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 Jaroslav G. Vostal and Traci Heath Mondoro

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

B

From the Laboratory of Cellular Hematology, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD. Address reprint requests to Jaroslav G. Vestal, MD, PhD, Division of Hematology, CBER-FDA, Bldg 29, Rm 323, HFM335, 8800 Rockville Pike, Bethesda, MD 20892. This is a US government work. There are no restrictions on its l~Se. 0887-7963/97/1104-000550.00/0 286

advances in understanding the mechanisms of cold temperature effects on platelets have led to novel strategies to prevent their occurrence and have again raised the possibility of an effective liquid cold storage of platelets. This article will summarize the effects of cold temperature on platelets and the mechanisms of cold-induced platelet responses. The article will also discuss methods that may be used for prevention of cold-induced platelet activation that provide the potential for development of platelet cold storage. EFFECTS OF COLD TEMPERATURE ON PLATELETS

In Vivo Studies of Cold-treated Platelets There have been few studies involving transfusion of cold-treated platelets in vivo. The studies that have been done show decreased circulation time of platelets stored at refrigerator temperatures for relatively short periods of time. Transfusion of thrombocytopenic patients 5 and aspirin-treated normal subjects 5,6 with platelets stored at temperatures of 0~ to 4~ for 24 to 72 hours showed correction of prolonged bleeding time almost immediately on transfusion, even more rapidly than observed with room temperature-stored platelets. However, although the recovery of 24-hour cold-stored versus room temperature-stored platelets was fairly similar (51% v 65% respectively), the survival of cold-stored versus room temperature-stored was significantly different (2 days v 8 days respectively). 7 In addition, platelets stored in the cold for up to 24 hours were found to be effective in correcting bleeding times in thrombocytopenic patients, but cold storage of platelets for longer periods made them ineffective) These finding are in accord with the earlier observations of Murphy and Gardner 9 who found that, after 8 hours of storage at 4~ there was a 27% reduction in platelet life span versus a 14% reduction after room temperature storage. 9

Morphological and Cytoskeletal Alterations Cold storage affects virtually every aspect of platelet morphology and physiology (Table 1).

Transfusion Medicine Reviews, Vol 11, No 4 (October), 1997: pp 286-295

LIQUID COLD STORAGE OF PLATELETS Table 1. Effects of Cold Exposure on Liquid-Stored Platelets

Morphology Sphering with increased volume Pseudopod formation Microparticle formation Loss of circumferential microtubule band (changes reversible with rewarming up to 18 hours in the cold) Physiological responses on rewarming Spontaneous aggregation with stirring Increased sensitivity to agonists in inducing aggregation Increased P-selectin expression in response to agonists Decreased secretion of granule content Increased binding of fibrinogen Cold-induced biochemical changes Increase in actin filament assembly Cytosolic calcium elevation Increased tyrosine phosphory]ation Changes in GPIIb-Illa to bind fibrinogen Changes in membrane lipids arrangement because of liquid to gel transition state of membranes

Platelet responses to cold temperatures are analogous to activation of platelets with physiological agonists. In the early 1900s Aynaud, 1~ using a simple microscope, described changes in the appearance of platelets exposed to cold temperatures as similar to that produced by coagulation. Other early studies of cold-induced alterations described morphological changes such as sphering accompanied by "spiny processes" and an increase of 15% to 30% in platelet volume, n,12 These effects were observed to resemble the transformations seen on the recalcification of platelet-rich plasma. 13 Years later, White and Krivit 13 further examined these phenomena using electron microscopy. Resting platelets have a circumferential band of microtubules, which is thought to contribute to the maintenance of the discoid shape. Cold-induced changes in platelet morphology are accompanied by the complete depolymerization of microtubules. On rewarming, the marginal band of microtubules reappears, and the discoid appearance can be recovered. 13More recently, White 14 also found that the assembly of new actin filaments and formation of pseudopods is even more important to shape change than the loss of microtubules. However, he concluded that the recovery of discoid shape required the reassembly of microtubules. A very recent study compared the extent of shape change in platelets that had been exposed to temperatures above 22~ or below 20~ to mimic conditions that might occur during shipping or storage. 15 They observed a gradual loss of platelet discoid shape at temperatures less than 20~ The

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greatest differences between control and test platelets were found at storage temperatures of 4~ and worsened as the storage time progressed. Based on these results, a nonlinear mathematical model was developed by multiple regression, which relates platelet discoid shape to temperature and time of exposure. The authors also formulated an equation that best fit the data, which may be useful for determining the quality of platelets that have been stored at cold temperatures for a given period of time. is In resting platelets, negatively charged phospholipids are preferentially located on the cytoplasmic side of the plasma membrane. During physiological platelet activation, the negatively charged phospholipids become localized on the outside of the plasma membrane where they serve as an assembly site for activated plasma clotting factors V and X.16 Historically, this is referred to as platelet factor 3 activity. As platelets proceed through shape change and develop pseudopods, the terminal ends of these membrane extensions are shed to form free-floating membrane microparticles that possess procoagulant properties. 17 Microparticles also contain the platelet surface glycoproteins (GP)Ib and IIb. 17 Cold-treated platelets have been shown to have a greater loss of total GPIb, less binding of monoclonal antibodies to GPIb, 45% more microparticles, and 64% more platelet factor 3 activity in the supernatant plasma than platelets stored at room temperature.18 These differences increase by warming platelets to 37~ It has been suggested that this increase in microparticle formation in cold-stored platelet concentrates accounts for the increased hemostatic effectiveness reported for supernatant platelet-poor plasma. 19

