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Marked increase of fibrin gel permeability with very low dose ASA treatment

https://doi.org/10.1016/j.thromres.2005.02.007Get rights and content

Introduction

Previous data from our group show that acetylsalicylic acid (ASA), especially at low dose, alters the network structure of fibrin, rendering it more porous. The present study was performed to extend the dose–response curve for effects of ASA on fibrinogen clotting properties and to examine the variability of these effects during a 24-h dose interval.

Material and Methods

Fifteen healthy volunteers received ASA 37.5 mg daily (low dose) for 10 days and, after an interval of 2 weeks, 320 mg daily (medium dose) for 7 days, followed by a single bolus dose of 640 mg (high dose). The plasma fibrinogen concentrations were determined and the permeability of fibrin gels (Ks) was assayed with a recently modified flow measurement technique. Three-dimensional (3D) structure of the fibrin network was studied by confocal microscopy.

Results

ASA therapy did not influence fibrinogen concentrations. Compared to baseline, Ks levels were increased by 21% and 31% in samples during medium and high dose ASA treatment (p < 0.01) and, even more markedly, by 44% (p < 0.0001) with very low dose ASA treatment (p < 0.01, compared to the higher doses). The effects of ASA on fibrin gel permeability were stable over a 24-h dose interval. During ASA treatment, thicker fibrin fibers and larger network pores with irregular structure were observed by confocal microscopy.

Conclusions

Acetylation of lysine residues in the fibrinogen molecule may explain the alterations in its clotting property, resulting in altered fibrin gel permeability. The mechanism(s) behind the greater increase in fibrin gel permeability and alterations in 3D structure of the fibrin network observed, and why this phenomenon is more pronounced at low compared to intermediate or high ASA doses, deserve further investigations.

Introduction

Acetylsalicylic acid (ASA) is an effective antithrombotic agent, which prevents a variety of cardiovascular diseases. This effect is commonly explained by acetylation of a serine residue of platelet cyclooxygenase (COX-1), with consequent inhibition of the biosynthesis of the platelet activating and vasoconstricting compound thromboxane A2 (TXA2) [1], [2], [3], [4].

However, it has been reported that ASA also acetylates lysine residues of several proteins in the coagulation system, such as in procoagulants like fibrinogen [5] and prothrombin [6] on the one hand, and anticoagulants like antithrombin on the other [7]. In the fibrinogen molecule, the lysine residues are involved in the cross-linking reaction of fibrin fibers promoted by Factor XIII, leading to formation of a mechanically stable clot that is more resistant to fibrinolysis. It may be hypothesized that ASA induced acetylation of lysine residues of the fibrinogen molecule interferes with the effects of FXIII, thus altering the structure and stability of the clot formed.

Previously, we have used a flow measurement technique developed by Blomback et al. [8] to determine the permeability of fibrin networks. We have found that subjects at risk of atherothrombotic complications, such as males with a previous myocardial infarction at young age [9] and patients with insulin-dependent diabetes mellitus [10], form a tighter fibrin gel.

In a pilot study in patients with stable angina pectoris, we observed a significantly higher porosity of the fibrin network during treatment with ASA as compared to after withdrawal [11]. In another pilot investigation in healthy subjects, we found a greater increase in fibrin gel permeability during a daily intake of 75 mg compared to 320 mg [12]. These findings regarding fibrin gel porosity imply that low dose ASA may give a better protective effect than high dose ASA treatment. In fact, there is evidence from clinical investigations suggesting better effects of ASA therapy with low doses (75–150 mg daily) compared to higher doses [13], but the mechanisms involved are not yet clear.

We have recently modified the flow measurement assay to make it easier to perform in any research laboratory and more similar to in vivo conditions. The method was originally based on the formation of fibrin gel in plasma in the presence of a high concentration of exogenous thrombin, aiming at initiating clotting and forming a stable clot [8]. We observed however that the addition of small amounts of thrombin (0.05 IU/mL) will not immediately initiate clotting, but will cause a feedback reaction stimulating thrombin generation and subsequent clot formation [14]. The measurement of fibrin gel porosity in this reaction system will thus show the combined effect of fibrin generation potential and fibrinogen clotting properties. However, if there is a need to measure changes in fibrinogen clotting ability as a single parameter determining the permeability of the fibrin gel, the influence of endogenous thrombin formation must be excluded. For this purpose, we suggested the use of a higher concentration of exogenous thrombin, i.e. 0.4 IU/mL, than that used in the system by Blomback et al. [8].

In the present study, we used flow measurements in the presence of 0.4 IU/mL of exogenous thrombin to further determine the dose-dependence of effects of ASA on fibrinogen clotting. Thus, we investigated the effects of treatment with a very low (37.5 mg), a medium (320 mg) and a high dose of ASA (960 mg) on the permeability through the clot with our new technique. We also wanted to investigate if the impairment in fibrinogen clotting decayed within 24 h after intake of the drug, i.e. during the normal interval between daily intakes of ASA. We also verified our findings by investigating the fibrin network changes with 3D confocal microscopy.

Section snippets

Reagents

Bovine thrombin was obtained from Sigma Chemical, St. Louis, MO, USA. Aprotinin (trasylol) was from Bayer, Levekusen, Germany. Human fibrinogen (free of plasminogen, fibronectin and FXIII, coagulability > 96% without addition of FXIII) was from American Diagnostica, Greenwich, USA.

Subjects

Fifteen male healthy volunteers, aged 22–39 years, participated in the study. They were nonsmokers and had body mass index (BMI) of 22.9 (range 20–26 kg/m2). The volunteers were instructed to avoid antiplatelet drugs

Results

The fibrinogen concentrations in plasma were within the reference range of our laboratory (2–4 g/L). They did not change over time and were not affected by ASA treatment at any dose (Table 1).

The permeability of fibrin gels (Ks) was similar in all three samples obtained during baseline conditions (Table 1, Fig. 2a), and Ks was inversely correlated to fibrinogen concentrations (r = 0.66, P < 0.01) (Fig. 3a). The thickness of the fibrin fibers (expressed as the fiber mass/length ratio [μ]) was

Discussion

The available evidence from clinical trials encourages the use of antiplatelet therapy with 75–150 mg ASA daily for patients with an elevated risk of occlusive vascular events [3], [4], [13]. The present study confirms with a simpler and more adequate technique our earlier results of a more favorable effect on the permeability of the fibrin network during treatment with low, compared to intermediate or high doses of ASA (37.5 mg vs. 320–960 mg daily in the present and 75 mg vs. 320 mg daily in

Acknowledgements

This study was supported by the Swedish Heart-Lung Foundation, the Magnus Bergvall Foundation, the Swedish Society for Medical Research, the Swedish Research Council (5930) and Karolinska Institutet. The authors are grateful for the technical assistance of Maud Daleskog and Maj-Christina Johansson.

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