Figure 2. Scatter plot of 24 hours increase in sCD40L levels vs 24 hours IL-6 levels in non-treated patients. (r = 0.54; p < 0.01).
Figure 3. Scatter plot of 24 hours IL-6 levels vs 24 hours C-reactive protein levels in non-treated patients. (r = 0.49; p < 0.05).
3.3. Antithrombotic Treatment and Inflammation
3.3.1. C-Reactive Protein
After angioplasty CRP increased significantly in control patients. This rise was blocked by GP IIb-IIIa treatment (control basal median (25th, 75th percentiles): 0.37 (0.16, 0.64) vs. 24 h: 0.68 (0.45, 1.16) mg/dL; p < 0.05) (treated basal 0.25 (0.14, 0.69) vs 24 h: 0.37 (0.19, 0.79) mg/dL; p = ns). When we analyzed separatedly patients with and without thrombus (Figure 4), GP IIb-IIIa inhibition was effective in patients with angiographic thrombus.
IL-6 showed its typical 24 hour post-angioplasty peak (control basal: 4.6 (2.4, 6.9) vs. 24 h: 13.7 (8, 20.1) ng/dL; p < 0.001), which was not modified by GPIIb-IIIa treatment (treated basal 3.7 (2.6, 6.9) vs. 24 h: 8.6 (6.5, 19.6) ng/dL; p < 0.01). When we analyzed separatedly patients with and without thrombus (Figure 5), GP IIb-IIIa inhibition was effective in patients with angiographic thrombus.
Absolute sCD40L showed a significant decrease 24 hr after angioplasty which is consistent with the downregulation of the CD40L receptor observed after platelet aggregation in experimental studies. GP IIb-IIIa inhibittion reduced absolute sCD40L decrease in all patients by sparing sCD40L consumption induced by platelet aggregation (control basal: 781 (0, 2140) vs. 24h: 298(57, 816) pg/ml; p < 0.05)(treated basal 605(169, 876) vs. 24h: 722(273, 1998) pg/ml; p = ns). When we analyzed separatedly patients with and without thrombus (Figure 6), GP IIb-IIIa inhibition was effective only in patients without angiographic thrombus.
3.4. Thrombus Formation and the Inflammatory Response
Patients carrying the −174 GG (high IL-6 production) gene promoter polymorphism had more angiographic thrombus (65%) than GC heterozygotes (42%) or patients with the CC (low IL-6 production) polymorphism, who only 10% of them had angiographic thrombus (p = 0.016).
Although the link between inflammation and thrombosis has been extensively studied, both in “in-vitro” and “in-vivo” studies, the influence of thrombosis on inflammation has yield controversial findings in clinical studies. Lincoff et al.  and ourselves [20,21] found an inhibition of inflammatory markers rise after angioplasty in patients who had intensive antiplatelet treatment. James et al., in a GUSTO IV substudy, fail to show a relationship between antithrombotic treatment and inflammation inhibition . That was probably due to the confounding effect of other variables such as myocardial damage which was observed in over 70% of the substudy population, and/or uncontrolled thrombus formation (they did not perform early coronary angiography). The population choosed for this particular study were unstable angina patients with elevated myocardial necrosis markers and recommended delayed coronary intervenetion until the end of the study. The patients selected for our experiment were unstable angina patients with no myocardial enzyme elevation at the time of angioplasty,
Figure 4. Box-and-whisker plot of changes in CRP levels from baseline to 24 hours, and from 24 to 48 hours postangioplasty in the treated and control groups. The box spans the interquartile range (25th to 75th percentiles), and the line within the box denotes the median. Whiskers extend from the 10th to the 90th percentiles. (a) Patients without angiographic thrombus: control basal median (25th, 75th percentiles): 0.33 (0.1, 0.65) vs. 24 h: 0.58 (0.41, 1.00) mg/dL; p < 0.01; treated basal 0.23 (0.13, 1.00) vs. 24 h: 0.59 (0.19, 1.19) mg/dL; p = ns. (b) Patients with angiographic thrombus: Control basal 0.52 (0.28, 0.71) vs. 24 h: 0.93 (0.44, 1.49) mg/dL; p < 0.05; treated basal 0.25 (0.13, 0.61) vs. 24 h: 0.27 (0.12, 0.54) mg/dL; p = ns.
