Thrombosis In Paroxysmal Nocturnal Hemoglobinuria Research Articles

Anticoagulation Use and Patient Outcomes in Paroxysmal Nocturnal Hemoglobinuria: A Province-Wide Retrospective Review

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disorder caused by somatic mutations in hematopoietic stem cells and is characterized by hemolysis, bone marrow failure, and thrombosis. Thrombotic events are the leading cause of morbidity and mortality in PNH, but little has been published about the role of prophylactic anticoagulation to prevent thrombotic events, and the ideal anticoagulant for patients who do experience thrombosis. Additionally, to our knowledge, bleeding outcomes in patients on prophylactic and therapeutic dose anticoagulants have not been examined. Our objective was therefore to examine our practice patterns regarding the use and outcomes of anticoagulation in PNH patients in our province. Methods: This multi-center, retrospective cohort study examined the epidemiology, management, thrombotic and bleeding complications of 31 adult patients with PNH in the province of Alberta, Canada during the period of January 1, 2000, to December 31, 2022. Patients were identified by International Classification of Diseases (ICD)-9 codes from hospital discharge summaries and ambulatory clinic records. Chart reviews were performed to collect patient demographics, clinical and laboratory characteristics, use of anticoagulation including agent and dose, and thrombotic and bleeding complications. Results: The median age of the cohort was 50 (interquartile range, IQR 35-70) years and 10 (32%) patients were male. Concurrent aplastic anemia and myelodysplastic syndrome were present in 15 (48%) and 3 (10%) patients, respectively. Disease presentation included thrombosis in 4 (13%) patients and bleeding in 5 (16%) patients. The majority of patients (74%) were treated with a complement inhibitor, most commonly eculizumab. Over the study period, 7 (23%) patients experienced 10 thrombotic events, including venous thromboembolism in 6 (60%) patients and arterial thromboembolism in 4 (40%) patients. The most common vascular sites of thrombosis were deep vein thrombosis of the lower extremity (3 patients) and stroke (2 patients). Of the 10 thrombotic events, 3 (30%) occurred while the patient was receiving a complement inhibitor. Thrombosis occurred at a median of 4.5 months from PNH diagnosis and the average granulocyte clone size at the time of thrombosis was 40.1% (IQR 11.3-82.9%). Of these 10 thrombotic events, 2 occurred on therapeutic-dose anticoagulation. In total, 8 (26%) patients experienced 13 bleeding events, 4 (13%) meeting criteria for ISTH major bleeding. The average platelet count at the time of bleeding was 96 (IQR 79-139). Of the 13 bleeding events, 5 occurred on an antiplatelet, 2 occurred on prophylactic-dose anticoagulation, and 2 occurred on therapeutic-dose anticoagulation. Thirteen (42%) patients were on anticoagulation: 7 (23%) patients on primary prophylaxis and 6 (19%) patients on secondary prophylaxis. For primary prophylaxis, 4 patients received a direct oral anticoagulant (DOAC), 3 at a prophylactic dose and 1 at a therapeutic dose, and 3 patients received warfarin at a therapeutic dose. Six (86%) patients on primary prophylaxis were also receiving a complement inhibitor. With regards to secondary prophylaxis, 2 patients received a DOAC, 3 patients received warfarin, and 1 patient received low molecular weight heparin. All secondary prophylaxis was given at a therapeutic dose. Five (83%) patients on secondary prophylaxis were also receiving a complement inhibitor. Of patients on a DOAC, 1 patient experienced a breakthrough arterial thromboembolism (stroke), and no patients experienced a breakthrough venous thromboembolism. Conclusion: Thrombosis in PNH is common and prevention is key to prevent long-term morbidity and mortality. Our practice patterns indicate that DOACs are being used in our province as primary prophylaxis, even in patients who are concurrently being treated with a complement inhibitor. DOACs are also being used more frequently than other anticoagulants as secondary prophylaxis, despite there not being robust data for DOAC use in this patient population. DOACs appear to be effective against VTE with no occurrence of breakthrough venous thrombosis. Major bleeding complications were observed at a higher rate than has been described in the literature and therefore the use of anticoagulants in this patient population must be weighed against the bleeding risk.

Thromboembolic events in patients with paroxysmal nocturnal hemoglobinuria (PNH): Real world data of a Greek nationwide multicenter retrospective study.

Thrombosis is the most common and a life-threatening complication in patients with Paroxysmal Nocturnal Hemoglobinuria. One-third of patients with PNH experience at least one thromboembolic event during the course of the disease, with thrombosis being the most common cause of death in these patients. The mechanism of thrombosis in PNH is complex and continues to be of great research interest. Since the introduction of C5 complement inhibitors in the treatment of PNH, the incidence of thromboembolic events has decreased substantially. We retrospectively analyzed data concerning the thrombotic episodes of 41 patients with PNH from 14 different national hematology centers in Greece. Sixteen patients (39%) experienced at least one episode of thrombosis, including, seven (43.8%) at diagnosis, seven (43.8%) during the course of the disease and two (12.5%) patients prior to PNH diagnosis. Nearly half of these individuals (n=7, 43.8%) had multiple episodes of thrombosis during the course of their disease. The most common sites of thrombosis were intra-abdominal veins. Three out of 26 patients developed thrombosis while on eculizumab. In none of the 16 patients, the thrombotic event was fatal. Our findings, despite the small number of patients, confirmed that thrombosis continues to be a significant complication of PNH affecting more than one third of the patients.

