Galactosylation does not prevent the rapid clearance of long-term, 4°C-stored platelets

In contrast to red cells, platelets do not tolerate cold storage (4°C), and refrigerated platelets are rapidly cleared from circulation following transfusion.^1,2  Therefore, platelets are stored at room temperature, with the optimal storage temperature determined to be 22°C (± 2°C).³  Because room temperature storage is favorable for the growth of bacteria and conducive to detrimental changes in platelet structure and function,^4-7  platelets are currently licensed for only 5 to 7 days of storage. This short shelf-life severely limits available platelet inventory and drives donor recruitment efforts.

Platelets stored in the cold lose their characteristic discoid shape, which was originally thought to be the reason for their rapid clearance after transfusion.¹  However, prevention of the cold-induced shape change with EGTA and cytochalasin did not prevent clearance of platelets chilled at 0°C for 2 hours, suggesting that other mechanisms were involved.^8,9  A new mechanism for the clearance of cold-stored platelets emerged when we demonstrated that chilling causes clustering of the GPIb/V/IX complex on the surface of platelets, resulting in recognition of β-N-acetylglucosamine (βGlcNAc)–terminating immature glycans by the lectin domain of the α_Mβ₂ integrin receptor (CR3, Mac-1) present on hepatic macrophages.^8,10  Accordingly, hepatic macrophages were demonstrated to selectively phagocytose chilled murine platelets, leading to their rapid clearance.⁸  A potential method for preventing the rapid clearance of 4°C-stored platelets was envisioned to be enzymatic galactosylation of surface βGlcNAc residues on platelet glycoproteins with endogenous enzymes. The galactosylation process was shown to prevent the phagocytosis of chilled murine platelets.¹⁰  Furthermore, in vitro studies demonstrated that human platelets could be galactosylated by the addition of UDP-galactose to platelets stored in plasma. This galactosylation process inhibited the in vitro phagocytosis of human galactosylated platelets by THP-1 cells, suggesting that galactosylated platelets stored at 4°C might circulate when transfused into humans.¹¹  Reported here are the results of a phase 1 clinical trial in which autologous apheresis platelets obtained from healthy volunteers were stored for 48 hours using different storage conditions before reinfusion: galactosylated platelets stored at 4°C, nongalactosylated platelets stored at 4°C, or nongalactosylated platelets stored at room temperature (22°C). In addition, similar platelet storage studies were performed using murine platelets in a mouse model.

Aliquots (2 mL) of nonwashed human apheresis platelets (1- 2 × 10⁹ platelets/mL) were incubated with increasing concentrations (0.00 mM to 1.50 mM at 0.25 mM increments) of UDP-galactose for time periods of 0 to 90 minutes at 15 minute increments at 37°C. Platelets were washed, and the galactosylation efficiency was monitored with fluorescently labeled succinyl wheat germ agglutinin (sWGA) and ricinus communis agglutinin (RCA-1) as previously described by FACS analysis (FACScalibur Flow Cytometer; Becton Dickinson Biosciences, San Jose, CA).¹⁰  Maximal RCA-1 binding and minimal sWGA binding was achieved using 1.2 mM UDP-galactose and an incubation time of 1 hour. This galactosylation protocol was scaled up and evaluated using apheresis platelet collections from 12 different donors.

Using differentiated THP-1 cells (ATCC number: TIB-202), in vitro phagocytic assays were performed with the galactosylated apheresis platelet collections as previously described.¹² 

