Rucha Patil

Elevated Procoagulant Endothelial and Tissue Factor Expressing Microparticles in Women with Recurrent Pregnancy Loss

Background 15% of reproducing couples suffer from pregnancy loss(PL) and recurs in 2-3%. One of the most frequently hypothesized causes of unexplained PL refers to a defective maternal haemostatic response leading to uteroplacental thrombosis. Hereditary thrombophilia and antiphospholipid antibodies have been extensively described as risk factors for PL in women with unknown aetiology. Recently, a new marker has emerged: the cell-derived procoagulant circulating microparticles(MPs) which have been reported to have a major role in many thrombosis complicated diseases. This study aims to analyze the significance of procoagulant MPs in women suffering from unexplained recurrent pregnancy loss(RPL), and characterize their cellular origin. Method and Findings 115 women with RPL were analyzed for common thrombophilia markers and different cell derived MPs-total annexinV, platelet(CD41a), endothelial(CD146,CD62e), leukocyte(CD45), erythrocyte(CD235a) and tissue factor(CD142)(TF) expressing MPs and were compared with 20 healthy non-pregnant women. Methodology for MP analysis was standardized by participating in the “Vascular Biology Scientific and Standardization Committee workshop”. Results Total annexinV, TF and endothelial MPs were found significantly increased(p<0.05, 95% confidence interval) in women with RPL. The procoagulant activity of MPs measured by STA-PPL clotting time assay was found in correspondence with annexinV MP levels, wherein the clot time was shortened in samples with increased MP levels. Differences in platelet, leukocyte and erythrocyte derived MPs were not significant. Thirty seven of 115 women were found to carry any of the acquired or hereditary thrombophilia markers. No significant differences were seen in the MP profile of women with and without thrombophilia marker. Conclusion The presence of elevated endothelial, TF and phosphatidylserine expressing MPs at a distance (at least 3 months) from the PL suggests a continued chronic endothelial damage/activation which may get exaggerated at the onset of pregnancy. The data suggests that MPs may contribute to uteroplacental thrombosis and are associated with the pathogenesis of RPL.

A simple clot based assay for detection of procoagulant cell-derived microparticles.

Abstract Background: Cell-derived microparticles (MPs) are important biomarkers in many facets of medicine. However, the MP detection methods used till date are costly and time consuming. The main aim of this study was to standardize an in-house clot based screening method for MP detection which would not only be specific and sensitive, but also inexpensive. Methods: Four different methods of MP assessment were performed and the results correlated. Using the flow cytometry technique as the gold standard, 25 samples with normal phosphatidylserine (PS) expressing MP levels and 25 samples with elevated levels were selected, which was cross checked by the commercial STA Procoag PPL clotting time (CT) assay. A simple recalcification time and an in-house clot assay were the remaining two tests. The in-house test measures the CT after the addition of calcium chloride to MP rich plasma, following incubation with Russell viper venom and phospholipid free plasma. Results: The CT obtained by the in-house assay significantly correlated with the results obtained by flow cytometry (R2=0.87, p<0.01). Conclusions: Though preliminary, the in-house assay seems to be efficient, inexpensive and promising. It could definitely be utilized routinely for procoagulant MP assessment in various clinical settings.

Inherited and acquired thrombophilia in Indian women experiencing unexplained recurrent pregnancy loss

The most frequently hypothesized cause of unexplained recurrent pregnancy loss (RPL) refers to a defective maternal haemostatic response leading to uteroplacental thrombosis. Approximately 20% women suffering from pregnancy loss (PL) are associated with autoimmune disorders and more than 50% remain idiopathic after common traditional investigations. The present study aims to investigate the prevalence of different genetic and acquired thrombophilia markers in a large series of Indian women with RPL. Such studies will help analyze the markers which pose maximum risk and help in the appropriate treatment in subsequent pregnancies. The study comprised of 587 women with no apparent etiological causes of RPL and 115 healthy women controls. p values were calculated with two tailed Fishers exact test; statistical significance was assumed at p<0.05, 95% confidence interval. Relative risks were also calculated. Among genetic thrombophilia, the risk of PL was highest with protein S deficiency (16%, p=0.006) followed by plasminogen activator inhibitor-1 4G/4G (23%, p=0.007) polymorphism. Among acquired markers, the risk of PL was the highest in women with anti-cardiolipin antibodies (24%, p=0.0001), followed by anti-annexin V antibodies (23%, p=0.0009) and lupus anticoagulants (8%, p=0.02). Thrombophilia, inherited and acquired, is an important contributing factor in unexplained RPL and should be screened in the order of its prevalence.

Role of microparticles in recurrent miscarriages and other adverse pregnancies: a review

The multiple functions attributed to microparticles (MPs) include blood coagulation, inflammation, tumorigenesis, angiogenesis, immunomodulatory functions and intercellular cross talk. These have drawn considerable interest during the last few years. The prothrombotic nature of MPs has linked them with almost all groups of thrombotic disorders including recurrent miscarriage (RM) and other abnormal pregnancy outcomes. Two authors (SS and RP) conducted a search independently on the computerized databases MEDLINE and EMBASE using relevant key words. Contradictory reports were observed on the association of MPs with RM. While most of the reports showed increased prevalence of MPs, both platelet and endothelial cell derived, in RM, some did not show any association. Almost all the reports showed a strong association of MPs with preeclampsia (PE), while the association with other adverse pregnancy conditions was not very conclusive. It may be concluded that MPs by themselves may result in adverse conditions or that they may be additive factors to an already existing prothrombotic state due to acquired or genetic thrombophilia or some unknown thrombophilic condition, besides the pre-existing hypercoagulable status of pregnancy.

