DNA Polymorphisms in Pregnant Women with Sticky Platelet Syndrome.

Sticky platelet syndrome (SPS) is a thrombophilia caused by the increased aggregability of platelets in response to the addition of low concentrations of epinephrine (EPI) and/or adenosine diphosphate (ADP). Some of the single nucleotide polymorphisms (SNP), alleles and haplotypes of platelet glycoprotein receptors were proved to have a role in the etiology of thrombotic episodes When comparing SPS and the control group, in VEGFA rs3025039, the p value for both CC vs. TT and CT vs. TT analyses was <0.001. Interestingly, no minor TT genotype was present in the SPS group, suggesting the thrombotic pathogenesis of recurrent spontaneous abortions (RSA) in these patients. Moreover, we found a significant difference in the presence of AT containing a risky A allele and TT genotype of ALPP rs13026692 (p = 0.034) in SPS patients when compared with the controls. Additionally, we detected a decreased frequency of the GG (CC) genotype of FOXP3 rs3761548 in patients with SPS and RSA when compared with the control group (p value for the CC (GG) vs. AA (TT) 0.021). This might indicate an evolutionary protective mechanism of the A (T) allele in the SPS group against thrombotic complications in pregnancy. These results can be used for antithrombotic management in such pregnant patients.

Heterogeneity of Genotype-Phenotype in Congenital Hypofibrinogenemia-A Review of Case Reports Associated with Bleeding and Thrombosis.

Congenital fibrinogen disorders are diseases associated with a bleeding tendency; however, there are also reports of thrombotic events. Fibrinogen plays a role in the pathogenesis of thrombosis due to altered plasma concentrations or modifications to fibrinogen's structural properties, which affect clot permeability, resistance to lysis, and its stiffness. Several distinct types of genetic change and pathogenetic mechanism have been described in patients with bleeding and a thrombotic phenotype, including mutations affecting synthesis or processing in three fibrinogen genes. In this paper, we focused on familial hypofibrinogenemia, a rare inherited quantitative fibrinogen disorder characterized by decreased fibrinogen levels with a high phenotypic heterogeneity. To begin, we briefly review the basic information regarding fibrinogen's structure, its function, and the clinical consequences of low fibrinogen levels. Thereafter, we introduce 15 case reports with various gene mutations derived from the fibrinogen mutation database GFHT (French Study Group on Hemostasis and Thrombosis), which are associated with congenital hypofibrinogenemia with both bleeding and thrombosis. Predicting clinical presentations based on genotype data is difficult. Genotype-phenotype correlations would be of help to better understand the pathologic properties of this rare disease and to provide a valuable tool for the identification of patients who are not only at risk of bleeding, but also at risk of a thrombotic event.

How can Secondary Thromboprophylaxis in High-Risk Pregnant Patients be Improved?

Low-molecular-weight heparin (LMWH) is suggested for thromboprophylaxis in pregnant women with previous venous thromboembolism (VTE). Anyway, there is only limited amount of studies evaluating the effect of LMWH on hemostatic parameters during pregnancy of patients with previous VTE and the need of secondary thromboprophylaxis. We therefore provide results of prospective and longitudinal assessment of changes in hemostasis in high-risk pregnant women at four times during pregnancy (T1-T4) and one time after the postpartum period (T5) used for individualized modification of thromboprophylaxis. In this study, the results of coagulation factor VIII (FVIII) and protein S (PS) activity, ProC Global ratio and anti-Xa activity were evaluated. Despite the thromboprophylaxis, an increased predisposition to thromboembolic complications was detected (significant increase in FVIII activity and decrease in PS function, ProC Global ratio not normalized even after the postpartum period - p < .0001 between controls and T5 for PS and ProC Global). These results indicate that hemostasis may not be restored even 6 to 8 weeks after delivery and pose the question when is it safe to withdraw the anticoagulant thromboprophylaxis in high-risk patients with prior VTE.

Congenital Afibrinogenemia and Hypofibrinogenemia: Laboratory and Genetic Testing in Rare Bleeding Disorders with Life-Threatening Clinical Manifestations and Challenging Management.

Congenital fibrinogen disorders are rare pathologies of the hemostasis, comprising quantitative (afibrinogenemia, hypofibrinogenemia) and qualitative (dysfibrinogenemia and hypodysfibrinogenemia) disorders. The clinical phenotype is highly heterogeneous, being associated with bleeding, thrombosis, or absence of symptoms. Afibrinogenemia and hypofibrinogenemia are the consequence of mutations in the homozygous, heterozygous, or compound heterozygous state in one of three genes encoding the fibrinogen chains, which can affect the synthesis, assembly, intracellular processing, stability, or secretion of fibrinogen. In addition to standard coagulation tests depending on the formation of fibrin, diagnostics also includes global coagulation assays, which are effective in monitoring the management of replacement therapy. Genetic testing is a key point for confirming the clinical diagnosis. The identification of the precise genetic mutations of congenital fibrinogen disorders is of value to permit early testing of other at risk persons and better understand the correlation between clinical phenotype and genotype. Management of patients with afibrinogenemia is particularly challenging since there are no data from evidence-based medicine studies. Fibrinogen concentrate is used to treat bleeding, whereas for the treatment of thrombotic complications, administered low-molecular-weight heparin is most often. This review deals with updated information about afibrinogenemia and hypofibrinogenemia, contributing to the early diagnosis and effective treatment of these disorders.

