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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

BACKGROUND: Annexin V binding to platelets (PLTs) is considered the gold standard for monitoring phosphatidylserine (PS) exposure. However, recent comparison of annexin V with the new calcium-independent PS probe lactadherin revealed that annexin V requires a certain threshold of PS exposure (2%-8%) for binding to occur. The aim of this study was to compare annexin V and lactadherin labeling of PLTs in PLT concentrates (PCs).

STUDY DESIGN AND METHODS: Optimal labeling conditions for lactadherin and annexin V were established and then compared in either resting or calcium ionophore (CI)-activated PLTs from normal whole blood. Furthermore, 40 PCs (20 apheresis-derived and 20 pooled buffy coat–derived) were stored under standard blood bank conditions and PLT activation was monitored by measuring PS exposure with annexin V and lactadherin along with CD42b, CD61, and CD62P by flow cytometry on Days 1, 3, 5, and 7.

RESULTS: Lactadherin reported a higher exposure of PS than did annexin V in normal PLTs at submaximal doses of CI. PLTs from both types of concentrate, as expected, demonstrated evidence of increased activation during storage using annexin V, lactadherin, CD42b, or CD62P. However, a significantly higher percentage of PS-positive PLTs was found with lactadherin than annexin V.

CONCLUSION: PS exposure on the surface of stored PLTs has been previously underestimated due to the wide use of annexin V. Lactadherin provides a truer reflection of the degree of PS exposure and offers a new calcium-independent approach to studying PLT activation and/or apoptosis.

ABBREVIATIONS:
AP-PC(s)

apheresis-derived platelet concentrate(s)

BC-PC(s)

buffy coat–derived platelet concentrate(s)

CI

calcium ionophore

F-P ratio

fluorescein-to-protein ratio

MP(s)

microparticle(s)

PC(s)

platelet concentrate(s)

PRP

platelet-rich plasma

PS

phosphatidylserine

During storage under standard blood bank conditions, platelets (PLTs) undergo many alterations that may adversely affect their posttransfusion viability and function. A battery of tests have been applied to study the metabolic, structural, and functional changes that PLTs undergo during storage that are collectively known as the PLT storage lesion.1,2 The clinical significance of these tests, however, is unclear because there is no single test that can accurately predict the posttransfusion survival and/or recovery of PLTs.3 Therefore, the current gold standard for the evaluation of the clinical efficacy of transfused PLT concentrates (PCs) are in vivo survival studies of radiolabeled PLTs with all of their inherent limitations.

Among the important alterations that PLTs undergo during storage is the externalization of phosphatidylserine (PS).4,5 In resting PLTs, anionic phospholipids (e.g., PS) are predominantly asymmetrically distributed on the inner leaflet of the plasma membrane. However, when PLTs are activated with an agonist that induces a prolonged rise in intracytosolic calcium levels, PS is translocated to the outer leaflet. This process is also referred to as the PLT procoagulant response as it transforms the PLT membrane into a negatively charged surface where coagulation factors can assemble to efficiently generate thrombin.6 PS exposure is also one of the hallmarks of programmed cell death, or apoptosis, where it is widely believed to potentially serve as a recognition cue for engulfment by phagocytes.7

Lactadherin is an opsonin released by stimulated macrophages and characterized by a PS-binding motif and an integrin-binding motif. This structure allows lactadherin to bridge PS exposing apoptotic cells to macrophages (through binding αvβ3 or αvβ5) facilitating their engulfment.8,9 More recently, lactadherin has been used as a probe for the detection of PS on the surface of cells. It has been suggested that lactadherin is not only more sensitive as a PS-binding probe, but, unlike annexin V, it binds in a calcium independent manner. It has been shown using synthetic membranes with variable PS and phosphatidylethanolamine contents that lactadherin detects low levels of PS (0.5%) independently of phosphatidylethanolamine content. In contrast, annexin V binding was detected only when PS was 8 percent and in the presence of 2 percent phosphatidylethanolamine content.10 Furthermore, the actual binding properties of lactadherin and annexin V appear to be different because lactadherin, but not annexin V, also preferentially binds to highly curved membranes.11 More recently, it has been shown that lactadherin detects PS exposure earlier than annexin V on leukemic cells undergoing apoptosis and reports higher exposure of PS on the surface of either activated red blood cells (RBCs) or PLTs.10,12 Although PLTs express small amounts (several hundred copies) of the vitronectin receptor αvβ3 (but not αvβ5),13 lactadherin appears to bind selectively to PS on the surface of PLTs because it was inhibited by PS-containing phospholipid vesicles and by antibodies to the C2 domain.14

