Hematopoietic Cells from Pluripotent Stem Cells: Hope and Promise for the Treatment of Inherited Blood Disorders.

Inherited blood disorders comprise a large spectrum of diseases due to germline mutations in genes with key function in the hematopoietic system; they include immunodeficiencies, anemia or metabolic diseases. For most of them the only curative treatment is bone marrow transplantation, a procedure associated to severe complications; other therapies include red blood cell and platelet transfusions, which are dependent on donor availability. An alternative option is gene therapy, in which the wild-type form of the mutated gene is delivered into autologous hematopoietic stem cells using viral vectors. A more recent therapeutic perspective is gene correction through CRISPR/Cas9-mediated gene editing, that overcomes safety concerns due to insertional mutagenesis and allows correction of base substitutions in large size genes difficult to incorporate into vectors. However, applying this technique to genomic disorders caused by large gene deletions is challenging. Chromosomal transplantation has been proposed as a solution, using a universal source of wild-type chromosomes as donor, and induced pluripotent stem cells (iPSCs) as acceptor. One of the obstacles to be addressed for translating PSC research into clinical practice is the still unsatisfactory differentiation into transplantable hematopoietic stem or mature cells. We provide an overview of the recent progresses in this field and discuss challenges and potential of iPSC-based therapies for the treatment of inherited blood disorders.

Evaluation of Bleeding Phenotype of Inherited Factor VII Deficiency in Children With a Bleeding Assessment Tool and Global Assays.

INTRODUCTION: Inherited factor VII (FVII) deficiency is the most common of the rare bleeding disorders and shows a heterogenous distribution of bleeding phenotypes independent of factor activity level. The bleeding score (BS) evaluates the phenotype of patients with rare bleeding disorders. Thromboelastography (TEG) and thrombin generation assays (TGAs) are 2 methods to evaluate global hemostasis, and controversially both tests are useful for identifying different bleeding tendency phenotypes. The purpose of this study was to investigate the use of the BS and global assays (TEG and TGAs) to predict the bleeding phenotype of inherited FVII deficiency. MATERIALS AND METHODS: A total of 27 patients with FVII deficiency were evaluated with the BS and global hemostasis assays. RESULTS: The BS was compatible with disease severity according to the FVII activity level (P<0.05) but the BS and bleeding grade of patients did not show a statistically significant correlation with factor activity level (P>0.05). No significant correlation was observed between the factor activity level and any TEG parameter (P>0.05). The factor activity level was negatively correlated with the lag time of the TGA on the contrary positively correlated with the peak thrombin time of the TGA (P<0.05). CONCLUSIONS: The global assays do not successfully predict the bleeding phenotype. The BS is a more suitable tool than conventional and global assays for predicting the bleeding phenotype.

An Unexpectedly High Rate of Thrombophilia Disorders in Patients with Superficial Vein Thrombosis of the Lower Extremities.

BACKGROUND: Superficial vein thrombosis (SVT) is a common venous condition. Recent studies have shown that SVT is associated with high frequency of thromboembolic complications: from 22-37% for deep venous thrombosis and up to 33% for pulmonary embolism. Our goal was to assess the prevalence of major hereditary and acquired thrombophilic factors in patients with SVT. METHODS: Sixty-six patients presenting with primary SVT underwent evaluation for thrombophilia: molecular testing for the factor V Leiden and factor II G20210 A (prothrombin) mutations, protein C, protein S, antithrombin deficiency, presence of lupus anticoagulant, as well as anticardiolipin antibody titers. Patients aged less than 18 years, with confirmed deep vein thrombosis, and pregnant women were excluded. RESULTS: 95.5% were Caucasian, and 62.1% were female gender. Age ranged from 21-88 years. Molecular testing showed that 34.2% of patients were heterozygous for factor V Leiden, 23.6% were heterozygous for the factor II mutation, 7.8% had antithrombin deficiency, 2.6% had protein S deficiency, and 2.1% had protein C deficiency. CONCLUSIONS: Our study showed that hereditary and acquired thrombophilias are higher than previously expected and reported.