A Case of PNH with Abdominal Pain, AKI and Hemolytic Anemia

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder affecting 1 to 10 people per million. With an associated 10-year mortality, PNH is a life-threatening disorder where the complement system of the body's innate immune system prematurely lyses red blood cells. It can be characterized by a number of clinical features, the most common being hemolytic anemia, pancytopenia, and thrombosis. 45 year old African American male presented with a chief complaint of shortness of breath x 2-3 days. Reports associated dry, nonproductive cough, congestion, myalgias onset one week prior. He was taking two tablets of Advil daily since symptom onset. He has a past medical history of right sided BKA after motor vehicle accident and currently uses a prosthetic leg. No other significant past medical history noted. On ED admission, the patient was febrile to 103.2F, tachycardic to 110 beats per minute, tachypneic to 26 breaths per minute, and normotensive to 128/64. On physical exam, there was conjunctival pallor, no scleral icterus, and right BKA. The remainder of the physical exam was unremarkable. Labs on admission were notable for anemia (Hgb 3.9, baseline of 9.2, 2 years prior), AKI (Cr 7.3, baseline of 0.9), elevated LDH 2336, and low Haptoglobin <10. Platelet count WNL throughout admission however there was a drop from 242 to 180 between Day 1 and 2 of admission. Peripheral smear did not show schistocytes however ADAMTS13 sent per heme/onc consult. Anemia improved to Hgb 7.6 on Day 3 of admission status post 4 units packed red blood cell transfusion. Workup for hemolytic anemia with AKI was pursued: Direct Coombs test negative, PNH screen sent. SPEP and UPEP unremarkable. On Day 6 of admission, flow cytometry showed evidence of PNH clone upon analysis of RBCs, granulocytes, and monocytes. AKI was attributed to ATN s/t heme-pigment deposition vs NSAID use and Cr trended down to 2.9 prior to discharge. On day 7 of admission, patient developed severe abdominal pain, not associated with fevers, chills, nausea, vomiting, or diarrhea. Pain moderately improved with Norco, ultrasound imaging was negative, and unable to CTA given renal function. Pt was started empirically on therapeutic anticoagulation with Lovenox, as the literature reports a high incidence of venous and/or arterial thrombosis in PNH w/granulocyte count >50% (89.8% in this patient). Patient was discharged the next day with close outpatient hematology follow-up with plan to start eculizumab. Given patient's presentation of fever, symptomatic anemia, and AKI the differential diagnosis included: hemoglobinopathies, red cell membrane disorders, PNH, DIC, TTP, HUS, AIHA, and infections such as malaria or HIV. It was difficult to identify PNH as the cause in this patient on ED presentation as there was no reported urine discoloration. Though classified as an acquired disorder through mutation of a gene on the X chromosome, it occurs in equal distribution among men and women. PNH specifically occurs through the mutation of a gene involved in the synthesis of glycosylphosphatidylinositol (GPI), an anchor protein for complement regulatory proteins that prevent complement-induced hemolysis of red blood cells. The resulting GPI deficiency leads to the unregulated formation of complement attack complexes that damage the cell membrane causing them to lyse. This intravascular hemolysis is seen as decreased levels of Hgb and hemoglobinuria that leads to elevated levels of LDH as seen with this patient. The degree of hemolysis and PNH clone size can increase risk of thrombosis, the leading cause of death in patients with PNH. Strong indicators for the start of PNH therapy are severe anemia, thrombosis, renal dysfunction, and dyspnea. The typical management of PNH in patients with thrombosis is shown in this case as the patient is put on therapeutic anticoagulation and plans for eculizumab, a monoclonal antibody that is a complement inhibitor used to prevent hemolysis. This PNH therapy will likely be administered lifelong and decrease the need for RBC transfusions as well as decrease thromboembolic rate. If the patient is unresponsive to eculizumab they become a strong candidate for allogeneic stem transplantation. This patient meets the popular standard for starting primary prophylaxis for management of thrombosis with a PNH clone size >50%.

Intestinal ischemia in paroxysmal nocturnal hemoglobinuria with associated aplastic anemia: a rare and challenging association (case report)

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematological disorder due to an acquired genetic mutation in phosphatidylinositol glycan anchor biosynthesis, class A (PIGA) gene. Paroxysmal nocturnal hemoglobinuria has different manifestations, the most common being hemolysis, thrombosis and aplastic anemia. For the latter, aplastic anemia can be both the cause and the consequence of PNH. Complement inhibitors, such as eculizumab and ravulizumab represents the cornerstone in the management of PNH as it is associated with fewer recurrence of hemolytic and thrombotic events. However, PNH associated aplastic anemia does not improve with complement inhibitor, and is managed either with allogeneic stem cell transplantation or immunosuppressive therapy. The major cause of death in PNH is thrombosis, which is often venous, and of unusual locations. Intestinal ischemia represents a rare location of thrombosis in PNH. There are a few cases reported, where the management was either surgical or conservative. Many of these reports showed a recurrence of thrombosis, and the outcome has been fatal in some. Severe aplastic anemia adds a level of difficulty to the therapeutic challenge as both resection surgery and efficient anticoagulation become infeasible or delicate. We report the case of a 24-year-old woman, who was diagnosed with severe aplastic anemia associated with PNH, for which she was put on triple immunosuppressive therapy. Eight months following the diagnosis, the patient complained of acute abdominal pain with vomiting. A contrast enhanced computed tomography scan showed a jejunal ischemia with signs of perforation. Due to the low platelet level, a conservative approach was taken consisting of oral feeding cessation, hydration, anticoagulation, and antibiotics therapy. This raises the question of whether a complement inhibitor therapy could have prevented this potentially fatal complication.