Most studies on PLT activation/apoptosis within PCs have traditionally used annexin V as the probe. Because of the apparent superior properties of lactadherin over annexin V for the detection of PS, we hypothesized that lactadherin could also be more sensitive than annexin V at the detection of PS exposure on PLTs within PCs. The main aim of this study was therefore to compare the amount of PS exposure reported by annexin V and lactadherin on the surface of PLTs stored under standard blood bank conditions for up to 7 days.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Reagents

Bovine serum albumin (BSA) and calcium ionophore (CI) were purchased from Sigma (Poole, Dorset, UK). Fluorescein isothiocyanate (FITC)-labeled anti-human CD61, phycoerythrin (PE)-labeled anti-human CD61, and PE-labeled anti-human CD62P, and their isotype controls FITC-labeled mouse immunoglobulin G1 (IgG1), PE-labeled mouse IgG2a, and PE-labeled mouse IgG1 were purchased from BD PharMingen (Oxford, Oxfordshire, UK). PE-labeled anti-human CD42b (SZD) and its isotype control PE-labeled mouse IgG1 were purchased from Beckman Coulter (High Wycombe, Buckinghamshire, UK). FITC-conjugated annexin V was from Roche Diagnostics Ltd (Burgess Hill, West Sussex, UK).

Lactadherin, free of lipids and protein contaminants, was purified from fresh bovine milk essentially as described previously.15 Purity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealing only two bands at Mr 47 and 52 kDa corresponding to the two glycosylations variants, and by N-terminal amino acid sequencing demonstrating more than 97 percent purity. Protein concentration was determined by amino acid analysis with postcolumn ortho-phthalaldehyde derivatization. Lactadherin was labeled with FITC using a FITC protein labeling kit as instructed (FluoReporter, Molecular Probes, Eugene, OR). A 20-fold molar excess of FITC was added to the lactadherin. After incubation for 1 hour at room temperature in the dark, free fluorescein was removed by gel filtration using a microspin column. Two batches of lactadherin were prepared with fluorescein-to-protein (F-P) ratios of 1.1 and 0.6, respectively.

PLT preparation

This study was approved by the Oxford Radcliffe Hospitals Ethics Committee (Oxford, UK). Pooled buffy coat–derived PCs (BC-PCs) and apheresis-derived PCs (AP-PCs) were prepared according to UK blood transfusion service guidelines.16 For the preparation of BC-PCs, 80 whole blood units (450 ± 50 mL) were collected on Day 0 using a bottom-and-top pack system into 63 mL of citrate-phosphate-dextrose anticoagulant. Whole blood was centrifuged at 2800 × g for 11.5 minutes (Sorval RC3BP centrifuge, Kendro, Inc., Newtown, CT) and processed into RBCs, plasma, and a buffy coat using an automated blood component extractor (Optipress II system, Baxter Healthcare, Newbury, UK). Buffy coats were stored overnight at 22°C before pooling 4 units with a unit of ABO matched plasma and centrifuged (700 × g, 5 min) on Day 1. PCs were filtered using an in-process leukoreduction filter (Autostop, Pall Biomedical, Portsmouth, UK) and stored in CLX-HP bags (PVC-TOTM, Pall Biomedical).

AP-PCs were collected using an apheresis machine with Version 5 software and citrate-plasticized PVC PLT bags (Trima Accel, Gambro BCT, Lakewood, CO). These machines perform leukoreduction during the donation process by passing the blood via a leukoreduction system chamber containing fluidized particle-bed technology (LRS chamber). PLTs were resuspended in 100 percent plasma containing acid-citrate-dextrose (ACD).

PCs were stored at 20 to 24°C under continuous gentle agitation in a monitored PLT storage incubator (Helmer PLT incubator, Noblesville, IN). Sampling was performed on Days 1, 3, 5, and 7 of storage where a 15-mL sample was taken under sterile conditions.

