Characterization of Shear Stress Mediated Platelet Dysfunction: Data from an Ex Vivo Model for Extracorporeal Circulation and a Prospective Clinical Study.

Extracorporeal circulation (ECC) is frequently used in intensive care patients with impaired lung or cardiac function. Despite being a life-saving therapeutic option, ECC is associated with increased risk for both bleeding and thrombosis. The management of bleeding and thromboembolic events in ECC patients is still challenging partly due to the lack of information on the pathophysiological changes in hemostasis and platelet function during the procedure. Using a combination of an ex vivo model for shear stress and a sensitive and easy-to-use laboratory method, we analyzed platelet responsiveness during ECC. After shear stress simulation in an ex vivo closed-loop ECC model, we found a significantly decreased response of α-granules after activation with adenosine diphosphate and thrombin receptor activating peptide (TRAP-6) and CD63 expression after activation with TRAP-6. Mepacrine uptake was also significantly reduced in the ex vivo shear stress model.In the same line, platelets from patients under ECC with venovenous systems and venoarterial systems showed impaired CD62P degranulation after stimulation with ADP and TRAP-6 compared with healthy control on day 1, 6, and 10 after implantation of ECC. However, no correlation between platelet degranulation and the occurrence of bleeding or thromboembolic events was observed.The used whole blood flow cytometry with immediate fixation after drawing introduces a sensitive and easy-to-use method to determine platelet activation status and our data confirm that increased shear stress conditions under ECC can cause impaired degranulation of platelet.

Blood donor-derived buffy coat to produce platelets in vitro.

BACKGROUND AND OBJECTIVES: Platelet transfusion is a standard medical therapy used to treat several bleeding disorders. However, a critical drawback is the dependency on donor-derived platelets, which leads to concerns like insufficient availability and immunological complications. In vitro platelet production from hematopoietic progenitor cells (CD34) may represent a reasonable solution. MATERIALS AND METHODS: CD34+ cells were isolated from either buffy coat or peripheral blood and compared in terms of platelet production in vitro. The number and the quality of magnetically isolated CD34+ cells and their capability to differentiate into mature megakaryocytes were investigated using flow cytometry. Additionally, the functionality of megakaryocytes in term of in vitro platelet production was tested. RESULTS: Similar purity and quantity of CD34+ cells was found after their isolation from both cell sources. In contrast, after 6 days of culture, enhanced number of CD34+ cells isolated from buffy coat compared with peripheral blood was observed (5·3 x 106 vs. 3·0 x 106, respectively). Interestingly, despite a comparable nuclear maturation phenotype, the yield of platelets released from buffy coat-derived megakaryocytes was significantly higher than from peripheral blood cells (platelet yield pro MK: 7·2 vs. 2·7, respectively). Importantly, platelets produced from buffy coat-derived cells could be activated by agonists. CONCLUSION: Haematopoietic progenitor cells isolated from buffy coat have increased yield of platelets released from mature megakaryocytes and enhanced in vitro functionality, compared with peripheral blood-derived cells. Our study, suggests that buffy coat, obtained during blood donation processing, might be a promising source of megakaryocytes for in vitro platelet production.