The association between EPCR gene p.Ser219Gly polymorphism and venous thromboembolism risk: a case-control study, meta-analysis, and a reproducibility study.

Background: The rs867186 single-nucleotide polymorphism in the PROCR gene (g.6936A > G, c.4600A > G) results in a serine-to-glycine substitution at codon 219 of endothelial protein C receptor (EPCR). We performed a case-control study followed by an updated meta-analysis of the association between this polymorphism and the risk of venous thromboembolism (VTE). Objective and methods: We enrolled 263 VTE patients and 320 unrelated healthy controls for the case-control study. The total number of cases and controls for the meta-analysis were 5,768 and 30,017, respectively. A new online MetaGenyo Statistical Analysis System software was used to perform the current meta-analysis. Furthermore, a reproducibility study was conducted to validate our results. Results: Among well-defined thrombosis risk factors, Factor V Leiden was more frequent in the VTE group (p < 0.001), while there was no difference in mutation frequency of prothrombin 20210G>A polymorphism between the two groups. There was no difference in the mutation frequency of Factor V Leiden and prothrombin 20210G>A between cases with and without provoking factors and cases with and without VTE recurrence. The rs867186 "G" carriership did not influence the risk of VTE [odds ratio (OR) 1.339; 95% confidence interval (CI): 0.904-1.984] in our study. No significant differences could be demonstrated among the rs867186 genotype frequencies between VTE cases with and without provoking factors (p = 0.430). PROCR rs867186 was associated with an OR of 1.72 (95% CI: 0.95-3.13, p = 0.075) in terms of VTE recurrence. In the meta-analysis, a significant association was found between EPCR Ser219Gly polymorphism and VTE under the dominant model (OR = 1.27, 95% CI: 1.11-1.46, p = 0.0006), the recessive model (OR = 1.60, 95% CI: 1.26-2.04, p = 0.0001), the GG vs. AA contrast model (OR = 1.64, 95% CI: 1.28-2.09, p = 0.0001), and the GA vs. AA contrast model (OR = 1.24, 95% CI: 1.08-1.43, p = 0.002). Conclusion: The rs867186 was not associated with the first VTE risk in our case-control study; however, a tendency to VTE recurrence was observed. Based on the results of our reproducibility study, MetaGenyo is acceptable for meta-analysis in case of genetic epidemiology studies. Although the risk conferred by the rs867186 is mild in all meta-analyses, including ours, identifying patients carrying the minor allele might have an impact on personalized VTE risk assessment, risk-score calculation, and patient management.

Case report: Metastatic pancreatic neuroendocrine tumour associated with portal vein thrombosis; successful management with subsequent pregnancies.

Background: Splanchnic vein thrombosis due to co-existing metastatic pancreatic neuroendocrine tumour (pNET) and JAK2V617F mutation is a rare condition. Case report: Here we present a case of a young woman with complete remission of a non-functioning grade 2 pNET with unresectable liver metastases, coexisting with JAK2V617F mutation. Splenectomy and distal pancreatectomy were performed. Neither surgical removal, nor radiofrequency ablation of the liver metastases was possible. Therefore, somatostatin analogue (SSA) and enoxaparine were started. Peptide receptor radionuclide therapy (PRRT) was given in 3 cycles 6-8 weeks apart. Genetic testing revealed no multiple endocrine neoplasia type 1 (MEN-1) gene mutations. After shared decision making with the patient, she gave birth to two healthy children, currently 2 and 4 years old. On pregnancy confirmation, SSA treatment was interrupted and resumed after each delivery. Ten years after the diagnosis of pNET, no tumour is detectable by MRI or somatostatin receptor scintigraphy. PRRT followed by continuous SSA therapy, interrupted only during pregnancies, resulted in complete remission and enabled the patient to complete two successful pregnancies.

Concentration and Subclass Distribution of Anti-ADAMTS13 IgG Autoantibodies in Different Stages of Acquired Idiopathic Thrombotic Thrombocytopenic Purpura.

