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European Journal of Heart Failure 1999 1(3):243-249; doi:10.1016/S1388-9842(99)00029-X
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© 1999 European Society of Cardiology

Increased soluble platelet / endothelial cellular adhesion molecule-1 and osteonectin levels in patients with severe congestive heart failure. Independence of disease etiology, and antecedent aspirin therapy

Victor L. Serebruanya,*, Selva R. Murugesana, Anitha Pothulaa, Dan Atarb, David R. Lowrya, Christopher M. O'Connorc and Paul A. Gurbela

a Sinai Hospital Baltimore, MD, USA
b University of Zurich Hospital Zurich, Switzerland
c Duke Clinical Research Institute Durham, NC, USA

* Corresponding author. Center for Thrombosis Research, Sinai Hospital of Baltimore, 2401 West Belvedere Avenue, Schapiro Research Building-R 202, Baltimore, MD 21215, USA. Tel.: +1-410-601-5266; fax: +1-410-601-9061. E-mail address: heartdrug{at}aol.com (V.L. Serebruany)


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background: Platelet–endothelial interactions modulated by adhesion molecules, may play an important role in the pathogenesis of congestive heart failure (CHF). Soluble levels of these molecules and platelet-derived substances are reportedly elevated in patients with CHF. However, no data are available on the plasma levels of Platelet / Endothelial Cell Adhesion Molecule-1 (PECAM-1), and platelet-derived osteonectin in this growing population.

Methods and Results: Soluble levels by ELISA were prospectively determined in patients with severe CHF (n = 37) and correlated to etiology and antecedent aspirin use, and compared with 14 healthy control subjects. Left ventricular dysfunction was attributed to idiopathic dilated cardiomyopathy in 18 and coronary artery disease in 19 patients. Twenty-one patients were aspirin-free and 16 patients were using aspirin (81–500 mg daily). Elevated soluble PECAM-1 (51.31 ± 2.44 ng/ml, P = 0.0001), and osteonectin (826.27 ± 22.37 ng/ml, P = 0.0001) were observed in patients with CHF, as compared to healthy controls (32.56 ± 1.21 ng/ml, and 478.02 ± 31.32 ng/ml, respectively). Neither etiology of CHF, nor antecedent aspirin therapy significantly affects the levels of PECAM-1 or osteonectin.

Conclusions: Despite long-term aspirin therapy and independently of the etiology of the disease, soluble PECAM-1 and osteonectin were elevated in the majority of patients with severe CHF, suggesting platelet–endothelial activation. The present data provide additional evidence that more potent anti-platelet and endothelial preservation regimens deserve further study in the heart failure population.

Key Words: PECAM- 1 • Osteonectin • Aspirin • Congestive heart failure

Accepted June 23, 1999


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The important role of platelets and vascular endothelium in patients with CHF has received increasing recognition in recent years. Most researchers agree that platelet–endothelium interactions could affect both short- and long-term outcome in patients with CHF [1]. Heightened platelet activity, if indeed present, could be a factor in the increased occurrence of thrombotic events observed in this population. However, there is an obvious lack of information available about the status of platelet activity in patients with CHF.

It has been reported that cellular adhesion molecules participate in the pathogenesis of CHF via the modulation of platelet–leukocyte–endothelial interactions [2,3]. Platelet/Endothelial Cell Adhesion Molecule-1 (PECAM-1, CD 31), a membrane glycoprotein, is an immunoglobulin that is found on the surface of platelets and leukocytes, and at the intercellular junctions of the endothelial cells [4,5]. PECAM-1 is the most prevalent endothelial antigen known, and also as an {alpha}-granule constituent, PECAM-1 is a distinct, well-defined component of the platelet plasma membrane with the intracellular distribution identical to glycoprotein IIb/IIIa [6,7]. Osteonectin (SPARC, BM40), is a small membrane phosphoglycoprotein, and a major protein constituent of the mineral matrix of bone [8]. It is present at the internal face of the platelet {alpha}-granules [9], and is known to be different both structurally and immunologically from the bone-derived form [10]. In contrast to PECAM-1, osteonectin has an intracellular distribution similar to P-selectin, but not to glycoprotein IIb/IIIa [11]. In spite of the proposed importance of osteonectin as an ideally positioned marker of platelet activation, the role of osteonectin in patients with CHF has never been explored.

