Hemostatic phenotype of thrombi derived from STEMI patients on cardiovascular prevention therapy :Jeske J. K van Diemen, Bernard J Smilde, Wessel W Fuijkschot, P Stefan Biesbroek, Yolande E Appelman, Paul A. J Krijnen, Yvo M Smulders, Hans W. M Niessen, Abel Thijs, Journal of the Practice of Cardiovascular Sciences

ORIGINAL ARTICLE
Year : 2019 | Volume : 5 | Issue : 2 | Page : 86--90

Hemostatic phenotype of thrombi derived from STEMI patients on cardiovascular prevention therapy

Jeske J. K van Diemen¹, Bernard J Smilde², Wessel W Fuijkschot¹, P Stefan Biesbroek³, Yolande E Appelman³, Paul A. J Krijnen², Yvo M Smulders¹, Hans W. M Niessen², Abel Thijs¹,
¹ Department of Internal Medicine, Amsterdam UMC, Location VU University, Amsterdam, The Netherlands
² Department of Cardiovascular Pathology, Amsterdam UMC, Location VU University, Amsterdam, The Netherlands
³ Department of Cardiology, Amsterdam UMC, Location VU University, Amsterdam, The Netherlands

Correspondence Address:
Miss Jeske J. K van Diemen
De Boelelaan 1118 (4A-45), 1081 HZ Amsterdam
The Netherlands

Abstract

Introduction: Aspirin and statin therapy are the basis of cardiovascular disease (CVD) prevention therapy. Recent data have suggested new additional preventive mechanisms: both aspirin and statin exhibit anti-inflammatory effects within intracoronary thrombi. As inflammation, aggregation, and thrombosis are closely intertwined, aspirin and subsequent statin therapy might influence the hemostatic content within intracoronary thrombi. Aim: The aim of the study is to explore the spectrum of CVD prevention therapy on intracoronary thrombus composition by analyzing main hemostasis components in patients with ST-segment elevation myocardial infarction (STEMI). Materials and Methods: We performed a cross-sectional histological pilot study with intracoronary thrombi derived from STEMI patients on CVD prevention therapy without usage of another anticoagulant agent other than aspirin. They were actively matched with intracoronary thrombi in a control group derived from STEMI patients not on any therapy – without a previous CVD history – based on thrombus age (fresh), sex, and age of the participant. Immunohistochemistry was performed with primary antibodies of factor XII (F-XII), tissue plasminogen activator (tPA), and factor VII (F-VII). Results: Four of the 13 thrombi derived from patients on CVD prevention therapy were not characterized as fresh and subsequently excluded. Moreover, one participant in the patient group had to be excluded post hoc. The thrombi in the patient group had a significantly more F-XII (27.7%) and tPA (10.1%) positive area versus the controls (17.5%; 4.5%), respectively. The F-VII-positive thrombus area was similar in both groups. Conclusion: These exploratory data demonstrate an increased procoagulant F-XII and fibrinolytic tPA content within intracoronary thrombi derived from patients on CVD prevention therapy compared with controls.

How to cite this article:
van Diemen JJ, Smilde BJ, Fuijkschot WW, Biesbroek P S, Appelman YE, Krijnen PA, Smulders YM, Niessen HW, Thijs A. Hemostatic phenotype of thrombi derived from STEMI patients on cardiovascular prevention therapy.J Pract Cardiovasc Sci 2019;5:86-90

How to cite this URL:
van Diemen JJ, Smilde BJ, Fuijkschot WW, Biesbroek P S, Appelman YE, Krijnen PA, Smulders YM, Niessen HW, Thijs A. Hemostatic phenotype of thrombi derived from STEMI patients on cardiovascular prevention therapy. J Pract Cardiovasc Sci [serial online] 2019 [cited 2023 Mar 23 ];5:86-90
Available from: https://www.j-pcs.org/text.asp?2019/5/2/86/264626

Full Text

Introduction

Aspirin (acetylsalicylic acid) and statin therapy are at the base of cardiovascular prevention therapy.[1] Aspirin in a low dose is thought to primarily act through platelet inhibition,[2] whereas statins lower cholesterol and stabilize atherosclerotic plaques.[3] Interestingly, recent data in patients with myocardial infarction suggest additional preventive mechanisms: both aspirin and statin exhibit anti-inflammatory effects within intracoronary thrombi.[3],[4] Moreover, statin therapy also inhibits platelets.[3],[5]

