EXPERIMENTAL STUDY
Lack of effect of glycoprotein IIb/IIIa blockade on myocardial platelet or polymorphonuclear leukocyte accumulation and on infarct size after transient coronary occlusion in pigs
José A. Barrabés, MD*,
David Garcia-Dorado, MD, FACC*,*,
Maribel Mirabet, PhD*,
Rosa-Maria Lidón, MD*,
Bernat Soriano, PharmD ,
Marisol Ruiz-Meana, PhD*,
Pilar Pizcueta, PhD ,
José Blanco, MD*,
Yolanda Puigfel, RN* and
Jordi Soler-Soler, MD, FACC*
* Servicios de Cardiología, Hospital Universitari Vall d’Hebron, Barcelona, Spain
Medicina Nuclear, Hospital Universitari Vall d’Hebron, Barcelona, Spain
Institut de Malalties Digestives, Hospital Clínic, Barcelona, Spain
Manuscript received December 20, 2000;
revised manuscript received August 21, 2001,
accepted October 11, 2001.
* Reprint requests and correspondence: Dr. David Garcia-Dorado, Servicio de Cardiología, Hospital Universitari Vall d’Hebron, Pg. Vall d’Hebron 119-129, 08035 Barcelona, Spain.
dgdorado{at}hg.vhebron.es
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Abstract
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OBJECTIVES: We sought to assess the effect of glycoprotein (GP) IIb/IIIa blockade on myocardial platelet and polymorphonuclear leukocyte accumulation and on infarct size after coronary injury and transient coronary occlusion (CO) in pigs.
BACKGROUND: It has been suggested that platelet GP IIb/IIIa blockade might reduce the severity of microvascular damage after reperfusion.
METHODS: Sixteen thiopental-anesthetized, open-chest pigs, in whom platelets had been labeled with technetium-99m (99mTc) on the previous day, were submitted to catheter-induced left anterior descending coronary artery (LAD) injury followed by 55 min of CO and 5 h of reperfusion. Five minutes before reflow, the animals were blindly allocated to receive lamifiban (intravenous bolus of 250 µg/kg body weight and continuous infusion of 3 µg/kg per min) or saline.
RESULTS: Lamifiban had a rapid and potent platelet anti-aggregatory effect, as demonstrated by significant prolongation of the bleeding time and profound ( 90%) inhibition of ex vivo platelet aggregation, and completely prevented the development of cyclic flow reductions of the LAD (0 vs. 5 ± 1, one of them followed by re-occlusion, in control animals, p = 0.005). However, compared with animals receiving placebo, those treated with lamifiban had a similar (p = NS) content of 99mTc platelets in the reperfused myocardium (288 ± 40% vs. 205 ± 27% of the value in the control region, respectively) and similar myeloperoxidase activity (0.50 ± 0.17 U/g vs. 0.47 ± 0.17 U/g, respectively) and infarct size (46.8 ± 12.0% vs. 49.8 ± 10.5% of the area at risk, respectively). Arteriolar platelet thromboemboli were very rarely seen on histologic analysis. Lamifiban did not modify platelet P-selectin expression in additional studies.
CONCLUSIONS: Platelet GP IIb/IIIa blockade has a potent antithrombotic effect at the culprit lesion, but does not significantly reduce the magnitude of microvascular platelet accumulation or myocardial damage after transient CO.
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Abbreviations and Acronyms
| | ADP | | adenosine diphosphate | | CFR | | cyclic flow reduction | | CO | | coronary occlusion | | FBS | | fetal bovine serum | | GP | | glycoprotein | | HMPAO | | hexamethylpropyleneamineoxime | | LAD | | left anterior descending coronary artery | | MPO | | myeloperoxidase | | PBS | | phosphate-buffered saline | | PMNs | | polymorphonuclear leukocytes | | 99mTc | | technetium-99m |
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Reperfusion strategies have led to a dramatic improvement in the prognosis of patients with acute myocardial infarction. However, the beneficial effect of successful restoration of coronary flow on myocardial salvage may be hampered by inadequate tissue perfusion (1–3). Recovery of myocardial flow after coronary reperfusion may be compromised, especially in areas of severe ischemic damage, by intravascular plugging of polymorphonuclear leukocytes (PMNs), deposition and aggregation of platelets or edema (4–10). In addition, periodic dislodgment of the culprit coronary thrombus might result in distal embolization, causing additional microvessel obstruction (11,12).