Multimer Analysis of Von Willebrand Factor in Von Willebrand Disease with a Hydrasys Semi-Automatic Analyzer-Single-Center Experience.

von Willebrand disease (VWD) is reportedly the most common inherited bleeding disorder. This disorder develops as a result of defects and/or deficiency of the plasma protein von Willebrand factor (VWF). Laboratory testing for VWF-related disorders requires the assessment of both VWF level and VWF activity, the latter requiring multiple assays. As an additional step, an evaluation of VWF structural features by multimer analysis is useful in selective investigations. Multimer analysis is also important for the selection of a suitable VWF therapy preparation (desmopressin, VWF/FVIII concentrate, recombinant VWF) and the determination of the correct dose for the patient. Based on clinical and laboratory findings, including the analysis of VWF multimers, we classified our patients into individual types of VWD. Our study group included 58 patients. The study group consisted of 66% (38 patients) with VWD type 1, 5% (3 patients) with VWD type 2, 7% (4 patients) with VWD type 3, 5% (3 patients) with mixed type 1/2A VWD, and 17% (10 patients) comprising an unclassified group. In this article, we provide an overview of our practical experience using a new complementary method-the analysis of von Willebrand factor multimers with a semi-automatic analyzer Hydrasys 2 scan. We explain the principle, procedure, advantages, and pitfalls associated with the introduction of the VWF multimer analysis methodology into standard VWD diagnostics.

How Can Rotational Thromboelastometry as a Point-of-Care Method Be Useful for the Management of Secondary Thromboprophylaxis in High-Risk Pregnant Patients?

Thromboprophylaxis with low-molecular-weight heparin (LMWH) for patients with a history of venous thromboembolism (VTE) is suggested. Rotational thromboelastometry (ROTEM®) represents an innovative point-of-care method enabling the complex and quick evaluation of hemostasis. However, there are only episodic cases of its use for hemostasis assessment and guidance of LMWH in pregnancy. Therefore, we provide the results of unique prospective and longitudinal monitoring of hemostasis in high-risk pregnant women, which we used for the individualized optimalization of secondary thromboprophylaxis. According to the shortening of clot formation time (CFT) in EXTEM (p = 0.0007 from the 26th gestational week vs. controls) and INTEM (p = 0.002 from the 35th gestational week), increase in alpha angle (AA) in EXTEM, INTEM, and HEPTEM, and the persistence of increase in maximum clot firmness (MCF) in EXTEM, INTEM, and HEPTEM (p < 0.001 from the 26th and 35th gestational week vs. controls for EXTEM and INTEM, p = 0.0012 from the 26th gestational week in HEPTEM), LMWH dose was modified. Even after the postpartum period, AA in EXTEM was steeper than in controls (p = 0.0007), indicating that hemostasis is not fully normalized after 6-8 weeks following delivery. Therefore, ROTEM may be a useful tool for the individual evaluation of the termination of anticoagulant thromboprophylaxis.

A Novel Nonsense Mutation in FGB (c.1421G>A; p.Trp474Ter) in the Beta Chain of Fibrinogen Causing Hypofibrinogenemia with Bleeding Phenotype.

Congenital hypofibrinogenemia is a rare bleeding disorder characterized by a proportional decrease of functional and antigenic fibrinogen levels. Hypofibrinogenemia can be considered the phenotypic expression of heterozygous loss of function mutations occurring within one of the three fibrinogen genes (FGA, FGB, and FGG). Clinical manifestations are highly variable; most patients are usually asymptomatic, but may appear with mild to severe bleeding or thrombotic complications. We have sequenced all exons of the FGA, FGB, and FGG genes using the DNA isolated from the peripheral blood in two unrelated probands with mild hypofibrinogenemia. Coagulation screening, global hemostasis, and functional analysis tests were performed. Molecular modeling was used to predict the defect of synthesis and structural changes of the identified mutation. DNA sequencing revealed a novel heterozygous variant c.1421G>A in exon 8 of the FGB gene encoding a Bß chain (p.Trp474Ter) in both patients. Clinical data from patients showed bleeding episodes. Protein modelling confirmed changes in the secondary structure of the molecule, with the loss of three ß sheet arrangements. As expected by the low fibrinogen levels, turbidity analyses showed a reduced fibrin polymerisation and imaging difference in thickness fibrin fibers. We have to emphasize that our patients have a quantitative fibrinogen disorder; therefore, the reduced function is due to the reduced concentration of fibrinogen, since the Bß chains carrying the mutation predicted to be retained inside the cell. The study of fibrinogen molecules using protein modelling may help us to understand causality and effect of novel genetic mutations.

