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Cardiovascular Research 1999 43(1):183-191; doi:10.1016/S0008-6363(99)00078-4
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Copyright © 1999, European Society of Cardiology

Platelet aggregatory response to platelet activating factor (PAF), ex vivo, and PAF-acetylhydrolase activity in patients with unstable angina: effect of c7E3 Fab (abciximab) therapy

Alexandros D. Tselepisa, John A. Goudevenosb, Afroditi P. Tambakia, Lambros Michalisb, Christos S. Stroumbisb, Demokritos C. Tsoukatosa, Moses Elisafc and Dimitris A. Siderisb,*

aLaboratory of Biochemistry, Department of Chemistry, Medical School, University of Ioannina, 45110 Ioannina, Greece
bDepartment of Cardiology, Medical School, University of Ioannina, 45110 Ioannina, Greece
cDepartment of Internal Medicine, Medical School, University of Ioannina, 45110 Ioannina, Greece

* Correspondence author. Tel.: +30-6519-7533; fax: +30-6514-8103 dsideris{at}cc.uoi.gr

Received 16 November 1998; accepted 14 January 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Platelet activation and aggregation is a dominant feature in the pathophysiology of unstable angina. The final step of platelet aggregation is mediated through the platelet integrin glycoprotein IIb/IIIa (GP IIb/IIIa), while abciximab (ReoPro) is one of the most potent inhibitors of this receptor. Platelet-activating factor (PAF) is a potent platelet agonist which is degraded and inactivated by PAF-acetylhydrolase (PAF-AH). The plasma form of PAF-AH is associated with lipoproteins. We studied the platelet response to the aggregatory effect of PAF, ex vivo, in relation to the plasma PAF-AH activity in 32 patients with unstable angina, as well as the effect of abciximab therapy on the above parameters. Methods: Thirty two patients with unstable angina and 25 sex- and age-matched healthy controls participated in the study. On the day of admission (day 1) 17 patients received a bolus of abciximab (0.25 mg/kg) followed by a 12-h infusion (10 µg/min). Platelet aggregation to both PAF and ADP, in platelet rich plasma, was successively studied in both patients receiving abciximab or remaining untreated. The plasma and HDL-associated PAF-AH activity was also determined at the same times. Results: In the untreated patients, the PAF EC50 values were significantly lower on the day of admission, whereas the maximal percentage of aggregation was significantly higher compared to controls (p<0.01 for both comparisons). Similar behaviour of the platelets was observed in the aggregatory effect of ADP. This aggregatory response was not significantly altered 4 days, 7 days or 1 month afterwards. In the 17 patients who received abciximab, platelet aggregation to both PAF and ADP was inhibited by 90±5 and 96±3%, respectively, 1 h after bolus. At 2 and 3 days after treatment, platelet aggregation to both agonists was significantly recovered being similar to controls. However, it was fully restored 6 days after bolus, still being significantly higher compared to controls (p<0.01 for PAF and p<0.003 for ADP). The total plasma PAF-AH activity in both patient groups was not different from that of controls, whereas the HDL-associated PAF-AH activity was significantly lower. The total plasma or HDL-associated enzyme activity was not altered at any time interval studied, and it was not influenced by abciximab. Conclusions: The increased aggregatory response of platelets to PAF and the low plasma levels of HDL-cholesterol and HDL-associated PAF-AH activity in patients with unstable angina may contribute to the severe atherosclerosis and to acute thrombosis found in these patients. Abciximab therapy may protect platelets from PAF action in vivo the first days after drug administration, but it fails to permanently restore the enhanced aggregatory response observed.

KEYWORDS Platelet; Platelet activating factor; PAF-acetylhydrolase; Unstable angina; abciximab


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
In patients with coronary atherosclerosis unstable angina may develop in association with plaque fissuring or rupture with subsequent aggregation, platelet adhesion, and intracoronary thrombosis [1,2]. Platelet activation and aggregation is a dominant feature in the pathophysiology of unstable angina [3]. The crucial role of platelets in unstable angina is supported by the existence of platelet granule constituents, such as β-thromboglobulin and platelet factor 4 in the plasma of these patients [4]. More importantly, the platelets from patients with unstable angina exhibit a higher sensitivity compared to those with stable angina or to normal individuals, as it is indicated by their hyperaggregability to various agonists ex vivo, as well as by the demonstration of higher plasma concentrations of platelet granule constituents, such as fibrinopeptide A and β-thromboglobulin [5,6].

The final step of platelet aggregation is mediated by the binding of fibrinogen to the activated platelet integrin glycoprotein IIb/IIIa (GP IIb/IIIa) [7]. GP IIb/IIIa is a heterodimer of two subunits, unique to platelets and the most abundant platelet surface glycoprotein. In its resting form this intergrin is unable to bind any ligand. When the platelet is stimulated by an agonist (such as ADP, thrombin or arachidonic acid) however, GP IIb/IIIa undergoes a conformational change and is capable of binding mainly fibrinogen, although it recognizes as ligands von Willebrand factor, fibronectin, and vitronectin [8].