Among several strategies that have been developed to increase the benefits of coronary reperfusion by reducing the severity of microvascular obstruction, platelet glycoprotein (GP) IIb/IIIa blockade appears to be one of the most promising. Besides promoting a more rapid and stable infarct artery patency when administered as an adjunct to reperfusion therapy (13,14), recent clinical data suggest that GP IIb/IIIa inhibitors could directly improve microvascular perfusion (15,16). However, experimental data supporting this hypothesis are scarce (17–20), and little is known about the effects of GP IIb/IIIa blockade on microvascular blood cell deposition and on myocardial salvage after transient coronary occlusion (CO) in vivo.
Accordingly, the present study tested whether lamifiban, a potent and selective GP IIb/IIIa inhibitor (13), administered during reperfusion, was able to reduce the magnitude of myocardial platelet or PMN accumulation or infarct size after coronary injury and transient CO in swine.
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Methods
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Animal preparation.
After approval by the Institutional Research Commission, and in accordance with the Position of American Heart Association on Research Animal Use, adopted by the Association in November 1984, 19 farm pigs of either gender, weighing 25 ± 1 kg, were sedated with 10 mg/kg of azaperone intramuscularly, anesthetized with 10 mg/kg of thiopental intravenously, intubated and connected to a mechanical ventilator supplying room air. Anesthesia was maintained with a continuous infusion of thiopental. One femoral artery and both femoral veins were catheterized; the right carotid artery was dissected; a sternotomy was performed; and the pericardium was opened. The left anterior descending coronary artery (LAD) was dissected free at its mid segment and surrounded by a snare.
Study groups and protocol.
The LAD was injured, as previously described (10,21), by means of an intracoronary Cordis 2.5F catheter (Miami, Florida) introduced through the carotid artery, to stimulate the development of mural thrombosis. After this injury, the LAD was ligated for 55 min, followed by 5 h of reflow. Five minutes before reperfusion, the animals were randomly allocated to receive—blindly to the investigators performing the experiments—lamifiban (250 µg/kg body weight IV followed by a continuous infusion of 3 µg/kg per min, a dose that had shown to cause profound inhibition of platelet aggregation in this model) or saline. Three animals were excluded before randomization: two due to severe hypotension and one due to occlusion of the left main coronary artery during catheterization. Therefore, 16 animals, 8 receiving lamifiban and 8 receiving placebo, were included.
Study monitoring.
Serial arterial blood gases were obtained to adjust the ventilatory variables. Aortic blood pressure was continuously monitored with a crystal quartz transducer and LAD flow with an electromagnetic flowmeter (MDL-1401, Skalar, Holland). These signals, along with lead II of the electrocardiogram, were amplified and continuously recorded at a sampling rate of 200 Hz per channel on a thermic pad recorder (Astro-Med MT9500, West Warwick, Rhode Island). Cyclic flow reductions (CFRs) were defined as progressive reductions in blood flow at the mid LAD over a period of minutes, followed by abrupt increases in blood flow with an amplitude of at least 25% of the maximal blood flow value (22). When ventricular fibrillation occurred, it was converted to sinus rhythm by internal shocks of 10 to 20 J.
Bleeding time and ex vivo platelet aggregometry.
Serial measurements of bleeding time and platelet aggregation were performed to assess the effect of lamifiban. A small incision was made on one ear of the animal, as previously described (21), and the time until bleeding ceased was measured. Two measurements were performed at each one of these time points: before CO, before treatment initiation, and 15 min, 1 h, and 2 h after treatment initiation. The mean value of each pair was calculated and used for analysis. At these time points, venous blood was withdrawn and processed as previously described (21) to obtain platelet-rich plasma. The platelet count (System-8000, Baker Instruments, Allentown, Pennsylvania) in platelet-rich plasma was maintained <350 x 103 platelets/µl by adding platelet-poor plasma. The platelet-rich plasma was distributed into 0.45-ml siliconized cuvettes and warmed to 37°C. Platelet aggregation was induced by addition of 10 µmol/l of adenosine diphosphate (ADP) in saline (21) and was measured in a four-channel aggregometer (Chrono-log 570VS, Havertown, Pennsylvania) as the maximal increase in light transmittance, expressed as a percentage of that observed with 0.5-ml platelet-poor plasma.
