Hereditary thrombotic thrombocytopenic purpura and the hereditary TTP registry.

Hereditary thrombotic thrombocytopenic purpura, Upshaw-Schulman syndrome, ADAMTS13 Hereditary thrombotic thrombocytopenic purpura (TTP), also known as Upshaw-Schulman syndrome, is a rare recessively inherited disease. Underlying is a severe constitutional deficiency of the von Willebrand factor-cleaving protease, ADAMTS13, due to compound heterozygous or homozygous mutations in the ADAMTS13 gene. The clinical picture is variable and more and more patients with an adult-onset are diagnosed. In the majority of countries the only available treatment is plasma, which when administered regularly can efficiently prevent acute disease bouts. The decision to initiate regular prophylaxis is often not easy, as evidence based guidelines and long term outcome data are lacking. Through the hereditary TTP registry (www.ttpregistry.net, ClinicalTrials.gov identifier: NCT01257269), which was initiated in 2006 and is open to all patients diagnosed with Upshaw-Schulman syndrome and their family members, we aim to gain further information and insights into this rare disease, which eventually will help to improve clinical management of affected patients.

[Thrombophilic mutation by women with serious pregnancy complications]. / Výskyt trombofilních mutací zen se závaznými tehotenskými komplikacemi.

OBJECTIVE: The purpose of this study was to determine whether maternal or fetal genotype frequencies of the inherited thrombophilic gene mutation (F V Leiden, F II) are altered in adverse pregnancy outcomes - severe preeclampsia, IUGR, abruption of placenta and stillbirth. DESIGN OF THE STUDY: Retrospective study. SETTING: Department of Gynecology and Obstetrics of the Teaching Hospital and the 2nd Medical Faculty of the Charles University in Prague. METHODS: We studied 232 women who had pregnancy complications. All women were tested postpartum for mutation of factor V Leiden and G20210A prothrombine gene. At the same time were tested the newborns of those women. RESULTS: In the group of women with preeklampsia (n=141) we have demonstrated 5 women with mutation encoding for F V, 5 women with mutation encoding for F II and 1 combination of both. In the group of IUGR 2 women with mutation F V, 1 with mutation F II a 1 combination of both were found. In women after stillbirth occure two mutation of F V, one mutation of F II and one combination of both. In the group with abruptio of placenta was 1 case of mutation F V and 3 cases of mutation F II. When we tested a newborn we found 4 cases of mutation F V and 3 cases of F II in the group with preeclampsia, 4 cases of mutation F V 3 cases od mutation of F II in the group with IUGR, no case in the group with abruptio of placenta and 1 case in a death fetus. There was no assotiation between any severe pregnancy complications and any of the maternal or fetal inherited thrombophilia. CONCLUSION: Factor V Leiden and prothrombin gene mutations did not seem play a significant role in adverse pregnancy outcome in our population.

[Does the asymptomatic carriage of FV Leiden and FII prothrombin in heterozygous configuration represent the increased risk of thrombembolic complications during pregnancy, childbirth and postpartum?]. / Predstavuje asymptomatické nosicství mutace FV Leiden a FII protrombin v heterozygotní. Konfiguraci zvýsené riziko trombembolických komplikací v prubehu tehotenství, porodu a sestinedelí?

OBJECTIVE: To evaluate the course of pregnancy and puerperium in asymptomatic carriers of FV Leiden and FII prothrombin mutation in heterozygous configuration in terms of risk of thrombembolic disease and late pregnancy complications. To evaluate whether global prophylactic LMWH administration already during pregnancy has brought some benefit to these women. TYPE OF STUDY: Prospective study. METHODS: From June 2007 to June 2011, we monitored the incidence of thrombembolic events (TED) and severe late pregnancy complications in 473 asymptomatic carriers of FV Leiden and FII prothrombin mutation in heterozygous configuration. We also compared the ongoing changes of commonly clinically available hemocoagulation tests. In selected women, we added to coagulation tests a thrombin generation test (TGA) and thrombin-antithrombin test (TAT). In 253 women (Group A), preventive LMWH application was introduced already during pregnancy. In 220 women (Group B), the application of LMWH was commenced as late as on the delivery day. In both groups application of LMWH continued during the puerperium. RESULTS: The incidence of TED in the whole group of carriers of thrombophylic mutations accounted for 0.19%. The incidence of severe late pregnancy complications was very low - 3%. Medians of the monitored parameters of the hemocoagulation in compared groups and 'healthy' controls did not show statistically significant differences at any stage of pregnancy, labor or end of puerperium, with the exception of the results of TAT test at the end of puerperium. CONCLUSIONS: No direct causal relationship has been established between asymptomatic carriage of Leiden and prothrombin mutation in heterozygous configuration and the occurrence of severe late pregnancy complications. These types of mutation represent only a slightly increased risk in terms of development of thrombophylic events. General LMWH prophylaxis during pregnancy is not indicated. However, individual careful monitoring of hemocoagulation changes and early detection of associated transient situations potentiating risk of thrombembolic events is desirable. Statistically significant differences in the TAT results between group A and B at the end of puerperium revealed that the recommended extended LMWH prophylaxis until the end of puerperium was not followed by a number of women who started the prophylaxis on the date of labor.

