Increased Bleeding Tendency Research Articles

A CASE REPORT OF BERNARD-SOULIER SYNDROME IN DIFFERENTIAL DIAGNOSIS OF IMMUNE THROMBOCYTOPENIC PURPURA.

Bernard-Soulier Syndrome (BSS)is a rare hereditary disorder.Platelets in patients with BSS are unable to adhere, leading to an increased bleeding tendency. BSS cases are often misdiagnosed as idiopathic thrombocytopenic purpura (ITP). We report here a seven years old girl diagnosed as Bernard-Soulier syndrome with homozygous deletion of 39 nucleotides in the exon 2 of GP1BA. Bernard–Soulier syndrome should be considered before the patient is diagnosed with immune thrombocytopenia.

Improved anticoagulant effect of fucosylated chondroitin sulfate orally administered as gastro-resistant tablets.

Fucosylated chondroitin sulfate (FucCS) is a potent anticoagulant polysaccharide extracted from sea cucumber. Its anticoagulant activity is attributed to the presence of unique branches of sulfated fucose. Although this glycosaminoglycan exerts an antithrombotic effect following oral administration, high doses are necessary to achieve the maximum effect. The diminished activity of FucCS following oral administration is likely due to its degradation in the gastrointestinal tract and its limited ability to cross the intestinal cell membranes. The latter aspect is particularly difficult to overcome. However, gastro-resistant tablet formulation may help limit the degradation of FucCS in the gastrointestinal tract. In the present work, we found that the oral administration of FucCS as gastro-resistant tablets produces a more potent and prolonged anticoagulant effect compared with its administration as an aqueous solution, with no significant changes in the bleeding tendency or arterial blood pressure. Experiments using animal models of arterial thrombosis initiated by endothelial injury demonstrated that FucCS delivered as gastro-protective tablets produced a potent antithrombotic effect, whereas its aqueous solution was ineffective. However, there was no significant difference between the effects of FucCS delivered as gastro-resistant tablets or as aqueous solution in a venous thrombosis model, likely due to the high dose of thromboplastin used. New oral anticoagulants tested in these experimental models for comparison showed significantly increased bleeding tendencies. Our study provides a framework for developing effective oral anticoagulants based on sulfated polysaccharides from marine organisms. The present results suggest that FucCS is a promising oral anticoagulant.

Desialylation of Megakaryocytes Diminishes Platelet Production By Disrupting Megakaryocyte Adhesion, Migration and Proplatelet Formation in Chronic Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is a common autoimmune disorder characterized by increased bleeding tendency and isolated thrombocytopenia. The precise pathogenesis of the decreased thrombopoiesis in chronic ITP (cITP) is poorly understood. Megakaryocytes (MKs) in cITP show impaired maturation and thrombopoiesis, which are correlated with numerous glycoproteins on the surface of MKs. Different types of sialoglycoproteins are expressed on the surface of megakaryocytes, including GPIbα and platelet endothelial cell adhesion molecule-1 (PECAM-1), both of which participate in megakaryocyte migration to the vascular niche in the bone marrow (BM) and in proplatelet formation. Desialylation has recently been identified a contributor to the pathogenesis of thrombocytopenia. Our previous study has demonstrated that desialylation of GPIbα is related to increased apoptosis and phagocytosis of platelets in cases of prolonged isolated thrombocytopenia after allogeneic hematopoietic stem cell transplantation (Zhang et al., J Hematol Oncol, 2015). Because MKs are heavily sialylated cells, we raised the question whether the desialylation of megakaryocytes contributes to the defective thrombopoiesis in patients with cITP through impaired MK migration, adhesion and proplatelet formation in the vascular niche.MK desialylation was analyzed by flow cytometry using lectins. Desialylated glycoproteins were measured using selective exo-enzymatic labeling. Protein expression, distribution and interaction were measured using the following techniques: immunofluorescence, flow cytometry, western blot and immunoprecipitation. cITP MKs exhibited increased β-galactose exposure compared to the control MKs, indicating excessive desialylation. Desialylation was correlated with decreased platelet production of MKs. We further explored the cause of desialylation and found that the sialidase NEU1 was over-expressed in MKs. Treatment with the sialidase inhibitor DANA ameliorated the loss of sialic acids. These results indicated that NEU1 contributed to the desialylation of MKs in cITP. Altered MK distribution in the BM niche was exhibited upon BM biopsy of cITP patients. The ratio of perivascular MKs was markedly decreased in cITP patients. Defective adhesion and transmigration behaviors were also discovered in desialylated cITP MKs. The motility of cITP MKs through stromal cell monolayers driven by stromal cell derived factor 1 (SDF1) was decreased. Adherence to fibronectin, collagen and fibrinogen was assessed, and desialylated cITP MKs exhibited an increase in adhesion with these macromolecules. Similar abnormalities were observed in the BM niche of ST6Gal1-/- mice, and treatment with ST6Gal1 and CMP-SA augmented the ratio of MKs in the BM vascular niche in ST6Gal1-/-mice, indicating that desialylation impaired the MK migration and adhesion. Additional experiments focused on which specific sialoglycoproteins are excessively desialylated. As detected by SEEL, PECAM-1 exhibited excessive desialylation in cITP MKs, which was related to impaired CXCR4 polarization in response to SDF1. Inhibition of sialidase using DANA partially restored this polarization, demonstrating that desialylation of PECAM-1 was responsible for the defect in MK migration to the vascular niche. Meanwhile, PECAM-1 desialylation was associated with GPIIb/IIIa overactivation, which correlated to the increased adhesion of MKs. This increased adhesion was reversed by the GPIIb/IIIa inhibitor lotrafiban, indicating that desialylated PECAM-1 contributed to the abnormal adhesion via overactivation of GPIIb/IIIa. Desialylation of GPIbα was found on the surface of MKs from cITP patients and was associated with abnormal microtubule formation and increased MK apoptosis through altered 14-3-3ζ distribution, which led to the impediment of proplatelet formation in the vascular niche.In conclusion, our results demonstrate that MKs are desialylated by NEU1 in cITP patients, and desialylated MKs present with defective migration towards the vascular niche, abnormal adhesion to the extracellular matrix and impaired proplatelet formation. Desialylation of PECAM-1 and GPIbα have been demonstrated to be responsible for these abnormal behaviors of MKs. Sialidase inhibitor shows an improvement in the thrombopoiesis of cITP MKs; therefore, our study implies a novel potential approach for the treatment of cITP. DisclosuresNo relevant conflicts of interest to declare.