The identification of GP IIb/IIIa as the pivotal receptor for platelet aggregation independently of the aggregating agonist has made this integrin a logical target to control the platelet response to vascular injury. Thus, one significant development of the last years has been the introduction of a new pharmacologic approach that inhibits fibrinogen binding to GP IIb/IIIa, thus preventing platelet aggregation [9]. One of the potent inhibitors of the platelet GP IIb/IIIa receptor is abciximab (ReoPro), the human–murine chimeric Fab fragment of the monoclonal antibody c7E3 directed against the GP IIb/IIIa receptor [10]. The beneficial effect of abciximab in patients with unstable angina has been shown mainly in two large trials. Among the 2099 patients forming the population of the EPIC trial, 489 patients enrolled with the diagnosis of unstable angina. These patients underwent percutaneous transluminal coronary angioplasty (PTCA) and received placebo, a bolus of abciximab immediately before the intervention or a bolus which was followed by a 12-h infusion. The primary endpoints were a composite of death, myocardial infarction or urgent intervention within 30 days after random assignment. Abciximab administration significantly reduced the incidence of all these primary endpoints [11]. The largest trial for abciximab in unstable angina was the CAPTURE (c7E3 Fab antiplatelet therapy in unstable refractory angina) [12]. This study showed that abciximab substantially reduced the rate of thrombotic complications, in particular myocardial infarction, in patients with unstable refractory angina undergoing PTCA, before, during, and after PTCA. However, abciximab did not influence the rate of myocardial infarction or the need for subsequent reintervention, after the first few days of administration.

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent phospholipid mediator of inflammation that exhibits a wide range of biological activities both in vivo and in vitro [13]. Among the most sensitive cells to PAF action are platelets which can aggregate, degranulate and release biologically potent agents through a specific PAF receptor-mediated mechanism [14,15]. It has been shown that PAF induces a transient exposure of binding sites of the GP IIb/IIIa receptor, possibly by stimulating the phosphorylation of the receptor β3 subunit [16]. Several lines of evidence suggest that PAF may additionally play an important role in atherogenesis [17]. Furthermore, this bioactive phospholipid may be implicated in acute myocardial ischemia [18] either directly, playing a role as proadhesive signalling molecule [19], or indirectly, through other mediators released by cells activated by PAF (e.g. platelets and leukocytes) [20,21]. The various pathophysiological effects of PAF in vivo are mainly regulated by PAF-acetylhydrolase (PAF-AH) (EC 3.1.1.47 [EC] ), which inactivates PAF by hydrolyzing its sn-2 acetate group and thus converting it into the inactive metabolite lyso-PAF [22]. PAF-AH is present in plasma and in several cells and tissues [22,23]. Human plasma PAF-AH is a lipophilic enzyme associated with lipoproteins, mainly with low-density lipoprotein (LDL) and high-density lipoprotein (HDL) [24,25]. In addition to PAF, this enzyme can effectively hydrolyze oxidized phospholipids containing short-chain peroxidized sn-2 residues and exhibiting a wide spectrum of biological activities, some of which resemble PAF (i.e. PAF-like phospolipids) [26,27]. Such oxidized phospholipids are formed on LDL during oxidation and may play an important role in the inflammatory and atherogenic effects of oxidized LDL [28].

In previous studies we have shown that platelets from patients with acute myocardial infarction exhibit an increased aggregatory response to PAF, ex vivo, the first 48 h following the onset of the symptoms [29]. Additionally, we reported increased platelet aggregability in response to PAF in patients with coronary artery disease and positive exercise tests, but no effect of exercise on platelet responsiveness, ex vivo [30]. To the best of our knowledge, there is a paucity of data concerning the behaviour of platelets from patients with unstable angina to the aggregatory effect of PAF. Additionally, there is no data describing the effect of abciximab therapy in these patients on the aggregatory effect of PAF ex vivo. Therefore, the aim of the present study was to investigate the platelet response to the aggregatory effect of PAF in relation to the plasma PAF-AH activity in patients with unstable angina. The effect of abciximab therapy on the above parameters was also studied in these patients.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Study population
We studied 32 consecutive patients, 28 men and four women, (mean age 64±10 years, range 45–80 years) hospitalized in the coronary care unit of the Cardiology Department of the University Hospital of Ioannina with the diagnosis of unstable angina (class IIIb) [31]. Unstable angina was defined as a new onset or a sudden worsening of angina occurring at rest or on minimal exertion within the past 24 h, with ST-segment depression or T-wave inversion. Patients who presented with ST-segment elevation or new Q waves on the ECG or if they had a suspected myocardial infarction in evolution were not included in the study. Patients were also excluded from the study if they had contraindication to anticoagulation, a history of a platelet disorder or thrombocytopenia, a history of stroke or other intracranial pathology likely to predispose to bleeding or if they presented with severe hypertension (>180/120 mmHg) or renal insufficiency. A medical history regarding previous myocardial infarction, hypertension, smoking, and diabetes mellitus was carefully obtained in all patients. Standard medications, including nitrates, calcium channel blockers, β-blockers, and aspirin were routinely started and continued throughout the study period. On admission, heparin was administered in all patients for 2 days and adjusted to achieve an activated partial prothrombin time between 2 and 2.5 times the normal. No thrombolytic agents were used and none of the patients received ticlopidine during the study. After enrollment, 17 patients were randomly assigned abciximab (0.25 mg/kg bolus followed by a continuous infusion of 10 µg/min for 12 h), whereas the remaining 15 patients continued with the standard treatment alone. We followed a simple randomization method using a table of random numbers as described [32]. During hospitalization and 30 days afterwards all events and medications were recorded with special attention to bleeding complications and recurrent ischaemic symptoms. Five patients (three from the untreated group and two received abciximab developed non-Q wave myocardial infarction. Coronary arteriography was performed by a standard technique within 1–2 weeks after admission in nine untreated patients and 12 patients receiving abciximab. A population of 25 normolipidemic volunteers with a mean age of 52±12 years (range 40–64 years) was selected from the hospital staff and served as the control group. Volunteers had no evidence of cardiovascular disease as defined by a normal electrocardiogram at rest, negative exercise test and no history of coronary artery disease or chest pain. None of the volunteers had also a history of cancer, inflammatory disorder, or infection. The investigation conforms with the principles outlined in the Declaration of Helsinki (Cardiovascular Research 1997;35:2–3), and has been approved by the Ethics Committee of the University Hospital of Ioannina. All studied individuals gave written informed consent for the participation in the study.