Effect of warfarin anticoagulation on thrombin generation in patients with idiopathic pulmonary arterial hypertension.

BACKGROUND: Oral anticoagulant therapy is recommended for patients with pulmonary arterial hypertension (PAH). The rationale for the use of anticoagulant treatment is based on thrombophylic predisposition in PAH and improvement of survival in patients treated with anticoagulation. However, the target INR value has not been evaluated. The aim of this study was to analyze thrombin generation in patients with PAH treated with warfarin anticoagulation. METHODS: The study was performed in 58 patients with idiopathic PAH treated with warfarin at stable doses. Thrombin generation assay was performed in all subjects and three parameters were derived from the thrombin generation curves: lag time, maximal concentration of formed thrombin (peak thrombin) and area under the curve (AUC). Thrombin generation parameters were correlated with INR and compared between the patient groups with different intensity of anticoagulant therapy. RESULTS: Significant correlation between the lag time and INR was observed (r = 0.495, p < 0.001). Significant negative correlation between the maximal concentration of formed thrombin and INR and between the area under the curve of thrombin generation and INR was observed (r = -0.709, p < 0.001 and r = -0.784, p < 0.001, respectively). Thrombin generation was significantly reduced in patients with INR between 1.5 and 2.5. CONCLUSIONS: Low-intensity warfarin anticoagulation with target INR between 1.5 and 2.5 could be effective and sufficient to suppress thrombin generation in patients with idiopathic PAH (Fig. 3, Tab. 4, Ref. 12). Full Text in free PDF www.bmj.sk.

Thrombotic thrombocytopenic purpura: incidence of congenital form of disease in north Moravia (region Moravia-Silesia).

Thrombotic thrombocytopenic purpura (TTP) was first described by Eli Moschcowitz in 1924. The pathophysiology of this disease is related to unusual, large multimers of von Willebrand factor in microcirculation, that are insufficiently cleaved by ADAMTS13 protease (a disintegrin-like and metalloprotease with thrombospondin type 1motif,13). Congenital TTP/Upshaw-Schulman syndrome is less frequent than acquired one TTP/HUS (haemolytic-ureamic syndrome). Short characteristic of patients with inherited form of TTP is reported as well as their clinical and laboratory features and management of treatment.

The role of tissue factor in thrombosis and hemostasis.

The tissue factor (TF) is one of the most important regulators of arterial thrombosis. Because arterial thrombosis is the pathophysiologic background of acute coronary syndrome, the possible impact of blocking the arterial thrombosis on its onset is a challenging problem. The investigations of TF brought a new concept of "cell-based coagulation model" which highlighted the question of blood-borne TF as a source of TF in circulating blood. In this review we summarize essential information on the pathophysiology, molecular structure, expression and distribution of TF and we propose a novel concept of blood-borne TF, suggesting the possibilities of inhibition of the coagulation cascade with newly synthetized drugs.

[Evaluation of DHPLC analysis for mutation detection in haemophilia A]. / Testování DHPLC analýzy pro detekci mutací u hemofilie A.