Idelalisib Rapidly Improves Platelets Function in Patients with Chronic Lymphocytic Leukemia (CLL)

INTRODUCTIONPlatelet function has never been studied systematically in patients with CLL. Novel drugs are now available for CLL treatment that may impact on platelet functions. Fifty per cent of patients treated with Ibrutinib suffered from minor bleedings and only 5% from major bleedings, partly caused by the drug driven inhibition of platelet glycoprotein VI signaling. No data on bleeding tendency has yet emerged on patient treated with Idelalisib, the first specific inhibitor of PI3K δ p110 approved for the treatment of relapsed/refractory CLL or for patients with del17p or TP53 as first line therapy. In animal models a reduction of p110δ on platelets (PLT) does not increase bleeding, causing only a slight reduction of platelet aggregation and activation. Knowledge about potential bleeding complications associated with the use of small molecules may be relevant in older patients and those at increased bleeding risk due to concomitant therapies.PATIENTS AND METHODSTen patients with CLL (M/F: 6/4; median age: 71 years, range 47-82) who started therapy with Idelalisib were enrolled in a prospective observational pilot study. The Bleeding Severity Score (BSS), a validated questionnaire, was administrated to patients to estimate bleeding before and during idelalisib therapy. All patients underwent coagulation tests and platelet aggregation/secretion studies with different aggregating agents before starting therapy with Idelalisib, after 28 + 7 days and after 3 months. Patients with a platelet count less than 80.000/mm3, in antiplatelet or anticoagulant therapy, with recent use (within 7 days) of NSAIDs and a diagnosis of hereditary thrombocytopenia/pathy were excluded. We defined complete haematological remission (CHR) as Hb more than 11g/dl, PLT more than 100.000/mm3, lymphocyte less than 5.000/mm3 and partial haematological remission (PHR) as a response not fulfilling criteria for CHR.RESULTSNo cases of hemorrhagic complications or increased bleeding tendency were observed in patients with CLL and no patients had a pathologic BSS (>5) at enrolment. All patients had coagulation tests within normal limits at baseline and after 28 days. Platelet count was below 100.000/mm3 in 5 patients. In 9 out of 10 patients platelet aggregation was pathological with at least 2 of the 4 aggregating agents tested. Platelet secretion before initiation of treatment with Idelalisib was particularly impaired with ADP (8/10 patients), while was pathological with collagen, a strong agonist, in only 2 patients. In 8 patients intraplatelet ATP/ADP ratio was pathological, as observed in delta storage pool disease. After 28 days of treatment 4 of 10 patients were in CHR and 3 in PHR. Platelets count was still below 100.000/mm3 in 2 subjects. At 28 days in 5 out of the 9 patients with pathological baseline test, platelet aggregation improved, while 3 remained unchanged and in one worsened. Even ADP secretion normalized in 4 patients. ATP/ADP ratio did not significantly change. At three months 7 patients reached CR and 2 reached PR. At three months platelets count was still below 100000/mm3 in 2 patients. In 3 patients platelet aggregation further ameliorated.CONCLUSIONSIn this pilot study, treatment with idelalisib improved platelet aggregation tests in most of the CLL patients who presented a pathological test before starting therapy. It’s unlikely that the drug has a direct effect on platelets, given their low expression of PI3Kδ; therefore our results are probably due to the rapid idealisib effect on CLL clone. Based on these preliminary data, Idelalisib seems to be safe in patients with an increased bleeding risk. DisclosuresReda:Roche: Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding. Peyvandi:Alexion: Other: research funding paid to Luigi Villa Foundation, Research Funding; Ablynx: Membership on an entity's Board of Directors or advisory committees, Other: research funding paid to Luigi Villa Foundation, Research Funding; CSL Behring: Speakers Bureau; Biotest: Other: research funding paid to Luigi Villa Foundation, Research Funding, Speakers Bureau; Octapharma: Consultancy; Kedrion Biopharma: Consultancy, Other: research funding paid to Luigi Villa Foundation, Research Funding; LFB: Consultancy; Grifols: Speakers Bureau; Novo Nordisk: Other: research funding paid to Luigi Villa Foundation, Research Funding, Speakers Bureau; SOBI: Speakers Bureau; Bayer: Speakers Bureau.

