Blood Cells, Molecules and Diseases 57 (2016) 115–117
Contents lists available at ScienceDirect
Blood Cells, Molecules and Diseases journal homepage: www.elsevier.com/locate/bcmd
Letter to the Editor Does consumption of platelet-derived microparticles in the fibrin clot explain the decrease in their percentage during pregnancy?☆
Keywords: Platelet-derived microparticles Pregnancy Recurrent pregnancy loss
To the Editor Platelet-derived microparticles (PMPs) are submicronic phospholipid vesicles, derived from platelets during activation, stress or apoptosis. PMPs are the most abundant MPs expressing procoagulant phosphatidylserine (PS) and functional receptors from platelet membranes on their surface giving them their prothrombotic nature. PMPs can therefore act as a pathophysiological threat in certain conditions and cause vascular dysfunction especially in thrombosis complicated diseases [1]. The clinical significance of these procoagulant MPs is many. There are many studies which show a strong association of elevated MPs with different thrombotic diseases including recurrent miscarriages and bad obstetric history (BOH) [2]. We analyzed the percentage of PMPs out of the total PS-expressing MPs in 25 healthy pregnant women by flow cytometry. The method for MP analysis (standardized by participating in the International Society of Thrombosis and Haemostasis workshop) was carried out as described earlier [2]. In this study, Annexin V binding, platelet and endothelial MPs were assessed using Annexin V-fluorescein isothiocyanate (FITC), phycoerythrin (PE) labeled specific monoclonal antibody against platelet antigen (CD41-PE, IgG1, κ, clone HIP8) and activated endothelial antigen (CD 62e-PE, IgG1, κ, clone 68-5H11). All the antibodies and buffers were provided by BD Biosciences, United States. In our study, all the samples were processed uniformly with regard to sample collection, time spent between sample collection and processing, the sample processing and time period for storage at −80 °C. The mean %PMPs was found to be 20.7%, 17% and 20% in the 1st, 2nd and 3rd trimesters and 32% post-delivery respectively. In another group of 14 women with a history of unexplained pregnancy loss (PL) on heparin and aspirin during pregnancy, it was observed that elevated PSexpressing MPs at the start of pregnancy decreased as anticoagulant
☆ Author statement: KG and SS conceived and designed the experiments. RP performed the experiments, analyzed the data and wrote the manuscript. SM assisted in the flow cytometry technique for MP detection. The clinical evaluation was done by KG. Critical revision of manuscript for important intellectual content and final approval was done by SS.
http://dx.doi.org/10.1016/j.bcmd.2015.12.007 1079-9796/© 2015 Elsevier Inc. All rights reserved.
therapy (ACT) progressed and normalized in women who had live birth outcome, possibly as a result of ACT [3]. On analysis of the %PMPs, it was found that it increased from 13% to 23% and then 19% in the 1st, 2nd and 3rd trimesters respectively, as pregnancy with ACT progressed. The %PMPs post-delivery was found to be 28%, again similar to healthy controls. There were 3 women with a history of unexplained recurrent PL without any thrombophilia markers, thus given no treatment in their subsequent pregnancies. The mean %PMPs in only these 3 women was 6% and 7% in the 1st and 2nd trimesters after which they suffered from PL. The %PMP was 27% post-PL similar to that as healthy controls. This data shows a significant decrease in %PMP in such women. The mean ± Standard deviation (SD) of %PMPs along with the mean ± SD PMP levels and PS expressing MP levels of all groups are given in Table 1. Fig. 1 shows the graph of the mean %PMPs in recurrent PL patients initiated on ACT and those not on any therapy along with controls as pregnancy progresses and Fig. 2 shows the scatter diagram of %PMPs of different groups in different trimesters. As it can be seen in Table 1 and Fig. 1, the %PMPs rather than the actual PMP levels seems to be a clinically interesting marker along with the PS expressing MP levels. Statistical significance was assumed at p b 0.05, 95% confidence