Pathogenesis Of Thrombocytopenia In Patients Research Articles

Abstract 13784: Thrombopoietin Restores Megakaryopoiesis By Protecting Bone Marrow Endothelial Progenitor Cells Of Patients With Hematologic Malignancies Undergoing Chemotherapy

Introduction and Objective: Thrombocytopenia is the most common side effect of patients with hematologic malignancies undergoing chemotherapy. Platelet production depends on a good bone marrow (BM) microenvironment, especially the endothelial progenitor cells (EPC), which play an important role in angiogenesis and thus supporting megakaryopoiesis. However, little is known regarding the function of EPC in patients with hematologic malignancies. Therefore, the present study aimed to examine the role of EPC in angiogenesis and megakaryopoiesis, and the effect of thrombopoietin (TPO) on EPC in patients with hematologic malignancies. Methods: Twenty-three patients with ALL or AML, and ten age-matched healthy donors were recruited in the present study. EPCs were isolated from BM mononuclear cells. The cell count, proliferation, migration, and tube formation assays were conducted. Megakaryocytes were quantified by flow cytometry after coculturing with EPC in the absence and presence of TPO (100 ng/ml). In addition, transgenic zebrafish with vascular endothelial cells expressing EGFP [Tg(fli-1:EGFP)] were used to examine the angiogenic effect of TPO. Simvastatin was used to induce vascular defects in zebrafish embryos. The embryos were then treated with or without TPO (10 -3 U/μl) by intravenous injection before fluorescent images were captured. Results: The number of BM EPC was significantly smaller in patients with ALL and AML than in healthy donors. The BM EPCs from patients showed significantly reduced proliferation, migration and tube formation when compared to those from healthy donors. TPO (100 ng/ml) significantly improved those functions of EPC, and significantly increased the number of megakaryocytes after coculture. In Tg(fli-1:EGFP) zebrafish embryos, TPO (10 -3 U/μl) significantly increased the SIV vascularized area in the presence of simvastatin. Conclusion: The present study shows that dysfunctional BM EPC contributes to the pathogenesis of thrombocytopenia in patients with hematologic malignancies undergoing chemotherapy; and such impaired functions of BM EPC could be restored by TPO. In addition, TPO increases the number of megakaryocytes after coculture with EPC in vitro and promotes angiogenesis in zebrafish embryos.

Time course of immature platelet count and its relation to thrombocytopenia and mortality in patients with sepsis.

IntroductionThe pathogenesis of thrombocytopenia in patients with sepsis is not fully understood. The aims of this study were to investigate changes in thrombopoietic activity over time by using absolute immature platelet counts (AIPC) and to examine the impact of platelet production on thrombocytopenia and mortality in patients with sepsis.MethodsThis retrospective observational study included adult patients with sepsis admitted to the intensive care unit at a university hospital. Two hundred five consecutive sepsis patients were stratified into four groups according to nadir platelet count: severe (nadir ≤40×103/μL), moderate (41–80×103/μL), or mild thrombocytopenia (81–120×103/μL), or normal-increased platelet count (>120×103/μL). The development of thrombocytopenia was assessed during the first week; mortality was assessed at day 28.ResultOf the 205 patients included, 61 (29.8%) developed severe thrombocytopenia. On admission, AIPC did not differ among the four groups. In patients with severe thrombocytopenia, AIPC decreased significantly from days 2 to 7, but remained within or above the normal range in the other three groups (overall group comparison, P<0.0001). Multivariate analysis including coagulation biomarkers revealed that AIPC was independently associated with the development of severe thrombocytopenia (day 3 AIPC, odds ratio 0.49 [95% confidence interval (CI) 0.35–0.66], P<0.0001; day 5 AIPC, 0.59 [95% CI 0.45–0.75], P<0.0001). AIPC was a significant predictor of 28-day mortality in Cox hazard models adjusted for Acute Physiology and Chronic Health Evaluation II and Sequential Organ Failure Assessment scores (day 3 AIPC, hazard ratio 0.70 [95% CI 0.52–0.89], P = 0.0029; day 5 AIPC, 0.68 [95% CI 0.49–0.87], P = 0.0012).ConclusionsThrombopoietic activity was generally maintained in the acute phase of sepsis. However, a decrease in AIPC after admission was independently associated with the development of severe thrombocytopenia and mortality, suggesting the importance of suppressed thrombopoiesis in the pathophysiology of sepsis-induced thrombocytopenia.

