Aspirin and the fibrinolytic response

Thrombosis Research 110 (2003) 331 – 334

Regular Article

Aspirin and the fibrinolytic response WlAodzimierz Buczko *, Andrzej Mogielnicki, Karol Kramkowski, Ewa Chabielska Department of Pharmacodynamics, Medical University of Bialystok, 15-089 Bialystok, Mickiewicza Str. 2C, Poland

Abstract Apart from the anti-inflammatory action, aspirin (ASA) by inhibiting thromboxane A2 synthesis, decreases platelets activity and possesses the antithrombotic action. However, an ASA effect on fibrinolysis has not been yet finally established. Menon [Lancet 1 (1970) 364] reported increased fibrinolytic response in patients treated with high doses of ASA and this observation started a series of studies to find the relation between aspirin and fibrinolysis. This review comprises the results of those studies, divided into in vitro and in vivo, animal and human experiments. The results of our animal studies are also included. Data survey shows that the ASA effect on fibrinolysis depends on experimental conditions, the dose and the time of drug administration. The results of our study indicate the essential role of plasma components in the fibrinolysis regulation by ASA. D 2003 Elsevier Ltd. All rights reserved. Keywords: Aspirin; Fibrinolytic response; ASA

1. Haemostasis

2. Fibrinolysis

Physiological haemostasis is a complex phenomenon that involves the vascular wall (endothelium), platelets, coagulation, and fibrinolytic systems interactions. Endothelium, lining the vascular wall interior, plays crucial roles in the control of local hemodynamics and blood coagulability. It releases prostacyclin (PGI2) and nitric oxide—the most potent vasodilating and antithrombotic factor, but also other anticoagulants like tissue factor pathway inhibitor and fibrinolysis activators: tissue plasminogen activator (t-PA) and urokinase type plasminogen activator (u-PA). The protective properties of endothelium can be abolished under the pathological conditions and may lead to platelets and coagulation system activation. During the first phase of aggregation, platelet release granule contents affecting coagulation and fibrinolysis, such as a2-antiplasmin, plasminogen activator inhibitor type 1 (PAI-1), fibrinogen, serotonin and others. The second step of platelet activation is related to the secretion of de novo synthesized thrombaxane A2 (TxA2). Coagulation factors and platelets transform fibrinogen into fibrin which consolidates the thrombus. This process is simultaneously limited by the fibrinolytic system—the enzymatic system that limits fibrin generation.

The key enzyme of the fibrinolytic system is plasmin which is formed from plasminogen. One of its main roles is digesting fibrin and limiting thrombus formation. Conversion of plasminogen to plasmin is controlled by various factors. The balance between group of activators (t-PA, uPA) and inhibitors (PAI-1, antiplasmin) determines plasma fibrinolytic activity (Fig. 1).

* Corresponding author. Tel./fax: +48-85-7425601. E-mail address: [email protected] (W. Buczko). 0049-3848/$ - see front matter D 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2003.08.006

3. Aspirin and fibrinolysis Although it is well known that aspirin (ASA) decreases platelets activity and possesses antithrombotic action, its effect on fibrinolysis has not been yet entirely established. In 1966 Gryglewski in in vitro experiment demonstrated that ASA enhances fibrinolysis [1]. Menon [2] was the first who reported increased fibrinolytic response in healthy volunteers treated with high doses of ASA. This observation stimulated series of studies undertaken to find the relation between ASA and fibrinolysis. The enhancing effect of high-dose ASA on fibrinolysis in normal plasma was confirmed by Moroz [3]. Experiments carried out on animals also proved profibrinolytic activity of ASA administered in high doses [4,5]. In vitro study showed that acetylation of fibrinogen caused dose-related enhanced rate of fibrinolysis [6]. Subsequently it has been demon-

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W. Buczko et al. / Thrombosis Research 110 (2003) 331–334

Fig. 1. Fibrinolysis activation.

strated that also low-dose ASA treatment decreased PAI-1 level [7,8]. Thus, it may be speculated that ASA increased fibrinolytic activity. But on the other hand, when fibrinolysis was activated by venous occlusion, high dose of ASA inhibited fibrinolytic response and low doses did not affect fibrinolysis [9,10]. In addition, some in vitro studies showed that low concentration of ASA did not change fibrinolytic activity [3], whereas ASA at high concentration inhibited fibrinolysis [11]. The overall data concerning the effect of ASA on fibrinolysis are presented in Table 1. From the preformed studies it may be concluded that aspirin modifies fibrinolytic response and this effect depends on the initial plasma fibrinolytic activity, experimental conditions, and the dose and the time of aspirin administration. Generally, high doses of ASA enhances fibrinolysis in normal plasma. However, in subjects where fibrinolytic activity is increased, high-dose ASA inhibits the fibrinolytic response. The results of the clinical studies also indicate that low doses of ASA administered acutely do not affect fibrinolysis, whereas administered chronically enhance fibrinolytic activity decreasing PAI-1 concentration.

