http://informahealthcare.com/plt ISSN: 0953-7104 (print), 1369-1635 (electronic) Platelets, 2015; 26(2): 119–126 ! 2015 Informa UK Ltd. DOI: 10.3109/09537104.2014.888546
ORIGINAL ARTICLE
Effect of steroids on the activation status of platelets in patients with Immune thrombocytopenia (ITP) Preeti Bhoria1*, Saniya Sharma2*, Neelam Varma3, Pankaj Malhotra1, Subhash Varma1, & Manni Luthra-Guptasarma2 1
Departments of Internal Medicine, 2Department of Immunopathology, and 3Department of Hematology, Postgraduate Institute of Medical Education and Research, Chandigarh, India Abstract
Keywords
The activation status of platelets in Immune Thrombocytopenia (ITP) patients – which is still somewhat controversial – is of potential interest, because activated platelets tend to aggregate (leading to excessive clotting or thromboembolic events) but cannot do so when platelet numbers are low, as in ITP. Although corticosteroids are the first line of therapy in ITP, the effect of steroids on activation of platelets has not been evaluated so far. We examined the status of platelet activation (with and without stimulation with ADP) in ITP patients, at the start of therapy (pre-steroid treatment, naive) and post-steroid treatment (classified on the basis of steroid responsiveness). We used flow cytometry to evaluate the levels of expression of P-selectin, and PAC-1 binding to platelets of 55 ITP patients and a similar number of healthy controls, treated with and without ADP. We found that platelets in ITP patients exist in an activated state. In patients who are responsive to steroids, the treatment reverses this situation. Also, the fold activation of platelets upon treatment with ADP is more in healthy controls than in ITP patients; treatment with steroids causes platelets in steroid-responsive patients to become more responsive to ADP-activation, similar to healthy controls. Thus steroids may cause changes in the ability of platelets to get activated with an agonist like ADP. Our results provide new insights into how, and why, steroid therapy helps in the treatment of ITP.
ADP, ITP, PAC-1, platelets, P-selectin
Introduction Immune thrombocytopenia (ITP) is a common acquired autoimmune disorder characterised by low platelet count, either due to accelerated platelet destruction, or abnormal platelet function or impaired production under the effect of antiplatelet antibodies. ITP can be classified on the basis of absence or presence of other diseases (primary or secondary), age (adult or childhood ITP) and duration of thrombocytopenia (acute, persistent or chronic). Antiplatelet auto-antibodies in ITP are generally targeted against the GPIIb/IIIa platelet glycoprotein receptor [1–3]. GP IIb/IIIa is a dimeric receptor specific to megakaryocyte lineage [4]. It is in a state of low affinity for the ligands in resting platelets but upon activation of platelets with agonists, the receptor undergoes conformational changes, leading to exposure of binding sites for ligands e.g. fibrinogen, thereby causing platelet adhesion and aggregation [5]. This also exposes an epitope close to the ligand binding site on the GPIIb monomer that is recognised by auto-antibodies resulting in platelet destruction [6]. Other antibodies found to have a pathogenic role in ITP include those against GPIa/IIa, GPIV, GPIb/IX, GPV, and the thrombopoeitin receptor (TPO) [7–9].
*These authors have contributed equally to the work Correspondence: Manni Luthra-Guptasarma, Department of Immunopathology, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India. Tel: 91 172 2755196. Fax: 91 172 2744401. E-mail: [email protected]
History Received 25 November 2013 Revised 9 January 2014 Accepted 25 January 2014 Published online 11 March 2014
The receptor most read out during in vitro studies of platelet activation is the P-selectin, which is the ligand for the leukocyte receptor PSGL-1 (important for the release of platelet granules) [10]. In addition, full activation requires the platelets to bind to their ligands e.g. fibrinogen for inducing platelet aggregation and thrombosis. This as already highlighted requires a conformational change in GPIIb/IIIa receptor. This interaction of the ligand can be studied by PAC-1 binding closely to fibrinogen binding site [11]. Currently, data regarding the activation status of platelets in chronic ITP patients is rather scarce, and contradictory in nature. Various therapeutic modalities exist for ITP, including glucocorticosteroids, splenectomy, intravenous immunoglobulin and thrombopoietin receptor agonists. Among these, corticosteroids remain the first line therapy for ITP; however, no systematic study has been done to investigate the effect of corticosteroids on platelet activation status. We wanted to test the hypothesis that steroids may affect the activation status of platelets (contributing to their clinical response), by examining the status of platelet activation in different categories of patients: pre-treatment (i.e. naive patients not yet started on steroids), and post-treatment (patients showing complete or partial response, as well as patients who were non-responsive to steroids).
Materials and methods Study design Sixty patients diagnosed with chronic ITP in the age range of 16 years to 67 years and 50 age-matched healthy controls with
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platelet count in the normal range of 150 000 to 450 000/ml were recruited in this prospective study from August 2012 to June 2013. Informed consent was taken from each patient and the study was approved by the Institutional Ethics Committee of PGIMER. The patients who were on drugs other than steroids, or with other autoimmune disorders, infections and below 15 years of age were considered inappropriate for the study. Five patients were excluded from the study due to procedure related technical error. Finally, 55 patients entered the study. The indication of treatment was ongoing bleeding in the form of purpuric spots on the body or mucosal bleed (gums, hematuria, menorrhagia) or platelet count less than 30 000/ml. Blood samples were drawn at the start of therapy in 13 cases, which constituted the treatment naive group, while in all other cases samples were drawn after 4 weeks of therapy, and patients were categorized (according to the ASH 2011 practice guidelines for ITP [12]) into three groups on the basis of steroid response and platelet count.
Activation fold for P-selectin expression and PAC-1 binding was determined by dividing the post-activation MFI value in each case, with pre-activation MFI value. No fixative solutions were used in the assay before and after antibody binding because use of a fixative can inhibit antibody binding before staining and artificially activate platelets after antibody binding. After incubation with antibodies, samples were resuspended in 200 ml of PBS and analyzed on the flow cytometer. Isotype matched controls (BD Biosciences, Heidelberg, Germany) were used for the determination of non-specific binding. Platelets were identified by gating on side scatter and forward scatter, followed by CD-41-FITC fluorescence. Samples were analyzed with the acquisition of 10 000 events. The fluorescence data, obtained in the logarithmic mode, was analyzed in terms of mean (geometric) fluorescence intensity (MFI).
Clinical evaluation
Non-parametric tests (Mann–Whitney U-test) were applied to the data, since the data was skewed, as determined by using the Kolmogorov–Smirnov test. Comparisons were made between two groups at a time, and results were declared significant if p value 50.05. Significance was denoted as p50.05(*); 50.001(**); 50.0001(***). Since the expression of activation markers was significant between the control (healthy) population and the ITP population, we calculated the area under the receiver-operating characteristic (ROC) curves which plots sensitivity (true-positive fraction) versus 1-specificity (false-positive fraction), for the MFI values to determine the cut-off MFI value. To check the correlation between activation marker and platelet count, scatter plots were drawn and non-parametric Spearman test was applied. Results were represented as Mean ± SEM. Statistical analysis was performed with SPSS software (version 17, Chicago, IL), for Windows.