The human studies were performed at the Puget Sound Blood Center, Seattle, Washington. These studies were conducted under an investigational new drug application, and they were approved by an institutional review board and a radiation safety committee. Informed consent was obtained from all volunteers in accordance with the Declaration of Helsinki, they met standard donation criteria, and they had not taken any medication known to alter platelet function for 14 days before platelet donation. The study was an open-label, controlled phase 1 study using standard radiolabeled autologous platelet transfusion protocols to determine platelet recoveries and survivals.¹³  Three platelet products—each stored for 48 hours—were evaluated: (1) platelets stored at 4°C that were galactosylated with UDP-galactose; (2) platelets stored at 4°C without galactosylation; and (3) platelets stored at 22°C without galactosylation. Four healthy volunteers were enrolled, and each donated apheresis platelets on a Haemonetics MCS+ apheresis machine (Haemonetics, Braintree, MA). Platelets were collected into one bag, and then divided into 2 bags of approximately 120 mL. UDP-galactose was supplied by a commercial supplier, and prepared for use under contract with Cytosol Laboratories by reconstituting the UDP-galactose in 0.9% saline to produce a 40 mM sterile filtered solution filled into 5-mL plastic syringes. Aseptic media fill validation was conducted as a part of the controls on the fill and finish operation. The stock solution was kept at 4°C throughout the process and storage and stability of the UDP-galactose was verified using a pre-validated high-performance liquid chromatography (HPLC) quantification procedure performed at ZymeQuest (Beverly, MA). Immediately after collection, one bag of platelets from each donor was treated with UDP-galactose by sterile docking the UDP-galactose container onto a platelet storage bag. After addition of UDP-galactose the platelets were incubated for 1 hour at 37°C with agitation and then stored at 4°C for 36 to 48 hours without agitation. The other bag of platelets served as a control and was incubated without UDP-galactose for 1 hour at 37°C with agitation, followed by storage for 36 to 48 hours either without agitation at 4°C (n = 2) or with agitation at 22°C (n = 2).

The 2 bags of platelets from each individual donor were radiolabeled as previously described¹³  with a different radioactive isotope, either ⁵¹Chromium or ¹¹¹Indium, and 5 mL to 10 mL of both the radiolabeled test and control platelets were simultaneously transfused back to their donor. Radioisotopes used for labeling were alternated between test and control platelets to avoid bias related to the isotope used for radiolabeling. Blood samples were drawn before and at 2 hours, 1, 2, 3, 5, 7, and 10 days after transfusion, and the posttransfusion recovery and survival of the platelets were determined using the COST program.¹⁴  Samples were obtained to correct for elution of either radioisotopic label and for any residual radioactivity bound to red cells. The recovery and survival data are reported both uncorrected for label elution or residual activity, as well as corrected for these 2 parameters.

During and for 2 hours after each platelet infusion, subjects were carefully monitored for vital signs and potential adverse reactions. Follow-up visits were conducted at days 1, 2, 3, 5, 7, 10, 14, and 90. Vital signs were obtained at each visit, and the subjects were queried about the occurrence of any adverse events. Additional telephone interviews to document any long-term adverse events were conducted on days 28, 42, 56, and 70 after infusion.

Baseline and at days 14 and 90 after infusion, samples were taken from each volunteer to detect IgG and IgM antiplatelet antibodies. Platelets with and without galactosylation were incubated with each volunteer's plasma and with the Fab'2 fraction of FITC-conjugated goat antibody to the Fc chain of human IgG and IgM (Jackson Laboratories, Bar Harbor, ME). Binding of conjugated Fab'2 fragments was monitored by FACS analysis (FACScan; Becton Dickinson Biosciences). Plasma with known HLA antibodies was used as a positive control.

Samples were collected on days 0 and 2 from the stored platelets for the following measurements. Blood gas and pH measurements using a blood gas analyzer (Bayer, East Walpole, MA). Glucose and lactate were measured using an Abbott Aeroset Analyzer (Abbott, Round Lake, IL). Platelet counts and mean platelet volume (MPV) were performed on an ABX Micros particle counter (ABX, Montpellier, France). Morphology score was performed by the method of Kunicki.¹⁵  Hypertonic shock response (HSR) and extent of shape change (ESC) were performed as previously described¹⁶ ; CD62P expression was measured by FITC-labeled CD62P-specific monoclonal antibody S-12 using FACS analysis. Annexin V binding was determined by FACS analysis using fluorescently labeled annexin-V (Vybrant Assay Kit [V-13240]; Molecular Probes, Eugene, OR). Galactosylation of the samples was verified using fluorescently labeled RCA-1 and sWGA lectins with FACS analysis as previously described.¹⁰  Platelet aggregation and agglutination experiments were performed with a PLT aggregation profiler (Model PAP-; Bio/Data, Horsham, PA). Platelets were washed and resuspended as previously described¹¹  and activated by adding 0.1 U to 1 U thrombin (Sigma-Aldrich, St Louis, MO) per mL; platelet-rich plasma (PRP) was mixed with platelet-poor plasma (PPP) in the ratio 1:1 and was then activated through the addition of 1.5 mg/mL ristocetin (Sigma-Aldrich) for 3 minutes at 37°C under constant stirring (1000 rpm). Resuspension buffer for washed PLTs and PPP for PRP were set as maximum of light transmission.

