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Circulation. 1996;93:229-237

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(Circulation. 1996;93:229-237.)
© 1996 American Heart Association, Inc.

Articles

Platelet Function in Acute Myocardial Infarction Treated With Direct Angioplasty

Presented in part at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 1994.

Meinrad Gawaz, MD; Franz-Josef Neumann, MD; Ilka Ott, MD; Alexander Schiessler, MD; Albert Schömig, MD

From the 1. Medizinische Klinik der Technischen Universität München, Germany.

Correspondence to Dr Meinrad Gawaz, 1. Medizinische Klinik der Technischen Universität München, Klinikum rechts der Isar, Ismaninger Straße 22, 81675 München, Germany.


*    Abstract
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 *Abstract
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Background In acute myocardial infarction (AMI), platelets play a key role in thrombotic processes that limit the patency of the recanalized, infarct-related coronary artery and contribute to reperfusion injury. Platelet function in the course of AMI treated by direct percutaneous transluminal coronary angioplasty (PTCA) has not been evaluated.

Methods and Results In 15 patients with anterior AMI, peripheral venous blood samples were obtained before and 4, 8, 24, and 48 hours after recanalization of the occluded artery by PTCA. Fifteen patients who had stable coronary heart disease and were undergoing elective balloon angioplasty served as control subjects. Fibrinogen receptor function and surface expression of P-selectin on platelets were determined by flow cytometry. In addition, we evaluated generation of platelet-derived microparticles and the effect of systemic plasma from patients with AMI on normal platelet function and on platelet adhesion to human endothelial cells in culture. We found fibrinogen receptor activity and P-selectin expression on circulating platelets 8 hours after direct PTCA are decreased (P<.01). This coincided with a decrease in peripheral platelet count (P<.05) and an increase in generation of microparticles (P<.002). Twenty-four to 48 hours after PTCA, fibrinogen receptor activity and P-selectin expression increased again. Systemic plasma obtained before and after direct PTCA sensitized normal platelets to hyperaggregate in vitro (P<.001) and stimulated platelet adhesion to endothelial cells in culture (P<.01). None of the changes found in AMI were detectable in the control group.

Conclusions After transient apparent deactivation of circulating platelets, probably caused by sequestration of hyperactive platelets, the level of platelet activation increases in patients with AMI treated by direct PTCA. These findings underscore the need for novel antiplatelet strategies in AMI.


Key Words: platelets • revascularization • myocardial infarction • thrombosis • angioplasty


*    Introduction
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 up arrowAbstract
 *Introduction
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down arrowResults
down arrowDiscussion
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Direct PTCA in AMI is more effective than thrombolysis in restoring patency and preventing reocclusion of infarct-related artery.1 Recent randomized studies suggest that these advantages translate into an improved clinical outcome.2 3 4 However, clinical success of direct PTCA continues to be limited by platelet activation that contributes to infarct extension through thrombotic events. Furthermore, platelet-mediated impairment of microcirculation advances reperfusion injury of the area at risk.5 6

It is well recognized that platelets play a key role in arterial thrombosis and in acute ischemic coronary syndromes.7 8 9 10 11 12 13 The sequence of events leading to thrombosis is initiated by the adhesion of platelets to the vessel wall.14 15 Once contact has occurred, platelets become activated and surface expose fibrinogen binding sites on the membrane glycoprotein complex GPIIb/IIIa.16 17 18 19 Plasmatic fibrinogen can then bind to the platelet surface, enabling platelets to form microaggregates via fibrinogen bridging.19 Thereafter, platelets degranulate and the {alpha}-granule glycoprotein P-selectin is translocated to the activated platelet surface and serves to consolidate the thrombotic plug.16 During activation, platelets also form microparticles that are shed from the plasma membrane and released into the extracellular environment.20 21 22 Platelet-derived microparticles exhibit significant procoagulant activity, and their occurrence has been associated with thrombotic disease states.23 24

Thrombolysis in AMI has significant impact on platelet function.25 26 The effect of direct PTCA on platelet function has not been evaluated. In the present study, we elucidated various aspects of platelet function in patients with AMI undergoing direct PTCA. Specifically, we investigated surface receptors (GPIIb/IIIa, P-selectin) on circulating platelets, formation of platelet-derived microparticles, platelet aggregability, and platelet adhesion to cultured endothelial cells.