Clinical history, physical examination (petechie, purpura, ecchymosis, mucosal bleeding, menorrhagia, internal bleeding) and laboratory investigations (haemogram, bone-marrow findings) were recorded. Patients positive for auto-immune markers for other auto-immune disorders were excluded from the study. Sample preparation and reagents Five milliliters of peripheral whole blood sample was collected, either pre-steroid therapy (in the naive group), or post-therapy (after 4 weeks of steroid treatment), by venepuncture using 21 gauge needle in 3.2% trisodium citrate vacutainers (Becton Dickinson, San Diego, CA). Complete blood count and other hematological tests were measured immediately following the blood draw, taking precautions by not using first 2 ml of blood for flow analysis. The patient and control samples were processed within 1 hour of PRP (platelet rich plasma) preparation. FITC conjugated CD41 antibody specific for GPIIb (Cat No. 41F-100T from Immuno step, Spain) was used to confirm the platelet population in PRP preparation; FITC-conjugated PAC-1 antibody specific for activated GPIIb/IIIa complex (Cat No. 340507) and PE-conjugated CD62P antibody specific for P-selectin (Cat No.555524) were obtained from BD Biosciences, Heidelberg, Germany. ADP agonist was purchased from Chrono-Log (Havertown, PA) and used at 20 mM concentration for stimulating platelets. Phosphate-buffered saline (PBS) was used as diluting buffer. Flow-cytometry based analysis of platelet activation In all study groups and controls, the same methodology was used to determine the platelet activation status. Platelet activation was assessed in platelet-rich plasma (PRP) instead of whole blood [13] to prevent non-specific binding of antibodies on RBCs. PRP was prepared by centrifugation at 150 g at 22 C for 10 minutes. In each case, the total platelet count was maintained at 100 000 platelets in 50 ml PBS; e.g. if the platelet count in samples was 10 000/ml, a volume of 10 ml of PRP was used, along with 50 ml of PBS. Activation status on resting platelets (baseline values) was determined by incubating the PRP samples with antibodies specific for activation markers P-selectin (PE-CD62P) and FITCPAC-1, for 45 minutes in dark at 30 C, respectively. Both antibodies were used at optimal concentration, as determined by titration. Activation status on ADP-stimulated PRP samples was assessed by activating platelets with 20 mM ADP followed by gentle mixing for 2 minutes at room temperature (22–25 C), and incubated with antibodies (P-selectin (PE-CD62P) and FITC-PAC-1 for 45 minutes in dark at 30 C, respectively.
Statistical analysis
Results Patient characteristics A total of 55 patients were recruited for the study; the diagnosis of ITP was based on established clinical criteria [12–15]. Blood samples from a comparable number of healthy controls were also obtained and included in the study. As seen from Table I, there was a clear preponderance of females over males in our ITP population (2.1:1). The mean age of the patients was 32 years (range 16–67). All the ITP patients (except the treatment naı¨ve group) were classified after 4 weeks of steroid therapy, based on the ASH 2011 practice guidelines for ITP [12], as either complete or good responders (CR; 100 109/l, and absence of bleeding), responders (R; 30–100 109/l, and a greater than two-fold increase in platelet count from baseline, and the absence of bleeding), and non-responders (NR 530 109/l, or less than twofold increase in platelet count from baseline, or the presence of bleeding). In our study, the classification resulted in the following grouping: CR: 18 patients; NR: 9 patients; R: 15 patients. Blood samples were drawn at the start of therapy in 13 patients (which constituted the treatment naive group). Platelets were gated based on side scatter and forward scatter; unstained cells and isotype controls were used to determine the autofluorescence and non-specific binding respectively (Supplementary Figure S1). Platelets were stained with CD41FITC (activation-independent platelet marker), P-selectin-PE or PAC-1 FITC, and binding was checked by flow cytometry. Mean fluorescence intensity (MFI) was used to determine the expression of activation markers. A representative case of a healthy control, with and without stimulation with ADP, is illustrated in Figure 1.
Steroids and ITP platelet activation status
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Table I. Demographics data of ITP patients.
Patient n ¼ 18
Sex
Age (Years)
(i) completely responsive (CR) to steroid therapy 1 F 52 2 F 25 3 F 36 4 F 24 5 M 40 6 M 36 7 F 40 8 M 33 9 F 50 10 M 24 11 F 34 12 F 37 13 F 38 14 F 33 15 M 21 16 F 33 17 F 34 18 F 30
Platelet count post-steroid therapy ( 109/l) 342 454 194 140 111 231 249 145 115 131 141 200 169 218 235 153 267 252
(ii) partially responsive (R) to steroid therapy 1 F 55 2 F 20 3 F 36 4 F 18 5 F 40 6 M 46 7 F 19 8 F 41 9 M 18 10 F 40 11 F 27 12 F 25 13 M 33 14 M 46 15 F 47
78 69 53 57 91 54 54 68 83 29 45 42 31 15 21
(iii) non-responsive (NR) to steroid therapy 1 F 40 2 M 46 3 F 16 4 F 19 5 F 17 6 F 16 7 F 18 8 F 36 9 F 30
18 20 9 7 24 23 22 10 8
(iv) treatment-naive group of patients 1 M 16 2 M 42 3 M 18 4 F 42 5 F 40 6 F 33 7 F 32 8 M 38 9 F 27 10 M 67 11 F 23 12 M 18 13 M 53
14 17 42 15 39 4 45 67 80 50 30 6 8
Figure 2(A and B) show that the MFI values for expression of both the markers – P-selectin and PAC-1, is more in the case of platelets from ITP patients, than platelets derived from healthy controls (p value 50.001). The area under the curve (AUC) in ROC analysis for P-selectin expression was 0.698 and 95% CI 0.598–0.798 (Figure 1C). In 63.6% of ITP patients (35/55),
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P-selectin expression was more than the cut off value (1630); in the case of healthy controls, 32% (16/50) of individuals showed values above this cut-off MFI value. AUC for PAC-1 binding was 0.771 and 95% CI 0.683–0.858 (Figure 1D). PAC-1 binding was similarly found to be more in greater number of ITP patients (63%; 35/55) than in healthy controls (24%; 12/50), with the cut-off value determined to be 636. Comparison of activation status of platelets among various subgroups of ITP patients An evaluation of activation markers to measure activation status of platelets among the various sub-groups of ITP patients classified on the basis of steroid responsiveness is shown in Figure 3. It is clear from Panel A that expression of P-selectin is significantly more in platelets from treatment naive patients diagnosed as having ITP, as compared to controls (p value ¼ 0.002); P-selectin expression is also more in CR and NR cases as compared to controls (p ¼ 0.02 and p ¼ 0.006 respectively). Expression of the markers in the non-responsive (NR) cases is similar to that in the naı¨ve cases. Panel B showed that in the three groups, NR, R and naive, PAC-1 binding was significantly more than in controls (p50.001). A comparison of PAC-1 binding