Targeted Whole Exome Sequencing of 87 Predisposition Genes in 156 Patients from the Oresund Region with Bleeding Disorders

Introduction Rare inherited bleeding disorders are a common cause of bleeding tendency. To ensure a correct diagnosis, specialized laboratory analyses are required, and the mere complexity of these investigations renders a significant part of patients without proper diagnosis. Several inherited platelet disorders are part of a syndrome, and thus a correct diagnosis may have clinical consequences, beyond genetic counseling. The aim was to report the results of up front targeted whole exome sequencing (WES).Methods Patients from the Öresund Region referred for an inherited bleeding disorder, were screened for plasma coagulation factor deficiencies and scored by ISTH Bleeding assessment tool (BAT) score. Patients with a significant BAT score and/or chronic thrombocytopenia and without evidence of a coagulation factor deficiency were included. After informed consent, blood samples for genetic screening were obtained. When genetic analysis revealed a mutation or variant of uncertain significance (VUS), further evaluation with specific functional testing and transmission electron microscopy were done, followed by a concluding multidisciplinary conference.WES and variant filtering WES was selected to identify germline mutations, as well as enabling extending the number of genes analyzed. Genomic DNA was purified from whole blood. Illumina‘s HiSeq 2500 platform was used for sequencing. The exome was covered with an average of 84x, where 97 % was covered with at least 9 x. Based on literature search, 87 genes related to bleeding, thrombocytopathia, and thrombocytopenia were analyzed. Common single-nucleotide polymorphisms were subtracted. Variant Classification:The integrated Alamut Visual software (v.2.6.1) (http://www.interactive-biosoftware.com) was used to predict the pathogenicity of specific protein variants. To designate the clinical significance, a clinical score was developed, the Öresund score, combining inheritance pattern of known diseases within the Silico based prediction. Following classification of each variant according to the five-tier scheme, patients were individually evaluated for genetic predisposition.Results 156 patients were included in the study, age 38.6±15 years (mean +/- 1SD), whereof 25 were male (16%) and 131 (84%) were female. The majority of patients had a family history of increased bleeding tendency (111 patients, 71%) and 47 patients (30%) had thrombocytopenia (a value below the normal range). The majority of patients were of European ethnicity (90%). Mean BAT score was 8 +/- 6 (1SD). A total of 421 germline mutations/variants (2.7 variant pr. patient) were found. In 28 patients (18%) a class 4-5 mutation (previously published and/or confirmed by specific functional testing) were found. In 21 patients (13%), a possible contributing class 3 VUS was found. In 21/47 patients (45%) with thrombocytopenia and 27/119 patients (23%) with normal platelet counts (p < 0.0001), a pathogenic or possibly pathogenic class 3 VUS was detected. Incidental findings occured i 3 patients (2%).Discussion and conclusion We implemented targeted WES of 87 genes up front to diagnose rare inherited bleeding disorders. In 156 patients we report the finding of 4 previously published gene mutations and 42 previously non reported mutations or class 3 VUS in 25 genes. The success rate of finding a diagnostic or possible pathogenic VUS was significantly higher in patients with thrombocytopenia. The substantial finding of additional 368 variants required clinical designation and extensive functional testing, before final classification could be made. Our finding of multiple deleterious class 3 variants in COL3A1, COL5A2 and COL5A1 warrant further investigations into the role of collagen deficiency. Only 1 patient had a (tolerated) variant in protein disulfide isomerase (P4HB), a newly discovered gene described to play a key role in initiation of clot formation. In our hands, WES was an efficient tool in defining the underlying cause of bleeding disorders. However, the large number of previously non described variants required substantial efforts to sort out. In patients with rare inherited bleeding disorders, targeted WES is a feasible method to discover possible pathogenic mutations or VUS to direct the specific functional testing. DisclosuresNo relevant conflicts of interest to declare.