Immature platelet fraction (IPF) as a predictive value for thrombopoietic recovery after allogeneic stem cell transplantation

We consecutively examined the utility of measurements of percentage of immature platelet fraction (IPF%) and absolute IPF number (A-IPF) in predicting thrombopoietic recovery in 15 adult patients who underwent allogeneic hematopoietic stem cell transplantation (allo-SCT). Four patients were excluded from the evaluation due to insufficient data. Platelet count and IPF were measured by Sysmex XN-1000 (XN), a newer generation analyzer. First, we confirmed that platelet count measured by XN was more accurate than by XE-2100 (XE). IPF measurement was effective to predict the recovery in 7 of the 11 patients examined. Moreover, IPF measurement, especially IPF% measurement, suggested accelerated platelet turnover in two patients who failed to achieve platelet recovery by day 60. In addition to IPF%, A-IPF showed a complementary role on the prediction of thrombopoietic recovery. The increase in IPF% was only transient, while A-IPF values showed lasting increase during platelet recovery. In two patients (cases 6 and 7) an increase in A-IPF, but not in IPF%, was observed during platelet recovery. Our data suggest that IPF% and A-IPF measured by XN are useful for the prediction of thrombopoietic recovery and the assessment of pathogenesis of thrombocytopenia in patients after allo-SCT.

Prolactin contributes to the pathogenesis of thrombocytopenia in patients with hepatitis C virus

Prolactin contributes to the pathogenesis of thrombocytopenia in patients with hepatitis C virus

Serum Cytokine Profile in Patients with Septic Shock/Severe Sepsis and Thrombocytopenia

IntroductionSepsis is the result of an uncontrolled inflammatory response to various stimuli such as bacterial infections. Sepsis can be complicated by hemodynamic instability, multi-organ dysfunction and hematological abnormalities. Patients with severe sepsis usually develop thrombocytopenia due to various reasons, and the presence of thrombocytopenia is considered as a negative prognostic marker. The aim of the present study was to determine the cytokine profile in serum of patients with severe sepsis and thrombocytopenia.Patients and MethodsSerum cytokine profile was analyzed in a cohort of 112 consecutive patients with severe sepsis and/or septic shock treated in the intensive care unit (ICU) of our institute from October/2009 to September/2012. Patients were divided in two groups (A and B). Group A consisted of 43 patients with severe sepsis and thrombocytopenia (platelet count below 70X103/ìl), while group B consisted of 69 patients without thrombocytopenia. Patients with thrombocytopenia due to disseminated intravascular coagulation (DIC), suspected drug etiology, or due to any obvious etiology such as hematologic malignancy were excluded from our analysis. A cohort of 10 healthy volunteers served as control group.Serum levels of IFNã, IL-8, ICAM, VCAM, and SUPAR (soluble urokinase plasminogen activation receptor) were estimated by using Luminex xMAP technology. Statistical analyses were performed using NCSS software. The following variables were entered in a multiple logistic regression model: 1) age and sex, 2) active malignancy vs. not, 3) septic shock vs. severe sepsis, 4) APACHEII score, 5) SOFA score, 6) serum IFNã, ICAM, VCAM, IL-8, and SUPAR levels, and 7) platelet number. All parameters were estimated on day of admission.ResultsHospital Mortality: Overall 65 out of 112 patients died during their hospital stay. The overall hospital mortality was 58%. In multivariate analysis non-survivors had higher APACHE score (p=0.01) and had lower platelet counts (p<0.001) as compared with survivors.Plasma cytokine levels in patients with and without thrombocytopenia: Patients in group A had a different cytokine profile as compared with patients in group B. Serum levels of VCAM and IFNã were not different between group A and B. However, patients with sepsis and thrombocytopenia had statistically significantly higher serum levels of ICAM, IL-8 and SUPAR (p<0.0001) in comparison with patients with severe sepsis and normal PLT count.Predictive value of serum cytokine for thrombocytopenia: Serum levels of ICAM, IL-8, and SUPAR were good predictors of the presence of thrombocytopenia in patients with sepsis. The ROC curves of ICAM, IL-8, and SUPAR serum levels in predicting thrombocytopenia in patients with sepsis are shown in Figure 1. The AUCs were 0.785 (95% CI, 0.696 – 0.857, p<0.001), 0.729 (95% CI, 0.637 – 0.809, p<0.001), 0.789 (95% CI, 0.701 – 0.861, p<0.001) for ICAM, IL-8 and SUPAR levels respectively. However in multiple logistic regression analysis serum levels of ICAM was the most powerful predictor of thrombocytopenia.ConclusionThrombocytopenia is not an uncommon finding in patients with severe sepsis. Although DIC or drug reactions are well known causes of low PLT counts, the pathogenesis of thrombocytopenia in patients with sepsis remains poorly understood. High levels of ICAM, IL-8 and SUPAR are usually associated with severe endothelial dysfunction. In our study, we showed that patients with thrombocytopenia have a specific serum cytokine profile expression consistent with the presence of endothelial damage. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Carmustine induces platelet apoptosis