*    Methods
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 *Methods
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Patients
Thirty patients were included in the study (Table 1Down). All patients gave informed consent, and the study was approved by the institutional ethics committee. Fifteen patients presenting with anterior AMI were treated with direct PTCA for an occluded LAD that was judged suitable for recanalization by balloon angioplasty. In all patients with AMI, recanalization of the occluded LAD was successful, achieving TIMI 3 flow. Fifteen patients selected for elective PTCA for symptomatic high-grade LAD stenosis served as the control group. In addition, 20 healthy volunteers (mean age, 27 years; range, 24 to 40 years) were recruited from the hospital staff to establish normal range values for flow cytometry. Neither patients nor control subjects had a history of cancer, inflammatory disorders, or infection.


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  		Table 1. Clinical Characteristics of Study Patients

Study Design
All patients with AMI were administered a bolus injection of 5000 IU unfractionated heparin, 500 mg aspirin, and 5 mg (1x to 3x) metoprolol depending on individual response. Coronary angiography was performed via the transfemoral approach. Before angioplasty, an additional bolus of 10 000 IU heparin was administered intra-arterially. In control patients, PTCA was performed via the same approach after the administration of 15 000 IU heparin and 500 mg aspirin. Patients in both study groups were maintained on intravenous heparin for 24 hours after PTCA to obtain an activated partial thrombin time of two to three times normal.

Specimen Collection
Peripheral venous blood samples were taken by applying a mild tourniquet and then inserting a Luer-lock 18-gauge intravenous cannula in a forearm vein just before and then 4, 8, 24, and 48 hours after PTCA. With a multiple-syringe sampling technique, the first 2 mL of blood was discarded. Thereafter, 2.5 mL of blood collected in tubes containing EDTA was used for determination of platelet and white blood cell count, hemoglobin, and hematocrit by the use of a Sysmex counter (model F800, Digitana). Next, 4 mL was collected in tubes containing 3.8% citrate to determine coagulation parameters, and 5 mL was collected for serum chemistry profiles. Then, for flow cytometric analysis 1.6 mL of blood was collected into a polypropylene syringe containing 0.4 mL of CPDA. For determination of platelet-derived microparticles and for platelet testing in vitro, 10 mL of additional CPDA-anticoagulated blood was immediately placed on crushed ice, and plasma was stored at -120°C after centrifugation at 1500g for 20 minutes.

Preparation of Samples for Platelet Flow Cytometric Analysis
Preparation and immunolabeling of platelets with MAbs for flow cytometric analysis were performed immediately after blood was drawn as described.27 28 29 30 The platelet assay that we used provides reproducible results without significant artifactual platelet activation and has proved to be suitable for platelet analysis in a variety of clinical settings.27 28 29 30 To establish reference values, control samples obtained from healthy individuals were always run and processed simultaneously with patient samples. Antibody binding was expressed as relative mean particle fluorescence intensity of total platelet population and was used as quantitative measure for glycoprotein surface expression. The platelet population evaluated was found to be >=98% positive for the platelet-specific CD41 antigen.

MAbs
All MAbs used in the present study were either commercially obtained as FITC-conjugates or labeled with fluorescent dye according to standard methods. Anti-CD41 (Dianova) is raised against the GPIIb/IIIa complex and detects the receptor regardless of whether it is in its resting or activated form. Anti-LIBS1 MAb (generously provided by Dr Mark Ginsberg, Scripps Clinic) recognizes a cryptic epitope on GPIIIa that becomes exposed only on the activated and ligand-occupied GPIIb/IIIa complex.31 Thus, anti-LIBS1 binding indicates fibrinogen receptor activity on the platelet surface. Antifibrinogen MAb recognizes the E-fragment of the fibrinogen molecule and was used to detect fibrinogen molecules bound to the activated platelet surface. Anti-CD62P recognizes the {alpha}-granule membrane glycoprotein P-selectin (GMP-140, PADGEM) that is exclusively surface exposed on the activated platelet surface and was used as a marker for {alpha}-degranulation.16

Microparticles
Purified human fibrinogen (Sigma Chemical Co) was incubated with small latex beads (105 beads/mL) (diameter, 2.96 µm) at a final concentration of 1 g/L at room temperature for 2 hours. Thereafter, beads were washed once with Tyrode's buffer and incubated with 2% bovine serum albumin in Tyrode's buffer for an additional 1 hour. The particles were then passed over a PD10 column (Pharmacia) to separate latex beads from unbound fibrinogen. The final preparation of fibrinogen-coated particles was resuspended in Tyrode's buffer to obtain a particle count of 108/mL. To evaluate the presence of platelet-derived microparticles, 5 µL of fibrinogen-coated particles and 5 µL of CaCl2 (50 mmol/L) were added to 190 µL of CPDA-plasma and stirred constantly at a rate of 1000 rpm at 37°C for 30 minutes with the use of a platelet aggregometer (Chronolog). Thereafter, beads were immunostained with anti-CD41 as described above for platelets. CD41 immunofluorescence was used as parameter of microparticle binding to fibrinogen-coated beads.