Recent epidemiological studies have consistently shown that antecedent aspirin therapy is associated with a reduced mortality risk in patients with CHF [12,13]. However, the effect of long-term aspirin therapy on plasma PECAM-1 and osteonectin in CHF patients is unknown. The present study defines the levels of soluble PECAM-1 and osteonectin in patients with severe heart failure, and correlates these values with the disease etiology, and aspirin use.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Patients
Thirty-seven patients (17 men, 20 women) with severe CHF (New York Heart Association functional classes III and IV), typical physical exam findings, and supportive radiographic data were included in this study. All patients exhibited symptoms for at least 3 months, and had no change in medication during the last month. Left ventricular dysfunction was attributed to idiopathic dilated cardiomyopathy in 18, and coronary artery disease in 19 patients.

Information on antecedent aspirin use was obtained as part of the study protocol. Aspirin use was defined as self-reported daily consumption of at least 81 mg of acetylsalicylic acid containing medication (coated or uncoated). The regional ethics committee approved the study and all subjects gave informed consent.

2.2. Controls
Fourteen non-smoking, non-diabetic subjects (eight men, six women) without systemic hypertension or cardiovascular disease, and, for at least 2 weeks, free of pharmacological agent use, served as a control group. Volunteers were matched for age, sex, and smoking history.

2.3. Samples
Blood samples for enzyme-linked immunosorbent (ELISA) studies were obtained using a 19-gauge needle by direct venipuncture, and drawn into a 5-ml Vacutainer® citrate–theophylline–adenosine–dypiridamole (CTAD) tube. Samples were collected before applying any additional therapy, and processed within 1 h after blood drawing. Blood samples were coded and blinded. Sampling procedures and ELISA studies were performed by individuals unaware of the protocol. Plasma was obtained by centrifugation of the whole blood–CTAD mixture at +4°C at (1800xg) for 10 min. Samples were stored at –80°C before final determination of antigen levels. ELISA assays for PECAM-1 (<0.8 ng/ml sensitivity, 3.2% intra-assay coefficient of variation, 92% spike recovery, and no cross-reactivity with other vascular adhesion molecules and selectins), were obtained from Bender MedSystems, (Vienna, Austria). ELISA for osteonectin (overall intra-assay coefficient of variation was 5.4%±0.8%, with a plasma recovery rate of 94.6%) was purchased from Haemostatic Technologies, Inc. (Essex Junction, VT, USA). The osteonectin assay utilizes a purified protein standard derived from human platelets. Kits were used according to manufacturer's instructions. Each sample was measured in duplicate.

2.4. Statistical analysis
All comparisons were made using repeated measures ANOVA with the Scheffe's correction. Values are expressed as mean±S.D.), and P<0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
The clinical and laboratory characteristics of the CHF patients are shown in Table 1. There were no important differences in the idiopathic and ischemic cardiomyopathy groups apart from a gender bias, when women prevailed in the idiopathic cardiomyopathy group. Patients in this group were slightly older, smoked less, and exhibited similar patterns of hypertension and diabetes, but a higher incidence of hypercholesterolemia as compared to patients with an ischemic origin of heart failure. As expected, the ischemic cardiomyopathy group was treated more aggressively, especially with calcium channel blockers and nitrates. Angiotensin-converting enzyme inhibitors were clearly equally underused in both groups (18 and 19%, respectively). This pattern is in agreement with our previous observation [14] that for no apparent reason, angiotensin-converting enzyme inhibitors are rarely prescribed medications in patients with CHF. None of the patients had other significant concomitant diseases such as infection, pulmonary disease, malignancy, or collagen vascular disorder. Twenty-one patients did not use aspirin and 16 patients were treated with aspirin daily for more than 1 year. None of the aspirin-using patients were on chronic non-steroidal anti-inflammatory drugs. Laboratory findings were similar between groups.


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  		Table 1 Characteristics of the patients

 
Table 2 summarizes the data on soluble PECAM-1 and osteonectin levels in the CHF population and in healthy control subjects. Individual data are shown in Fig. 1A, B. Plasma levels of PECAM-1 were consistently almost twofold higher in patients with CHF than in control subjects. Soluble osteonectin was variable in both groups, but overall, significantly higher in the patients with CHF. Individually, certain patients exhibited similar PECAM-1 and osteonectin levels to control subjects, however, the highest concentrations were observed among patients with CHF. There was no correlation between soluble PECAM-1 and osteonectin measurements, with a statistically non-significant low curve fit regression rate (r2=0.006). There were no differences in the soluble antigen levels dependent on the etiology of the CHF or antecedent aspirin therapy.


