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Heparin-induced thrombocytopenia (HIT) is one of the most common immune-mediated drug reactions. Immunoglobulin G-type antibodies against platelet factor 4(PF4)/heparin complexes are known to play a key role in the pathogenesis of HIT. Rapid-onset HIT is caused by the presence of circulating HIT antibodies at the time of heparin readministration. These antibodies are generally resulted from a recent immunizing exposure to heparin. Here we report a case of rapid-onset HIT developed after a septicemia without previous heparin exposure. The diagnosis of HIT as well as the presence of platelet activating and heparin-dependent antibodies was confirmed by ELISA and flow cytometric functional assays. Our case report reinforces that rapid-onset HIT cannot be excluded only based on the absence of previous heparin exposure. In addition, it may support the new theory of pre-immunization by PF4-coated bacteria in the pathomechanism of HIT. We also call the attention that venous limb gangrene can be rarely associated with HIT and thrombosis even in the absence of coumarin therapy. Furthermore, transient presence of anti-phospholipid antibodies can cause a differential diagnostic problem in the cases of HIT.

Aim To investiate the presence of antinuclear and anticytoskeletal autoantibodies in patients with systemic autoimmune and connective tissue diseases. Setting 646 sera of 322 patients with systemic sclerosis, systemic lupus eryhtematosus, mixed connective tissue disease, rheumatoid arthritis with or without secondary Sjogren's syndrome, and localized scleroderma, and 127 healthy controls were tested. Methods ‘Routine’ indirect immunofluorescence technique on HEp-2 cells. Results Antinuclear antibodies were found in 45–91.1% of the sera. Anti-intermediate filament antibodies were detected in 24 patients. Eight of the 24 cases had another (second) organ-specific autoimmune disease, mainly chronic active hepatitis or autoimmune thyroid disease.

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.

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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. Summary. Background: von Willebrand disease (VWD) Vicenza is characterized by low plasma von Willebrand factor (VWF) levels, the presence of ultra-large (UL) VWF multimers and less prominent satellite bands on multimer gels, and the heterozygous amino acid substitution R1205H in the VWF gene. The pathogenesis of VWD Vicenza has been elusive. Accelerated clearance is implicated as a cause of low VWF level. Objectives: We addressed the question, whether the presence of ultra-large multimers is a cause, or a result of accelerated VWF clearance, or whether it is an unrelated phenomenon. Patients/methods: We studied the detailed phenotype of three Hungarian patients with VWD Vicenza, expressed the mutant VWF-R1205H in 293T cells and developed a mathematical model to simulate VWF synthesis and catabolism. Results: We found that the half-life of VWF after DDAVP was approximately one-tenth of that after the administration of Haemate P, a source of exogenous wild-type (WT) VWF (0.81 ± 0.2 vs. 7.25 ± 2.38 h). An analysis of recombinant mutant VWF-R1205H showed that the biosynthesis and multimer structure of WT and mutant VWF were indistinguishable. A mathematical model of the complex interplay of VWF synthesis, clearance and cleavage showed that decreasing VWF half-life to one-tenth of normal reproduced all features of VWD Vicenza including low VWF level, ultra-large multimers and a decrease of satellite band intensity. Conclusion: We conclude that accelerated clearance alone may explain all features of VWD Vicenza.

Temporarily successful eradication therapy in acquired haemophilia with high inhibitor titer: A case report with a new protocol

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.

Type 2B von Willebrand disease in seven individuals from three different families: Phenotypic and genotypic characterization

SummaryMany 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 polymorphis mA/T1381 in the VWF A1-domain would affect VWF binding to platelet GPIbα. 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 GPIbα 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 GPIbα 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 GPIbα, 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.

SummaryWe 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 ofVWF to a captured recombinant fragment of GPIb α (rfGPIb α ,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 GPIb α . We therefore studied whether non-purified plasma glycocalicin can replace rfGPIbα in our ELISA. Of 42 anti-GPIbα monoclonal antibodies (MAbs) capable of binding plasma glycocalicin, only one MAb captured glycocalicin in a spatial orientation exposing theVWF-binding site in glycocalicin,allowing a specific and dosedependent ristocetin-mediated VWF-binding. Intra- and interassay variability were comparable with those for the rfGPIbα basedVWF:RCo ELISA.TheVWF: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 ofVWD 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 rfGPIb α basedVWF: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.