Platelet Aggregation In Vitro
The effect of plasma collected from infarct and control subjects on aggregation of normal platelets obtained from healthy volunteers was evaluated in a suspension of washed platelets at 37°C using a two-channel Chronolog aggregometer. Washed platelets were prepared from platelet-rich plasma by centrifugation (200g for 20 minutes) of citrated whole blood as described.32 The final platelet count was adjusted to 2x108 platelets/mL with Tyrode's buffer containing 1 mmol/L CaCl2. Fifty microliters of platelet suspension were added to 150 µL of plasma and incubated under a constant stirring rate of 800 rpm at 37°C for 5 minutes. Thereafter, 5 µL of ADP (final concentration, 5 µmol/L) was added, and aggregation was recorded for 5 minutes. For flow cytometric analysis, platelets were incubated with plasma for 5 minutes without agitation before the addition of ADP.

HUVEC Culture
Primary HUVEC were harvested using collagenase digestion (Worthington) as described.33 Cells were grown in 96-well microtiter plates (Nunc) in complete media composed of M199 (Sigma), 10% FCS, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 mg/L streptomycin and immediately used for experiments as soon as they reached confluency after 4 to 8 days. The cells were identified as endothelial cells by their typical cobblestone morphology and by immunostaining for von Willebrand factor.

Platelet/Endothelium Adhesion
Monolayers of HUVEC were washed twice with M199. Platelet adhesion was evaluated by the addition of washed platelets (10 µL) to the endothelial cell–coated 96-microtiter wells (final density, 108 platelets/mL) supplemented with 100 µL of CPDA-plasma, 5 µL of 50 mmol/L CaCl2, and 85 µL of M199 (reaction volume, 200 µL). After a 30-minute incubation at 37°C, the nonadherent platelets were removed by two rounds of gentle washing with M199. After a 15-minute incubation with PE-conjugated anti-CD41 and FITC-conjugated anti-CD62P at 37°C, endothelial cells were detached and separated by the addition of 200 µL of 0.05% trypsin in 0.02% EDTA containing 1.67 mg/mL LDS-751 (Styry 18, Exciton Inc) antagonized by immediate addition of FCS. LDS-751 was used to label the nuclei of living endothelial cells; it does not require the cells to be permeabilized. For flow cytometric analysis, single separated HUVEC were identified by size and LDS-751 fluorescence. Five thousand events were evaluated, and mean intensity of CD41 immunofluorescence was used as a parameter of platelet/endothelium adhesion. Platelet adhesion was verified by immunofluorescence microscopy with a confocal laser microscope (Axiovert 35, Zeiss).

Statistical Analysis
The Kolmogorov-Smirnov test showed that the data were not normally distributed. Thus, results are reported as median (quartiles) values unless otherwise indicated. Differences between more than two matched samples were tested by Friedman's test followed by Wilcoxon's matched-pairs signed rank test, and differences between the study and the control groups were tested by the Mann-Whitney-Wilcoxon rank sum test or by Fisher's exact test, as appropriate. A value of P<.05 in the two-tailed test was regarded as significant.


*    Results
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*Results
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Baseline Characteristics
There were no significant differences between the infarction group (n=15) and the control group (n=15) with regard to age, sex, or coronary risk factor (Table 1Up). Mean time from the onset of symptoms in the infarct group and the start of PTCA was 4.5 hours (range, 3 to 9 hours). In all control patients, reconstruction of the stenosed artery was successful, resulting in a residual stenosis of <25%.