interval (CI) and was calculated using paired Student's t test using SPSS statistical software. Though the endothelial MP levels were found elevated in the patients when compared to controls and decreased as ACT progressed; no statistically significant difference was obtained with regards to %endothelial MPs. Recently another report [4] also found that the %PMPs in laboring healthy women was 8.5% in the 3rd trimester before delivery and 20.5% after delivery. The %PMPs in non-pregnant healthy controls was also found to be the same as post-delivery i.e. 18%. However, they concluded that labor and delivery are associated with an increase in platelet activation and thus the increase in %PMPs. Rather than an increase in the %PMPs, the trend seems to be that %PMPs decrease during pregnancy and normalize post-delivery. In women with recurrent PL the decrease is highly significant. The data showed that in the first trimester the %PMPs was statistically reduced in patients with unexplained RPL when compared to healthy pregnant women (p b 0.01). When these patients are put on ACT, the %PMPs, though highly decreased in the 1st trimester, yet it increased gradually and normalized like healthy pregnancy. The %PMPs in the 2nd and 3rd trimesters was statistically the same as controls, thus showing that due to the ACT the PMPs in fibrin clot may be released, thus achieving normal values and leading to successful live birth. In the 3 women where no treatment was given, the %PMPs remained decreased and they suffered from PL. On comparing the %PMP in RPL patients on ACT and those not on ACT, though statistically the difference is not seen as the number is extremely small (n = 3) for patients not on ACT, but on observation it may suggest that in patients not on ACT, the PMPs remain in the clot which may lead to uteroplacental thrombosis and subsequent PL as seen in these 3 patients, thus the extremely low %PMP in these patients in both 1st and 2nd trimesters.
116
Letter to the Editor
Table 1 Microparticle levels along with %platelet MPs in different groups. Patients
MP levels
1st trismester
2nd trimester
3rd trimester
3 months later
Normal pregnancy n = 25
PS expressing MP levels (MPs/μl) Mean ± SD PMP levels (MPs/μl) Mean ± SD Mean %PMPs PS expressing MP levels (MPs/μl) Mean ± SD PMP levels (MPs/μl) Mean ± SD Mean %PMPs PS expressing MP levels (MPs/μl) Mean ± SD PMP levels (MPs/μl) Mean ± SD Mean %PMPs
1473 ± 489.3
1865.9 ± 590.4
2755.6 ± 771
1894.7 ± 488
304.6 ± 220.8
315 ± 218.1
552.1 ± 348.8
731.5 ± 377
20.7 12,911.9 ± 5911.2
17 6101.7 ± 1979
20 3045.4 ± 905
39 4294.1 ± 3549
1678.6 ± 691
1403 ± 1165
706.5 ± 515
1168.2 ± 2640
13 8333 ± 602
23 14,113.5 ± 2385
23 -
27 4294.1 ± 3549
463 ± 22.6
944.5 ± 686
-
1168.2 ± 2640
6
7
-
27
Women with unexplained PL on anticoagulant therapy n = 14
Women with unexplained PL not on anticoagulant therapy n=3
Thus, we believe that one possible reason for this decrease in the percentage during pregnancy may be the removal of PMPs from peripheral circulation due to consumption in initiating or favoring the clot formation which occurs in the placental beds, thus trapping the PMPs. Fibrin deposits have been evidenced in placental vasculature in women with BOH and PMPs have been shown to adhere to fibrin. There have been other hypotheses that MPs may be cleared from plasma by adhering to blood or vascular cells or their binding to first complement protein–C1q [5,6]. However, PMP consumption in fibrin deposits in the hypercoagulable state of pregnancy seems to be the most apt explanation. MPs derived from other cell types may also have a role in accelerating the clotting cascade. This may promote further clot formation and uteroplacental thrombosis thus aiding in the PL. In patients on ACT, the anticoagulants may be preventing the clot formation, explaining the gradual increase in %PMP in such patients. Conclusion Thus based on our preliminary data along with few other reports [5,6] in this context, it may be stated that the percentage of PMPs is an important marker to assess, diagnose and predict the pregnancy outcome. A decrease in the %PMPs may suggest their consumption in fibrin deposits.