Figure 5. Box-and-whisker plot of changes in IL-6 levels from baseline to 24 hours, and from 24 to 48 hours postangioplasty in the treated and control groups. (a) Patients without angiographic thrombus: control basal median (25th, 75th percentiles): 3.1 (2.2, 5.6) vs. 24 h: 13.5 (6.4, 20.7) ng/ml; p < 0.01); treated basal 3.7 (2.5, 7) vs. 24 h: 14.6 (7.1, 26.1) ng/ml; p < 0.001. (b) Patients with angiographic thrombus: Control basal 6.7 (3, 9.3) vs. 24 h: 14.9 (11.5, 19.8) ng/ml; p < 0.01; treated basal 3.6 (2.2, 5.0) vs. 24 h: 7 (5.8, 9.5) ng/ml; p = ns.
Figure 6. Box-and-whisker plot of changes in sCD40L levels from baseline to 24 hours, and from 24 to 48 hours postangioplasty in the treated and control groups. (a) Patients without angiographic thrombus: Control basal median (25th, 75th percentiles): 1017 (12, 2140) vs. 24 h: 198 (16, 728) pg/ml; p < 0.05; treated basal 693 (196, 895) vs. 24 h: 582 (255, 2040) pg/ml; p = ns. (b) Patients with angiographic thrombus: Control basal 686 (0, 3936) vs. 24 h: 649 (86, 986) pg/ml; p = ns; treated basal 546 (80, 680) vs. 24 h: 811 (658, 1577) pg/ml; p = ns.
so that the effect of stent implantation was not confounded by even subtle myocardial necrosis. Thus, we excluded patients with complicated procedures, acute myocardial infarction and any with periprocedural enzyme elevation.
Platelets are a key step in the arterial thrombotic complications of atherosclerosis and are known to carry many pro-inflammatory substances. So platelets can well be the link between thrombosis and inflammation. More than 300 proteins have been identified in human platelets . Among them, some data point to the CD40L/ sCD40L system as playing the principal role between intra-arterial thrombosis and the increase in inflammatory markers [7-11].
We designed this experiment to assess if there is a relationship between intra-arterial thrombosis and inflamematory markers elevation after a pro-thrombotic event such as coronary stent implantation. We found that in non-treated patients: 1) There is a linear relationship between platelet number and sCD40L concentration, between sCD40L post-angioplasty increase and IL-6 postangioplasty concentration, and between IL-6 post-angioplasty concentration and CRP post-angioplasty concentration; 2) A GP IIb-IIIa inhibitor blocked CRP and IL-6 increase particularly in patients with angiographic thrombus, and preserved sCD40L consumption in all patients.
There are several issues to be discussed in relation to methodology, the findings themselves and their pathophysiological implications.
4.1. Methodological Issues
We did not perform randomization of patients in this study, although patients were consecutively assigned to treated and control groups. We controlled all the variables which may have altered the results of the experiment: Cardiovascular risk factors, metabolic indexes, and rheological and anatomical features. Second, the study of the pathophysiology of acute coronary syndromes is limited by the lack of an exact relationship between the actual intracoronary events and the patient’s clinical picture. We have used coronary angioplasty as a model of acute coronary syndrome. Both conditions share a common pathophysiological sequence after plaque rupture, and in the case of angioplasty, balloon inflation determines the exact time of plaque rupture. Other authors and ourselves have demonstrated an increase in CRP and IL-6 after angioplasty-induced plaque rupture, which peaks 24 - 48 hours after the procedure [19-21]. Percutaneous coronary intervention with stent implantation increases intra-arterial thrombosis in such a magnitude that requires intense antithrombotic treatment . Some will argue that coronary intervention with stent implantation is not a pro-thrombotic condition, but experiments performed in primates clearly show that platelets form a mural thrombus inside the stent immediately after implantation, producing a thin layer which covers the stent’s luminal surface . Abciximab, another GP IIbIIIa platelet receptor blocker, inhibits the formation of this platelet mural thrombus . Third, there have been reports of the anti-inflammatory effects of Eptifibatide, but Eptifibatide has no direct anti-inflammatory effects. It is a cyclic heptapeptide which contains a KGD sequence which competitively blocks the platelet GP IIb-IIIa receptor and has no other known effects . Forth, we classified patients having “angiographic thrombus” those who presented the features of Ambrose/ Fuster type II lesions as described in the Methods section. Angiographically complex lesions were described two decades ago and reflect the platelet rich thrombus image [12-14]. Finally, samples were collected at room temperature because we decided to analyze “absolute” sCD40L as explained in the Methods section. The rationale for “absolute” sCD40L determination is based on a practical as well as a physiological ground. First, it is much easier to achieve reproducible results because the samples must not be immediately frozen after blood collection, and the protocol used for sample freezing and storing may alter the results. Second, it seems more pathophysiologically suited to analyze the absolute and future potential of sCD40L release, than only the past sCD40L released which is responsible for only part of the pro-inflammatory reaction after a pro-thrombotic situation such as coronary stent implantation.