Von Willebrand's disease is caused by the quantitative or qualitative deficiency of von Willebrand factor. It is the most common congenital bleeding disorder affecting 1% of the population. The disease has a deep impact on the quality of life of the patients and in severe cases it may lead to life-threatening bleeding complications or may be fatal. A number of therapeutic options are available nowadays, and by the prophylactic use of these drugs before surgery, delivery etc., severe haemorrhagic complications can be avoided. Therefore it is extremely important to establish the correct diagnosis in time. There is no single diagnostic test due to the high variability of the disease. A number of assays are to be carried out in order to confirm the diagnosis apd identify the subtype of the disease. In this review we describe a simple approach to get to the correct diagnosis of this common bleeding disorder.

Introduction: congenital FXIII deficiency is a rare bleeding disorder with a high risk of spontaneous intracranial haemorrhage. Life-long preventive supplementation is mandatory if FXIII activity is Aim: to report the effective use of rFXIII for the treatment of a major bleeding in a patient with severe inherited FXIII-A deficiency. Patient and methods: a 19-year-old man exhibiting severe bleeding phenotype without clear diagnosis was referred to our haemostasis centre. After presenting negative haemostasis screening tests he was evaluated for FXIII deficiency by the determination of FXIII activity, FXIII-A2B2, FXIII-A and FXIII-B concentrations and by mutation analysis. Results: severe FXIII-A deficiency was established and prophylaxis with rFXIII was planned. Prior to the start of the prophylaxis he was hospitalized due to a large intramuscular hematoma (19.0x8.3x7.7 cm) in the right thigh and rFXIII, in the prophylactic dose (35 IU/kg) was administered for therapy. Following the administration of rFXIII, the pain disappeared and the swelling of the thigh considerable decreased. Complete resolution of the hematoma occurred without any additional FXIII supplementation. Results of the pharmacokinetic assessment were comparable with those described in the literature. Conclusion: rFXIII can be successfully used in acute bleeding episode of severe congenital FXIII deficiency. Disclosures No relevant conflicts of interest to declare.

Von Willebrand's disease (vWD) is the most common congenital haemorrhagic diathesis, characterized by the quantitative or qualitative disorder of von Willebrand factor (vWF). A number of methods have been used for the diagnosis of the disease, and the bleeding time determination is widely accepted as a screening test in spite of its low sensitivity. Our aim was to evaluate and compare the performance of two high shear systems (the O'Brien filter test and the PFA-100 device) in the screening and diagnosis of vWD. Thirty patients (n = 13 type 1 with mild symptoms, n = 9 type 1 with severe symptoms, n = 2 type 2A, n = 3 type 2B and n = 3 type 3 vWD) and twenty controls were investigated. In mild vWD the platelet retention in the second phase of the filter test with citrated blood showed the highest sensitivity (91.6%). The sensitivity of the PFA-100 method with collagen-epinephrine cartridges in this group was 76.9%, while the bleeding time was prolonged only in 15.4% of the cases. In severe type 1, in type 2A and type 3 all functional tests reflected the bleeding tendency of the patients. In type 2B disease the bleeding time was prolonged only when the patient was thrombocytopenic, but both high shear systems revealed the disease independently of the presence of thrombocytopenia. The overall sensitivity of the bleeding time determination was 50% compared to the 80-90% sensitivity of the O'Brien filter test and the PFA-100 system. The sensitivity values of the filter test and the PFA-100 device with collagen-epinephrine cartridges were in the same range, but the collagen-ADP cartridges showed a lower (65.5%) sensitivity, though the results were specific and had high positive predictive value. We conclude that both high shear systems are suitable for the screening of vWD, and that they are superior to the traditional bleeding time determination in case of mild disease or type 2B vWD.

SummaryIn this paper we describe two pathways leading to platelet activation by crosslinking glycoprotein (GP) Ib to the platelet Fc-receptor (FcγRII). First the monoclonal antibody (MoAb) 9C8, raised against human platelet GPIbα, dose-dependently induced platelet aggregation of citrate-anticoagulated platelet-rich plasma, an effect that can be inhibited by several activation inhibitors. The FcγRII-inhibitory MoAb IV.3 was able to prevent the aggregatory effects of MoAb 9C8, indicating that crosslinking of the antigen GPIbαto the FcγII-receptor is necessary for the activating effect. Secondly we observed a synergistic activating effect of two anti-von Willebrand factor (vWF) MoAbs 1C1E7 and B724, both known to enhance vWF binding to GPIbαin the presence of shear or ristocetin. When these antibodies are added together to PRP, platelet aggregation is induced without further need for an additional modulator. This effect can be blocked by either MoAb IV.3 or an inhibitory anti-GPIbαMoAb, indicating that again the platelet activation results from signaling through FcγRII crosslinked to vWF bound to GPIbα. In addition, both the anti-GPIbαMoAb 9C8, or the two anti-vWF MoAbs 1C1E7 and B724 induce genuine platelet activation, as evidenced by the secretion of ATP and protein tyrosine phosphorylation. These findings with both anti-GPIbαand anti-vWF MoAbs add further proof to recent reports demonstrating an interaction between the platelet receptors GPIbαand FcγRII, suggesting a role for the FcγII-receptor in GPIb-related signaling.