The number of volunteers enrolled was believed to be sufficient to prove or disprove the hypothesis that platelet galactosylation prevents rapid clearance of 4°C-stored human platelets and also to preliminarily assess feasibility and safety. However, the number of subjects enrolled was too small for any application of statistical assessments.

All of the murine studies were performed at Brigham and Women's Hospital, Boston, MA. Mice were maintained and treated as approved by the Harvard Medical Area Standing Committee on Animals according to National Institutes of Health standards as set forth in “The Guide for the Care and Use of Laboratory Animals.”¹⁷ 

Murine blood was obtained and platelets were separated from plasma, washed, and resuspended at a concentration of 10⁹/mL in 140 mM NaCl, 3 mM KCl, 0.5 mM MgCl₂, 5 mM NaHCO₃, 10 mM glucose, and 10 mM Hepes, pH 7.4 (buffer A), as previously described.¹⁰  Platelets were stained with 5 μM CMFDA (5-chloromethylfluorescein diacetate) for 20 minutes at 37°C, sedimented by centrifugation at 1200g for 5 minutes, resuspended in their original plasma, and allowed to rest for 60 minutes at 37°C. The platelets were injected into mini transfusion bags (0.9 mL/transfusion bag) using a 27.5-gauge needle, sealed with a heat sealer (SEBRA, Tucson, AZ), and placed in the dark for 6, 48, or 72 hours at 4°C without agitation or for 6 hours at room temperature (22°C) on a horizontal table-top shaker rotating at 60 cycles/minute. Platelet mini-bags with a storage volume of 1 mL to 2 mL were made by heat-sealing 20-mm by 30-mm squares from gas-permeable platelet storage bags (Gambro BCT, Lakewood, CO). Freshly isolated platelets stained with 5 μM CMFDA, as described, were used as controls to determine normal platelet recoveries and survivals. Platelet counts were adjusted to 2 × 10⁸ before injection into a mouse.

Murine PRP was galactosylated by incubation with 1.2 mM UDP-galactose or control uridine 5′-diphosphoglucose (UDP-glucose) for 60 minutes at 37°C, labeled with 5 μM CMFDA, and stored for 48 hours at 4°C. The importance of the timing of the galactosylation reaction was tested by performing the galactosylation process after the storage period in a subset of experiments.

Recoveries and survivals of murine platelets were performed in age-, strain-, and sex-matched C57BL/J6 WT mice (Jackson Laboratories). Mice lacking αM (CR3) were of the C57BL/6 × 129/sv genetic background.¹⁸ 

CMFDA-labeled fresh or murine platelets (2 × 10⁸) stored at 4°C for 4, 6, 24, and 48 hours or platelets stored at room temperature for 6 hours were injected via the retroorbital venous plexus into anesthetized (2.5% Avertin [Fluka Chemie, Steinham, Switzerland]) mice of the same strain and age. Blood samples were collected before or at 5 minutes, 30 minutes, and 2, 24, 48, and 72 hours after transfusion into 0.1 volume of acid-citrate-dextrose (ACD) anticoagulant. The percentage of CMFDA-positive circulating platelets, as determined by flow cytometry, was used to determine platelet recoveries and survivals.¹⁹  The number of CMFDA-positive fresh platelets circulating immediately after transfusion (≤ 5 minutes) was set as the 100% reference recovery value.

Transfusion of UDP-galactose– and UDP-glucose–treated murine platelets was performed after 48 hours of storage at 4°C and recovery and survival determined as described. In order to test for the efficacy of platelet galactosylation directly in plasma, PRP was treated with 1.2 mM UDP-galactose or control UDP-glucose for 1 hour at 37°C, followed by washing, storage at 0°C for 4 hours, and transfused as previously described.¹⁰  Transfusion of platelets stored at 4°C for 48 hours compared with fresh platelets into α_Mβ₂ integrin knockout mice was also performed as previously described.⁸ 

CD62P expression on platelets was measured by FITC-labeled CD62P-specific monoclonal antibody S-12 using flow cytometry, and annexin V binding was determined using FITC-labeled annexin V (Vybrant Assay Kit; Molecular Probes). Results are expressed as percentage of positive cells compared with the appropriate controls.

