Sodium–Calcium Exchanger Reverse Mode Sustains Dichotomous Ion Fluxes Required for Procoagulant COAT Platelet Formation

Abstract

Procoagulant collagen-and-thrombin (COAT)-activated platelets represent a subpopulation of activated platelets, which retain a coat of prohemostatic proteins and express phosphatidylserine on their surface. Dichotomous intracellular signaling generating procoagulant platelet activity instead of traditional aggregating endpoints is still not fully elucidated. It has been demonstrated that secondary messengers such as calcium and sodium play a critical role in platelet activation. Therefore, we developed a flow cytometric analysis to investigate intracellular ion fluxes simultaneously during generation of aggregating and procoagulant platelets. Human platelets were activated by convulxin-plus-thrombin. Cytosolic calcium, sodium, and potassium ion fluxes were visualized by specific ion probes and analyzed by flow cytometry. We observed high and prolonged intracellular calcium concentration, transient sodium increase, and fast potassium efflux in COAT platelets, whereas aggregating non-COAT platelets rapidly decreased their calcium content, maintaining higher cytosolic sodium, and experiencing lower and slower potassium depletion. Considering these antithetical patterns, we investigated the role of the sodium–calcium exchanger (NCX) during convulxin-plus-thrombin activation. NCX inhibitors, CBDMB and ORM-10103, dose-dependently reduced the global calcium mobilization induced by convulxin-plus-thrombin activation and dose-dependently prevented formation of procoagulant COAT platelets. Our data demonstrate that both NCX modes are used after convulxin-plus-thrombin-induced platelet activation. Non-COAT platelets use forward-mode NCX, thus pumping calcium out and moving sodium in, while COAT platelets rely on reverse NCX function, which pumps additional calcium into the cytosol, by extruding sodium. In conclusion, we described for the first time the critical and dichotomous role of NCX function during convulxin-plus-thrombin-induced platelet activation.

Characterization of Procoagulant COAT Platelets in Patients with Glanzmann Thrombasthenia

“…It has been described that procoagulant COAT platelets are generated in the order of 1–2 min after combined CVX-plus-THR activation [ 18 , 27 ]. Since GT patients revealed the ability to generate a higher proportion of procoagulant COAT platelets with a global higher calcium mobilization, we decided to better dissect the calcium kinetics in each GT patient individually with a special focus on this early time frame.…”

“…To this end, we used a flow-cytometry based approach to monitor ion fluxes (notably calcium, sodium, and potassium) in our three patients affected by GT. Moreover, flow cytometry allowed for the subsequent differentiation and selective analysis of procoagulant COAT platelets formed from non-COAT platelets [ 18 , 27 ].…”

“…An important pivot point that influences the generation of procoagulant COAT platelets is the high and sustained calcium level reached after combined activation by CVX-plus-THR [ 18 , 26 , 27 , 46 , 47 ]. Procoagulant platelets are generated in the time frame of about 1–2 min after strong activation [ 18 , 27 ]. For that reason, we focused our kinetics on the calcium mobilization within the first three minutes ( Figure 3 ).…”

“…Further detailed method is described in our previous publications [ 26 , 27 ]. In summary, PRP was adjusted to 30 × 10 9 /L in Tyrode’s buffer and was incubated in the dark with either Fluo-3 AM (final concentration 2 μM), ING-2 AM (7.5 μM) or IPG-2 AM (0.1 μM) at RT for 60 min.…”

High-Dose Epinephrine Enhances Platelet Aggregation at the Expense of Procoagulant Activity

“…To compare the reduction of procoagulant activity induced by the inhibition of the sodium-calcium exchanger (NCX) 28 with the EPI effect, we pre-treated platelet with NCX inhibitor CBDMB or its vehicle DMSO and we activated them with CVX-plus-THR in presence of increasing doses of EPI ( Figure 5). CBDMB significantly reduced the procoagulant response assessed by the expression of PS on the platelet surface.…”