In the differential diagnosis of primary and secondary thrombocytosis platelet function tests may play an important role. We examined the applicability of a platelet filter test (shear-dependent platelet aggregation) as a tool, to differentiate primary thrombocytosis (cases with myeloproliferative disorders) from secondary (reactive) thrombocytosis. The test was carried out in 53 patients suffering from myeloproliferative disorders associated with primary thrombocytosis and in 21 patients with other diseases complicated by secondary thrombocytosis. Using citrate as anticoagulant, the sensitivity of the O'Brien's test proved to be 77.1%, and its specificity 94.4%. Using heparin as anticoagulant the sensitivity and specificity of the test were found to be also reliably high. Based on these studies we suggest the use of the O'Brien's filterometer as a screening test in the differential diagnosis in patients with elevated platelet count. In the case of normal results, the causes of reactive thrombocytosis should be clarified first, while with abnormal results, haematological examination of the patients should be performed.

Von Willebrand's disease (vWD) is the most common congenital haemorrhagic diathesis, characterized by the quantitative or qualitative disorder of von Willebrand factor (vWF). A number of methods have been used for the diagnosis of the disease, and the bleeding time determination is widely accepted as a screening test in spite of its low sensitivity. Our aim was to evaluate and compare the performance of two high shear systems (the O'Brien filter test and the PFA-100 device) in the screening and diagnosis of vWD. Thirty patients (n=13 type 1 with mild symptoms, n = 9 type 1 with severe symptoms, n = 2 type 2A, n = 3 type 2B and n = 3 type 3 vWD) and twenty controls were investigated. In mild vWD the platelet retention in the second phase of the filter test with citrated blood showed the highest sensitivity (91.6%). The sensitivity of the PFA-100 method with collagen-epinephrine cartridges in this group was 76.9%, while the bleeding time was prolonged only in 15.4% of the cases. In severe type 1, in type 2A and type 3 all functional tests reflected the bleeding tendency of the patients. In type 2B disease the bleeding time was prolonged only when the patient was thrombocytopenic, but both high shear systems revealed the disease independently of the presence of thrombocytopenia. The overall sensitivity of the bleeding time determination was 50% compared to the 80-90% sensitivity of the O'Brien filter test and the PFA-100 system. The sensitivity values of the filter test and the PFA-100 device with collagen-epinephrine cartridges were in the same range, but the collagen-ADP cartridges showed a lower (65.5%) sensitivity, though the results were specific and had high positive predictive value. We conclude that both high shear systems are suitable for the screening of vWD, and that they are superior to the traditional bleeding time determination in case of mild disease or type 2B vWD.

We describe a patient with positive antinuclear antibodies, polyclonal gammopathy and high level of circulating immunocomplexes, resulting in vascular purpura. In addition, the patient had a slightly prolonged bleeding time and an isolated defect of ristocetin-induced platelet aggregation (RIPA) in platelet-rich plasma (PRP). The patient’s plasma also inhibited RIPA in normal PRP and in normal platelet suspension. The activity and multimeric structure of plasmatic von Willebrand factor showed no alteration. We could demonstrate an autoantibody against platelet membrane glycoprotein (GP) Ib, using an ELISA-type assay. These data suggest an acquired Bernard-Soulier syndrome. We suggest that the patient had an immunocomplex-mediated leukocytoclastic vasculitis accompanied by production of antinuclear autoantibodies as well as the presence of an autoantibody against GPIb. The titer of the anti-GPIb antibody, however, was too low to induce significant platelet-type bleeding tendency, only laboratory alterations were found. Moreover, in a later stage of her disease, she developed a severe necrotizing vasculitis which was followed by a deep venous thrombosis.