Hyperfibrinolysis increases blood–brain barrier permeability by a plasmin- and bradykinin-dependent mechanism

AbstractHyperfibrinolysis is a systemic condition occurring in various clinical disorders such as trauma, liver cirrhosis, and leukemia. Apart from increased bleeding tendency, the pathophysiological consequences of hyperfibrinolysis remain largely unknown. Our aim was to develop an experimental model of hyperfibrinolysis and to study its effects on the homeostasis of the blood–brain barrier (BBB). We induced a sustained hyperfibrinolytic state in mice by hydrodynamic transfection of a plasmid encoding for tissue-type plasminogen activator (tPA). As revealed by near-infrared fluorescence imaging, hyperfibrinolytic mice presented a significant increase in BBB permeability. Using a set of deletion variants of tPA and pharmacological approaches, we demonstrated that this effect was independent of N-methyl-D-aspartate receptor, low-density lipoprotein–related protein, protease-activated receptor-1, or matrix metalloproteinases. In contrast, we provide evidence that hyperfibrinolysis-induced BBB leakage is dependent on plasmin-mediated generation of bradykinin and subsequent activation of bradykinin B2 receptors. Accordingly, this effect was prevented by icatibant, a clinically available B2 receptor antagonist. In agreement with these preclinical data, bradykinin generation was also observed in humans in a context of acute pharmacological hyperfibrinolysis. Altogether, these results suggest that B2 receptor blockade may be a promising strategy to prevent the deleterious effects of hyperfibrinolysis on the homeostasis of the BBB.

Further evidence of the limitations of Activated Partial Thromboplastin Time to monitor Argatroban

Further evidence of the limitations of Activated Partial Thromboplastin Time to monitor Argatroban

Atherogenesis in hypothyroid patients

Hypothyroidism may lead to circulatory system impairment, which could appear as atherosclerotic lesions in blood vessels and ischemic heart disease. Thyroid hormones deficiency directly affects on heart and blood vessels functions, or they may promote metabolic changes, which affect atherosclerosis. Hypothyroidism influences in decreasing stroke volume and cardiac contractility, arterial media intima thickness, and arterial smooth muscles diastole disorders. Atherogenesis is characterized by lipoprotein metabolism disorders, which increase total cholesterol, LDL cholesterol, apoprotein B–100 and triglycerides. High LDL levels during hypothyroidism may be the result of LDL receptors reduction. The lipid profile alterations could be the consequence of specific enzymes and proteins decreased activity: hepatic lipase, lipoprotein lipase and cholesteryl ester transfer protein, which maintain correct lipid metabolism. Hypothyroidism may contribute to oxidative LDL modifications, thus tyroxine has antioxidant effect on lipoproteins. Increased atherosclerosis risk in the course of hypothyroidism may be caused also by elevated lipoprotein (a) level. Atherogenesis is often associated with chronic inflammation, which leads to increased C-reactive protein level. Moreover, it may be connected with hyperhomocysteinemia, which is caused by decreasing activity of enzymes remethylating homocysteine, and by reducing renal clearance of homocysteine. Regarding to hemostatic system, patients suffering from hypothyroidism are characterized by impaired coagulation factors synthesis and by intensified fibrynolysis, which manifest as increased bleeding tendency. Atherogenesis in hypothyroid patients is a compound mechanism. The coexistence of lipid abnormalities, intensified inflammation, and hyperhomocysteinemia amplifies atherogenesis in the course of hypothyroidism. However changes, which are observed in hemostatic system may prevent escalating of atherosclerosis.

ALG6-CDG: a recognizable phenotype with epilepsy, proximal muscle weakness, ataxia and behavioral and limb anomalies.

Alpha-1,3-glucosyltransferase congenital disorder of glycosylation (ALG6-CDG) is a congenital disorder of glycosylation. The original patients were described with hypotonia, developmental disability, epilepsy, and increased bleeding tendency. Based on Euroglycan database registration, we approached referring clinicians and collected comprehensive data on 41 patients. We found hypotonia and developmental delay in all ALG6-CDG patients and epilepsy, ataxia, proximal muscle weakness, and, in the majority of cases, failure to thrive. Nine patients developed intractable seizures. Coagulation anomalies were present in <50% of cases, without spontaneous bleedings. Facial dysmorphism was rare, but seven patients showed missing phalanges and brachydactyly. Cyclic behavioral change, with autistic features and depressive episodes, was one of the most significant complaints. Eleven children died before the age of 4years due to protein losing enteropathy (PLE), sepsis, or seizures. The oldest patient was a 40year-old Dutch woman. The most common pathogenic protein alterations were p.A333V and p.I299Del, without any clear genotype-phenotype correlation. ALG6-CDG has been now described in 89 patients, making it the second most common type of CDG. It has a recognizable phenotype and a primary neurologic presentation.

Bone marrow niche in immune thrombocytopenia: a focus on megakaryopoiesis.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by increased bleeding tendency and thrombocytopenia. In fact, the precise pathogenesis of this disease is still not clear. Megakaryopoiesis involves complete differentiation of megakaryocyte (MK) progenitors to functional platelets. This complex process occurs in specific bone marrow (BM) niches composed of several hematopoietic and non-hematopoietic cell types, soluble factors, and extracellular matrix proteins. These specialized microenvironments sustain MK maturation and localization to sinusoids as well as platelet release into circulation. However, MKs in ITP patients show impaired maturation and signs of degradation. Intrinsic defects in MKs and their extrinsic environment have been implicated in altered megakaryopoiesis in this disease. In particular, aberrant expression of miRNAs directing MK proliferation, differentiation, and platelet production; defective MK apoptosis; and reduced proliferation and differentiation rate of the MSC compartment observed in these patients may account for BM defects in ITP. Furthermore, insufficient production of thrombopoietin is another likely reason for ITP development. Therefore, identifying the signaling pathways and transcription factors influencing the interaction between MKs and BM niche in ITP patients will contribute to increased platelet production in order to prevent incomplete MK maturation and destruction as well as BM fibrosis and apoptosis in ITP. In this review, we will examine the interaction and role of BM niches in orchestrating megakaryopoiesis in ITP patients and discuss how these factors can be exploited to improve the quality of patient treatment and prognosis.

