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A.S. Macwan et al.
unresponsiveness to further homologous stimulation. In contrast, in the case of PAR receptor stimulation, our find- ings show that GDI is strictly gradient-dependent, as platelet reactivity to instantaneous additions of agonists was found to be unaffected by GDI in our study. One important exception from this rule was observed for ADP, as ADP-induced GDI rendered platelets unresponsive to further stimulation in our experiments. This finding sug- gests that desensitization is an important mechanism of ADP-induced GDI, in agreement with previous findings showing that desensitization of P2Y12 receptors occurs rapidly enough to affect ADP signaling during the time- scales relevant for this study.12 In contrast, desensitization of PAR receptors has previously only been reported to occur upon prolonged exposure (typically 10-60 min) to subthreshold concentrations of agonists.17,26,27 This is con- trasted by the rapid and dynamic effects of GDI on throm- bin-induced platelet activation observed in our study, as an infusion time of 80 s was sufficient to completely inhibit platelet aggregation as a response to stimulation with 1 U/mL thrombin. The finding that PAR1 is the receptor most prominently affected by GDI is not surpris- ing when considering that the unique enzymatic activa- tion mechanism of the PAR receptors, where one throm- bin molecule theoretically could activate a large number of PAR receptors, puts high demands on balancing inhibitory signaling machinery, most particularly in the case of a high-affinity receptor such as PAR1, requiring only sub- nanomolar concentrations of thrombin to effect signifi- cant receptor cleavage over time.17,27,28
Whereas GPCR desensitization typically involves slow cellular processes such as altered protein translation and receptor internalization,17-20,29 we provide evidence against internalization or decoupling as a primary cause of GDI. Firstly, in accordance with previous results from our group,26 PAR1 receptor density was found to be unaffected
AB
Figure 6. Gradient-triggered activation of the cAMP-dependent pathway controls signaling from stimulatory G protein-coupled receptors. Signaling pathways with a net stimulatory effect on platelet activation are colored red and signaling pathways with a net inhibitory effect are colored blue. (A) In the presence of high agonist concentration gradients, strong activation of multiple stimulatory pathways will produce Akt phosphorylation, which in itself results in inhibition of the cAMP depend- ent pathway by stimulation of PDE3 activity.39 (B) In the presence of low agonist concentration gradients, gradient-dependent activation of the cAMP-dependent path- way will counteract stimulatory signaling from G protein-coupled receptors (GPCR), resulting in a refractory state characterized by an absence of Akt phosphorylation but prominent VASP phosphorylation and non-responsiveness to high concentrations of agonists. As proof of the involvement of the cAMP-dependent pathway in gra- dient-dependent inhibition (GDI), we found that GDI was effectively shut off by inhibition of adenylyl cyclase with epinephrine, and partially reversed by inhibition of PKA with H89, while GDI was potentiated by stimulation of the cAMP-dependent pathway with PGI2 or milrinone.
in GDI-platelets (Online Supplementary Figure S9). Secondly, inhibiting dynamin had no effect on GDI (Online Supplementary Figure S10), excluding a role for clathrin-mediated receptor endocytosis. As additional lines of evidence supporting a conceptual distinction between GDI and desensitization, we present multiple observations strongly supporting a mechanistic link between GDI and activation of cAMP-dependent signal- ing (Figure 6). This link provides mechanistic insight into how GDI can be abolished by stimulation with inhibitors of adenylyl cyclase such as ADP or epinephrine, empha- sizing the important role of these agonists in GPCR signal- ing. The unexpected observation that inhibition of paracrine signaling potentiates GDI lends further support to our notion that GDI is mechanistically related to the
Table 1. Summary of measures characterizing gradient-dependent inhibition of G protein-coupled receptor-mediated platelet aggregation.
Agonist
PAR1-AP
+ inhibitors*
PAR4-AP
C (μM) agg
30 30
300
300 2
0.16§
ΔC (s) nres
C (μM) res
+ inhibitors* U46619†
CRP-XL
320 160
1280
640 640 1280
t (s) 50
151 60 77 ND
607 300
322 ND 485 NA ND 2‡
ADP
NA NA NA
Cagg: the minimal agonist concentrations required to induce strong aggregation (>65%) in all
tested samples (n≥5); ΔCnres: the highest agonist concentration gradient for which >75% of sam-
ples are unresponsive (maximal aggregation <25 %) to Cagg; t50: the shortest infusion time at
which ≥50% of the samples ceased to aggregate; Cres: the lowest agonist dose required to
induce strong activation (>65% aggregation) when added at an infusion time of 2 s in all sam-
ples (n>5) after rendering platelets unresponsive to Cagg added with the gradient ΔCnres. *For
PAR1 and PAR4, ΔCnres and t50 were also determined in the presence of P2Y1, P2Y12, and throm-
boxane synthesis inhibitors,shown here as‘+ inhibitors’.†t50 was not determined since GDI was
not observed for TPa in all experiments. ‡The Cres measurement for U46619 was calculated §
from experiments in which gradient dependent inhibition was observed. μg/mL.NA:not appli- cable; ND: not determined.
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