Human mesenchymal stromal cells inhibit platelet activation and aggregation involving CD73-converted adenosine

Background: Mesenchymal stromal cells (MSCs) are promising cell therapy candidates. Clinical application is considered safe. However, minor side effects have included thromboembolism and instant blood-mediated inflammatory reactions suggesting an effect of MSC infusion on hemostasis. Previous studies focusing on plasmatic coagulation as a secondary hemostasis step detected both procoagulatory and anticoagulatory activities of MSCs. We now focus on primary hemostasis and analyzed whether MSCs can promote or inhibit platelet activation. Methods: Effects of MSCs and MSC supernatant on platelet activation and function were studied using flow cytometry and further platelet function analyses. MSCs from bone marrow (BM), lipoaspirate (LA) and cord blood (CB) were compared to human umbilical vein endothelial cells or HeLa tumor cells as inhibitory or activating cells, respectively. Results: BM-MSCs and LA-MSCs inhibited activation and aggregation of stimulated platelets independent of the agonist used. This inhibitory effect was confirmed in diagnostic point-of-care platelet function analyses in platelet-rich plasma and whole blood. Using inhibitors of the CD39–CD73–adenosine axis, we showed that adenosine produced by CD73 ectonucleotidase activity was largely responsible for the LA-MSC and BM-MSC platelet inhibitory action. With CB-MSCs, batch-dependent responses were obvious, with some batches exerting inhibition and others lacking this effect. Conclusions: Studies focusing on plasmatic coagulation suggested both procoagulatory and anticoagulatory activities of MSCs. We now show that MSCs can, dependent on their tissue origin, inhibit platelet activation involving adenosine converted from adenosine monophosphate by CD73 ectonucleotidase activity. These data may have strong implications for safety and risk/benefit assessment regarding MSCs from different tissue sources and may help to explain the tissue protective mode of action of MSCs. The adenosinergic pathway emerges as a key mechanism by which MSCs exert hemostatic and immunomodulatory functions

Additional file 2: of Human mesenchymal stromal cells inhibit platelet activation and aggregation involving CD73-converted adenosine

Figure S1. Effect of MSCs on platelet adhesion and aggregation under shear flow conditions. To assess effect of MSCs on platelet activation under shear flow conditions, we performed microfluidic experiments using a pneumatically driven channel system (BioFlux, San Francisco, CA, USA) mounted on an inverted microscope capable of live cell reflectance interference contrast microscopy (RICM) as described previously [31]. Briefly, channels were coated with 10 μg/cm2 fibronectin (from human plasma F2006; Sigma Aldrich, St. Louis, MO, USA). The coated channels were filled with 300 μl of native whole blood with and without 1.5 × 105 BM-MSCs upon hematocrit adjustment and perfused with a constant shear stress of 5 dyne/cm2. At indicated points in time, RICM photographs of channel footprints were taken and analyzed by counting the number of adherent/aggregated platelets. BM-MSCs n = 3. (TIF 1135 kb

Additional file 5: of Human mesenchymal stromal cells inhibit platelet activation and aggregation involving CD73-converted adenosine

Figure S4. Effect of MSCs on platelet activation using different agonists and pathway inhibitors. A, B Effect of 105 LA-MSCs/ml on platelet activation after stimulation with different agonists ADP, TRAP-6 and U46619 (n = 4). Expression of two different activation markers shown: A CD62P and B PAC-1 binding. *p < 0.05. C, D Effect of AK4 and indomethacin on platelet inhibition by 5 × 105 BM-MSCs/ml. Platelets stimulated with TRAP-6. x axis, PAC-1 fluorescence intensity; y, axis, platelet count. One of two experiments shown: C AK4 to block CD62P and D MSC preculture in indomethacin to block COX. (TIF 176 kb