Myocardial platelet content.
The previous day, the animals were anesthetized and ventilated as described; one femoral vein was catheterized and 51 ml of blood was withdrawn. The blood was processed as previously described (10,21), and platelets were isolated and labeled with 99mTc-hexamethylpropyleneamineoxime (HMPAO) and re-injected into the animal. Then, anesthesia was stopped, the femoral cannula was withdrawn and the animals were allowed to recover, after which they were extubated and returned to the animal room until the next day. Assuming that the total blood pool in swine represents 7% of the animal weight, the labeling fraction in animals allocated to lamifiban or placebo averaged 3.3 ± 0.2% and 2.9 ± 0.2% of total blood, respectively (p = NS). The labeling efficiency averaged 55.1 ± 3.2%, and 16.1 ± 1.2 mCi of 99mTc was injected into each animal (both p = NS between groups).
After 5 h of reperfusion, the LAD was re-occluded, 5 ml of 10% fluorescein was injected into the left atrium and the heart was excised, immersed in Ringer’s solution at 4°C and cut in 5- to 7-mm slices. The slices were weighed, and transmural myocardial fragments (0.78 ± 0.06 g) were obtained from the area at risk and the inferior region in the third slice. The radioactivity of these fragments was counted for 20 s in a gamma counter (1282-Compugamma, Wallac, Turku, Finland). Specific activity in reperfused myocardium (counts/g) was calculated and expressed as a percentage of the specific activity in the control myocardium.
Myocardial PMN accumulation and infarct size.
After gamma counting, the myocardial samples were stored at –80°C. The samples were processed as previously described (10,21), and myeloperoxidase (MPO) activity was quantified after addition of o-dianisidine dihydrochloride and H2O2, expressed in units defined as the quantity of enzyme degrading 1 µmol peroxide/l per min at 25°C.
The remaining slices were weighed and imaged from the basal side with a video camera under ultraviolet light. Instead of the third slice, the apical surface of the fourth slice was imaged. The slices were then incubated at 37°C in 1% triphenyltetrazolium chloride, at pH 7.4, for 10 min, and imaged under white light. The images were digitized into 768 x 576 pixel images (Matrox-IP8 card, Dorval, Quebec, Canada), and the area at risk and the area of necrosis were measured semi-automatically (Image Pro-Plus software, Media Cybernetics, Silver Spring, Maryland) in the digital images, and infarct size was calculated from these measurements and the weight of the slices.
Histologic analysis.
The slices were fixed in 10% formaldehyde, and the fourth slice was processed for histologic analysis (10,23), stained with hematoxylin and eosin and examined with an Olympus IMT2 microscope. Infiltration of PMNs was graded according to a previously described score (23): 0 = absent; 1 = scant; 2 = intravascular plugs; and 3 = intravascular plugs and PMNs present in the interstitium. Arteriolar platelet plugs >50 µm in diameter were considered as microemboli (10–12).
Platelet P-selectin expression.
In five additional experiments, aliquots of platelet suspensions were incubated for 25 min at room temperature with the anti–P-selectin monoclonal antibody KO.2.9 (24), washed with phosphate-buffered saline (PBS)/0.5% fetal bovine serum (FBS) and further incubated for 25 min with F(ab")2 fragments of a goat anti-mouse fluorescein isothiocyanate-conjugated antibody (3.5 µg/ml, Caltag, Burlingame, California). Platelets were washed again and resuspended in PBS/0.5% FBS for flow cytometric analysis. Negative control studies were performed using an equivalent volume of PBS/2% FBS instead of the anti–P-selectin antibody. The platelet population was analyzed at a low flow rate in a FACScaslibur flow cytometer (Becton Dickinson, San Jose, California). Platelets were identified on the basis of forward and sideward scatter variables in the logarithmic mode. For each sample, 20 x 103 platelets were collected. Data were analyzed with CELLQuest software (Becton Dickinson, Mansfield, Massachusetts), and the results were expressed as a percentage of specific antibody-positive platelets, defined as those with a fluorescence intensity exceeding that of 99% of negative control platelets.
Stability of labeling with 99mTc/HMPAO and effects on platelet function.