Lactate dehydrogenase versus haemoglobin: which one is the better marker in paroxysmal nocturnal haemoglobinuria?

Lactate dehydrogenase versus haemoglobin: which one is the better marker in paroxysmal nocturnal haemoglobinuria?

Thrombosis in Non-Hemolytic Paroxysmal Nocturnal Haemogloinuria

▪Paroxysmal Nocturnal Hemoglobinuria (PNH), a rare hematological condition, presents in patients with a variety of symptoms. The risk of thrombosis is high, with a cumulative thrombosis incidence over an eight to ten year period of between 23% and 30% (in the pre-eculizumab era). 20% of patients have multi-site thrombosis and 10% of patients present with thrombosis. Thrombosis in PNH is a clinical emergency, due to risk of death, thrombosis extension and long term morbidity.The complement and coagulation systems are intimately linked, and explain some of the mechanisms for thrombosis in PNH. Intravascular haemolysis is associated with thrombosis risk however, thrombosis has been observed in patients without hemolysis, most likely related to white cell and platelet factors.The proportion of granulocyte PNH cells correlates with thrombosis risk; those with cell proportions over 50% have a cumulative ten-year incidence of thrombosis of 34.5% compared to 5.3-7% in those with a clone of <50%. Thus in the UK, those with PNH cellular proportions over 50% commence prophylactic anticoagulation.The Leeds (UK) PNH service manages 394 patients with PNH, and has observed PNH patients with minimal hemolysis suffer thrombosis. Predicting those at highest risk is difficult. A retrospective review to identify rates of thrombosis in patients with no/minimal hemolysis was undertakenMethodsThe Leeds PNH database was interrogated, and identified patients with a PNH granulocyte or monocyte clone of over 10% and an LDH less than twice the upper limit of normal for the laboratory in which the sample was tested. Anonymised clinical data was then analysed to determine risk factors for thrombosis. Comparisons between those with and without thrombosis were assessed.All patients who fulfilled the above requirements were then subdivided into groups as below:Group 1: White cell clone >30%, red cell clone <10%, LDH <2xULNGroup 2: White cell clone >30%, red cell clone >10% with higher proportion of type II red cells than type III red cells, LDH <2xULNGroup 3: White cell clone: 10-30%, red cell clone <10%, LDH <2xULNResults29 patients were identified who fit the criteria. Median age of the patient cohort was 62 years (range 22-80 years). 12 patients fit the criteria for group 1, of whom 6 (50%) had thrombosis, 12 patients fit the criteria for group 2 of whom 2 (16.6%) had thrombosis and 5 patients fit the criteria for group 3, with no thrombosis. All 8 patients with thrombosis are detailed in the Table7 patients were not on anticoagulation at the time of the thrombosis, with on patient extending their thrombosis whilst on warfarin (patient 1).Discussion and conclusionsWe report the first case series of patients with non-hemolytic PNH who experience thrombosis. Patients at higher risk of thrombosis in this cohort were those with non-hemolytic PNH, high white cell clones and low red cell clones (group I). However those with a greater proportion of type II red cell clones compared to type III red cells and LDH <2x ULN were also at risk of thrombosis. This is an under-recognised risk for patients with PNH cells.Whilst the risk of arterial thrombosis is around 20%, interestingly and surprisingly, arterial thrombosis in this patient cohort was disproportionately high, with 62.5% of patients with either arterial or mixed venous and arterial thrombosis. It should also be noted that only one patient was anticoagulated at the time of their CVA extension. This was due to a combination of patients not being diagnosed with PNH at the time of thrombosis, or thought to be unsuitable for anticoagulation due to thrombocytopenia.This study supports the theory that while hemolysis may be associated with a thrombotic event, white cell and platelet factors have a more pivotal role than previously thought in the mechanisms of thrombosis in PNH. As per the National recommendations in England, thrombosis thought to be due to PNH regardless of LDH level, is an indication to commence treatment [Display omitted] DisclosuresGriffin:Alexion Pharmaceuticals, Inc.: Honoraria. Munir:Gilled: Honoraria; Alexion Pharmaceuticals, Inc.: Honoraria; Janssen: Honoraria; Roche: Honoraria; AbbVie: Honoraria. Hillmen:Pharmacyclics LLC, an AbbVie Company: Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Celgene: Research Funding; GSK: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Arnold:Alexion Pharmaceuticals, Inc.: Honoraria. Richards:Alexion Pharmaceuticals, Inc.: Honoraria. Hill:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Fibrinogen and Clot Structure Is Affected By Eculizumab in Patients with Paroxysmal Nocturnal Haemoglobinuria