Fig. 1. Percentage mean platelet microparticles in different groups as pregnancy progresses. The blue line shows the mean %PMPs in healthy pregnancy, the green line shows that in women suffering from recurrent pregnancy loss, mean %PMPs is decreased at the beginning, but after anticoagulant therapy is initiated, the % increases and normalizes. The red line shows the highly decreased %PMPs as pregnancy progresses in patients not on any therapy with the outcome being pregnancy loss.
Such cases may be dealt by ACT which will prevent such clot formation. More studies are required to explain these observations and strengthen this hypothesis. Ethics approval The study was approved by the Institutional Ethics Review Board i.e. “Institutional Committee for Research on Human Subjects, National Institute of Immunohaematology (ICMR)” and written informed consent was obtained from all participants. All investigations were conducted according to the principles expressed in the Declaration of Helsinki. Disclosure RP, KG, SB and SS declare that they have no conflict of interest. Acknowledgment The study was supported by the Council of Scientific and Industrial Research (CSIR) (Grant no.: File No.: 09/859 (0001)/2011- EMR-I) and
Fig. 2. Scatter diagram of the %platelet microparticles in different groups as pregnancy progresses. The green marker shows the %PMPs in healthy pregnancy in 3 trimesters; the blue marker shows the %PMPs in women suffering from recurrent pregnancy loss on anticoagulant therapy (ACT) and the red marker shows those not on any therapy. It clearly shows that %PMPs in RPL women is decreased at the start of pregnancy and when ACT is initiated the %PMPs normalize with successful pregnancy outcome. However, in 3 patients who were not on therapy, the %PMPs remains decreased and the outcome was pregnancy loss.
Letter to the Editor
Department of Science and Technology (DST) (Grant no. SR/ SO/ HS/ 150/ 2012). References [1] T. Burnouf, H.A. Goubran, M.L. Chou, D. Devos, M. Radosevic, Platelet microparticles: detection and assessment of their paradoxical functional roles in disease and regenerative medicine, Blood Rev. 28 (2014) 155–166. [2] R. Patil, K. Ghosh, P. Satoskar, S. Shetty, Elevated procoagulant endothelial and tissue factor expressing microparticles in women with recurrent pregnancy loss, PLoS One 20 (2013), e81407. [3] R. Patil, K. Ghosh, K. Damania, V. Bansal, P. Satoskar, A. Darekar, S. Shetty, Effect of anticoagulant therapy on cell-derived microparticles (MP) and pregnancy outcome in women with pregnancy loss, Br. J. Haematol. (2015) (Epub ahead of print). [4] A.E. Wong, H.C. Kwaan, W.A. Grobman, I. Weiss, C.A. Wong, Microparticle source and tissue factor expression in pregnancy, Ann. Hematol. 94 (2015) 1285–1290. [5] J. Alijotas-Reig, C. Palacio-Garcia, I. Farran-Codina, C. Zarzoso, L. Cabero-Roura, M. Vilardell-Tarres, Circulating cell-derived microparticles in women with pregnancy loss, Am. J. Reprod. Immunol. 66 (2011) 199–208. [6] F. Bretelle, F. Sabatier, D. Desprez, L. Camoin, L. Grunebaum, V. Combes, et al., Circulating microparticles: a marker of procoagulant state in normal pregnancy and pregnancy complicated by preeclampsia or intrauterine growth restriction, Thromb. Haemost. 89 (2003) 486–492.
