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Thrombophilic markers in patients with congenital bleeding disorders.

We have studied the presence or absence of thrombophilic markers in 59 patients with congenital bleeding disorders. Theoretically, the coexistence of coagulation factor deficiency and thrombophilia in patients with haemophilia may cause the bleeding tendency to decline. In 8 cases we have found thrombophilic marker positivity, in 7 patients APC resistance and in 1 patient protein S deficiency has been revealed. The presence of thrombophilic markers did not affect the frequency of bleeding episodes or the development of haemophilic arthropathy. The factor concentrate utilization, however, was lower in the group carrying thrombophilic markers, which indicates that the bleeding episodes were less severe. Relying on the strength of these findings, the medicinal influencing of inhibitor mechanisms may present itself as a potential therapy.

Activated Protein C Resistance↗

Proteins invoked by vitamin K absence and clotting times in clinically ill cats.

The clinical utility of the Thrombotest, a method for determining the prothrombin time that is uniquely sensitive to the presence of proteins invoked by vitamin K absence (PIVKA), was prospectively evaluated and compared to routine coagulation tests in cats with clinically suspected bleeding tendencies. Abnormal PIVKA clotting values were determined by comparison to results of a concurrently evaluated pooled feline plasma sample and by use of an absolute cutoff value of 25.2 seconds. To be recognized as abnormal, PIVKA clotting values had to be >20% of the pooled feline plasma PIVKA clotting time (the "20% rule") or > or =25.2 seconds (mean + 2 standard deviations of 150 different pooled feline plasma samples). Among the disorders in the population examined were 74 cats with liver disease and 19 cats with severe inflammatory bowel disease. Overall, a prolonged PIVKA clotting time based on the 25.2-second cutoff was found in 39.3% of cats, and based on the 20% rule in 40.7% of cats. An abnormal prothrombin time (PT) developed in 5.8% of cats, an abnormal APTT in 14% of cats, subnormal fibrinogen in 8.8% of cats, and thrombocytopenia in 3.3% of cats. Bleeding tendencies were confirmed in 22 cats, of which abnormal PIVKA clotting times were recognized in 95.5%, abnormal PT in 21%, abnormal activated partial thromboplastin time in 25%, hypofibrinogenemia in 16.7%, and thrombocytopenia in 4.5%. Response to treatment with vitamin K was demonstrated in 21 of 24 cats with an abnormal PIVKA clotting time. In these cats, an abnormal PIVKA clotting time normalized within 3 to 5 days of parenteral vitamin K administration. Cats responding to vitamin K administration had hepatic lipidosis (n = 7), severe inflammatory bowel disease (n = 4), severe inflammatory bowel disease associated with cholangiohepatitis (n = 5), and miscellaneous disorders (n = 5). Using either endpoint, the PIVKA clotting time is more sensitive for the detection of cats with coagulopathies than routinely used coagulation assessments in our hospital. Our findings confirm that cats with hepatic lipidosis, severe cholangiohepatitis, and severe inflammatory bowel disease develop coagulopathies responsive to vitamin K administration.

Animals↗

Valproate-associated coagulopathies are frequent and variable in children.

PURPOSE: Valproic acid (VPA) is an antiepileptic drug (AED) commonly used for generalized and focal epilepsies. The clinical relevance of coagulopathies, known as side effects of VPA therapy, especially thrombocytopenia, von Willebrand disease, and a decrease of factor XIII, is still unclear. METHODS: In our institute, we noticed a high incidence of clinically relevant coagulation problems related to VPA in eight patients within 1 year only and a further seven children with significant coagulopathy were identified in the context of planned surgery. RESULTS: We provide an overview of these patients and all six VPA-induced coagulopathies. CONCLUSIONS: At this time, it cannot be recommended to control all hemostatic parameters in every patient. Whenever an increased bleeding tendency is observed, or before surgical procedures, a platelet count, thrombelastography, prothrombin time, activated partial thromboplastin time, TT, fibrinogen, von Willebrand factor, and factor XIII should be examined. With 385 VPA-treated patients per year and 15 cases of coagulation disorders in this period, we estimate the incidence of coagulation disorders related to VPA in children to be nearly 4%.

