Fibrin biofilm can be detected on intracoronary thrombi aspirated from patients with acute myocardial infarction

.Fibrin constitutes a major protein component of intravascular thrombi in all locations.1 In patients with ST-elevation myocardial infarction (STEMI), fibrin represents 60% of thrombus mass, which increases with time since symptom onset.1 Fibrin forms a three-dimensional porous network, which is more compact in plasma clots in STEMI patients.2 Recently, it has been demonstrated that fibrin at the blood–air interface can form a biofilm that helps retain blood cells and protect against microbial infections.3 The fibrin film encapsulating a clot at early stages of tissue repair is a thin continuous layer connected to the fibrin network through tethering fibres.3 The external surface of the biofilm is composed of ordered half-staggered fibrin molecules with few very small pores.3 To our knowledge, the fibrin film on human thrombi formed in vivo has not been demonstrated yet. Therefore, we investigated whether the fibrin film can be observed on the surface of intracoronary thrombi aspirated from coronary arteries of STEMI patients. We studied 47 STEMI patients, aged 61.5 [interquartile range (IQR) 51–71.5] years (32 men) with median peak creatine kinase (CK) MB of 384 (IQR 198–531) U/L who were recruited within 12 h since the chest pain onset and treated with primary percutaneous coronary intervention (PCI) preceded by aspiration thrombectomy. Median time since the chest pain onset to PCI was 230 (IQR 166–355) min. All patients received 300 mg of aspirin and 600 mg of clopidogrel prior to PCI. Patients were eligible if infarct-related artery was ≥2.5 mm in diameter and TIMI flow grade was ≤1. The exclusion criteria were cardiogenic shock, previous MI, previous cardiac surgery, malignancy, previous thromboembolism, severe liver or kidney insufficiency and current oral anticoagulation. Before PCI patients received weight-adjusted bolus of unfractionated heparin to obtain activated clotting time (ACT) of 200–250 s. Thrombotic material was successfully removed with the Export® Aspiration Catheter (Medtronic Vascular, Santa Rosa, CA, USA).4 The study protocol complied with the Declaration of Helsinki was approved by the Ethics Committee of the Jagiellonian University. All patients gave written informed consent. Preparation and analysis of the intracoronary thrombi were described previously.1 Briefly, after a median of 10 min (5–30 min) of air contact since aspiration, fresh thrombi were fixed with 2.5% glutaraldehyde, rinsed in phosphate-buffered saline, dehydrated in ethanol and then placed into tert-Butyl alcohol. After drying procedure thrombi were coated with gold using JEOL JFC-1300 Ion Sputter (JEOL, Tokyo, Japan) and 40 randomly selected external areas were scanned using the scanning electron microscope (SEM) JCM 6000 (JEOL). In each of 400 squares of the grid superimposed on each SEM image, the dominant component of the thrombus was scored1 (Figure 1). SEM images of intracoronary thrombi of STEMI patients with the fibrin biofilm present on the surface. ↑fibrin film, *red blood cells, †platelets, and ‡fibrin network. Magnification 2500–5000×. SEM analysis revealed the fibrin biofilm on the surface of 7 (14.9%) thrombi (Figure 1), including two thrombi aspirated from the right coronary artery and five from the left anterior descending artery. The estimated film thickness was 0.6 µm (range 0.21–1.11 µm) similar to that by Macrae et al.,3 and tended to be associated with plasma fibrinogen (r = −0.71, P = 0.068), but not with age or clinical variables. There were no differences in demographic, clinical or laboratory parameters, including ACT between patients forming the biofilm and those without such structures, except higher plasma fibrinogen in the former subgroup [4.63 (4.10–4.98) vs. 3.46 (3.03–3.85) g/L, P = 0.022; fibrinogen >4 g/L vs. ≤4 g/L, odds ratio for the film formation, 6.48, 95% confidence interval 1.18–35.58]. The presence of the biofilm was not associated with the time of air-contact or the content of fibrin, erythrocytes, and platelets. Our study is the first-in-man evidence that fibrin biofilm can be detected on ∼15% of human thrombi aspirated from epicardial arteries of STEMI patients. Elevated fibrinogen known to lead to dense fibrin networks formation1,2 appears to be the key predictor of the biofilm occurrence on coronary thrombi. The lack of association with the time between thrombus retrieval and fixation suggests that the biofilm can be formed in vivo. However, it is still unclear whether air contact is necessary to create such structures.3 Langmuir–Blodgett films were previously observed also on liquid–solid interfaces, indicating that such films may occur intravascularly.3 Not surprisingly, the films were not detected on all thrombi given heterogeneous STEMI patient population. Possibly, heparin infusion during PCI facilitated lysis of the biofilm resulting in its absence on most thrombi. It should be acknowledged that the number of thrombi with the film on the surface could be underestimated because we did re-analysis of the images which had been performed previously. It might be hypothesized that the fibrin film can limit the intravascular expansion of thrombi, but on the other hand the compact film may impair mechanical or enzymatic thrombus disintegration. Resistance of thrombi with biofilm to fibrinolysis is worth investigating since different binding of fibrinolytic proteins on a film vs. fibre network is possible. The mechanisms underlying the fibrin biofilm formation in human thrombosis and its relevance need further investigation. Conflict of interest: none declared. This work was supported by a grant from Jagiellonian University Medical College (K/ZDS/007717 to A.U.).