Cold-lnduced Alterations in Physiological Platelet Responses Exposure of platelets to cold temperatures can lead to the induction of spontaneous aggregation after the platelets have been rewarmed and stirred, z~ When warming is prolonged, the spontaneous aggregation ceases, but the previously chilled platelets have a higher aggregation response to agonists than control platelets that were never chilled. 2a In contrast, platelets stored in the cold have reduced agglutination in response to ristocetin. This is a result of a loss of GPIb during platelet microparticle formation in the cold.18 Platelet aggregation occurs when the adhesive

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protein fibrinogen binds to its platelet receptor, GPIIb-IIIa. The fibrinogen binding site of GPIIbIIIa on resting platelets remains cryptic so that the ligand does not bind until its binding pocket is exposed. 22 There are two mechanisms by which GPIIb-IIIa binds fibrinogen. The first is a consequence of platelet activation. A platelet agonist, such as adenosine diphosphate (ADP) or thrombin, binds to its platelet receptor. This interaction initiates signaling pathways, which result in platelet shape change and ultimately in fibrinogen binding and platelet aggregation. When this occurs, the balance between effectors and second messengers is shifted towards platelet activation and suppression of inhibitory pathways. 23 Second messengers that are involved in the exposure of GPIIb-IIIa include calcium and protein kinase C, whereas inhibition is mediated by an increase in cyclic adenosine monophosphate (cAMP). 24 The second mechanism involves the direct alteration of the GPIIb-IIIa complex through a conformational change, or a change in the membrane microenvironment, which leads to a receptor that is competent for binding fibrinogen. 22Typically, the activation of GPIIb-IlIa in the absence of platelet activation can be induced by certain monoclonal antibodies, which are known as complex activating antibodies. Cold-induced fibrinogen binding to platelets starts to occur after chilling for 20 minutes; whereas, spontaneous aggregation occurs only when the platelets are warmed to ambient temperature and stirred. 25 Aggregation is followed by disaggregation, 25 unless the platelets are kept at 4~ for 24 hours or more. 26 Cold temperatures expose fibrinogen binding sites in a manner similar to that caused by ADP; however, there is no measurable ADP in platelet-rich plasma before the initiation of stirring, suggesting that the aggregation observed is not ADP-mediated. Furthermore, ADP-induced platelet responses were shown to be decreased by metabolic inhibitors antimycin A and 2-deoxy-Dglucose, whereas cold-induced responses were not affected. 25 Like ADP-induced fibrinogen binding, cold-induced binding was shown to be inhibited by prostaglandin E1 and by dibutyryl cAME both of which increase the cAMP concentration in the cell. Similar results were observed in a study that found a cAMP inhibitory step to be critical for the exposure of fibrinogen binding sites. 27 At a low cAMP concentration, there was a correlation between protein kinase C activity and fibrinogen

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binding to GPIIb-IIIa. When the platelets were chilled, fibrinogen binding increased, but the level of protein kinase C activity remained stable. 27 We have found that fibrinogen binding as well as aggregation, secretion, and P-selectin expression were increased in cold-stored platelets when compared with room temperature controls. 28 However, this was not caused by a change in the affinity of fibrinogen for GPIIb-IIIa or an increase in the exposure of new GPIIb-IIIa complexes on the platelet surface. 28 These results suggest that cold temperatures increase the percentage of activated GPIIb-IIIa complexes among the population of surface expressed GPIIb-IIIa. Another study also found increased P-selectin expression in cold-stored platelets. Platelet concentrates that were either stored at 4~ for 16 hours, treated with EDTA, or stored at a pH below 6.2 showed an irreversible increase in P-selectin. 29 These authors also found a fairly good correlation between P-selectin expression and in vivo survival, but the extent of shape change, lactate levels, and hypotonic shock response were better indicators of platelet survival. 29 Additional evidence also suggests that increased P-selectin levels may not be a clear indicator of decreased in vivo circulation time. 3~ Studies performed in baboons have shown that transfused activated platelets have similar recovery values to transfused resting platelets. Activated platelets shed their surface expressed P-selectin on transfusion and remain in circulation as P-selectin-negative platelets. These degranulated platelets also continue to function in vivo as seen by their participation in bleeding time wounds, binding to Dacron in an arteriovenous shunt, and the generation of microparticles. 3~Thus, P-selectin expression is unlikely to fully explain the decreased circulation of cold-treated platelets. Not only do cold-stored platelets increase their aggregation response to agonists, but they also exhibit an increased sensitivity to other platelet responses, most notably when activated by thrombin. 31 At low concentrations of thrombin, coldstored platelets release more serotonin and calcium than room temperature-stored platelets. This is not the result of an increased uptake of serotonin during storage. The authors of this study hypothesized that storage at 4~ may alter the number or type of thrombin binding sites, which may change the turnover rate of thrombin. They also suggested

