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Ann Thorac Surg 2006;81:619-624
© 2006 The Society of Thoracic Surgeons

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Original article: Cardiovascular

Effect of Aprotinin and Recombinant Variants on Platelet Protease-Activated Receptor 1 Activation

Eric Bywaters Centre, British Heart Foundation Cardiovascular Medicine Unit, Imperial College, Hammersmith Hospital, London, United Kingdom

Accepted for publication July 18, 2005.

^_* Address correspondence to Dr Day, Eric Bywaters Centre, British Heart Foundation Cardiovascular Medicine Unit, Imperial College, Hammersmith Hospital, London W12 ONN, United Kingdom (Email: j.day{at}imperial.ac.uk).

	Abstract

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BACKGROUND: Thrombin generated during cardiopulmonary bypass activates the high-affinity thrombin receptor, protease-activated receptor 1 (PAR1), causing platelet dysfunction and excessive bleeding. The serine protease inhibitor aprotinin protects platelets against thrombin-mediated PAR1 activation in vitro and in vivo. Here we have investigated three novel recombinant aprotinin variants with specific modifications to the active site lysine at amino acid position 15 (arginine-15, arginine-15-alanine-17, and valine-15-leucine-17) for their effect on PAR1-mediated platelet aggregation in vitro.

METHODS: Aggregation studies were carried out using washed human platelets (n = 9) or platelet rich plasma (n = 7) from healthy volunteers activated with 1 or 5 nM thrombin. Recombinant aprotinin variants were used at the molar equivalent to 50 KIU/mL of the parent compound. The PAR1-specific antagonist peptide, FLLRN, was used at 500 µM.

RESULTS: Platelet aggregation at low concentrations of thrombin (1 nM) was mediated exclusively through PAR1, as shown by inhibition of aggregation in the presence of FLLRN. At 1 nM thrombin, the mean percentage ± SD aggregation of washed platelets was 68.6% ± 12.3%. This was suppressed by each aprotinin variant at the 50 KIU/mL equivalent dose: arginine-15 (23.0% ± 17.5%, p < 0.001); arginine-15-alanine-17 (33.3% ± 22.9%, p < 0.01); aprotinin (37.5% ± 19.4%, p < 0.05); valine-15-leucine-17 (50.0% ± 16.1%, not significant)). At 5 nM thrombin, which activates both high (PAR1) and low-affinity (PAR4) thrombin receptors on platelets, FLLRN and aprotinin failed to block aggregation: this finding indicates that aprotinin selectively targeted PAR1. In platelet-rich plasma, aggregation at 1 nM thrombin was 77.1% ± 10.0%, and this was inhibited in the following order: arginine-15 (30.1% ± 9.6%, p < 0.001); arginine-15-alanine-17 (52.3% ± 9.7%, p > 0.001); aprotinin (55.9% ± 6.2%, p > 0.001); valine-15-leucine-17 (73.7% ± 7.1%, not significant).

CONCLUSIONS: Aprotinin variants differentially inhibit PAR1-mediated platelet aggregation. With more understanding of the mechanisms of action of aprotinin and its derivatives, safer and more efficacious aprotinin variants may become available for clinical use.

	Introduction

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Thrombin generation occurs during cardiopulmonary bypass despite heparinization [1–3]. Although platelet function is critical for normal hemostasis, thrombin generated in the bypass circuit activates platelets systemically, thus rendering them insensitive to stimulation in surgical wounds and leading to bleeding problems [4]. The main thrombin receptors on human platelets are the high-affinity thrombin receptor, protease activated receptor-1 (PAR1), and a closely related low-affinity receptor, PAR4, both of which require proteolytic cleavage to become activated [5, 6]. Platelet dysfunction after cardiopulmonary bypass is due to the selective activation of the high affinity PAR1 receptor by thrombin in the bypass circuit [4].

Aprotinin (Trasylol; Bayer AG, Wuppertal, Germany) was originally introduced into cardiac surgical practice in the 1980s, owing to its ability to reduce blood loss [7, 8]. Its hemostatic mechanism of action is based, in part, upon an inhibition of plasmin, thus resulting in an antifibrinolytic effect [9]. However, compared with other antifibrinolytics (eg, the lysine analogs tranexamic acid and -amino caproic acid), aprotinin was recognized to provide additional patient benefits including improved platelet function [10–12], diminished inflammatory response to bypass [13–15], and reduction in perioperative stroke [16–18].

