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VASCULAR BIOLOGY
1Faculty of Medical Sciences, Framlington Place, Newcastle University, Newcastle-Upon-Tyne, United Kingdom; and 2Gambro Corporate Research, Hechingen, Germany
Submitted 3 November 2006 ; accepted in final form 30 March 2007
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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inhibitor rottlerin and the serotonin [5-hydroxytryptamine (5-HT)]2A/2C receptor antagonist ritanserin in a dose-dependent manner. Moreover, the 5-HT2A/2C receptor agonist 1,2,5-dimethoxy-4-iodophenyl-2-aminopropane mimicked the effect of HSA-AGE on PS externalization. These data demonstrate, for the first time, that HSA-AGE stimulates PS externalization in a subpopulation of platelets via the 5-HT2A/2C receptor. This may have important consequences for platelet involvement in inflammatory responses and the increased cardiovascular risk observed in individuals with diabetes and/or CKD. signal transduction; thrombosis; caspase-3
The membrane phospholipid phosphatidylserine (PS) resides in the inner leaflet of the plasma membrane of resting platelets (3), but, following activation of scramblase by PKC-, PS translocates to the outer leaflet of the plasma membrane (3, 6). A number of studies have demonstrated that chronically elevated or prolonged exposure of PS on the cell surface both increases vascular damage and results in the formation of a hypercoagulable environment by stimulating adherence of inflammatory cells to the vascular endothelium (e.g., Ref. 16) and providing a catalytic surface for assembly of the prothrombinase complex (22) that accelerates the generation of thrombin (26, 41). Interestingly, a recent study (40) has demonstrated that platelet agonists elicit PS externalization in a subpopulation of these cells and that the size of this population correlates with prothrombinase activity.
Increased platelet PS externalization has also been reported in patients with diabetes (35). Moreover, when platelets isolated from healthy controls were reconstituted with serum prepared from individuals with CKD, a rapid increase in PS externalization in a small, subpopulation was observed (37). However, the factor(s) responsible for this response is unknown.
Advanced glycation end products (AGE) arise by covalent modification of cellular and plasma proteins and form a series of heterogeneous compounds (15). AGE are substantially increased in individuals with either diabetes or CKD compared with the general population (18, 36) and may contribute to the development and progression of cardiovascular disease in these patient groups (30, 32). Although the mechanism(s) by which AGE influence the progression of atherogenesis is not completely understood, it is thought that, either by acting via specific "receptors for AGE" (RAGE) or through other mechanisms (13, 23), they can promote vascular damage, fibrosis, and inflammation, which are associated with accelerated atherogenesis (32).
The goals of our laboratory were to identify the serum factor(s) responsible for increased platelet PS externalization and investigate the potential involvement of this process in the increased cardiovascular risk observed in those individuals with diabetes/CKD. To address the first of these aims, we examined the ability of human serum albumin (HSA)-AGE to elicit platelet PS externalization and the mechanism involved.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Annexin V-FITC was purchased from Immunotech (Marseille, France), and fluorospheres for FACScan calibration were obtained from DakoCytomation (Glostrup, Denmark). The YSI 2300 STAT Plus Analyzer was purchased from Yellow Springs Instruments (Yellow Springs, OH), and the ENDOSAFE LAL Gel Clot Test was obtained from Charles River (Charleston, NC). The Superdex HR75 10/30 column was purchased from Amersham Pharmacia Biotech (Freiburg, Germany), the LS 50B Spectrofluorimeter was obtained from Perkin-Elmer (Oberlingen, Germany), and the protein standards for molecular weight calibration were from Sigma (Deisenhofen, Germany). The fluorescence-activated cell scanner (FACScan) CD61-PerCP, the caspase-3 inhibitor Z-DEVD-FMK, and the negative control Z-FAD-FMK were purchased from Becton Dickinson (Oxford, UK). Rottlerin, bisindolylmaleimide I, and Ca2+ ionophore A23187 [GenBank] were from Merck Biosciences (Nottingham, UK). HSA, ADP, penicillin, gentamycin, the 5-HT2A2C receptor antagonist ritanserin, the 5-HT2A2C receptor agonist 1,2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI), and all general chemicals were obtained from Sigma-Aldrich (Dorset, UK). The low-flux Fresenius polysulfone hemodialysis membrane was purchased from Fresenius Medical Care (Bad Homburg, Germany).