LIQUID COLD STORAGE OF PLATELETS

289

that cold temperatures have an overall effect on the regulation of the internal environment of the cell including ion fluxes and concentration, cyclic nucleotide concentration, and the polymerization state of structural proteins. 31 Cold-stored platelets have an increased conversion of adenine to its metabolites and ultimately to hypoxanthine, but the amount of released ADP is equal in room temperature-stored and cold-stored platelets. 3: This could not be attributed to increased Na+-K § ATPase activity because ouabain had no effect on the increased metabolism of adenine. It has been suggested that cold temperatures may fix immunoglobulin M (IgM) or complement onto the platelet membrane, which could induce agglutination and increase adenine metabolism. This did not appear to occur because anti-IgM does not prevent this effect. 32 With regard to platelet secretion, it has been reported that cold- and room temperature-stored platelets had differing amounts of adenine nucleotides and that cold platelets possess a secretion defect. 33 The total adenosine triphosphate (ATP) and ADP content of stored platelets decreases over time, but a larger decrease occurs in the room temperature-stored platelets. Further measurements showed that cold-stored platelets have a higher total content of ATP and ADP, but secrete significantly less of these nucleotides on agonist stimulation. 33 The investigators attributed this secretion defect to an inability of cold platelets to maintain ATP homeostasis. Figure 1 is an illustration showing the steps of cold-induced platelet activation superimposed on a

increasedactin Nament

time line. This time line records the chronological order of the morphological and biochemical events that occur on the chilling of platelets and summarizes the results of the studies discussed in this section. MECHANISMS OF COLD INDUCED PLATELET RESPONSES

Four cold-induced biochemical events have been implicated as part of the mechanism involved in platelet responses to chilling (Table 2). These include (1) actin filament assembly, 34 (2) calcium elevation, 34,35 (3) disassembly of microtubule bands, 13 and (4) packing defects between gel and liquid crystalline phases of the plasma membrane. 36

Actin Filament Assembly A recent study 34 investigated cytoskeletal alterations involved in the mechanism of shape change in chilled platelets. The transformation from a discoid appearance to the formation of pseudopods appears to be because of actin-mediated movement of the cytoskeleton. In resting platelets, the majority of actin filaments are crosslinked throughout the cytoplasm and concentrated toward the periphery of the cell. 37 On platelet activation, the amount of actin filament formation increases from 30% to 40% to 60% to 70%. The cytoskeleton undergoes a reassembly of actin, which acts as a scaffolding for the transformed platelet, with the newly formed filaments involved in forming pseudopods. 38 There are at least two mechanisms that prevent the polymerization of actin in resting platelets. 39 First, monomeric actin is bound to a protein called

reversib{eloss of microtubuleband I ~gen

30% lifespanreductionin vivo rnfc~3tub'dteband

corrects bleedingtime in thrombocytopenics 90% Iffespanreductionin vivo no longer able to correct bleedingtime in thrombocytopenics I

decieasein total GPIb, increased PF3 activity and microparfieleformation

t spo~aneous aggregation secretion defect I sphering in,creasedadeninemetabolism increasedcytesolic i~reased platelet volume calcium irreversibleshape change and spontaneousaggregation spontaneousfibdnogenbir~d{ng Fig 1.

33me line of the cold-induced effects on stored platelets

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VOSTAL AND MONDORO Table 2. Mechanisms of Cold-induced Platelet Effects

Mechanism Growth of actin filaments

Elevation of cytosolic calcium

Increase membrane permeability Dissolution of microtubular ring

Cause

Consequence

Release and/or aggregation of polyphosphoinositides, uncapping of actin filament Inhibition of Ca++ ATPase, leakage of stored calcium and of extracellular calcium Membranes passing through liquid-gel transition temperature Unknown

Assembly of cytoskeleton, formation of pseudopods

[~4-thymosin, which serves as a storage site to prevent actin monomers from binding to existing filaments. In the second mechanism, the growing end of the filamentous actin is covered by capping proteins such as gelsolin or capZ to inhibit actin monomers from binding. The capping proteins are removed from the actin filaments during activation by a mechanism that may involve aggregated polyphosphoinositide lipids. 34 Once the cap is removed, the high affinity of monomeric actin for the growing filament shifts the equilibrium to remove actin from its binding site on [M-thymosin, and actin begins to incorporate into the filamentous chain. It is postulated that the rapidly growing actin filaments force platelet shape change by filling in the pseudopod protrusions that platelets generate. Cold temperatures induce assembly of actin filaments in platelet lysates as well as in intact platelets. It has been suggested that the coldinduced changes in the platelet membranes could aggregate polyphosphoinositide lipids and, thus, mediate the removal of capping proteins in chilled platelets. 34 Further studies by another group 4~ reported that temperature-dependent shape change correlates with the phosphorylation of the 20-kD myosin light chain, which interacts with actin to cause contraction of the cytoskeleton. The light chain is phosphorylated to regulate its association with actin. 41 This suggests that the plasma membrane and cytoskeleton act in concert to initiate cold-induced platelet activation.

