Coagulation factor activity and clinical bleeding severity in rare bleeding disorders: results from the European Network of Rare Bleeding Disorders

Summary. Background: The European Network of Rare Bleeding Disorders (EN‐RBD) was established to bridge the gap between knowledge and practise in the care of patients with RBDs. Objectives: To explore the relationship between coagulation factor activity level and bleeding severity in patients with RBDs. Patients/Methods: Cross‐sectional study using data from 489 patients registered in the EN‐RBD. Coagulation factor activity levels were retrieved. Clinical bleeding episodes were classified into four categories according to severity. Results: The mean age of patients at data collection was 31 years (range, 7 months to 95 years), with an equal sex distribution. On linear regression analysis, there was a strong association between coagulation factor activity level and clinical bleeding severity for fibrinogen, factor (F) X, FXIII, and combined FV and FVIII deficiencies. A weaker association was present for FV and FVII deficiencies. There was no association between coagulation factor activity level and clinical bleeding severity for FXI. The coagulation factor activity levels that were necessary for patients to remain asymptomatic were: fibrinogen, > 100 mg dL^?1; FV, 12 U dL^?1; combined FV + VIII, 43 U dL^?1; FVII, 25 U dL^?1; FX, 56 U dL^?1; FXI, 26 U dL^?1; FXIII, 31 U dL^?1. Moreover, coagulation factor activity levels that corresponded with Grade III bleeding were: undetectable levels for fibrinogen, FV and FXIII, < 15 U dL^?1 for combined FV + VIII; < 8 U dL^?1 for FVI; < 10 U dL^?1 for FX; and < 25 U dL^?1 for FXI. Conclusions: There is a heterogeneous association between coagulation factor activity level and clinical bleeding severity in different RBDs. A strong association is only observed in fibrinogen, FX and FXIII deficiencies.     

Gastric retention of sucralfate gel and suspension in upper gastrointestinal diseases

This study was designed to compare by scintigraphy the gastric retention of a new dosage form of sucralfate as gel (Gastrogel) with that of sulcralfate suspension in 25 patients with upper gastrointestinal symptoms referred for routine endoscopy. After endoscopy 4 subgroups were defined: macroscopically normal mucosa (n= 7), antral gastritis and/or erosions (n= 6), gastric ulcer (n= 6) and duodenal ulcer (n= 6). Each patient received either sucralfate gel or sucralfate suspension in equivalent doses (5 ml containing 1 g sucralfate). Both formulations were labelled with 111 MBq ^99m Tc-DTPA before administration. The mean value of t_½ in the total group was significantly longer when patients were taking sucralfate gel (61.6 min) compared to sucralfate suspension (33.8 min) (P < 0.001). The mean values of t_½ were significantly longer for sucralfate gel compared to sucralfate suspension also among the subgroups (macroscopically normal P < 0.02, antral gastritis P < 0.05, gastric ulcer P < 0.02 and duodenal ulcer P < 0.05). After 2 and 3 hours, the percentage residual activity in the gastric area was significantly higher following administration of sucralfate gel compared to sucralfate suspension. This study has shown that, compared to sucralfate suspension, sucralfate gel persists longer in the stomach of patients with gastritis and peptic ulcer.     

Trypsin-pancreatic secretory isoinhibitor A from bovine pancreas (Kazal type)

The secondary and tertiary structure of isoinhibitor A from bovine pancreas secretion (Kazal inhibitor) was investigated by circular dichroism (CD) and fluorescence measurements. The protein shows noteworthy thermal stability as seen by the temperature dependence of the CD spectra and the intensity of emission fluorescence at different pH values.     

Genetic diagnosis of haemophilia and other inherited bleeding disorders

Summary.  Inherited deficiencies of plasma proteins involved in blood coagulation generally lead to lifelong bleeding disorders, whose severity is inversely proportional to the degree of factor deficiency. Haemophilia A and B, inherited as X-linked recessive traits, are the most common hereditary hemorrhagic disorders caused by a deficiency or dysfunction of blood coagulation factor VIII (FVIII) and factor IX (FIX). Together with von Willebrand's disease, a defect of primary haemostasis, these X-linked disorders include 95% to 97% of all the inherited deficiencies of coagulation factors. The remaining defects, generally transmitted as autosomal recessive traits, are rare with prevalence of the presumably homozygous forms in the general population of 1:500.000 for FVII deficiency and 1 in 2 million for prothrombin (FII) and factor XIII (FXIII) deficiency. Molecular characterization, carrier detection and prenatal diagnosis remain the key steps for the prevention of the birth of children affected by coagulation disorders in developing countries, where patients with these deficiencies rarely live beyond childhood and where management is still largely inadequate. These characterizations are possible by direct or indirect genetic analysis of genes involved in these diseases, and the choice of the strategy depends on the effective available budget and facilities to achieve a large benefit. In countries with more advanced molecular facilities and higher budget resources, the most appropriate choice in general is a direct strategy for mutation detection. However, in countries with limited facilities and low budget resources, carrier detection and prenatal diagnosis are usually performed by linkage analysis with genetic markers. This article reviews the genetic diagnosis of haemophilia, genetics and inhibitor development, genetics of von Willebrand's disease and of rare bleeding disorders.     

