Enhanced shear-induced platelet aggregation due to low temperature storage

Platelets, though miniscule in size (∼2--5 microm) are vital to survival. Anucleate cell fragments of hematopoietic progenitors, megakaryocytes, platelets circulate in numbers of 150,000--400,000 /mul of blood with an average lifespan of 7--10 days [1]. As first responders to sites of vascular damage and initiators of clot formation, their function is crucial in hemostasis. Platelets circulate in a resting state and upon exposure to stimuli which occur in vascular injury, platelets get activated and secrete soluble factors, including ADP, thrombin and thromboxane A2 (TxA 2). These activate neighboring platelets in a paracrine fashion resulting in aggregation and clot formation.

A number of medical conditions in which platelet counts go down to as low as 10,000 /mul, are treated with platelet transfusion. These include cardiovascular surgery (particularly when bypass pumps are used, hematologic abnormalities, intensive cancer therapies, surgery or trauma [2]. Platelets for transfusion are stored in gas-permeable bags with constant agitation for up to 5 days [3]. Although currently used platelet storage techniques have facilitated millions of transfusions every year, these banking conditions are far from ideal. The problems associated with the current storage methods are viral and bacterial contamination, loss in platelet functionality, and the inability to match supply with demand. Low temperature storage of platelets (4°C) could potentially mitigate some of these issues [4]. However, low temperature storage produces a number of physical effects on platelets which in turn could affect their function. Further, understanding the mechanisms of chilling on platelet structure and function can help in overcoming the disadvantages associated with refrigeration. In this study, we compared shear-induced aggregation in platelets stored for 2 or 7 days at either 4°Cor 22°C with that of fresh platelets.

Washed platelets, either fresh or stored for 2 or 7 days at room temperature (RT) or refrigerated (4°C) without agitation, were mixed with 5 mug/ml VWF and 2 mg/ml fibrinogen and subjected to a shear rate of 0, 500s -1, 2500s-1 or 10000s-1 for 2 min at 37°C in a cone-and-plate viscometer. As expected, platelet aggregation increased with increasing shear in both fresh and stored samples. Surprisingly, at 10,000s-1 4°C-stored platelets showed significantly enhanced aggregation (8.46-fold over static) compared to room temperature storage (3.79-fold) or fresh platelets (3.78-fold) (P < 0.004, n = 9). Such enhancement was maintained even when the cell concentration and viscosity were varied. However, compared to fresh or RT-stored platelets, 4°C-stored platelets showed lower GP Ib expression levels when measured by the binding of AK2, SZ2 and VM16d antibodies. We found that 4°C-stored platelets are more activated by shear stress and also bind more VWF. We speculate that the increased binding of VWF could be due to cold-induced clustering of GP Ib on platelet surfaces.

We also observed that when platelets were stored for 7 days, both RT-stored and 4°C-stored platelets are equally less responsive to shear-induced aggregation, but 4°C-stored platelets display better GP Ib receptor expression levels. However, the platelets were highly activated as indicated by increased P-selectin levels.