PB0412 - Simulating the Impact of Thrombin on Platelet Function Requires Simultaneous Activation of Both PAR-1 and PAR-4

Background:

Background: The platelet PAR-1 receptor can be activated by small peptides representing the tethered ligand SFLLRN (T6) produced by thrombin cleavage of its N-terminal domain. The later-discovered PAR-4 receptor was also shown to be activated by peptides simulating its tethered ligand sequence (AYPGKF), leading to platelet aggregation. Ofosu et al. reported that activation of PAR-1 with SFLLRN led to cleavage of both PAR-1 and PAR-4 activation peptides within 10 s. This suggests that a PAR-1 peptide should produce the same response as a combination of PAR-1 and PAR-4 peptides, and that in vivo thrombin activation of platelets can be simulated ex vivo with a PAR-1 peptide alone.

Aims: Aims and

Methods:

Methods: To test this hypothesis, we: 1. Identified submaximal platelet aggregation concentrations for citrated platelet-rich plasma (PRP) for SFLLRN or AYPGKF (n=6 healthy donors who gave consent under an IRB-approved protocol). 2. Tested the effect of combining the submaximal PAR-1 and PAR-4 concentrations. 3. Measured both the primary slope (PS) and the maximal aggregation (MA).

Results:

Results: The submaximal concentrations of SFLLRN ranged from 5.5 to 10.9 (mean 8.6±1.1) uM and those for AYPGKF ranged from between 32.5 to 85 (mean 52.1±5.3) uM (p=0.005). As shown in the Figure, the submaximal concentrations produced PS values (AU) of 42±4.2 for SFLLRN and 25±5.6 for AYPGKF (p=0.15); the corresponding values for MA (%) were 34.5±0.7 for SFLLRN and 22.5±3.5 for AYPGKF (p=0.17). In sharp contrast, the PS and MA values for the combination of peptides were 72.5±0.7 and 92±5.6 (p=0.005 versus the submaximal values of either SFLLRN or AYPGKF for both PS and MA).

Conclusion(s):

Conclusion: We conclude that simultaneous activation of PAR-1 and PAR-4 elicits a more robust aggregation response than either alone and that this needs to be considered when trying to simulate the in vivo impact of thrombin on platelet function.