SUMMARY The appearance of free DNA ends in the chromatin is usually considered an indication of advanced apoptosis. Unexpectedly, the nuclei of non-apoptotic cells derived from mouse thymuses could be specifically labeled by terminal transferase after proteinase K treatment of the fixed, cytocentrifuged samples. Artifactual mechanical or contaminating nucleolytic factors have been ruled out as players in the generation of free DNA ends. The phenomenon was detected in both formaldehyde- and ethanol-fixed specimens, in agarose-embedded fixed cells, and in chromatin spreads. By urea‐agarose gel electrophoresis, the average single-strand size of the DNA molecules carrying the free ends was found between 50 and 250 kb. We suggest that ss discontinuities preexisting in the fixed normal cells are unmasked by protease treatment eliciting TUNEL (terminal transferasemediated nick end-labeling) positivity. (J Histochem Cytochem 48:963‐969, 2000)

Thromboembolic complications during pregnancy are the most common causes of maternal death. Here we report on thromboembolic prophylaxis of 60 pregnancies of 32 pregnant women with familial thrombophilia. Long-term Fraxiparine (Sanofi-Chinoin) as thromboprophylaxis was applied in 26 cases throughout pregnancy. UFH (Heparin-Ca inj.) was used in 11 cases, and there were 23 pregnancies without thromboembolic prophylaxis in our patient's case histories. Artificial abortions were not included in this paper. The ratio of successful pregnancies were: with Fraxiparine: 24/26 (92.3%), with UFH (Heparin-Ca): 8/11 (72.7%), without prophylaxis: 4/23 (17.4%). In the patient group treated with Fraxiparine there were no foetopathy, thrombocytopenia or bleeding complication. LMWH is recommended for pregnant women with familial thrombophilia. According to literature data and our own experiences the doses of LMWH in patients with familial thrombophilia, and -antiphospholipid syndrome, and -artificial heart value are suggested.

In the differential diagnosis of primary and secondary thrombocytosis, platelet function test can be used. We have examined the possible role of O'Brien's filter test in the differentiation of primary and secondary thrombocytosis in 53 patients with myeloproliferative diseases with primary thrombocytosis and in 21 patients with other disorders complicated by secondary thrombocytosis. By using heparin as an anticoagulant, the sensitivity of O'Brien's filter test proved to be 75%, and it's specificity was 85.7%. In blood samples anticoagulated with citrate, the sensitivity was 100% and specificity 83.3%. Based on these studies we suggest the use of O'Brien's filterometer as a screening test in the differential diagnosis in patients with elevated (400 x 10(9)/L) platelet count. In case of normal results, the causes of reactive thrombocytosis should be cleared first, while with pathologic results, haematological examination of the patients should be performed.

The single stranded [3H]oligonucleotide uptake by HL-60 human promyelocyte and K562 human erythroleukemia cells was stimulated 20–45-fold by DUSF (DNA uptake stimulating protein), and this effect was drastically reduced (to 1.6–13×) if the cells were induced to differentiate. The oligonucleotide uptake stimulating effect of DUSF was not altered in HL-60 and K562 cells, if the proliferation of the cells was inhibited by hydroxyurea (HU) treatment. The oligonucleotide uptake by separated granulocytes and mononuclear cells from healthy donors was not stimulated by DUSF, while the uptake of oligonucleotides by myeloid and lymphoid leukemic cells was greatly stimulated (10–15×). The uptake of oligonucleotides by differentiated mononuclear cells of healthy donors could not be stimulated by DUSF, but the oligonucleotide uptake was greatly increased (11×) by DUSF if the cells were subjected to blast transformation.

The presence of the DNA uptake stimulating protein (designated earlier as DUSF by Schablik and Szabo (1981) FEMS Microbiol. Lett. 10, 395-397) was demonstrated on the surface of Neurospora crassa (FGSC 1118, slime) fungal cells as well as on HEp-2 and K562 human and F4N mouse tumor cells by immunofluorescence microscopy. DUSF markedly enhanced the uptake of both macromolecular [3H]DNA and of [3H]oligonucleotides by K562, HL-60 and also by DD human leukemia cells. Polyclonal anti-DUSF antibodies inhibited both the basal and stimulated [3H]oligonucleotide uptake by K562 cells. DNA-DNA hybridization has shown that the uptake of linearized pBR322 plasmid DNA into N. crassa and K562 cells was stimulated by DUSF, increasing proportionally with the length of incubation time. Unfragmented plasmid molecules were recovered from the cells. It is assumed that DUSF (or related proteins) may play a role in the physiological uptake of oligonucleotides and DNA not only in N. crassa but also in--at least some--mammalian cells.