4. Effect of aspirin on overall fibrinolysis potential Recently He et al. [12] described a new method for the measurement of plasma hemostatic potential. Using this method in our modification the overall potential in the entire system in rats can be assessed. Taking into account that ASA may influence fibrinolysis in different ways, the aim of our study was to find if ASA in ex vivo and in vitro conditions affects plasma fibrinolytic potential. Male Wistar rats, anesthetized with pentobarbital (45 mg/kg ip), were used in the experiments. Blood samples were taken from the heart and mixed with 3.13% trisodium

citrate in a volume ratio 9:1, centrifuged at 2000  g, 4 jC for 20 min to obtain platelet poor plasma (PPP). Briefly, two fibrin time curves, changing during fibrin generation and clot formation, were made by the registration of optical density (OD) using microplate reader (Dynex Tech). To make the first one, determining Overall Haemostasis Potential (OHP), CaCl2 (final conc. 36 mmol/l), thrombin (0.09 IU/ml) and t-PA (3200 ng/ml) were added to the Tris buffer (66 mmol/l Tris and 130 mmol/l NaCl, pH 7) and 100 Al of the prepared buffer was mixed with 120 Al PPP in each well. The second fibrin time curve was created without adding t-PA and illustrated Overall Coagulation Potential (OCP). Values of OD were recorded every minute from time zero until 30 min. Based on the principle of integrals, the area under the curve, illustrating OHP and OCP, was expressed by summation of the OD values. The difference between these two curves equals Overall Fibrinolysis Potential (OFP) in plasma calculated as: [(OCP OHP)/OCP]  100%. The data are shown as mean F SEM. Two-tailed Mann – Whitney test was used Table 1 Aspirin and fibrinolysis Increased fibrinolytic activity

No change in fibrinolytic activity

Ex vivo studies (human) Menon IS, 1970 Keber I et al., 1985 Moroz LA, 1977 Bounameaux H et al., 1985 Green D et al., 1983 de Gaetano et al., 1986 Hammouda MW et al., Hampton KK 1986 et al., 1990 Bjornsson TD et al., Husted SE et al., 1989 1992 Cogo H et al., 1990 Winther K et al., 1994 Basin´ski A et al., 1991 Tohgi H et al., 1993 Bremer HA et al., 1995

Ex vivo studies (animal) Rosenior JC et al., Iacoviello L et al., 1974 1992 Housholder GT et al., 1980 Cattaneo M et al., 1983 Garabedian HD et al., 1991 In vitro studies Gryglewski R, 1966 Bjornsson TD et al., 1989 Mildwidsky A et al., 1991

Moroz LA et al., 1977 Woods AI et al., 1987, 1988

Decreased fibrinolytic activity Ghezzo F et al., 1981 Levin RI et al., 1984 Hammouda MW et al., 1986 De Gaetano et al., 1986 Keber I et al., 1987 Kowalski ML et al., 1987 Bertele V et al., 1989 Hampton KK et al., 1990 Iacoviello L et al., 1992 Winther K et al., 1994

Iacoviello L et al., 1994

Iatridis PG et al., 1974

W. Buczko et al. / Thrombosis Research 110 (2003) 331–334

consistently throughout the study. p-values less than 0.05 were considered significant. Effect of ASA on OCP, OHP and OFP are presented in Table 2. In the first part of our study we tried to estimate the effect of ASA on fibrinolysis in ex vivo conditions, when endothelium, platelet, plasma coagulation and fibrinolytic systems were involved. ASA was administered to rats intravenously or orally according to Iacoviello et al. [18] and Housholder and Moorrees [5]. The blood was collected, then OCP, OHP and OFP were measured in PPP. ASA administrated intravenously or orally in a dose of 100 mg/kg decreased significantly OCP and OFP by 43%, 15% (iv) and 41%, 34% (po), respectively (Table 2). ASA in dose of 50 mg/kg administered orally significantly decreased OFP by 37%, whereas administered iv in the same dose did not change any of haemostasis potentials. The results of our study show that ASA disturbs the balance between coagulation and fibrinolysis. Although it decreases fibrin generation at the same time the haemostatic potential is increased and fibrinolytic potential decreased. High-dose ASA administered intravenously or orally into rats inhibits both coagulation and fibrinolysis. The decrease in OCP involved probably the thrombin activity inhibition that has been shown previously [13]. One may suggest that the reduced fibrinolytic activity following high-dose ASA depends on the diminished endothelium derived PGI2 production. Indeed, iloprost (PGI2 analogue) reverses the inhibitory effect of ASA on fibrinolysis [14]. It is also in line with the studies showing that low-dose ASA, while significantly reducing platelet TxA2 synthesis, leaves unaltered the PGI2 synthesizing activity of endothelium cells. It is also hypothesized that ASA antagonism of fibrinolysis is related to inhibition of nitric oxide and may be reversed by providing a nitric oxide donor or L-arginine [15]. In our experiment low dose of ASA shows less potent fibrinolytic inhibitory effect (50