Age Factors↗

A missense mutation in gamma-glutamyl carboxylase gene causes combined deficiency of all vitamin K-dependent blood coagulation factors.

To identify potential mutations in the gamma-glutamyl carboxylase gene, the sequence of all exons and intron/exon borders was determined in 4 patients from a consanguineous kindred with combined deficiency of all vitamin K-dependent procoagulants and anticoagulants and results were compared with normal genomic sequence. All 4 patients were homozygous for a point mutation in exon 9 that resulted in the conversion of an arginine codon (CTG) to leucine codon (CGG) at residue 394. Screening of this mutation based on introduction of Alu I site in amplified fragment from normal allele but not from the mutated allele showed that 13 asymptomatic members of the kindred were heterozygous for the mutation. The mutation was not found in 340 unrelated normal chromosomes. The segregation pattern of the mutation which is the first reported in the gamma-glutamyl carboxylase gene fits perfectly with phenotype of the disorder and confirms the suggested autosomal recessive pattern of inheritance of combined deficiency of all vitamin K-dependent procoagulants and anticoagulants in this kindred. The mutated carboxylase protein expressed in Drosophila cells was stable but demonstrated threefold reduced activity compared with WT carboxylase, confirming that the L394R mutation results in a defective carboxylase.

Animals↗

Testing of platelet deposition on polystyrene surface under flow conditions by the cone and plate(let) analyzer: role of platelet activation, fibrinogen and von Willebrand factor.

Recently, we described a method of testing platelet deposition on extracellular matrix under flow conditions. The method was used for assessment of platelet function in various platelet disorders, for monitoring of replacement and anti-platelet therapy. In the present study, we investigated platelet deposition on a polystyrene surface compared with that on extracellular matrix, under defined shear rates, using the original Cone and Plate(let) Analyzer. A correlation of adhesion rate (surface coverage) and aggregate formation (average size) of platelets from normal citrated blood between polystyrene and extracellular matrix was observed. Blocking of von Willebrand factor binding to glycoprotein Ib by a recombinant von Willebrand factor fragment substantially decreased platelet adhesion to both surfaces. Blocking of GPIIb-IIIa by Arg-Gly-Asp-Ser peptide prevented platelet adhesion to the polystyrene while an extensive adhesion of single platelets to extracellular matrix was observed. Furthermore, platelet adhesion to polystyrene but not to extracellular matrix was completely inhibited by platelet inactivation with prostaglandin E(1). Platelets from patients with severe von Willebrand disease yielded very low adhesion to both polystyrene and extracellular matrix. The addition of von Willebrand factor to the blood of these patients or pre-coating of polystyrene surface with von Willebrand factor restored the ability of platelets to adhere and aggregate on the surface. Platelets from patients with Glanzmann's thrombasthenia and afibrinogenemia adhered to extracellular matrix (with defective aggregate formation), while they failed to adhere to the polystyrene. Fibrinogen added to afibrinogenemia blood or pre-coating of the polystyrene with fibrinogen restored the ability of platelets to adhere and aggregate on the surface. In conclusion, the polystyrene surface, like extracellular matrix, can be used to assess platelet function disorders taking in account that platelet deposition on polystyrene under flow is absolutely dependent on platelet activation and on the presence of fibrinogen, von Willebrand factor, and their receptors.

Afibrinogenemia↗

[Extrahepatic portal hypertension: spleno-portal thrombosis secondary to protein C deficiency].

