Biomedical subjects

U Budde

Publications and source records attributed to U Budde.

At least 55 records · Page 3Linked to original sources

PubMed1997

Acquired von Willebrand disease in patients with high platelet counts.

For patients with high platelet counts an inverse relationship has been established between platelet counts and large vWf multimers in plasma. Platelets are currently thought to be primarily involved in the pathogenesis of this decrease in plasma large vWf multimers, presumably by binding large vWf multimers, resulting in their effective removal from the circulation and/or by degrading (platelet-bound) vWf. In patients with myeloproliferative disorders associated with high platelet counts, a decrease in large vWf multimers in plasma may potentially compromise primary hemostasis. Patients with reactive thrombocytosis exhibit a similar reduction in large vWf multimers in plasma, but their clinical course is usually not complicated by bleeding, probably as a consequence of increased circulating vWf levels due to the behavior of vWf as a reactive protein, which compensates for the relatively decreased levels of large vWf multimers in plasma.

Adult↗

PubMed1996

Defective dimerization of von Willebrand factor subunits due to a Cys-> Arg mutation in type IID von Willebrand disease.

The same heterozygous T -> C transition at nt 8567 of the von Willebrand factor (vWF) transcript was found in two unrelated patients with type III) von Willebrand disease, with no other apparent abnormality. In one family, both alleles were normal in the parents and one sister; thus, the mutation originated de novo in the proposita. The second patient also had asymptomatic parents who, however, were not available for study. The structural consequences of the identified mutation, resulting in the CyS2010 -> Arg substitution, were evaluated by expression of the vWF carboxyl-terminal domain containing residues 1366-2050. Insect cells infected with recombinant baculovirus expressing normal vWF sequence secreted a disulfide linked dimeric molecule with an apparent molecular mass of 150 kDa before reduction, yielding a single band of 80 kDa after disulfide bond reduction. In contrast, cells expressing the mutant fragment secreted a monomeric molecule of apparent molecular mass of 80 kDa, which remained unchanged after reduction. We conclude that CyS2010 is essential for normal dimerization of vWF subunits through disulfide bonding of carboxyl-terminal domains and that a heterozygous mutation in the corresponding codon is responsible for defective multimer formation in type III) von Willebrand disease.

Animals↗

PubMed1996

Prevalence of factor V Leiden in children with thrombo-embolism.

UNLABELLED: Hereditary resistance to the anticoagulatory action of activated protein C (APC resistance, APCR) was identified as a possible new thrombophilic factor in a high percentage (17%-60%) of young adults with thrombotic events. A single missense mutation (R506Q) due to a G/A transition (G1691A) in exon 10 of the factor V gene is regarded as the causative molecular defect, resulting in factor V Leiden which is correlated with APCR. Identification of this mutation by polymerase chain reaction-based methods is easy to perform and prevents pre-analytical and analytical errors in the coagulometric assay for APCR. Since the impact of this mutation in children with thrombo-embolic disease has not been determined to date, we initiated a multi centre prevalence study in two paediatric populations, with and without thrombo-embolic events. We compared 125 paediatric patients with thrombosis, divided into three different age groups (0 to < 0.5 years; > 0.5 to < 10 years; > 10 to < 18 years) with a normal population of 159 children. Although the mutation G1691A was found with an unexpectedly high prevalence of 12% in our normal controls, the prevalence was significantly higher in the age groups; 0 to < 0.5 years (26%) and > 10 to < 18 years (30%). In patients between > 0.5 and < 10 years the overall prevalence was similar to that of the control group (13%). However, in patients of this age with spontaneous thrombosis, G1691A was also a significant risk factor (5/17 approximately equal to 29%). Homozygosity for G1691A was detected in three patients but not in the control group. Including deficiencies of protein C, protein S, antithrombin, and the presence of anti-phospholipid antibodies, thrombosis was correlated with endogenous thrombophilic factors in 38/125 patients (30.4%). CONCLUSION: Our results emphasize the impact of factor V Leiden on thrombogenesis in children. However, the significance is age-dependent and may reflect the different physiology of haemostasis in the three age groups. The diagnostic workup of children with thrombosis should include tests for factor V Leiden. The correlation of factor V Leiden with the clinical course of thrombo-embolism in children is essential to establish rational guidelines for therapy and prophylaxis of APCR-related thrombosis which are not yet available.

