the Journal of Coagulation Disorders Vol 2 Issue 3

Construction and Expression of a New Modified Coagulation FVIII cDNA In NIH3T3, CHO, and HepG2 Cell Lines

Hemophilia A or factor VIII deficiency is a common X-linked genetic bleeding disorder in humans. The FVIII contains a domain sequence organization designated A1-A2-B-A3-C1-C2, which the B-domain is not necessary in coagulation activity. We constructed a new B-domain deleted FVIII cDNA and cloned it into the N-terminal His tagged expression vector via the Gateway technology. This vector transfected into three cell lines: NIH3T3, CHO, and HepG2. The rFVIII extracted was purified and detected with SDS PAGE and western blot using anti-His tag and anti-FVIII antibodies and the rFVIII activity was measured using the ST4 kit. The results showed high expression and activity in NIH3T3 and CHO cell lines. The B-domain containing glycosylation sites was removed in this construct. As a result of this diminished glycosylation heterogeneity, B-domain truncated variants of FVIII may also be a more suitable precursor for making well-characterized, long-acting FVIII variants.

A Case of Severe Disseminated Intravascular Coagulation During Pneumococcal Septic Shock

A 55-year-old man presented with a pneumococcal pneumonia that evolved into a septic shock. This pneumococcal infection was complicated by severe disseminated intravascular coagulation (DIC) leading to necrosis of several fingertips. The current management of DIC consists of treatment of the underlying disease. In the case of infection, this means fluid resuscitation and broad spectrum antibiotic therapy. In addition, the coagulation cascade is often disturbed by excessive activation and consumption of coagulation proteins and platelets. This requires supportive hemostatic therapy. Recently, the administration of activated protein C (APC) to support the impaired coagulation pathway during sepsis is studied in a number of randomized controlled trials. Although this drug is already approved by the US Food and Drug Administration and the European Community for treatment of patients with severe sepsis, in clinical practice the use of APC is very rare.

Risk of Venous and Arterial Thrombosis in HIV-infected Patients: A Narrative Review

With the increasing number of patients living with HIV-infection who are successfully treated with antiretroviral therapy (ART), mortality has declined drastically. Either through this prolonged survival or by the introduction of ART, other underlying diseases or risks for such diseases could become clinically relevant such as venous and arterial thrombosis. Prothrombotic abnormalities have consistently been noted amongst HIV-infected patients, who are at a 4- to 6-fold increased risk to develop venous or arterial thrombosis compared to the normal population. This risk is partially due to HIV-infection itself, as prothrombotic abnormalities and thrombotic risk increase when patients develop AIDS. The use of ART, and in particular the use of protease inhibitors (PI), seems to contribute to this risk as well. Nevertheless, even if ART contributes to this risk, this does not outweigh the benefits of ART (ie, reducing mortality and increasing AIDS-free survival). As interruption of ART seems to increase the risk of death from cardiovascular disease or any other cause, as compared with continuous ART use, this implies that once a patient starts with ART it should not be discontinued.

Tissue Factor Expression by Malignant Cells Contributes to Tumor Progression

Tissue factor (TF) is a transmembrane protein that binds its ligand factor VII/VIIa (FVII/FVIIa) and initiates the coagulation protease cascade. The TF is essential for hemostasis. In addition, TF has been implicated in several pathological processes, including venous thromboembolism, sepsis, inflammation, and cancer. Aside from their primary roles in coagulation, FVIIa and the downstream proteases FXa and FIIa (thrombin) can also cleave and activate protease-activated receptors (PARs) on cells to induce intracellular signaling. In this review, we will summarize the proposed mechanisms by which TF contributes to tumor progression. Specifically, we will focus on how TF expression by cancer cells and host cells promotes malignancy by increasing angiogenesis, tumor growth, and metastasis.

Incidence and Risk Factors of Severe Obstetric Hemorrhage

Severe obstetric hemorrhage is the main cause of severe maternal morbidity and mortality worldwide. The incidence of severe hemorrhage varies considerably, even among countries with high resource settings. Uterine atony is the main cause of severe obstetric hemorrhage, followed by retained placental tissues and trauma of the genital tract. Preexisting or acquired coagulopathy accounted for almost 1% of severe obstetric hemorrhage. The mode of delivery is the most important risk factor for severe obstetric hemorrhage, especially an emergency cesarean section (CS), followed by elective CS. Other important risk factors included multiple pregnancy, von Willebrand's disease, HELLP syndrome, anemia, macrosomia, cardiac disease, chorioamnionitis, induction, prolonged labor, and older age. Mothers with a history of unexplained severe obstetric hemorrhage should be investigated for preexisting or acquired bleeding disorders, and should receive conjoined care by a hematologist, obstetrician, and anesthesiologist during pregnancy and labor as well as in the postpartum period. Severe obstetric hemorrhage is a relatively frequent complication. Increasing knowledge of risk factors may contribute to reducing its incidence and impact. The findings call for reviewing labor management, as well as screening for different bleeding disorders.

The Changing Coagulation Profile of Females: can Ultrastructure of Fibrin Teach Us Something?