Membrane Glycoproteins on Peripheral Platelets
In patients with AMI before PTCA, we found a significantly increased platelet surface expression of activated fibrinogen receptor (LIBS1 immunofluorescence) and of membrane-bound fibrinogen compared with the control group (Fig 1Down) (activated fibrinogen receptor, P<.01; bound fibrinogen, P<.03). P-selectin associated with the platelet surface was also significantly higher in the AMI group than in the control group (P<.02) (Fig 1Down). The increase in fibrinogen receptor activity was not due to an overall increase in GPIIb/IIIa molecules present on the platelet surface because anti-CD41 immunofluorescence was not significantly different between the two groups (Fig 1Down).



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  		Figure 1. Plots of fibrinogen receptor function and surface expression of P-selectin showing individual values of surface expression of GPIIb-IIIa complex (A), activated fibrinogen receptor (B), bound fibrinogen (C), and P-selectin (D) on circulating platelets in patients with AMI before direct PTCA was performed (n=15) and in patients with stable angina (n=15) selected for elective PTCA (control subjects). Determination of receptors present on the platelet surface was performed with specific MAbs (anti-CD41, anti-LIBS1, anti-fibrinogen, and anti-CD62P) and flow cytometry. Relative mean intensity of immunofluorescence is shown. Horizontal lines indicate median values.

Effect of PTCA on Platelet Membrane Glycoproteins
After successful PTCA in AMI, surface expression of activated fibrinogen receptor, P-selectin, and bound fibrinogen decreased on circulating platelets, reaching statistical significance at 4 to 8 hours after the intervention (Fig 2Down). Thereafter, fibrinogen receptor activity and P-selectin expression rose again at 24 to 48 hours (Fig 2Down). No significant changes in platelet membrane glycoproteins after PTCA occurred in the control group (Fig 2Down). Surface densities of GPIIb/IIIa (anti-CD41 signals) were not altered throughout the observation period in both the infarct and the control groups (Fig 2Down). Thus, fibrinogen receptor activity in AMI was not due to an overall change in surface density of the GPIIb/IIIa complex.



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  		Figure 2. Plots of time course of platelet surface markers after direct angioplasty showing surface expression of GPIIb-IIIa (A), P-selectin (B), bound fibrinogen (C), and activated fibrinogen receptor (D) on circulating platelets in AMI patients (n=15) before and after (4, 8, 24, and 48 hours) successful recanalization of the infarct-related vessel ({bullet}). Patients undergoing elective angioplasty (n=15) served as controls ({circ}). * and # indicate significance compared with time before angioplasty (*) and compared with control group (#). Shaded boxes indicate median (quartiles) of relative immunofluorescence of normal individuals (n=20). Circles indicate median values; horizontal lines, quartiles.

Platelet Count and Microparticles
The decrease in platelet surface glycoproteins after PTCA in AMI coincided with a significant drop in peripheral platelet count 8 hours after the intervention (P<.05) (Fig 3Down). At the same time, the number of platelet-derived microparticles present in plasma was significantly increased (P<.002) (Fig 3Down). No change in platelet count or microparticle formation was found in the control group (Fig 3Down).



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  		Figure 3. Plots of platelet count and platelet-derived microparticles showing peripheral platelet count (A) and formation of platelet-derived microparticles (B) in patients with AMI treated by PTCA (n=15) ({bullet}). Fifteen patients undergoing elective PTCA served as controls ({circ}). * and # indicate significance compared with time before angioplasty (*) and compared with controls (#). Circles indicate median values; horizontal lines, quartiles.

Effect of Patient Plasma on Normal Platelet Function
In the presence of AMI, ADP-induced aggregation of normal platelets was significantly enhanced in plasma obtained before and after PTCA compared with control plasma (P<.001) (Fig 4Down). Similarly, ADP-induced surface expression of fibrinogen receptor and of P-selectin on normal platelets was enhanced in AMI compared with control samples (P<.001) (Fig 4Down).



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  		Figure 4. Plots of hyperaggregability of normal platelets showing the effect of plasma derived from AMI patients ({bullet}) and controls ({circ}) on aggregation (A), fibrinogen receptor activation (B), and surface expression of P-selectin (C) on normal platelets. Washed platelets isolated from healthy volunteers were reconstituted in patient plasma and incubated for 5 minutes at 37°C. After the addition of ADP (5 µmol/L), aggregation, surface expression of fibrinogen receptor, and P-selectin were determined as described in `Methods.' Significance of P<.01 was obtained for all tested time points.

