstrated that 'although the area of mitral valve increased significantly and the mean left atrial and pulmonary arterial pressures fell significantly and immediately 'after PTMV, there were no significant changes in the plasma endothelin-1 concentrations in the femoral vein immediately after PTMV. Therefore, the persistently increased plasma P-selectin after PTMV might be partly attributed to the persistent endothelial dysfunction after PTMV in our patients.
Plasma Level of Soluble Y-Selectin as a Marker of Platelet Activation
In another study, (6) we demonstrated that in patients with rheumatic MS, increased regional left atrial platelet P-selectin expression has a significantly direct relationship with the severity of MS, and the increased regional left atrial platelet P-selectin expression is not reflected in peripheral venous blood samples. However, in the present study, the plasma soluble P-selectin concentrations in the left atrium did not significantly differ from those in the right atrium, femoral vein, and femoral artery. These findings indicated that plasma soluble P-selectin levels could not reflect changes in individual platelets, and these findings also accounted for the absence of significant correlation between the plasma levels of soluble P-selectin in the left atrium and the severity of MS in the present study. Mow, over, endothelial dysfunction also contributed to the increased plasma levels of soluble P-selectin in patients with MS. Therefore, plasma level of soluble P-selectin is less specific than platelet P-selectin expression in terms of platelet activity, and should not be used as the sole marker of platelet activation.
Study Limitations
There were several limitations in this study. First, although warfarin therapy was discontinued for at least 3 days in patients in groups 1 and 3, we could not completely exclude the potential late effect of warfarin on platelet activity. However, the plasma levels of soluble P-selectin in group 1 patients were significantly higher than those in groups 2 and 3, and the plasma levels of soluble P-selectin in group 2 patients did not differ from those in group 3. These should exclude the potential late effect of warfarin on platelet activity, as most of the group 1 and group 3 patients had warfarin therapy before entering the study. Second, in the present study, we demonstrated that the plasma levels of soluble P-selectin among patients with lone atrial fibrillation did not differ from those of healthy volunteers. Our results should be viewed as preliminary and await confirmation by larger clinical study. Finally, as the number of patients having a history of systemic arterial thromboembolism was small, it was not our aim to study the difference in the plasma levels of soluble P-selectin between patients with and without a history of systemic arterial thromboembolism.
In conclusion, the venous plasma levels of soluble P-selectin in patients with moderate-to-severe MS were significantly higher than those in healthy volunteers or patients with lone atrial fibrillation. In addition, in patients with MS, there was no difference in the plasma levels of soluble P-selectin between the left and right atrial blood and between peripheral and atrial blood. Moreover, there was no change in soluble P-selectin levels as a result of PTMV.
ACKNOWLEDGMENT: We thank Ms. Hsiu-Chin Tsai for technical assistance.
* From the Division of Cardiology (Drs. Chen, Yip, and Wu), Department of Internal Medicine Chang Gung Memorial Hospital, Kaohsiung; Department of Biological Sciences (Dr. Chang), National Sun Yat-Sen University. Kaohsiung; and Chia Nan University of Pharmacy and Science (Mr. Juang), Tainan, Taiwan, Republic of China.
This study was supported by a grant from Chang Gung Memorial Hospital, Chang Gung University (CMRP1139).
Manuscript received August 4, 55003; revision accepted January 19, 2004.
Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: permissions@chestnet.org).
Correspondence to: Mien-Cheng Chen, MD, Division of Cardiology, Department of Internal Medicine, Chang Gung Memorial Hospital, 123, Ta Pei Rd, Niao Sung Hsiang, Kaohsiung Hsien 83301, Taiwan, ROC; e-mail: chenmien@ms76.hinet.net
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