BACKGROUND: The acquired form of idiopathic thrombotic thrombocytopenic purpura (TTP) is an autoimmune disease, in which the underlying deficiency of the ADAMTS13 protease is caused by autoantibodies, predominantly of the IgG isotype. Certain HLA-DR-DQ haplotypes were associated with the risk of developing TTP. OBJECTIVES: To investigate the development of the ADAMTS13-specific antibody response during the course of the disease, we analyzed the concentration, subclass distribution, and inhibitory potential of anti-ADAMTS13 IgG autoantibodies in samples of TTP patients drawn during the first acute phase, in remission, and during relapse. Additionally, we compared the anti-ADAMTS13 IgG levels between patients carrying and not carrying risk and protective HLA-DR-DQ haplotypes. PATIENTS AND METHODS: We determined the anti-ADAMTS13 IgG concentration and subclass distribution in 101 antibody-positive samples of 81 acquired TTP patients by ELISA methods. The presence and semi-quantitative amount of anti-ADAMTS13 inhibitors were determined in 97 of 100 deficient samples, and the specific inhibitory potential of anti-ADAMTS13 autoantibodies was determined in 49 selected samples, by mixing ADAMTS13-activity assays. HLA-DR-DQ typing and haplotype prediction were performed in 70 of the above patients. RESULTS: We found that IgG1 and IgG4 were the predominant subclasses, present in almost all samples. While IgG1 was the dominant subclass in almost half of the samples taken during the first acute episode, IgG4 was dominant in all samples taken during or following a relapse. The inhibitory potential of the samples correlated with levels of the IgG4 subclass. Anti-ADAMTS13 antibodies of IgG4-dominant samples had higher specific inhibitory potentials than IgG1-dominant samples, independently of disease stage. Interestingly, we found that patients carrying the protective DR7-DQ2 and DR13-DQ6 haplotypes had higher anti-ADAMTS13 IgG levels. CONCLUSION: Our results indicate that IgG4 becomes the dominant subtype at some point of the disease course, apparently before the first relapse, parallel to the increase in inhibitory potential of the anti-ADAMTS13 autoantibodies. Furthermore, we found an association between the genetic background and the antibody response in TTP.

A decade-long clinical experience on the prophylactic use of activated prothrombin complex concentrate in acquired haemophilia A: a case series from a tertiary care centre.

: In acquired haemophilia A (AHA), risk for recurrent bleeding exists until the inhibitor is detectable. Thus, patients with persisting inhibitor may benefit from prophylaxis with activated prothrombin complex concentrate (aPCC). Potential thromboembolic complications and cost are also factors to consider. Today, no high level evidence or clear recommendations are available on aPCC prophylaxis in AHA. Recently, a small prospective study demonstrated a favourable outcome with short-term, daily administered aPCC infusion. Here we report a retrospective case series of 19 patients with AHA to demonstrate our practice on aPCC prophylaxis. In our practice, clinical bleeding tendency guided our decision on the initiation of aPCC prophylaxis. In patients with serious bleeding tendency, aPCC infusion was prolonged beyond bleeding resolution in a twice-weekly or thrice-weekly regimen. Serious bleeding phenotype included a single episode of life-threatening bleeding or recurrent, severe haemorrhages. Patients who did not present such events were treated on-demand. The preventive dose of aPCC was equal with the lowest effective therapeutic dose. Prophylaxis was continued until the inhibitor disappeared. Eleven patients received aPCC prophylaxis. In nine cases, prophylaxis lasted beyond two months. No severe bleeding developed spontaneously and no thromboembolic complication occurred in the median 16 weeks (interquartile range 9-34) duration of prophylaxis. Eight patients of the nonprophylaxis group did not present any severe haemorrhage. According to our experience, we consider prophylaxis with aPCC effective and well tolerated for patients with AHA and serious bleeding tendency, until the acquired inhibitor persists.