Comparison of clinical phenotype with genetic and laboratory results in 31 patients with congenital dysfibrinogenemia in northern Slovakia.

Congenital dysfibrinogenemia (CD) is a rare disorder of hemostasis. The majority of cases are caused by heterozygous missense mutations in one of the three fibrinogen genes. Patients with CD may experience bleeding and thrombosis, but many are asymptomatic. To better describe the clinical, laboratory, and genotypic picture of CD, we evaluated 31 patients from seven unrelated families using standard coagulation tests and genetic analysis. The clinical phenotype consisted of bleeding in 13/31 (42%) patients; other patients (18/31; 58%) were asymptomatic. Among patients with bleeding, symptoms were mostly in single anatomical sites, with variable intensity of bleeding. Compared to results from a previous large systematic survey, our results showed a similar mean bleeding score, but a higher incidence of bleeding episodes without thrombotic complications. In the present study, we identified three known pathogenic point mutations in the FGA (c.95G > A, c.104G > A) and FGB (c.586C > T) genes. The variants of CD identified in this cross-sectional study were either asymptomatic or had bleeding manifestations and showed similar laboratory features, irrespective of genotype. Results from genetic and clinical studies will continue to yield valuable information on the structure and function of the fibrinogen molecule.

Genetic Background of von Willebrand Disease: History, Current State, and Future Perspectives.

Sequencing of the gene encoding for von Willebrand factor (VWF) has brought new insight into the physiology of VWF as well as its pathophysiology in the context of von Willebrand disease (VWD). Molecular testing in VWD patients has shown high variability in the overall genetic background of this condition. Almost 600 mutations and many disease-causing mechanisms have been described in the 35 years since the VWF gene was identified. Genetic testing in VWD patients is now available in many centers as a part of the VWD diagnostic algorithm. Molecular mechanisms leading to types 2 and 3 VWD are well characterized; thus, information from genetic analysis in these VWD types may be beneficial for their correct classification. However, the molecular basis of type 1 VWD is still not fully elucidated and most likely represents a multifactorial disorder reflecting a combined impact of environmental and genetic factors within and outside of VWF. Regarding sequencing methods, the previous gold-standard Sanger sequencing is gradually being replaced with next-generation sequencing methods that are more cost- and time-effective. Instead of gene-by-gene approaches, gene panels of genes for coagulation factors and related proteins have recently become a center of attention in patients with inherited bleeding disorders, especially because a high proportion of VWD patients, mainly those with low VWF plasma levels (type 1), appear to be free of mutations in VWF. Whole-exome sequencing (WES) and whole-genome sequencing (WGS) are accessible in a very limited number of laboratories. Results from these studies have presented several genes other than VWF or ABO possibly affecting VWF levels, and such findings will need further validation studies.

Apixaban - Metabolism, Pharmacologic Properties and Drug Interactions.

BACKGROUND: Apixaban is an oral, potent, highly selective, reversible and direct inhibitor of activated coagulation factor X, that is the end point of the intrinsic and extrinsic coagulation pathway. Additionally, apixaban has the capacity to indirectly inhibit thrombin-induced platelet aggregation. This new oral anticoagulant represents an immediate-release form of peroral drug with quick dissolution, linear pharmacokinetics, good bioavailability and rapid onset and offset of action. No clinically relevant age- or sex-dependent difference in the apixaban pharmacokinetics and pharmacodynamics which would lead to the modification of the dose exists, apixaban may even be administered with or without food. Its elimination is mediated by metabolism, renal elimination of unmodified drug and excretion in the gastrointestinal tract. OBJECTIVE: The authors aim to provide a review of currently available literature about apixaban. METHOD: The authors summed-up the data from the scientific journals related to thrombosis and hemostasis and searched the available databases. RESULTS AND CONCLUSION: Apixaban has many advantages including predictable pharmacokinetics and pharmacodynamics, low number of drug and food interactions, and relatively wide therapeutic window.

Congenital afibrinogenemia: from etiopathogenesis to challenging clinical management.