For the preparation of fresh PLT-rich plasma (PRP), whole blood from healthy volunteers was collected into 4.5-mL blood collection tubes (Vacutainer, Becton Dickinson, Oxford, UK) containing 0.105 mol per L trisodium citrate (1:9, vol/vol) and centrifuged at 200 × g for 10 minutes at room temperature.

PLT counting and pH measurement

PLT counts were measured using a hematology full blood analyzer (XE-2100, Sysmex, Milton Keynes, UK). Measurement of pH was performed in an open system at 22°C on a pH meter (PHA 230, CAMLAB, Cambridge, UK).

Flow cytometry measurements

All flow cytometric analysis was performed on PC or PRP samples diluted in Isoton (Becton Dickinson) to obtain a PLT count of 100 × 109 per L. Measurement of PS by annexin V was performed by incubating 5 µL of diluted PLTs at room temperature with 2 µL of FITC-labeled annexin V (final concentration, 100 nmol/L) and then diluting in 43 µL of N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES)-buffered saline (0.145 mol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgSO4, 10 mmol/L HEPES, pH 7.4; HBS) containing 2.5 mmol per L calcium or 5 mmol per L K2-ethylenediaminetetraacetate. The latter was used to determine the level of nonspecific binding as annexin V binding to PS is calcium-dependent. After incubation for 10 minutes at room temperature, 1 mL of the appropriate buffer was added and samples were analyzed immediately. Detection of PS by lactadherin was performed by incubating 5 µL of diluted PLTs for 10 minutes at room temperature with 10 µL of FITC-labeled lactadherin (100 nmol/L final concentration) followed by dilution in 35 µL HBS containing 0.1 percent BSA without calcium. To adjust for any autofluorescence, a sample was prepared exactly as the above but without the addition of lactadherin. Samples were then resuspended in 1 mL of the same buffer and analyzed immediately. In some experiments, double labeling with PE-CD61 and FITC-lactadherin or FITC-annexin V was undertaken. In these experiments, 5 µL of diluted PLTs was incubated with 5 µL of PE-CD61 for 10 minutes at room temperature and made up to 50 µL with the appropriate buffer before labeling with lactadherin or annexin V as described previously. In activation experiments, 5 µL of diluted PRP was first incubated with 5 µL of CI (final dilution, 0.1, 0.5, or 10 µmol/L) in the appropriate buffer at 37°C for 15 minutes before labeling with annexin V or lactadherin. Analysis of PLT activation markers was performed by incubating 5 µL of diluted PLTs for 10 minutes at room temperature with 5 µL of PE-conjugated anti-CD42b, 5 µL of FITC-conjugated anti-CD61, 5 µL of PE-conjugated anti-CD62P, or 5 µL of the appropriate isotype control and made up to 50 µL with Isoton. Samples were then resuspended in 1 mL of the same buffer and analyzed immediately.

Flow cytometric analyses were performed using a flow cytometer equipped with its accompanying software (FACSCalibur and CellQuestPro, respectively, Becton Dickinson). Measurements were made under logarithmic gain and at least 10,000 PLT events were collected. PLTs were identified based on their characteristic log forward and side scatter. An electronic gate was placed around the PLT cloud and the identity of events was verified on a separate sample labeled with FITC-conjugated anti-CD61. It was ensured that the events in this gate are more than 95 percent positive for CD61. Suitable controls were used as appropriate to set the background noise at less than 2 percent. Percentages of events appearing above the background level were then recorded. The mean fluorescence intensity (MFI) of the total population was also recorded. In experiments involving double labeling with PE-CD61 and FITC-annexin V or FITC-lactadherin, color compensation was performed to eliminate signal overlap. In these experiments, no electronic threshold was imposed and events were identified based on their positivity for CD61. This is done to include PLT-derived microparticles (MPs) in the analysis. The percentages of events positive for CD61 and annexin V or lactadherin were then recorded.