Paradoxical bleeding and thrombotic episodes of dysprothrombinaemia due to a homozygous Arg382His mutation.

We have characterised the pathogenic basis of dysprothrombinaemia in a patient exhibiting paradoxical bleeding and thrombotic defects during pregnancy and postpartum. Genetic analysis revealed that the proband is homozygous for the prothrombin Arg382His mutation, possessing only ~1 % clotting activity. The proband experienced severe bleeding episodes during her pregnancy, which required treatment with prothrombin complex concentrates, and then pulmonary embolism and deep-vein thrombosis at 28 days postpartum, which required treatment with LMWH and fresh frozen plasma. Analysis of haemostatic parameters revealed that the subject had elevated FDP and DD and decreased fibrinogen levels, indicating the presence of hyperfibrinolysis. Thrombin generation and clotting assays with the proband's plasma in the presence of soluble thrombomodulin and tissue-type plasminogen activator indicated a defect in activation of both protein C and thrombin activatable fibrinolysis inhibitor (TAFI). Unlike normal plasma, no TAFI activation could be detected in the patient's plasma. The expression and characterisation of recombinant prothrombin Arg382His indicated that zymogen activation by prothrombinase was markedly impaired and the activation of protein C and TAFI by thrombin-Arg382His was impaired 600-fold and 2500-fold, respectively. The recombinant thrombin mutant exhibited impaired catalytic activity toward both fibrinogen and PAR1 as determined by clotting and signalling assays. However, the mutant activated factor XI normally in both the absence and presence of polyphosphates. Arg382 is a key residue on (pro)exosite-1 of prothrombin and kinetic analysis of substrate activation suggested that the poor zymogenic activity of the mutant is due to its inability to bind factor Va in the prothrombinase complex.

Bleeding related to disturbed fibrinolysis.

The components and reactions of the fibrinolysis system are well understood. The pathway has fewer reactants and interactions than coagulation, but the generation of a complete quantitative model is complicated by the need to work at the solid-liquid interface of fibrin. Diagnostic tools to detect disease states due to malfunctions in the fibrinolysis pathway are also not so well developed as is the case with coagulation. However, there are clearly a number of inherited or acquired pathologies where hyperfibrinolysis is a serious, potentially life-threatening problem and a number of antifibrinolytc drugs are available to treat hyperfibrinolysis. These topics will be covered in the following review.

Detection of mild inherited disorders of blood coagulation: current options and personal recommendations.

Although assessment of prior personal and familial bleeding history is an important aspect of the diagnosis of bleeding disorders, patients with mild inherited bleeding disorders are sometimes clinically asymptomatic until presented with a hemostatic challenge. However, bleeding may occur after incursion of trauma or surgery, so detection of these conditions reflects an important facet of clinical and laboratory practice. Mild bleeding disorders may be detected as a result of family studies or following identification of abnormal values in first-line screening tests such as activated partial thromboplastin time, prothrombin time, fibrinogen and global platelet function screen testing, such as the platelet function analyzer. Following determination of abnormal screening tests, subsequent investigation should follow a systematic approach that targets specific diagnostic tests, and including factor assays, full platelet function assays and more extensive specialized hemostasis testing. The current report provides a personal overview on inherited disorders of blood coagulation and their detection.

Myocardial infarctions and other acute coronary syndromes in rare congenital bleeding disorders: a critical analysis of all reported cases.