117
Rucha Patil Kanjaksha Ghosh National Institute of Immunohaematology (ICMR), 13th floor, KEM Hospital, Parel, Mumbai 400012, India Srabani Mukherjee National Institute for Research in Reproductive Health (NIRRH), Jehangir Merwanji Street, Parel, Mumbai 400012, India Shrimati Shetty National Institute of Immunohaematology (ICMR), 13th floor, KEM Hospital, Parel, Mumbai 400012, India Corresponding author at: Department of Haemostasis and Thrombosis, National Institute of Immunohaematology (ICMR), KEM Hospital, Parel, Mumbai, India. E-mail address: [email protected]. 15 December 2015
Contents lists available at ScienceDirect
Blood Cells, Molecules and Diseases journal homepage: www.elsevier.com/locate/bcmd
Letter to the Editor Does consumption of platelet-derived microparticles in the fibrin clot explain the decrease in their percentage during pregnancy?☆
Keywords: Platelet-derived microparticles Pregnancy Recurrent pregnancy loss
To the Editor Platelet-derived microparticles (PMPs) are submicronic phospholipid vesicles, derived from platelets during activation, stress or apoptosis. PMPs are the most abundant MPs expressing procoagulant phosphatidylserine (PS) and functional receptors from platelet membranes on their surface giving them their prothrombotic nature. PMPs can therefore act as a pathophysiological threat in certain conditions and cause vascular dysfunction especially in thrombosis complicated diseases [1]. The clinical significance of these procoagulant MPs is many. There are many studies which show a strong association of elevated MPs with different thrombotic diseases including recurrent miscarriages and bad obstetric history (BOH) [2]. We analyzed the percentage of PMPs out of the total PS-expressing MPs in 25 healthy pregnant women by flow cytometry. The method for MP analysis (standardized by participating in the International Society of Thrombosis and Haemostasis workshop) was carried out as described earlier [2]. In this study, Annexin V binding, platelet and endothelial MPs were assessed using Annexin V-fluorescein isothiocyanate (FITC), phycoerythrin (PE) labeled specific monoclonal antibody against platelet antigen (CD41-PE, IgG1, κ, clone HIP8) and activated endothelial antigen (CD 62e-PE, IgG1, κ, clone 68-5H11). All the antibodies and buffers were provided by BD Biosciences, United States. In our study, all the samples were processed uniformly with regard to sample collection, time spent between sample collection and processing, the sample processing and time period for storage at −80 °C. The mean %PMPs was found to be 20.7%, 17% and 20% in the 1st, 2nd and 3rd trimesters and 32% post-delivery respectively. In another group of 14 women with a history of unexplained pregnancy loss (PL) on heparin and aspirin during pregnancy, it was observed that elevated PSexpressing MPs at the start of pregnancy decreased as anticoagulant
☆ Author statement: KG and SS conceived and designed the experiments. RP performed the experiments, analyzed the data and wrote the manuscript. SM assisted in the flow cytometry technique for MP detection. The clinical evaluation was done by KG. Critical revision of manuscript for important intellectual content and final approval was done by SS.
http://dx.doi.org/10.1016/j.bcmd.2015.12.007 1079-9796/© 2015 Elsevier Inc. All rights reserved.
therapy (ACT) progressed and normalized in women who had live birth outcome, possibly as a result of ACT [3]. On analysis of the %PMPs, it was found that it increased from 13% to 23% and then 19% in the 1st, 2nd and 3rd trimesters respectively, as pregnancy with ACT progressed. The %PMPs post-delivery was found to be 28%, again similar to healthy controls. There were 3 women with a history of unexplained recurrent PL without any thrombophilia markers, thus given no treatment in their subsequent pregnancies. The mean %PMPs in only these 3 women was 6% and 7% in the 1st and 2nd trimesters after which they suffered from PL. The %PMP was 27% post-PL similar to that as healthy controls. This data shows a significant decrease in %PMP in such women. The mean ± Standard deviation (SD) of %PMPs along with the mean ± SD PMP levels and PS expressing MP levels of all groups are given in Table 1. Fig. 1 shows the graph of the mean %PMPs in recurrent PL patients initiated on ACT and those not on any therapy along with controls as pregnancy progresses and Fig. 2 shows the scatter diagram of %PMPs of