Enhanced shear-induced platelet aggregation due to low-temperature storage

Platelets (PLTs) are transfused to prevent bleeding due to thrombocytopenia associated with hematologic malignancies or to manage severe blood loss during surgery or trauma. PLTs are stored at room temperature in gas-permeable bags with constant agitation for up to 5 days.1 Although millions of PLT transfusions are performed every year, supply does not match the demand. PLTs stored under current practices undergo a gradual decline in function and viability, which presumably is a result of progressive activation and an accumulation of deleterious metabolic byproducts.2,3 Other major problems associated with current storage techniques that limit the relatively short shelf life include viral and bacterial contamination despite improvements in bacterial detection and pathogen inactivation technologies.4,5 In principle, storage of PLTs under refrigeration (4°C), which is standard practice for red blood cells (RBCs), can overcome the problems associated with room temperature storage since refrigeration drastically impedes bacterial growth and reduces PLT metabolism, thus alleviating these aspects of the storage lesion.6 In addition, refrigeration would also simplify the storage and transportation of blood products in emergency use settings, such as military hospitals and civilian emergency departments, as only one storage technology would be needed for RBCs, PLTs, and thawed plasma. However, Murphy and Gardner in 19696,7 showed that the recovery and survival half-life of PLTs after 18 hours of storage at room temperature were similar to fresh PLTs at 55% and 4.0 days, respectively, while the corresponding values for storage at 4°C were 40% and 1.3 days. Several other studies have confirmed poor survival and half-life of refrigerated PLTs, leading to the current practice of storage at room temperature.8–10 PLTs stored for either short-term (1–4 hr) or long-term (2–14 days) at 4°C undergo a number of morphologic, biochemical, and functional changes collectively called the “cold storage lesion.”11 Exposure of PLTs to low temperature for 1 to 4 hours results in the loss of discoid shape due to the loss of circumferential microtubular rings around the periphery of disc-shaped PLTs12 and uncapping of actin filaments.13 Long-term refrigeration results in a number of progressive changes in PLTs: change in glycoprotein receptor (GP Ib and GP IIb/IIIa) levels,14 up regulation of PLT activation markers such as P-selectin and annexin V,15 changes in fluidity of the plasma membrane,16 altered responses to aggregating17 and disaggregating18 agents, increase in intracellular calcium concentration,19 and decreased adhesion to sub-endothelium in vivo.20 Upon transfusion, PLTs stored at 4°C for short and long term are cleared rapidly by macrophages and hepatocytes, respectively.21,22 The clearance processes are attributed to clustering and different degrees of desialylation of PLT receptor (GP) Ibα.21,23 While these studies have greatly improved our understanding of the effect of low temperature on PLT morphology and biochemistry, the effect on hemostatic function is still an unanswered question. In this article, we have examined the effect of long-term refrigeration on in vitro PLT hemostatic function under flow. PLTs are captured from flowing blood on to injured surfaces to form a hemostatic plug through a process initiated by the binding between PLT GP Ib-IX-V complex on the PLT surface and exposed von Willebrand factor (VWF) bound to the subendothelial matrix. After this initial PLT adhesion, aggregates form through the binding of GP IIb/IIIa to fibrinogen. We hypothesized that PLTs stored at low temperature for long periods (48 hr) will function differently from fresh PLTs under shear conditions. To test this hypothesis, we evaluated PLT aggregation and activation in vitro. Our results indicate that low-temperature-stored PLTs aggregate more than fresh PLTs under shear conditions but not under static conditions. Similar phenomena may modulate hemostatic function in vivo, in response to colder temperature exposure of PLTs in peripheral circulation, as others have suggested.22