Carmustine is one of the alkylating chemotherapeutic agents, which are used to treat various types of cancers, such as brain tumors, Hodgkins and non-Hodgkins lymphoma and multiple myeloma. However, carmustine has the side effect of thrombocytopenia, and the mechanism is not completely understood. In this study, we show that carmustine dose-dependently induced depolarization of mitochondrial inner transmembrane potential (ΔΨm), up-regulation of Bax, down-regulation of Bcl-2 and caspase-3 activation. Carmustine did not induce surface expression of P-selectin or PAC-1 binding, whereas, obviously reduced collagen and thrombin-induced platelet aggregation. Dicumarol, c-Jun NH2-terminal kinase-specific inhibitor, reduced carmustine-induced ΔΨm depolarization in platelets. The numbers of circulating platelets were reduced, and the tail bleeding time was significantly increased in mice that were injected with carmustine. Taken together, these data indicate that carmustine induced platelet apoptosis, suggesting the possible pathogenesis of thrombocytopenia in patients treated with carmustine.

Arsenic Trioxide Induces Apoptosis in Human Platelets via C-Jun NH2-Terminal Kinase Activation

Arsenic trioxide (ATO), one of the oldest drugs in both Western and traditional Chinese medicine, has become an effective anticancer drug, especially in the treatment of acute promyelocytic leukemia (APL). However, thrombocytopenia occurred in most of ATO-treated patients with APL or other malignant diseases, and the pathogenesis remains unclear. Here we show that ATO dose-dependently induces depolarization of mitochondrial inner transmembrane potential (ΔΨm), up-regulation of Bax and down-regulation of Bcl-2 and Bcl-XL, caspase-3 activation, and phosphotidylserine (PS) exposure in platelets. ATO did not induce surface expression of P-selectin and PAC-1 binding, whereas, obviously reduced collagen, ADP, and thrombin induced platelet aggregation. ATO dose-dependently induced c-Jun NH2-terminal kinase (JNK) activation, and JNK specific inhibitor dicumarol obviously reduced ATO-induced ΔΨm depolarization in platelets. Clinical therapeutic dosage of ATO was intraperitoneally injected into C57 mice, and the numbers of circulating platelets were significantly reduced after five days of continuous injection. The data demonstrate that ATO induces caspase-dependent apoptosis via JNK activation in platelets. ATO does not incur platelet activation, whereas, it not only impairs platelet function but also reduces circulating platelets in vivo, suggesting the possible pathogenesis of thrombocytopenia in patients treated with ATO.