2.2 Chemicals
PAF (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) purchased from Bachem (Switzerland) was dissolved in ethanol (80% v/v) at a final concentration of 20 mM. Aliquots of 100 µl from this solution were mixed with 30 µl of 1-O-hexadecyl-2-[3H]acetyl-sn-glycero-3-phosphocholine (10 Ci/mmol; Du Pont–New England Nuclear, Boston MA), dried under a stream of nitrogen, and redissolved in 2 ml of a solution containing bovine serum albumin (BSA)/saline (0.25%) to obtain a [3H]PAF solution with a concentration of 1 mM (specific activity: 2000 cpm/nmol) which was used as a substrate for PAF-AH. For the aggregation studies, aliquots of PAF solution in ethanol were evaporated under a stream of nitrogen, and redissolved in a solution containing BSA/saline (0.25%) to obtain PAF solutions with concentrations ranging from 0.1 to 10 µM. ADP, creatine kinase (CK), creatine phosphate (CP), BSA and trichloroacetic acid (TCA) were obtained from Sigma (St. Louis, MO, USA). Aspirin as a lysine soluble salt was from Galenica (Greece). ACD solution, consisting of 0.07 M citric acid, 0.11 M sodium citrate and 0.11 M D(+)glucose was used as anticoagulant. HEPES buffer, pH 7.4, consisted of 4.2 mM HEPES, 137 mM NaCl, 2.6 mM KCl and 2 mM EDTA.

2.3 Platelet aggregometry
Platelet aggregometry was performed using platelet-rich plasma (PRP) essentially as previously described [30] with some modifications. Briefly, the PRP containing 250 000 platelets/µl was pre-incubated with the cyclooxygenase inhibitor aspirin, 0.1 mM, 15 min before the aggregation in order to inhibit platelet aggregation due to thromboxane A2 (TxA2) production. Under these conditions platelet aggregation was performed by adding either PAF or ADP at a final concentration of 100 nM or 10 µM, respectively. The maximal aggregation achieved within 2 min after the addition of the agonist, expressed as a percentage of 100% light transmission calibrated for each specimen (maximal percentage of aggregation) was determined. The remaining aspirin-treated PRP was used for the determination of the PAF EC50 values (concentration which induces 50% of maximal aggregation). In these experiments, PRP was further treated with the combination of CP/CK (a scavenger of ADP) in a concentration of 5 mM and 40 U/ml, respectively, 1 min before the aggregation induced by different PAF concentrations. All aggregation studies were performed in a Chronolog aggregometer (model 330) at 37°C with stirring at 1200 rpm and completed within 2 h after venepuncture.

2.4 PAF-AH assay
PAF-AH activity was measured by the trichloroacetic acid (TCA) precipitation procedure as described [25,32]. For the routine assay, 50 µl of either plasma (diluted 1/50 v/v with HEPES buffer) or the HDL-containing supernatant after treatment of plasma with magnesium-dextran sulphate (diluted 1/5 v/v with the same buffer), were mixed with HEPES buffer in a final volume of 90 µl. The reaction was initiated by adding 10 µl of the 1 mM [3H]PAF solution in BSA/saline (100 µM final concentration in the reaction mixture). The PAF-AH assay was performed for 10 min, at 37°C, and the enzyme activity was expressed as nmol of PAF degraded per min per ml of plasma [33].