Background Paroxysmal nocturnal haemoglobinuria (PNH) is a haematopoietic stem cell disorder in which an acquired somatic mutation of the PIGA gene in a haematopoietic stem cell results in the deficiency of key complement regulatory molecules, namely CD59 and CD55, from the surface of blood cells leading to an increased susceptibility of blood cells to complement-mediated activation +/- intravascular haemolysis and thrombosis. Thrombosis has been the leading cause of death in PNH. Multiple mechanisms of thrombosis in PNH have been proposed, including cross talk between the coagulation and complement system. This may lead to denser clots which are more resistant to fibrinolysis. Treatment with eculizumab, which inhibits complement factor C5 and thereby prevents terminal complement activation, reduces thromboembolic events in PNH, indicating that complement activation plays a significant role in the pathophysiology of thrombosis in this disease. Patients with other thrombotic disorders are known to form denser fibrin clots that incur greater resistance to fibrinolysis. It has not previously been explored whether changes to clot architecture contribute to thrombosis in PNH. Furthermore, the role of eculizumab in clot architecture is undocumented.Aims Characterise fibrin clot structure in patients with PNH and determine the effect of eculizumab on clot structure.Methods Informed consent was obtained from all participants. 53 patients from the PNH National Service (Leeds) were recruited. They were grouped according to the proportion of PNH granulocytes, <40% (n=18), 40> - < 90 %(n=18) and >90% (n=17) and reanalysed according to treatment with (n=19) or without (n=32) eculizumab. Plasma samples were obtained and ex vivo fibrin clot structure and function was analysed by permeation analysis of clot pore structure, turbidity analysis of clot formation and fibrinolysis, and fibrin ultrastructure by laser scanning confocal microscopy and scanning electron microscopy, according to methodology and protocols well established in our laboratory. The proportion of PNH granulocytes was measured by flow cytometry and fibrinogen levels by Clauss. P values of <0.05 were taken to indicate significance.Results The proportion of PNH granulocytes correlated with fibrinogen (r= -0.35, p=0.048), maximum absorbency (r= -0.34, p=0.046) and clot density (r= -0.36, p=0.04). Seventy per cent of patients with large proportions of PNH granulocytes were on eculizumab. Analysis of patients not on eculizumab showed a trend towards proportion of PNH granulocytes correlating positively with fibrinogen levels (r=0.42, p=ns). Patients on eculizumab had lower fibrinogen levels (3.1±0.2 v 3.6±0.1g/L, P=0.02), maximum absorbance (0.31±0.1 v 0.40±0.1, p=0.028) and fibre density/100µm (18.5±4.4 v 23.9±4.2, p=0.006), indicative of a less dense clot.Summary/Conclusions This is the first study to demonstrate that patients on eculizumab showed reduced fibrinogen, fibre thickness and fibre density. Our data indicate that the beneficial effects of eculizumab in PNH is partly due to reduced complement activation and attenuated inflammatory response, leading to lower fibrinogen levels and a less thrombotic fibrin clot structure. These novel data help to explain the impact of complement inhibition in this disease. [Display omitted] DisclosuresArnold:Alexion Pharmaceuticals, Inc.: Honoraria. Griffin:Alexion Pharmaceuticals, Inc.: Honoraria. Munir:Janssen: Honoraria; Alexion Pharmaceuticals, Inc.: Honoraria; Roche: Honoraria; AbbVie: Honoraria; Gilled: Honoraria. Hillmen:Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie Company: Honoraria, Research Funding; Celgene: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding. Richards:Alexion Pharmaceuticals, Inc.: Honoraria. Hill:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Circulating Endothelial Progenitor Cells and Their Relation to Thrombosis in Paroxysmal Nocturnal Hemoglobinuria and Aplastic Anemia.

Thrombosis is a leading cause of morbidity and mortality in paroxysmal nocturnal hemoglobinuria (PNH). Multiple factors are responsible for the thrombotic tendency in these patients. Endothelial progenitorcells (EPCs) originate from primitive hematopoietic stem cells. The EPC count is considered indicative of potential damage and restoration capacity in vascular disease; lower EPC counts are deemed as a risk factor in cardiovascular diseases. We aimed to investigate the count of circulating EPCs in PNH and aplastic anemia (AA) patients receiving eculizumab treatment or not receiving treatment and their relationship with thrombosis. Seventeen PNH patients, 18 AA patients, and 10 healthy volunteers were included in the study. The CD309, CD133, and CD34 antibodies were used to determine counts of circulating EPCs using flowcytometry. EPC levels were compared between the PNH, AA, and healthy control groups. Kolmogorov-Smirnov test. ANOVA, Kruskal-Wallis, and Mann-Whitney U testswereperformedto analyze the quantitative data, while χ2 testing was performed to analyze the qualitative data. Therewasnosignificantdifference in EPC levelsbetweenpatientswithandwithout a history of thrombosis (P > 0.05). Further, therewasnosignificantdifference in thelevels of EPCsbetweenthe AA and PNH groups (P > 0.05). However, there was a significant positive correlation between levels of EPCs and lactate dehydrogenase (LDH) in multivariate analysis (P < 0.05). The study findings suggest that hemolysis promotes vascular endothelial and new blood vessel formation. Increased EPCs in PNH may indirectly indicatevascular endothelial damage in PNH.

PNH phenotypes and their genesis.

PNH phenotypes and their genesis.