BACKGROUND: Haemophilia A is one of the most prevalent inherited bleeding disorders. Causal mutations in the factor VIII gene are detected to facilitate the genetic counselling and to estimate the risk of serious complication associated with standard treatment (factor VIII inhibitor). Wide range of mutations located across the entire length of the factor VIII gene underlies the factor VIII deficiency of variable severity. The only two common recurrent mutations in the factor VIII gene are intron 22 and intron I inversions. In the remaining cases it is necessary to screen all 26 exons encoding 9kb mRNA together with adjacent nonncoding sequences. In order to speed up genotyping in haemophilia A families in the Czech Republic we evaluated DHPLC-based screening technique. METHODS AND RESULTS: We tested sensitivity of the analysis on a panel of DNA samples containing 49 different sequence variations distributed over 21 exons. All the genetic alterations were readily detected. Analysis of family members has shown good reproducibility of the respective elution patterns. DHPLC analysis detected mutations in 4 out of 5 samples from apparently unrelated haemophilia patients, where previously applied multiplex CSGE was not successful. CONCLUSIONS: Establishing of DHPLC analysis will substantially speed up the genotyping of haemophilia A families in the Czech Republic.

[Hereditary form of thrombotic thrombocytopenic purpura]. / Vrozená forma trombotickté trombocytopenické purpury.

BACKGROUND: Thrombotic thrombocytopenic purpura is characterized by microvascular platelet clumping resulting in thrombocytopenia, microangiopathic hemolysis, neurological abnormality, and renal dysfunction. Similar manifestations also occur in patients with the hemolytic uremic syndrome or other types of disorders. Recent studies demonstrate that severe deficiency of the von Willebrand factor cleaving metalloprotease, ADAMTS 13, causes thrombotic thrombocytopenic purpura. Aim of our study was to characterize gene defects causing inherited type of disease. METHODS AND RESULTS: We investigated nine patients with recurrent type of disease with familiar origin and twelve relatives. Samples were taken in a remission of disease. We measured activity of ADAMTS13 (vWF-CP) with modified method of the quantitative immunoblotting of degraded vWF multimers. Mutation screening was carried out by sequencing all 29 exons and flanking intron regions of the ADAMTS13 gene. Five distinct mutations were found. Three of them are novel. CONCLUSIONS: Mutation analysis of the ADAMTS 13 gene brought interesting results in eight patients. We found a one single base frameshift insertion, 4143insA in 8 of 9 unrelated individuals. This investigation represents an advantage in the differential diagnosis of disease since the thrombotic thrombocytopenic purpura phenotype in childhood can be variable and rapid detection of mutation is helpful for the recurrence prevention.

[Hematological aspects of gestational cholestatic hepatosis (ICP)]. / Hematologické aspekty tehotenské cholestatické hepatózy (ICP).

AIM OF THE STUDY: To assess the changes of hemocoagulative parametres induced by ICP from the view of posssible affection of the hemostasis. SEATING: Department of Obstet. Gynecol. 2nd Medical School Charles University and Teaching Hospital Motol, Prague, Institute of Haematology and Blood Transfusion Prague. METHODOLOGY: 20 blood samples of the pregnant with severe signs of ICP underwent the precise hemocoagulative analysis. The control group was composed from 12 women with physiological course of pregnancy comparable in terms of gestational week, age and parity. RESULTS: The routine hemocoagulative tests did show any statistical significant difference between both groups of examined women. The statisticaly significant elevation was found in women with ICP in factor VIII (fVIII:C), vWf and in the serum levels of fibrinogen. The elevated levels of fibrinogen did not influence the function of platelets and even the analysis of fibrinogen itself did not show any significant differences. Clinically assessed peripartal blood loss did not deviate from average of loss during the spontaneous labor of healthy women. CONCLUSIONS: The changes in hemocoagulative parametres in patients with ICP do not increase the risk of thromboembolic events during pregnancy and labor. We also did not approve the risk of the higher peripartal blood loss in these patinets.

Thrombelastography monitoring of platelet substitution therapy and rFVIIa administration in haemato-oncological patients with severe thrombocytopenia.