2.5 Other biochemical parameters
Total plasma triglyceride and cholesterol levels were measured by enzymatic colorimetric assays using commercially available enzymic reagents (Bio-Merieux). HDL-cholesterol levels were also determined by the same method after precipitation of apolipoprotein B (apoB)-containing lipoproteins with magnesium-dextran sulphate. LDL-cholesterol levels were calculated using the Friedewald formula.

2.6 Statistical analysis
The data were expressed as mean values±S.D. All measurements were made in duplicate and the average of the two values was used to compute the mean. To assess statistically significant differences among the studied groups we performed chi-square test, one-way analysis of variance (ANOVA) followed by Fisher’s least-significant of difference (LSD) method for those data presenting a normal distribution, and Kruskall–Wallis test followed by Mann–Whitney U-test for non-normally distributed data. Friedman one-way analysis of variance (Friedman ANOVA) followed by Wilcoxon signed-rank test was used to assess differences in measured parameters among the various time intervals of the study. A p-value of less than 0.05 was considered as statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Characteristics of the subjects investigated
Thirty two patients with unstable angina and 25 normolipidaemic control subjects participated in the study. Seventeen of the patients received abciximab therapy on the day of admission, whereas 15 were left untreated. As shown in Table 1, there were no differences in the age, body mass index or gender distribution among the three groups. No difference was also observed in prevalence of the major risk factors between the two patient groups. A history of myocardial infarction was noted in five untreated patients and in three patients receiving abciximab. The angiographic characteristics were also similar between the two patient groups. They did not differ in the use of β-blockers, nitrates, calcium channel blockers, or aspirin. No difference was also observed in the plasma triglyceride as well as the total- and LDL-cholesterol levels among the three groups. By contrast, the HDL-cholesterol levels and consequently the LDL-cholesterol to HDL-cholesterol ratio were significantly higher in both patient groups compared to control subjects (p<0.01). Within 1 month follow-up one untreated patient died. Angioplasty was required in three abciximab-treated patients and in two of the untreated ones. Bypass surgery was needed in one abximab-treated patient. Additionally, medication within 1 month follow-up was similar in the two groups. Finally, no bleeding complications occurred in the two patient groups.


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  		Table 1 Characteristics of the unstable angina patients and normolipidaemic control subjectsa

 
3.2 Platelet aggregatory response
The platelet aggregatory response to both PAF and ADP was determined in the control group as well as in the 15 untreated patients with unstable angina, within the first 24 h from the admission (day 1), as well as 4 days, 7 days and 1 month afterwards. Platelet aggregation in response to PAF in all samples of both groups was monophasic and fully reversible. As shown in Table 2, the PAF EC50 values of platelets treated with aspirin and CP/CK were significantly lower in the patient group on day 1 compared with the control group (13.7±7.3 and 28.2±9.4 nM, respectively, p<0.01). Fig. 1 shows typical sigmoidal curves for the estimation of the PAF EC50 values of platelet aggregation in both groups; the displacement to the left of the sigmoidal curve of platelet aggregation in the patient group (day 1) compared to the controls is apparent. The maximal percentage of aggregation to 100 nM PAF (measured in PRP treated with aspirin) was found to be significantly higher in the patient group on day 1 compared to controls (25.8±5.5 versus 14.8±4.1%, respectively, p<0.01) (Table 2). The platelet aggregatory response to PAF expressed either as EC50 values or maximal percentage of aggregation in the patient group was not significantly altered 4 days, 7 days or 1 month afterwards (Table 2). Similar behaviour was observed to the aggregatory effect of ADP. Thus, platelets from the patient group on day 1 were more sensitive to 10 µM ADP, since the maximal percentage of aggregation (measured in PRP treated with aspirin) was significantly higher compared to controls (54.6±15.3 versus 36.5±11.1%, respectively, p<0.003) (Table 2). Similarly to PAF, the platelet response to ADP was not significantly altered 4 days, 7 days or 1 month afterwards (Table 2).


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  		Table 2 Platelet aggregatory response and PAF-AH activity in patients with unstable anginaa

 

Figure 1
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  		Fig. 1 Typical sigmoidal curves for the estimation of the PAF EC50 values of platelet aggregation in patients with unstable angina and in normolipidaemic controls.