A procedure for the direct galactosylation of PRP from apheresis platelet collections was developed. Optimal transfer of galactose onto exposed βGlcNAc on platelets was obtained by incubating the PRP with 1.2 mM UDP-galactose for 60 minutes at 37°C. This galactosylation protocol was field tested by galactosylating all the platelets obtained from 12 apheresis collections directly in the collection bag. Lectin FACS profiling verified a uniform platelet modification in all 12 apheresis units (Figure 1A). The modification of exposed βGlcNAc by UDP-galactose treatment was further verified by the prevention of platelet phagocytosis by THP-1 cells, which express the α_Mβ₂ integrin receptor recognizing exposed βGlcNAc (Figure 1B).¹²  Galactosylated platelets were fully functional as determined by ristocetin- and thrombin-induced agglutination/aggregation assays, and no increase in P-selectin or phosphatidyl serine exposure was induced by the treatment when compared with nongalactosylated cold-stored platelets (data not shown).

There were 4 subjects (3 males and 1 female) enrolled in the study, and all completed the study according to protocol. All the infused units (treated and control) had in vivo platelet recoveries within acceptable limits, and the mean recoveries in each group did not differ regardless of the treatment or storage temperature (Table 1). Although the recovery (26%) of the galactosylated platelets from subject 4 (unit A) was at the lower end of the expected range, the corresponding room temperature (RT)–stored control platelets (unit B) from the same subject had a recovery value of only 34%. This suggests that the low recovery of the galactosylated platelets was donor-related rather than an effect of the galactosylation process.

The platelet survival data are shown in Figure 2 and Table 1. The survivals of the 2 nongalactosylated control units stored at RT for 48 hours from subjects 2 and 4 (B units) were within the expected normal range (ie, 6.8 and 6.9 days, respectively). The platelets in the control units stored at 4°C for 48 hours without galactosylation from subjects 1 and 3 (B units) were cleared rapidly with survival times of 2.9 and 2.8 days, respectively. In contrast to what was predicted from the previous murine data,¹⁰  the human UDP-galactosylated platelet units from subjects 1 through 4 (A units) stored at 4°C for 48 hours were also cleared rapidly with survival times of 2.8, 2.2, 2.8, and 1.0 days, respectively. Therefore, no difference was observed between the mean survivals of the treated and untreated platelets stored at 4°C (2.2 ± 0.9 [SD] days and 2.9 ± 0.1 days, respectively). These results demonstrate that galactosylation of human platelets stored for 48 hours at 4°C did not improve survivals. Lectin profiling of samples obtained from all units incubated with UDP-galactose (A units) with RCA-1 and sWGA verified the galactosylation of the treated platelets (data not shown).

The safety of transfused chilled galactosylated platelets, along with any residual UDP-galactose, was evaluated by regular clinical assessments, follow-up phone interviews, monitoring the subjects' platelet counts, and testing for the presence of antibodies against both nongalactosylated and galactosylated platelets. No adverse events were observed or reported by the subjects either during the administration of the UDP-galactose–treated platelets or during the 90-day follow-up period. Platelet counts remained normal (Table 2), and there was no evidence of antibody formation against either the subjects' treated or unmodified autologous platelets (data not shown). In conclusion, there was no evidence of any adverse effects following the administration of up to 10 mL of galactosylated platelets, together with any residual UDP-galactose.

Platelet count in the storage bags remained stable. Before and afterstorage platelet counts averaged 1.57 × 10¹¹ (± 4.78 × 10¹⁰) versus 1.61 × 10¹¹ (± 3.45 × 10¹⁰), 1.97 × 10¹¹ (± 2.29 × 10¹⁰) versus 1.63 × 10¹¹ (± 3.39 × 10¹⁰), and 1.85 × 10¹¹ (± 4.12 × 10⁹) versus 1.93 × 10¹¹ (± 3.79 × 10⁹) for the galactosylated 4°C-stored, the control 4°C-stored, and the control 22°C-stored platelets, respectively.