Cytosolic Calcium Elevation When cells are incubated at 37~ an increase in cytosolic calcium mediates a great variety of cellular processes and is a unifying signal that simultaneously sets all of them into play. All strong platelet agonists elevate cytosolic calcium as a part of their activation pathway. 42 Calcium levels can increase in the platelet cytosol from two different sources,

Activation of signal transduction pathways, gelsolin clipping of actin filaments Increased ion fluxes, cytosolic calcium elevation Loss of discoid shape

one of which is the release from internal stores located in the dense tubular system, and the other is the influx of extracellular calcium. Internal stores are refilled during the resting state by the action of a calcium ATPase pump that lowers the resting concentration in the cytosol to about 1 • 10 7 mol/L calcium and concentrates the calcium in the stores to approximately 5 • 10 .3 mol/L. 42 With such a gradient difference, it is presumed that the pump is continuously working against a persistent calcium leak into the cytosol. A plasma membrane calcium ATPase also works to pump out cytosolic calcium into the plasma. Agonist-induced activation is accompanied by the elevation of inositol phosphate metabolites with inositol-1,4,5-trisphosphate being the molecule that releases the calcium from the internal stores. 43 As the internal stores empty of calcium, an unidentified signal is generated, which alters the permeability of plasma membrane for calcium and allows extracellular calcium to come into the platelet. 42 Thus, emptying the internal stores also initiates an influx of extracellular calcium. Two independent observations suggest that cytosolic calcium becomes elevated with platelet chilling. One is a direct measurement of calcium with fluorescent dyes, which approximates the increase in calcium from 80 nmol/L at rest to 200 nmol/L when chilled. 34 The other observation is the occurrence of calcium-dependent tyrosine phosphorylation with platelet chilling. 35 At room temperature, this tyrosine phosphorylation occurs at approximately 300 nmol/L cytosolic calcium. The mechanism of cold-induced cytosolic calcium rise is likely caused by the inhibition of the calcium ATPase, which pumps calcium into the stores, and by the calcium ATPase, which pumps calcium out of the cell across the plasma membrane. Inhibition of these pumps will allow stored calcium to leak out into the cytosol and potentially induce the

LIQUID COLD STORAGE OF PLATELETS

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signal for the increase of extracellular calcium influx. Without active removal of calcium, prolonged elevation of cytosolic calcium, even at levels lower than those reached with agonist activation, will likely induce some aspects of platelet activation that could include granule secretion and expression of GPUb-IIIa. Calcium is also required for gelsolin to hydrolyze filamentous actin. This protein can clip the growing actin chain to allow for branching and remodeling of the growing actin filament structure. 34

Microtubule Disassembly Resting ptatelets have a circumferential band of microtubules that disappear when platelets are chilled. 13 It has been thought that the microtubule band maintains the platelet discoid shape because disruption of this band with vincristine is associated with loss of the platelet discoid form. 44 Rewarming of the platelets can lead to the reassembly of the microtubules with the reestablishment of the discoid shape, although this reversibility depends on the length of time the platelets were subjected to the cold.

Lipid Phase Transition It is likely that the phase transition of lipid membranes has a major role in the initiation of the cold activation of platelets. 36 Cold-induced effects on platelets occur at temperatures in which platelet

membrane lipids pass through the liquid-gel transition. The alterations in lipid membrane packing induced by the phase transition could change the ion permeability properties of the membrane and/or mediate the release of polyphosphoinositides. 36 METHODS FOR THE PREVENTION OF COLD-INDUCED PLATELET ACTIVATION

Physical Methods Approaches to preventing the effects of cold on platelets have used either physical or biochemical means (Table 3). One of the early attempts at preventing cold-induced changes in liquid-stored platelets used increases in atmospheric pressure. 45 Pressures of 2,500 to 9,000 lb/in 2 prevented coldinduced platelet shape change on platelet concentrates stored at 2~ for 2 hours. Increased pressure, before or immediately after cooling, prevented the sphering of platelets and pseudopod formation but did not prevent disappearance of the microtubule circumferential band. Equal pressure applied to platelets at room temperature did not cause the disappearance of the microtubule band indicating that its disappearance was mediated by the cold. It was not shown whether pressure alone or pressure and chilling had lasting effects on physiological responses of platelets returned to atmospheric pressures and room temperatures. Another purely physical method involved cy-

Table 3. Approaches to the Prevention of Cold-Induced Platelet Activation Mechanism of Action Physical methods Increased atmospheric pressures Temperature cycling

Biochemical methods Taxol

Cytochalasin/Quin2 Antifreeze glycoprotein

Signal transduction ]nhibitors

Unknown Presumed reversal of cytoskeletel assembly, reactivation of Ca++ ATPases and decrease in cytosolic calcium Microtubule stabilization