Markers of Thrombin Generation and Inflammation in Patients with Paroxysmal Nocturnal Hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) presents with intravascular hemolysis, bone marrow failure and thrombosis. Various studies have reported geographic and ethnic variation in prevalence of thrombosis in PNH. There is limited data on thrombosis in PNH from the Indian subcontinent. In this study we describe disease burden and risk factors for thrombosis in 18 Indian PNH patients. We studied markers of thrombin generation (Thrombin-antithrombin complexes; TAT and D-Dimer), endothelium and platelet activation (soluble P-selectin) and inflammation (interleukin-6; IL-6) in PNH patients and compared their levels with healthy controls. Thrombosis was identified in 17% of PNH patients. TAT, sP-selectin and D-Dimer levels were significantly elevated in PNH patients (TAT: 5.06 ± 1.08ng/ml; sP-selectin: 80.57 ± 19.5ng/ml; D-Dimer mean: 936ng/ml 95% CI 559, 1310) compared to control population (TAT: 3.39 ± 0.769ng/ml P = 0.016; sP-selectin: 44.67 ± 5.17ng/ml P = 0.002). Using Youden's J statistic, the cut-off values for TAT and sP-selectin in our cohort of PNH patients were 2.90ng/ml and 58.41ng/ml respectively. TAT, sP-selectin and D-Dimer levels were elevated beyond the cut-off values in PNH patients with thrombosis compared to those without thrombosis. A positive correlation was noted between TAT, sP-selectin and D-Dimer levels. Increased TAT, sP-selectin, and D-Dimer levels may indicate impending thrombosis in PNH.

Gene mutations associated with thrombosis detected by whole-exome sequencing in paroxysmal nocturnal hemoglobinuria.

Thrombosis is a most common and lethal complication of paroxysmal nocturnal hemoglobinuria (PNH), which is a complex progression and its mechanism remains unclear. We tried to explore the possible genetic background of thrombosis in PNH patients and provide potential gene mutations associated with thrombosis in PNH patients. The CD59- cells of 7 PNH and 6 PNH- aplastic anemia (AA) patients were sorted by flow cytometry and sequenced by whole-exome sequencing (WES). The sequencing results and target mutation genes were analyzed and screened, respectively, and Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analysis was carried out. Finally, the expression of target genes was detected in 22 PNH (including seven cases with thrombus) and 20 normal controls, and the correlation between the expression of mRNA and the clinical thrombus-related indexes was analyzed. The mutation genes screened from 4 PNH with thrombus were BMPR2, F8, ITGA2B, THBD, and THBS1. The pathways enriched by these genes included Notch, Wnt, and arachidonic acid metabolism signaling pathways, which may be related to the pathogenesis of thrombosis in PNH. The BMPR2, THBD, and THBS1 gene expression was significantly different between PNH with and without thrombus group, and the THBS1 gene expression was positively correlated with D-Dimer and su-PAR levels. Genetic defects have a non-negligible effect on the incidence of thrombosis, and therefore, gene mutations maybe a genetic risk factor in PNH, which increase the incidence of thrombosis.

Differential miRNA expression profile and proteome in plasma exosomes from patients with paroxysmal nocturnal hemoglobinuria

Paroxysmal Nocturnal Hemoglobinuria (PNH) is a clonal disease of blood cells caused by the lack of glycosyl phosphatidyl inositol anchored proteins bound to the cell membrane. In consequence, erythrocytes lead to intravascular hemolysis upon complement activation, which promotes high risk of thrombosis, intravascular hemolytic anemia, and bone marrow failure in patients. The mechanisms of thrombosis in PNH are still poorly understood. Treatment with eculizumab reduces intravascular hemolysis and thrombotic risk, but not in all cases. Exosomes are extracellular vesicles released by cells and whose secretion is closely related to the inflammatory status. They participate in cell communication by activating signaling pathways and transferring genetic material and proteins to host cells. In consequence, exosomes may serve as surrogate biomarkers for the prognosis and/or diagnosis of a disease. Isolation of exosomes was carried out from healthy controls and from three groups of PNH patients, i.e. i) with no eculizumab treatment; ii) under treatment with eculizumab that have not suffered thrombosis; and iii) under treatment with eculizumab but that have suffered thrombosis. The miRNAome and proteome was analyzed using plasma focus miRNAs PCR panel and LC-MS analysis respectively. We found differential expression of miRNAs miR-148b-3p, miR-423-3p, miR29b-3p, miR15b-5p, let-7e-5p, miR126-3p, miR-125b-5p and miR-376c-3p as well as hemoglobin, haptoglobin, protein S and C4-binding protein in healthy controls vs PNH patients. Our results warrant further research and provide new information on the content of exosomes that could play a role in the hypercoagulable state in this disease.

