High-Dose Epinephrine Enhances Platelet Aggregation at the Expense of Procoagulant Activity.

Platelet activation is characterized by shape change, granule secretion, activation of fibrinogen receptor (glycoprotein IIb/IIIa) sustaining platelet aggregation, and externalization of negatively charged aminophospholipids contributing to platelet procoagulant activity. Epinephrine (EPI) alone is a weak platelet activator. However, it is able to potentiate platelet activation initiated by other agonists. In this work, we investigated the role of EPI in the generation of procoagulant platelets. Human platelets were activated with convulxin (CVX), thrombin (THR) or protease-activated receptor (PAR) agonists, EPI, and combination thereof. Platelet aggregation was assessed by light transmission aggregometry or with PAC-1 binding by flow cytometry. Procoagulant collagen-and-THR (COAT) platelets, induced by combined activation with CVX-and-THR, were visualized by flow cytometry as Annexin-V-positive and PAC-1-negative platelets. Cytosolic calcium fluxes were monitored by flow cytometry using Fluo-3 indicator. EPI increased platelet aggregation induced by all agonist combinations tested. On the other hand, EPI dose-dependently reduced the formation of procoagulant COAT platelets generated by combined CVX-and-THR activation. We observed a decreased Annexin-V-positivity and increased binding of PAC-1 with the triple activation (CVX + THR + EPI) compared with CVX + THR. Calcium mobilization with triple activation was decreased with the higher EPI dose (1,000 µM) compared with CVX + THR calcium kinetics. In conclusion, when platelets are activated with CVX-and-THR, the addition of increasing concentrations of EPI (triple stimulation) modulates platelet response reducing cytosolic calcium mobilization, decreasing procoagulant activity, and enhancing platelet aggregation.

Roles of the C-terminal tyrosine residues of LAT in GPVI-induced platelet activation: insights into the mechanism of PLC gamma 2 activation.

Linker for activation of T cells (LAT) is an adaptor protein required for organization of the signaling machinery downstream of the platelet collagen receptor, the glycoprotein VI (GPVI). Here, we investigated the effect of LAT mutations on specific signaling pathways and on platelet functions in response to GPVI triggering by convulxin (Cvx). Using mice containing tyrosine to phenylalanine mutations of the adaptor, we show the crucial role played by the tyrosine residues at positions 175, 195, and 235 in the phosphorylation of LAT and in the whole pattern of protein tyrosine phosphorylation in response to Cvx. These 3 C-terminal tyrosine residues are important to recruit the tyrosine kinase Fyn, which may be involved in LAT phosphorylation. Efficient phosphoinositide 3-kinase (PI3K) activation requires the 3 C-terminal tyrosine residues of LAT but not its tyrosine 136. Interestingly, single mutation of the tyrosine 136 results in the loss of phospholipase C gamma2 (PLCgamma2) activation without affecting its PI3K-dependent membrane association, and is sufficient to impair platelet responses to Cvx. Thus, activation of PLCgamma2 via GPVI is dependent on 2 complementary events: its interaction with the tyrosine 136 of LAT and its membrane location, which itself requires events mediated by the 3 C-terminal tyrosines of LAT.