Cold Storage Of Platelets Research Articles

Hepatocytes Ingest Longterm Refrigerated Platelets: A Novel Platelet Clearance Mechanism.

We previously reported that the lectin domain of αMβ2 receptors on hepatic macrophages mediates rapid clearance of washed murine platelets transfused after refrigeration for 2 hours, recognizing exposed βN-acetylglucosamine (βGlcNAc) residues of N-linked glycans on clustered platelet GPIbα molecules and that the same receptors elicit phagocytosis of refrigerated human platelets human macrophages in vitro. A platelet-associated galactosyltransferase catalyzes the covering of βGlcNAc residues with galactose in the presence of UDP-galactose, thereby blocking clearance of cold mouse platelets in vivo and phagocytosis of human platelets in vitro. These intriguing findings contradicted earlier evidence that refrigeration of human platelets procured for transfusion only promotes their rapid clearance after prolonged (>8h) incubation and also are inconsistent with the well-known recognition system for exposed galactose residues through asialoglycoprotein (ASGP) receptors. Reconciling these contradictions, we report that the absence of plasma during storage accounts for the differences in time of exposure to cold to promote clearance and that mouse platelets cold-stored in plasma also only clear rapidly after long-term (48h) storage. We also found that hepatic clearance of long-term cold-stored (LTCS) mouse platelets occurs in hepatocytes. Streptavidin-POD staining revealed abundant LTCS biotinylated platelets in hepatocyte phagosomes. Furthermore, cells of the hepatocyte HepG2 line avidly ingest fluorescently-labeled LTCS human platelets (7-fold above the baseline of room-temperature-stored platelets), as evidenced by flow cytometry, fluorescent microscopy and by time-laps video microscopy. Long-term cold storage increases by ~1.7-fold platelet binding of the galactose-specific lectin RCA I, implying that with long-term cold storage, exposed galactose residues cluster sufficiently to induce recognition by hepatocyte ASGPR receptors. The results define a new clearance mechanism, representing the first example of blood cell removal by a non-myeloid cell. Since we find that human platelets also express a cell surface sialotransferase that adds sialic acid to galactose residues, we suggest that a combination of sialylation and glactosylation, achievable by addition of sugar substrates alone, might accommodate long-term cold storage of platelets for transfusion.

An Externally Active Endogenous Platelet Galactosyl Trasferase (Gal-T).

Over 30 years ago, Jamison and Barber proposed that externally disposed glycosyl transferase (GT) activity mediates platelet adhesion and other functions. Subsequent work ruled out ecto-GT activity in nucleated cells and established the Golgi as the site of such enzymes, although no further studies examined platelets. We recently reported a platelet-associated galactosyl transferase (β4Gal-T) catalyzing galactose coupling in a β1,4 linkage to exposed N-acetylglucosamine (GlcNAc) residues on N-linked glycans of the GPIbα subunit of the von Willebrand factor receptor complex. This activity, also present in plasma, mediates galactose transfer to GPIbα following addition of UDP-galactose (UDP-Gal) alone. Since galactosylation of GPIbα prevents clearance of chilled platelets by liver macrophages after transfusion, sufficiently active endogenous endogenous β4Gal-T could facilitate platelet cold storage after the mere addition of substrate UDP-Gal (Science 301: 1531–1534, 2003). We now give evidence that human platelets contain a highly active (0.05 nmol/min) plasma membrane-associated ecto-β4Gal-T with properties of an isoform designated as type 1 (β4Gal-T1). A specific β4Gal-T1 antibody immunoprecipitates β4Gal-T1 activity from platelet lysates and the inhibition profile activity by α-lactalbumin and benzyl-β-D-GlcNAc are characteristic of β4Gal-T1. β4Gal-T1 has a transmembrane domain, and platelet β4Gal-T, although releasable by non-ionic detergent, remains cell- and membrane associated despite extensive washing. The platelet enzyme incorporates UDP-[C14]Gal into a single polypeptide with the apparent Mr of GPIbα, identifying this protein as its unique endogenous target. Nevertheless, washed platelets are as active as recombinant β4Gal-T1 in galactosylating exogenous substrates, benzyl-β-D-GlcNAc and ovalbumin. In conclusion, we have revived the concept of an ecto-glycosyl transferase on blood platelets and demonstrated that platelet β4Gal-T is probably sufficiently active to accommodate reliable enzymatic conversion of platelets by simple addition of UDP-Gal.