within the different groups shows that binding in NR group4CR group (p50.001); naı¨ve group4CR group (p50.05). From Panel A, it was clear that P-selectin expression within the different subgroups also shows a similar trend to PAC-1 binding, i.e. there was a decreased expression of P-selectin in platelets derived from CR group as compared to the NR or naive groups, although the data did not reach statistical significance. In other words, patients who were completely responsive (CR) to steroid therapy in terms of normal platelet numbers, also respond by changing their activation status (based on comparison of CR group with naive, R and NR groups). Comparison of fold increase in activation upon treatment with an agonist (ADP) in ITP patients and controls The mean fluorescence intensity for P-selectin expression and PAC-1 binding was recorded for each patient as well as control after activation with ADP. The fold increase in activation (value of MFI after ADP activation/ MFI pre-activation) is shown in Figure 4, for healthy controls, as well as ITP patients. Fold increase in ADP-activation of platelets derived from healthy controls were significantly more than when the platelets from ITP patients were treated similarly, as judged by P-selectin expression, as well as PAC-1 binding (p50.001). Therefore, platelets from normal controls can be activated significantly more with ADP, than platelets from ITP patients. Comparison of fold increase in activation upon treatment with ADP among the various subgroups of ITP patients An evaluation of activation markers to measure activation status of platelets among the various sub-groups of ITP patients, after treatment with ADP, is shown in Figure 5. It is clear from Panel A that in terms of P-selectin expression, there is a significant difference in the fold activation status of platelets in the completely responsive group of patients, when compared to the platelets derived from the non-responsive group (p value50.001); the platelets of the NR category are not activable by ADP. Similarly, there is a significant difference in the ability of platelets to be activated (by ADP) in the naive group versus the completely responsive (p50.006). In terms of PAC-1 marker expression, a similar analysis shows that like P-selectin expression, platelets of patients in the steroid responsive group (CR) show increased expression of PAC-1 upon ADP-activation, as compared to
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Figure 1. Flow cytometric quantification of platelet activation markers, PAC-1 and P-selectin. Platelets were either stimulated with ADP (lower panel) or not (upper panel), stained with CD41-FITC (activation-independent platelet marker), P-selectin-PE and PAC-1-FITC, and binding was checked by flow cytometry. Mean fluorescence intensity (MFI) was used to determine the expression of the markers.
platelets of the non-responsive group (p value50.001); here too, it emerges that the platelets of the NR category are functionally not responding to activation by ADP. Further, expression of PAC-1 is significantly more by platelets in the completely responsive group vs. those belonging to the naive group (p ¼ 0.014), after activation with ADP. Upon ADP-activation, the fold increase in expression of P-selectin as well the binding of PAC-1, is maximal in the healthy control group (Panels A and B), in line with data shown in Figure 4, suggesting that healthy platelets respond strongly to platelet activation. It is logical to conclude from this set of data that (i) treatment with steroids leads to change in the ability of platelets to respond to activation by ADP (since the CR group displays maximal fold activation among the ITP patients), (ii) in patients who are non-responsive to steroids, neither P-selectin nor PAC-1 marker expression increases upon ADP activation, (iii) platelets of naive ITP patients, similarly do not get activated upon treatment with ADP (with respect to either P-selectin or PAC-1 marker expression). It would mean therefore, that steroids help to increase the platelet count of ITP patients, during which another effect is on the change in the ability of platelets to get activated with an agonist like ADP. Correlation of platelet count with expression of P-selectin and PAC-1 binding An examination of platelet count with expression of P-selectin (Figure 6A) shows that there was a negative correlation between platelet count and expression of P-selectin (r ¼ –0.175). Figure 6(B) shows that the binding of PAC-1 was significantly negatively correlated with platelet numbers (r ¼ –0.486; p50.0002).
Discussion The status of platelets in ITP patients, in terms of their number, their functional competence, and their behaviour, remains enigmatic. Canonically, activated platelets must tend to aggregate, and this should lead to excessive clotting or thromboembolic events; however, reduced platelet numbers, as in ITP would promote bleeding. The results of these situations are likely to be dependent on the dynamic interplay of platelet activation and platelet destruction in each ITP patient, and this may explain the confusing literature that reports both increased emboli [16,17], and the increased bleeding (which defines ITP). We evaluated the baseline status of platelets in ITP patients by studying the markers associated with activation; these include the change in conformation of GPIIb/IIIa, assessed by binding of a conformation-specific antibody, PAC-1, and the activationdependent release of P-selectin (CD62P) from the platelet granules. We observed that both the activation markers, P-selectin, and PAC-1 had significantly increased in ITP patients than in healthy controls. With respect to platelet numbers, all ITP studies performed since the disease was identified report that ITP patients had lower number of circulating platelets. The assumption was that some mechanism of cellular destruction operates to sequester and destroy platelets. The causative factors that elicit platelet destruction remained contentious and unestablished. However, in respect of the status of platelets with regard to activation, the subject had been contentious for three reasons: Firstly, all reports do not suggest that platelets in ITP patients were activated [13,18]. Secondly, reports that suggested platelet activation, were not always talking about the same thing; some mentioned
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Figure 2. Platelet activation markers in healthy controls and ITP patients. Baseline expression of P-selectin (Panel A) and binding of PAC-1 (Panel B) in healthy controls (n ¼ 50) and ITP patients (n ¼ 55), determined by flow cytometry. MFI: mean fluorescence intensity. Panels C and D represent ROC curves corresponding to the above data for P-selectin expression (Panel C) and PAC-1 binding (Panel D) respectively. The straight diagonal greenlines in both C and D, indicate an area under the curve (AUC) of 0.698 and 0.771 respectively. ***P5 0.0001.