As inflammation, aggregation, and thrombosis are closely intertwined,[4],[6] we therefore hypothesized that aspirin and subsequent statin therapy might also induce additional anti-hemostatic effects within intracoronary thrombi. In this, we focused on factor XII (F-XII) and factor VII (F-VII) as these factors are the starting point of the intrinsic and extrinsic pathway, respectively. Furthermore, we studied thrombus tissue plasminogen activator (tPA) expression, which plays a central role in natural thrombolysis and is homologous to F-XII.[7]

Materials and Methods

Thrombus aspiration and clinical data

We conducted a cross-sectional histological pilot study within a subcohort of the study by Fuijkschot et al.[4] The original study was designed to analyze inflammatory cells in intracoronary thrombi derived from 113 patients with ST-segment elevation myocardial infarction (STEMI) in relation to histologically classified thrombus age. The study was conducted in accordance with the Helsinki II Declaration. In total, 13 of these patients were on cardiovascular prevention therapy. All 13 patients presented with STEMI (defined according to the European Society of Cardiology guidelines for STEMI) at the VU University Medical Center.[8] Their thrombi were actively matched with intracoronary thrombi derived from STEMI patients – without a previous cardiovascular history – based on thrombus age (fresh), sex, and age of the participant (±2 years). We excluded patients with an intracoronary thrombus age other than fresh (i.e., lytic or organized), usage of an antiplatelet agent other than aspirin, usage of chronic coagulation therapy, and diabetes mellitus. Diabetes mellitus was excluded due to its known prothrombotic state, including endothelial dysfunction, coagulative activation, and platelet hyperreactivity.[9] The method of coronary thrombus aspiration in STEMI patients has been previously described.[4] In brief, after pretreatment with a heparin bolus, a guidewire was used intracoronary to insert an aspiration catheter and a thrombectomy by aspiration was performed.

Histology

Immediately after aspiration, the aspirated thrombi were fixed overnight with 4% formalin and afterward embedded in paraffin. Tissue sections (4 μm thick) were cut using an HM335E Microtome (MICROM GmbH, Germany), mounted on microscope slides (Superfrost plus, Gerhard Menzel GmbH, Germany), and deparaffinized and rehydrated using xylene and ethanol, respectively. For thrombus classification, a standard hematoxylin-eosin stain was used. Two independent observers classified the thrombi as fresh, lytic, or organized according to the criteria previously described.[4]

Immunohistochemistry

To characterize the hemostatic content, we applied primary antibodies of F-XII (Sanquin Research, Amsterdam, noncommercial antibody F1, dilution 1:50),[10] tPA (Sanquin Research, Amsterdam, noncommercial antibody a-tPA, dilution 1:100), and F-VII (Sanquin Research, Amsterdam, noncommercial antibody VII-8, dilution 1:250). Deparaffinized and rehydrated tissue sections were submerged in methanol/hydrogen peroxide (0.3%) for 30 min to block endogenous peroxidase activity. As a preparatory step for antigen retrieval, boiling in citrate pH 6.0 buffer was performed. Next, primary antibodies were applied for 60 min, labeled with α-mouse/rabbit Envision (Dako) for 30 min, and visualized with 3.30-diaminobenzidine (0.1 mg/ml, 0.02% H2O2) for 10 min, all at room temperature. The tissue slides were then counterstained with hematoxylin, dehydrated, and covered. Phosphate-buffered saline (PBS) was used to wash slides between steps. To prevent false positives, PBS control was included in every staining sequence. None of these slides demonstrated a positive result (not shown).

Quantitative analysis of tissue slides

Tissue slides were scanned with MIRAX scan and total thrombus surface area was calculated with MIRAX Viewer software (3DHISTECH Ltd, Budapest, Hungary). FXII-, tPA-, and FVII-stained slides were analyzed with ImageJ software (Bethesda, MD, USA) using color thresholding to determine the positive surface area. Furthermore, the analyzist was blinded for both type of antibody and group allocation during quantification. Finally, to calculate interobserver variability, a subset of 10 slides was re-analyzed by another researcher. The Spearman's rho correlation coefficient indicated low interobserver variability in between both raters (all r ≥ 0.84, P = 0.04).

Statistical analysis

Due to the small sample size, all variables were analyzed nonparametrically, using the Mann–Whitney U-test. A P < 0.05 was considered statistically significant. All statistical analyses were performed with SPSS software (Windows version 22, IBM corp., Armonk, NY, USA).