Novel platelet products, substitutes and alternatives

Despite the many advances in the safety, processing, and storage of conventional 22 °C liquid-stored allogeneic platelet concentrates, there still are significant drawbacks to the use of such products. Efforts to overcome these shortcomings have resulted in an array of novel platelet products, substitutes, and alternatives; which are currently at various stages of development. This review summarizes the recent developments in the frozen and cold storage of platelets; their pathogen inactivation; as well as the status of lyophilized platelets, infusible platelet membranes (IPMs), red cells bearing arginine–glycine–aspartic acid (RGD) ligands, fibrinogen-coated albumin microcapsules, and liposome-based agents; as potential alternatives to the use of conventional platelet transfusions. Pre-clinical studies have been encouraging for several of these novel products; however, to date, very few have entered human trials. Nonetheless, with the ongoing development of diverse products, those properties that may be necessary for their hemostatic effectiveness will become apparent. However, safety and efficacy must be demonstrated in pre-clinical and phase I to III clinical trials before these novel agents, substitutes and alternatives can be used clinically for patients with thrombocytopenia.

Animal models for studies on cold-induced platelet activation in human beings

When human platelets are chilled below about 20°C, they spontaneously activate, a phenomenon that limits their storage lifetime. We have previously shown that this activation in chilled human platelets is associated with passage through a lipid phase transition. Because animal models are necessary for investigating methods for cold storage of platelets, it is essential to determine whether such phase transitions and chilling-induced activation are found in these models. In this study we examined platelets from some commonly used animal models—pigs, rhesus monkeys, mice, dogs, and rabbits. Using Fourier transform infrared spectroscopy (FTIR), we detected the thermotropic membrane phase transition in intact platelets and assessed the morphologic response of the platelets to chilling. Statistical analysis of both FTIR and shape change show that of the animal models tested, pig platelets are most similar to human platelets. These studies suggest that pigs and pig platelets are the models of choice for the study of cold-induced platelet activation in human beings. (J Lab Clin Med 2000;135:339-46)

This month in J Lab Clin Med: Issue highlights for April 2000

This month in J Lab Clin Med: Issue highlights for April 2000

Liquid cold storage of platelets: A revitalized possible alternative for limiting bacterial contamination of platelet products

ACTERIAL CONTAMINATION of blood products is a recurring problem that can pose a significant risk to the transfusion recipient. With the advent of the closed system for platelet apheresis collections, the cause for concern has decreased, 1 but it has not been completely alleviated. For platelet products, bacterial contamination is especially problematic because they are stored in plasma at room temperature where bacterial pathogens can rapidly multiply to produce a significant bioburden. This was highlighted in 1986 when a group of cases of platelet transfusion-associated bacteremia was reported, and the storage period, which previously had been increased to 7 days, was reduced to 5 days to limit the time for bacterial growth in inadvertently contaminated units. 2 This has not fully eliminated the problem, and in 1990, the Food and Drug Administration received reports of six deaths as a result of bacterial sepsis; five were caused by contaminated platelets and one was caused by contaminated red blood cells) In addition, there may be the occurrence of nonfatal sepsis in as many as 1 in 1,700 transfusions of pooled platelet concentrates, 1 making platelet transfusionassociated bacteremia the most common transfusion-associated infection challenge today. 4 A simple way to decrease the growth of bacteria in stored platelets would be to store them at 4~ which retards the growth of most strains of bacteria. Although these conditions were easily adapted for storage of red blood cells, platelets were found to be significantly and irreversibly affected by exposure to cold temperatures. The coldqnduced changes negatively impact on in vivo platelet recovery and survival. It was for this reason that cold storage of platelets was abandoned. Recent

Liquid cold storage of platelets: A revitalized possible alternative for limiting bacterial contamination of platelet products

Liquid cold storage of platelets: A revitalized possible alternative for limiting bacterial contamination of platelet products

Treatment of Refractory Ventricular Tachycardia and Fibrillation by the Administration of Potassium and Quinidine: Report of a Case

Platelet transfusions are a life-saving medical intervention used for the treatment of thrombocytopenia or hemorrhage. Extensive research has gone into trying to understand how to store platelets prior to the transfusion event. Much has been learned about storage bag materials, synthetic solutions, and how temperature impacts platelet viability and function. While room temperature storage of platelets preserves 24-hour in vivo platelet recovery and survival there is a greater risk for bacterial growth. Therefore, cold storage of platelets has become attractive due to the reduction in potential bacterial proliferation and the maintenance of platelet function beyond 5 days of storage. Cold stored platelets, however, have their own set of challenges. Cold stored platelets become activated through several mechanisms. The morphological and molecular changes that occur due to cold exposure enhance their ability to participate in the hemostatic process at the cost of rapid clearance from circulation. This review focuses on the underlying mechanisms leading to cold platelet activation and the receptor modifications involved in platelet clearance.