The stability of platelet labeling was assessed in five additional experiments. Labeled platelets were resuspended in platelet-poor plasma and distributed into 1-ml aliquots that were incubated at 33°C. Ninety minutes, 17 h and 22 h after labeling, these samples were centrifuged, and the radioactivity in the platelet pellet and in platelet-poor plasma was counted. To confirm that free 99mTc/HMPAO is rapidly cleared from the blood in our model, venous blood was withdrawn at these same time points after 99mTc/platelet re-injection in three additional animals, and the radioactivity in platelet-poor plasma was quantified and compared with that in platelet-rich plasma. Finally, in three animals, the influence of labeling on platelet P-selectin expression and on platelet aggregation induced by 10 µM ADP was investigated.
Statistical analysis.
The study was intended to detect a large, clinically relevant reduction of infarct size by lamifiban. According to the variability of infarct size in the same model in previous studies in our laboratory, the sample size was powered to detect, with alpha and beta probabilities of 0.05 and 0.2, respectively, a reduction in infarct size of 15% of the area at risk. Statistical analysis was performed using SPSS software (SPSS Inc., Chicago, Illinois). Comparisons between groups were performed by the Student t test, with the exception of those involving histologic scores, which were subjected to the Mann-Whitney U test. General linear model analysis of variance for repeated measures was used to evaluate intra-subject factors (e.g., changes in physiologic variables along time, differences between control and ischemic myocardium), inter-subject factors (e.g., effect of treatment) and the interaction between both. Data are expressed as the mean value ± SEM. A p value <0.05 was considered statistically significant.
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Results
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Hemodynamic data and ventricular arrhythmias.
Hemodynamic variables were similar in animals receiving lamifiban or placebo (Table 1). Mean aortic pressure remained stable during the experiment. At baseline, mean blood flow at the mid LAD in animals allocated to lamifiban or placebo was 12 ± 1 ml/min and 15 ± 2 ml/min, respectively (p = NS). One animal from the placebo group had ventricular fibrillation during CO, and three (one receiving lamifiban and two on placebo) had ventricular fibrillation immediately after reflow.
Antithrombotic effect of lamifiban.
At baseline, the bleeding time in animals allocated to lamifiban or placebo was 80 ± 8 s and 91 ± 7 s, respectively (p = NS). In animals receiving placebo, the bleeding time and ADP-induced platelet aggregation did not change appreciably during the experiment. In contrast, lamifiban rapidly produced a significant prolongation of the bleeding time and a profound (90% of baseline values) inhibition of platelet aggregation (Fig. 1). In animals treated with lamifiban, the development of CFRs after reperfusion was completely prevented, whereas animals receiving placebo had an average of 5 ± 1 CFRs (p = 0.005). One of the animals in the placebo group had LAD re-occlusion and was excluded for further analysis.
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Figure 1 Bleeding time (top) and ex vivo aggregation induced by 10 µmol/l of adenosine diphosphate (ADP) (bottom) at several time points of the experiment. P values refer to the effect of treatment on bleeding time and aggregation according to general linear model analysis of variance for repeated measures.
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Platelet accumulation in reperfused myocardium.
Specific 99mTc/platelet activity in the reperfused myocardium was 249 ± 26% of that in the control zone (p < 0.001). The magnitude of platelet accumulation in the reperfused myocardium was not correlated with the number of CFRs (r = –0.04) and was similar in animals treated with lamifiban than in those receiving placebo (288 ± 40% vs. 205 ± 27%, respectively, p = NS).
Myeloperoxidase activity and infarct size.
Activity of MPO averaged 0.11 ± 0.03 U/g in the control region and 0.46 ± 0.14 U/g in the reperfused myocardium (p = 0.037). Activity of MPO was also not correlated with the number of CFRs (r = 0.05) and was similar in animals receiving lamifiban and in those receiving placebo (Fig. 2).
The area at risk averaged 11.7 ± 0.9 g, involving 10.0 ± 0.6% of the ventricular mass, without inter-group differences. Infarct size averaged 5.6 ± 1.0 g (48.2 ± 7.8% of the area at risk) and was significantly correlated with specific 99mTc activity (r = 0.52, p = 0.045) and MPO activity (r = 0.66, p = 0.01) (Fig. 3), but not with the number of CFRs (r = 0.18, p = NS). Infarct size was also similar in animals treated with lamifiban and in those receiving placebo (Fig. 4).
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Figure 3 Correlation between specific technetium-99m (99mTc) activity (top) and myeloperoxidase (MPO) activity (bottom) in reperfused myocardium, reflecting platelet and polymorphonuclear leukocytes accumulation, respectively, and infarct size.