Estrogen plays an important role during the life of a healthy female and impacts on the coagulation profile. Products that impact on the hemostatic health of women include contraception, as well as using hormone replacement later in life. Also, lifestyle habits like smoking are known to have an impact on coagulation and hemostasis. Because hemostasis and estrogen are so closely linked to these lifestyle choices of women, it is suggested that an ultrastructure of fibrin networks may provide relevant insight into the changing coagulation profile of females. In the current research, the fibrin network in females, under different conditions, is studied using a scanning electron microscope. Control female profiles are compared with pregnancy, contraceptive use, hormone replacement, females who smoke, as well as females during old age. Here, we argue that the changes in the different stages of womanhood have a direct impact on the health and possibly thrombotic status in women. It is concluded that information provided by ultrastructural analysis during routine clinical history taking, might provide useful information regarding the most appropriate management or regime, particularly for women.

An Approach to the Diagnosis of Mild and Moderate Bleeding Disorders in Children

Most cases of severe inherited defects of hemostasis will be diagnosed early in life, however, moderate and mild bleeding disorders, either coagulation factor deficiencies or disorders of primary hemostasis, may not be detected. Children presenting with mild bleeding symptoms can pose a significant diagnostic challenge, as the bleeding symptoms with which they present, such as recurrent epistaxis or bruising, are frequent among healthy children. Thus, the clinical distinction between normal children and those with mild/moderate bleeding disorders can be complex. The first step in the diagnosis of bleeding disorders, therefore, is the identification of whether a child has significant bleeding symptoms. Useful in this regard is a recently described, validated, standardized pediatric bleeding questionnaire. Identification of a family history of bleeding is also important, and the severity of bleeding symptoms of family members could be quantitated with a validated, standardized bleeding questionnaire. If a bleeding disorder is suspected based on a personal and/or family bleeding history, specific laboratory testing is needed for attaining a diagnosis. The screening tests for coagulation factor deficiencies are considered reliable and are routinely available. However, for primary hemostatic defects, the various screening tests have not yet proven to be reliable and referral for a specialized hematology consultation and specific laboratory testing is needed. Perhaps a Bayes theorem approach, combining the personal and family bleeding history with the results of a screening test for a primary hemostatic defect would prove to be useful in the identification of those children who will most benefit from additional testing.

Bernard Soulier Syndrome: A Rare Bleeding Disorder with a Wide Range of Genetic Defects

Bernard Soulier Syndrome (BSS) is a rare inherited bleeding disorder caused by a quantitative or qualitative defect in glycoprotein (GP)Ib-IX-V complex, the receptor for the von Willebrand factor (vWF). This complex plays a major role in platelet adhesion at sites of vascular injury through binding to vWF. It is also involved in the thrombin induced platelet activation. BSS is usually inherited in an autosomal recessive manner and is characterized by a prolonged bleeding time, thrombocytopenia, and giant platelets. Clinical manifestations include a series of purpura, epistaxis, menorrhagia, gingival, and gastric bleeding. Diagnosis is based on prolonged skin bleeding time, macrothrombocytopenia, defective ristocetin-induced platelet aggregation, and the exploration of GPIb-IX-V complex expression. The complex is composed of four subunits: GPIbα, GPIbβ, GPIX, and GPV, but the principal candidate genes for this disease are GPIbα, GPIbβ, and GPIX. These genes are unique in the genome and share the scarcity of introns with one or two introns per gene. In this review, we give an overview of the mutations occurring in the candidate genes with an emphasis on compound heterozygous mutations as well as on founder mutations.

Decreased Bone Mineral Density in Adult Patients with Hemophilia: Clinical Significance, Prevalence and Risk Factors

Osteoporosis is widely recognized as a major health problem, and considerable resources are spent screening, diagnosing, and treating this disease in postmenopausal women. Adults with hemophilia have unique factors that place them at an increased risk for developing osteoporosis including periods of immobilization after bleeds, impaired joint mobility that may limit weight bearing physical activity, and infection with Hepatitis C virus (HCV) and/or human immunodeficiency virus (HIV). Several publications report the prevalence of osteoporosis in adults with hemophilia to be in the range of 25% and 50%. The severity of hemophilic joint arthropathy appears to be the strongest predictor of significant bone loss in patients with hemophilia. The relative contribution of other risk factors, including HIV and HCV infection are less well defined. The clinical significance of decreased bone mineral density as measured by dual energy X-ray absorptiometry (DXA) to fracture risk in men, and in particular younger men, is also unclear. We recommend screening adult patients with hemophilia with documented fragility fractures, in those with severe joint arthropathy, and in those patients >50 years of age with additional risk factors such as HIV/HCV infection or low BMI. Adequate vitamin D and calcium intake, weight bearing physical activity, and interventions to maintain joint mobility are recommended for all hemophilia patients. The role of pharmacologic agents, including bisphosphonates, is untested, and they should be reserved for those with documented osteoporosis and fragility fractures or additional risk factors that place them at high risk for developing fragility fractures.