 
Seventeen patients received a bolus of abciximab (0.25 mg/kg) followed by a 12-h infusion (10 µg/min) on the day of admission. Platelet aggregation to PAF and ADP was studied before and 1 h, 2 days, 3 days, 6 days and 1 month after the bolus administration. As shown in Table 3, 1 h after the bolus administration of abciximab, platelet aggregation to both PAF and ADP (measured as % of maximum) was completely inhibited by 90±5 and 96±3%, respectively (p<0.001). Two days after bolus administration there was a significant recovery of the platelet aggregatory response to both agonists, although it was still significantly lower compared to the baseline values (p<0.01 for PAF and p<0.003 for ADP). On day 3, the platelet aggregatory response to PAF did not significantly change compared to that of day 2, whereas the response to ADP was further increased (p<0.04 compared to that of day 2), although it was still significantly lower compared to the baseline values (p<0.01). Platelet aggregation to both PAF and ADP was fully reversed 6 days after bolus (Table 3). It is noteworthy that the platelet aggregatory response to both PAF and ADP on the 6th day or 1 month after abciximab bolus was similar to that before treatment, thus it was still significantly higher compared to that of controls (p<0.01 for PAF and p<0.003 for ADP) (Tables 2 and 3Go). An important observation is that 2 days after abciximab bolus a significant difference between PAF and ADP in the inhibition of platelet aggregation, was observed (p<0.04). Thus, as it is shown in Fig. 2, platelet aggregation to PAF was inhibited by 35±6.3%, whereas platelet aggregation to ADP was inhibited by 44.6±9.6%. It is interesting to mention that 3 days after bolus the inhibition of platelet aggregation to PAF persisted, since it was inhibited by 38.7±12%, whereas the platelet aggregatory response to ADP was further restored, since the inhibition was reduced to 31±8% (p<0.04 compared to that measured on day 2) (Fig. 2). Another important observation is that the PAF EC50 values were affected by abciximab. Thus, as it is shown in Table 3, the PAF EC50 values 2 days and 3 days after bolus were significantly higher compared to those before administration (p<0.01 and p<0.03, respectively), and returned to the baseline values on day 6 after bolus administration.


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  		Table 3 Platelet aggregatory response and PAF-AH activity in patients with unstable angina received abciximaba

 

Figure 2
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  		Fig. 2 Inhibition of platelet aggregation induced by PAF and ADP in patients with unstable angina received the 0.25 mg/kg bolus and a 12-h infusion (10 µg/min) of abciximab. Results represent the mean values at each time point.

 
3.3 PAF-AH activity in plasma and HDL
The total plasma PAF-AH activity in both unstable angina patient groups measured within 24 h after admission to the hospital was not different to that of controls (Tables 2 and 3Go). By contrast, the HDL-associated PAF-AH activity in both patient groups was significantly lower compared to controls (3.5±0.8 nmol/ml/min for untreated patients and 4.0±0.9 nmol/ml/min for patients treated with abciximab, versus 5.0±1.4 nmol/ml/min for controls, p<0.01 and p<0.03, respectively) (Tables 2 and 3Go). The total plasma or HDL-associated enzyme activity was not significantly altered at any time interval studied (Table 2). Finally, it was not influenced by abciximab treatment (Table 3).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The results of the present study indicate that the platelet response to the aggregatory effect of PAF as well as to ADP, ex vivo, is significantly increased in patients with unstable angina compared to controls the first 24 h after admission to the hospital, and persists for at least 1 month afterwards. Furthermore, we show for the first time that bolus abciximab administration followed by a 12-h infusion completely inhibits platelet aggregation to PAF. It is remarkable that 2 and 3 days after bolus the platelet aggregatory response to PAF as well as to ADP was similar to that of the control group. However, 6 days after bolus administration the platelet aggregation to both agonists was similar to the baseline values, indicating that platelets become again more sensitive compared to controls.

Platelet activation and aggregation with resultant arterial thrombus formation are pivotal in the pathophysiology of unstable angina [3,6]. Platelets from patients with unstable angina exhibit a hyperreactivity compared to healthy individuals, while one of the features of this hyperreactivity is the enhanced aggregatory response to various agonists ex vivo [5,34]. The platelet hyperaggregability to PAF observed in our study could be of clinical importance and may be an additional factor contributing to the higher atherogenicity and to the acute coronary events observed in this population. Firstly, it has been established that the oxidized form of LDL plays an important role in the development of atherosclerosis [35]. Oxidized LDL is enriched with PAF [36] as well as with oxidized phospholipids [28,36], which exhibit PAF-like biological potency [28], including platelet aggregatory activity [37,38]. Thus, the increased platelet aggregatory response to PAF found in our unstable angina patients could take part in the evolution of the severe atherosclerosis observed in this population. Secondly, it has been shown that acute coronary events are associated with leukocyte and platelet activation, and increased leukocyte-platelet adhesion, which could play an important role in the acute inflammation and thrombosis occurring after plaque rupture [39,40]. Recently, it was suggested that PAF may be involved in a juxtacrine mechanism for neutrophil adhesion on platelets [41]. Additionally, stimulated endothelial cells synthesize PAF, which is transiently expressed on their surface playing a role as a proadhesive signalling molecule [42,43]. Since PAF is a potent agonist for neutrophils and platelets, which can also synthesize PAF upon stimulation [13], this mediator could contribute to the pathophysiological events associated with plaque fissuring and ulceration and thus to the induction of acute coronary events [44].