Statistical analysis

Experiments involving PCs were performed in singleton and on all PCs (n = 40), apart from lactadherin where each batch was used to test 20 PCs (10 AP-PCs and 10 BC-PCs). Experiments on PRP have been carried out at least three times. Data were analyzed using a statistical package (SPSS, SPSS, Inc., Chicago, IL) and a biostatistics, curve fitting, and scientific graphing program (GraphPad Prism, GraphPad Prism Software, Inc., San Diego, CA). Statistical differences were measured using a t test. Only CD62P results were abnormally distributed and, therefore, a nonparametric test was used. A p value of less than 0.05 was considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Lactadherin binding to PS on the surface of PLTs

Preliminary studies were conducted to optimize lactadherin binding to PS on the surface of normal PLTs. Titration experiments on PLTs treated with 10 µmol per L CI, conditions that induce PS externalization in more than 90 percent of PLTs, have revealed that the minimal saturating concentration of lactadherin was 100 nmol per L. Moreover, preliminary experiments on PLTs expressing PS, either spontaneously during storage or activated by CI, have shown that lactadherin binding is the same in the presence or absence of calcium (data not shown). These results are in agreement with those reported previously.12 In addition, binding of FITC-lactadherin or FITC-annexin V to fully activated PLTs (91 ± 2 and 90 ± 4, respectively) was inhibited in the presence of 50-fold molar excess of unlabeled lactadherin (45 ± 9 and 50 ± 8, respectively; p < 0.05; Fig. 1). It is unclear why 50-fold molar excess of unlabeled lactadherin did not block binding of labeled annexin V or lactadherin completely but Dasgupta and colleagues12 also reported similar findings using the same concentration. All experiments thereafter were conducted using lactadherin at 100 nmol per L final concentration and in the absence of calcium.

image

Figure 1. Inhibition of labeled annexin V (AV) and lactadherin binding to fully activated PLTs in the presence of unlabeled lactadherin. Fresh PRP (n = 3) activated with CI (10 µmol/L) were incubated in the presence (▪) or absence (□) of 50-fold molar excess of unlabeled lactadherin for 20 minutes at room temperature. Subsequently, FITC-lactadherin or FITC-annexin V was added. Unlabeled lactadherin significantly inhibited binding of FITC-annexin V (p = 0.01) and FITC-lactadherin (p = 0.007) to PS on activated PLTs. (Data presented as mean ± SD.)

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PS exposure on the surface of resting and CI-activated PRP

PRP, prepared from freshly drawn whole blood, was treated with 0.1, 0.5, or 10 µmol per L CI at 37°C before labeling with annexin V or lactadherin. Lactadherin detected a significantly higher exposure of PS on resting PLTs and on the surface of PLTs treated with 0.1 µmol per L but not with 0.5 or 10 µmol per L CI (Fig. 2).

image

Figure 2. Binding of annexin V and lactadherin to resting and CI- activated PLTs. Lactadherin (▪; F-P ratio, 1.1) detected significantly a higher percentage of PS-positive PLTs than did annexin V (□) in resting (9.4 ± 2.8 vs. 1.8 ± 0.7, p < 0.01) and PLTs treated with 0.1 µmol per L CI (18.4 ± 6.3 vs. 11 ± 4.1, p = 0.029; n = 3). (Data presented as mean ± SD.)

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Detection of PLT-derived MPs within PCs

In a subset of experiments, the percentages of CD61/PS-positive PLTs and PLT-derived MPs were measured within Day 6 and Day 8 AP-PCs. Samples were either 1) singly labeled with annexin V or lactadherin or 2) double labeled with CD61 and annexin V or lactadherin. In single-labeling experiments, only PLTs were included in the analysis whereas in double-labeling experiments MPs were also included. In double-labeling experiments, the inclusion of MPs in the analysis resulted, as expected, in increase in the percentage of PS-positive events detected by both annexin V and lactadherin. However, lactadherin detected a significantly higher percentage of CD61/PS-positive events than did annexin V on Day 6 but not on Day 8 (Fig. 3).

image

Figure 3. Measurement of MPs within Day 6 and Day 8 PCs by lactadherin (▪) and annexin V (□). Representative flow plot showing the gating strategy for MPs. Forward scatter threshold was removed and MPs were identified as those particles positive for CD61 but below the PLT cloud (A). Double labeling (DL) of PLTs with CD61 and lactadherin or annexin V showed that lactadherin reports a significantly higher percentage of PS/CD61-positive events than did AV on Day 6 (21.5 ± 6.5 vs. 13.8 ± 6, p = 0.02) but not on Day 8 (28 ± 11 vs. 22.2 ± 15, p > 0.05). Similar to other days, lactadherin detected a significantly higher percentage of PS than did annexin V on both Day 6 (17.5 ± 3.8 vs. 9.7 ± 4.5, p < 0.05) and Day 8 (21.4 ± 2.8 vs. 13.7 ± 2.3, p < 0.05; n = 5) (B). (Data presented as mean ± SD.)