activation of GPIIb/IIIa integrin through binding of PAC-1 antibody (which purports to assay the activation status of GPIIb/IIIa, through a single immunodiagnostic method, without fully establishing conformational change by other methods), while others focused on markers such as P-selectin and GPIb and GP53 (CD63) [13,18–20]. Furthermore, the assays performed to check the activation status tend to assay ‘‘activability’’ as well, and different studies used different activating reagents such as thrombin and ADP. One interesting study [18] draws a difference between activation owing to GPIIb/IIIa conformational changes leading to exposure of fibrinogen-binding site (sometimes indirectly assayed by reactivity of platelets to PAC-1 antibody), from activation assayed by for example, P-selectin. Indeed, this study proposes that the latter was not activation at all, but rather a state of higher competence of activation. In the light of the above, our results are different; our data shows simultaneously increased P-selectin expression, as well as increased PAC-1 binding in ITP patients, when compared with platelets in normal individuals. Since the first line of therapy for ITP patients is steroid treatment, it is important to assess whether there is any change in the status of ITP patients after treatment with steroids. We knew that corticosteroids improve platelet counts in ITP patients, not only by suppressing the reticuloendothelial system function, but also by reducing autoantibody
production [21]. It has also been shown that steroids can shift the monocyte FcgR balance toward the inhibitory FcgRIIb type, in patients with ITP [22]. However, the effect of steroids with respect to activation status of platelets has not been investigated. Our data suggests that except for the CR group, all the other groups (NR, R and naive) continue to show significantly increased PAC-1 binding, compared to healthy controls, suggesting that patients who responded to steroids in terms of increasing their platelet numbers to normal values (i.e. the CR group), also showed lesser activation of platelets. With respect to P-selectin expression, the trend is similar to PAC-1 binding; i.e. the CR group showed reduced expression of P-selectin compared to the NR and naive groups, although the data did not reach statistical significance. An important point to note is that although the R group reached statistical significance compared to healthy control in case of PAC-1 binding, it did not reach statistical significance in case of P-selectin expression. It would appear to be because of any of the following considerations: i) the R group may be considered to be intermediate between CR and NR groups; ii) PAC-1 and P-selectin are both activation markers; however, when one is positive, the other marker need not be so. This is because the kinetics of activation of the two markers is different; PAC-1 is an early activation marker and P-selectin is a late activation marker [23]. The data in Figure 3(A) suggests that P-selectin expression for the R group was not elevated enough (although the
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Figure 3. Platelet activation markers in healthy controls and different subgroups of ITP patients. Baseline expression of P-selectin (Panel A) and binding of PAC-1 (Panel B) in healthy controls (n ¼ 50) and different subgroups of ITP patients, classified on the basis of steroid responsiveness as complete responders (CR; n ¼ 18), non-responsive patients (NR; n ¼ 9), partially responsive patients (R; n ¼ 15) and treatment naive patients (without steroid treatment, n ¼ 13). *P50.05; **P50.001; ***P50.0001.
Figure 4. Fold increase in activation upon treatment with ADP, in healthy controls and ITP patients. Fold increase in MFI was measured for expression of P-selectin (Panel A) and binding of PAC-1 (Panel B) in healthy controls (n ¼ 50) and in ITP patients (n ¼ 55), in response to treatment with an agonist, ADP. Activation fold was determined for both the activation markers, by dividing the MFI value of ADP-treated platelets by the baseline MFI value in each case.
Figure 5. Fold increase in activation upon treatment with ADP, in healthy controls and different subgroups of ITP patients. Fold increase in MFI was measured (as mentioned in Figure 4) for expression of P-selectin (Panel A) and binding of PAC-1 (Panel B) in healthy controls (n ¼ 50) and different subgroups of ITP patients, classified on the basis of steroid responsiveness as complete responders (CR; n ¼ 18), non-responsive patients (NR; n ¼ 9), partially responsive patients(R; n ¼ 15) and treatment naive patients (without steroid treatment, n ¼ 13).
PAC-1 binding was high as seen from Figure 3B), which might explain the reason why the statistical significance for R vs. control group was not reached. Data correlating the platelet numbers with activation status also corroborates this data showing that when the platelet numbers are more (as in CR cases), the activation of platelets (with respect to either P-selectin expression or PAC-1 binding) is less. It may be noted here that, although there exists a negative correlation of platelet count with P-selectin and PAC-1 levels, it is known that platelet responses to ADP are unaffected by the platelet count [20]. In response to an agonist, ADP, platelets from healthy controls were capable of being activated significantly more than platelets of ITP patients. Among the various treatment groups based on responsiveness to steroid therapy, we observed that platelets from completely responsive groups of patients (CR) showed significant
increase in P-selectin expression upon activation with ADP, than platelets from NR or naive groups of patients (p50.001); similarly the CR group of patients also showed increase in PAC-1 binding (upon ADP treatment), as compared to the NR or naive groups of patients. This suggests that treatment with steroids in the steroid responsive (CR) category of patients, leads to the platelets becoming activable (in respect of both, P-selectin expression, as well as PAC-1 binding), similar to the platelets of healthy control individuals. As discussed above, ADP-activation data (Figure 5) again suggests that the R group does not behave like the NR or naive groups, at least where P-selectin expression is concerned. Overall, the picture that emerges from Panels A and B is that the platelets in NR and naive groups are less able to be activated upon ADP stimulation (for both P-selectin and PAC-1 markers), presumably because in the resting state both of these groups have high activation levels, and therefore their threshold
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Figure 6. Correlation between platelet count and activation markers in ITP patients. Negative correlation between platelet count and baseline expression of P-selectin (Panel A); negative correlation of platelet count with baseline MFI values for binding of PAC-1 (Panel B) in ITP patients (n ¼ 55).
for activation is already reached. This point was very important, i.e. the background itself was high, causing the phenomenon to not be seen in the case of the NR and naive groups. The case of the R group was somewhat intermediate in that for one marker, i.e. PAC-1 binding, this situation was true but not for P-selectin expression (and these may behave differently for reasons explained above). In conclusion, it emerges that the (i) platelets in ITP patients are already in an activated state (as gauged by expression of Pselectin and PAC-1 binding); (ii) the fold activation of platelets upon treatment with ADP is more in healthy controls than in ITP patients; (iii) treatment with steroids leads to decrease in activation status of platelets; and (iv) treatment with steroids leads to change in the ability of platelets to respond to activation with ADP. Thus steroids may cause changes in the ability of platelets to get activated with an agonist like ADP.
Authorship contributions Contribution: PB and SS collected and analyzed data; NM, PM and SV participated in the diagnosis, management and recruitment of patients; MLG designed research, obtained funding, analyzed and interpreted the data and wrote the manuscript; all authors revised the manuscript, and reviewed and approved the final version of the manuscript.
Declaration of interest The authors declare no competing financial interests. MLG would like to thank the Department of Biotechnology (DBT), Govt. of India, for funding the study. PB thanks the UGC for providing fellowship.
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22. Liu XG, Ma SH, Sun JZ, Ren J, Shi Y, Sun L, Dong XY, Qin P, Guo CS, Hou M, Peng J. High-dose dexamethasone shifts the balance of stimulatory and inhibitory Fc gamma receptors on monocytes in patients with primary immune thrombocytopenia. Blood 2011;117: 2061–2069. 23. Merten M, Thiagarajan P. P-selectin expression on platelets determines size and stability of platelet aggregates. Circulation 2000;102:1931–1936.