Arsenic Trioxide Induces Platelet Apoptosis

Background Arsenic trioxide (ATO), a component of Traditional Chinese Medicine, is known as an effective anticancer drug especially in the treatment of acute promyelocytic leukaemia (APL). APL has emerged as the most curable subtype of acute myeloid leukaemia since the widely use of arsenic trioxide-based chemotherapy. However, recent researches showed that thrombocytopenia occurred in 79% of the relapsed or refractory APL patients treated with arsenic trioxide, and part of the APL patients had to be stopped treatment because of catastrophic bleeding, such as intracranial and pulmonary haemorrhage. Thrombocytopenia also occurred in 43% of the myelodysplastic syndrome patients treated with arsenic trioxide. Recently, arsenic trioxide has been proved to have a pro-apoptotic effect on various kinds of nucleated tumour cells or non-tumour cells. The effect of ATO on enucleated platelet, however, still remains unclear. The aim of current study is to investigate whether ATO induces platelet apoptosis. Methods Washed platelets (3 × 108/ml) were incubated with different concentrations of ATO or vehicle at 37°C for 4 hours. Then, mitochondrial inner transmembrane potential (ΔΨm), phosphatidylserine (PS) exposure, P-selection and PAC-1 binding were tested by flow cytometry. In the mean time, the treated platelets were analyzed by western blot for the expression levels of pro-apoptotic protein (Bax), and anti-apoptotic proteins (Bcl-2 and Bcl-XL). Activation of caspase-3 and c-jun NH2-terminal kinase (JNK) was also examined by western blot. To test whether inhibition of JNK can attenuate ATO-induced platelet apoptosis, JNK specific inhibitor dicumarol was used and ΔΨm was analysis by flow cytometry. For platelet aggregation analysis, PRP was incubated with ATO (2 μM) for 1 hr and then subjected to platelet aggregation assay by collagen. To further investigate whether ATO incurs thrombocytopenia in vivo, clinical therapeutic dosage of ATO (or vehicle (0.9% NS) control) was intraperitoneally injected into C57 mice, and the numbers of circulating platelets were counted. Results and Conclusions ATO dose-dependently induces depolarization of mitochondrial inner ΔΨm, up-regulation of Bax and down-regulation of Bcl-2 and Bcl-XL, caspase-3 activation, and PS exposure in platelets. ATO did not induce surface expression of P-selectin and PAC-1 binding, whereas, obviously reduced collagen-induced platelet aggregation. ATO dose-dependently induced JNK activation, and JNK specific inhibitor dicumarol obviously reduced ATO-induced ΔΨm depolarization in platelets. Clinical therapeutic dosage of ATO was intraperitoneally injected into C57 mice, and the numbers of circulating platelets were significantly reduced after 12 and 24 hours of injection. Taken together, the data demonstrate that ATO induces caspase-dependent apoptosis via mitochondria-mediated intrinsic pathway in platelets. ATO does not incur platelet activation, whereas, it not only impairs platelet function but also incurs thrombocytopenia in vivo, suggesting the possible pathogenesis of thrombocytopenia in patients treated with ATO. Disclosures: No relevant conflicts of interest to declare.

Antiphospholipid antibody associated thrombocytopenia and the paradoxical risk of thrombosis

The pathogenesis of thrombocytopenia in patients with antiphospholipid syndrome (APS) is heterogeneous. Patients with antiphospholipid antibodies (aPL) and thrombocytopenia in the absence of clinical manifestations of APS will be diagnosed and treated as idiopathic thrombocytopenic purpura. However, the presence of aPL places those individuals at particular risk for developing both bleeding and thrombotic complications. Therefore, we propose the inclusion of such patients in the subgroup 'aPL-associated thrombocytopenia'. More attention should be devoted to this subgroup of patients to elucidate the role of aPL in the development of thrombocytopenia and to facilitate the adequate monitoring of its potential thrombotic risk.

Hepatic fibrosis plays a central role in the pathogenesis of thrombocytopenia in patients with chronic viral hepatitis.