Platelet/Endothelium Adhesion
Compared with control plasma, plasma from patients with AMI stimulated adhesion of normal platelets to cultured HUVEC (P<.01). This was demonstrated by enhanced binding of platelet-specific MAb anti-CD41 to endothelial cells (Fig 5Down). Maximal platelet/endothelium adhesion was found in the presence of AMI plasma obtained 48 hours after PTCA (Fig 5Down). Concomitantly, P-selectin associated with the endothelial surface was also significantly increased in infarct compared with control samples (P<.01) (Fig 5Down). Only background fluorescence on endothelial cells was noted in the absence of platelets (Fig 5Down). Platelet adhesion to endothelial cells was verified by the use of confocal laser microscopy (Fig 6Down).



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  		Figure 5. Plots showing the effect of patient plasma on platelet adhesion to cultured HUVEC. Normal platelets were resuspended in plasma and incubated for 30 minutes with confluent monolayers of HUVEC. After two rounds of washes to remove nonadherent platelets, endothelial monolayers were incubated with saturating concentrations of MAbs (anti-CD41, anti-CD62P). Anti-CD41 immunofluorescence present on endothelial cells was determined by flow cytometry and was used as an index of platelet to endothelium adhesion. Plots show platelet adhesion (A) and P-selectin surface exposure (B) on endothelial cells in the presence of AMI or control plasma. C and D, Effects of patient plasma in the absence of platelets on CD41 and CD62P immunofluorescence.




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  		Figure 6. Photomicrographs depicting platelet adhesion to cultured HUVEC. Washed platelets from healthy volunteers were reconstituted in plasma obtained from a patient with AMI, incubated with confluent cultured HUVEC, and immunostained with anti-CD41 as described in `Methods.' After two rounds of washings, samples were fixed with 4% paraformaldehyde in phosphate-buffered saline and evaluated by confocal laser microscopy. B, Corresponding CD41 immunofluorescence micrograph of A.

Effects of Medications
Ten of 15 patients who presented with AMI and 13 of the control patients were receiving aspirin on a long-term basis with no significant differences between the two groups (P=.388) (Table 2Down). Likewise, antianginal pretreatment did not differ significantly between the two study groups (P>.5) (Table 2Down). In patients with AMI, no significant differences in platelet function were shown between patients receiving long-term pretreatment with aspirin or ß-blockers and patients not receiving long-term cardiac medication before AMI (data not shown).


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  		Table 2. Pre- and Post-PTCA Medication

In the observation time (48 hours) after PTCA, all patients of both groups received intravenous heparin for 24 hours, and all received antiplatelet therapy on an indefinite basis (Table 2Up). Except for a trend toward a more frequent use of intravenous nitrates (P=.11), antianginal treatment after PTCA was essentially the same in the two study groups (P=1.0) (Table 2Up).


*    Discussion
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 up arrowIntroduction
 up arrowMethods
up arrowResults
*Discussion
down arrowReferences
 
Platelet Activation Before and in Early Hours After Direct PTCA
The present study is the first to evaluate the effect of direct PTCA on platelet activation in AMI. We found that platelet fibrinogen receptor activity and surface exposure of P-selectin are significantly enhanced in AMI. At 4 to 8 hours after successful PTCA, surface densities of both receptors on circulating platelets transiently declined. This observation was not procedure related because no significant change in membrane glycoproteins was seen in the control group. Peripheral platelet count also fell below starting values in the early hours after direct PTCA.

The decrease in membrane-exposed glycoproteins may, thus, reflect sequestration of highly adhesive circulating platelets rather than platelet deactivation. This explanation is supported by recent findings that P-selectin expression is correlated with decreased platelet survival.34 Furthermore, studies in animal showed that platelet adhesion to blood vessels damaged by angioplasty is maximal after 4 to 8 hours.35 In addition, the decrease in surface glycoproteins may be caused by the generation that we describe of microparticles that are shed from the platelet surface during the activation process, resulting in loss of membrane glycoproteins. Thus, we conclude that early after direct PTCA, platelet activation is significantly enhanced, as reflected by increased platelet consumption and microparticle formation.

Platelet Activation 24 to 48 Hours After Direct PTCA
We found that the decrease in fibrinogen receptor activity and P-selectin expression on circulating platelets early after PTCA is transient and that surface exposure of both receptors is increased again after 24 to 48 hours. This increase coincides with a rise in platelet count and a decrease in microparticle formation, suggesting that population and function of platelets in the circulation have changed. Function of circulating platelets may have been altered by the appearance of new platelets released from bone marrow. AMI is associated with an increased ploidy of megakaryocytes,36 and acute inflammatory phase products (eg, interleukin 6) that are present in AMI37 have been shown to stimulate thrombopoiesis.