OBJECTIVE: To investigate the occurrence of myocardial infarction or other acute coronary syndromes in rare congenital bleeding disorders. PATIENTS: All patients with factor I (FI), factor II (FII), factor V (FV), factor VII (FVII), factor X (FX), factor XI (FXI), or factor XIII (FXIII) deficiency or abnormality reported to have presented a myocardial infarction or another acute coronary syndrome were investigated. The condition had to be demonstrated by objective means, including a coronary/angiography. Cases of stable angina were excluded. RESULTS: A total of 53 patients (4 had FI, 2 had FV, 2 had FVII, 36 had FXI, 1 had FXIII deficiency, and 8 patients had platelet disorders) met the inclusion criteria . No patient with FII or FX deficiency and acute coronary disease met the inclusion criteria. In the majority of patients, common risk factors were present, namely hypertension, hypercholesterolemia, smoking, and diabetes. Replacement therapy was involved in 5 cases. CONCLUSION: The congenital hypocoagulability present in these patients was unable to allow a protection from acute coronary diseases. The significance of the findings is discussed.

A hereditary bleeding disorder resulting from a premature stop codon in thrombomodulin (p.Cys537Stop).

In this study, we describe a novel thrombomodulin (TM) mutation (c.1611C>A) that codes for a change from cysteine 537 to a premature stop codon (p.Cys537Stop). Three members of a family with a history of posttraumatic bleeding were identified to be heterozygous for this TM mutation. All coagulation screening tests, coagulation factor assays, and platelet function test results were within normal limits. However, the endogenous thrombin potential was markedly reduced at low-tissue factor concentration, and failure to correct with normal plasma indicated the presence of a coagulation inhibitor. Plasma TM levels were highly elevated (433-845 ng/ml, normal range 2-8 ng/ml, equating to 5 to 10 nM), and the addition of exogenous protein C further decreased thrombin generation. The mutation, p.Cys537Stop, results in a truncation within the carboxyl-terminal transmembrane helix. We predict that as a consequence of the truncation, the variant TM is shed from the endothelial surface into the blood plasma. This would promote systemic protein C activation and early cessation of thrombin generation within a developing hemostatic clot, thereby explaining the phenotype of posttraumatic bleeding observed within this family.

A self-controlled comparative clinical trial to explore the effectiveness of three topical hemostatic agents for stopping severe epistaxis in pediatrics with inherited coagulopathies.

OBJECTIVE: The aim of this study was to assess the effectiveness of localized treatments to persistently stop epistaxis in patients with inherited bleeding disorders. METHODS: In a self-controlled comparative clinical trial, to offer the best solution to stop epistaxis at home (within 10 minutes), patients with inherited bleeding disorders were treated using three different topical hemostatic agents, including Tranexamic acid impregnated tampon, EpiCell tampon prepared from oxidized regenerated cellulose pad, and ChitoHem tampon (reinforced with chitosan). The results of using these different products on three groups of randomly selected patients were ultimately compared using the χ(2) and Fisher's exact test statistics. RESULTS: A total of 31 patients, 5 females and 26 males with a mean age of 5.6 years, were included in the study. Twenty-three patients had Glanzmann disease, four had von-Willebrand disease, two had Bernard soulier syndrome, two had activated factor VII deficiency, and one patient had impaired secretion of adenosine deaminase. The study exhibited that statistically there was no significant difference between EpiCell tampon and Tranexamic acid impregnated tampon treatments with respect to the hemostasis duration. However, ChitoHem tampon was more efficient than Tranexamic acid impregnated tampon (P value <0.001) and EpiCell tampon (P value < 0.05). CONCLUSION: ChitoHem tampon, the chitosan-reinforced product, was the best therapy solution to stop epistaxis. We recommend further research on the use of other hemostatic agents for localized bleeding in patients with inherited bleeding disorders.

Desmopressin (DDAVP) in the management of patients with congenital bleeding disorders.

Bleeding disorders, including haemophilia, von Willebrand disease, and platelet function abnormalities pose a substantial, ongoing management challenge. Patients with these disorders not only require treatment during bleeding events but also need effective management strategies to prepare for events ranging from minor dental procedures to major surgery and childbirth. Moreover, women with bleeding disorders often require ongoing treatment to prevent menorrhagia during childbearing years. Desmopressin (DDAVP), a synthetic derivative of the antidiuretic hormone l-arginine vasopressin, has become a well-established tool for the management of patients with bleeding disorders in a variety of clinical settings. However, despite the widespread use of DDAVP, the available clinical evidence on its efficacy and safety in these settings is limited, and there has not been a recent comprehensive review of its role in the clinical management of patients with bleeding disorders. As such, this article provides a review of the mechanism of action and pharmacokinetic properties of DDAVP, followed by a concise summary of the available evidence for its use in the treatment and prevention of bleeding.