Preparation of Platelet-Rich Plasma
Blood was obtained from healthy control subjects who had no history of cardiovascular disease or hypertension and were not using any medication. Ten milliliters of blood were withdrawn using a standard 16-gauge hypodermic needle into acid citrate dextrose and then immediately centrifuged at 100 g for 15 min at 20°C (Beckman J6-MC). The platelet-rich plasma (PRP) fraction was removed and subsequently incubated under various treatment conditions as described below.
Preparation of HSA-AGE
This was prepared by incubating 1 M glucose with 50 mg/ml HSA in 100 mM PBS (pH 7.4) in the presence of 1.5 mM PMSF, 1 mM EDTA, 100 µg/ml penicillin, and 40 µg/ml gentamicin for 2, 4, or 6 wk in the dark at 37°C under sterile conditions as described previously (34). An unmodified protein control was prepared in parallel, and, at the end of the incubation, all proteins were extensively dialyzed (3 washes/18 h at 4°C) against PBS (pH 7.4, 4°C) to remove unreacted sugar, separated into aliquots, and stored at –20°C before use.
Analysis of HSA-AGE Proteins
Total HSA-AGE content was determined as arbitrary fluorescence intensity per milligram of HSA. Briefly, HSA concentration was determined by size exclusion chromatography. Protein samples were initially diluted 1:10 in 10 mM PBS containing 300 mM NaCl; 20 µl were then injected onto a Superdex HR75 10/30 HPLC column, and samples were separated using 10 mM PBS containing 300 mM NaCl. The column was calibrated using a set of proteins in the molecular weight range of 6.5–67 kDa (aprotinin, myoglobin, carbonic anhydrase, ovalbumin, and HSA). Proteins were detected by absorbance at 220 nm, and HSA was used for quantitative calibration using peak areas. AGE formation was measured by spectrofluorometry as previously described (14). Briefly, protein samples were diluted 1:1,000 in PBS (pH 7.4), and fluorescence emission was detected at 430 nm after excitation at 350 nm using a LS 50B spectrofluorimeter. Glucose concentration was determined using a Yellow Springs YSI 2300 STAT Plus Analyzer, and endotoxin levels were assessed using the ENDOSAFE LAL Gel Clot Test.
Measurement of PS Externalization in PRP
PRP was prepared as described above and then incubated with either 1) 0–2 mg/ml of 2-, 4-, or 6-wk HSA-AGE or HSA control for 10 min or 2) 0.5 mg/ml of 6-wk HSA-AGE or HSA control for increasing times up to 30 min. For comparison purposes, we also examined the effect of ADP (0.5–100 nM), a classic physiological activator of human platelets (7). PS externalization was then determined using cell membrane-impermeant, PS-specific, annexin V-FITC conjugate and CD61-PerCP as a platelet-specific marker with subsequent quantification by a FACScan supporting Lysis II software. Briefly, 7.5 µl Ca2+-binding buffer (10 mM HEPES, 140 mM NaCl, 1% BSA, and 3 mM CaCl2; pH 7.4), 3 µl of 10 mM glycine-proline-arginine-proline (GPRP), 5 µl PerCP-labeled CD-61 (glycoprotein-llb/llla) monoclonal antibody, 4 µl FITC-labeled annexin-V, and 3 µl PRP were added to FACScan-specific tubes. Blank samples were prepared as above without FITC-annexin V. Each sample was then left on ice for 15 min in the dark prior to analysis, which counted 10,000 cells from the total platelet population. The FACScan was calibrated monthly with Dako fluorospheres, and a standard curve fit procedure was used to enable data to be expressed as total numbers of binding sites.
Effect of Inhibition of Caspase 3
PRP was preincubated with 20 µM of the caspase-3 inhibitor Z-DEVD-FMK or its negative control Z-FAD-FMK (8) for 30 min and then either 0.5 mg/ml of 6-wk HSA-AGE were added for 10 min or 100 nM of ADP were added for 1 min. The extent of PS externalization was determined as described above.