Role and Relevance of Factor VIII Gene Haplotyping for the Indirect Genetic Analysis of Carrier Diagnosis in Hemophilia

Hemophilia A is an X-linked recessive coagulation disorder because of the quantitative or qualitative deficiency of blood coagulation factor VIII (FVIII). Molecular genetic analysis; that is, direct mutation detection and linkage analysis of restriction markers, contribute effectively for the carrier detection in hemophilia. Due to the large size of FVIII gene (∼186 Kb), direct mutation analysis is not handy. Therefore, linkage analysis using informative markers has been considered as the pragmatic approach where the propositus, his heterozygous mother, and suspected carrier are available. For the better assessment of carriership, haplotypes of various genomic markers have obvious advantage over the determination of allelic frequencies of individual polymorphisms. This review examines the existing repertoire of useful markers, current advancements and trail along the feasibility of using haplotypes and linkage disequilibrium strategies for the indirect genetic analysis in the direction of efficient carrier testing.

Current Practice in Clinical Thrombosis and Hemostasis

Social Survey of Cases With Hemophilia A and Their Families in North India and Their Attitudes to Carrier Testing

ORIGINAL ARTICLE

Syed Tasleem Raza^1, Nuzhat J Fareedi² and Nuzhat Husain²

Affiliations: ¹Department of Biotechnology, Manipal University Dubai Campus, Block-7 Academic City Dubai, UAE and ²Genetics Lab, Department of Pathology, CSM Medical University, Lucknow 226003, Uttar Pradesh, India

ABSTRACT

OBJECTIVES
Implementation of a DNA-based carrier testing for the prevention of genetic diseases in a population is targeted toward technology development, personnel training, and technique perfection. In the current study we had focused on a group of cases with hemophilia A and their relatives at risk from North India who were being subjected to counseling for a carrier diagnosis.

METHODS
A cross-sectional study was conducted. The data was collected from different Chapters of the Hemophilia Federation of India (HFI) including Lucknow, Jammu, Ahmedabad, Baroda, Surat, and Rajkot. The study population was divided into two groups. Group 1, 60 cases with hemophilia A (Table 1) and Group 2, family members including relatives at risk. Group 2 comprises 199 females and 74 males (Table 2).

RESULTS
About 73% of patients had discussed hemophilia A inheritance with someone in the past. A majority of patients’ (48%) belief that, by chance, relatives could have a child with hemophilia were medium. Nearly 71% had discussed carrier testing with relatives in the past and over 90% realized the obligation to inform relatives about the availability of genetic testing. About 88% of cases considered hemophilia A as a severe or very severe disease. Only 25% of the patients felt that queries from relatives were an invasion of their privacy. Most of the relatives were married (100% in males and 73% in females). The majority (58%) of relatives has household income of less than 25,000 INR per annum.

CONCLUSION
It appears that this participation helped in increased awareness as well as acceptance of the disease, generated confidence in patients and families, and developed an open approach to newer modalities of treatment and prevention.

Keywords: hemophilia A, social awareness, genetic counseling, carrier diagnosis

Correspondence: Syed Tasleem Raza, Department of Biotechnology, Manipal University Dubai Campus, PO Box 345050, Dubai, UAE. Tel: +971 4 4291214; Fax: +971 4 522 3694541; e-mail: tasleem24@gmail.com

Pulmonary Complications Caused by Acute Leukemia

REVIEW ARTICLE

Kazuhiko Kakihana, Kazuteru Ohashi and Hideki Akiyama

Affiliation: Hematology Division, Tokyo Metropolitan Cancer and Infectious Diseases Center, Komagome Hospital, Tokyo, Japan

ABSTRACT

Infectious and noninfectious pulmonary complications represent a critical problem for patients with leukemia, which itself can be the direct cause of pulmonary leukostasis, pulmonary leukemic infiltration (PLI), and leukemic cell lysis pneumopathy. These disorders are usually more frequent in patients with hyperleukocytic leukemia. Pulmonary leukostasis is characterized by occlusion of the pulmonary capillaries and arterioles by leukemic cells. Neurological signs can also simultaneously arise if the central nervous system is affected. The number of circulating leukemic cells as well as interactions between leukemic and epithelial cells play important roles in the progression of leukostasis. Although PLI is the most common form of pulmonary involvement, antemortem diagnosis is very difficult. High resolution computed tomography is a useful, noninvasive tool that can identify PLI as abnormal shadows distributed along the bronchovascular bundle. Transbronchoscopic biopsy can help to correctly diagnose patients who undergo stem cell transplantation, as graft versus host disease renders such diagnosis far more difficult. Leukemic cell lysis pneumopathy is characterized by acute respiratory failure relatively early during the course of chemotherapy. Some enzymatic proteins released by degenerate leukemic cells might be potential agents involved in the development of pneumopathy. This report reviews the clinical manifestations of pulmonary complications associated with leukemia.

Keywords: pulmonary leukostasis, pulmonary leukemic infiltration, leukemic cell lysis pneumopathy, hyperleukocytosis

Correspondence: Kazuhiko Kakihana, Hematology Division, Tokyo Metropolitan Cancer and Infectious Disease Center, Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8677, Japan. Tel: +81 3823 2101; Fax: +81 3 3824 1552; e-mail: kakihana@cick.jp