Bioenergetic profiling of platelet mitochondria during storage: 4°C storage extends platelet mitochondrial function and viability.

BACKGROUND: Platelets (PLTs) are stored at room temperature (RT) to preserve in vivo circulation time, but PLT quality is degraded. The PLT storage lesion is mitigated by refrigeration, but questions remain regarding effects of cold storage (4°C) on mitochondrial function. Mitochondrial reactive oxygen species (ROS) generation may adversely affect PLT function and viability during storage, and refrigeration may mitigate these effects. STUDY DESIGN AND METHODS: PLTs were stored under two temperature conditions (RT, 20-24°C; or 4°C, 1-6°C) and four storage durations (baseline [BL] and Days 3, 5, and 7). Mitochondrial respiration and maximal oxygen utilization were assessed with high-resolution respirometry. Mitochondrial ROS generation was assessed using a superoxide stain. Rotational thromboelastometry (ROTEM) was performed at BL and on Day 5 to assess PLT function. Collagen-induced PLT aggregation was measured by impedance aggregometry. RESULTS: Mitochondrial ROS in 4°C-stored samples were lower compared to RT and retained a greater capacity to generate ROS after activation. Mitochondrial respiration and maximal mitochondrial utilization was conserved in PLTs stored at 4°C. ROTEM data demonstrated that net maximum clot firmness was higher in 4°C samples compared to RT and prevented fibrinolysis. The aggregation response to collagen was preserved in the 4°C samples versus RT-stored PLTs. Aggregation impairment correlated well with attenuated mitochondrial respiration and elevated production of intracellular mitochondrial ROS in the RT PLTs. CONCLUSION: Mitochondrial damage and ROS production may contribute to loss of PLT viability during storage, whereas cold storage is known to preserve PLT function. Here we demonstrate that 4°C storage results in less oxidant stress and preserves mitochondrial function and potential compared to RT.

Primary hemostatic capacity of whole blood: a comprehensive analysis of pathogen reduction and refrigeration effects over time.

BACKGROUND: Whole blood (WB) has been used in combat since World War I as it is readily available and replaces every element of shed blood. Component therapy has become standard; however, recent military successes with WB resuscitation have revived the debate regarding wider WB use. Characterization of optimal WB storage is needed. We hypothesized that refrigeration preserves WB function and that a pathogen reduction technology (PRT) based on riboflavin and ultraviolet light has no deleterious effect over 21 days of storage. STUDY DESIGN AND METHODS: WB units were stored for 21 days either at 4°C or 22°C. Half of each temperature group underwent PRT, yielding four final treatment groups (n = 8 each): CON 4 (WB at 4°C); CON 22 (WB at 22°C); PRT 4 (PRT WB at 4°C); and PRT 22 (PRT WB at 22°C). Testing was at baseline, Days 1-7, 10, 14, and 21. Assays included coagulation factors; platelet activation, aggregation, and adhesion; and thromboelastography (TEG). RESULTS: Prothrombin time (PT) and partial thromboplastin time increased over time; refrigeration attenuated the effects on PT (p ≤ 0.009). Aggregation decreased over time (p ≤ 0.001); losses were attenuated by refrigeration (p ≤ 0.001). Refrigeration preserved TEG parameters (p ≤ 0.001) and PRT 4 samples remained within normal limits throughout the study. Refrigeration in combination with PRT inhibited fibrinolysis (p ≤ 0.001) and microparticle formation (p ≤ 0.031). Cold storage increased shear-induced platelet aggregation and ristocetin-induced platelet agglutination (p ≥ 0.032), as well as GPIb-expressing platelets (p ≤ 0.009). CONCLUSION: The in vitro hemostatic function of WB is largely unaffected by PRT treatment and better preserved by cold storage over 21 days. Refrigerated PRT WB may be suitable for trauma resuscitation. Clinical studies are warranted.

ENDOTHELIUM-DERIVED INHIBITORS EFFICIENTLY ATTENUATE THE AGGREGATION AND ADHESION RESPONSES OF REFRIGERATED PLATELETS.

Refrigeration of platelets (4°C) provides the possibility of improving transfusion practice over the current standard-of-care, room temperature (RT) storage. However, the increased level of platelet activation observed at 4°C in vitro is cause for concern of uncontrolled thrombosis in vivo. In this study, we assessed the safety of 4°C-stored platelets by evaluating their response to physiologic inhibitors prostacyclin (PGI2) and nitric oxide (NO). Apheresis platelets were collected from healthy donors (n = 4) and tested on Day 1 (fresh) or Day 5 (RT- and 4°C-stored) after treatment with PGI2 and NO or not for: thrombin generation; factor V (FV) activity; intracellular free calcium, cAMP and cGMP; ATP release; TRAP-induced activation; aggregation to ADP, collagen, and TRAP, and adhesion to collagen under arterial flow. Data were analyzed using two-way ANOVA and post-hoc Tukey test for multiple comparisons, with significance set at P < 0.05. Treatment with inhibitors increased intracellular cAMP and cGMP levels in fresh and stored platelets. Thrombin generation was significantly accelerated in stored platelets consistent with increased factor V levels, PS exposure, CD62P expression, intracellular free calcium, and ATP release. While treatment with inhibitors did not attenuate thrombin generation in stored platelets, activation, aggregation, and adhesion responses were inhibited by both PGI2 and NO in 4°C-stored platelets. In contrast, though RT-stored platelets were activated, they did not adhere or aggregate in response to agonists. Thus, refrigerated platelets maintain their intracellular machinery, are responsive to agonists and platelet function inhibitors, and perform hemostatically better than RT-stored platelets.

Both acute delivery of and storage with magnesium sulfate promote cold-stored platelet aggregation and coagulation function.

BACKGROUND: The platelet storage lesion causes loss of function and viability over time. A new paradigm for platelet storage is desired to enable safer, more effective transfusions while reducing waste. We hypothesized that repletion of Mg, which is chelated by citrate anticoagulant, could reduce platelet storage lesion severity when given in conjunction with storage at a refrigerated temperature. METHODS: Apheresis platelet units were collected from healthy donors and stored at 22°C or 4°C. On Days 0, 2, 4, and 8, samples were collected for analyses of receptor-mediated aggregation, coagulation, adhesion to collagen under flow, and viability. In the first series, samples were given an acute dose of MgSO4 before testing; in the second series, storage bags were supplemented with 0-, 3-, or 6-mM MgSO4. RESULTS: Acutely delivered MgSO4 induced a more rapid coagulation time in apheresis platelets, further enhanced by storage at 4°C. Platelet adhesion to a collagen surface while exposed to arterial shear rates (920 s) was enhanced by MgSO4 supplementation-acute MgSO4 had a large effect on adhesion of fresh platelets, which diminished more rapidly in 22°C samples, while storage with MgSO4 showed significant benefits even out to Day 4 at both temperatures. Although 4°C storage improves the longevity of platelet aggregation responses to agonists, MgSO4 supplementation did not change those responses. CONCLUSION: Acute MgSO4 reduces clot time likely through the transient increase of free Ca. Limited differences between platelet function in acute delivery of and storage with MgSO4 diminish the possibility that Mg-induced metabolic inhibition of platelets synergizes with 4°C storage. Regardless, magnesium supplementation to platelets is an exciting possibility in transfusion because the adhesion response of 22°C-stored platelets on Day 4 is significantly enhanced when stored with 6-mM MgSO4.