Association of Genetic Polymorphisms of CYP2C9 and VKORC1 with Bleeding Following Warfarin: A Case-Control Study.

Various factors have been shown to increase the risk of bleeding with warfarin. This study aimed to assess the association of CYP2C9 and VKORC1 with the development of bleeding following warfarin. A case control study was initiated after obtaining institutional ethics committee clearance and written informed consent from patients. Cases were defined as those who bled within three months of warfarin initiation and controls as those who did not have any episode of bleeding within three months. Genotyping for CYP2C9 (*1, *2, *3) and VKORC1 1639 (GG, GA and AA) was performed by PCRRFLP. Chi square test was used to find out the association and trend of CYP2C9 and VKORC1 genotypes with odds ratio (95% CI) for strength of association. A binary logistic regression model was developed associating age, body weight, sex, CYP2C9 and VKORC1 status with risk of bleeding. A total of 100 controls and 38 cases were studied from Oct 2009 to July 2011. A significant association (P < 0.0001) and trend (P = 0.027) of mutant alleles of CYP2C9 and VKORC1 were noted with bleeding with odds ratios of 7.8 [3.4, 17.9] and 2.7 [1.3, 5.7] respectively. Weekly dose requirement was significantly lower with the presence of *3 allele relative to *1 in CYP2C9 (P < 0.001). The regression model showed an accuracy of 80% and could explain 35.3% of the variability. A significant association between CYP2C9 (*1,*2,*3) genotype and VKORC1 (1639 G>A) haplotype status has been found with increased bleeding tendency to warfarin. This may help to individualize therapy.

Meningioma with purely intratumoral hemorrhage mimicked intracerebral hemorrhage: Case report and literature review

Meningiomas are the most common non-glial intracranial tumor with an incidence of 2.3-5.5/100,000 people, accounting for 20-30% of all primary adult brain tumor diagnoses. Meningiomas associated with hemorrhage are rare; the reported incidence is 0.5-2.4%. We share a case experience of meningioma with purely intratumoral hemorrhage. The initial image study of the intratumoral hemorrhage mimicked intracerebral hemorrhage. A 63-year-old woman with initial presentation of headache, dizziness, nausea, and vomiting has brought to the emergent department. The brain computed tomography showed a 5.3 cm hyperdense lesion over the left occipitotemporal area. Under the impression of intracerebral hemorrhage with unknown etiology, the brain magnetic resonance imaging was done for tumor survey and showed lobulated enhanced mass over left T-O area. Tumor with bleeding with skull bone invasion was first considered. The surgical pathology revealed atypical meningioma with focal clear cell change (World Health Organization Grade II), which is separated with the brain by pia meter. Meningiomas associated with hemorrhage are rare; the reported incidence is 0.5-2.4%. The clinical features about increasing bleeding tendency: (i) age >70 years old or <30 years old; (ii) the location of meningiomas locate at intraventricle or convexity; (iii) histopathalogical type: Meningotheliomatous, malignant, fibrous, and angioblastic type. Most reported intracranial hemorrhages associated with meningiomas are found in the subarachnoid and subdural spaces. There are several hypotheses for the possible mechanism of tumoral bleeding. The tumoral bleeding in meningioma is a rare event. The incidence depended on the location and the histopathological finding. There might be other indicators but still need more research.

Anticoagulation Therapy in Patients with Chronic Kidney Disease.

Patients with chronic kidney disease (CKD) are at increased risk for both thrombotic events and bleeding. The early stages of CKD are mainly associated with prothrombotic tendency, whereas in its more advanced stages, beside the prothrombotic state, platelets can become dysfunctional due to uremic-related toxin exposure leading to an increased bleeding tendency. Patients with CKD usually require anticoagulation therapy for treatment or prevention of thromboembolic diseases. However, this benefit could easily be offset by the risk of anticoagulant-induced bleeding. Treatment of patients with CKD should be based on evidence from randomized clinical trials, but usually CKD patients are excluded from these trials. In the past, unfractionated heparins were the anticoagulant of choice for patients with CKD because of its independence of kidney elimination. However, currently low-molecular-weight heparins have largely replaced the use of unfractionated heparins owing to fewer incidences of heparin-induced thrombocytopenia and bleeding. We undertook this review in order to explain the practical considerations for the management of anticoagulation in these high risk population.

Management of perioperative bleeding associated with the use of selective serotonin reuptake inhibitors

Increased bleeding tendency is associated with the use of selective serotonin reuptake inhibitors. In this study, we report a case of perioperative bleeding in patient on selective serotonin reuptake inhibitors. The management options for such cases are discussed.