Sustained platelet activation in the course of AMI treated by direct PTCA is further substantiated by our in vitro experiments. We found that normal platelets responded hyperreactively in the presence of plasma obtained from patients with AMI. This was not related to the PTCA procedure as demonstrated by our experiments with control plasma from patients who underwent elective PTCA. Thus, plasma-derived mediators that sensitize platelet reactivity are present in AMI, which results in overexpression of surface receptors. The nature of these compounds remains speculative. However, an enhanced sympathoadrenal activation has been suggested to play a role in thrombosis,38 and an increased concentration of circulating catecholamines may contribute to the increased platelet aggregation seen in myocardial infarction.39 40 Moreover, enhanced platelet responsiveness may be a consequence of systemic inflammatory response syndrome that evolves in AMI. Acute inflammatory phase products such as fibrinogen or interleukin 6 are increased in AMI37 and have been shown to modulate platelet function.41 Plasmatic fibrinogen is a prerequisite for platelet aggregation, and concentration of this glycoprotein directly correlates with aggregation.42 Interleukin 6 has been found to augment platelet aggregation in vitro.43 Furthermore, platelet products (eg, ADP or serotonin) released during the activation process might contribute to the observed hyperresponsiveness in vitro.40

Platelet Adhesion to Endothelium in AMI
Enhanced interaction of activated platelets with endothelium has been suggested to play a role in the development of reperfusion injury and impairment of coronary vasomotor and endothelial function.6 44 45 We found that adhesion of activated platelets to cultured endothelial cells (HUVEC) is increased in the presence of AMI plasma, whereas only marginal platelet adhesion was found in the presence of control plasma. Thus, the above-described hyperresponsiveness of platelets in AMI is paralleled by a significant platelet adhesion to cultured endothelium. Furthermore, we found that platelet/endothelium adhesion was accompanied by enhanced surface exposure of P-selectin. Together with P-selectin, potent vasoactive compounds (eg, adenine nucleotides, thromboxane A2, serotonin, and thrombin) are likely to be released at the adhesion site. This may lead to local accumulation of platelet-derived substances. The presence of thromboxane, serotonin, and other platelet-derived mediators has been previously shown to promote platelet activation, coronary artery vasoconstriction, and reduction in coronary blood flow.12 46 47 Thus, platelet/endothelium adhesion and microcirculatory entrapment of platelets in AMI may impair microcirculation and may limit myocardial salvage by reducing collateral flow to the area at risk. This conclusion is further supported by the observation that platelet consumption contributes to the extent of myocardium necrosis.48 Moreover, the present findings may help to explain impaired coronary endothelium-dependent vasodilator responses, even in areas of myocardium not directly supplied by the infarct-related artery.5

P-selectin has been found to be a specific receptor for the attachment of neutrophils to activated platelets and to activated endothelium.16 Moreover, adherent platelets to the vessel wall have been shown to promote leukocyte-dependent fibrin deposition via P-selectin.49 Thus, our data may offer a novel platelet-mediated mechanism for leukocyte-dependent tissue injury and disturbance of microcirculation during reperfusion. This may reflect one aspect of why platelets can contribute to arrhythmogenic, hemodynamic, and necrotic effects in myocardial ischemia.

Study Limitations
The findings of the present study indicate enhanced platelet/endothelium adhesion induced by plasma constituents in AMI. However, we do not provide data that this phenomenon also occurs in the circulation. The pathophysiological importance in respect to myocardial salvage or occurrence of reperfusion arrhythmia remains to be assessed.

The purpose of the study was to test platelet function in the setting of currently established therapeutic regimens. Therefore, medication, in particular, aspirin and ß-blockers, may have affected the results.50 However, in the time period after direct PTCA, no significant change in platelet function was noted between patients who had been receiving long-term cardiac medication, including aspirin and ß-blockers. Differences between study and control patients cannot be attributed to differences in medication because neither preinterventional nor postinterventional treatment differed significantly between the two study groups.