Effect of Inhibition of PKC-
Two inhibitors were used in these experiments. First, PRP was preincubated for 10 min with 10 nM of the PKC inhibitor bisindolylmaleimide I; this inhibits both the classical PKC (PKC-
, -
, and -
) and novel PKC (PKC- and -
) isoforms (33, 38). Six-week HSA-AGE (0.5 mg/ml) was then added for 10 min, and PS externalization was determined as described above. Second, PRP was preincubated with 10 µM of the PKC- inhibitor rottlerin for 5 min. At this concentration, the drug has been reported to block PKC- activity by 
80–90% while having no effect on representative examples of either the cPKC isoforms e.g., PKC-, or atypical PKC isoforms, e.g., PKC-
(6). Then, either 0.5 mg/ml of 6-wk HSA-AGE were added for 10 min or 100 nM of ADP were added for 1 min. PS externalization was determined as described above.
Effect of a 5-HT2A/2C Receptor Antagonist
PRP was preincubated for 30 min with increasing concentrations (10 nM–1 µM) of the 5-HT2A/2C receptor antagonist ritanserin (19). Then, either 0.5 mg/ml of 6-wk HSA-AGE were added for 10 min or 100 nM of ADP were added for 1 min. The extent of PS externalization was determined as described above.
Effect of a 5-HT2A/2C Receptor Agonist
PRP was incubated with either 1) 1 nM–10 µM of the 5-HT2A/2C receptor agonist DOI for 10 min or 2) 1 µM DOI, a concentration previously shown to optimally activate extracellular signal-regulated kinases in healthy human platelets (9, 20) for increasing times up to 30 min. We also investigated the ability of the 5-HT2A/2C receptor antagonist ritanserin to block the effect of DOI. This agonist is classified as a category 1 drug and was used as specified in a licence issued by the Home Office. PS externalization was determined as described above.
Collection of Ultrafiltrate From Individuals With CKD or CKD and Type 2 Diabetes
Eight individuals (4 individuals with CKD and 4 individuals with CKD and Type 2 diabetes) gave their informed consent to participate in an initial study and were subject to conventional hemodialysis using a low-flux Fresenius polysulfone membrane. To collect pure ultrafiltrate, the dialysis fluid was disconnected from the dialyser for a short period 20 min after the start of treatment. The dialysis fluid pathway was evacuated, the ultrafiltrate was allowed to flow for 2–3 min, and 3 ml of ultrafiltrate were collected into an endotoxin-free tube. The system was then reconnected, and the treatment was continued.
Response to a 5-HT2A/2C receptor antagonist. PRP was preincubated with or without the 5-HT2A/2C receptor antagonist ritanserin (1 µM) for 30 min at 37°C. Ultrafiltrate (10 µl) from individuals with either CKD or CKD and Type 2 diabetes was then added, and the incubation was continued for a further 10 min. PS externalization was determined using annexin V-FITC and CD61-PerCP with subsequent quantification by FACScan. Data were expressed as total binding sites and are shown as means ± SE of four independent experiments with statistical analysis by Student's t-test.
Statistical Analysis
Statistical analysis was undertaken using Minitab 14 (Minitab, State College, PA). Experiments with ADP, caspase-3/PKC- inhibitors, the 5-HT2A/2C receptor antagonist, and ultrafiltrate were analyzed using Student's t-test and are presented as means ± SE, where P < 0.05, P < 0.01, and P < 0.001 with respect to the relevant control. Serial measurements with HSA-AGE and the 5-HT2A/2C receptor agonist were analyzed as summary measures (21), i.e., the areas under the concentration or time curves (AUC). All experiments were performed on at least three occasions.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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1 EU/ml, corresponding to <0.2 ng/ml.
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When PRP from a healthy control was incubated with both annexin V-FITC and CD61-PerCP and then analysed by flow cytometry, a single large platelet population was observed (Fig. 2, A and B). However, in the presence of 0.5 mg/ml of 6-wk HSA-AGE, a small (2%) subpopulation, expressing elevated levels of PS externalization, became apparent (Fig. 2, E and F), which was not observed with the corresponding HSA control (Fig. 2, C and D) prepared in the absence of glucose. Moreover, a small platelet subpopulation was also observed in individuals with diabetes and CKD (Fig. 2, G and H). In addition, a similar response was also seen when scramblase was activated directly (3) with a suboptimal concentration (2 µM) of the Ca2+ ionophore A23187
[GenBank]
(Fig. 2, I and J). Interestingly, the mean fluorescence intensity of the platelet subpopulation induced by HSA-AGE, A23187
[GenBank]
and that present in individuals with diabetes and CKD were comparable. We have therefore subsequently characterized the response of this small population to HSA-AGE in detail.