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Figure 4 Area at risk, expressed as a percentage of ventricular mass (left axis) and infarct size, expressed as a percentage of the area at risk (right axis).
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Histologic analysis.
In the control region, the histologic appearance of the myocardium was normal in all animals. In the area at risk, histologic analysis showed the presence of myocardial edema, as well as cellular infiltration at the microvessels or in the interstitial space. Histologic scores for PMN infiltration were similar in animals receiving lamifiban or placebo (1.8 ± 0.5 vs. 1.7 ± 0.3, respectively, p = NS). There were two arteriolar occlusive plugs in only one animal from the lamifiban group, suggestive of platelet microemboli.
Platelet P-selectin expression.
In control platelets, P-selectin expression averaged 8.7 ± 3.2%. Platelets from the same animals incubated with 100 or 1,000 ng/ml of lamifiban showed a similar level of P-selectin expression (9.5 ± 3.6% and 6.8 ± 2.5%, respectively, p = NS), despite a dose-dependent inhibition—virtually complete at the highest concentration—of ADP-induced aggregation.
Stability of labeling with 99mTc/HMPAO and effects on platelet function.
The in vitro elution of 99mTc from labeled platelets was 11 ± 1% at 90 min, 30 ± 3% at 17 h and 35 ± 7% at 22 h after labeling. At these same time points, the content of free 99mTc in platelet-poor plasma in vivo was only 7 ± 1%, 5 ± 1% and 6 ± 1%, respectively, of that in platelet-rich plasma.
Platelet labeling did not influence platelet aggregation stimulated with 10 µmol/l of ADP (98 ± 8% of that obtained before labeling, p = NS) and induced a moderate increase in the proportion of platelets expressing P-selectin (18.8 ± 4.1% vs. 9.9 ± 2.4% before labeling, p = 0.09).
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Discussion
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This study tested the effect of GP IIb/IIIa blockade with lamifiban administered during reperfusion on myocardial platelet and PMN accumulation and on infarct size after coronary injury followed by transient CO. Although lamifiban produced a potent inhibition of platelet function and prevented the development of CFRs at the injured vessel, it had no influence on the magnitude of platelet or PMN accumulation in the reperfused myocardium, nor on infarct size.
Effect of GP IIb/IIIa blockade on microvascular injury.
Since the description of the "no-reflow" phenomenon after transient CO (1), attempts have been made to reduce the severity of microvascular injury during reperfusion, with disparate results (25–28). Abcximab, administered as an adjunct of coronary stenting in patients with acute myocardial infarction, has improved the recovery of tissue perfusion and regional contractile function (15). Moreover, treatment with abcximab plus reduced-dose alteplase in patients with ST-segment elevation infarction has been associated with an increased rate of ST-segment resolution, a surrogate for the integrity of microvascular perfusion (3), as compared with alteplase alone (16).
Some laboratory findings also suggest that platelet GP IIb/IIIa inhibitors could have direct salutary effects on tissue perfusion, as they have been shown to preserve the distal vasodilatory response after coronary injury and progressive stenosis (19) and to improve coronary flow and contractile recovery and reduce PMN accumulation and platelet/PMN aggregation in reperfused isolated hearts (17,18). Recently, treatment with the GP IIb/IIIa inhibitor MK-0852 during coronary reperfusion reduced infarct size in dogs, although neither contractile function nor blood flow in the area at risk or the peak reactive hyperemic response of the infarct-related artery was improved by the treatment (20).
There are several explanations for the discrepancy between some of these observations and the present results. First, it is possible that the benefit of GP IIb/IIIa blockade as an adjunct to stenting (15) was due in part to reduced embolization of atherothrombotic material, a complication that may be important after percutaneous intervention (29), but that is probably irrelevant in our model (10). Second, it cannot be ruled out that some of the benefit of GP IIb/IIIa blockade was mediated by a faster recanalization of the culprit CO and reduced myocardial injury (15,16). Finally, it is also possible that the beneficial effect of some of these drugs was due to their ability to interact with receptors other than GP IIb/IIIa that mediate platelet or PMN adhesion to the endothelium (30), or even to a cytoprotective mechanism (20).
Mechanisms of platelet deposition in reperfused myocardium.