One-Stage FVIII Activity Is Inversely Correlated with Heavy Menstrual Bleeding in Hemophilia a Carriers

IntroductionThe majority of carriers of hemophilia A have historically been considered to have normal hemostasis (FVIII:C >50%) and thus not have an increased bleeding tendency. However, our research group has demonstrated that some hemophilia A carriers can experience increased bleeding compared to normal women despite this normal FVIII activity. In our recently published study in adult hemophilia A carriers there was no correlation between FVIII:C, as measured by a one-stage coagulation assay, and bleeding phenotype, as measured by the MCMDM-1 VWD bleeding score. In this follow up study we sought to determine which of these hemophilia A carriers with normal FVIII:C are at risk for bleeding. The goal of this study is to determine the relationship between FVIII assays (one-stage and chromogenic) and thrombin generation with both menstrual bleeding and an overall bleeding tendency. MethodsWe recruited mothers of children with hemophilia A in the Emory University pediatric bleeding disorders clinic. We included only adult (>18 year of age) obligate hemophilia A carriers that did not have a concomitant bleeding disorder or chronic disease that would increase their bleeding tendency. We gathered basic demographic information and evaluated the overall bleeding tendency using the ISTH Bleeding Assessment Tool (ISTH BAT), a semi-quantitative assessment of bleeding scored from 0 to 56. We considered a score of 6 or higher consistent with pathologic bleeding. In addition, we inventoried menstrual bleeding with the Pictorial Bleeding Assessment Chart (PBAC), a visual representation of menstrual blood loss. We considered a PBAC greater than 100 consistent with heavy menstrual bleeding. Plasma samples were collected from each subject at the time of the survey in sodium citrate tubes without corn trypsin inhibitor. Samples were double spun at 2,500 rpm for 15 minutes and stored at -80°C.Laboratory evaluation for each subject included FVIII:C, measured by one-stage coagulation assay (aPTT reagent with micronized silica) and chromogenic assay (COATEST SP4 FVIII kit, Chromogenix), as well as thrombin generation (Calibrated Automated Thrombogram, PPP-Reagent Low, FluCa Kit) to evaluate endogenous thrombin potential (ETP, nM*min), peak thrombin (nM) and lag time (min). We performed linear regression using GraphPad Prism; p<0.05 was considered significant. ResultsOver a three-month period, we approached 32 adult obligate hemophilia A carriers; 23 agreed to participate in the survey, 16 consented to blood draw. One carrier was excluded in extreme outlier analysis and due to an autoimmune disorder, leaving 15 evaluable subjects. Our cohort is relatively young with a median age of 41 years (range 23-51), predominantly Caucasian (53%, 8/15) and the majority carry a severe mutation (10/15 severe, 2/15 moderate, 3/15 mild).Reproductive bleeding (post-partum hemorrhage and/or menstrual bleeding) was commonly reported (93%, 14/15). The median ISTH BAT bleeding score for subjects was 2 (range 1-8). The median PBAC score was 148 (range 10-608). The carriers had a relatively normal FVIII:C by one-stage assay with a median of 0.78 U/mL (range 0.30 - 1.06) and by chromogenic assay with a median of 1.15 U/mL (range 0.66 - 1.9). FVIIIC by one-stage assay was inversely correlated to PBAC (r2 =0.4; p=0.01, figure 1A). Conversely, FVIII:C by chromogenic assay did not correlate with PBAC, however trended toward significance (r2 = 0.19; p=0.10, figure 1A). Additionally, there was no correlation found between FVIII:C (one-stage or chromogenic assay) and ISTH BAT bleeding score (figure 1B). There was also no correlation found between bleeding score or PBAC and the parameters of the thrombin generation assay or between severity of the closest male relative's hemophilia and any of the assays performed. ConclusionsIn our cohort of adult obligate hemophilia A carriers, as the FVIII:C (one-stage assay) decreased, the menstrual bleeding tendency increased as measured by the PBAC. We were unable to find a correlation between other measures of hemostasis, including the chromogenic assay and thrombin generation, and commonly used bleeding assessment tools. Further larger studies are warranted to help determine which hemophilia A carriers would be at risk for bleeding. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Defining the Risk: Benefit Ratio of Venous Thromboembolism (VTE) Prophylaxis in Hospitalized Cancer Patients