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Exposure of PS on the surface of PLTs within PCs during storage

PLT contents (median, range) in AP-PCs and BC-PCs were 279 (260-510) × 109 and 324 (244-395) × 109, respectively. The pH remained essentially unchanged during storage (Table 3). The PS exposure on the surface of PLTs within PCs was measured during storage for up to 7 days. We first measured PS exposure in 20 PCs (10 AP-PCs and 10 BC-PCs) using annexin V and lactadherin with high F-P ratio (1.1) and then measured an additional 20 PCs (10 AP-PCs and 10 BC-PCs) using the same annexin V but this time using lactadherin with low F-P ratio (0.6). The difference between the F-P ratios of the two batches of lactadherin was likely to be caused by a change in the kit labeling performance with time as the same batch of purified protein was used.

Table 3. Characteristics of PLTs during storage
Variable*Day 1Day 3Day 5Day 7
  • * 

    Data are reported as mean ± SD apart from CD62 percent and MFI, which is reported as median (range).

  • † 

    p < 0.05 compared to Day 1.

  • ‡ 

    p < 0.05 AP-PC compared to BC-PC at the same day.

pH (22°C)    
 AP-PC7.4 ± 0.097.5 ± 0.087.4 ± 0.17.2 ± 0.2
 BC-PC7.1 ± 0.67.4 ± 0.67.4 ± 0.67.3 ± 0.5
CD42b %    
 AP-PC97.2 ± 2.897.6 ± 1.796.9 ± 2.694.1 ± 6.1
 BC-PC98.3 ± 1.797.6 ± 1.797.7 ± 2.196.5 ± 2.8
CD42b MFI    
 AP-PC207.7 ± 25.5194 ± 37.7160.3 ± 25.1158.9 ± 28.9
 BC-PC164.1 ± 30146.2 ± 18.7140.2 ± 22.1132.2 ± 16.9
CD62P %    
 AP-PC8.1 (2.6-24.4)13.3 (5.4-22.6)18.8 (5.5-38.8)28.8 (9.3-46.8)
 BC-PC8.5 (2.4-36)10.2 (5.9-32.9)23.8 (7.3-43.3)22.2 (12.5-57.5)
CD62P MFI    
 AP-PC7.1 (3.9-13.5)10.2 (6-15.5)12.2 (5.8-24.8)14.1 (6.7-29.9)
 BC-PC6.1 (3.8-18.6)8.4 (5.9-17)15.2 (6.4-22.1)12.4 (7.9-21.8)

There were no significant differences on most days between PS exposure, as reported by either annexin V or lactadherin, in AP-PCs or BC-PCs. However, although annexin V and lactadherin both detected a significant gradual increase in PS exposure during storage, lactadherin (both high and low F-P ratio) always reported a significantly higher percentage of PS-positive PLTs at all time points and irrespective of production method (apart from AP-PC Day 1 using low F-P ratio lactadherin, p = 0.08; Tables 1 and 2 and Fig. 4). The MFI reported by lactadherin, on the other hand, was dependent on its F-P ratio. Whereas high F-P ratio lactadherin always reported significantly higher MFI than annexin V, low F-P ratio lactadherin reported either similar or lower MFI than annexin V (data not shown). The difference in the MFI reported by the two batches of lactadherin is expected as the higher the F-P ratio the higher is the amount of fluorescence (or MFI). This difference in F-P ratio, however, should not affect the binding of the probe to cells. Hence, both batches of lactadherin detected higher percentages of PS-positive PLTs than annexin V, despite the significant differences in the MFI caused by the difference in F-P ratio. Although comparing the percentages of PS reported by the two batches revealed differences on some days, this is not likely caused by the difference in F-P ratio because it was variable and, on some occasions, it was matched by annexin V results, suggesting that this may have been caused by variability in PS exposure between PCs (Tables 1 and 2).