Portal vein thrombosis (PVT) is the most frequent cause of hypertension portal extrahepatic. It is a rare disorder an the main risk factors are cirrhosis, hepatobiliary malignancies and prothrombotic disorders, which have been identified as major risk. Therapy with anticoagulants must to be considered in acute portal thrombosis or chronic one and proven hypercoagulability. We present the case of a twenty-nine years old patient, with extrahepatic portal hypertension secondary to portal and splenic vein thrombosis, who was diagnosed because of splenomegaly and a coagulation disorder. A protein C deficiency were discovered and anticoagulation and beta-blocker therapy were initiated. One year later the patient had not presented complications concerning to the disease or to the treatment.

Adult↗

Italian guidelines for the diagnosis and treatment of patients with haemophilia and inhibitors.

The Italian Association of Haemophilia Centres reviewed and finally approved in November 2004 the new Italian Guidelines for the diagnosis and treatment of patients with clotting factor inhibitors. The recommendations have been based on the identification of levels of clinical evidence derived from the systematic review carried out in 2003 by the School of Health and Related Research, the University of Sheffield, UK, and further integrated by clinical studies published from 2003 to 2004. The Italian guidelines consist of six major domains concerning inhibitor definition, epidemiology, risk factors, diagnosis, inhibitor eradication, management of bleeding episodes, in patients with congenital and acquired coagulation disorders, with 121 statements, 59 synthesis and 54 recommendations. We report here recommendations and open issues concerning the diagnosis and monitoring of inhibitors, inhibitor eradication and the management of bleeding in patients with haemophilia A and B.

Algorithms↗

Orphan drugs: legal aspects, current situation.

'Rare disorders' is the name given to those diseases, of very varied aetiology, whose common denominator is that they are low-prevalence diseases, and for the majority of which there is no treatment available. The World Health Organization defines a rare disorder as all pathological conditions affecting 0.65-1 out of every 1000 inhabitants. The European Union understands a rare disorder to be one with a prevalence of 5 : 10 000 Europeans; the USA defines it as an ailment affecting fewer than 200 000 Americans; Japan sets the limit at 50 000 Japanese patients; Australia at 2000 Australian patients. According to the aforementioned ratios, haemophilia, von Willebrand disease and other congenital coagulation disorders could be considered to be rare disorders. 'Orphan drugs' is the term given to those drugs intended for the treatment of rare disorders, because the number of patients affected is so small that it is not profitable to invest in research and development or to market them. Although the ratio of people affected by haemophilia means it could be classed as a rare disorder and its treatment could therefore be given orphan designation, haemophilia does have an established treatment, and research and development in this area proves profitable for the pharmaceutical companies. Haemophilia is therefore singular in that pharmaceutical companies can file an application for orphan designation with all the benefits deriving from this, while the treatment of haemophilia is profitable in its own right for companies carrying out research and development for the products involved.

Coagulation Protein Disorders↗

[Pharmacotherapeutic choices for correction of disorders of primary hemostasis].

A defective primary haemostatic system is a relatively frequent disorder, due to the increasing use of aspirin and other non-steroid anti-inflammatory agents (NSAIDs) by the general population. Impaired primary haemostasis may result in enhanced perioperative bleeding. Administration of desmopressin is an effective pharmacotherapeutic intervention that will improve the process of primary haemostasis in many cases, with or without additional interventions in the clotting system.

Adult↗

[Portal vein thrombosis: causes and treatment].

Portal vein thrombosis (PVT) has a heterogeneous aetiology. Recently discovered thrombotic risk factors such as latent myeloproliferative disorders and prothrombotic genetic defects are important in the aetiology of PVT. At least one-third of the PVT patients exhibit a combination of thrombotic risk factors. Treatment with anticoagulant drugs has not proved beneficial for most PVT patients. Therapy with anticoagulants is only recommended in those with acute PVT, in those who recently underwent a portosystemic shunt procedure and in those with other thrombotic manifestations, particularly in case of proved hypercoagulability. Due to intact coagulation, oesophageal varices bleeding in patients with PVT has a better prognosis than in cirrhotics.