Inhibition of actin filament growth, chelation of cytosolic calcium Unknown, may prevent increase in membrane permeability

Inhibition of Na/H exchanger, elevation of cAMP and cGMP, inhibition of phospholipase A2

Prevention of Cold-Induced PlateletResponses No pseudopod formation, did not prevent loss of microtubular band Reassembly of microtubular band with warming up to 72 hours after cold, intact hypo-osmotic, and shape change responses Discoid shape not preserved, aberrant reassembly of microtubules in center of platelet Preservation of discoid shape Prevented cold-induced activation (Gp53 expression), preserved thrombin (1 U/mL)-induced activation up to 21 days cold storage Preservation of ADP-induced shape change and aggregation up to 9 days cold storage

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cling of platelets between cold and warm temperattires. 26 Platelets were stored for 12 hours at 4~ then rewarmed for 30 minutes at 37~ and then again placed at 4~ for further storage. This temperature cycling allowed spontaneous platelet microtubule assembly on rewarming, even after 72 hours of storage. In comparison, platelets stored continuously at 4~ lost the ability to reassemble the microtubular band after only 24 hours of storage. Moreover, temperature-cycled platelets responded to ADP-induced activation, shape change, and to hypo-osmotic shock as well as fresh platelets. Thus, temperature cycling of stored platelets appears to preserve some aspects of in vitro platelet responses.

VOSTAL AND MONDORO

buffered by the intracellular chelator, although at cold temperatures (4~ the affinity of the calcium chelators for calcium is decreased by about twofold to threefold so that higher concentrations of the chelator are required to overcome the low temperatures effects. 47 In the absence of a cytosolic calcium rise, chilled platelets can still develop actin filament assembly and form pseudopods. Thus, both calcium chelation and cytochalasin are necessary to maintain the discoid shape. 34 Whether cytochalasin along with calcium chelation prevents the appearance of activation surface markers and whether physiological platelet responses such as agonistinduced aggregation and secretion can also be returned to normal levels is not known.

Cytoskeletal Stabilizers Biochemical approaches to preventing coldinduced platelet activation have used drugs that can specifically prevent some of the changes associated with platelet chilling. One of the early attempts was to use the microtubule stabilizing agent taxol. 46 When added to platelets before chilling, taxol prevented the disappearance of the microtubule band, although it did not uniformly preserve the discoid shape. Approximately half of taxol-treated platelets did assume irregular shapes when subjected to cold. Adding taxol after platelet chilling caused the reassembly of microtubules, but not into the formation of a circumferential band. Instead, the microtubules organized in the center of the cell with tubules radiating in all directions. Moreover, taxol did not restore the discoid shape to spherical chilled cells. Thus, it is not clear whether taxol can protect platelet physiological responses in coldstored platelets. A recent approach has been to prevent coldinduced actin filament elongation with the combination of the drug cytochalasin and to chelate cytosolic calcium with an intracellular chelator such as Quin 2 (Molecular Probes, Eugene, OR).34 Cytochalasin binds to the growing actin filaments and prevents further monomeric actin addition. Preventing the growth of actin filaments prevents the generation of pseudopods with subsequent platelet activation, although such platelets do transform from the discoid state into spheres. Cytochalasin also does not prevent the disappearance of the circumferential microtubule band. Cytosolic elevation of calcium associated with platelet chilling is

Antifreeze Proteins Antifreeze proteins and glycoproteins have been isolated from fish that have adapted to survive under extremely cold conditions. 48 These proteins can prevent ice formation in blood and tissue by directly binding to ice crystals. Attempts have been made to use these unique proteins for platelet cold storage. 36 It has been shown recently that the presence of the antifreeze glycoproteins decreases the platelet activation associated with chilling in a concentration-dependent manner. Antifreeze glycoproteins from three species of Antarctic fish prevented cold-induced platelet activation as measured by the surface expression of the lysosomeassociated membrane protein (known as LAMP, CD63, or GP53). The presence of the antifreeze glycoproteins preserved the activation response of the stored platelets to thrombin (1 U/mL) for up to 21 days of storage in 4~ The mechanism of this protection is not clear but appears not to involve changing the crystalline liquid to gel transition temperature (Tin) of platelet membranes. It may be associated with preventing ion flux across membranes which are going through their Tm. It has also been suggested that the antifreeze glycoproteins can interact with specific ion channels. 49 More recently these proteins were shown to decrease leakage of fluorescent probes from liposomes. 5~ Because these liposomes do not have ion channels, this experiment suggests that the interaction between the antifreeze glycoproteins and phospholipid membranes is less specific. This observation requires further investigation.