INTRODUCTION: Congenital afibrinogenemia belongs to the group of autosomal recessive bleeding disorders and represents the absolute deficiency of fibrinogen detected by an antigenic test. This can lead to severe clinical manifestations of the disorder. Therefore, it is very important to take afibrinogenemia into account in the process of the differential diagnostics of the patients. AREAS COVERED: The authors provide a summary of currently available literature about afibrinogenemia. They collected the information from the scientific journals dedicated to thrombosis and hemostasis and searched world-wide databases. Expert commentary: The most frequent clinical manifestation of this disorder is mucosal bleeding, but musculoskeletal bleeding pattern, gynecologic and obstetric issues, spontaneous bleeding, episodes provoked by minor injury or any other intervention, and even paradoxical thromboembolic events have been published. Afibrinogenemia is the consequence of mutations of the homozygous or compound heterozygous type in gene FGA, FGB or FGG encoding fibrinogen. Pregnant women with a family history, or with a history of consanguinity ought to be properly counselled. However, primary prophylaxis of bleeding events is not suggested. The article deals with actual information about afibrinogenemia contributing to its early diagnosis and effective treatment, which in many cases requires multidisciplinary approach.

Monitoring of Hemostasis and Management of Anticoagulant Thromboprophylaxis in Pregnant Women with Increased Risk of Fetal Loss.

Physiological prothrombotic changes during pregnancy and the postpartum period, along with other preexisting maternal risk factors, increase the risk of both venous thromboembolism (VTE) and adverse pregnancy outcomes. Pregnancy complications that develop due to placental insufficiency as a result of inappropriate activation of coagulation are present in more than 5% of pregnancies and can contribute to significant maternal morbidity and mortality. Therefore, anticoagulant prophylaxis in women with congenital and acquired thrombophilic conditions should be actively considered. According to the Guidelines of American College of Chest Physicians, the use of low-molecular-weight heparin is suggested for prophylaxis of VTE and pregnancy complications in high-risk pregnant women. However, personalized refinements of such thromboprophylaxis remains unspecified, despite the necessity of better targeted recommendations for life-threatening conditions. We, therefore, review the possibilities of longitudinal monitoring and comprehensive assessment of changes in hemostasis in the group of high-risk pregnant women, which can then be used for early prediction and individualization of the optimal anticoagulant thromboprophylaxis of pregnancy complications. Simultaneously, we present our single-center experience with such monitoring and our first series of results.

Sticky platelet syndrome: an important cause of life-threatening thrombotic complications.

Sticky platelet syndrome (SPS) is a prothrombotic thrombocytopathy with familial occurrence, characterized by hyperaggregability of platelets in response to adenosine diphosphate (ADP), epinephrine (EPI) or both. The syndrome has been identified in approximately 21% of unexplained arterial thrombotic episodes, regarded to be the most common thrombophilia in arterial thrombosis and 13.2% of unexplained venous thromboembolism (VTE). The relatively young age at the first manifestation, relation to fertility and pregnancy, seriousness of the symptoms, easy and effective management of the disorder indicate to the necessity to take it into account in the differential diagnosis of the underlying cause of the thrombotic event. As the various localizations of the thrombosis in SPS have been reported, its management often requires a multidisciplinary approach. This review deals with the clinical aspects of thrombophilia, its etiopathogenesis, diagnosis as well as novel advances in the treatment and outlines the challenges for the further research.

Monitoring of dabigatran therapy using Hemoclot(®) Thrombin Inhibitor assay in patients with atrial fibrillation.

Dabigatran, a new direct thrombin inhibitor, achieves strong anticoagulation that is more predictable than warfarin. Nevertheless, a patient on dabigatran therapy (DT) may suffer from thrombotic or bleeding events. The routine monitoring of DT is not recommended, and standard coagulation tests are not sensitive enough for the assessment of DT activity. The aim of this study was to examine the clinical usefulness of the Hemoclot(®) Thrombin Inhibitor (HTI) assay in the assessment of dabigatran plasma levels in patients with non-valvular AF. Nineteen patients (12 men, 7 women) on DT were included in this preliminary prospective observational study. Dabigatran was administrated twice daily in a two dose regimens: 150 mg (5 patients) and 110 mg (14 patients). Blood samples were taken for the assessment of trough and peak levels of dabigatran. Dabigatran concentrations were measured with the HTI assay. The average dabigatran trough level was 69.3 ± 55.5 ng/ml and the average dabigatran peak level was 112.7 ± 66.6 ng/ml. The dabigatran trough plasma concentration was in the established reference range in 15 patients and the dabigatran peak plasma concentration was in the established reference range in 9 patients, respectively. Despite the fact that the activated partial thromboplastin and thrombin times were generally changed (prolonged), these tests failed to identify the patients with too low or too high dabigatran concentrations. The study confirmed the high sensitivity of the HTI assay for the assessment of dabigatran plasma levels. When compared to standard coagulation tests, the HTI is a more suitable assay for the monitoring of patients treated with dabigatran. Monitoring of DT may be beneficial in selected patients; however, further studies will be needed for the final clarification of this issue.