Table 1. Percentages of PS exposure in AP-PCs*
 Annexin V (n = 10)Lactadherin, high (n = 10)p ValueAnnexin V (n = 10)Lactadherin, low (n = 10)p Valuep Value (high vs. low lactadherin)
  • * 

    Each batch of lactadherin (high and low F-P ratio) was used to measure PS exposure in 10 PCs and the results were compared with annexin V. Data are presented as mean ± SD.

  • a

    p < 0.05,

  • b

    p < 0.01, or

  • c

    p < 0.001 compared to Day 1 for the same probe.

Day 15.7 ± 2.215.6 ± 6.50.00035.2 ± 2.57 ± 40.080.003
Day 39.2 ± 4.1a21.8 ± 8<0.00016.8 ± 3.713 ± 6a0.0050.02
Day 515.2 ± 3.5c32.2 ± 10.8b0.00112.2 ± 4.2b37.5 ± 5.6c<0.00010.2
Day 719.4 ± 5.8c30.2 ± 7.7b0.000119.5 ± 5.4c34.3 ± 14.3c0.0030.4
Table 2. Percentages of PS exposure in BC-PCs*
 Annexin V (n = 10)Lactadherin, high (n = 10)p ValueAnnexin V (n = 10)Lactadherin, low (n = 10)p Valuep Value (high vs. low lactadherin)
  • * 

    Each batch of lactadherin (high and low F-P ratio) was used to measure PS exposure in 10 PCs and the results were compared with annexin V. Data presented as mean ± SD.

  • a

    p < 0.05,

  • b

    p < 0.01, or

  • c

    p < 0.001 compared to Day 1 for the same probe.

Day 110.1 ± 3.713.6 ± 4.80.024.4 ± 2.67.1 ± 1.30.030.007
Day 310.9 ± 620 ± 7.3a0.0015.7 ± 2.6a13.3 ± 4.8b0.0050.02
Day 514.8 ± 5.7b22.5 ± 7.4a0.00914.8 ± 6.1c34 ± 17.3b0.00070.07
Day 722.8 ± 7.5c34.9 ± 11.5c0.0414.7 ± 6.5b36 ± 12.3c<0.00010.8
image

Figure 4. Detection of PS-positive PLTs by annexin V and lactadherin using flow cytometry. Representative flow plots showing PLT gating (A) and labeling of PLTs from Day 7 PCs with annexin V and lactadherin and their controls (B).

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PLT activation and glycoprotein expression

Although the percentage of CD42b (GPIb) remained unchanged, the MFI decreased significantly during storage in both AP-PCs and BC-PCs. However, BC-PCs showed significantly lower CD42b MFI than AP-PCs on all days. In both types of PCs, a fraction of PLTs expressed CD62P (P-selectin) on Day 1. This increased significantly during storage, reaching a median of more than 20 percent on Day 7. Similarly, the MFI of CD62P-positive PLTs increased significantly during storage (Table 3). In contrast, there was no appreciable change on the expression of CD61 (GPIIIa) in both types of PCs throughout storage (data not shown).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

This study compared the exposure of PS on the surface of PLTs by the widely used calcium-dependent probe annexin V and the new probe lactadherin. We confirmed that lactadherin binding is calcium-independent and is more sensitive than annexin V at detecting low levels of PS. We have also extended these observations to PCs produced by either apheresis or buffy coat methods. We report here for the first time that lactadherin detects a significantly higher percentage of PS exposing PLTs in PCs than annexin V. The results with annexin V are also comparable to those published previously which show that after 5 or 7 days of storage approximately 20 percent of PLTs become PS-positive.4,5 The new data with lactadherin now suggest that more than 30 percent of PLTs at the end of their storage time become PS-positive. These results indicate that PS exposure on the surface of stored PLTs has been previously underestimated due to the wide use of annexin V. Given the likely important role of PS in both PLT procoagulant activity and clearance, more accurate detection of its exposure is likely to be of clinical significance. For example, a recent study has shown that most adherent PLTs at the site of vascular damage bound lactadherin but not annexin V. This suggests that these PLTs expressed PS levels below the detection limit of annexin V, yet this exposure was sufficient to induce thrombosis in an animal model. Furthermore, in vitro results also suggest that PLTs may express low levels of PS that are sufficient to induce thrombin generation and can be blocked by lactadherin but not annexin V.17

