The ATP-gated P2X1 Receptor Plays a Pivotal Role in Activation of Aspirin-treated Platelets by Thrombin and Epinephrine

Abstract

Human platelets express protease-activated receptor 1 (PAR1) and PAR4 but limited data indicate for differences in signal transduction. We studied the involvement of PAR1 and PAR4 in the cross-talk between thrombin and epinephrine. The results show that epinephrine acted via ␣ 2A -adrenergic receptors to provoke aggregation, secretion, and Ca 2؉ mobilization in aspirin-treated platelets pre-stimulated with subthreshold concentrations of thrombin. Incubating platelets with antibodies against PAR4 or the PAR4-specific inhibitor pepducin P4pal-i1 abolished the aggregation. Furthermore, platelets pre-exposed to the PAR4-activating peptide AYPGKF, but not to the PAR1-activating peptide SFLLRN, were aggregated by epinephrine, whereas both AYPGKF and SFLLRN synergized with epinephrine in the absence of aspirin. The roles of released ATP and ADP were elucidated by using antagonists of the purinergic receptors P2X 1 , P2Y 1 , and P2Y 12 (i.e. NF449, MRS2159, MRS2179, and cangrelor). Intriguingly, ATP, but not ADP, was required for the epinephrine/thrombin-induced aggregation. In Western blot analysis, a low concentration of AYPGKF, but not SFLLRN, stimulated phosphorylation of Akt on serine 473. Moreover, the phosphatidyl inositide 3-kinase inhibitor LY294002 antagonized the effect of epinephrine combined with thrombin or AYPGKF. Thus, in aspirin-treated platelets, PAR4, but not PAR1, interacts synergistically with ␣ 2A -adrenergic receptors, and the PI3-kinase/Akt pathway is involved in this cross-talk. Furthermore, in PAR4-pretreated platelets, epinephrine caused dense granule secretion, and subsequent signaling from the ATP-gated P2X 1 -receptor and the ␣ 2A -adrenergic receptor induced aggregation. These results suggest a new mechanism that has ATP as a key element and circumvents the action of aspirin on epinephrine-facilitated PAR4-mediated platelet activation.

The neutrophil serine protease PR3 induces shape change of platelets via the Rho/Rho kinase and Ca2+ signaling pathways

“…Therefore, we conclude that PR3 stimulates shape change of human platelets through activation of the Rho/Rho kinase and Ca 2+ signaling pathways. It is well established that adrenaline enhances platelet activation by other, more efficient activators [27]. We found that adrenaline reinforced platelet shape change by PR3 but this was not followed by aggregation.…”

“…There is a large body of evidence that positive modulators, such as adrenaline, facilitate for secretion and aggregation when platelets are subsequently exposed to strong activators like thrombin, ADP and collagen [26,27]. However, PR3 was not shown to alter activation of the fibrinogen receptor in response to thrombin, PAR1-or PAR4-activating peptides at any concentrations, nor did it change the magnitude of ADP-induced aggregation (in the absence of apyrase).…”

ATP secreted by endothelial cells blocks CX3CL1-elicited natural killer cell chemotaxis and cytotoxicity via P2Y11 receptor activation

“…We then studied ATP inhibition of NK chemotaxis to CX 3 CL1 in the presence of NF157, or MRS2159 and NF023, 2 different P2X 1 inhibitors. [19][20][21][22] Although NF157 restored NK chemotaxis abolished by ATP, neither MRS2159 nor NF023 modified NK-cell migration to CX 3 CL1 in the presence of ATP ( Figure 5A). Similarly, NK-cell killing of HUVECs that was elicited by CX 3 CL1 and blocked by ATP was restored by NF157 but not by P2X 1 antagonists ( Figure 5B).…”

Increased plasma cathepsin S and trombospondin-1 in patients with acute ST-segment elevation myocardial infarction

“…Activation of platelet by the thrombin mimetic hexapeptide SFLLRN was marginally affected by aspirin and clopidogrel treatments. This is probably explained by the efficiency of thrombin receptors in inducing intracellular signaling [32]. Specifically, this means that thrombin activation of platelets does not rely on secondary positive feed-back loops such as thromboxane A2 and ADP.…”