The results concur with a previous study in our laboratory (10) in which myocardial platelet accumulation was not associated with the number of CFRs and was also not lessened by antithrombotic treatment—in that case, aspirin—despite a reduction in the number of CFRs. These findings are consistent with a rapid de-aggregation of the platelets released during CFRs and suggest that microembolization from a mural coronary thrombus is not a prominent cause of platelet deposition in downstream myocardium, and that alternative mechanisms, such as distal vasoconstriction (31,32), may mediate the deleterious influence of nonocclusive coronary thrombosis on distal tissue perfusion, as observed previously (21,33,34).
The finding of an increased platelet content in the reperfused myocardium and its correlation with infarct size concur with previous studies (4,5,7,9,10), and the lack of effect of lamifiban on platelet deposition is in agreement with previous observations that aspirin failed to reduce the magnitude of myocardial platelet content after transient CO (9,10). However, the present finding is at variance with a previous study showing that GP IIb/IIIa plays a prominent role in mediating platelet deposition on reperfused mesenteric vessels (35). This discrepancy could be explained by methodologic differences, as well as by the time when platelet deposition was analyzed. Five hours after reperfusion, when myocardial platelet accumulation is still ongoing (5), mechanisms other than the interaction between GP IIb/IIIa and fibrinogen might be involved in the microvascular deposition of platelets. In this respect, a role for endothelial P-selectin has been demonstrated (36), and other studies suggest that a platelet–leukocyte interaction might contribute as well (4,7,10). The fact that lamifiban apparently had no effect on platelet P-selectin expression suggests that its failure in reducing myocardial platelet deposition was not due to a dual effect of the drug on platelet function (37).
Methodologic considerations and limitations.
Platelet labeling resulted in some degree of activation. However, virtually complete clearance of altered platelets from the circulation is expected to occur during the 20 h between labeling and CO. It also seems unlikely that the temporal gap between platelet labeling and counting had affected the results, as additional experiments showed that, after this period, two-thirds of 99mTc continued to be bound to platelets, and the content of free plasmatic 99mTc was negligible.
Because the LAD was re-occluded to ensure an accurate delineation of the area at risk, the coronary vessels could not be washed to reduce the background activity. However, several reasons make it unlikely that this strategy prevented us from detecting a relevant reduction in myocardial platelet content. First, the ratio of platelet content between the reperfused and remote myocardium in controls was not very different to that observed previously (10). Second, the specific activity in reperfused myocardium after subtracting the activity in the remote zone was similar in animals receiving lamifiban or placebo (419 ± 179 counts/g/mCi vs. 345 ± 144 counts/g/mCi of 99mTc injected, respectively, p = NS). Finally, lamifiban also failed to reduce MPO activity and infarct size, variables that are closely correlated with platelet content (4,5,7,9,10).
In the present study, 99mTc and MPO activities were analyzed in a small myocardial sample. Although a more comprehensive sampling would have been preferable, this sampling was representative of a central slice, allowed accurate analysis of infarct size and spared an additional central slice for histologic analysis. Moreover, the results on platelet and leukocyte content were consistent with each other and with infarct size.
Finally, although it cannot be excluded that with a substantially larger sample, a significant reduction in infarct size with lamifiban may have been disclosed, a clinically relevant effect of this drug under the present experimental conditions seems unlikely, because infarct size was virtually identical in both groups, and no trend toward a lower myocardial platelet or leukocyte content was observed in the lamifiban-treated animals.
Clinical implications.
The present results contradict a significant beneficial effect of GP IIb/IIIa blockade on microvascular obstruction after transient CO and support the hypothesis that the benefits of GP IIb/IIIa inhibitors, as observed in previous clinical studies, are mainly due to more rapid coronary reperfusion and reduced myocardial injury or to a concomitant interaction with receptors other than GP IIb/IIIa. The results stress the convenience of further studies exploring alternative mechanisms of platelet accumulation in reperfused microvessels and the effect of their pharmacologic modification on myocardial salvage after reperfusion.
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Acknowledgments
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The authors are indebted to Dr. Pierre Théroux for his contribution to the discussion of the present findings.
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Footnotes
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This study was supported in part by a grant from the Fondo de Investigación Sanitaria de la Seguridad Social (FIS 99/1059) and by the Becas Marató TV3 (nos. 0021/1997 and 4310/2000). Lamifiban was kindly provided by F. Hoffmann-La Roche Ltd., Basel, Switzerland.
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