Clinical and laboratory characteristics of antithrombin deficiencies: A large cohort study from a single diagnostic center.

INTRODUCTION: Inherited antithrombin (AT) deficiency is a heterogeneous disease. Due to low prevalence, only a few studies are available concerning genotype-phenotype associations. The aim was to describe the clinical, laboratory and genetic characteristics of AT deficiency in a large cohort including children and to add further laboratory data on the different sensitivity of functional AT assays. PATIENTS AND METHODS: Non-related AT deficient patients (n=156) and their family members (total n=246) were recruited. Clinical and laboratory data were collected, the mutation spectrum of SERPINC1 was described. Three different AT functional assays were explored. RESULTS: Thirty-one SERPINC1 mutations including 11 novel ones and high mutation detection rate (98%) were detected. Heparin binding site deficiency (type IIHBS) was the most frequent (75.6%) including AT Budapest3 (ATBp3), AT Padua I and AT Basel (86%, 9% and 4% of type IIHBS, respectively). Clinical and laboratory phenotypes of IIHBS were heterogeneous and dependent on the specific mutation. Arterial thrombosis and pregnancy complications were the most frequent in AT Basel and AT Padua I, respectively. Median age at the time of thrombosis was the lowest in ATBp3 homozygotes. The functional assay with high heparin concentration and pH7.4 as assay conditions had low (44%) sensitivity for ATBp3 and it was insensitive for AT Basel and Padua I. CONCLUSION: Type IIHBS deficiencies behave differently in clinical and laboratory phenotypes from each other and from other AT deficiencies. Heparin concentration and pH seem to be the key factors influencing the sensitivity of AT functional assays to IIHBS.

Antithrombin Debrecen (p.Leu205Pro) - Clinical and molecular characterization of a novel mutation associated with severe thrombotic tendency.

INTRODUCTION: Hereditary antithrombin (AT) deficiency is a rare thrombophilic disorder with heterogeneous genetic background and various clinical presentations. In this study we identified a novel AT mutation. Genotype-phenotype correlations, molecular characteristics and thrombotic manifestations of the mutation were investigated. MATERIALS AND METHODS: Thirty-one members of a single family were included. Clinical data was collected regarding thrombotic history. The mutation was identified by direct sequencing of the SERPINC1 gene. HEK293 cells were transfected with wild type and mutant SERPINC1 plasmids. Western blotting, ELISA and functional amidolytic assay were used to detect wild type and mutant AT. After double immunostaining, confocal laser scanning microscopy was used to localize mutant AT in the cells. Molecular modeling was carried out to study the structural-functional consequences of the mutation. RESULTS: Unprovoked venous thrombotic events at early age, fatal first episodes and recurrences were observed in the affected individuals. The median AT activity was 59%. Genetic analysis revealed heterozygous form of the novel mutation p.Leu205Pro (AT Debrecen). The mutant AT was expressed and synthesized in HEK293 cells but only a small amount was secreted. The majority was trapped intracellularly in the trans­Golgi and 26S proteasome. The mutation is suspected to cause considerable structural distortion of the protein. The low specific activity of the mutant AT suggested functional abnormality. CONCLUSIONS: AT Debrecen was associated with very severe thrombotic tendency. The mutation led to misfolded AT, impaired secretion and altered function. Detailed clinical and molecular characterization of a pathogenic mutation might provide valuable information for individualized management.

The role of human leukocyte antigen DRB1-DQB1 haplotypes in the susceptibility to acquired idiopathic thrombotic thrombocytopenic purpura.