Results

Population

[Table 1] describes the major characteristics of both study groups. In a total of the 13 thrombi derived from patients on CVD therapy that were screened, four were not characterized as fresh and subsequently excluded. Moreover, one patient in the CVD therapy group was excluded post hoc due to a missed chronic usage of a Vitamin K antagonist. Not surprisingly, patients and controls differed in cardiovascular risk factors. In specific, controls included more current smokers (44.4% vs. 25%), but less participants with diagnosed hypertension (22.2% vs. 62.5%).{Table 1}

Thrombus characterization

First, the thrombi in the patients on CVD therapy group (27.7%; interquartile range [IQR]: 21.8–39.3) had a significantly larger F-XII-positive area, in comparison with the controls (17.5%; IQR: 8.0–27.3; P = 0.02). Second, the F-VII-positive thrombus area was similar in the CVD therapy group (46.4%; IQR: 41.0–61.0), in comparison with the controls (56.7%; IQR: 39.3–75.0). At last, the area of the thrombus positive for tPA was significantly larger in the patients on on CVD therapy group (10.1%; IQR: 9.4–17.3), than in the control group (4.5%; IQR: 3.4–4.5; P ≤ 0.01). In [Figure 1] and [Figure 2], images showing slides derived from patient on and patients off cardiovascular prevention therapy are depicted.{Figure 1}{Figure 2}

Discussion

This study sets out to explore the spectrum of cardiovascular prevention therapy on intracoronary thrombus composition by analyzing main components of secondary hemostasis in patients with ST-segment elevation myocardial infarction (STEMI) while on cardiovascular prevention therapy. The most obvious finding to emerge from this analysis is the increased procoagulant F-XII and fibrinolytic tPA content of intracoronary thrombi derived from patients receiving secondary cardiovascular prevention therapy.

The present findings extend earlier data; however, to understand these ambiguous results, we considered some possible explanations. First, although the binding sites of F-XIIa on platelets have not been elucidated, there is emerging evidence that platelets also support the intrinsic coagulation pathway mediated by F-XII and XI,[11],[12] albeit the exact mechanism is still unclear. Second, an explanation for the increased procoagulant F-XII and fibrinolytic tPA could be the interaction between inflammation and hemostasis. Although discovered for its role in the intrinsic coagulation cascade, F-XII also plays a central role in the inflammation process. For example, activation can initiate the pro-inflammatory kallikrein–kinin Pathway.[13] Consequently, as both aspirin and statin exhibit anti-inflammatory properties,[14] they might induce a response in F-XII. Finally, another possible explanation could be through an indirect aspirin and/or statin–hemostasis interaction as both exhibit anti-platelet properties.[2],[5] Moreover, aspirin acetylates fibrinogen consequently enhancing fibrinolysis and increasing tPA release.[15]

Our study has some limitations. An important limitation of this study is the small sample size with a considerable variation in patient characteristics. Consequently, it is not possible to exclude confounding or effect modification. Moreover, we are unable to determine which precise factor is causing the difference (i.e., aspirin or statins usage), or perhaps whether it is due to synergism of several factors associated with a secondary cardiovascular prevention profile. Furthermore, there are no data on the compliance of prescribed therapy. Therefore, we do not know if the patients on secondary prevention used aspirin and statin as prescribed by their cardiologist. However, smoking, for example, has been demonstrated to increase F-XII activity,[16] as well as tPA.[17] Hence, if smoking is a confounder, the illustrated difference in platelet (re-) activity might be underestimated.

Conclusion

Intracoronary thrombi derived from patients presenting with STEMI while on cardiovascular prevention therapy with aspirin and statin demonstrate an increased procoagulant F-XII and fibrinolytic tPA content. However, these preliminary findings are somewhat limited, and the clinical impact and/or meaning of these findings cannot yet be stated. Therefore, to establish the precise role of aspirin and statin therapy and their possible complementary and/or synergistic anti-hemostatic effects in thrombus formation in patients with STEMI, future studies should be undertaken, for example, an experimental–animal study where thrombi are developed under either aspirin, statin, both medicines, and placebo.

Financial support and sponsorship

The work from the original study by Fuijkschot et al. was supported by a grant from the ICaR VU Institute, Amsterdam, the Netherlands.

Conflicts of interest

There are no conflicts of interest.

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