An antithrombotic property of aprotinin has been discovered in platelets through inhibition of the high-affinity thrombin receptor, PAR1, in vitro and in vivo [19, 20]. Vascular thrombosis of the carotid artery in rabbits is also attenuated by aprotinin through a PAR-dependent mechanism [21]. However, since protease-independent pathways of platelet activation (namely, through adenosine diphosphate, collagen, or epinephrine expressed at wound sites) remain intact in the presence of aprotinin, platelet activation in the chest cavity and bleeding times in the rabbit model remain unaffected [21, 22]. Given that aprotinin preserves platelet function and exerts a net hemostatic effect clinically, a "hemostatic yet antithrombotic" paradigm has therefore been proposed to govern its mechanism of action on platelets [23].

Serious issues surround the continued use of pharmacologic agents derived from animal tissue [24]. Because aprotinin is extracted from bovine lung tissue, there are concerns associated with the prion diseases bovine spongiform encephalopathy and new variant Creutzfeldt-Jakob disease. Notwithstanding current data showing that any risk of transmission is infinitesimally small [25], there is interest in the development of safer and more effective recombinant aprotinin variants. Through recombinant approaches, not only can one eliminate the risk of prion transfection, but it is also possible to change or enhance the serine protease inhibition profile, with alterations to the lysine at position-15 of the active center [26].

Here, we have investigated three novel aprotinin variants for their ability to block PAR1-mediated platelet aggregation in vitro. Two concentrations of thrombin were used to dissect the molecular pathway of receptor activation in platelets: 1 nM, which activates only the high affinity PAR1 receptor, and 5 nM, which additionally activates the low-affinity thrombin receptor, PAR4 [6]. We demonstrate that aprotinin and three novel aprotinin variants selectively inhibit platelet aggregation at 1 nM thrombin (namely, through blockade of PAR1) and that two of the aprotinin variants, arginine-15-aprotinin and arginine-15-alanine-17-aprotinin, achieve this more effectively than the parent compound.

	Material and Methods

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Reagents
Thrombin was obtained from Sigma Diagnostics (Poole, United Kingdom). The PAR1 antagonist peptide FLLRN was synthesised by the Advanced Biotechnology Centre (Imperial College, London, United Kingdom). Aprotinin (Trasylol, molecular weight 6.512 kDa), and the recombinant aprotinin variants Arg-15-aprotinin (molecular weight 6.540 kDa), Arg-15-Ala-17-aprotinin (molecular weight 6.455 kDa), and Val-15-Leu-17-aprotinin (molecular weight 6.440 kDa) were provided by Bayer AG (Wuppertal, Germany).

Platelet Preparation
Platelets were isolated from peripheral blood samples of normal healthy human volunteers. Blood was collected into a prewarmed syringe, containing sodium citrate (10 mM/L) in a 1:10 ratio and 2% dextrose, by antecubital venepuncture in the absence of a tourniquet to avoid platelet activation. Prostaglandin I₂ (prostacyclin) was added at a dose of 1 µM/L and platelet-rich plasma (PRP) was decanted off after centrifugation at 1,100 rpm for 10 minutes. Washed platelets were prepared from PRP by recentrifugation at 2,100 rpm for 12 minutes at room temperature, and the resulting pellet gently resuspended at 2 x 10⁸ platelets/mL in a platelet buffer containing bovine serum albumin 0.2% and Hanks balanced salt solution (Sigma, Poole, United Kingdom) with NaHCO₃ 25mM/L, and without calcium chloride and magnesium sulphate. The platelets were then incubated for 30 minutes at 37°C, to allow the effects of prostacyclin to wear off. Absence of clotting factors in washed platelets was demonstrated by lack of detectable antithrombin III (<0.01 units/mL), protein C (<0.01 units/mL) and protein S (<0.01 units/mL) in the platelet suspension. Absence of activation in resting samples was determined by demonstrating only baseline expression of surface P-selectin, assessed by flow cytometric analysis, as previously described [27].