Paroxysmal nocturnal hemoglobinuria and thrombosis in the era of eculizumab

Paroxysmal nocturnal hemoglobinuria (PNH) arises as a consequence of clonal expansion of hematopoietic stem cells that have acquired a somatic mutation in the PIGA gene. The resulting hematopoietic cells have deficiencies in the GPI-anchored complement regulatory proteins CD55 and CD59, which account for the intravascular hemolysis that is the primary clinical manifestation of PNH. Thromboembolism is a major cause of morbidity and mortality in PNH, particularly in Caucasian patients. In a previous report on the clinical course of PNH patients in the United States and Japan, we showed that thrombosis was significantly more prevalent in white PNH patients than in Asian PNH patients. The pathophysiological mechanisms underlying thrombosis in PNH have not been fully clarified, and multiple factors are likely to be involved. Eculizumab, a humanized monoclonal antibody, targets the terminal complement protein C5 and inhibits terminal complement-mediated hemolysis associated with PNH. Brodsky et al. reported that eculizumab treatment reduces the risk of clinical thromboembolism in patients with PNH. These facts strongly suggest that the main cause of thrombosis in PNH is complement activation and/or hemolysis. In this review, the pathophysiology of thrombosis in PNH is discussed in the context of observations in PNH patients treated with eculizumab.

Proteinase 3 expression on the neutrophils of patients with paroxysmal nocturnal hemoglobinuria.

Proteinase 3 (PR3) is released from neutrophils and regulates platelet activity, which is associated with cluster of differentiation (CD)177 antigen (NB1), a glycosylphosphatidylinositol-linked protein. In the present study, the effect of PR3 on thrombosis in paroxysmal nocturnal hemoglobinuria (PNH) and PNH-aplastic anemia (AA) syndrome was explored. The expression of PR3 and NB1 on CD59- neutrophils was detected by flow cytometry, immunofluorescence (IF), reverse transcription-quantitative polymerase chain reaction analysis and western blotting. Serum levels of PR3, proteinase-activated receptor 1 (PAR1) and D-Dimer were measured using ELISAs. The expression of PR3 and NB1 on the plasma membrane of CD59- neutrophils in patients with PNH/PNH-AA was significantly lower compared with their expression on CD59+ neutrophils in patients and controls (P=0.001). However, no correlation between PR3 and NB1 expression was identified. IF staining further demonstrated partially positive PR3 expression on CD59- neutrophils. The serum level of PR3 in patients was identified to be significantly decreased compared with healthy controls (P<0.0001), and significantly negatively correlated with PAR1 (r=-0.456; P=0.043) and D-Dimer (r=-0.503; P=0.028) levels. The mRNA and protein levels of PR3 on PNH clones did not change significantly compared with the control group. In conclusion, PR3 expression on the plasma membrane of neutrophils and in the serum of patients with PNH/PNH-AA decreased, which may result in increased PAR1 expression and increased clotting. The present study provides the basis for further study on platelets in PNH.

The prothrombotic state in paroxysmal nocturnal hemoglobinuria: a multifaceted source.

Paroxysmal nocturnal hemoglobinuria is a rare acquired hematologic disorder, the most serious complication of which is thrombosis. The increased incidence of thrombosis in paroxysmal nocturnal hemoglobinuria is still poorly understood, but unlike many other thrombotic disorders, predominantly involves complement-mediated mechanisms. This review article discusses the different factors that contribute to the increased risk of thrombosis in paroxysmal nocturnal hemoglobinuria. Paroxysmal nocturnal hemoglobinuria leads to a complex and multifaceted prothrombotic state due to the pathological effects of platelet activation, intravascular hemolysis and neutrophil/monocyte activation. Platelet and endothelial microparticles as well as oxidative stress may play a role. Impaired fibrinolysis has also been observed and may be caused by several mechanisms involving interactions between complement activation, coagulation and fibrinolysis. While many factors may affect thrombosis in paroxysmal nocturnal hemoglobinuria, the relative contribution of each mechanism that has been implicated is difficult to quantify. Further studies, including novel in vivo and in vitro thrombosis models, are required in order to define the role of the individual mechanisms contributing to thrombosis, impaired fibrinolysis and clarify other complement-driven prothrombotic mechanisms in paroxysmal nocturnal hemoglobinuria.

Polymorphism of the ABO gene associate with thrombosis risk in patients with paroxysmal nocturnal hemoglobinuria.

Thrombosis is one of the most common causes of mortality in Paroxysmal nocturnal hemoglobinuria (PNH), but the predisposing factors for thrombosis are yet to be defined. In this study, we outline the clinical characters and the susceptible genes which lead to thrombotic formation in 104 patients with PNH. The results displayed that the genotypes with minor alleles of rs495828 or rs2519093 in the ABO gene were associated with high risk to thrombus formation (OR 5.95, 95% CI 1.90-18.65 and OR 6.3, 95% CI 2.01-19.79, respectively). Further, the TT haplotype was associated with a significant increased risk of thrombosis (OR=3.25, 95%CI 1.42-7.39). Multivariate regression analysis showed larger PNH clone and genotypes with rs495828/rs2519093 minor allele as independent risk factors for thrombosis in PNH. Some patients who came back for follow-up were tested for the plasma levels of vWF and factor VIII. Patients carrying the rs495828/rs2519093 minor allele had a significant higher level of vWF and factor VIII compared with those carrying the major allele. Therefore, we found for the first time that the rs495828/rs2519093 polymorphism represent an independent prognostic factor in PNH patients for thrombus formation, probably by increasing the vWF and factor VIII.