The increased aggregability of platelets to PAF in unstable angina is in accordance to our previously published results from patients with acute myocardial infarction, another acute coronary syndrome [30]. The biochemical or pathophysiological basis of the enhanced aggregatory response to PAF is not provided in our study. However, the displacement to the left of the sigmoidal curve of platelet aggregation to PAF in our patients favours the hypothesis that these platelets express a high density/affinity of receptors to PAF. Furthermore, we can exclude the possibility that the enhanced PAF aggregatory effect on platelets in our patients could be due to a platelet sensitivity to ADP or TxA2 secreted from platelets during aggregation, since both the cyclooxygenase inhibitor (aspirin) and the ADP scavenger (CP/CK) were present in our in vitro study. Additionally, it should be mentioned that only primary aggregation curves were obtained in our study, which is a further evidence that aggregation was not affected by any secreted endogenous agonist. Another factor which could contribute to the platelet hyperreactivity to PAF would be a low plasma PAF-AH activity. However, our results show that the plasma PAF-AH activity in the unstable angina patients is not different from that of controls, a finding which supports the suggestion that the higher aggregatory response to PAF in PRP is not due to any decrease in plasma PAF-AH activity.

Several studies have shown that human plasma PAF-AH may exert both antiinflammatory and antiatherogenic activity, since PAF-AH inactivates PAF and oxidized phospholipids [26], and inhibits the oxidative modification of LDL [45]. Our results show that although the PAF-AH activity in the plasma of our patients is not different compared to controls, the HDL-associated enzyme activity is significantly reduced in both patient groups. The importance of the HDL-associated PAF-AH activity in the HDL-mediated protection against (or reversal of) LDL oxidation has been demonstrated in previous studies [45,46]. Thus, when LDL is oxidized in the presence of HDL, the oxidized LDL-associated PAF and oxidized phospholipids may be transferred to HDL and then they are hydrolyzed by the HDL-associated PAF-AH. Furthermore, HDL could act as a source for replenishing PAF-AH in oxidized LDL, in which the enzyme activity is substantially decreased during oxidation [36,46]. Since the total cholesterol and LDL-cholesterol plasma levels in our unstable angina patients were similar to those of controls, we may suggest that the low plasma HDL cholesterol levels and the low HDL-associated PAF-AH activity could be important factors contributing to the higher atherogenicity observed in this population.

Among the new antiplatelet agents, abciximab is very effective in inhibiting platelet aggregation and is currently used in clinical practice [9]. Our study demonstrates for the first time that abciximab administration to unstable angina patients completely inhibits platelet aggregation to PAF, ex vivo, 1 h after bolus administration, while it is effective in regulating the platelet aggregatory response in the control levels 2 or 3 days afterwards. However, 6 days after bolus the platelet hypereaggregability is completely restored and persists for at least 1 month. This finding, as well as the unaltered, for at least 1 month, enhanced platelet aggregatory response of untreated patients, accord with the direct angioscopic observations of the lesion causing an acute coronary syndrome. These observations performed between 1 week and 1 month after the event showed no difference in the appearance of adherent mural thrombus overlying the culprit yellow plaque or in its incidence (70–83%) [47]. The longer duration of inhibition of PAF-induced platelet aggregation, as compared to ADP could be due to the fact that abciximab increases the PAF EC50 values. This observation allows us to speculate that abciximab could interfere with the platelet PAF receptor, a suggestion which needs further elucidation.

In conclusion, our study suggests that the increased aggregatory response of platelets to PAF and the low plasma levels of HDL-cholesterol and HDL-associated PAF-AH activity in patients with unstable angina may contribute to the severe atherosclerosis and to acute thrombosis found in these patients. Abciximab therapy may protect platelets from PAF action in vivo the first days after drug administration, but it fails to permanently restore the enhanced aggregatory response observed.

Time for primary review 32 days.


   Acknowledgements
 
This work was partially supported by a research grant from the European Community (BIOMED 2 programme PL 963191).