Controlled proteolysis of mouse epidermal growth factor An RP-HPLC and 1H-n.m.r. study

Tryptic digestion of the mouse epidermal growth factor (mEGF) and the chromatographic separation of its proteolytic fragments by RP-HPLC affords the isolation of the pure hormone, of its 1–48 (Des(49–53)mEGF) and 1–45 (Des(46–53)mEGF) derivatives, and of the carboxyl-terminal pentapeptide W49–W50-E51-L52-R53. Kinetics of mEGF proteolytic degradation follows a two-state time-course: native mEGF being converted into Des(49–53)mEGF with an apparent half-time of 10min; and Des(49–53)mEGF subsequently hydrolyzed to Des(46–53)mEGF with an apparent half-time of 7 h. Native mEGF and its proteolytic fragments have been characterized by ¹H-n.m.r. spectroscopy. In the aromatic and aliphatic regions, the ¹H-n.m.r. spectrum proved to be a sufficiently sensitive probe for following controlled proteolysis, and for analyzing the influence of the carboxyl-terminal sequence on the hormone conformation and stability.     

Efficacy of prophylaxis and genotype‐phenotype correlation in patients with severe Factor X deficiency in Iran

Summary. We aimed to evaluate the effect of regular prophylaxis with a Factor X (FX) concentrate for patients with severe FXD in Iran and to assess the correlation of the genotype and phenotype in these patients. Ten patients with severe FXD (FX activity <1%) were enrolled and characterized during 2010–2011. Prophylaxis with 20 IU FX P Behring per kg body weight was administered once a week. FX levels, were monitored at baseline, 15 and 30 min, 1, 3, 6, 12, 24, 48, 72 and 96 h after starting prophylaxis. All patients were followed for 1 year. The mean age of the patients was 15 ± 7.8 years (age range of: 6–27 years). One patient had anaphylactic reaction after the first infusion, and the treatment was stopped. During one‐year follow‐up after starting prophylaxis, no bleeding symptoms occurred in any patient who tolerated and remained on the prophylaxis programme and all of them had a FX level of 1% or above. The maximum level of FX activity has been observed at 15 min after starting prophylaxis. A level of 1.5–3.5% was detected after 96 h. Homozygous mutations p.Arg40Thr (Arg‐1Thr), p.Gly51Arg and p.Glu69Lys were detected in patients with intracranial haemorrhage. In our patients, significant decrease in symptoms without any complication after administration of FX, was demonstrated in all except one patient who had an anaphylactic reaction. It seems that the dose of 20 IU kg^?1 could be probably the best choice for patients with severe FXD, who require regular prophylaxis.     

Effectiveness of intraaortic balloon counterpulsation in the elderly

The effectiveness of intraaortic balloon counterpulsation (IABP)in the elderly was evaluated in a group of 63 patients withacute myocardial infarction (AMI) complicated by refractoryleft ventricular failure (LVF, n = 16), cardiogenic shock (CS,n = 32) or acute ventricular septal defect (VSD, n = 15). Themean age was 61 years (range: 32–78 years); 35 patientswere under and 28 over the age of 65. Failures of insertion of the IABP catheter were significantlymore frequent in the elderly (37.5 v. 11.4%, P <0.05). The rate of major complications, which was 12.7% in the wholeseries, did not significantly differ between the two age groups. Patients with medical indications for IABP (LVF, CS) had anoverall in-hospital mortality of 68.8%: no difference was observedbetween young (67.7%) and elderly (70.6%) patients. In elderlypatients with VSD, IABP was ineffective even when reparativesurgery could be performed. Haemodynamic effects of IABP were evaluated in 32 medical patientswho survived the 4th hour of treatment. All haemodynamic parametersimproved in the early phase of pumping (within 4h) except cardiacindex and total systemic resistance, which were significantlymodified after 12 and 24 h of treatment, respectively. An earlyimprovement in stroke index proved to be predictive of survival.No haemodynamic difference was found between the age groupseither in the early or in the late phase of cardiocirculatorymechanical assistance. Fifteen patients (10 CS, 3 LVF, 2 VSD) were discharged fromthe hospital and 14 were long-term survivors (more than 60 days). It was concluded that age cannot be considered as a contraindicationto IABP during complicated AMI, since the only difference betweenthe two age groups consisted in a higher rate of failure inintroducing the catheter in the elderly.     

Rare bleeding disorders

Summary.  Deficiencies of coagulation factors other than factor VIII and factor IX (afibrinogenemia, FII, FV, FV+FVIII, FVII, FX, FXI, FXIII) that cause bleeding disorders (RBDs) are inherited as autosomal recessive traits and are rare, with prevalences in the general population varying between 1 in 500.000 and 1 in 2 million for the homozygous forms. As a consequence of the rarity of these deficiencies, the type and severity of bleeding symptoms, the underlying molecular defects, and the actual management of bleeding episodes are not as well established as for hemophilia A and B. The study of the genetic basis of these disorders could represent an important tool for prevention through prenatal diagnosis. Treatment of patients with RBDs during bleeding episodes or surgery is a challenge because of the lack of experience and the paucity of data. For some deficiency factor concentrates are still non available and severe complications can occur. These complications can be minimized by assessment of risks of bleeding and thrombosis, use of haemostatic means other than blood components or no therapy at all. The RBDs pose a problem for guideline writers because there are no suitable clinical trials to supply good evidence for how these people are best treated. The lack of adequate information on clinical manifestations, treatment and genetic basis of RBDs could be improved by the collection of data in an International Database ( http://www.rbdd.org ), linkable to others previously published. This could be a useful tool to fill the gap between clinical data and clinical practice. This article reviews the genetic basis of RBDs, problems and complications of treatment, problems in the preparation of suitable guidelines for treatment and the future perspectives of the International Registry on RBDs.     

CONFORMATIONAL STABILITY OF PORCINE-PANCREATIC SECRETORY TRYPSIN INHIBITORS (KAZAL INHIBITORS)

The presence of secondary structure and the strong conformational resistance of porcine pancreatic trypsin inhibitors (Kazal inhibitors) PSTI I and PSTI II to denaturant agents have been investigated by circular dichroism (CD) and fluorescence measurements. These proteins heated for 6 h at 85° are still highly active.