Possible Risk Factors of Thrombosis in Chinese Patients with Paroxysmal Nocturnal Hemoglobinuria

▪Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease caused by an acquired mutation of the X-linked PIGA gene on the hematopoietic stem cell. Thrombosis is one of the most common causes of mortality in PNH, but the predisposing factors for thrombosis are yet to be defined. In this study, we outline the clinical characters of thrombotic PNH and detect the susceptible genes which may lead to thrombotic formation.Methods: There were totally 94 patients with PNH diagnosed between 2011 and 2015 enrolled in the study. Clinical data like sex, age, hemoglobin level, reticular cell percentage, white blood cell and platelet count, LDH level, CD59- and FLAER granulocytes percentage, thrombophilia risk factors like level of protein C, protein S, antithrombin III, APC resistance, blood fat, phospholipid antibody were evaluated. Samples from patients were genotyped for the reported 33 alleles in 21 genes including MTHFR, PROC, PROS, F2, F5, ABO, prothrombin and other genes which are reported as high risk factors for venous thromboembolism (VTE) by polymerase chain reaction fragment length polymorphism methods (PCR-RFLP). Furthermore, we detected plasma VIII factor and vWF levels which were affect by ABO polymorphism.Results:Of the 94 PNH patients, 16 (17%) patients had at least 1 episode of thrombotic event. Only 2 patients had arterial thrombosis and 14 patients had venous thrombosis. The medium age of patients with thrombosis was 42-year old, similar to those without (43-year-old, p=0.199). Male : female ratio was 1.29 in thrombosis group and 1.16 in non-thrombosis group (p=1.000). Although there was no difference in level of hemoglobin, white blood cell count, platelet count, reticular cell count, LDH, protein C, protein S, antithrombin III, APC resistance, blood fat, phospholipid antibody level (p>0.05) between patients with thrombosis and those without, the percentage of CD59- erythrocytes( p=0.001 ), CD59- granulocytes ( p=0.004 ) and FLAER- granulocytes (p=0.003) was higher in thrombotic patients. Patients with the TT genotype (rs495828 in the ABO gene) were approximately 3.03 folds prone to thrombus formation than those with the GG genotype (p=0.0204 ), and patients with the TC genotype (rs2519093 in the ABO gene) were approximately 4.24 folds prone to thrombus formation than those with the CC genotype ( p=0.0352 ). In addition, minor allele frequencies of rs495828 or rs2519093 and CD59- erythrocytes were independent risk factor for thrombosis in PNH patients. No association was detected between other SNPs and risk to thrombosis. There was no difference in level of vWF-Ag (156% vs 152%, p=0.870) and VIII factor (190% vs 156%, p=0.198) between patients with or without thrombosis, although the level was much higher in PNH patients than normal controls (178% vs 107%, p=0.005 and 203% vs 128%, p=0.003 respectively).Conclusion: Compared with non-thrombotic patients, thrombotic PNH patients have have bigger PNH clone. And for the first time, our results suggested that the rs495828/rs2519093 in the ABO gene confers risk to thrombosis in PNH. Therefore, rs495828/rs2519093 polymorphism may represent a potential genetic biomarker in PNH patients for thrombus formation. DisclosuresNo relevant conflicts of interest to declare.

The dysfunction of platelets in paroxysmal nocturnal hemoglobinuria

IntroductionThrombosis is a dangerous complication of paroxysmal nocturnal hemoglobinuria (PNH) and has a high mortality rate. However, the mechanism underlying the development of thrombosis in PNH remains unclear. To explore this, platelet function and serum complement activity were investigated in 14 patients with classical PNH, 11 with PNH aplastic anemia (AA) and 30 healthy controls. Material and methodsSerum concentrations of the terminal complement complex (sC5b-9) were determined by enzyme-linked immunofluorescence assay (ELISA), and the levels of C5b-9, CD61 and CD62p on platelet membranes were determined by flow cytometry. Clinical parameters were assessed, including D-dimer and platelet aggregation induced by adenosine diphosphate (ADP) and arachidonic acid (ARA). ResultsSerum sC5b-9 concentrations were significantly lower in the PNH/PNH-AA than in the control group (P<0.01). C5b-9 deposition was significantly higher on CD59− platelets than on CD59+ platelets in PNH/PNH-AA patients and healthy controls (P<0.01 for each). D-dimer concentration was significantly higher in PNH/PNH-AA patients – especially those with lactate dehydrogenase (LDH) concentrations>1000U/L – than in controls (P<0.05). CD61 (P<0.05) expression was lower on CD59+ platelets in PNH than in controls and CD5− platelets in PNH. Expression of CD62p (P<0.01) was lower on CD59− and CD59+ platelets (P<0.01) in PNH cases than in controls. Platelet aggregation stimulated by the agonists ADP and ARA in the PNH/PNH-AA patients was significantly lower than that in controls (P<0.05). ConclusionsThe adhesion and aggregation of platelets, especially of CD59+ platelets, were compensatively decreased in PNH/PNH-AA patients without active thrombosis.