Aggregometry
Platelet aggregation was monitored by light transmission using an APACT 4 platelet aggregometer (Helena Biosciences, Sunderland, United Kingdom). Aggregometry was carried out on washed platelets in a 200 µL volume, at 2 x 10⁸ platelets/mL, adjusted to a 1.2 mM/L concentration of CaCl₂ immediately before addition of thrombin (1 or 5 nM/L). The PRP was diluted in platelet-poor plasma (PPP) to achieve a final concentration of 2 x 10⁸ platelets/mL, and aggregation was carried out in a 200 µL volume in the presence of 1.0 mM synthetic GPRP peptide to prevent fibrin clot formation. Platelet antagonists, such as FLLRN (500 µM) or aprotinin variants were added together with thrombin and aggregometry was carried out immediately. Aprotinin variants were added at the molar equivalent to 50 KIU/mL dose of the parent compound.

Statistics
Statistical comparisons between groups were carried out using a one-way analysis of variance (ANOVA) with a Newman Keuls post-test. Significance was assumed at p less than 0.05.

	Results

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Aprotinin Inhibits Platelet Aggregation Mediated Through PAR1
Initial experiments were carried out to verify that platelet aggregation at 1 nM thrombin was exclusively mediated through PAR1. Proof of this was provided by complete ablation of aggregation in the presence of the synthetic PAR1-specific antagonist pentapeptide, FLLRN (Fig 1). At this concentration of thrombin, native aprotinin dose-dependently inhibited aggregation in washed platelets, consistent with previous results. However, at the high dose of thrombin (5 nM), neither FLLRN nor aprotinin inhibited platelet aggregation (Fig 2). These experiments therefore demonstrate that aprotinin specifically inhibited PAR1-mediated platelet aggregation.

View larger version (26K): [in this window] [in a new window]   Fig 1. Representative platelet aggregometry traces (of n = 4) using washed platelets (2 x 108/mL) showing effects of the PAR1-specific peptide antagonist, FLLRN, (500 µM/L) on platelet aggregation induced by (A) low-dose thrombin (1 nM), and (B) high-dose thrombin (5 nM).

View larger version (27K): [in this window] [in a new window]   Fig 2. Representative platelet aggregometry traces (of n = 9) showing effects of clinically relevant concentrations of aprotinin (50, 100, 200 KIU) on platelet aggregation induced by (A) low-dose thrombin (1 nM), and (B) high-dose thrombin (5 nM). Washed platelets (2 x 108/mL) were preincubated with aprotinin for 5 minutes before the addition of thrombin.

View larger version (51K): [in this window] [in a new window]   Fig 3. Effect of aprotinin variants on thrombin-induced aggregation of washed platelets. Histogram showing the order of efficacy of recombinant variants is (from highest to lowest) Arg-15-aprotinin (p< 0.001) greater than Arg-15-Ala-17-aprotinin (p < 0.01) greater than aprotinin (p < 0.05) greater than Val-15-Leu-17-aprotinin (not significant [NS]). Results are mean ± SD from a total of n = 9 experiments. Washed platelets (2 x 108/mL) were preincubated with aprotinin or one of its variants for 5 minutes before the addition of thrombin. The aprotinin variants were used at the molar equivalent of 50 KIU/mL doses for the parent compound.

View larger version (61K): [in this window] [in a new window]   Fig 4. Effect of aprotinin variants on thrombin-induced aggregation of platelet-rich plasma. Histogram showing the order of efficacy of recombinant variants is (from highest to lowest) Arg-15-aprotinin (p < 0.001) greater than Arg-15-Ala-17-aprotinin (p < 0.001) greater than aprotinin (p < 0.001) greater than Val-15-Leu-17-aprotinin (not significant [NS]). Results are mean ± SD from a total of n= 7 experiments. Platelet-rich plasma (2 x 108 platelets/mL containing 1.0 mM GPRP to prevent fibrin polymerization) was preincubated with aprotinin or one of its variants for 5 minutes before the addition of thrombin. The aprotinin variants were used at the molar equivalent of 50 KIU/mL for the parent compound.