▪Introduction: VTE affects 1.6-1.8/1000 hospitalized patients per year; active cancer increases the rate of VTE 6-fold. Despite the high risk of VTE, studies show cancer patients receive inpatient VTE prophylaxis at a lower rate than general medical patients. In addition, VTE prophylaxis is often held for a platelet value of <50x109/L, despite a lack of evidence. Current recommendations for all inpatients with cancer (and no contraindication to blood thinners) are to receive prophylactic anticoagulation, regardless of an increased bleeding tendency in this population. A formal risk:benefit ratio of inpatient VTE prophylaxis for cancer patients has not been evaluated to date.Methods: The Michigan Hospital Medicine Safety Consortium, a 49 hospital quality collaborative, has prospectively collected data on VTE risk factors and outcomes in medical patients > 18 years of age. Exclusion criteria include surgical patients, pregnancy, admission to the ICU or for palliative care, therapeutic anticoagulation, diagnosis of acute thrombus, history of VTE within 6 months, and length of stay < 2 days. We compared rates of VTE prophylaxis, bleeding and new VTE between cancer and general medicine patients who were eligible for prophylaxis (i.e. no contraindications including active bleed within 3 months, coagulopathy, or high risk brain metastasis). Student's t-test was used for continuous variables and chi-square for categorical data. Logistic regression was used to calculate odds ratio (OR). The number needed to treat and number needed to harm were used to derive a risk:benefit ratio.Results: Between 7/2012-7/2015, 86,634 admissions were captured in the cohort; 70,086 were eligible for VTE prophylaxis and included in this analysis. 22% of cases had a diagnosis of cancer.Table 1Demographics on AdmissionCancer (n= 15,166), %General Medicine (n=54,920), %pRace (Caucasian)82.374.3<0.001Age mean yrs (SD)72.5 (14.4)63.7 (18.8)<0.001Gender (female)53.656.5<0.001Central Line17.84.1<0.001History of VTE7.65.3<0.001Caprini risk score ≥ 588.135.8<0.001Charleston Index mean (SD)5.1 (2.5)2.1 (2.0)<0.001Ever Smoked59.656.3<0.001Obese29.337.1<0.001Surgery w/in 30 days3.62.1<0.001Trauma w/in 30 days0.30.40.01Of cancer admissions, 89.5% had solid tumors, 13.5% hematologic malignancies, 3.4% both and 20% metastatic disease. Active treatment for cancer was delivered <6 months from index admission in 32.1%, within 6-12 mo in 3.2%, >12 mo in 44.4% and no treatment or unknown in 20.3%.When compared to general medical admissions, cancer admissions were more likely to receive VTE prophylaxis (72.16% vs 69.21%, p<0.001), and have a new VTE out to 90 days post discharge despite prophylaxis (0.91% vs 0.45%, p<0.001, unadjusted OR 2.07 (95% CI 1.6-2.7)). There was no difference in VTE rate with regard to platelet count in cancer cases (0.43% for plt < 50 vs 1.08% for plt ≥ 50, p=0.10). Among all patients receiving VTE prophylaxis, bleeding was more common in cancer cases (major bleeding 0.84% vs 0.58%, p=0.005; minor bleeding 1.80% vs 1.36%, p=0.002). Among cancer cases, bleeding rates were higher in patients with platelet <50 vs ≥ 50 (major bleed 4.86% vs 1.88%, p<0.001; minor bleed 2.88% vs 1.7%, p=0.04).Table 2The Number Needed to Treat (NNT) to Prevent One VTE During Admission or 90 Days Post Discharge and Number Needed to Harm (NNH) to Cause One Major Bleed During Admission with Risk:Benefit Ratio (NNH:NNT)General MedicineCancerNNT 1428NNT 1000NNH 2500NNH 277.9NNH:NNT 1.75NNH:NNT 0.28Conclusions: In this prospective inpatient cohort, we compared general medicine to cancer cases and found cancer admissions received VTE prophylaxis at a higher rate. This is different than previously reported data, likely due to the exclusion of patients with contraindications to prophylaxis. However, despite prophylaxis, cancer patients had a higher rate of VTE during admission and 90 days post discharge as well as more bleeding complications. The risk:benefit ratio of VTE prophylaxis is 6 times worse in cancer patients due to bleeding. While bleeding occurs more frequently in cancer patients with platelet count <50x109/L, VTE occurs at a similar rate regardless of platelet count, meaning that cancer patients are at high risk of both clotting and bleeding. Recommendations for inpatient VTE prophylaxis for cancer patients require a targeted approach to identify a subset who would most benefit from VTE prophylaxis. DisclosuresFlanders:Institute for Healthcare Improvement and the Society of Hospital Medicine: Consultancy; Wiley Publishing: Patents & Royalties; CDC Foundation: Research Funding; Blue Cross Blue Shield of Michigan: Research Funding; Michigan Hospital Association: Research Funding. Sood:Bayer: Research Funding.

Association between bleeding tendency and health‐related quality of life in carriers of moderate and severe haemophilia

Carriers of severe and moderate haemophilia A and B are expected to have approximately 50% of the normal level of factors VIII and IX. However, due to X chromosome inactivation in early embryonic life, factor levels can vary considerably. This can lead to increased bleeding tendency, which may in turn impact on health-related quality of life (HRQOL). The aim of this study was to assess HRQOL in carriers of severe and moderate haemophilia with and without increased bleeding tendency. One hundred and twenty-four adult carriers and 90 controls were recruited. Bleeding tendency was evaluated using a structured bleeding assessment tool. HRQOL was measured by the short form 36 (SF-36) questionnaire. The SF-36 scores were compared with Swedish normative age-matched data and reported as Z scores. There was no significant difference between the whole groups of carriers and controls in the Z scores of the eight SF-36 domains. The mental component summary (MCS) was lower in carriers, compared with controls (P=0.048). The subgroup of carriers with an increased bleeding tendency had significantly lower Z scores compared to controls regarding the General Health (P=0.008), the Social Functioning (P=0.040) and the Mental Health (P=0.048) domains. The MCS was significantly lower in this carrier subgroup than in controls (P=0.033). We conclude that the subgroup of carriers of haemophilia with increased bleeding tendency have impaired HRQOL. The SF-36 results indicate that this condition affects mental rather than physical health.

