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

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


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

Date of Submission17-Apr-2019
Date of Decision28-May-2019
Date of Acceptance14-Jun-2019
Date of Web Publication19-Aug-2019

Correspondence Address:
Miss Jeske J. K van Diemen
De Boelelaan 1118 (4A-45), 1081 HZ Amsterdam
The Netherlands
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jpcs.jpcs_30_19

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  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.

Keywords: Aspirin therapy, coagulation, coronary thrombi, fibrinolysis, statin therapy


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 2019 Dec 11];5:86-90. Available from: http://www.j-pcs.org/text.asp?2019/5/2/86/264626




  Introduction Top


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 Top


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 Top


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: Baseline characteristics

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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: Example of images of slides derived from one patients off CVD prevention therapy; (a) Factor XII, (b) Factor VII, (c) Tissue plasminogen activator

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Figure 2: Example of images of slides derived from one patients on CVD prevention therapy; (a) Factor XII, (b) Factor VII, (c) Tissue plasminogen activator

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  Discussion Top


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 Top


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.



 
  References Top

1.
Piepoli MF, Hoes AW, Agewall S, Albus C, Brotons C, Catapano AL, et al. 2016 European Guidelines on cardiovascular disease prevention in clinical practice: The sixth joint task force of the European Society of Cardiology and other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by representatives of 10 societies and by invited experts) developed with the special contribution of the European Association for Cardiovascular Prevention & Rehabilitation (EACPR). Eur Heart J 2016;37:2315-81.  Back to cited text no. 1
    
2.
Vane JR, Botting RM. The mechanism of action of aspirin. Thromb Res 2003;110:255-8.  Back to cited text no. 2
    
3.
Sexton T, Wallace EL, Smyth SS. Anti-thrombotic effects of statins in acute coronary syndromes: At the intersection of thrombosis, inflammation, and platelet-leukocyte interactions. Curr Cardiol Rev 2016;12:324-9.  Back to cited text no. 3
    
4.
Fuijkschot WW, Groothuizen WE, Appelman Y, Radonic T, van Royen N, van Leeuwen MA, et al. Inflammatory cell content of coronary thrombi is dependent on thrombus age in patients with ST-elevation myocardial infarction. J Cardiol 2017;69:394-400.  Back to cited text no. 4
    
5.
Mitsios JV, Papathanasiou AI, Goudevenos JA, Tselepis AD. The antiplatelet and antithrombotic actions of statins. Curr Pharm Des 2010;16:3808-14.  Back to cited text no. 5
    
6.
Verhamme P, Hoylaerts MF. Hemostasis and inflammation: Two of a kind? Thromb J 2009;7:15.  Back to cited text no. 6
    
7.
Ponczek MB, Gailani D, Doolittle RF. Evolution of the contact phase of vertebrate blood coagulation. J Thromb Haemost 2008;6:1876-83.  Back to cited text no. 7
    
8.
Ibanez B, James S, Agewall S, Antunes MJ, Bucciarelli-Ducci C, Bueno H, et al. 2017 ESC guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The task force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC). Eur Heart J 2018;39:119-77.  Back to cited text no. 8
    
9.
Vazzana N, Ranalli P, Cuccurullo C, Davì G. Diabetes mellitus and thrombosis. Thromb Res 2012;129:371-7.  Back to cited text no. 9
    
10.
Citarella F, Ravon DM, Pascucci B, Felici A, Fantoni A, Hack CE. Structure/function analysis of human factor XII using recombinant deletion mutants. Evidence for an additional region involved in the binding to negatively charged surfaces. Eur J Biochem 1996;238:240-9.  Back to cited text no. 10
    
11.
Renné T, Pozgajová M, Grüner S, Schuh K, Pauer HU, Burfeind P, et al. Defective thrombus formation in mice lacking coagulation factor XII. J Exp Med 2005;202:271-81.  Back to cited text no. 11
    
12.
Cheng Q, Tucker EI, Pine MS, Sisler I, Matafonov A, Sun MF, et al. Arole for factor XIIa-mediated factor XI activation in thrombus formation in vivo. Blood 2010;116:3981-9.  Back to cited text no. 12
    
13.
Long AT, Kenne E, Jung R, Fuchs TA, Renné T. Contact system revisited: An interface between inflammation, coagulation, and innate immunity. J Thromb Haemost 2016;14:427-37.  Back to cited text no. 13
    
14.
Hohlfeld T, Schrör K. Antiinflammatory effects of aspirin in ACS: Relevant to its cardiocoronary actions? Thromb Haemost 2015;114:469-77.  Back to cited text no. 14
    
15.
Hammouda MW, Moroz LA. Aspirin and venous occlusion: Effects on blood fibrinolytic activity and tissue-type plasminogen activator levels. Thromb Res 1986;42:73-82.  Back to cited text no. 15
    
16.
Becker CG, Dubin T. Activation of factor XII by tobacco glycoprotein. J Exp Med 1977;146:457-67.  Back to cited text no. 16
    
17.
Allen RA, Kluft C, Brommer EJ. Acute effect of smoking on fibrinolysis: Increase in the activity level of circulating extrinsic (tissue-type) plasminogen activator. Eur J Clin Invest 1984;14:354-61.  Back to cited text no. 17
    


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