The acquired form of idiopathic thrombotic thrombocytopenic purpura (TTP) is an autoimmune disease, in which the underlying ADAMTS13-deficiency is caused by inhibitory autoantibodies against the protease. Human leukocyte antigens (HLA), responsible for antigen presentation, play an important role in the development of antibodies. The loci coding HLA DR and DQ molecules are inherited in linkage as haplotypes. The c.1858C>T polymorphism of the PTPN22 gene, which codes a protein tyrosine phosphatase important in lymphocyte activation, predisposes to a number of autoimmune diseases. We determined the HLA-DRB1-DQB1 haplotypes and the PTPN22 c.1858C>T genotypes in 75 patients with acquired idiopathic TTP and in healthy controls, in order to assess the role of these genetic factors and their interactions in the susceptibility to TTP. We found that the carrier frequencies of the DRB1∗11-DQB1∗03 and DRB1∗15-DQB1∗06 haplotypes were higher, while those of the DRB1∗07-DQB1∗02 and DRB1∗13-DQB1∗06 haplotypes were lower in TTP patients. There was no difference in the overall frequency of the PTPN22 c.1858T allele between TTP patients and controls. In conclusion, we identified four HLA-DRB1-DQB1 haplotypes associated with an increased (DRB1∗11-DQB1∗03 and DRB1∗15-DQB1∗06) or a decreased (DRB1∗07-DQB1∗02 and DRB1∗13-DQB1∗06) susceptibility to acquired idiopathic TTP.

Carboxiterminal pro-endothelin-1 as an endothelial cell biomarker in thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is characterised by the deficiency of the von Willebrand factor (VWF) cleaving protease (ADAMTS-13). Although several observations indicate an important role of endothelial activation in the pathogenesis of TTP, no reliable endothelial activation markers are available in the clinical management of TTP. Our aim was to investigate the presence of endothelial activation in TTP and to determine its connections with disease activity, therapy and complement activation. We enrolled 54 patients (median age 40.5; 44 females) and 57 healthy controls (median age 34; 30 females),VWF antigen, carboxiterminal-pro-endothelin-1 (CT-proET-1), complement Factor H and complement activation products (C3bBbP and SC5b-9) were measured. In both the acute and remission phase of TTP we found increased CT-proET-1 and VWF levels, while Factor H levels decreased compared with healthy controls. In remission, however, the elevated CT-proET-1 levels showed 22 % decrease when compared with the acute phase in paired samples (p=0.0031), whereas no changes for VWF and Factor H levels were observed. We also found positive correlations between CT-proET-1 levels and alternative pathway activation markers (C3bBbP; p=0.0360; r=0.4299). The data we present here demonstrate a role of endothelium activation in patients with acute TTP. The finding that CT-proET-1 levels decreased in remission compared with the acute phase further supports endothelial involvement. In addition, we show that endothelial activation also correlated with the activation of the alternative complement pathway. The data suggest that complement and endothelium activation jointly contribute to the development of TTP episodes in patients with predisposition to TTP.

Elevated plasma neutrophil elastase concentration is associated with disease activity in patients with thrombotic thrombocytopenic purpura.

INTRODUCTION: Genetic and autoimmune risk factors contribute to the development of thrombotic thrombocytopenic purpura (TTP) but triggers are needed to bring about acute disease. The aim of the study was to investigate the association of neutrophil activation with acute TTP, to assess whether neutrophil activation changes during plasma exchange therapy and to show if complement- and neutrophil activation are parallel, characteristic processes in acute TTP. MATERIALS AND METHODS: Altogether 49 EDTA-plasma samples of 21 TTP patients with acute disease and 17 in remission were investigated along with 20 healthy controls. A stable complex of PMNE-proteinase-inhibitor was measured by ELISA (Calbiochem, Merck-Millipore, Darmstadt, Germany). RESULTS: Acute disease was associated with significantly increased PMNE levels, the group medians were similarly low in TTP patients in remission and in healthy controls. Increased PMNE levels were characteristic for hematologically active and ADAMTS13 deficient form of TTP. PMNE concentration inversely correlated to disease activity markers platelet count (r=-0.349, p=0.032) and hemoglobin levels (p=-0.382 p=0.018). Achievement of remission was associated with significant reduction of plasma PMNE levels (p=0.031, Wilcoxon test). There was positive correlation between PMNE levels and complement activation markers C3a and Bb. CONCLUSIONS: We report increased PMNE levels in acute TTP and showed its association to activity markers of acute TTP and complement activation. Effective treatment of an acute TTP episode resulted in marked decrease in PMNE levels. Our data support and extend previous observations that neutrophil extracellular traps may be released in acute TTP and potentially contribute to the pathophysiology of this disease.