Overall, pH, PCO₂, HCO₃, glucose, P-selectin, and annexin V binding measurements (except for unit 4A [UDP-galactose, vol 4]) were very similar among the groups (Table 3). The platelets in Unit 4A demonstrated a high annexin V binding, indicating phosphatidyl serine exposure that may have correlated with the low in vivo recovery of these platelets (26%). PO₂ values tended to be lower, whereas ESC, HSR, and morphology scores were higher after storage in the 22°C-stored platelets compared with the 4°C-stored platelets. However, there were no differences among the 4°C-stored platelets either treated or control for any of the assays.

The discrepancy between the human studies and the previous preclinical murine studies with platelets stored at 0°C for 2 hours¹⁰  was unexpected. However, 2 potentially important methodologic changes were introduced in the human study: (1) the procedure used to galactosylate the platelets, and (2) the duration of cold storage. The murine platelets in our previous study were washed and treated with 0.2 mM UDP-galactose in a resuspension buffer.¹⁰  In the present study, human PRP was directly treated with 1.2 mM UDP-galactose. However, both lectin profiling and the THP-1 phagocytosis assay confirmed efficient galactosylation of the human platelets and loss of macrophage Mac-1 binding, respectively (Figure 1).

To evaluate if the UDP-galactose treatment procedure was related to the different results, murine platelets in PRP were treated with 1.2 mM UDP-galactose or 1.2 mM UDP-glucose (as a control) for 1 hour at 37°C, followed by a washing procedure and storage of the washed platelets at 0°C for 4 hours. The short-term chilled murine platelets galactosylated directly in plasma had a similar increase in survival (Figure 3A and Table 4) as short-term, chilled, washed platelets galactosylated with 0.2 mM in resuspension buffer, as performed in our previously published murine study.¹⁰  In contrast, the control UDP-glucose–treated platelets were rapidly cleared following even short-term 4°C storage.

Another substantial difference between the human and the prior murine studies was the duration of platelet storage at 4°C. The human platelets were stored for 48 hours at 4°C before transfusion. This storage time was chosen because it had previously been demonstrated that human platelets are irreversibly damaged, resulting in markedly shortened platelet survivals after only 18 to 24 hours of 4°C storage,²⁰  suggesting that extended storage time at 4°C would be important in a human feasibility trial.

Since methods for extended storage of murine platelets were unavailable, we developed a mini-bag methodology to support the storage of murine PRP. Surprisingly, both platelet recoveries and survivals after 4 and 6 hours of cooling in plasma were similar to fresh platelets (Figure 3B and Table 4). However, there were progressive decreases in both platelet recoveries and survivals with extended storage times of 24 to 48 hours at 4°C (Figure 3B and Table 4). We documented that the reduced platelet recoveries and survivals seen after 48 hours of 4°C storage were not due to loss of the CMFDA label during storage (Figure 3B inset panel). In contrast, platelet recovery and survival were greatly diminished following only 6 hours of RT storage. We therefore compared all murine 4°C-stored platelet data to the recovery and survival of freshly isolated platelets. In vitro characterization of murine platelets stored at 4°C for 48 hours demonstrated stable pH, but some increase in P-selectin expression (18.1% ± 2.8% compared with 5.9% ± 1.4% for fresh platelets,; n = 3) and a moderate increase in annexin V binding (5.9% ± 1.4% compared with 2.3% ± 0.6% for fresh platelets; n = 3).

The recoveries and survivals of murine platelets galactosylated in plasma and stored for 48 hours at 4°C were then tested to mimic the conditions used in the human studies. Compared with fresh or 4-hour, 4°C-stored murine platelets galactosylated in plasma, murine galactosylated platelets stored for 48 hours at 4°C were rapidly cleared (Figure 3A) similar to the results found in the human studies (Figure 2). In addition, the murine galactosylated platelets were cleared at the same rate as the control UDP-glucose–treated murine platelets when both were stored for 48 hours at 4°C (Figure 3A).

Since galactosylation is aimed at eliminating recognition by the α_Mβ₂ integrin receptor, we evaluated whether 48-hour galactosylated cold-stored platelets would survive in mice lacking the α_Mβ₂ integrin receptor. Surprisingly, both wild-type and Mac-1–deficient mice cleared 48-hour extended cold-stored galactosylated platelets similarly (Figure 3C and Table 4), indicating that the α_Mβ₂ integrin receptor is not the main receptor responsible for the clearance of extended 4°C-stored platelets. Interestingly, we found increased survival of freshly prepared RT platelets after transfusion into α_Mβ₂ integrin receptor knockout mice when compared with their survival in wild-type mice (Table 4).