	Comment

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Kallikrein inhibitory units (KIU) were not used in comparing variants, since these reflect functional activity, and the inhibitory constant (Ki) for kallikrein has been shown to be altered by the active site mutations under study [26]. Comparisons between variants were therefore made using molar equivalents to the parent compound of 50 KIU/mL. Using either washed platelets or platelet-rich plasma, the same order of inhibition of thrombin-induced platelet activation was observed: arginine-15-aprotinin and arginine-15-alanine-17-aprotinin were more efficacious than the parent compound, whereas valine-15-leucine-17-aprotinin was least efficacious. Experiments using platelet-rich plasma confirmed and extended the washed platelet work in a model that includes fibrinogen and other plasma proteins that can promote platelet activation.

The present study has demonstrated that second-generation recombinant aprotinin variants inhibit PAR1-mediated platelet activation, in a mechanism similar to the parent compound. These in vitro findings are likely to be of physiologic relevance in light of the fact that, clinically, it is PAR1 that is selectively activated and internalised during bypass [4]. We predict that, through protection of thrombin activation of PAR1, these new variants, especially arginine-15-aprotinin and arginine-15-alanine-17-aprotinin, would exert a similar hemostatic yet antithrombotic platelet protective mechanism of action, as proposed for the parent compound [23]. Thus, for aprotinin, blockade of platelet PAR1 has been shown not to exacerbate bleeding, since platelets maintain their ability to be activated by other stimuli present in the chest cavity, including collagen and adenosine diphosphate [21, 22]. The net effect is that aprotinin minimizes participation of thrombin-activated platelets in the coagulation cascade, thereby exerting an antithrombotic effect, while the hemostatic capacity of platelets in surgical wounds is maintained.

Several lines of evidence suggest that aprotinin targets the proteolytic cleavage step in PAR1 activation. We have previously shown that thrombin receptor activating peptide (TRAP)-6-dependent PAR1 activation, which bypasses the need for receptor proteolysis, is not blocked by aprotinin [19]. Furthermore, the mechanism of PAR1 sparing by aprotinin is distinct from peptide-mimetic antagonists (like FLLRN): thus, whereas peptide antagonists inhibit signaling but not receptor cleavage, aprotinin inhibits signaling and receptor cleavage in response to thrombin. This was shown using an anti-PAR1 antibody, SPAN12, which detected only uncleaved receptors [20]. The obvious hypothesis, that aprotinin as a serine protease inhibitor directly inhibits the catalytic activity of thrombin in solution, is unlikely as the systemic concentration of aprotinin achievable during bypass is approximately 60-fold below the inhibitory constant for thrombin [28]. The precise mechanism by which aprotinin, or its recombinant analogs, inhibits PAR1 receptor cleavage is under investigation.

The recombinant aprotinin variants described in this study, despite exhibiting some improvement in their PAR1-protecting abilities, have been primarily developed to increase their inhibitory constants toward plasmin and kallikrein and thereby have improved hemostatic properties. Our data indicate that arginine-15 aprotinin is more potent at inhibiting platelet aggregation than native aprotinin, and may therefore also be more effective as a hemostatic agent.

It is possible that PAR1 expressed elsewhere within the vasculature or central nervous system may be similarly targeted by aprotinin. For instance, protection of PAR1 on the vascular endothelium may explain the mechanism behind reduced systemic inflammation in the presence of aprotinin after cardiopulmonary bypass. Moreover, the reported significant reduction in perioperative stroke may also involve protection of PAR1 in the central nervous system [29, 30]. The growing appreciation of the key role played by the PARs in the host response to infection and injury should stimulate renewed interest in the development of second-generation aprotinin analogs and other serine protease antagonists for introduction into cardiac surgery.

	Acknowledgments

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This study was supported by grants from the British Heart Foundation.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Jonathan R.S. Day Kenneth M. Taylor Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Day, J. R.S. Articles by Landis, R. C. Search for Related Content PubMed PubMed Citation Articles by Day, J. R.S. Articles by Landis, R. C. Related Collections Extracorporeal circulation