Acute Disease↗

Unexpected bleeding disorders: Algorithm for approach to therapy.

The management of unexpected bleeding must be directed at the specific abnormality identified, as there is no universally effective and safe procoagulant product. Where practical, a purely pharmaceutical approach obviates the residual risks of exposure to plasma-derived products. Desmopressin is often effective in bleeding due to mild haemophilia A, Type I von Willebrand's disease and some platelet function disorders. Where replacement therapy is necessary, it should be as specific as possible, preferably using purified components singly or in combination. Recombinant proteins provide the greatest margin of safety, but it must be borne in mind that these are biologicals, and that they may contain human and animal plasma-derived proteins. Where specific replacement is unavailable or impractical, plasma or crudely fractionated plasma derivatives may be used. In the case of inhibitor antibodies to factor VIII, high dose human factor VIII or porcine factor VIII may be used. Where replacement therapy is impossible due to a high inhibitory titre, it may be necessary to bypass the specific haemostatic defect using activated prothrombin complex concentrates or recombinant activated factor VIIa. The latter product is being studied in patients with various disorders of platelet function, and in the more global haemostatic failure that accompanies end-stage liver disease. Ancillary methods are often of great value in securing haemostasis. These may be derived from pharmacological or biological sources, and their sites of action may be systemic or topical. Examples include antifibrinolytic lysine analogues, corticosteroids where inflammation accompanies bleeding, and the topical application of fibrin sealants or thrombin. Simple physical measures such as pressure, ice, or splinting are also valuable adjunctive measures. Finally, it must be emphasized that the ultimate control of bleeding often depends upon effective management of the inciting cause, such as eliminating the trigger for DIC, or suppressing the causative antibody of ITP. These principles will be presented using a practical algorithmic approach. The initial question when considering treatment should be whether or not the patient is acutely unstable. Instability may be due to one of two causes: the volume of blood loss leading to a compromised cardio-vascular status, or the site of the bleed. The relevance of the site of the bleed is independent of the volume of blood loss, so for example, a closed bleed into CNS will cause critical functional compromise even though the volume of bleeding may be minimal. Similarly bleeding into a compartment, such as into a forearm or a calf will cause critical functional compromise irrespective of the volume of bleeding.

Algorithms↗

Gene therapy for bleeding disorders.

The goal of gene therapy for bleeding disorders is to provide stable insertion and expression of a particular gene whose absence is responsible for a particular disease. The bleeding disorders for which the most basic and clinical research has been completed are the hemophilias factor VIII deficiency and factor IX deficiency. These X-linked diseases are caused by single-gene mutations; replacement of the defective gene has not only resulted in clinical and biochemical improvement in animal models but also provided promising results in phase I clinical trials. An ideal gene transfer approach to the treatment of hemophilia would require a minimally invasive procedure for gene delivery, have minimal associated morbidity, and result in long-term transgene expression, ideally yielding factor levels in the therapeutic range. Multiple approaches to gene transfer in the hemophilias are currently under investigation.

Animals↗

Advances in clotting factor treatment for congenital hemorrhagic disorders.

During the last 50 years, clotting factor replacement has evolved from the use of frozen plasma in the 1950s, through the serendipitous discovery of cryoprecipitate in the 1960s and the development of purified clotting factors in the 1970s and 1980s, to the era of recombinant clotting factors beginning in the 1990s. The dawn of the new millennium has seen the refinement of recombinant factor (rF) VIII with enhanced safety via the elimination of plasma-derived culture media or product stabilizers. During the last decade of the 20th century, a cure for hemophilia through gene therapy became a possibility. This was, in part, facilitated by availability of large (dogs) and small (mice) animal models for hemophilia A and B. Although this review will focus primarily on clotting factor replacement, the reader may refer to recent discourse on gene therapy for hemophilia.