Acquired haemophilia: an often overlooked cause of bleeding - experience from a Hungarian tertiary care centre.

Acquired haemophilia is a potentially life-threatening bleeding disorder. Its early diagnosis and treatment is of major importance. We evaluated the elapsed time between the first presentation of the bleeding symptoms and the correct diagnosis in the cases of the acquired haemophilia patients referred to our centre between 1999 and 2011. The causes and consequences of the often delayed diagnosis were also examined. The clinical and laboratory data of 13 patients with acquired haemophilia were analysed. Eleven patients had inhibitors to factor VIII (FVIII), in one case the autoantibody developed to factor XIII (FXIII) and in one to factor V (FV). The median period between the onset of the bleeding symptoms and the correct diagnosis was 1.5 months (3.0 days-9.0 months). In four cases 4.0-9.0 months were needed to establish the diagnosis. The main reason of this delay was that either the prothrombin time was used exclusively to evaluate haemostasis in primary care and also in some secondary care centres, or the prolonged activated partial thromboplastin time went unnoticed despite the obvious bleeding symptoms. Our observation underlines the importance of early referral of patients with unexplained bleeding symptoms to centres with appropriate laboratory facilities and experience in the diagnosis of bleeding disorders.

Rapid-onset heparin-induced thrombocytopenia without previous heparin exposure.

Heparin-induced thrombocytopenia (HIT) is one of the most common immune-mediated drug reactions. Immunoglobulin G-type antibodies against platelet factor 4(PF4)/heparin complexes are known to play a key role in the pathogenesis of HIT. Rapid-onset HIT is caused by the presence of circulating HIT antibodies at the time of heparin readministration. These antibodies are generally resulted from a recent immunizing exposure to heparin. Here we report a case of rapid-onset HIT developed after a septicemia without previous heparin exposure. The diagnosis of HIT as well as the presence of platelet activating and heparin-dependent antibodies was confirmed by ELISA and flow cytometric functional assays. Our case report reinforces that rapid-onset HIT cannot be excluded only based on the absence of previous heparin exposure. In addition, it may support the new theory of pre-immunization by PF4-coated bacteria in the pathomechanism of HIT. We also call the attention that venous limb gangrene can be rarely associated with HIT and thrombosis even in the absence of coumarin therapy. Furthermore, transient presence of anti-phospholipid antibodies can cause a differential diagnostic problem in the cases of HIT.

Stem cell therapy: a promising and prospective approach in the treatment of patients with severe Buerger's disease.

No effective blood-flow enhancement therapies are available for patients with severe peripheral arterial disease (SPAD), thus amputation remains the only option for relief of rest pain or gangrene. Autologous bone marrow-derived stem cell therapy (ABMSCT) is an emerging modality to induce angiogenesis from endothelial progenitors. A total of 5 patients with SPAD were treated by ABMSCT using isolated CD34+ cells with characterized phenotype administered by intramuscular injections. The follow-up before and 1, 3, 6, 9, and 12 months after ABMSCT was based on clinical (rest pain, walking distance without pain, nonhealing ulcers, ankle-brachial index [ABI]) and laboratory (angiography, duplex and laser ultrasonography, TcPO(2)) parameters. Significant improvement of pain and walking distance was observed in all patients. Nonhealing ulcers disappeared in 3 patients and became smaller and thinner in 1 patient. The average of ABI improved significantly on the treated limb but did not change on the contralateral limb. New collaterals were detected by angiography in 3 patients, but duplex ultrasonography detected improvement in one patient only. Laser ultrasonography showed a mild significant change, TcPO(2) values improved mainly on the foot. Severe adverse events were not observed. We conclude that ABMSCT with isolated CD34+ cells is safe, effective, and results in sustained clinical benefit for patients with SPAD.

