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A.S. Macwan et al.
AP to mimic the composite stimulus of PAR obtained with thrombin (Online Supplementary Figure S4).
The secondary mediators ADP and thromboxane A2 play an important role in amplifying stimulatory signaling from PAR receptors and have previously been shown to be sus- ceptible to desensitization.11-13 To assess how ADP and thromboxane A2 affect GDI of PAR receptor-mediated platelet activation, we performed additional experiments in which PAR-APs were infused in the presence of inhibitors of P2Y1, P2Y12, and thromboxane synthesis. Surprisingly, the effects of GDI on PAR receptor-mediated platelet activation was enhanced in the absence of paracrine signaling, as the t50 wasreducedfrom151sto77sandfrom607to320sfor PAR1 and PAR4, respectively (Figure 2D-F).
Gradient-dependent inhibition of platelet alpha-granule exocytosis and intracellular calcium mobilization
To test whether GDI was a general feature of stimulato- ry GPCR signaling and not restricted to platelet aggrega- tion, we investigated the gradient-dependent effects on a- granule exocytosis, measured by flow cytometry as P- selectin exposure (Fig. 2G). Using the highest concentra- tion gradient with an infusion time of 2 s, all agonists, except ADP, induced strong P-selectin expression (>80%), in accordance with in vitro observations by other groups showing that stimulation with ADP is not sufficient to evoke a robust paracrine response in platelets.14-16 Interestingly, a striking difference was observed in the effects of GDI between PAR4 and PAR1-mediated activa- tion, as PAR4-AP continued to produce a virtually intact P- selectin exposure (>90 %) until the gradient was lowered to an infusion time of 640 s, whereas GDI of PAR1-AP- induced P-selectin exposure was evident already when using the 80 s infusion time (Figure 2G). In line with the results from the aggregometry experiments described above, glycoprotein VI (GPVI)-mediated platelet activa- tion by CRP-XL showed no signs of GDI, producing a high P-selectin exposure that remained >80% even at the lowest concentration gradient tested (infusion time 1,280 s). Although the large inter-individual differences observed for U46619 in the aggregometry experiments were still evident to some extent, the effects of GDI on a- granule release were evident at longer infusion times, i.e., 640 s and 1,280 s.
While the effects of GDI on GPCR signaling were prominent also when measuring platelet intracellular cal- cium concentrations (Online Supplementary Figure S5), a comparative quantitative analysis of GDI was not feasi- ble because of differences in the ability of each receptor to generate a robust calcium response when exposed to a high agonist concentration gradient (2 s infusion time). However, a transient and immediate calcium “spike” of progressively smaller amplitude was obtained for PAR1- AP and ADP even with medium and low agonist gradi- ents, whereas longer infusion times generated prolonged calcium mobilization with a temporal shift in [Ca2+]max for PAR4-AP and U46619. To examine whether this phe- nomenon was a unique feature of platelets or whether it could be generalized to other cell types, we character- ized the effects of GDI on PAR1 signaling in epithelial cells, revealing calcium transients similar to those observed in platelets, with diminishing calcium mobi- lization with increasing infusion times (Online Supplementary Figure S6).
Gradient-dependent inhibition of G protein-coupled receptor-signaling leads to different levels of refractoriness to subsequent stimulation
With the presence of a gradient-dependent mechanism for platelet activation verified in the above experiments, we asked to what extent the unresponsive state induced by low agonist gradients made platelets refractory to subsequent stimulation with high gradients of the same agonist. To answer this question, we performed experi- ments on platelets that had been rendered unresponsive to Cagg added with the concentration gradient ΔCnres (here- inafter called GDI-platelets). We defined Cres as the min- imal concentration required to achieve aggregation as a response to instantaneous (2 s infusion time) addition of the same agonist in GDI-platelets (algorithm in Figure 3A). As shown in Figure 3B and Table 1, GDI-platelets could be activated by immediate addition of Cagg x 2 for PAR1-AP and Cagg x 1 for PAR4-AP, clearly demonstrating that GDI did not render platelets refractory to subse- quent stimulation with the same agonist. In contrast, for ADP, GDI induced a state of pronounced unresponsive- ness to subsequent activation, as we were unable to identify a concentration of ADP that could induce platelet aggregation in GDI-platelets, even when reach- ing concentrations exceeding 20 x Cagg, a result consistent with previous findings.11,12 Additional experiments shown in Online Supplementary Figure S7 demonstrate that GDI is strictly agonist-specific, as the aggregatory response to heterologous stimulation of GDI-platelets with another agonist (e.g. PAR4, ADP or U46619 in the case of PAR1-induced GDI) was identical to that of untreated platelets.
Platelet activation via PAR1 and PAR4 is gradient-dependent and not concentration-dependent
To confirm that the determinant of the aggregatory response to the instantaneous addition of Cres was the ago- nist concentration gradient and not the final agonist con- centration, we investigated whether adding Cres with the gradient ΔCnres to GDI-platelets could elicit the same aggre- gation response as adding Cres instantaneously (Figure 3C). In these experiments, GDI-platelets were exposed to either instantaneous or prolonged gradient infusion to reach the final concentration Cres. In contrast to the 2 s infusion, no aggregation was observed when adding Cres with the ΔCnres gradient using the agonists for which ΔCnres and Cres could be defined (PAR1-AP and PAR4-AP). These results show that the platelet response to these agonists is independent of the final agonist concentration but highly dependent on the agonist concentration gradient.
Gradient-dependent inhibition is regulated by a cAMP- dependent pathway
A comparison of total serine phosphorylation levels in GDI-platelets with those of resting and activated platelets for the agonists PAR1-AP, PAR4-AP and ADP (Online Supplementary Figure S8) showed that GDI involves specif- ic phosphorylation events which are not observed in either resting or activated platelets. In further explorations into the mechanisms involved in GDI, we used PAR1 as the model receptor as it was the receptor most prominent- ly affected by GDI in our study. Since receptor internaliza- tion has been reported as a common mechanism of desen- sitization for GPCR,17-20 we compared platelet PAR1 recep- tor density in resting platelets and GDI-platelets using
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haematologica | 2019; 104(7)