Currently there is no consensus on the best in vitro test that can predict PLT viability after transfusion.18 Exposure of CD62P has been suggested to play a role in the removal of PLTs after transfusion, and therefore it may be a useful predictor of posttransfusion PLT viability.19 However, thrombin-activated PLTs expressing CD62P circulate and function normally after transfusion as was shown in animal models.20,21 Tests such as extent of shape change, morphology scores, and hypotonic shock response show reasonable correlation with posttransfusion PLT viability.22 However, there is evidence to suggest that PLT shape change, for example, may be reversible under certain conditions.3,23 This reversibility of many of the alterations that PLTs undergo during storage is partly responsible for the lack of correlation of many in vitro tests with posttransfusion PLT viability. Rinder and coworkers24 have shown that alterations induced by a transient metabolic stress (e.g., α-granule release and aggregation responsiveness) were reversible upon metabolic rescue. Importantly, this stress, however, did not induce PS externalization. This suggests that PS exposure may be associated with more permanent damage to PLT viability. Therefore, if indeed PS exposure plays a role in the clearance of stored PLTs, its measurement may provide a more reliable approach to predicting the posttransfusion viability of PLTs. Furthermore, the use of lactadherin will provide a more accurate measurement of PS exposure. Although there is some evidence to suggest an association between PS exposure and clearance of PLTs,25,26 more in vivo studies will be needed to examine the role of PS in the removal of PLTs stored under standard blood bank conditions.

Lactadherin provides two important advantages for the assessment of PS exposure over annexin V. First, it is more sensitive than annexin V when PLTs are minimally activated or at an early stage of apoptosis. Of note, this higher sensitivity was not affected by differences in F-P ratio because reduction of nearly 50 percent of bound fluorescein to lactadherin did not largely influence its ability to detect PS exposing PLTs. However, lactadherin appears to be equivalent to annexin V when PLTs are fully activated or express a sufficient concentration of PS for annexin V binding (Fig. 1). Second, whereas annexin V requires the presence of near physiologic levels of calcium for binding, lactadherin binds to PS in a calcium-independent manner.

The externalization of PS is also intimately related to shedding of MPs.27 Owing to their small surface area, it is expected that some MPs express a limited number of PS copies that are below the binding threshold of annexin V. Our results suggest that lactadherin is either equivalent or more sensitive than annexin V at detecting PS exposing PLT-derived MPs (Fig. 3). The higher sensitivity of lactadherin and/or its calcium independence makes it a more attractive probe for the detection of PS-positive MPs than annexin V. Those procoagulant MPs may improve the overall hemostatic potential of PCs. However, MPs in PCs may also exert pathologic effects.27,28 Therefore, more accurate measurement of MPs in blood products may be of important clinical significance. We are currently performing more detailed studies to confirm these findings.

PLT activation during collection and storage of PLTs is well documented19,29 and the degree of activation may depend on the mode of production.30 In this study, the degree of CD62P and PS exposure in both AP-PCs and BC-PCs was similar. Although previous studies have found that BC-PCs show lower levels of activation than AP-PCs,4 this may depend on the type of apheresis machine used, because the newer generation of machines may theoretically induce lower activation levels. Similar to some previous studies on PCs stored up to 7 days,4,31 we observed a decrease in GPIb expression without significant change in the percentage of PLTs expressing this glycoprotein. On the other hand, CD61 expression remained essentially unchanged, which is in line with many previous studies.4,32

The clinical significance of measuring PS exposure by lactadherin during storage of PCs will need to be verified by in vivo studies. However, with methods of pathogen inactivation that allow safe prolongation of the PC shelf life moving toward wider use, the need for tests that can predict the quality of PCs is becoming increasingly important. Because of its established role in the removal of apoptotic cells and its likely irreversible nature, measurement of PS exposure may provide useful information on PLT viability after transfusion and the use of lactadherin may improve the sensitivity of this approach.

ACKNOWLEDGMENTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

We thank staff at issue departments of National Blood Service—Oxford and Bristol for technical help with collection and preparation of platelet concentrates. The authors declare no conflict of interests with this study.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES