Ágota Schlammadinger

Complement activation in thrombotic thrombocytopenic purpura

Summary.  Background:  Ultra‐large von Willebrand factor and deficiency of its cleaving protease are important factors in the events leading to thrombotic microangiopathy; however, the mechanisms involved are only partly understood. Whereas pathological activation of the alternative complement pathway is linked to atypical hemolytic uremic syndrome, the role of complement activation in thrombotic thrombocytopenic purpura (TTP) is unknown. The aim of this study was to investigate whether signs of complement activation are characteristic of TTP.

Severe bleeding complications caused by an autoantibody against the B subunit of plasma factor XIII: a novel form of acquired factor XIII deficiency

Acquired factor XIII (FXIII) deficiency due to autoantibody against FXIII is a very rare severe hemorrhagic diathesis. Antibodies directed against the A subunit of FXIII, which interfere with different functions of FXIII, have been described. Here, for the first time, we report an autoantibody against the B subunit of FXIII (FXIII-B) that caused life-threatening bleeding in a patient with systemic lupus erythematosus. FXIII activity, FXIII-A(2)B(2) complex, and individual FXIII subunits were undetectable in the plasma, whereas platelet FXIII activity and antigen were normal. Neither FXIII activation nor its activity was inhibited by the antibody, which bound to structural epitope(s) on both free and complexed FXIII-B. The autoantibody highly accelerated the elimination of FXIII from the circulation. FXIII supplementation combined with immunosuppressive therapy, plasmapheresis, immunoglobulin, and anti-CD20 treatment resulted in the patients recovery. FXIII levels returned to around 20% at discharge and after gradual increase the levels stabilized above 50%.

Comparison of PFA-100 Closure Time and Template Bleeding Time of Patients with Inherited Disorders Causing Defective Platelet Function

MINI REPORT Comparison of PFA-100 Closure Time and Template Bleeding Time of Patients with Inherited Disorders Causing Defective Platelet Function Adrienne Kerenyi1, Agota Schlammadinger2, Eva Ajzner1, Istvan Szegedi3, Csongor Kiss3, Zoltan Pap4, Zoltan Boda2 and Laszlo Muszbek1 1Department of Clinical Biochemistry and Molecular Pathology, 2Second Department of Medicine, 3Department of Pediatrics, University Medical School of Debrecen, Debrecen, 4City Health Service, Ophthalmology Unit, Debrecen, Hungary.

Plasma glycocalicin as a source of GPIbα in the von Willebrand factor ristocetin cofactor ELISA

We have previously demonstrated that the von Willebrand factor ristocetin cofactor activity (VWF:RCo), used in the diagnosis of vonWillebrand disease (VWD), can be accurately determined via ELISA by measuring the ristocetin-induced binding of VWF to a captured recombinant fragment of GPIbalpha (rfGPIbalpha, AA 1-289) (Vanhoorelbeke et al., Thromb Haemost 2000; 83: 107-13). This ELISA is more reliable than the currently used platelet agglutination test. Normal plasma contains relatively high concentrations of glycocalicin, a proteolytic fragment of GPIbalpha. We therefore studied whether non-purified plasma glycocalicin can replace rfGPIbalpha in our ELISA. Of 42 anti-GPIbalpha monoclonal antibodies (MAbs) capable of binding plasma glycocalicin, only one MAb captured glycocalicin in a spatial orientation exposing the VWF-binding site in glycocalicin, allowing a specific and dose-dependent ristocetin-mediated VWF-binding. Intra- and interassay variability were comparable with those for the rfGPIbalpha based VWF:RCo ELISA. The VWF:RCo activity of plasma from 33 normal individuals, 19 type 1, 16 type 2A, 9 type 2B, 8 type 2M and 7 type 3VWD patients was determined with this ELISA and allowed a clear identification of VWD patients. Furthermore, determination of the VWF:RCo/VWF:Ag ratio resulted in the discrimination between type 1 and type 2 VWD patients. Results for the glycocalicin based and the rfGPIbalpha based VWF:RCo ELISAs were in good agreement (r=0.943). There was also a good correlation between the glycocalicin based ELISA and the standard platelet agglutination test (r=0.963). In conclusion, to diagnose VWD, a VWF:RCo ELISA based on antibody immobilized plasma glycocalicin can be performed reliably.

Occurrence of the Asn45Ser mutation in the GPIX gene in a Belgian patient with Bernard Soulier syndrome.

Bernard Soulier Syndrome (BSS) is a rare inherited bleeding disorder caused by a defect in the glycoprotein (GP)Ib/IX/V complex. A patient with a bleeding problem was diagnosed as having BSS based on the prolonged bleeding time, the absence of ristocetin induced platelet aggregations, thrombocytopenia and the presence of giant platelets. Analysis of the platelets of the propositus, a 39-year-old Belgian female, by flow cytometry revealed a decreased expression of the GPIb/IX polypeptides. Western blotting confirmed these results and showed moreover that there was a decreased disulfide bridge formation between GPIb f and GPIb g . After sequence analysis of the GPIb f , GPIb g and GPIX genes, only a mutation in the GPIX gene at position 1826 (A M G) was identified, changing Asn45 M Ser. Restriction analysis with Fnu 4H1 demonstrated that the patient was homozygous for this mutation. As this Asn45 M Ser mutation in the GPIX gene was already found in four unrelated families, i.e. in a British, Austrian, Swedish and Finnish one, the occurrence of this mutation in a Belgian patient supports the hypothesis of Koskela et al . (1999) that the Asn45Ser mutation in GPIX appears to be an ancient mutation shared by northern and central European populations. Our present observation of a decreased disulfide bridge formation between GPIb f and GPIb g shows that GPIX is not only needed for the correct assembly of the complex but might also be needed for the disulfide bridge formation between GPIb f and GPIb g .

Accelerated clearance alone explains ultra-large multimers in von Willebrand disease Vicenza.

See also Goodeve AC. Vicenza deciphered: modeling the von Willebrand disease enigma: commentary on accelerated clearance alone explains ultralarge multimers in VWD Vicenza. This issue, pp 1271–2.

Accelerated clearance alone explains ultralarge multimers in VWD Vicenza

See also Goodeve AC. Vicenza deciphered: modeling the von Willebrand disease enigma: commentary on accelerated clearance alone explains ultralarge multimers in VWD Vicenza. This issue, pp 1271–2.

Elevated plasma neutrophil elastase concentration is associated with disease activity in patients with thrombotic thrombocytopenic purpura

INTRODUCTION Genetic and autoimmune risk factors contribute to the development of thrombotic thrombocytopenic purpura (TTP) but triggers are needed to bring about acute disease. The aim of the study was to investigate the association of neutrophil activation with acute TTP, to assess whether neutrophil activation changes during plasma exchange therapy and to show if complement- and neutrophil activation are parallel, characteristic processes in acute TTP. MATERIALS AND METHODS Altogether 49 EDTA-plasma samples of 21 TTP patients with acute disease and 17 in remission were investigated along with 20 healthy controls. A stable complex of PMNE-proteinase-inhibitor was measured by ELISA (Calbiochem, Merck-Millipore, Darmstadt, Germany). RESULTS Acute disease was associated with significantly increased PMNE levels, the group medians were similarly low in TTP patients in remission and in healthy controls. Increased PMNE levels were characteristic for hematologically active and ADAMTS13 deficient form of TTP. PMNE concentration inversely correlated to disease activity markers platelet count (r=-0.349, p=0.032) and hemoglobin levels (p=-0.382 p=0.018). Achievement of remission was associated with significant reduction of plasma PMNE levels (p=0.031, Wilcoxon test). There was positive correlation between PMNE levels and complement activation markers C3a and Bb. CONCLUSIONS We report increased PMNE levels in acute TTP and showed its association to activity markers of acute TTP and complement activation. Effective treatment of an acute TTP episode resulted in marked decrease in PMNE levels. Our data support and extend previous observations that neutrophil extracellular traps may be released in acute TTP and potentially contribute to the pathophysiology of this disease.

Common large partial VWF gene deletion does not cause alloantibody formation in the Hungarian type 3 von Willebrand disease population

Summary.  Background: Type 3 von Willebrand disease (VWD) is an autosomal recessive bleeding disorder, characterized by virtually undetectable plasma von Willebrand factor (VWF) and consequently reduced plasma factor VIII levels. Genetic mutations responsible for type 3 VWD are very heterogeneous, scattered throughout the VWF gene and show high variability among different populations. Methods: Twenty‐five severe VWD patients were studied by direct sequencing of the 51 coding exons of the VWF gene. The total number of VWD type 3 families in Hungary is 24, of which 23 were investigated. Results: Fifteen novel mutations were identified in 31 alleles, five being nonsense mutations (p.Q1238X, p.Q1898X, p.Q1931X, p.S2505X and p.S2568X), four small deletions and insertions resulting in frame shifts (c.1992insC, c.3622delT, c.5315insGA and c.7333delG), one a large partial deletion (delExon1‐3) of the 5′‐region, four candidate missense mutations (p.C35R, p.R81G, p.C295S, p.C623T) and one a candidate splice site mutation (c.1730–10C>A). Six previously described mutations were detected in 17 alleles, including the repeatedly found c.2435delC, p.R1659X and p.R1853X. Only one patient developed alloantibodies to VWF, carrying a homozygous c.3622delT. Conclusion: We report the genetic background of the entire Hungarian type 3 VWD population. A large novel deletion, most probably due to a founder effect, seems to be unique to Hungarian type 3 VWD patients with high allele frequency. In contrast to previous reports, none of the five patients homozygous for the large partial deletion developed inhibitors to VWF. This discrepancy raises the possibility of selection bias in some of the reports.

The A/T1381 polymorphism in the A1-domain of von Willebrand factor influences the affinity of von Willebrand factor for platelet glycoprotein Ibα

Many polymorphisms in vonWillebrand factor (VWF) have been reported and their association with VWF plasma levels or cardiovascular diseases has been investigated. The aim of this study was to examine whether the amino acid polymorphism A/T1381 in the VWF A1-domain would affect VWF binding to platelet GPIbalpha. Sixty-one normal individuals were genotyped at the A/T1381 locus. Twenty-one A/A1381 homozygotes, 30 A/T1381 heterozygotes and 10 T/T1381 homozygotes were identified. Remarkably, when compared to VWF of A/T1381 and A/A1381 individuals, VWF of individuals carrying the T/T1381 variant showed an increased affinity for its platelet receptor GPIbalpha under static conditions, as reflected by an increased sensitivity to low concentrations of ristocetin or botrocetin. In addition, also the rVWF-T1381 demonstrated a higher affinity for GPIbalpha than rVWF-A1381. Interestingly, this enhanced affinity of the T/T variant over the A/T and A/A variant was, however, too subtle to affect platelet adhesion under physiological flow conditions, which fully corroborates the normal haemostatic phenotype of all individuals. We demonstrate that the VWF A/T1381 polymorphism plays an important role in inter-individual variability of the affinity of VWF for GPIbalpha, with T/T variants having a higher affinity than A/A and A/T variants, at least under static conditions in vitro. Further genetic linkage and association studies are necessary to establish whether the A/T1381 polymorphism could correlate with an increased risk of thrombotic events.