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Gradient-dependent inhibition of GPCR
Vascular damage is associated with a localized rapid increase in the concentrations of soluble agonists acting on platelet stimulatory GPCR. By contrast, concentrations of such agonists outside the core of a forming hemostatic plug change slowly due to dilution, mechanically restrict- ed diffusion and agonist degradation.3 Recent studies of intra-thrombus architecture have shown that spatial dif- ferences in thrombus porosity result in distinct diffusion rates of solutes,4 leading to heterogeneous concentration gradients of soluble agonists in different regions inside and outside a developing thrombus. In pathological conditions that affect thrombus consolidation and contraction, diffu- sion of soluble agonists to regions outside the thrombus core is increased,5 resulting in altered spatial and temporal distributions of agonists.
In this study, we hypothesized the presence of a gradi- ent-dependent gating mechanism for platelet activation by soluble agonists. Gradient-sensing mechanisms are used in other cell types to regulate dynamic and complex cellu- lar processes such as chemotaxis,6,7 and can be predicted to enhance the information processing ability of cells in rela- tion to changes in the ambient stimulation level.8,9 For platelets, gradient-sensing could hypothetically enable dynamic modification of hemostatic responses according to the type of precipitating event and the relative position of a platelet in a developing thrombus. Gradient-depen- dent activation could ensure a robust activation response under conditions of rapidly increasing agonist concentra- tions, such as those encountered when a platelet is recruit- ed from the blood stream to the core regions of a hemo- static plug. At the other end of the spectrum, gradient- dependent activation could also provide a mechanism for ensuring relative inertia in the face of a slow rise of agonist concentrations, as exemplified by platelets attaching to the peripheral shell regions of a consolidating thrombus.10 Such a mechanism could conceivably be of particular importance for regulating the platelet response to throm- bin stimulation via the protease-activated receptors (PAR1 and PAR4), since one thrombin molecule is capable of irre- versibly activating an indeterminate number of PAR recep- tors by enzymatic receptor cleavage. Gradient-dependent modulation of PAR signaling could thus constitute a previ- ously unidentified mechanism for equilibrating a signaling machinery otherwise inherently tilted towards unchecked platelet activation.
To test our hypothesis, we used novel instrumental setups to continuously monitor the platelet response to temporal agonist gradients (Online Supplementary Figure S1), enabling us to verify the presence of a mechanism for gradient-dependent inhibition (GDI) of platelet activation involving activation of cyclic adenosine monophosphate (cAMP)-dependent signaling mechanisms.
Methods
Blood collection and sample preparation
Whole blood from healthy adult volunteers was collected into tubes containing hirudin, sodium citrate or acid-citrate-dextrose as per the local Ethics Committee of Linköping University Hospital and platelet-rich plasma or washed platelets were prepared using standard procedures as described in the Online Supplement.
Light transmission aggregometry
Platelet aggregation was measured by light transmission
aggregometry using a Chronolog Corporation model 490-X, Haverton, USA aggregometer. A pump controlled agonist infu- sion system (Online Supplementary Figure S1) was developed to generate constant temporal agonist concentration gradients and allow for continuous monitoring of platelet aggregation. In this system, 1 mL disposable plastic syringes were used in the syringe pumps and connected by fine tubing, of which the other end was directly immersed (~3 mm) into the platelet-rich plas- ma in the aggregometer cuvette via a custom-made cuvette adapter. A Matlab (MathWorks, Natick, USA) program was cre- ated in-house for controlling infusion rates and agonist loading (Online Supplementary Figure S1D). Predefined algorithms were followed for the parameters in the aggregometry experiments (Figures 1A, 3A and Online Supplementary Figure S2A) to avoid the potential for bias associated with the ad hoc experimental design. Based on that, aggregation was measured after infusing the same volume and concentration of agonists for 2, 40, 80, 160, 320, 640 or 1,280 s. The details of the experimental conditions, including the use of various inhibitors and the stability of all the agonists used in the study under experimental conditions, are described in the Online Supplement.
Flow cytometry
Western blotting
Levels of total serine phosphorylation, total and phosphory- lated VASP (at S-157) or total and phosphorylated AKT (at S-473) were assessed by western blotting using standard procedures as described in the Online Supplement.
Fluorescence microscopy
Resting platelets, platelets activated by PAR1 activating pep- tide (PAR1-AP) and platelets with induced GDI were visualized by fluorescence microscopy after staining F-actin according to the manufacturer’s protocol, as described in the Online Supplement.
Electron microscopy
Transmission electron microscopy was used to visualize sub- cellular differences between resting, activated platelets and platelets with induced GDI, as described in the Online Supplement.
Results
A gradient-dependent mechanism modulates G protein-coupled receptor-mediated platelet activation
The minimal agonist concentration (Cagg) required to induce strong aggregation (>65%) in all samples (n≥5) with an infusion time of 2 s was determined (Table 1) using the algorithm shown in Online Supplementary Figure S2A (results in Online Supplementary Figure S2B). To verify the presence of GDI, we then sought to identify the high- est concentration gradient (ΔCnres) at which no significant aggregation (<25%) was observed in ≥75% of samples at a final agonist concentration of Cagg (algorithm in Figure
haematologica | 2019; 104(7)
The effect of agonist gradients on platelet a-granule release was assessed by taking aliquots from samples identical to those used in the aggregometry experiments except for the inclusion of a step in which samples were pre-incubated with 1 μM tirofiban for 10 min at room temperature to prevent aggregation. Samples were collected 1 min after completion of agonist infu- sion, labeled and analyzed by flow cytometry as described in the Online Supplement.
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