The preliminary research in paroxysmal nocturnal hemoglobinuria with thrombosis

目的探讨阵发性睡眠性血红蛋白尿症（PNH）患者易发血栓的可能因素。方法回顾性分析142例PNH患者的临床资料，对3个已知血栓相关基因位点[PROC c.574_576del（rs199469469）、PROC c.565C>T（rs146922325）和THBD c.-151G>T（rs1698852）]采用Sanger法进行DNA序列检测，以性别、年龄相匹配的100名健康志愿者为正常对照。结果142例患者中，合并血栓者21例（14.8%），其中2例为动脉血栓，19例为静脉血栓，血栓组患者的中位年龄为35（20~67）岁，与非血栓组[40（7~79）岁]比较差异无统计学意义（P=0.687）；两组患者的男女比例、PNH临床分类、HGB、WBC、PLT、LDH水平差异均无统计学意义（P值均>0.05）。血栓组RBC、粒细胞CD59−、红细胞CD59−及嗜水气单胞菌溶素变异体阴性细胞（Flear−）百分率均高于非血栓组（P值分别为0.015、0.005、0.004和0.026）。PNH患者组与正常对照组、PNH血栓组与非血栓组之间蛋白C、蛋白S、抗凝血酶Ⅲ、活化蛋白C抵抗、血脂、狼疮抗凝物等常见易栓因素差异均无统计学意义（P值均>0.05）。PNH患者中，rs199469469位点突变1例（0.704%）、rs16984852位点突变2例（1.408%）、无一例发生rs146922325位点突变，所有突变者均无血栓形成；正常对照组3个位点突变率分别为2.424%、0.873%和0.974%。PNH患者组与正常对照组、PNH血栓组与非血栓组之间各位点突变率差异均无统计学意义（P值均>0.05）。结论RBC与PNH克隆大小是PNH患者发生血栓的独立危险因素。PNH患者与正常人、PHN合并血栓患者与未合并血栓患者相比，常见遗传性和后天获得性易栓因素、常见的静脉血栓3个易感基因的突变率差异均无统计学意义。

Circulating Endothelial Progenitor Cells and Their Relation to Thrombosis in Paroxysmal Nocturnal Hemoglobinuria

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disorder. Chronic intravascular hemolytic anemia, bone marrow failure and thrombophilia are the main clinical findings. Thrombosis is one of the most important cause of morbidity and mortality of this disease. Multiple factors are held responsible for thrombotic tendency in these patients. Endothelial progenitor cells (EPCs) originate from primitive hematopoietic stem cells and are able to turn into endothelial cells. There are extremely small numbers of EPCs in the circulation under normal conditions. The level of EPCs is considered to be indicative of restoration capacity in case of vascular disease and potential damage. Lower EPC levels are also considered as a risk factor in cardiovascular diseases. In this study, our aim was to investigate circulating EPCs in PNH and their relationship with thrombosis. Seventeen patients with PNH, 18 patients with aplastic anemia and 10 healthy volunteers were included in the study. CD309, CD133 and CD34 antibodies were used in order to determine circulating EPCs by flow cytometry and cells which expressed all three antibodies were analyzed as EPC. Prepared samples were read using a prepared list mode software for endothelial progenitor cells on FACS Diva software in BD FACS Canto II device with 6 color lasers and a total of 1,000,000 cells per analysis were evaluated. EPC levels were compared between untreated PNH patients and who were on eculizumab therapy. Statistical analysis was performed using SPSS 22.0 software. The distribution of variables was evaluated by Kolmogorov-Smirnov test, the analysis of quantitative data was evaluated by ANOVA, Kruskal-Wallis, Mann-Whitney U tests and the analysis of the qualitative data was evaluated by chi-square test.Findings and Discussion: The thrombotic complications were observed in five PNH patients. All of these patients had a history of portal vein thrombosis. One of them also had a history of peripheral arterial disease and amputation related to this. There was not a significant difference in EPC levels between patients with and without a history of thrombosis (p>0,05). We also did not find any significant difference between levels of EPC's in PNH groups with or without eculizumab therapy (p˃0,05). There was no significant difference in levels of EPC between aplastic anemia and PNH groups (p ˃ 0,05). However, we found a significant positive correlation between the levels of EPC and LDH in multivariate analysis (p < 0,05). This finding suggests that hemolysis causes vascular endothelium and promotes new blood vessel formations. Increased EPCs in PNH might be an indirect indicator for vascular endothelium damage in PNH.TableGeneral Features and Rates of EPC of PNH, AA, Healthy Volunteers GroupsAplastic Anemia groupPNH groupControl grouppAgemean±s.smedian (min-max)40.0±14.7 37.5 (20.0-67.0)41.9±13.9 43.0 (19.0-78.0)29.3±3.5 29.5(24.0-34.0)0.047SexFemale n-% Male n-%7 38,9% 11 61.1%9 52.9% 8 47.1%5 50% 5 50%0.687EPC(%)mean±s.s median (min-max)0.2% 0.2% 0.1% (0.0-0.6%)0.3%±0.3% 0.1% (0.0-0.9%)0.1%±0.0% %0,0(%0,0-0,2)0.393All Events (x1000)mean±s.s median (min-max)617±172* 565 (360-914)588±255* 471 (250-1000)878±143 950(655-1000)0.003CD309 and CD34mean±s.s median (min-max)0.003±0.002 0.002 (0.001-0.007)0.005±0.004 0.000-0.011)0.001±0.001 0.001 (0.000-0.002)0.009CD133mean±s.s median (min-max)45.8±36.3 58.3 (0.0-88.2)45.8±39.6 60 (0.0-94.7)42.0±19.4 46.4 (11.1-81.3)0.867ANOVA / Kruskal-Wallis / Mann-Whitney U test / Chi-square test*The difference with the control group, p <0.05EPCs: Endothelial progenitor cells DisclosuresYenerel:Alexion: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.