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

  1. Davies M.J., Thomas A.C. Plaque fissuring: the cause of acute myocardial infarction, sudden ischaemic death, and crescendo angina. Br Heart J (1985) 53:363–373.[Free Full Text]
  2. Falk E. Unstable angina pectoris with fatal outcome: dynamic coronary thrombosis leading to infarction and/or sudden death: autopsy evidence of recurrent mural thrombosis with peripheral embolization culminating in total vascular occlusion. Circulation (1985) 71:699–705.[Abstract/Free Full Text]
  3. Bashour T.T., Myler R.K., Andreae G.E., Stertzer S.H., Clark D.A., Ryan C.J.M. Current concepts in unstable myocardial ischemia. Am Heart J (1988) 115:850–861.[CrossRef][Web of Science][Medline]
  4. Sobel M., Salzman E.W., Davies G.C., et al. Circulating platelet products in unstable angina. Circulation (1981) 63:300–308.[Abstract/Free Full Text]
  5. Fitzgerald D.J., Roy L., Catella F., Fitzgerald G.A. Platelet activation in unstable coronary disease. N Engl J Med (1986) 315:983–989.[Abstract]
  6. Willerson J.T., Golino P., Eidt J., Campbell W.B., Buja L.M. Specific platelet mediators and unstable coronary artery lesions. Experimental evidence and potential clinical implications. Circulation (1989) 80:198–205.[Abstract/Free Full Text]
  7. Phillips D.R., Charo I.F., Parise L.V., Fitzgerald L.A. The platelet membrane glycoprotein IIb/IIIa complex. Blood (1988) 71:831–843.[Free Full Text]
  8. Lefkovits J., Plow E.F., Topol E.J. Platelet glycoprotein IIb/IIIa receptors in cardiovascular medicine. N Engl J Med (1995) 332:1553–1559.[Free Full Text]
  9. Coller B.S. Monitoring platelet GP IIb/IIIa antagonist therapy. Circulation (1997) 96:3828–3832.[Web of Science][Medline]
  10. Coller B.S. Blockade of platelet GP IIb/IIIa receptors as an antithrombotic strategy. Circulation (1995) 92:2373–2380.[Free Full Text]
  11. Lincoff A.M., Califf R.M., Andrerson K.M. The EPIC Investigators. Evidence for prevention of death and myocardial infarction with platelet membrane glycoprotein IIb/IIIa receptor blockade by abciximab (c7E3) among patients with unstable angina undergoing percutaneous coronary intervention. J Am Coll Cardiol (1997) 30:149–156.[Abstract]
  12. The CAPTURE investigators. Randomised placebo-controlled trial of abciximab before and during coronary intervention in refractory unstable angina: the CAPTURE study. Lancet (1997) 349:1429–1435.[CrossRef][Web of Science][Medline]
  13. Zimmerman G.A., Prescott S.M., McIntyre T.M. Inflammation, Basic Principles and Clinical Correlates. Gallin J.I., Goldstein I.M., Snyderman R., eds. (1993) New York: Raven Press. 149–175.
  14. Marcus A.J., Safier L.B., Ullman H.L., et al. Effects of acetyl glyceryl ether phosphorylcholine on human platelet function in vitro. Blood (1981) 58:1027–1031.[Abstract/Free Full Text]
  15. Braquet P., Godfroid J. Platelet-activating Factor and Related Lipid Mediators. Snyder F., ed. (1987) New York: Plenum Press. 153–190.
  16. van Willigen G., Hers I., Gorter G., Akkerman J.-W.N. Exposure of ligand-binding sites on platelet integrin {alpha}IIβ/β3 by phosphorylation of the β3 subunit. Biochem J (1996) 314:769–779.[Web of Science][Medline]
  17. Koltai M., Hosford D., Bourgain R.H., Braquet P. The role of PAF in blood cell-vessel wall interactions and atherosclerosis. Thromb Haemorrh Disord (1990) 2:47–60.
  18. Lepran J., Lefer A.M. Ischemia aggravating effects of platelet-activating factor in acute myocardial ischemia. Basic Res Cardiol (1985) 80:135–141.[CrossRef][Medline]
  19. Zimmerman G.A., McIntyre T.M., Prescott S.M. Perspectives series: Cell adhesion in vascular biology. Adhesion and signaling in vascular cell-cell interactions. J. Clin Invest (1996) 98:1699–1702.[Web of Science][Medline]
  20. Schwinger R.H.G., Bohm M., La Rosee K., Erdmann E. Existence of PAF receptors in human platelets and human lung tissue but not in the human myocardium. Am Heart J (1992) 124:320–330.[CrossRef][Web of Science][Medline]
  21. Siminiak T., Flores N.A., Sheridan D.J. Neutrophil interactions with endothelium and platelets: possible role in the development of cardiovascular injury. Eur Heart J (1995) 16:160–170.[Abstract/Free Full Text]
  22. Tselepis A.D., Pinckard R.N. PAF-acetylhydrolase: A novel enzyme with biochemical and clinical interest. Clin Chem Enzym Commun (1992) 4:279–290.
  23. Stafforini D.M., Prescott S.M., Zimmermann G.A., McIntyre T.M. Platelet-activating factor acetylhydrolase in human tissues and blood cells. Lipids (1991) 26:979–985.[Web of Science][Medline]
  24. Stafforini D.M., McIntyre T.M., Carter M.E., Prescott S.M. Human plasma platelet-activating factor acetylhydrolase. Association with lipoprotein particles and role in the degradation of platelet-activating factor. J Biol Chem (1987) 262:4215–4222.[Abstract/Free Full Text]
  25. Tselepis A.D., Dentan C., Karabina S.-A.P., Chapman M.J., Ninio E. PAF-degrading acetylhydrolase is preferentially associated with dense LDL and VHDL-1 in human plasma. Catalytic characteristics and relation to the monocyte-derived enzyme. Arterioscler Thromb Vasc Biol (1995) 15:1764–1773.[Abstract/Free Full Text]