The pathogenesis of thrombocytopenia in chronic hepatitis is not well known. This study evaluated the relationship between liver injury, serum thrombopoietin, splenomegaly and thrombocytopenia in chronic viral hepatitis. Two hundred and nine patients were enrolled, 85 with splenomegaly and 124 without. Thrombocytopenia was present in 71% and 23% of patients with or without splenomegaly respectively. In subjects with low platelet count, those with splenomegaly showed significantly lower platelet numbers than those without splenomegaly. The spleen size correlated with portal hypertension. An inverse correlation between spleen size and platelet count was observed (r = -0.54; P < 0.0001). In patients without splenomegaly, thrombocytopenia was associated with the grade of fibrosis; platelet counts were the highest in patients with fibrosis 0-2, lower in those with grade 3 (P < 0.008) and lowest in those with grade 4 (P < 0.05). These findings were independent of demographic and biochemical characteristics, hepatic necroinflammatory activity, portal hypertension and splenomegaly. Patients with normal platelet counts showed higher thrombopoietin levels than those with low platelet counts (P < 0.0001). An inverse correlation between thrombopoietin levels and fibrosis grade was observed (r = - 0.50; P < 0.0001). Median thrombopoietin levels were 58 and 27 pg/ml for fibrosis grade 0-1 and grade 4 respectively (P < 0.001). These data indicate that advanced hepatic fibrosis, causing an altered production of thrombopoietin and portal hypertension, plays the central role in the pathogenesis of thrombocytopenia in chronic viral hepatitis.

Persistence of hepatitis C virus in a human megakaryoblastic leukaemia cell line.

Thrombocytopenia is a frequent clinical finding in patients with hepatitis C virus (HCV) infection. Platelets from patients with HCV infection have been identified as carriers of HCV RNA in our previous studies. The present study was designed to further investigate the possibility of HCV replication in megakaryoblasts from which platelets are eventually released. A megakaryoblastic cell line (MEG-01), established from a chronic myelogenous leukaemia patient 13 years ago, was used for this study. The MEG-01 cells were inoculated with fresh serum from a patient with HCV infection and renamed MEG-01-I cells. Surprisingly, both MEG-01 and MEG-01-I were positive by HCV reverse transcription-polymerase chain reaction (RT-PCR) for the existence of HCV RNA and minus-strand HCV RNA, regardless of inoculation. This was further confirmed by in situ RT-PCR. The HCV antigens, such as core, envelope, and non-structural (NS)3 and NS4, were also present in both cell lines, as identified by Western blotting and indirect immunofluorescence staining. In addition, virus-like particles were observed by electron microscopy in the MEG-01 cell line as well as in the MEG-01-I cell line. These findings indicate that the megakaryoblasts are vulnerable to HCV infection and that replication of HCV can occur in these cells. This may help us to better understand the pathogenesis of thrombocytopenia in patients with HCV infection. The MEG-01 cell line, which may have been continuously shedding HCV for years, should be a useful model for experimental research into HCV.

Indium 111-labeled platelet kinetic studies and platelet-associated IgG in hairy cell leukemia.

In order to study the pathogenesis of thrombocytopenia in patients with hairy cell leukemia (HCL), levels of platelet-associated IgG (PAIgG), platelet life span (MLS), and the sequestration site of autologous 111In-labeled platelets were measured in nine patients with HCL. Splenectomized patients (n = 4) had a higher platelet count (x = 122.5 X 10(9)/l; range, 80-190 X 10(9)/l) as well as higher levels of PAIgG (x = 10.7%; range, 5.8-16.9%), than nonsplenectomized patients (platelets x = 76 X 10(9)/l, range 40-100 X 10(9)/l; PAIgG x = 3.2%, range 2.2-4.2%). A normal recovery of 111In-labeled platelets was found in splenectomized patients, whereas a very low recovery was observed in the nonsplenectomized group (x = 70.2%, range, 50-82.5%, versus x = 22.4%, range, 15-28.2%). The MLS was borderline normal in all patients. The site of sequestration was the spleen in nonsplenectomized patients. The low recovery of 111In-labeled platelets in nonsplenectomized patients suggests "hypersplenism" with pooling as a major cause of thrombocytopenia, in addition to impaired thrombocytopoiesis and possible immune-mediated platelet destruction.