4.2. Questions Related To the Findings
Our study addresses for the first time the issue of how arterial thrombosis may induce a pro-inflammatory reaction in patients. The potential role of CD40L/sCD40L as a link between inflammation, atherosclerosis and thrombosis has been postulated for at least a decade [6,8,27,28]. However, it has not been established a relationship between platelet number and the inflammatory response. Platelets express CD40L within seconds of activation in vitro and in the process of thrombus formation in vivo [10,11]. Soluble CD40L increase was related to postangioplasty IL-6 levels in non-treated patients, showing a link between thrombosis and an amplification of the inflammatory response by the “messenger cytokine” (IL-6). This finding supports the pro-inflammatory effect of the CD40L/sCD40L system on several cell types involved in atherosclerosis reported in experimental studies [9,11,23]. Finally, as expected, since IL-6 is the most potent inductor of CRP production, IL-6 levels were related to CRP levels 24 hours post-angioplasty. This relationship applied only to non-treated patients, since GP IIb-IIIa inhibition blocked the inflammatory response.
There is no clear explanation for the lack of inflammatory response blockade by GPIIb-IIIa inhibitors in patients without angiographic thrombus. It seems that there act other unknown mechanisms which could induce inflammation, but the fact that platelet GP IIb-IIIa receptor inhibition acts in patients with thrombus opens to some discussion.
First, patients carrying the high IL-6 production polymorphism had more angiographic thrombus than those with the low IL-6 production polymorphism. This particular polymorphism modulates the response of IL-6 to arterial thrombosis , and it could explain why the GP IIb-IIIa inhibitor blocked CRP, IL-6 and sCD40L increase only in patients with angiographic thrombus. But in second place, platelets release all the sCD40L they carry and cannot produce more. This fact may explain why CRP and IL-6 increase was blocked by the GP IIb-IIIa agent in patients with complex lesions. Patients without complex lesions could retain more sCD40L/ CD40L potential able to induce CRP and IL-6 production.
4.3. Questions Related to Pathophysiology
Patients with Acute Coronary Syndromes and after coronary angioplasty show an increase in CRP and IL-6 levels that peaks 24 - 72 hours after the procedure, and then return to previous levels [3-5,19-22]. The current explanation is that there is a pro-inflammatory effect of coronary interventions, but we have shown that a return pathway exists from thrombosis to inflammation and explain the rise in inflammatory markers in these patients Platelets are the main source of sCD40L in the vascular system. We found a direct relationship between platelet number and sCD40L levels.
Soluble CD40L release from the platelet surface is the sole mechanism for the down-regulation of CD40L. Transmembrane CD40L from platelets is fully released as sCD40L and it has a half-life of approximately six hours [10,11]. These experimental data are fully translated to our clinical data. We found a decrease of sCD40L blood levels after coronary angioplasty, which was spared by platelet GP IIb-IIIa inhibitor Eptifibatide (Figure 6(a)). The change in sCD40L levels after angioplasty was related to IL-6 levels, and in turn, IL-6 post-angioplasty levels were related to post-angioplasty CRP levels (Figures 1-3). This chain of events may explain the increase in IL-6 and CRP that are observed in patients with Acute Coronary Syndromes and after angioplasty. Thus, CRP could be a marker of the thrombotic status of a single patient in the setting of an ACS.
Not only platelets but other major cells involved in the atherosclerotic process, such as smooth muscle cells and leukocytes participate in this closed loop. Inflammation can induce mural thrombus formation, and formed thrombus may amplify inflammation. Inflammation and thrombosis are so deeply interrelated that we could consider antithrombotic drugs as anti-inflammatory agents and conversely [23,29-32]. Patients who have a pro-inflammatory status have an increased risk of thrombotic complications, and in turn, thrombosis increases their pro-inflammatory conditions, closing a vicious circle which induces thrombotic complications. Antithrombotic treatment can block the inflammatory response and break this systemic amplification loop.
Inflammatory response after angioplasty is produced by a thrombotic sequence which relates platelet aggregation to sCD40L, to IL-6 and, finally to CRP increase. Platelet GP IIb-IIIa receptor inhibitor Eptifibatide block this inflamematory burst, particularly when angiographic thrombus is present.
This study was funded by the Spanish Society of Cardiology.