Upper gastrointestinal bleeding in a young patient with Budd Chiari syndrome due to a mutation of factor V Leiden: a case report.

Budd Chiari syndrome or hepatic venous outflow obstruction is a complex entity with multiple etiologies and various clinical manifestations. It is often difficult to establish the diagnosis. The most common cause is a hypercoagulable state due to either genetic disorders of blood coagulation or several acquired conditions such as hematological diseases, tumors, infections, chronic inflammatory diseases, pregnancy. The most common clinical presentation is hepatomegaly, abdominal pain and ascites, but the onset can also be dramatical and life threatening with upper digestive bleeding due to portal hypertension through postsinusoidal blockage. We report the case of a young patient with a coagulation disorder secondary to a mutation of factor V Leiden, who presented with upper digestive bleeding as the first manifestation of Budd Chiari syndrome and who also was associated with myocardial infarction in his past medical history.

Factor deficiencies in pregnancy.

Pregnancy, childbirth, and the puerperium are hemostatically challenging to women with bleeding disorders. This article provides general recommendations for the management of pregnant women with inherited coagulation disorders. Each factor deficiency is discussed, providing an up-to-date review of the literature and, where possible, guidance about how to manage patients throughout pregnancy, delivery, and the puerperium. The factor deficiencies covered are inherited abnormalities of fibrinogen; deficiencies of prothrombin, factor (F)V, FVII, FX, FXI, FXIII; combined deficiencies of FV and FVIII; and the inherited deficiency of vitamin K-dependent clotting factors. The management of carriers of hemophilia A and B is also discussed.

Acquired von Willebrand syndrome associated with hypothyroidism: a mild bleeding disorder to be further investigated.

Acquired von Willebrand syndrome (AVWS) is a rare bleeding disorder with laboratory findings similar to those for congenital von Willebrand disease (VWD). Unlike VWD, AVWS usually occurs in individuals with no personal or family history of bleeding. The prevalence of AVWS in the general population is unknown because data from large prospective studies of this syndrome are not available. Although AVWS is more frequently associated with lympho-myeloproliferative and cardiovascular disorders, it can also occur in solid tumors and in immunological and other miscellaneous conditions. Among these miscellaneous conditions, hypothyroidism has been associated with AVWS type 1 with a frequency of ~2 to 5%. In the 47 cases reported in the literature, the low VWF is apparently due to a reduction in its synthesis and/or secretion. Diagnosis of AVWS is based on assays measuring the level and activity of von Willebrand factor (VWF). These tend to be low, whereas factor VIII (FVIII) coagulant activity can be normal. In patients with AVWS associated with hypothyroidism, mucocutaneous bleeding episodes are the most frequent and can be managed with local therapy and/or systemic administrations of antifibrinolytic agents and desmopressin. VWF/FVIII concentrates have been used in only a few patients. The use of thyroid hormones can reverse this abnormality.

Extensive arterial thrombosis in a patient with factor V Leiden mutation.

Ascending aorta and aortic arch thrombosis is rare in a young man with no risk factor. Here, we report the case of a young male patient with factor V Leiden mutation who developed ascending aorta and aortic arch thrombosis and subsequent emboli.

Acute mesenteric and aortic thrombosis associated with antithrombin deficiency: a rare occurrence.

Antithrombin is a potent inhibitor of the coagulation cascade exerting its primary effects on activated factors X, IX and II. It is the mechanism by which heparin and low-molecular weight heparin cause anti-coagulation. Deficiency of antithrombin presents as a hypercoagulable state, and may result in unexplained deep venous thrombosis, arterial thrombosis and systemic embolization.