Supplementary material available online Figure S1. Platelets gated based on side scatter and forward scatter (left panel); unstained cells (middle panel) and isotype controls (right panel) were used to determine the autofluorescence and non-specific binding respectively. Supplementary material can be viewed and downloaded at http://informahealthcare.com/plt.
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ORIGINAL ARTICLE
Effect of steroids on the activation status of platelets in patients with Immune thrombocytopenia (ITP) Preeti Bhoria1*, Saniya Sharma2*, Neelam Varma3, Pankaj Malhotra1, Subhash Varma1, & Manni Luthra-Guptasarma2 1
Departments of Internal Medicine, 2Department of Immunopathology, and 3Department of Hematology, Postgraduate Institute of Medical Education and Research, Chandigarh, India Abstract
Keywords
The activation status of platelets in Immune Thrombocytopenia (ITP) patients – which is still somewhat controversial – is of potential interest, because activated platelets tend to aggregate (leading to excessive clotting or thromboembolic events) but cannot do so when platelet numbers are low, as in ITP. Although corticosteroids are the first line of therapy in ITP, the effect of steroids on activation of platelets has not been evaluated so far. We examined the status of platelet activation (with and without stimulation with ADP) in ITP patients, at the start of therapy (pre-steroid treatment, naive) and post-steroid treatment (classified on the basis of steroid responsiveness). We used flow cytometry to evaluate the levels of expression of P-selectin, and PAC-1 binding to platelets of 55 ITP patients and a similar number of healthy controls, treated with and without ADP. We found that platelets in ITP patients exist in an activated state. In patients who are responsive to steroids, the treatment reverses this situation. Also, the fold activation of platelets upon treatment with ADP is more in healthy controls than in ITP patients; treatment with steroids causes platelets in steroid-responsive patients to become more responsive to ADP-activation, similar to healthy controls. Thus steroids may cause changes in the ability of platelets to get activated with an agonist like ADP. Our results provide new insights into how, and why, steroid therapy helps in the treatment of ITP.
ADP, ITP, PAC-1, platelets, P-selectin
Introduction Immune thrombocytopenia (ITP) is a common acquired autoimmune disorder characterised by low platelet count, either due to accelerated platelet destruction, or abnormal platelet function or impaired production under the effect of antiplatelet antibodies. ITP can be classified on the basis of absence or presence of other diseases (primary or secondary), age (adult or childhood ITP) and duration of thrombocytopenia (acute, persistent or chronic). Antiplatelet auto-antibodies in ITP are generally targeted against the GPIIb/IIIa platelet glycoprotein receptor [1–3]. GP IIb/IIIa is a dimeric receptor specific to megakaryocyte lineage [4]. It is in a state of low affinity for the ligands in resting platelets but upon activation of platelets with agonists, the receptor undergoes conformational changes, leading to exposure of binding sites for ligands e.g. fibrinogen, thereby causing platelet adhesion and aggregation [5]. This also exposes an epitope close to the ligand binding site on the GPIIb monomer that is recognised by auto-antibodies resulting in platelet destruction [6]. Other antibodies found to have a pathogenic role in ITP include those against GPIa/IIa, GPIV, GPIb/IX, GPV, and the thrombopoeitin receptor (TPO) [7–9].
*These authors have contributed equally to the work Correspondence: Manni Luthra-Guptasarma, Department of Immunopathology, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India. Tel: 91 172 2755196. Fax: 91 172 2744401. E-mail: [email protected]
History Received 25 November 2013 Revised 9 January 2014 Accepted 25 January 2014 Published online 11 March 2014
The receptor most read out during in vitro studies of platelet activation is the P-selectin, which is the ligand for the leukocyte receptor PSGL-1 (important for the release of platelet granules) [10]. In addition, full activation requires the platelets to bind to their ligands e.g. fibrinogen for inducing platelet aggregation and thrombosis. This as already highlighted requires a conformational change in GPIIb/IIIa receptor. This interaction of the ligand can be studied by PAC-1 binding closely to fibrinogen binding site [11]. Currently, data regarding the activation status of platelets in chronic ITP patients is rather scarce, and contradictory in nature. Various therapeutic modalities exist for ITP, including glucocorticosteroids, splenectomy, intravenous immunoglobulin and thrombopoietin receptor agonists. Among these, corticosteroids remain the first line therapy for ITP; however, no systematic study has been done to investigate the effect of corticosteroids on platelet activation status. We wanted to test the hypothesis that steroids may affect the activation status of platelets (contributing to their clinical response), by examining the status of platelet activation in different categories of patients: pre-treatment (i.e. naive patients not yet started on steroids), and post-treatment (patients showing complete or partial response, as well as patients who were non-responsive to steroids).
Materials and methods Study design Sixty patients diagnosed with chronic ITP in the age range of 16 years to 67 years and 50 age-matched healthy controls with
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platelet count in the normal range of 150 000 to 450 000/ml were recruited in this prospective study from August 2012 to June 2013. Informed consent was taken from each patient and the study was approved by the Institutional Ethics Committee of PGIMER. The patients who were on drugs other than steroids, or with other autoimmune disorders, infections and below 15 years of age were considered inappropriate for the study. Five patients were excluded from the study due to procedure related technical error. Finally, 55 patients entered the study. The indication of treatment was ongoing bleeding in the form of purpuric spots on the body or mucosal bleed (gums, hematuria, menorrhagia) or platelet count less than 30 000/ml. Blood samples were drawn at the start of therapy in 13 cases, which constituted the treatment naive group, while in all other cases samples were drawn after 4 weeks of therapy, and patients were categorized (according to the ASH 2011 practice guidelines for ITP [12]) into three groups on the basis of steroid response and platelet count.
Activation fold for P-selectin expression and PAC-1 binding was determined by dividing the post-activation MFI value in each case, with pre-activation MFI value. No fixative solutions were used in the assay before and after antibody binding because use of a fixative can inhibit antibody binding before staining and artificially activate platelets after antibody binding. After incubation with antibodies, samples were resuspended in 200 ml of PBS and analyzed on the flow cytometer. Isotype matched controls (BD Biosciences, Heidelberg, Germany) were used for the determination of non-specific binding. Platelets were identified by gating on side scatter and forward scatter, followed by CD-41-FITC fluorescence. Samples were analyzed with the acquisition of 10 000 events. The fluorescence data, obtained in the logarithmic mode, was analyzed in terms of mean (geometric) fluorescence intensity (MFI).
