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- MATERIALS AND METHODS
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.
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.
- Top of page
- MATERIALS AND METHODS
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.