The primary aim of this study was to evaluate the use of enzymatic platelet galactosylation to prevent the rapid clearance of cold-stored (4°C) human platelets. The study was based on preclinical studies in a murine platelet transfusion model, which indicated that covering βGlcNAc residues on murine platelets prior to short-term (2-hour) cold storage prevented macrophage lectin-mediated platelet clearance.¹⁰  In contrast to these preclinical murine experiments, the human study demonstrated that galactosylation with UDP-galactose did not prevent the rapid clearance of 48-hour, 4°C cold-stored platelets in humans. However, the study did demonstrate that administration of up to 10 mL of galactosylated platelets, together with any residual UDP-galactose present in the platelet preparation, was safe. In addition, administration of the UDP-galactose–treated platelets did not result in the formation of any platelet antibodies directed to either galactosylated or unmodified autologous platelets. The subjects' platelet counts remained stable throughout the 90-day period of observation, providing further evidence that platelet autoantibodies had not developed.

The different outcomes of the human study and the previous preclinical murine study¹⁰  were unexpected and could potentially be attributed to several methodologic changes. One such change was the direct galactosylation of nonwashed human apheresis platelets in plasma in order to accommodate the adoption of the technology to the clinical setting. The feasibility of this approach was confirmed by lectin profiling, in vitro phagocytic assays, and by confirmation that direct galactosylation of murine PRP also prevented the in vivo clearance of 4-hour, 0°C-stored murine platelets. These data suggest that the change in the galactosylation procedure did not play a role. In addition, both the storage time and the storage conditions differed between the 2 studies. In the previously published murine study, nongalactosylated platelets were washed and stored for only 2 hours at 0°C, and this resulted in rapid platelet clearance.¹⁰  Because of prior studies suggesting that cold-induced damage to human platelets did not become irreversible until the platelets had been stored at 4°C for at least 18 hours,^1,20  we elected to store the human apheresis platelets for 48 hours at 4°C to be well beyond the period when cold-induced platelet damage might be reversible. As rapid platelet clearance was not prevented by galactosylation of human platelets that had been stored for 48 hours (Figure 2 and Table 1), we duplicated these experiments in our murine model by developing methods to support extended cold storage of murine PRP. When murine platelets were stored as PRP for 4 to 6 hours at 4°C, platelet survivals did not differ from fresh platelets, suggesting some protective effect of plasma on the cold-induced platelet injury. In order to achieve rapid platelet clearance,^8,10  extended storage of murine PRP for 24 to 48 hours at 4°C was required (Figure 3B and Table 4).

Similar to the human studies, we did not see any effect of the addition of UDP-galactose in preventing the rapid clearance of 48-hour, 4°C-stored murine platelets (Figure 3A and Table 4). We confirmed that the rapid loss of 48-hour, 4°C-stored murine platelets was not due to loss of the CMDF label during storage (Figure 3B inset panel). As an internal control, we repeated the original experiments in the mouse model and demonstrated that galactosylation prevented the accelerated clearance of murine washed platelets stored for 4 hours at 0°C (Figure 3A and Table 4). In conclusion, these results suggest that exposed βGlcNAc is not the sole factor determining the clearance of long-term 4°C-stored platelets in either mice or humans. This was confirmed by the rapid clearance of 48-hour, 4°C-stored galactosylated murine platelets in α_Mβ₂ integrin knockout mice (Figure 3C and Table 4), indicating that other receptors besides the α_Mβ₂ integrin play a role in the clearance of long-term 4°C-stored platelets.

In the murine studies performed in this report, we compared the results of 4°C storage of murine platelets to results obtained with fresh platelets. The explanation for the failure of murine platelets to demonstrate any recovery after only 6 hours of storage at 22°C is unexplained, and we are exploring potential reasons for this finding. Therefore, under our current conditions of storage, the mouse model does not duplicate results obtained with human platelets stored at 22°C.

The original finding that galactosylation rescued the circulation of short-term chilled platelets was rationalized by the hypothesis that exposed βGlcNAc represented only a minority of the exposed glycans on the surface of platelets. This minority of immature glycans is only critical for platelet clearance when the glycans cluster after cold storage. In contrast, the majority surface glycans on normal circulating platelets are fully sialylated, preventing their recognition by glycan scavenger receptor systems^21,22  Preliminary results show that exposure of immature glycans on platelets increases with storage time. Such glycans could act as ligands for carbohydrate receptors on hepatocytes and macrophages. One of these receptors, the asialoglycoprotein (ASGP) receptor, is known to recognize and mediate endocytosis of proteins and cells carrying exposed βgalactose. Interestingly, platelets lose sialic acid during aging and circulation, exposing βgalactose residues,^21,23  and in vitro desialylated platelets are cleared rapidly.^24,25  Furthermore, mice lacking the α2,3STGal-transferase IV (CMPNeuAc: βGal α2,3-sialyltransferase IV) are thrombocytopenic due to the recognition and removal of platelets with increased exposure of βgalactose.²⁶  Additionally, humans infected with the parasite Trypanosoma cruzi and other infectious agents suffer from severe thrombocytopenia due to mobilization of sialic acid from the circulating platelets.^27-29  In preliminary experiments, we have found that removal of the external domain of GP1bα carrying complex type N-linked glycans from the platelet surface by O-sialoglycoprotein endopeptidase decreased the clearance rate of transfused platelets stored at 4°C for 24 hours (data not shown). This may suggest that domains or glycans on the external part of GP1bα are important for the clearance of long-term 4°C-stored platelets. Future studies will investigate the importance of the asialo-glycoprotein and other carbohydrate receptors for the accelerated clearance of long-term cold-stored platelets.

Cold storage not only increases the exposure of immature glycans, but also causes a partial activation of the platelets as indicated by increases in several intracellular messengers and release of granule material resulting in surface P-selectin and phosphatidyl serine expression.^11,30  We observed some increase in P-selectin and phosphatidyl serine expression on murine platelets after 48 hours of cold storage. In contrast, P-selectin and phosphatidyl serine expression on the cold-stored human platelets were similar to those of platelets stored at RT (Table 3). Additionally, cold activation induces the formation of large raft aggregates, causing enrichment of several glycoproteins within detergent-resistant membranes (DRMs) of cold-activated platelets.^31-33  It is therefore plausible that DRMs may play a role in important changes, including the clustering of GP1bα, that lead to clearance of cold-stored platelets.

Due to the differences in human versus murine glycosylation it would be advantageous to further develop and test cold-stored human platelets in a humanized murine model. In this context it has been shown that the clearance of human platelets is slowed somewhat in severe combined immunodeficient (SCID) mice.³⁴  Another model could be transplantation of CD34⁺ cord blood cells into conditioned newborn Rag2^−/− gamma c^−/− mice or SCID/NOD/IL-2 receptor/gamma c–null mice, reconstituting the immune system with populations of human B, T, and dendritic cells.^35,36 

In conclusion, UDP-galactose treatment does not prevent the clearance of either human or murine platelets stored for 48 hours at 4°C. The development of a murine model supporting long-term storage of platelets could prove useful for the identification of the critical molecular mechanisms leading to the clearance of cold-stored platelets and in the development of methods to prevent such clearance.

The authors are indebted to Ginny Knight for excellent administrative support and to members of the Platelet Storage Group at the Puget Sound Blood Center: Mary Kay Jones, Todd Christoffel, Joe Valvo, and Jill Corson. We are also grateful to members of the Platelet Team at ZymeQuest: Cheryl Hill, Emily Rositter, and Marilyn Horowitz for their exceptional support. Finally we are grateful to Emma Josefsson for excellent help and discussions throughout the project.

The study was funded by ZymeQuest.

Contribution: H.H.W., A.L.S., K.M.H., V.R., H.C., and S.J.S. designed and performed experiments, analyzed and interpreted data; H.H.W., K.M.H., H.C., J.H.H., and S.J.S. wrote the paper.

Conflict of interest disclosure: H.H.W. and A.L.S. were employed by ZymeQuest. H.C. is a consultant for ZymeQuest. H.H.W. and H.C. have stock options in ZymeQuest. All other authors declare no competing financial interests.

Correspondence: Sherrill J. Slichter, Puget Sound Blood Center, 921 Terry Ave, Seattle, WA 98104-1256; e-mail: sjslichter@psbc.org.