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ADP is a classic physiological activator of human platelets (7), but its ability to elicit PS externalization in these cells has not been reported to date. The incubation of PRP with ADP also resulted in the appearance of a small subpopulation of platelets expressing elevated PS externalization. Moreover, the mean fluorescence intensity of this cell fraction was comparable with that observed with HSA-AGE and A23187 [GenBank] and in individuals with diabetes and CKD (data not shown). The effect of ADP was concentration dependent, with a maximal response at 5 nM (Fig. 4A), but, unlike HSA-AGE, it was rapid in onset, being optimal at 1 min and sustained for at least 10 min (Fig. 4B).
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(4, 25). When PRP was preincubated for 30 min with the caspase-3 inhibitor Z-DVED-FMK (20 µM) and then subsequently challenged for 10 min with 0.5 mg/ml of 6-wk HSA-AGE, there was no inhibition of the HSA-AGE effect on PS externalization (Fig. 5), suggesting that this response does not involve caspase-3. In contrast, the response to ADP (100 nM) added for 1 min was abolished by Z-DVED-FMK (Fig. 5). The negative control Z-FAD-FMK (20 µM) had no effect (data not shown).
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inhibitor rottlerin (10 µM) completely prevented the increase in PS externalization elicited by the addition of 0.5 mg/ml of 6-wk HSA-AGE for 10 min (Fig. 6), suggesting a role for PKC- in the HSA-AGE-mediated response. The effect of 100 nM ADP added for 1 min was also abolished (Fig. 6).
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45% and from individuals with both CKD and Type 2 diabetes by 35% (Fig. 9).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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We report here, for the first time, that HSA-AGE stimulates PS externalization in a small subpopulation of human platelets. PS externalization via scramblase activation fulfills a dual role. It is critical for the efficient propagation and control of coagulation and thrombosis (41) or can act as an important and necessary signal for apoptotic cell removal by phagocytes (4). In either case, PKC- activation is required, and our use of both a cPKC/nPKC inhibitor (bisindolylmaleimide I) and a specific inhibitor of this isoform (rottlerin) supports a role for this signaling molecule in HSA-AGE-mediated PS externalization in human platelets. Moreover, it has previously been shown in other cell types that 5-HT2A/2C receptor stimulation (see below) correlates with increased PKC- activity (31).
In nucleated cells, the time course of PS externalization relating to thrombin formation is very different compared with that linked to apoptosis, i.e., activation-induced scramblase activity is rapid and transient, whereas it is delayed and sustained during apoptosis (6). The time course we report here for HSA-AGE-mediated PS externalization correlates with cell activation rather than apoptosis in nucleated cells.
PKC- is cleaved and activated during apoptosis by caspase-3 (25). In contrast, cell activation-induced scramblase activity, although also dependent on PKC-, is independent of caspase-3 (6). Our demonstration that HSA-AGE-mediated PS externalization was not blocked by the caspase-3 inhibitor Z-DEVD-FMK also correlates with cell activation in nucleated cells.
It is however, possible that, in platelets, these two events are interrelated. When activated by a hemostatic agent, platelets undergo a number of changes, including cell shrinkage, PS externalization, and cell disintegration. These changes promote hemostasis by inducing platelet aggregation/adhesion and activation of the coagulation cascade (39). Recent studies (29, 39) have identified the presence of caspase activity (including caspase-3) in human platelets, although, at present, their precise role in these cells remains unclear. Our data imply that the effect of ADP on PS externalization, but not that of HSA-AGE, is mediated via caspase-3. The significance of this requires further investigation.
A heterogeneous response of platelets to PS externalization has been reported previously (2, 40). A recent study (40) demonstrated the effects of thrombin and collagen on PS externalization in a small subpopulation of human platelets. Moreover, these authors reported that the effect of thrombin (5 nM), for example, was mediated on 4% of the total platelet population (40), a value comparable with that we reported here with HSA-AGE. Wolfs et al. (40) also showed that the mean annexin V fluorescence intensity of the platelet subpopulation was comparable, regardless of the agonist (or combination of agonists) used, with that found upon stimulation with the Ca2+ ionophore, which elicited a maximal response. We report here a similar observation following incubation of platelets with either HSA-AGE, ADP, and DOI or in platelets from individuals with diabetes/CKD compared with the effect of A23187.