Deficiency of the human complement regulatory protein factor H associated with low levels of component C9.

We identified a 4-year-old Brazilian boy from a family of Japanese descent and history of consanguinity, who suffered from severe recurrent pneumonia. He carries factor H (FH) deficiency associated with reduced levels of component C9 and low serum levels of C3 and factor B. His mother also presented low levels of these proteins and factor I, while his father and sister had only lower levels of FH. Western blot assays confirmed the complete absence of FH and FHL-1 polypeptides in this patient. Sequencing of the proband's FH cDNA revealed a homozygous G453A substitution, encoding an Arg(127)His change. His mother, father and sister are heterozygous for this substitution. Despite the absence of FH in the plasma, this protein was detected in the patient's fibroblasts, suggesting that Arg(127) may be important for FH secretion. Low concentrations of C9 were detected in the proband serum but no mutations in the patient's C9 gene or promoter have been identified, suggesting that this is a consequence of uncontrolled complement activation and high C9 consumption.

Nonstroke arterial thrombosis in children: Hacettepe experience.

Of the 488 pediatric patients with thrombosis in a single center, 50 (10.2%) had nonstroke arterial thrombosis. Half of the nonstroke arterial thrombosis patients had a preceding arterial catheterization history. Infection was a common contributing underlying disorder among both catheterized and overall groups (28 and 42%, respectively). The third most common underlying risk factor for development of arterial thrombosis was associated cardiac disease, and 70% of these patients had congenital heart disease and 30% had dilated cardiomyopathy. The most common site of arterial thrombosis was the heart, and 22% of the patients developed arterial thrombosis following cardiac surgery for congenital heart diseases. The prothrombotic risk factors were also analyzed in the study group and factor V G1691A and PT G20210A heterozygous mutations were detected to be higher (16 and 4%, respectively) than those in healthy controls (7.4 and 2.3%, respectively). In addition to factor V G1691A and PT G20210A mutations, other prothrombotic risk factors including protein C and protein S deficiencies and elevated factor VIII levels were commonly associated with pediatric nonstroke arterial thrombosis cases in the present study. This finding indicates the importance of these factors in arterial thrombosis development, similar to venous thrombosis. Another striking factor of the study is the higher number of prothrombotic risk factors (more than three) in patients who had persistent thrombosis, indicating the need for more intensive treatment in these patients.

Mechanisms of the factor V Leiden paradox.

OBJECTIVE: Carriers of the factor V Leiden mutation (FVL-carriers) have a substantially increased risk of deep venous thrombosis (DVT), whereas the risk of pulmonary embolism (PE) is only mildly increased compared with noncarriers. So far few studies have investigated possible mechanisms for this so-called FVL paradox. METHODS AND RESULTS: Consecutive patients with a first DVT or PE were included in a large population-based case-control study (MEGA study). Patients, aged 18 to 70 years, provided a questionnaire, DNA (n=3313), or plasma (n=1474). Surgery, injury, and travel were considered thrombosis-provocative. Of 2063 patients with isolated DVT, 20% were FVL-carrier, as were 8% of the 885 patients with isolated PE. Among DVT patients, FVL-carriers had their thrombi more often proximal and a higher number of affected veins than noncarriers. No differences were observed between FVL-carriers and noncarriers in time between provocation and diagnosis, in vitro coagulation time, and thrombus density. Compared with patients with both DVT and PE, isolated DVT patients more often had thrombi located distally and had a similar number of affected veins. Compared with isolated PE patients, isolated DVT patients had a similar time between provocation and diagnosis, and similar in vitro coagulation time and thrombus density. CONCLUSIONS: Although some effects were differential for FVL-carriers and noncarriers, and some were differential for PE and DVT patients, none of the potential mechanisms offered a clear explanation.

Disturbances of coagulation in neonates with functionally univentricular physiology prior to the first stage of surgical reconstruction.