LIQUID COLD STORAGE OF PLATELETS

Signal Transduction Inhibitors The hypothesis that cold activation of platelets mimics agonist-induced activation is supported by reports that inhibition of particular signaling pathways protects platelets from the deleterious effects of the cold. 51 An inhibitory cocktail that prevents activation of various signal transduction pathways was reported to decrease the activating effects of cold on platelets. The preventive solution contained the inhibitors amiloride, adenosine, sodium nitroprusside, dipyridamole, ticlopidine, and quinacrine. Amiloride is an inhibitor of the plasma membrane Na+Prt + exchanger and when added to resting platelets, it rapidly decreases pHi by about 0.05 U. 51 Amiloride inhibits platelet aggregation and secretion induced by thrombin, 52 collagen, platelet activating factor, and epinephrine. 53 The amiloride analog, ethylisopropyl-amiloride, has been shown to prevent thrombin-induced release of calcium from internal stores and also to decrease the influx of extraceUular calcium. 54 Amiloride can also inhibit agonist-induced shape change, possibly by interfering with cytoskeletal assembly,s4 Adenosine, 55 sodium nitroprusside, 56 and dipyridamole 57 are drugs that elevate the concentrations of platelet cyclic nucleotides, cAMP and cGMP. cAMP is a general platelet inhibitor that increases the removal of calcium from the cytosoL thus making the platelet less responsive to activation. 24 cGMP has similar effects. 23 Adenosine stimulates adenylate cyclase to produce cAMP, 5s whereas sodium nitroprusside activates guanylate cyclase to generate cGMP. 56 Dipyrimadole potentiates the effect of the other two drugs by inhibiting phosphodiesterases, 57 which are the enzymes that break down cyclic nucleotides. Ticlopidine is a novel platelet inhibitor that inhibits ADP-induced platelet aggregation and secretion in vivo by blocking platelet surface ADP receptors. 58 It has been shown that ticlopidine requires hepatic activation in vivo and that its use in vitro does not prevent ADP activation of platelets. 58 Thus, its role in protecting platelets from cold activation in vitro is not clear. Quinacrine is a phospholipase A2 inhibitor that has been shown to inhibit thrombin-induced release of arachidonic acid at concentrations that are three orders of magnitude higher than what is used in prevention of cold activation. 59 It may be that the combination of all of the inhibitors together has an additive or a

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synergistic effect on prevention of activation, but this has not been shown with physiological platelet agonists. Such an inhibitory cocktail has been reported to protect platelets from the activating effects of cold storage for up to 9 days. 51 The cold-stored platelets, after being washed to remove the inhibitors, exhibit ADP and collagen-induced aggregation and shape change, which is equivalent or better than that found with platelets stored 9 days at room temperature. Only the hypo-osmotic recovery is less than found in control platelets after 9 days of storage. 51 Platelets treated with this additive solution and stored at 4~ have recently been shown to have lower levels of interleukins 6 and 113 and tumor necrosis factor o~ than control platelets stored at 22~ 6~ These cytokines are derived from residual white blood cells in platelet concentrates and are thought to participate in nonbacteremic febrile nonhemolytic transfusion reactions. Platelets inoculated with low levels of Staphylococcus aureus and stored for 5 days in the cold with this additive solution showed no increase in bacterial titer, thus showing the feasibility of preventing bacterial growth under these storage conditions. 6~

CONCLUSIONS Much has been learned about platelet physiology and subcellular molecular mechanisms in the 30 years since the early attempts at storing platelets in the cold. The shortcomings of platelet cold storage were quickly recognized, and this practice was abandoned in favor of room temperature storage in gas-permeable plastic bags. The recent awareness of increased risks of bacteremia associated with transfusion of room temperature-stored platelets has renewed interest in the possibility of cold storage. It is difficult to predict whether any of the methods described above will be safe, effective, and practical for the cold storage of platelets. To date, no in vitro test has been shown to correlate well with platelet in vivo survival and recovery. Thus, preservation of platelet performance in a particular in vitro test or in a group of tests does not guarantee adequate in vivo recovery and survival. 61 The demonstration of in vivo effectiveness and survival of cold-stored platelets will ultimately require human clinical trials. However, before we

294

VOSTAL AND MONDORO

c a n a d v a n c e to this step, n o v e l s t o r a g e m e t h o d s w i l l

d e s i g n e d to p e r m i t c o l d s t o r a g e o f p l a t e l e t s w i l l r e s t

n e e d to s h o w t h a t t h e r i s k to t h e r e c i p i e n t f r o m t h e

on such a risk:benefit analysis.

c h e m i c a l a n d / o r p h a r m a c o l o g i c a l a d d i t i v e s is c o u n terbalanced by decreasing the risk of the recipient receiving a bacterially contaminated platelet transfusion. The ultimate success of any technology

ACKNOWLEDGMENT

We thank Dr Joseph C. Fratantoni for a critical reading of the manuscript.

REFERENCES 1. Morrow JF, Braine HG, Kickler TS, et al: Septic reactions to platelet transfusions. A persistent problem. JAMA 266:555558, 1991 2. Goldman M, Blajchman MA: Blood product-associated bacterial sepsis. Transfus Med Rev 5:73-83, 1991 3. News Briefs, American Association of Blood Banks, Bethesda, MD, 1992, p 14 4. Chin EKW, Yuen KY, Lie AKW, et al: A prospective study of symptomatic bacteremia following platelet transfusion and of its management. Transfusion 34:950-954, 1994 5. Becker GA, Tuccelli M, Kunicki T, et al: Studies of platelet concentrates stored at 22 ~ and 4~ Transfusion 13:6168, 1973 6. Valeri CR: Hemostatic effectiveness of liquid-preserved and previously frozen human platelets. N Engl J Med 290:353358, 1974 7. Slichter SJ, Harker LA: Preparation and storage of platelet concentrates. II. Storage variables influencing platelet viability and function. Br J Hematol 34:403-419, 1976 8. Filip DJ, Aster RH: Relative hemostatic effectiveness of human platelets stored at 4 ~ and 22~ J Lab Clin Med 91:618-624, 1978 9. Murphy S, Gardner FH: Platelet preservation. Effect of storage temperature on maintenance of platelet viability-deleterious effect of refrigerated storage. New Engl J Med 280:1094-1098, 1969 10. Aynaud M: Le globulin de l'homme. Ann Inst Pasteur 25:56-63, 1911 11. Bull B, Zucker MB: Changes in platelet volume produced by temperature, metabolic inhibitors, and aggregating agents. Proc Soc Exp Biol Med 120:296-301, 1965 12. Zucker MB, Borrelli J: Reversible alterations in platelet morphology produced by anticoagulants. Blood 9:602-608, 1954 13. White JG, Krivit W: An ultrastructural basis for the shape changes induced in platelets by chilling. Blood 30:625-635, 1967 14. White JG, Krumwiede MD, Cocking-Johnson DJ: Coldinduced platelet shape change and recovery. Blood 88:49b, 1996 (abstr, suppl 1) 15. Holme S, Sawyer S, Heaton A, et al: Studies on platelets exposed to or stored at temperatures below 20~ or above 24~ Transfusion 37:5-11, 1997 16. Wiedmer T, Esmon CT, Sims PJ: Complement proteins C5b-9 stimulate procoagulant activity through platelet prothrombinase. Blood 68:875-880, 1986 17. Owens MR: The role of platelet microparticles in hemostasis. Transfns Med Rev 8:37-44, 1994 18. Bode AR Knupp CL: Effect of cold storage on platelet glycoprotein Ib and vesiculation. Transfusion 34:690-696, 1994 19. Kevy SV, Jacobson MS, Button LN, et al: The hemostatic

effectiveness of supernatant plasma of 4~ platelet concentrates. Blood 48:1004, 1976 (abstr, suppl 1) 20. Kattlove H, Alexander B: The effect of cold on platelets. I. Cold-induced platelet aggregation. Blood 38:39-48, 1971 21. Kattlove HE, Alexander B, White F: The effect of cold on platelets. II. Platelet function after short-term storage at cold temperatures. Blood 40:688-696, 1972 22. Kieffer N, Phillips DR: Platelet membrane glycoproteins: Functions in cellular interactions. Ann Rev Cell Bit 6:329-357, 1990 23. Brass LF: Molecular basis for platelet activation, in Hoffman R, Benz EJ, Shattil SJ, et al (eds): Hematology Basic Principles and Practice. New York, NY, Churchill Livingstone, 1995, pp 1536-1551 24. Siess W: Molecular mechanisms of platelet activation. Physiological Rev 69:58-178, 1989 25. Peerschke El, Zucker MB: Fibrinogen receptor exposure and aggregation of human blood platelets produced by ADP and chilling. Blood 57:663-670, 1981 26. McGill M: Temperature cycling preserves platelet shape change and enhances in vitro test scores during storage at 4 ~ J Lab Clin Med 92:971-982, 1978 27. van Willigen G, Akkerman JWN: Protein kinase C and cyclic AMP regulate reversible exposure of binding sites for fibrinogen on the glycoprotein lib-Ilia complex of human platelets. Biochem J 273:115-120, 1991 28. Mondoro TH, Vostal JG: GPIIb-IIIa dynamics in coldtreated platelets. Blood 88:53b, 1996 (abstr, suppl 1) 29. Holme S, Sweeney JD, Sawyer S, et al: The expression of P-selectin during collection, processing, and storage of platelet concentrates: Relationship to loss of in vivo viability. Transfusion 37:12-17, 1997 30. Michelson AD, Barnard MR, Hechtman HB, et al: In vivo tracking of platelets: Circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function. Proc Natl Acad Sci USA 93:11877-11882, 1996 31. Robblee LS, Sherpo D, Vecchione JJ, et al: Increased thrombin sensitivity of human platelets after storage at 4~ Transfusion 19:45-52, 1979 32. Kattlove HE: The effect of cold on platelets. III. Adenine nucleotide metabolism after brief storage at cold temperature. Blood 42:557-564, 1973 33. Rat AK, Murphy S: Secretion defect in platelets stored at 4~ Thromb Haemost 47:221-225, 1982 34. Winokur R, Hartwig JH: Mechanism of shape change in chilled human platelets. Blood 85:1796-1804, 1995 35. Vostal JG, Jackson WL, Shulman NR: Cytosolic and stored calcium antagonistically control tyrosine phosphorylation of specific platelet proteins. J Biol Chem 266:16911-16916, 1991 36. Tablin F, Oliver AE, Walker NJ, et al: Membrane phase