Clinical evaluation
Non-parametric tests (Mann–Whitney U-test) were applied to the data, since the data was skewed, as determined by using the Kolmogorov–Smirnov test. Comparisons were made between two groups at a time, and results were declared significant if p value 50.05. Significance was denoted as p50.05(*); 50.001(**); 50.0001(***). Since the expression of activation markers was significant between the control (healthy) population and the ITP population, we calculated the area under the receiver-operating characteristic (ROC) curves which plots sensitivity (true-positive fraction) versus 1-specificity (false-positive fraction), for the MFI values to determine the cut-off MFI value. To check the correlation between activation marker and platelet count, scatter plots were drawn and non-parametric Spearman test was applied. Results were represented as Mean ± SEM. Statistical analysis was performed with SPSS software (version 17, Chicago, IL), for Windows.
Clinical history, physical examination (petechie, purpura, ecchymosis, mucosal bleeding, menorrhagia, internal bleeding) and laboratory investigations (haemogram, bone-marrow findings) were recorded. Patients positive for auto-immune markers for other auto-immune disorders were excluded from the study. Sample preparation and reagents Five milliliters of peripheral whole blood sample was collected, either pre-steroid therapy (in the naive group), or post-therapy (after 4 weeks of steroid treatment), by venepuncture using 21 gauge needle in 3.2% trisodium citrate vacutainers (Becton Dickinson, San Diego, CA). Complete blood count and other hematological tests were measured immediately following the blood draw, taking precautions by not using first 2 ml of blood for flow analysis. The patient and control samples were processed within 1 hour of PRP (platelet rich plasma) preparation. FITC conjugated CD41 antibody specific for GPIIb (Cat No. 41F-100T from Immuno step, Spain) was used to confirm the platelet population in PRP preparation; FITC-conjugated PAC-1 antibody specific for activated GPIIb/IIIa complex (Cat No. 340507) and PE-conjugated CD62P antibody specific for P-selectin (Cat No.555524) were obtained from BD Biosciences, Heidelberg, Germany. ADP agonist was purchased from Chrono-Log (Havertown, PA) and used at 20 mM concentration for stimulating platelets. Phosphate-buffered saline (PBS) was used as diluting buffer. Flow-cytometry based analysis of platelet activation In all study groups and controls, the same methodology was used to determine the platelet activation status. Platelet activation was assessed in platelet-rich plasma (PRP) instead of whole blood [13] to prevent non-specific binding of antibodies on RBCs. PRP was prepared by centrifugation at 150 g at 22 C for 10 minutes. In each case, the total platelet count was maintained at 100 000 platelets in 50 ml PBS; e.g. if the platelet count in samples was 10 000/ml, a volume of 10 ml of PRP was used, along with 50 ml of PBS. Activation status on resting platelets (baseline values) was determined by incubating the PRP samples with antibodies specific for activation markers P-selectin (PE-CD62P) and FITCPAC-1, for 45 minutes in dark at 30 C, respectively. Both antibodies were used at optimal concentration, as determined by titration. Activation status on ADP-stimulated PRP samples was assessed by activating platelets with 20 mM ADP followed by gentle mixing for 2 minutes at room temperature (22–25 C), and incubated with antibodies (P-selectin (PE-CD62P) and FITC-PAC-1 for 45 minutes in dark at 30 C, respectively.
Statistical analysis
Results Patient characteristics A total of 55 patients were recruited for the study; the diagnosis of ITP was based on established clinical criteria [12–15]. Blood samples from a comparable number of healthy controls were also obtained and included in the study. As seen from Table I, there was a clear preponderance of females over males in our ITP population (2.1:1). The mean age of the patients was 32 years (range 16–67). All the ITP patients (except the treatment naı¨ve group) were classified after 4 weeks of steroid therapy, based on the ASH 2011 practice guidelines for ITP [12], as either complete or good responders (CR; 100 109/l, and absence of bleeding), responders (R; 30–100 109/l, and a greater than two-fold increase in platelet count from baseline, and the absence of bleeding), and non-responders (NR 530 109/l, or less than twofold increase in platelet count from baseline, or the presence of bleeding). In our study, the classification resulted in the following grouping: CR: 18 patients; NR: 9 patients; R: 15 patients. Blood samples were drawn at the start of therapy in 13 patients (which constituted the treatment naive group). Platelets were gated based on side scatter and forward scatter; unstained cells and isotype controls were used to determine the autofluorescence and non-specific binding respectively (Supplementary Figure S1). Platelets were stained with CD41FITC (activation-independent platelet marker), P-selectin-PE or PAC-1 FITC, and binding was checked by flow cytometry. Mean fluorescence intensity (MFI) was used to determine the expression of activation markers. A representative case of a healthy control, with and without stimulation with ADP, is illustrated in Figure 1.
Steroids and ITP platelet activation status
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Table I. Demographics data of ITP patients.
Patient n ¼ 18
Sex
Age (Years)
(i) completely responsive (CR) to steroid therapy 1 F 52 2 F 25 3 F 36 4 F 24 5 M 40 6 M 36 7 F 40 8 M 33 9 F 50 10 M 24 11 F 34 12 F 37 13 F 38 14 F 33 15 M 21 16 F 33 17 F 34 18 F 30
Platelet count post-steroid therapy ( 109/l) 342 454 194 140 111 231 249 145 115 131 141 200 169 218 235 153 267 252
(ii) partially responsive (R) to steroid therapy 1 F 55 2 F 20 3 F 36 4 F 18 5 F 40 6 M 46 7 F 19 8 F 41 9 M 18 10 F 40 11 F 27 12 F 25 13 M 33 14 M 46 15 F 47
78 69 53 57 91 54 54 68 83 29 45 42 31 15 21
(iii) non-responsive (NR) to steroid therapy 1 F 40 2 M 46 3 F 16 4 F 19 5 F 17 6 F 16 7 F 18 8 F 36 9 F 30
18 20 9 7 24 23 22 10 8
(iv) treatment-naive group of patients 1 M 16 2 M 42 3 M 18 4 F 42 5 F 40 6 F 33 7 F 32 8 M 38 9 F 27 10 M 67 11 F 23 12 M 18 13 M 53
14 17 42 15 39 4 45 67 80 50 30 6 8
Figure 2(A and B) show that the MFI values for expression of both the markers – P-selectin and PAC-1, is more in the case of platelets from ITP patients, than platelets derived from healthy controls (p value 50.001). The area under the curve (AUC) in ROC analysis for P-selectin expression was 0.698 and 95% CI 0.598–0.798 (Figure 1C). In 63.6% of ITP patients (35/55),