different groups in different trimesters. As it can be seen in Table 1 and Fig. 1, the %PMPs rather than the actual PMP levels seems to be a clinically interesting marker along with the PS expressing MP levels. Statistical significance was assumed at p b 0.05, 95% confidence interval (CI) and was calculated using paired Student's t test using SPSS statistical software. Though the endothelial MP levels were found elevated in the patients when compared to controls and decreased as ACT progressed; no statistically significant difference was obtained with regards to %endothelial MPs. Recently another report [4] also found that the %PMPs in laboring healthy women was 8.5% in the 3rd trimester before delivery and 20.5% after delivery. The %PMPs in non-pregnant healthy controls was also found to be the same as post-delivery i.e. 18%. However, they concluded that labor and delivery are associated with an increase in platelet activation and thus the increase in %PMPs. Rather than an increase in the %PMPs, the trend seems to be that %PMPs decrease during pregnancy and normalize post-delivery. In women with recurrent PL the decrease is highly significant. The data showed that in the first trimester the %PMPs was statistically reduced in patients with unexplained RPL when compared to healthy pregnant women (p b 0.01). When these patients are put on ACT, the %PMPs, though highly decreased in the 1st trimester, yet it increased gradually and normalized like healthy pregnancy. The %PMPs in the 2nd and 3rd trimesters was statistically the same as controls, thus showing that due to the ACT the PMPs in fibrin clot may be released, thus achieving normal values and leading to successful live birth. In the 3 women where no treatment was given, the %PMPs remained decreased and they suffered from PL. On comparing the %PMP in RPL patients on ACT and those not on ACT, though statistically the difference is not seen as the number is extremely small (n = 3) for patients not on ACT, but on observation it may suggest that in patients not on ACT, the PMPs remain in the clot which may lead to uteroplacental thrombosis and subsequent PL as seen in these 3 patients, thus the extremely low %PMP in these patients in both 1st and 2nd trimesters.
116
Letter to the Editor
Table 1 Microparticle levels along with %platelet MPs in different groups. Patients
MP levels
1st trismester
2nd trimester
3rd trimester
3 months later
Normal pregnancy n = 25
PS expressing MP levels (MPs/μl) Mean ± SD PMP levels (MPs/μl) Mean ± SD Mean %PMPs PS expressing MP levels (MPs/μl) Mean ± SD PMP levels (MPs/μl) Mean ± SD Mean %PMPs PS expressing MP levels (MPs/μl) Mean ± SD PMP levels (MPs/μl) Mean ± SD Mean %PMPs
1473 ± 489.3
1865.9 ± 590.4
2755.6 ± 771
1894.7 ± 488
304.6 ± 220.8
315 ± 218.1
552.1 ± 348.8
731.5 ± 377
20.7 12,911.9 ± 5911.2
17 6101.7 ± 1979
20 3045.4 ± 905
39 4294.1 ± 3549
1678.6 ± 691
1403 ± 1165
706.5 ± 515
1168.2 ± 2640
13 8333 ± 602
23 14,113.5 ± 2385
23 -
27 4294.1 ± 3549
463 ± 22.6
944.5 ± 686
-
1168.2 ± 2640
6
7
-
27
Women with unexplained PL on anticoagulant therapy n = 14
Women with unexplained PL not on anticoagulant therapy n=3
Thus, we believe that one possible reason for this decrease in the percentage during pregnancy may be the removal of PMPs from peripheral circulation due to consumption in initiating or favoring the clot formation which occurs in the placental beds, thus trapping the PMPs. Fibrin deposits have been evidenced in placental vasculature in women with BOH and PMPs have been shown to adhere to fibrin. There have been other hypotheses that MPs may be cleared from plasma by adhering to blood or vascular cells or their binding to first complement protein–C1q [5,6]. However, PMP consumption in fibrin deposits in the hypercoagulable state of pregnancy seems to be the most apt explanation. MPs derived from other cell types may also have a role in accelerating the clotting cascade. This may promote further clot formation and uteroplacental thrombosis thus aiding in the PL. In patients on ACT, the anticoagulants may be preventing the clot formation, explaining the gradual increase in %PMP in such patients. Conclusion Thus based on our preliminary data along with few other reports [5,6] in this context, it may be stated that the percentage of PMPs is an important marker to assess, diagnose and predict the pregnancy outcome. A decrease in the %PMPs may suggest their consumption in fibrin deposits.