LIQUID COLD STORAGE OF PLATELETS

transition of intact human platelets: Correlation with coldinduced activation. J Cell Physiol 168:305-313, 1996 37. Boyles J, Fox JEB, Phillips DR, et al: Organization of the cytoskeleton in resting, discoid platelets: Preservation of actin filaments by a modified fixation that prevents osmium damage. J Cell Biol 101:1463-1472, 1985 38. Jennings LK, Fox JEB, Edwards HH, et al: Changes in the cytoskeletal structure of human platelets following thrombin activation. J Biol Chem 256:6927-6932, 1981 39. Fox JEB: The platelet cytoskeleton. Thromb Haemost 70:884-893, 1993 40. Higashihara M, Miyazake K, Asano S, et al: The mechanism of chilling-induced platelet shape change. Blood 88:49b, 1996 (abstr, suppl 1) 41. Niederman R, Pollard T: Human platelet myosin II. In vitro assembly and structure of myosin filaments. J Cell Biol 67:72-92, 1975 42. Sargent R Sage SO: Calcium signalling in platelets and other nonexcitable cells. Pharmacol Ther 64:395-443, 1994 43. Streb H, Irvine RE Berridge MJ, et al: Release of Ca from a nonmitochondrial store in pancreatic cells by inositol1,4,5, trisphosphate. Nature 306:67-68, 1983 44. White JG: Effects of colchicine and vinca alkaloids on human platelets: I. Influence on platelet microtubules and contractile function. Am J Patho153:281-291, 1968 45. Rytting JH, Chatterji DC, Higuchi T, et al: Effects of temperature and pressure on short term storage of platelets. Nature 253:539-540, 1975 46. White JG: Influence of taxol on the response of platelets to chilling. Am J Pathol 108:184-195, 1982 47. Harrison SM, Bets DM: The effect of temperature and ionic strength on the apparent Ca-affinity of EGTA and the analogous Ca-chelators BAPTA and dibromo-BAPTA. Biochim Biophys Acta 925:133- t43, 1987 48. Sicheri F, Yang DSC: Ice-binding structure and mechanism of an antifreeze protein from winter flounder. Nature 375:427-431, 1995 49. Rubinsky B, Arav A, Hong JS, et al: Freezing of mammalian livers with glycerol and antifreeze proteins. Biophys Biochem Res Cotmnun 200:732-741, 1994 50. Hays LM, Feeney RE, Crowe LM, et al: Antifreeze glycoproteins inhibit leakage from liposomes during tbermo-

295

tropic phase transitions. Proc Natl Acad Sci USA 93:6835-6840, 1996 51. Connor J, Currie LM, Allan It, et al: Recovery of in vitro functional activity of platelet concentrates stored at 4~ and treated with second-messenger effectors. Transfusion 36:691698, 1996 52. Home WC, Simons ER: Effects of amiloride on the response of human platelets to bovine ~ thrombin. Thromb Res 13:599-607, 1978 53. Siffert W, Gengenbach S, Scheid P: Inhibition of platelet aggregation by amiloride. Thromb Res 44:235-240, 1986 54. Siffert W, Akkerman JWN: Na+/H+ exchange as a modulator of platelet activation. Trends Biochem Sci 13:148151, 1988 55. Faulds D, Chrisp P, Buckley MM: Adenosine: An evaluation of its use in cardiac diagnostic procedures, and in the treatment of paroxysmal supraventricular tachycardia. D~gs 4l:596-624, 1991 56. Hardy E, Rubin PC, Horn EH: Effects of nitric oxide donors in vitro on the arachidonic acid-induced platelet release reaction and platelet cyclic GMP concentration in preeclampsia. Clin Sci 86:195-202, 1994 57. Saniabadi AR, Lowe GD, Barbenel JC, et al: Effect of dipyridamole on spontaneous platelet aggregation in whole blood decreases with the time after venepuncture: Evidence for the role ofADR Thromb Haemost 58:744-748, 1987 58. Saltiel E, Ward A: Ticlopidine. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy in platelet-dependent disease states. Drugs 34:222-262, 1987 59. McCrea JM, Robinson E Genard JIM: Mepacrine (quinacrine) inhibition of thrombin-induced platelet responses can be overcome by lysophosphatidic acid. Biochim Biophys Acta 842:189-194, 1985 60. Currie LM, Harper JR, Allan H, et at: Inhibition of cytokine accumulation and bacterial growth during storage of platelet concentrates at 4~ with retention of in vitro functional activity. Transfusion 37:18-24, 1997 61. Slichter S/: In vitro measurements of platelet concentrates stored at 4 and 22~ Correlation with posttransfusion platelet viability and function. Vox Sang 40:72-86, 1981 (suppl)