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Gradient-dependent inhibition of GPCR
tain agonist and individual-specific threshold resulting in a complete inhibition of aggregation (Figure 1B,C). For example, in the case of PAR4-AP, the maximum aggrega- tion observed was either below 20% or above 60%, but not in between; in other words, the aggregation response was “on or off”. To account for this phenomenon, we used logistic regression to model the effects of GDI for each agonist, using a threshold of 25% reduction in absorbance to discriminate between “aggregation” and “no aggrega- tion” (Figure 1D, Table 1). In this model, we defined the measure t50 as the minimal infusion time at which ≥50% of the samples ceased to aggregate. Aggregation induced by PAR1-AP was most sensitive to GDI, with a t50 of 151 s, whereas the effects of GDI on the aggregatory response to stimulation with PAR4-AP required significantly longer infusion times, with a t50 of 607 s.
To investigate the impact of GDI on thrombin-induced platelet activation, we performed infusion experiments with thrombin (final concentration 1 U/mL) in citrated platelet-rich plasma treated with Gly-Pro-Arg-Pro (GPRP) to prevent fibrin polymerization (Figure 2A,B). Our results show that GDI has dramatic effects on the platelet aggre- gatory response to thrombin stimulation even at short infusion times, with almost complete inhibition at an infu- sion time of 80 s. The difference in the effect distribution of GDI between PAR and ADP receptors is illustrated by a comparison of the representative curves for thrombin (Figure 2A) with those for ADP (Figure 2C), showing a bimodal effect distribution for thrombin whereas GDI increases incrementally over a large range of gradients for ADP. Results obtained with thrombin were further con- firmed by simultaneous infusion of PAR1-AP and PAR4-
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G
Figure 2. Characterization of gradient-dependent inhibition using thrombin, ADP, paracrine signaling inhibition and α-granule secretion. (A,B) To quantify the effects of gradient-dependent inhibition (GDI) on platelet aggregation induced by thrombin, light transmission aggregometry was performed on platelet-rich plasma pre-incu- bated with 4 mM GPRP to prevent fibrin polymerization. Thrombin (1 U/mL) was added with different infusion times as indicated, n≥3. (C) Platelet aggregation induced by ADP added with different infusion times as indicated. (D-F) The role of paracrine stimulation in GDI of PAR signaling was quantified by performing light transmission aggregometry on platelet-rich plasma in the presence of P2Y1, P2Y12 and thromboxane synthesis inhibitors (MRS2179, cangrelor and ASA), using the agonists PAR1-AP (30 μM) and PAR4-AP (300 μM), n≥3 (G). The effects of GDI on a-granule release were analyzed by measuring the percentage of platelets positive for CD62P (P-selectin), using flow cytometry, at different infusion rates, n ≥5. Data represent mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001
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