Increased Bleeding Tendency from Interaction between Rivaroxaban and Topical Miconazole: Case Report

Novel oral anticoagulants (OAC) are being increasingly used. We describe a case of increased bleeding tendency resulting from probable drug interaction between rivaroxaban and topical miconazole. Recognition of this possible interaction is important during concomitant use to prevent adverse outcome arising from over-anticoagulation.

The Non-catalytic B Subunit of Coagulation Factor XIII Accelerates Fibrin Cross-linking

Covalent cross-linking of fibrin chains is required for stable blood clot formation, which is catalyzed by coagulation factor XIII (FXIII), a proenzyme of plasma transglutaminase consisting of catalytic A (FXIII-A) and non-catalytic B subunits (FXIII-B). Herein, we demonstrate that FXIII-B accelerates fibrin cross-linking. Depletion of FXIII-B from normal plasma supplemented with a physiological level of recombinant FXIII-A resulted in delayed fibrin cross-linking, reduced incorporation of FXIII-A into fibrin clots, and impaired activation peptide cleavage by thrombin; the addition of recombinant FXIII-B restored normal fibrin cross-linking, FXIII-A incorporation into fibrin clots, and activation peptide cleavage by thrombin. Immunoprecipitation with an anti-fibrinogen antibody revealed an interaction between the FXIII heterotetramer and fibrinogen mediated by FXIII-B and not FXIII-A. FXIII-B probably binds the γ-chain of fibrinogen with its D-domain, which is near the fibrin polymerization pockets, and dissociates from fibrin during or after cross-linking between γ-chains. Thus, FXIII-B plays important roles in the formation of a ternary complex between proenzyme FXIII, prosubstrate fibrinogen, and activator thrombin. Accordingly, congenital or acquired FXIII-B deficiency may result in increased bleeding tendency through impaired fibrin stabilization due to decreased FXIII-A activation by thrombin and secondary FXIII-A deficiency arising from enhanced circulatory clearance.

Extracorporeal membrane oxygenation induces short-term loss of high-molecular-weight von Willebrand factor multimers.

High-molecular-weight (HMW) von Willebrand factor (vWF) multimers are crucial for primary hemostasis. Increased shear stress from ventricular assist devices can provoke premature degradation of HMW vWF multimers. Whether similar loss of vWF multimers occurs during extracorporeal membrane oxygenation (ECMO) is not clear. We conducted a prospective observational study in a clinical cohort of patients who required ECMO for intractable cardiac and/or respiratory failure. The primary end point was the quantity and quality of HMW vWF multimer bands before, during, and after ECMO support. To investigate further changes in primary hemostasis, we also measured vWF antigen activity (vWF:Ag), vWF ristocetin cofactor activity (vWF:RCo), and factor VIII in 38 patients who required ECMO support before initiation of ECMO (baseline), after 24 and 48 hours on ECMO, and 24 hours after termination of ECMO therapy. Compared with baseline, vWF:Ag and vWF:RCo decreased after 24 hours of ECMO (mean ± SD, vWF:Ag, 307% ± 152% to 261% ± 138%, P = 0.002; vWF:RCo 282% ± 145% to 157% ± 103%, P < 0.0001) and remained lower during ongoing support (vWF:Ag 265% ± 128%, P = 0.025; vWF:RCo 163% ± 94%, P < 0.0001). After termination of ECMO, vWF:Ag was greater than baseline (359% ± 131%, P = 0.004) and vWF:RCo was similar to baseline levels (338% ± 142%, P = 0.046). Compared with baseline, the calculated vWF:RCo/vWF:Ag ratio decreased after 24 hours on support (0.96 ± 0.23 to 0.61 ± 0.17, P ≤ 0.0001) and remained lower during 48 hours on ECMO (0.63 ± 0.18, P ≤ 0.0001). After termination of ECMO support (0.94 ± 0.19, P = 0.437), values rapidly returned to baseline. The number of HMW vWF multimers (n) decreased from baseline after 24 hours on ECMO (21 ± 1.4 to 14 ± 1.8, P ≤ 0.0001) and after 48 hours on ECMO (15 ± 2.1, P ≤ 0.0001). Twenty-four hours after termination of ECMO support, HMW vWF multimeric pattern had returned to baseline values (21 ± 1.8, P = 0.551). Loss of HMW vWF multimer bands occurred in patients undergoing ECMO support and resolved after the termination of ECMO. Although not detectable with coagulation screening tests, a vWF:RCo/vWF:Ag ratio <0.7 during ECMO was highly indicative for loss of HMW vWF multimers. Our findings may at least in part explain increased bleeding tendency during ECMO therapy. Administration of vWF concentrates may support restoration of primary hemostasis in patients with relevant bleeding during ECMO support.