Thrombocytopenic patients refractory to platelet concentrates (PC) could be treated during bleeding episodes with the recombinant activated FVII (rFVIIa). However, monitoring of administration of the rFVIIa or a response to platelet substitution therapy in thrombocytopenia patients is not well documented so far. Using of whole blood ROTEG analysis we monitored the changes in haemostatic parameters following in vivo platelet concentrate administration compared to ex vivo rFVIIa administration in patients with a severe to mild thrombocytopenia secondary to haemato-oncological disease. We use non-activated thrombelastography (NATEG) and a mild intrinsic activation thrombelastography (INTEG). NATEG analysis was sufficiently sensitive to monitor changes following PC and rFVIIa administration. Both, platelet infusion and rFVIIa treatment induced significant shortening of clotting time (CT) and clot formation time (CFT) parameters (p<0.05). When we compared the effect of platelet vs. rFVIIa treated whole blood by NATEG analysis we did not found any significant difference. Analysis with INTEG system was less sensitive and changes in CT and CFT were not significant. The monitoring with thrombelastography could enable efficient application of platelet concentrate and furthermore the using of rFVIIa as an alternative treatment of patients refractory to platelet infusion or with allergic reactions.

[Molecular study of type 2 von Willebrand disease]. / Molekulární studie von Willebrandovy choroby typu 2.

BACKGROUND: von Willebrand disease is an inherited bleeding disorders caused by mutations in the von Willebrand factor gene. We attempted to characterise the phenotype and the genotype in the first five families in Czech Republic affected by this heterogeneous disorder. METHODS AND RESULTS: The level of FVIII was measured by the one stage assay, the vWF:Ag by the immunoelectrophoresis, vWF:RiCo by aggregometry. For the vWF multimer analysis a western blot based technique was used. The vWF binding to FVIII was evaluated by the ELISA method. Two families were classified as the type 2A, one as the type 2B and two as the combined type 1/2N. Based on that knowledge, parts of the vWF gene were selected for genetic analysis. The previously described mutations Arg1374His and Gly1579Arg were identified in two families with the type 2A. In the family with type 2B a substitution Arg1308Cys was detected. In one family with the type 1/2N, two different previously described defects were found on the separate alleles of the vWF gene: a deletion of cytosine 2435 and a polymorphism Arg854Gln. Compound heterzygotes had the type 1/2N phenotype, while a carriers of the deletion had type 1 phenotype. In the second type 1/2N family, only the amino acid substitutions Thr791Me was found explaining the qualitative defect. A mutation underlying the quantitative deficiency needs to be searched for throughout the entire vWF gene. CONCLUSIONS: Based on the characterisation of the phenotype and genotype, five apparently unrelated families with the von Willebrand disease were diagnosed according to the revised classification. Our work represents laboratory basis for further studies into von Willebrand disease in Czech Republic.

Pro-coagulant state resulting from high levels of soluble P-selectin in blood.

The plasma concentration of soluble adhesion receptors is increased under pathological circumstances, but their function remains enigmatic. Soluble P-selectin (sP-sel) is shed from activated platelets and endothelial cells. Mice genetically engineered to express P-selectin without the cytoplasmic tail (DeltaCT) constitutively show a 3- to 4-fold increase of sP-sel in plasma. We observed that the DeltaCT mice formed fibrin very readily. In an ex vivo perfusion chamber, there was more fibrin deposited at the site of platelet thrombus formation than in wild type (WT), whereas no fibrin deposits were detected using P-selectin-deficient blood during the same interval. Similarly, in vivo, the hemorrhage produced by local Shwartzman reaction was smaller in the DeltaCT mice than in WT. In contrast, we previously showed hemorrhage to be more prominent in P-selectin knock-out mice. Infusion of mouse P-sel-Ig chimera produced the same protective effect in WT mice as seen in the DeltaCT mice, indicating that the effect was due to increased levels of sP-sel. Mice infused with P-sel-Ig showed significantly more fibrin deposited on the luminal face of the injured vessels than control mice. Plasma from DeltaCT mice or mice infused with P-sel-Ig contained higher concentration of pro-coagulant microparticles and clotted one minute faster than WT. This pro-coagulant phenotype of DeltaCT mice could be reversed by a 4-day treatment with PSGL-Ig, a P-selectin inhibitor. We propose that sP-sel should no longer be considered only as a marker of inflammation or platelet activation, but also as a direct inducer of pro-coagulant activity associated with vascular and thrombotic diseases.

[Molecular basis of hereditary antithrombin defects in 10 Czech families]. / Molekulární podstata vrozeného defektu antitrombinu u deseti ceských rodin.