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P-selectin expression was more than the cut off value (1630); in the case of healthy controls, 32% (16/50) of individuals showed values above this cut-off MFI value. AUC for PAC-1 binding was 0.771 and 95% CI 0.683–0.858 (Figure 1D). PAC-1 binding was similarly found to be more in greater number of ITP patients (63%; 35/55) than in healthy controls (24%; 12/50), with the cut-off value determined to be 636. Comparison of activation status of platelets among various subgroups of ITP patients An evaluation of activation markers to measure activation status of platelets among the various sub-groups of ITP patients classified on the basis of steroid responsiveness is shown in Figure 3. It is clear from Panel A that expression of P-selectin is significantly more in platelets from treatment naive patients diagnosed as having ITP, as compared to controls (p value ¼ 0.002); P-selectin expression is also more in CR and NR cases as compared to controls (p ¼ 0.02 and p ¼ 0.006 respectively). Expression of the markers in the non-responsive (NR) cases is similar to that in the naı¨ve cases. Panel B showed that in the three groups, NR, R and naive, PAC-1 binding was significantly more than in controls (p50.001). A comparison of PAC-1 binding within the different groups shows that binding in NR group4CR group (p50.001); naı¨ve group4CR group (p50.05). From Panel A, it was clear that P-selectin expression within the different subgroups also shows a similar trend to PAC-1 binding, i.e. there was a decreased expression of P-selectin in platelets derived from CR group as compared to the NR or naive groups, although the data did not reach statistical significance. In other words, patients who were completely responsive (CR) to steroid therapy in terms of normal platelet numbers, also respond by changing their activation status (based on comparison of CR group with naive, R and NR groups). Comparison of fold increase in activation upon treatment with an agonist (ADP) in ITP patients and controls The mean fluorescence intensity for P-selectin expression and PAC-1 binding was recorded for each patient as well as control after activation with ADP. The fold increase in activation (value of MFI after ADP activation/ MFI pre-activation) is shown in Figure 4, for healthy controls, as well as ITP patients. Fold increase in ADP-activation of platelets derived from healthy controls were significantly more than when the platelets from ITP patients were treated similarly, as judged by P-selectin expression, as well as PAC-1 binding (p50.001). Therefore, platelets from normal controls can be activated significantly more with ADP, than platelets from ITP patients. Comparison of fold increase in activation upon treatment with ADP among the various subgroups of ITP patients An evaluation of activation markers to measure activation status of platelets among the various sub-groups of ITP patients, after treatment with ADP, is shown in Figure 5. It is clear from Panel A that in terms of P-selectin expression, there is a significant difference in the fold activation status of platelets in the completely responsive group of patients, when compared to the platelets derived from the non-responsive group (p value50.001); the platelets of the NR category are not activable by ADP. Similarly, there is a significant difference in the ability of platelets to be activated (by ADP) in the naive group versus the completely responsive (p50.006). In terms of PAC-1 marker expression, a similar analysis shows that like P-selectin expression, platelets of patients in the steroid responsive group (CR) show increased expression of PAC-1 upon ADP-activation, as compared to
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Figure 1. Flow cytometric quantification of platelet activation markers, PAC-1 and P-selectin. Platelets were either stimulated with ADP (lower panel) or not (upper panel), stained with CD41-FITC (activation-independent platelet marker), P-selectin-PE and PAC-1-FITC, and binding was checked by flow cytometry. Mean fluorescence intensity (MFI) was used to determine the expression of the markers.
platelets of the non-responsive group (p value50.001); here too, it emerges that the platelets of the NR category are functionally not responding to activation by ADP. Further, expression of PAC-1 is significantly more by platelets in the completely responsive group vs. those belonging to the naive group (p ¼ 0.014), after activation with ADP. Upon ADP-activation, the fold increase in expression of P-selectin as well the binding of PAC-1, is maximal in the healthy control group (Panels A and B), in line with data shown in Figure 4, suggesting that healthy platelets respond strongly to platelet activation. It is logical to conclude from this set of data that (i) treatment with steroids leads to change in the ability of platelets to respond to activation by ADP (since the CR group displays maximal fold activation among the ITP patients), (ii) in patients who are non-responsive to steroids, neither P-selectin nor PAC-1 marker expression increases upon ADP activation, (iii) platelets of naive ITP patients, similarly do not get activated upon treatment with ADP (with respect to either P-selectin or PAC-1 marker expression). It would mean therefore, that steroids help to increase the platelet count of ITP patients, during which another effect is on the change in the ability of platelets to get activated with an agonist like ADP. Correlation of platelet count with expression of P-selectin and PAC-1 binding An examination of platelet count with expression of P-selectin (Figure 6A) shows that there was a negative correlation between platelet count and expression of P-selectin (r ¼ –0.175). Figure 6(B) shows that the binding of PAC-1 was significantly negatively correlated with platelet numbers (r ¼ –0.486; p50.0002).
Discussion The status of platelets in ITP patients, in terms of their number, their functional competence, and their behaviour, remains enigmatic. Canonically, activated platelets must tend to aggregate, and this should lead to excessive clotting or thromboembolic events; however, reduced platelet numbers, as in ITP would promote bleeding. The results of these situations are likely to be dependent on the dynamic interplay of platelet activation and platelet destruction in each ITP patient, and this may explain the confusing literature that reports both increased emboli [16,17], and the increased bleeding (which defines ITP). We evaluated the baseline status of platelets in ITP patients by studying the markers associated with activation; these include the change in conformation of GPIIb/IIIa, assessed by binding of a conformation-specific antibody, PAC-1, and the activationdependent release of P-selectin (CD62P) from the platelet granules. We observed that both the activation markers, P-selectin, and PAC-1 had significantly increased in ITP patients than in healthy controls. With respect to platelet numbers, all ITP studies performed since the disease was identified report that ITP patients had lower number of circulating platelets. The assumption was that some mechanism of cellular destruction operates to sequester and destroy platelets. The causative factors that elicit platelet destruction remained contentious and unestablished. However, in respect of the status of platelets with regard to activation, the subject had been contentious for three reasons: Firstly, all reports do not suggest that platelets in ITP patients were activated [13,18]. Secondly, reports that suggested platelet activation, were not always talking about the same thing; some mentioned
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Figure 2. Platelet activation markers in healthy controls and ITP patients. Baseline expression of P-selectin (Panel A) and binding of PAC-1 (Panel B) in healthy controls (n ¼ 50) and ITP patients (n ¼ 55), determined by flow cytometry. MFI: mean fluorescence intensity. Panels C and D represent ROC curves corresponding to the above data for P-selectin expression (Panel C) and PAC-1 binding (Panel D) respectively. The straight diagonal greenlines in both C and D, indicate an area under the curve (AUC) of 0.698 and 0.771 respectively. ***P5 0.0001.
