Red blood cell supernatant increases activation and agonist-induced reactivity of blood platelets

Abstract

Introduction: Transfusion of "older" packed red blood cells (PRBCs) in patients with cardiovascular disorders (CVD) may be associated with an increased risk of pro-thrombotic events, but the underlying mechanisms are poorly understood. We hypothesized that the PRBC supernatant can activate blood platelets due to hemolysis-induced oxidative stress.

Methods: Effects of the PRBC supernatants, and their filtrates (containing the soluble substances of molecular weight <10 kDa) prepared at day 1 and 42 of storage, from non-leukoreduced (D1 NLR, D42 NLR) and leukoreduced (D1 LR, D42 LR) PRBCs on PLT activation/reactivity and collagen-induced aggregation were measured by flow cytometry and turbidimetry, respectively.

Results: Supernatants display a stimulating effect on PLTs, which was manifested by a release of PLT-derived microparticles, generation of PLT aggregates, increased P-selectin expression on the membrane surface, and activation of integrin αIIbβ3. Moreover, supernatants interacted in a way that may be additive or synergistic with collagen or with ADP. The pre-storage LR did not affect the levels of PLT activation markers. The enhanced PLT activation was presumably mediated by free hemoglobin and/or the products of its breakdown, accumulating in the PRBC milieu, and their ability to trigger the ROS generation. Additionally, collagen-induced PLT aggregation was increased by low molecular weight substances possibly derived from the residual leukocytes and PLTs present in PRBCs.

Conclusion: Transfusion of aged PRBCs may result in the hyper-activity of PLTs, which, at least in part, could be a cause of transfusion-related thrombotic complications reported in CVD patients.

Keywords: Blood platelets; Cardiovascular complications; Flow cytometry; Platelet aggregation; Red blood cell; Transfusion.