Table 2 Effect of ASA on OCP, OHP and OFP Group

n

OCP

OHP

OFP

Ex vivo assay VEH (0.9%NaCl) 9 1211.3 F 114.0 297.8 F 35.7 ASA 50 mg/kg iv 4 739.2 F 156.1 230.8 F 32.0 ASA 100 mg/kg iv 4 687.7 F 122.2* 249.4 F 17.8 VEH (aq. dest.) 11 1320.9 F 111.0 291.3 F 33.5 ASA 50 mg/kg po 3 874.0 F 118.7 448.4 F 59.0 ASA 100 mg/kg po 5 779.6 F 39.1* 420.9 F 35.4

74.5 F 2.1 68.9 F 8.3 63.7 F 6.8** 73.5 F 3.0 48.7 F 2.9** 46.0 F 3.3***

In vitro assay VEH (0.9%NaCl) ASA 1 Ag/ml ASA 10 Ag/ml ASA 100 Ag/ml ASA 1000 Ag/ml

58.3 F 6.7 63.5 F 5.2 65.6 F 3.3* 68.9 F 5.0* 76.4 F 2.1**

17 1201.0 F 94.5 7 1135.5 F 61.5 7 934.5 F 81.0 9 865.3 F 25.5* 7 826.2 F 67.5*

* p < 0.05 vs. VEH. ** p < 0.01 vs. VEH. *** p < 0.001 vs. VEH.

501.2 F 31.5 414.6 F 25.1 321.6 F 71.6 268.3 F 62.3* 195.2 F 41.1***

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mg/kg iv does not significantly influence OFP or other potentials) which is in accordance with studies indicating that low-dose ASA does not inhibit fibrinolysis [10,16]. It has been suggested that high-dose ASA inhibits t-PA release from endothelium [9,10,17]. However, these results need to be further elucidated, especially that Iacoviello et al. [18] did not find any effect of ASA on t-PA release. In the second part of our study we preformed in vitro experiments to find the ASA influence on plasma coagulation and fibrinolytic system, when endothelium and platelets response were excluded. ASA was added to PPP at concentration of 1, 10, 100, 1000 Ag/ml and OCP, OHP and OFP were measured (Table 2). The range of ASA concentrations used in our study was in accordance with the maximal plasma concentrations reached after oral administration (in humans the dose of 100– 1000 mg corresponds to 2.7 – 15.9 Ag/ml [19], whereas in rats after doses of 50 –200 mg/kg comprise between 2.2 and 7.9 Ag/ml [20]). ASA decreased OCP, OHP and increased OFP in dose-dependent manner. Since the same antithrombin effect was observed, in contrast to our ex vivo experiment, the fibrinolytic potential was increased. The opposite results coming from in vitro experiments indicate that ASA, when affects only plasma components of fibrinolytic system, enhances fibrinolysis. We suggest that enhancing effect of ASA on fibrinolysis in in vitro study is due to modification of fibrinogen structure by high concentration of ASA. It is consistent with other in vitro experiments showing that acetylation of fibrinogen by ASA resulted in dose-dependent decrease in ELT [6]. It is also possible that ASA increases OFP through direct stimulation of plasmin activity, which was previously demonstrated by others [21]. We cannot exclude the role of platelets for the mechanism of ASA action on the fibrinolytic system. It has been proposed that ASA could inhibit antiplasmin and PAI-1 release from platelets. Although Woods et al. [22,23] failed to prove this hypothesis in in vitro experiments it has been shown that administration of 500 mg/day of ASA inhibited PAI-1 release in healthy volunteers [24]. The results of the clinical studies also showed that 40 or 80 mg/kg po of ASA administered chronically decreased plasma PAI-1 level [7,8]. Even though, these results have not been confirmed by other clinical experiments [16,17], the involvement of platelets in the profibrinolytic action of ASA cannot be ruled out.

5. Summary Although 30 years have passed from the first study of Menon [2] the ASA influence on fibrinolysis is still not entirely understood. The experimental and clinical data and also our results indicate that: 1. The influence of ASA on fibrinolysis depends on experimental conditions (e.g., plasma initial fibrinolytic state), and the dose and the time of drug administration.

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2. The OFP estimation indicates the essential role of plasma components in the regulation of fibrinolysis by ASA. 3. ASA affects the most important components of the fibrinolytic system: fibrinogen, plasmin, t-PA and PAI-1. 4. In in vivo conditions modification of the fibrinolytic activity by ASA involves endothelium, platelet, plasma coagulation and fibrinolytic system interactions.

[11] [12]

[13] [14]

Acknowledgements This work was supported by Grants No. 3P05B 198 22 and 4 PO5F 018 19 from the State Committee for Scientific Research, Poland.

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