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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).

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.

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				H. M. Rinder Invited commentary Ann. Thorac. Surg., February 1, 2006; 81(2): 624 - 624. [Full Text] [PDF]