The study focuses on AMI as representing the most severe manifestation within the continuum of acute coronary syndromes.12 The data are compared with those of a group of patients with symptomatic but stable coronary vessel disease. Comparison with unstable angina may also be of intriguing interest but was beyond the scope of the current work. The role of platelets in unstable angina has, however, been extensively investigated in previous studies.8 12 46 The present study demonstrates that the concepts derived from previous work on unstable angina may also hold true for AMI. In addition, it broadens our view by focusing on platelet/endothelium adhesion.

Clinical and experimental studies have convincingly shown that platelet aggregation is involved in reocclusion after thrombolysis.51 Moreover, thrombolysis in AMI has been shown to activate platelets, as demonstrated by enhanced thromboxane A2 production.25 26 The present data emphasize the importance of platelets in the postinfarct period. However, differences in study design and in platelet assays do not allow comparison of the effect of thrombolysis with that of direct PTCA on platelet function in AMI.

Pathophysiological Considerations and Therapeutic Implications
The risk of reocclusion after PTCA in AMI is substantial. It has been reported that expression of platelet membrane glycoproteins is associated with increased risk of acute ischemic events after angioplasty.52 53 54 55 56 Furthermore, clinical studies suggest that PTCA-injured blood vessels are highly platelet thrombogenic for the first 2 to 3 days.57 Thus, enhanced surface expression of glycoproteins on circulating platelets found 24 to 48 hours after PTCA may increase the risk of thrombotic reocclusion of the recanalized infarct artery. The importance of platelets in the postinfarct period is substantiated by recent reports that persistent platelet activation is associated with recurrence of postinfarct angina.58

Microparticles are formed during platelet activation and have been associated with procoagulatory diseases, including idiopathic thrombocytopenia purpura, acute respiratory distress syndrome, and transient ischemic attacks.23 24 59 Thus, in addition to enhanced platelet reactivity, disseminated procoagulant activity around a platelet aggregate might trigger coagulation cascade and thrombin formation and, thus, vaso-occlusive thrombotic events in the course of AMI.

Despite administration of high doses of aspirin at the time of catheterization, we found significant platelet activation in our patients with AMI. Thus, the present study suggested that there is a need for effective adjunct antiplatelet therapy for direct PTCA in AMI. Inhibition of platelet fibrinogen receptor function has been found to reduce complications in high-risk PTCA.57 Furthermore, inhibition of fibrinogen receptor has been found in vitro to significantly reduce formation of procoagulant platelet microparticles.60 Recent studies in animals have shown that antibodies to P-selectin protect against attenuation of coronary flow reserve and myocardial contractile function after coronary occlusion/reperfusion.6 Whether inhibition of platelet/endothelium adhesion is of potential clinical interest remains to be shown. The present data provide a rationale for future clinical studies addressing novel antiplatelet strategies in AMI.


*    Selected Abbreviations and Acronyms
 
AMI = acute myocardial infarction
CPDA = citrate/phosphate/dextrose acid/adenine
FCS = fetal calf serum
FITC = fluorescein
HUVEC = human umbilical vein endothelial cells
LAD = left anterior descending coronary artery
M199 = medium 199
MAb = monoclonal antibody
PE = phycoerythrin
PTCA = percutaneous transluminal coronary angioplasty


*    Acknowledgments
 
This study was supported in part by a grant from the Deutsche Forschungsgemeinschaft (Ga 381/2-1). The authors appreciate the expert technical assistance of Caroline Bogner, Margit Huber, Stefan Mehringer, and Markus Schrödel. We thank Prof M. Schmitt for assisting us with confocal laser microscopy.

Received May 1, 1995; revision received July 18, 1995; accepted August 25, 1995.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
up arrowDiscussion
*References
 

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  4. Zjistra F, Jan de Boer M, Hoorntje JCA, Reiffers S, Reiber JHC, Suryapranata H. A comparison of immediate coronary angioplasty with intravenous streptokinase in acute myocardial infarction. N Engl J Med. 1993;328:680-684. [Abstract/Free Full Text]
  5. Uren NG, Crake T, Lefroy DC, DeSilva R, Davies GJ, Maseri A. Reduced coronary vasodilator function in infarcted and normal myocardium after myocardial infarction. N Engl J Med. 1994;331:222-227. [Abstract/Free Full Text]
  6. Chen LY, Nichols WW, Hendricks JB, Yang BC, Metha JL. Monoclonal antibody to P-selectin (PB1.3) protects against myocardial reperfusion injury in the dog. Cardiovasc Res. 1994;28:1414-1422. [Abstract/Free Full Text]
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