BACKGROUND: Molecular basis of antithrombin deficiency has not yet been studied in Czech Republic. We looked for the causal mutations throughout the antithrombin gene in 26 patients from 10 unrelated families with antithrombin defect. METHODS AND RESULTS: We screened the gene by conformation sensitive gel electrophoresis and sequenced the mismatched regions using fluorescence technology to characterise mutations and polymorphisms. Mutations were detected in all ten families. Four novel mutations were identified in four families with type I antithrombin defect: Trp-6Arg, 5386-5387delCT, Glu163Stop, and 13246-13248del TGA causing deletion of Glu377 with change of Asn376 to Lys. In other three type I families we found following mutations: splicing site mutation G2777C, Arg197Stop and entire gene deletion. In the family carrying Trp-6Arg mutation antithrombin Vienna (Gln118Pro) was also detected. Leu99Phe recurrent in south-eastern Europe was identified in three families with type II defect. Only the homozygous carries of the mutation were symptomatic, although the heterozygous carries had decreased functional levels. CONCLUSIONS: Four novel mutations in families with type I antithrombin deficiency were characterised. In one family two different genetic defects were identified to be responsible for type I and II phenotypes. Altogether our data agree with the expected heterogeneity of the AT genetic defect.

[Anti-Xa activity during administration of low-molecular weight heparin in the prevention of postoperative thromboembolic complications]. / Aktivita anti-Xa pri podávání nízkomolekulárních heparinu v profylaxi pooperacních tromboembolických komplikací.

The authors evaluate the follow-up of the anti-Xa level during the prophylactic administration of two different low molecular heparins (enoxaparin, reviparin) in 40 patients with a planned resection of the large intestine, resection of the pancreas, laparoscopic cholecystectomy and laparotomic cholecystectomy. The assessed amounts of anti-Xa did not reach recommended levels even during the perioperative period. The selection of the preparation and the extent of the operation did not influence the anti-Xa levels.

[Clinical findings in individuals with the factor V Leiden mutation]. / Klinické nálezy u jedincu s Leidenskou mutací faktoru V.

Leiden mutation of the coagulation factor V is the most frequent known congenital risk factor of thrombophilia. The authors examined a group of 440 subjects with thrombosis in the case- or family-history. The mutation was found in 146. In 94 thrombotic manifestations were recorded in the case-history, five women were examined because of repeated abortions. 52.74% carriers of FVL had venous thrombosis of the lower extremities and pelvis in their case-history, 19.18% had pulmonary embolism in the case-history. In 27.40% during the initial manifestations of thrombosis no other risk factor of thrombosis was detected. In 10.27% the first thrombosis developed after an injury or operation. In 22.22% women the thrombosis was manifested during pregnancy or the puerperal period. Due to the high incidence of this defect screening of the resistance to activated protein C should be an integral part of examination of thrombophil conditions.

[The human genome--chromosome 12]. / Lidský genom--chromozom c. 12.

One of the most rewarding examples for teaching hereditary metabolic disorders is classical phenylketonuria (PKU) caused by the deficient function of phenylalanine hydroxylase, the locus of which (PAH) is on the long arm of the twelfth chromosome. The twelfth chromosome has also the locus (VWF, F8VWF) the pathogenic alleles of which cause impaired blood clotting--Willebrand's disease and it is at the same time also the site of the family of keratin genes (KRT) responsible for epidermolysis bullosa simplex and other diseases. The question of the relationship between membrane glucose transmitters--GLUT and diabetes (NIDDM) is the subject of many investigations concerned with these loci.

[Detection of carriers of hemophilia B]. / Moznosti stanovení prenasecství hemofilie B.

Thirty-two families with haemophilia B were divided into sub-groups based on assessment of fIX:Ag, fIX:C and the thromboplastin time with bovine thromboplastin. In confirmed carriers from these families after assessment of fIX:Ag and, fIX:C the normal range of individual sub-groups of confirmed carriers was assessed and compared with values of fiX:Ag and fiX:C of the control group. It will be possible to use these groups after their extension to assess probable carriers in sub-groups CRM+ and CRM-. In the sub-group CRMR the only criterium of transmission is a reduced fIX:C level. A prolonged TP with bovine thromboplastin in families with BM haemophilia cannot be used to assess carriers.