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  		Table 2 Soluble PECAM-1, and osteonectin in patients with CHF, and in healthy controlsa

 


Figure 1
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  		Fig. 1 Individual and mean data of plasma PECAM-1 (A), and osteonectin (B) levels in patients with heart failure, and control subjects. Circles represent patients with the ischemic origin of heart failure. Triangles represent those with the idiopathic dilated cardiomyopathy.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
To the best of our knowledge, this is the first prospective pilot study to evaluate PECAM-1 and osteonectin as markers of platelet, and endothelial activation in patients with severe congestive heart failure. The present data demonstrate that plasma concentrations of these soluble substances are significantly higher in such patients.

Observations that heart failure is associated with disturbances in hemostasis including platelet abnormalities, are not new [1,15–17]. It has been reported that plasma levels of β-thromboglobulin were higher in CHF patients when compared to both controls and patients with stable coronary artery disease [18]. Increased platelet activity could be in fact attributed to the elevation of cytosolic free calcium concentrations in patients with CHF [19]. Platelet size [20] and circulating platelet-activating factor [21] have also been reported to be higher in the CHF population. In contrast, other investigators observed diminished serotonin and adrenaline sensitivity in platelets from CHF patients, and no differences in the platelet factor 4 release, platelet volume and size [18,22]. An inability of platelets from CHF patients to bind excessive concentrations of norepinephrine may be directly related to a reduced presence of {alpha}-2-adrenoreceptors on the platelet surface, thus resulting in hypofunction [23]. It has been proposed, but never proven, that the failing myocardium may affect platelets via direct sympathoadrenal activation and enhanced catecholamine release [24].

Platelet-related adhesion molecules, particularly glycoprotein IIb/IIIa and P-selectin, have been well characterized, and are activated in acute coronary syndromes [25,26]. Much less is understood about the role of PECAM-1 [27,28], and virtually nothing is known about osteonectin in the clinical setting of CHF. Our data showing increased PECAM-1 is in agreement with a single recent report that soluble adhesion molecule levels are elevated in patients with heart failure [17]. Similarly, PECAM-1 and osteonectin in our study were not uniformly increased in every individual: indeed, certain patients with CHF exhibited a threefold increase of PECAM-1, and osteonectin, whereas, some had plasma levels within the control range. On the other hand, soluble osteonectin in this study was a sensitive marker of platelet activation. The unquestionable platelet origin of this protein, and the elevated plasma levels of this {alpha}-granule constituent, provide additional evidence of the existence of activated platelets in patients with CHF. Moreover, we found no evidence that patients receiving long-term aspirin therapy exhibited diminished platelet activity as compared to aspirin absentees, suggesting that more potent adjunctive anti-platelet regiments deserve further study since they may be beneficial in some patients with heart failure. This finding is critical for the implementation of future strategies combining the family of platelet GP IIb/IIIa inhibitors and novel thienopyridines.

Our data concurs with the few available observations [15–17] that the etiology of heart failure itself does not affect platelet-related characteristics. Furthermore, the present study found no evidence that antecedent aspirin significantly affects the PECAM-1 levels in patients with CHF, and contrasts with our earlier findings of reduced PECAM-1 in aspirin treated patients with acute myocardial infarction [29]. The clinical features, if any, that may play a role in the modulation of platelets in CHF are yet to be determined.

Finally, it is possible that PECAM-1 [30,31], and osteonectin [32] are playing a certain role in leukocyte migration during acute inflammation, and that their plasma levels could be up-regulated by exposure to inflammatory mediators [33]. Considering that immunologic and inflammatory responses appear to be pivotal in the development of CHF [34,35], it is possible that higher levels of these substances reflect chronic inflammation, rather than deteriorating hemodynamic events.

In conclusion, despite antecedent aspirin therapy and independently of the etiology of the disease, soluble PECAM-1 and osteonectin were elevated in the majority of patients with severe CHF. These findings suggest persistent platelet and endothelial activation. The presented data provides evidence that more potent anti-platelet and endothelial preservation regimens deserve further study in the heart failure population.


   Acknowledgements
 
Supported in part by Centocor Diagnostics of Pennsylvania (Malvern, PA, USA). We are indebted to the staff of the Emergency Department of the Sinai Hospital (Baltimore, MD, USA).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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V.L. Serebruany, M.E. McKenzie, A.F. Meister, S.Y. Fuzaylov, P.A. Gurbel, D. Atar, W.A. Gattis, and C.M. O'Connor
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