activation of GPIIb/IIIa integrin through binding of PAC-1 antibody (which purports to assay the activation status of GPIIb/IIIa, through a single immunodiagnostic method, without fully establishing conformational change by other methods), while others focused on markers such as P-selectin and GPIb and GP53 (CD63) [13,18–20]. Furthermore, the assays performed to check the activation status tend to assay ‘‘activability’’ as well, and different studies used different activating reagents such as thrombin and ADP. One interesting study [18] draws a difference between activation owing to GPIIb/IIIa conformational changes leading to exposure of fibrinogen-binding site (sometimes indirectly assayed by reactivity of platelets to PAC-1 antibody), from activation assayed by for example, P-selectin. Indeed, this study proposes that the latter was not activation at all, but rather a state of higher competence of activation. In the light of the above, our results are different; our data shows simultaneously increased P-selectin expression, as well as increased PAC-1 binding in ITP patients, when compared with platelets in normal individuals. Since the first line of therapy for ITP patients is steroid treatment, it is important to assess whether there is any change in the status of ITP patients after treatment with steroids. We knew that corticosteroids improve platelet counts in ITP patients, not only by suppressing the reticuloendothelial system function, but also by reducing autoantibody
production [21]. It has also been shown that steroids can shift the monocyte FcgR balance toward the inhibitory FcgRIIb type, in patients with ITP [22]. However, the effect of steroids with respect to activation status of platelets has not been investigated. Our data suggests that except for the CR group, all the other groups (NR, R and naive) continue to show significantly increased PAC-1 binding, compared to healthy controls, suggesting that patients who responded to steroids in terms of increasing their platelet numbers to normal values (i.e. the CR group), also showed lesser activation of platelets. With respect to P-selectin expression, the trend is similar to PAC-1 binding; i.e. the CR group showed reduced expression of P-selectin compared to the NR and naive groups, although the data did not reach statistical significance. An important point to note is that although the R group reached statistical significance compared to healthy control in case of PAC-1 binding, it did not reach statistical significance in case of P-selectin expression. It would appear to be because of any of the following considerations: i) the R group may be considered to be intermediate between CR and NR groups; ii) PAC-1 and P-selectin are both activation markers; however, when one is positive, the other marker need not be so. This is because the kinetics of activation of the two markers is different; PAC-1 is an early activation marker and P-selectin is a late activation marker [23]. The data in Figure 3(A) suggests that P-selectin expression for the R group was not elevated enough (although the
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Figure 3. Platelet activation markers in healthy controls and different subgroups of ITP patients. Baseline expression of P-selectin (Panel A) and binding of PAC-1 (Panel B) in healthy controls (n ¼ 50) and different subgroups of ITP patients, classified on the basis of steroid responsiveness as complete responders (CR; n ¼ 18), non-responsive patients (NR; n ¼ 9), partially responsive patients (R; n ¼ 15) and treatment naive patients (without steroid treatment, n ¼ 13). *P50.05; **P50.001; ***P50.0001.
Figure 4. Fold increase in activation upon treatment with ADP, in healthy controls and ITP patients. Fold increase in MFI was measured for expression of P-selectin (Panel A) and binding of PAC-1 (Panel B) in healthy controls (n ¼ 50) and in ITP patients (n ¼ 55), in response to treatment with an agonist, ADP. Activation fold was determined for both the activation markers, by dividing the MFI value of ADP-treated platelets by the baseline MFI value in each case.
Figure 5. Fold increase in activation upon treatment with ADP, in healthy controls and different subgroups of ITP patients. Fold increase in MFI was measured (as mentioned in Figure 4) for expression of P-selectin (Panel A) and binding of PAC-1 (Panel B) in healthy controls (n ¼ 50) and different subgroups of ITP patients, classified on the basis of steroid responsiveness as complete responders (CR; n ¼ 18), non-responsive patients (NR; n ¼ 9), partially responsive patients(R; n ¼ 15) and treatment naive patients (without steroid treatment, n ¼ 13).
PAC-1 binding was high as seen from Figure 3B), which might explain the reason why the statistical significance for R vs. control group was not reached. Data correlating the platelet numbers with activation status also corroborates this data showing that when the platelet numbers are more (as in CR cases), the activation of platelets (with respect to either P-selectin expression or PAC-1 binding) is less. It may be noted here that, although there exists a negative correlation of platelet count with P-selectin and PAC-1 levels, it is known that platelet responses to ADP are unaffected by the platelet count [20]. In response to an agonist, ADP, platelets from healthy controls were capable of being activated significantly more than platelets of ITP patients. Among the various treatment groups based on responsiveness to steroid therapy, we observed that platelets from completely responsive groups of patients (CR) showed significant
increase in P-selectin expression upon activation with ADP, than platelets from NR or naive groups of patients (p50.001); similarly the CR group of patients also showed increase in PAC-1 binding (upon ADP treatment), as compared to the NR or naive groups of patients. This suggests that treatment with steroids in the steroid responsive (CR) category of patients, leads to the platelets becoming activable (in respect of both, P-selectin expression, as well as PAC-1 binding), similar to the platelets of healthy control individuals. As discussed above, ADP-activation data (Figure 5) again suggests that the R group does not behave like the NR or naive groups, at least where P-selectin expression is concerned. Overall, the picture that emerges from Panels A and B is that the platelets in NR and naive groups are less able to be activated upon ADP stimulation (for both P-selectin and PAC-1 markers), presumably because in the resting state both of these groups have high activation levels, and therefore their threshold
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Figure 6. Correlation between platelet count and activation markers in ITP patients. Negative correlation between platelet count and baseline expression of P-selectin (Panel A); negative correlation of platelet count with baseline MFI values for binding of PAC-1 (Panel B) in ITP patients (n ¼ 55).
for activation is already reached. This point was very important, i.e. the background itself was high, causing the phenomenon to not be seen in the case of the NR and naive groups. The case of the R group was somewhat intermediate in that for one marker, i.e. PAC-1 binding, this situation was true but not for P-selectin expression (and these may behave differently for reasons explained above). In conclusion, it emerges that the (i) platelets in ITP patients are already in an activated state (as gauged by expression of Pselectin and PAC-1 binding); (ii) the fold activation of platelets upon treatment with ADP is more in healthy controls than in ITP patients; (iii) treatment with steroids leads to decrease in activation status of platelets; and (iv) treatment with steroids leads to change in the ability of platelets to respond to activation with ADP. Thus steroids may cause changes in the ability of platelets to get activated with an agonist like ADP.
Authorship contributions Contribution: PB and SS collected and analyzed data; NM, PM and SV participated in the diagnosis, management and recruitment of patients; MLG designed research, obtained funding, analyzed and interpreted the data and wrote the manuscript; all authors revised the manuscript, and reviewed and approved the final version of the manuscript.
Declaration of interest The authors declare no competing financial interests. MLG would like to thank the Department of Biotechnology (DBT), Govt. of India, for funding the study. PB thanks the UGC for providing fellowship.
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Supplementary material available online Figure S1. Platelets gated based on side scatter and forward scatter (left panel); unstained cells (middle panel) and isotype controls (right panel) were used to determine the autofluorescence and non-specific binding respectively. Supplementary material can be viewed and downloaded at http://informahealthcare.com/plt.
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