Fig. 1. Percentage mean platelet microparticles in different groups as pregnancy progresses. The blue line shows the mean %PMPs in healthy pregnancy, the green line shows that in women suffering from recurrent pregnancy loss, mean %PMPs is decreased at the beginning, but after anticoagulant therapy is initiated, the % increases and normalizes. The red line shows the highly decreased %PMPs as pregnancy progresses in patients not on any therapy with the outcome being pregnancy loss.
Such cases may be dealt by ACT which will prevent such clot formation. More studies are required to explain these observations and strengthen this hypothesis. Ethics approval The study was approved by the Institutional Ethics Review Board i.e. “Institutional Committee for Research on Human Subjects, National Institute of Immunohaematology (ICMR)” and written informed consent was obtained from all participants. All investigations were conducted according to the principles expressed in the Declaration of Helsinki. Disclosure RP, KG, SB and SS declare that they have no conflict of interest. Acknowledgment The study was supported by the Council of Scientific and Industrial Research (CSIR) (Grant no.: File No.: 09/859 (0001)/2011- EMR-I) and
Fig. 2. Scatter diagram of the %platelet microparticles in different groups as pregnancy progresses. The green marker shows the %PMPs in healthy pregnancy in 3 trimesters; the blue marker shows the %PMPs in women suffering from recurrent pregnancy loss on anticoagulant therapy (ACT) and the red marker shows those not on any therapy. It clearly shows that %PMPs in RPL women is decreased at the start of pregnancy and when ACT is initiated the %PMPs normalize with successful pregnancy outcome. However, in 3 patients who were not on therapy, the %PMPs remains decreased and the outcome was pregnancy loss.
Letter to the Editor
Department of Science and Technology (DST) (Grant no. SR/ SO/ HS/ 150/ 2012). References [1] T. Burnouf, H.A. Goubran, M.L. Chou, D. Devos, M. Radosevic, Platelet microparticles: detection and assessment of their paradoxical functional roles in disease and regenerative medicine, Blood Rev. 28 (2014) 155–166. [2] R. Patil, K. Ghosh, P. Satoskar, S. Shetty, Elevated procoagulant endothelial and tissue factor expressing microparticles in women with recurrent pregnancy loss, PLoS One 20 (2013), e81407. [3] R. Patil, K. Ghosh, K. Damania, V. Bansal, P. Satoskar, A. Darekar, S. Shetty, Effect of anticoagulant therapy on cell-derived microparticles (MP) and pregnancy outcome in women with pregnancy loss, Br. J. Haematol. (2015) (Epub ahead of print). [4] A.E. Wong, H.C. Kwaan, W.A. Grobman, I. Weiss, C.A. Wong, Microparticle source and tissue factor expression in pregnancy, Ann. Hematol. 94 (2015) 1285–1290. [5] J. Alijotas-Reig, C. Palacio-Garcia, I. Farran-Codina, C. Zarzoso, L. Cabero-Roura, M. Vilardell-Tarres, Circulating cell-derived microparticles in women with pregnancy loss, Am. J. Reprod. Immunol. 66 (2011) 199–208. [6] F. Bretelle, F. Sabatier, D. Desprez, L. Camoin, L. Grunebaum, V. Combes, et al., Circulating microparticles: a marker of procoagulant state in normal pregnancy and pregnancy complicated by preeclampsia or intrauterine growth restriction, Thromb. Haemost. 89 (2003) 486–492.
117
Rucha Patil Kanjaksha Ghosh National Institute of Immunohaematology (ICMR), 13th floor, KEM Hospital, Parel, Mumbai 400012, India Srabani Mukherjee National Institute for Research in Reproductive Health (NIRRH), Jehangir Merwanji Street, Parel, Mumbai 400012, India Shrimati Shetty National Institute of Immunohaematology (ICMR), 13th floor, KEM Hospital, Parel, Mumbai 400012, India Corresponding author at: Department of Haemostasis and Thrombosis, National Institute of Immunohaematology (ICMR), KEM Hospital, Parel, Mumbai, India. E-mail address: [email protected]. 15 December 2015