[GenBank]
A good correlation (r = 0.99) was also seen between the size of the platelet subpopulation and procoagulant activity, and thus these authors concluded that the increased procoagulant activity of a platelet suspension is related to the increase in the fraction of platelets exhibiting maximal PS exposure rather than to the entire platelet suspension (40).
It is intriguing to speculate on the criteria that sensitize a subpopulation of cells (or desensitize the majority of platelets) to these agonists. Two possibilities are differences in Ca2+ homeostasis that may then be reflected in scramblase activity and/or in specific agonist receptor density. The relationship between platelet age and susceptibility to PS externalization is also unknown. The development of novel treatments for various pathophysiological states may be facilitated by a more detailed understanding of these phenomena.
It is clear that many effects of AGE on a number of cell types are mediated via RAGE (23). However, as far as we are aware, RAGE has not been identified on human platelets, and, in preliminary experiments with anti-RAGE antibody, using methodology that confirmed the presence of RAGE on mononuclear phagocytes (28), we were unable to detect this receptor on human platelets (unpublished observations). A previous study in human platelets demonstrated that ADP-induced aggregation could be enhanced by the addition of AGE but that this effect was diminished by the addition of sarpogrelate (13), a serotonin 5-HT2A receptor antagonist (24). This suggests that the effects of AGE on human platelets are mediated via the 5-HT2A receptor. As sarpogrelate is not available commercially, we employed ritanserin, a 5-HT2A/2C receptor antagonist (19), to establish that HSA-AGE-mediated PS externalization is mediated via this receptor in these cells. The presence of 5-HT2A/2C receptors on human platelets has long been established (e.g., Ref. 1). The inability of ritanserin to inhibit the response to ADP established that it does not act in a nonspecific manner on other components of the pathway such as PKC- or scramblase activity. To then confirm that activation of this receptor is indeed linked to PS externalization, we employed the 5-HT2A/2C agonist DOI (9) and demonstrated that this agent mimics the effect of HSA-AGE in eliciting a transient, dose-dependent stimulation of PS externalization in a subpopulation of human platelets that is maximal at 10 min. Thus, our data support a signaling pathway in which HSA-AGE binds to the 5-HT2A/2C receptor leading to PS externalization via a mechanism involving PKC- but that is independent of caspase-3.
Although we did not assess HSA-AGE in terms of units of AGE per milligram of protein, data from previous publications indicate that the concentration range we used is similar to that detected in disease states in vivo (13, 17). Similarly, we did not attempt to establish the active component(s) among those likely to be present in our HSA-AGE preparation, which was responsible for the effects we have described. However, a previous study (12) examining the ability of BSA-AGE to potentiate ADP-mediated platelet macroaggregation demonstrated that neither pentosidine nor N-(carboxymethyl)lysine was responsible (12). Thus, the epitope(s) of HSA-AGE responsible for mediating platelet PS externalization remains to be established.
Evidence that the pathway we have described is of physiological and clinical significance is provided by the demonstration that the 5-HT2A/2C receptor antagonist ritanserin, under conditions that abolished PS externalization in response to HSA-AGE, also caused substantial inhibition of the effect elicited by ultrafiltrate from individuals with CKD alone and from patients with both CKD and Type 2 diabetes. This suggests that a significant fraction of the elevated platelet PS externalization observed in these individuals is mediated via the same receptor as the HSA-AGE generated in vitro.
Our study represents an important first step toward identifying the serum factor(s)/mechanism(s) responsible for increased platelet PS externalization and potentially facilitates the development of novel treatments aimed at manipulating platelet responses in pathophysiological states such as diabetes, CKD, myocardial infarction, and stroke.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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  Y. Wang, W. Beck, R. Deppisch, S. M. Marshall, N. A. Hoenich, and M. G. Thompson Differential effects of dialysis and ultrafiltrate from individuals with CKD, with or without diabetes, on platelet phosphatidylserine externalization Am J Physiol Renal Physiol, January 1, 2008; 294(1): F220 - F228. [Abstract] [Full Text] [PDF]
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