  26. Stremler K.E., Stafforini D.M., Prescott S.M., McIntyre T.M. Human plasma platelet-activating factor acetylhydrolase: oxidatively fragmented phospholipids as substrates. J Biol Chem (1991) 266:11095–11103.[Abstract/Free Full Text]
  27. Smiley P.L., Stremler K.E., Prescott S.M., Zimmerman G.A., McIntyre T.M. Oxidatively fragmented phosphatidylcholines activate human neutrophils through the receptor for platelet-activating factor. J Biol Chem (1991) 266:11104–11110.[Abstract/Free Full Text]
  28. Heery J.M., Kozak M., Stafforini D.M., et al. Oxidatively modified LDL contains phospholipids with platelet activating factor-like activity and stimulates the growth of smooth muscle cells. J Clin Invest (1995) 96:2322–2330.[Web of Science][Medline]
  29. Tselepis A.D., Tsoukatos D., Droudes C., Donas A., Evangelou A. Platelet response to the aggregatory effect of platelet activating factor (PAF) ex vivo in patients with acute myocardial infarction. Eur J Clin Invest (1991) 21:490–496.[Web of Science][Medline]
  30. Goudevenos J., Tselepis A.D., Tsoukatos D., Grekas G., Kritikakos J., Sideris D. Platelet aggregability to platelet activating factor at rest and after exercise in patients with coronary artery disease. Eur Heart J (1995) 16:1036–1043.[Abstract/Free Full Text]
  31. Braunwald E. Unstable angina: a classification. Circulation (1989) 80:410–414.[Free Full Text]
  32. Pocock S.J., ed. Clinical trials. A practical approach. (1993) New York: Wiley. 66–89.
  33. Karabina S.-A.P., Elisaf M., Bairaktari E., Tzallas C., Siamopoulos K.C., Tselepis A.D. Increased activity of platelet-activating factor acetylhydrolase in low-density lipoprotein subfractions induces enhanced lysophosphatidylcholine production during oxidation in patients with heterozygous familial hypercholesterolaemia. Eur J Clin Invest (1997) 27:595–602.[CrossRef][Web of Science][Medline]
  34. Mehta J., Mehta P., Conti C.R. Platelet function studies in coronary artery disease. Am J Cardiol (1980) 46:943–947.[CrossRef][Web of Science][Medline]
  35. Witzum J.L., Steinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest (1991) 88:1785–1792.[Web of Science][Medline]
  36. Liapikos Th.A., Antonopoulou S., Karabina S.-A.P., Tsoukatos D.C., Demopoulos C.A., Tselepis A.D. Platelet-activating factor formation during oxidative modification of low density lipoprotein when PAF-acetylhydrolase has been inactivated. Biochim Biophys Acta (1994) 1212:353–360.[Medline]
  37. Ardlie N.G., Selley M.L., Simons L.A. Platelet activation by oxidatively modified low density lipoproteins. Atherosclerosis (1989) 76:117–124.[CrossRef][Web of Science][Medline]
  38. Tanaka T., Makoto I., Tsukatani H., Tokumura A. Platelet-aggregating effects of platelet-activating factor-like phospholipids formed by oxidation of phosphatidylcholines containing an sn-2 polyunsaturated fatty acyl group. Biochim Biophys Acta (1994) 1216:202–208.
  39. Siminiak T., Flores N.A., Sheridan D.J. Neutrophil interactions with endothelium and platelets: possible role in the development of cardiovascular injury. Eur Heart J (1995) 16:160–170.[Abstract/Free Full Text]
  40. Ott I., Neumann F.-J., Gawaz M., Schmitt M., Schömig A. Increased neutrophil-platelet adhesion in patients with unstable angina. Circulation (1996) 94:1239–1246.[Abstract/Free Full Text]
  41. Ostrovsky L., King A.J., Bind S., et al. A juxtacrine mechanism for neutrophil adhesion on platelets involves platelet-activating factor and a selectin-dependent activation process. Blood (1998) 91:3028–3036.[Abstract/Free Full Text]
  42. Prescott S.M., Zimmerman G.A., McIntyre T.M. Human endothelial cells in culture produce platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) when stimulated with thrombin. Proc Natl Acad Sci USA (1984) 81:3534–3538.[Abstract/Free Full Text]
  43. Zimmerman G.A., McIntyre T.M., Mehra M., Prescott S.M. Endothelial cell-associated platelet-activating factor: a novel mechanism for signaling intercellular adhesion. J Cell Biol (1990) 110:529–540.[Abstract/Free Full Text]
  44. Willerson J.T. Inhibitors of platelet glycoprotein IIb/IIIa receptors. Will they be useful when given chronically? Circulation (1996) 94:866–868.[Free Full Text]
  45. Stafforini D.M., Zimmerman G.A., McIntyre T.M., Prescott S.M. The platelet activating factor acetylhydrolase from human plasma prevents oxidative modification of low density lipoprotein. Trans Am Assoc Physiol (1993) 106:44–63.
  46. Watson A.D., Navab M., Hama S.Y., et al. Effect of platelet activating factor-acetylhydrolase on the formation and action of minimally oxidized-low density lipoprotein. J Clin Invest (1995) 95:774–782.[Web of Science][Medline]
  47. Van Belle E., Lablanche J.M., Bauters C., Renaud N., McFadden E.P., Bertrand M.E. Coronary angioscopic findings in the infarct-related vessel within 1 month of acute myocardial infarction: natural history and the effect of thrombolysis. Circulation (1998) 97:26–33.[Abstract/Free Full Text]

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