[Autologous bone marrow-derived stem cell therapy in patients with severe peripheral arterial disorder]. / Autológ csontvelôi eredetû ôssejtterápia eredménye elôrehaladott perifériás artériás érbetegségben.

BACKGROUND AND AIMS: Amputation is the only current option for relief of rest pain or gangrene in patients with severe peripheral arterial disease. Up to now, no effective blood-flow enhancement therapies are available. Autologous bone marrow-derived stem cell transplantation is an arising therapy modality with an option of building new blood vessels through endothelial stem and/or progenitor cells. PATIENTS AND METHODS: Five patients with severe peripheral arterial disorder were treated by autologous bone marrow-derived stem cell therapy. CD34+, CD133+ and CD45+/- cell number and ratio were determined. CD34+ cells were isolated by magnetic separation and collected into a 10 ml sample. The cell suspension was administered by local intramuscular injections (0.5-1.0 ml injections in the musculus gastrocnemius). The follow-up (before; 1, 3, 6, 9 and 12 months after the autologous bone marrow-derived stem cell therapy) based on clinical (rest pain, walking distance without pain, changes of non-healing ischaemic ulcers, ankle-brachial index) and laboratory (angiography, Color- and Laser-Doppler scan, measurement of transcutaneous oxygen tension and endothelial function test) parameters was documented and analyzed. RESULTS: Improvement of pain and walking distance was observed in all five cases. In three cases the non-healing ischaemic ulcers disappeared, in one other case they became smaller and thinner, and in one case no change was realized. The average of ankle-brachial index improved significantly (before: 0.41, twelve months after: 0.83). New collaterals were detected by angiography in three patients, but duplex ultrasonography detected improvement in one patient only. Before and 1, 6 and 12 months after stem cell therapy the transcutaneous oxygen tension changed on the foot from 18.80/16.78/23.83/37.50 mmHg, and on the calf from 36.66/31.25/45.00/37.30 mmHg. The macro- and microcirculation parameters did not show improvement after 1 month, however, after 3, 6, 9 and 12 months improved parameters were recorded. Severe adverse events were not observed. In one case elevated level of serum creatinine phosphokinase, and in another case a mild form of vasculitis were detected. CONCLUSION: autologous bone marrow-derived stem cell therapy with isolated CD34+ cells is effective, safe and results in sustained clinical benefit for patients with severe peripheral arterial disease.

[Successful high-dose low-molecular-weight heparin thromboprophylaxis in pregnant women with antiphospholipid syndrome]. / Nagy dózisú kis molekulatömegú heparinprofilaxis sikere antifoszfolipid szindrómás terhesekben.

INTRODUCTION: Antiphospholipid syndrome is an autoimmune disorder, defined as the association of antiphospholipid antibodies with manifestations of venous or arterial thrombosis or pregnancy loss. Primary antiphospholipid syndrome means that the patients have the same clinical symptoms and laboratory findings but they are not suffering from systemic lupus erythematosus or a closely related autoimmune diseases. Secondary antiphospholipid syndrome occurs in association with autoimmune or other diseases. AIM, METHODS, RESULTS: 31 pregnancies of 10 women are detailed. 22 pregnancies were without thromboembolic prophylaxis and only 2 pregnancies were successful (9.1%). Out of the 9 pregnancies with high dose low-molecular-weight heparin and low dose aspirin thromboprophylaxis throughout pregnancy 8 were successful (88.8%). All the newborns were healthy. In spite of the long-term low-molecular-weight heparin therapy side effects (osteoporosis or heparin-induced thrombocytopenia) were not observed. CONCLUSION: Considering these results high-dose low-molecular-weight heparin and low dose aspirin prophylaxis is recommended for pregnant women with antiphospholipid syndrome throughout pregnancy.