BACKGROUND: Altered levels of coagulation factors are reported in patients with functionally univentricular physiology before and following the second and third stages of reconstructive surgery. The aims of our study were to determine if such abnormalities are also present in newborns with this physiology prior to the first stage of surgical treatment. PATIENTS AND METHODS: We studied 20 neonates with functionally univentricular physiology admitted to the Children's Cardiac Centre in Slovakia, using 20 healthy neonates as age-matched controls. Demographic characteristics, and concentration of liver enzymes, serum albumin, and complete blood count, did not differ between the two groups. Concentrations of Factor II, V, VII, VIII, Protein C, Protein S and Antithrombin were compared between the groups, and assessed as variable factors for coagulation. RESULTS: In those with functionally univentricular physiology, procoagulation Factor II (p < 0.001), VII (p < 0.001), VIII (p < 0.01), anticoagulation Protein C (p < 0.001), Protein S (p < 0.001) and Antitrombin III (p < 0.001) all were present in significantly lower values compared with findings in the control group. D-dimer (p < 0.0001) and Fibrin Degradation Products (p < 0.0001) were present at significantly higher levels, but the concentration of plasminogen was significantly lower (p < 0.0001). The activated partial thromboplastin time (p < 0.012), and the prothrombin time (p < 0.0001), was significantly prolonged in those with functionally univentricular physiology compared with their controls. CONCLUSION: The presence of abnormal coagulation factors, markers of thrombolysis in the plasma, and increased risk of bleeding, suggests activation of haemostasis, and consumption of factors responsible for coagulation, in those with functionally univentricular physiology. The question arises whether the reported abnormalities are predictive of the known abnormalities of coagulation occurring during the second and third stages of surgical repair for patients with functionally univentricular hearts.

Thrombosis in children with cardiac pathology: analysis of acquired and inherited risk factors.

The present study was conducted to analyze the features and risk factors of childhood thrombotic events in patients with cardiac defect followed-up at our hospital. The clinical and laboratory findings of 59 patients diagnosed with cardiac defects and thromboses between 1997 and 2006 were retrospectively analyzed. Thirty-one children (52.5%) had venous system thromboses, 21 (35.6%) had arterial system thromboses, and seven (11.9%) had venous and arterial system thromboses. Presence of congenital heart disease and cardiomyopathy (CMP) were significant risk factors for developing intracardiac thrombosis. In addition, presence of congenital heart disease was the significant statistical risk factor for developing left atrium and right ventricle thromboses. Presence of congenital heart disease was a significant risk factor for developing a central nervous system thrombosis. Presence of pulmonary stenosis and aortic coarctation were significant risk factors for developing a peripheral arterial system thrombosis. Acquired risk factors including major surgery, angiography, central venous catheter, systemic infection, and hypoxia were identified in 49 of the 59 patients. Many patients had more than one of these acquired risk factors. Analysis of the relationship between thrombosis and type of major surgery demonstrated a statistically significant relationship between an intracardiac thrombosis and total correction of tetralogy of Fallot and a peripheral venous system thrombosis and a Blalock Taussig shunt. Twenty-three of the 52 patients (44.2%) had at least one thrombophilic mutation. Overall, a heterozygous factor V Leiden mutation was found in nine patients (17.3%), a methylenetetrahydrofolate reductase 677C-T mutation in 15 patients (28.8%), and a PT 20210G-A mutation in three patients (5.8%). Our data suggest that cardiac defects are common risk factors for developing a childhood thrombosis. The type of disorder determines the site of thrombosis. Acquired risk factors may contribute to the development of a thrombosis. The results of this study also indicate that to ensure early diagnosis, routine screening for thrombosis should be performed in patients with a cardiac defect and that screening for factor V Leiden and PT 20210G-A mutations and other genetic risk factors should be included when assessing all patients with cardiac defects who present with a thrombosis, whether or not a predisposing factor has been identified.