Comment on Salomon et al. Gestational Diabetes Mellitus Is Associated With Changes in the Concentration and Bioactivity of Placenta-Derived Exosomes in Maternal Circulation Across Gestation. Diabetes 2016;65:598-609.

We read with interest the well-designed study by Salomon et al. (1) in which the authors have shown a twofold increase in placenta-derived exosomes (PdEs) in women with gestational diabetes mellitus (GDM) as compared with those in normal pregnancy at three gestational points. They have also shown the bioactivity of these exosomes in terms of increased release of proinflammatory cytokines from endothelial cells. We believe that, along with PdEs, procoagulant microparticles (MPs) (total and placenta-derived) should also …

Paradoxical Bleeding and Thrombosis in a Patient With Afibrinogenemia and Fibrinogen Mumbai Mutation

OBJECTIVES Thrombosis is rarely reported in cases of afibrinogenemia and is generally associated with thrombophilia or replacement therapy. Often, it is difficult to predict whether the patients will bleed or whether they are exposed to the risk of thrombosis. METHODS We report a patient with afibrinogenemia who presented with complete thrombosis of right hepatic, portal, and splenic veins and who described a lifelong history of bleeding. Direct sequencing of the three fibrinogen genes was performed to identify the mutation. RESULTS DNA sequencing showed the presence of a homozygous for G8017A substitution in exon 8 of the fibrinogen β-chain gene, resulting in a G434D missense mutation (Fibrinogen Mumbai). CONCLUSIONS Presence of both bleeding and thrombotic manifestations in a patient with afibrinogenemia in the presence of other associated risk factors warrants a very careful individualized approach in the management of patients with afibrinogenemia.

Profibrinolytic microparticles are not adequately produced to compensate their prothrombotic effect

We read with interest the report by Lacroix et al .[1][1] on the fibrinolytic potential of cell-derived microparticles (MPs), i.e. specifically endothelial-and leukocyte-derived microparticles, in a very well designed set of in vitro experiments. They have reported elevated levels of profibrinolytic

Management of pregnancy in dysfibrinogenemia cases: a dilemma.

Dysfibrinogenemia is a very challenging disorder, and there are no firm guidelines on treatment for pregnant patients with dysfibrinogenemia. A 37-year-old patient with a history of six unexplained recurrent miscarriages was referred for thrombophilia testing. Elevated procoagulant microparticles were found, for which during her seventh pregnancy anticoagulant therapy was initiated. However, she again miscarried and bled excessively. She was then diagnosed with dysfibrinogenemia. DNA sequence analysis revealed a novel homozygous insertion-deletion in exon 7 in FGB. Dysfibrinogenemia is very difficult to diagnose and even after diagnosis, the treatment varies with the patients symptoms. In this case, anticoagulant therapy failed. With her history of recurrent miscarriages, it is clear that pregnancy without any treatment is not an option. Few reports suggest a combination of intravenous fibrinogen infusions along with anticoagulants in which successful pregnancy outcome was achieved. The present case thus stresses on the need for some treatment guidelines in such cases.

Annexin A5 levels or circulating microparticles: what we see depends mainly on what we look for

Click here to view the article Letter to the Editor by C. Reutelingsperger et al.

Role of lupus anticoagulants in immediate acting inhibitor positivity in congenital haemophilia A patients

OBJECTIVES Presence of lupus anticoagulants (LA) in haemophilia and their interference in coagulation assays is well-known. Factor VIII (FVIII) inhibitors are generally time and temperature dependent whereas LAs are immediate acting inhibitors (IAIs). The present study reports the challenges in laboratory detection of both progressive and non-progressive, specific FVIII inhibitors in the presence of LA. METHODS From 2012 through 2015, 4900 HA patients were screened for inhibitors. APTT based inhibitor screening tests and Nijmegen-modified Bethesda assay (NBA) were done in all samples. LA test and FVIII inhibitors by ELISA were done in patients with IAIs. RESULTS Out of 451 patients positive for inhibitors in the initial screening tests, classical and progressive FVIII inhibitors were observed in 398 patients while 53 had IAIs showing no/partial correction in 1:1 mixtures of NPP and patient plasma. In 27 patients, both FVIII and FIX activity levels were <1%, resulting in difficulty in diagnosis. In 48 HA patients with IAIs, 42 were LA positive. 4 patients were found to have only LA with false positive results in NBA while 38 had a combination of LA and FVIII inhibitors. Six patients were LA negative and had only FVIII IAIs. Five (62.5%) of 8 HA patients initiated on immune tolerance induction (ITI) also were positive for IAIs. CONCLUSION The findings emphasizes the presence of specific FVIII inhibitors in congenital HA with absence of time dependent inactivation kinetics in a small proportion of cases. ELISA or chromogenic assays along with LA testing can offer accurate laboratory diagnosis in patients with coexisting LA.