Animals↗

Genetics of thrombophilia: impact on atherogenesis.

PURPOSE OF REVIEW: The goal of this review is to present an update on basic and epidemiological findings associating variants in prothrombotic genes with atherogenesis and atherothrombotic disease. RECENT FINDINGS: The relation between atherosclerosis and thrombosis has long been recognized but only recently has it been understood that certain hemostatic factors affect not only thrombus formation, but also have a direct atherogenic role. Atherosclerosis is a complex disorder that results from the interaction of multiple genetic and environmental factors. Numerous polymorphisms and mutations in genes related to the hemostatic system and to vascular redox determinants that modulate nitric oxide bioavailability have been identified in the past decade; their role in atherogenesis and the risk of cardiovascular disease, however, remain uncertain. We will discuss the functional implications and association with disease risk of polymorphisms in coagulation factors (fibrinogen, prothrombin, and factor V); fibrinolytic factors (plasminogen activator inhibitor 1 and lipoprotein(a)); platelet surface receptors; and vascular redox determinants (methylenetetrahydrofolate reductase, endothelial nitric oxide synthase, and the antioxidant enzymes cellular glutathione peroxidase and paraoxonase). SUMMARY: Overall, these genetic variants have a modest effect on risk when considered individually but gain potency when acting synergistically with other genetic or environmental risk factors. We conclude that a better characterization of these interactions, in addition to the identification of potential novel genetic determinants, constitute key issues in the future understanding of the pathogenesis of atherothrombosis.

Arteriosclerosis↗

Commercial variants of the prothrombin time test as a screening test of acquired coagulation factor II, VII, and X deficiencies in dogs.

The aim of this study was to acquire the single factor sensitivity of three commercial variants of the prothrombin time test (PT(Va1)-Thrombotest, PT(Va2)-Normotest, PT(Va3)-Hepato Quick) for canine plasma. For this purpose, 38 samples from animals with coumarin intoxication followed by vitamin K(1) treatment who had a reduced activity of the coagulation factors II, VII, and/or X (experiment 1) as well as 25 plasma samples from animals with moderately reduced activity of the coagulation factors II, V, VII, and/or X caused by a hepatogenic synthesis disorder or disseminated intravascular coagulation (experiment 2), were examined. Measurements for all tests were performed according to the instructions of the manufacturer and also with higher plasma dilution. Furthermore, control measurements were performed with the conventional prothrombin time test (standard test; PT(ST)). The prothrombin time measured with the PT(Va3), which was prolonged in all samples from experiments 1 and 2, especially reflected with high sensitivity the reduced activity of the coagulation factors. The measurements with PT(Va2) and PT(Va1) showed only an insignificantly lower sensitivity (> or =0.88) with a maximum of 1 (experiment 1) and 3 (experiment 2) false negative test results, respectively. Compared to the already high sensitivity, a higher dilution of the sample material did not lead to a significantly higher sensitivity (P>0.05) for any of the tested commercial variants of the prothrombin time assay. While there was no significant difference (P>0.05) between the number of false negative test results in the variants of the prothrombin time assay, in both experiments a significantly higher number of false negative test results was found for the PT(ST) (P<0.001). The results show that, in contrast to the PT(ST), all three tested commercial prothrombin time variants are suitable for the detection of deficiencies of coagulation factors II, VII, and X in canine plasma.

Animals↗

Thrombotic dysfibrinogenemia. Fibrinogen "Caracas V" relation between very tight fibrin network and defective clot degradability.

Fibrinogen Caracas V is a thrombotic dysfibrinogenemia with an Aalpha 532 Ser-->Cys mutation characterized by a tight fibrin network formed of thin fibers responsible for a less porous clot than a normal one. In the present work, fibrinogen Caracas V is further characterized in order to understand the relationship between the structural defect and thrombophilia. This thrombotic disorder has been attributed to a tight fibrin network responsible for a decreased permeation of flow through the clot, leading to defective thrombus lysis due to a diminished availability of fibrinolytic enzymes to the inner fibrin surface. Correction of clot structure anomaly, by addition of dextran 40 to fibrinogen before clotting, induces an improvement in fibrin degradation that was attributed to an increase in porosity. The pulmonary embolism observed in this family has been related to an hyper rigidity of the clot, an anomaly that is also corrected by dextran. Furthermore, this abnormal fibrinogen binds more albumin than does normal fibrinogen, a phenomenon attributed to the mutation of serine in Aalpha-532 by cysteine. Therefore, this fibrinogen shows a striking similarity to the fibrinogen Dusart, allowing us to confirm that the alphaC-terminal part of fibrinogen plays an important role in fibrin structure, and to conclude that the anomaly of fibrin network observed in fibrinogen Caracas V is responsible for a deficient thrombus lysis.

Albumins↗

Pregnancy and oral contraceptives in congenital bleeding disorders of the vitamin K-dependent coagulation factors.

Pregnancies and deliveries represent important hemostatic challenges for congenital coagulation disorders. The same is true for the assumption of oral contraceptives. Available information mainly deals with von Willebrand's disease, factor XI (FXI) deficiency and carriers of hemophilia A. Data concerning patients with congenital prothrombin complex factor deficiencies are very scanty. In the present study, data of a total of 27 women are presented, 11 patients with homozygous or double heterozygous deficiencies of FII, FVII and FX, together with 16 cases of hemophilia B carriers. The patients with FII, FVII or FX defects had a total of 14 pregnancies and often needed transfusion therapy. Proper management resulted in a decrease in postpartum bleeding and satisfactory fetal outcome. Elective cesarean delivery seems indicated only in recent years. Carriers of hemophilia B had a total of 19 pregnancies but showed no bleeding and needed no substitutive therapy. Searching the literature, we discovered only 9 additional patients with prothrombin deficiency or FX deficiency, having a total of 16 pregnancies. On the contrary, there were at least 17 additional patients with FVII deficiency, with a total of 21 pregnancies. The management of the diseases has been variable, but in substantial agreement with the personal observations. Oral contraceptive therapy was administered in some of our patients and in a few additional cases described in the literature. Medication was always well tolerated and patients who took it for a long period of time showed a decrease in menometrorrhagia and an improvement in hematocrit levels. This led to a decrease in transfusional needs and to improved general conditions.

Adult↗

The thrombogram in rare inherited coagulation disorders: its relation to clinical bleeding.

We investigated the relation between clotting factor concentration, the parameters of the thrombin generation curve (the thrombogram) and the severity of clinically observed bleeding in patients with congenital deficiency of prothrombin (n = 21), factor V (n = 22), factor VII (n = 22), factor X (n = 10), factor XI (n = 7) and factor XII (n = 6). The parameters used were: area under the curve (endogenous thrombin potential, ETP), peak concentration of thrombin attained and lag time before manifest formation. Peak height and ETP varied linearly with the concentration of prothrombin. For the other factors these parameters hyperbolically approached to the 100% limit with increasing clotting factor concentration. Half normal ETP was seen at about the following concentrations: prothrombin (50%), factor V (1%), factor VII (2%), factor X (5%) and factor XI (1%). As a rule, the peak height was some-what more sensitive to clotting factor decrease than the ETP was. In all the patients with severe bleeding symptoms the ETP was less than 20% of normal. Bleeding tendency was absent or mild in patients with an ETP of 30% or higher. This value (except for prothrombin) is already obtained at concentrations of clotting factor of 1%-2%, which corroborates the clinical observation that a severe bleeding tendency is only seen in severe clotting factor deficiencies (less than 1%). The one exception was a patient with factor VII deficiency and severe bleeding, who showed a normal ETP value, albeit with a decreased peak height and a prolonged lag-time.

Blood Coagulation Tests↗