Adolescent↗

PubMed1996

The reduction of large von Willebrand factor multimers in plasma in essential thrombocythaemia is related to the platelet count.

We have investigated the relationship between platelet count and large VWF (von Willebrand factor) multimers in the plasma of 36 patients with essential thrombocythaemia (ET) and 26 patients with reactive thrombocytosis (RT). In both ET and RT patients an inverse relationship could be established between platelet count and large VWF multimers in plasma as well in relatively decreased ristocetin cofactor/von Willebrand factor antigen and collagen binding activity/von Willebrand factor antigen ratios. A normalization of the platelet count was accompanied by restoration of a normal plasma VWF multimeric distribution. Our data suggest that increasing numbers of platelets circulating in blood result in increased removal of large VWF multimers from plasma.

Adult↗

PubMed1996

Bleeding time and platelet function in essential thrombocythemia and other myeloproliferative syndromes.

Bleeding time (BT) and platelet function tests have been widely used in patients with essential thrombocythemia (ET), with the aim to support diagnosis and to identify laboratory predictors of haemorrhagic and thrombotic complications. BT is significantly prolonged in 7-19% of ET patients and several functional abnormalities have been observed in platelet structure, biochemistry and survival. However, the attempt to relate these in vivo and in vitro platelet dysfunctions with diagnosis or clinical sequelae has been generally disappointing. Therefore, BT and platelet function tests are currently not recommended in the initial evaluation or during the follow-up of patients with ET, unless in the setting of a clinical or biological study. A noteworthy exception is represented by a subset of patients characterized by very high platelet count (> 1500 x 10(9)/L) and bleeding symptoms, who can have an acquired von Willebrand disease. In these cases, prolonged BT and abnormal multimeric pattern of von Willebrand factor are useful for diagnosing and monitoring this acquired hemorrhagic disease. BT and platelet function tests should be included in the baseline evaluation of ET patients enrolled in prospective clinical trials aiming assess their predictive role on clinical end-points.

Arachidonic Acid↗

PubMed1996

Acquired von Willebrand disease in myeloproliferative disorders.

The laboratory features of acquired von Willebrand defect (AvWD) in myeloproliferative disorders (MPD) are characterized by a very high platelet count, a normal or prolonged bleeding time, a normal factor VIII and von Willebrand factor (vWF) antigen level, but a decreased vWF-ristocetine cofactor activity and collagen binding activity with a decrease or absence of large vWF-multimers simulating a type II von Willebrand factor deficiency. Although the pathogenesis of type II AvWD in MPD remains unclear, evidence is accumulating that the increased number of platelets in the circulating blood seems to be directly responsible for the observed decrease of large vWF-multimers in plasma leading to a spontaneous bleeding tendency at very high platelet counts. This observation may have clinical implications for the use of platelet anti-aggregants such as aspirin in MPD at platelet counts in excess of 1000 to 2000 x 10(9)/L.

Aspirin↗

PubMed1996

Results of a screening for von Willebrand disease type 2N in patients with suspected haemophilia A or von Willebrand disease type 1.

A screening program for the detection of patients with von Willebrand disease type 2N (VWD 2N) was carried out in 177 unrelated patients previously diagnosed with haemophilia A and in 199 unrelated patients with VWD type 1 in comparison. By measuring the factor VIII (FVIII) binding capacity of von Willebrand factor (VWF), we detected 13 patients with VWD 2N within 8 unrelated families. The former diagnosis has been haemophilia A in 5 index patients, and VWD in the remaining 3. Included in this study were 14 patients with suspected haemophilia A, whose molecular analysis for mutations in the FVIII gene was unsuccessful. Five of them had VWD 2N. In all patients with the VWD 2N phenotype we were able to identify specific molecular defects in the corresponding DNA sequence of the FVIII binding domain of VWF. The most common defect was R91Q. Four patients from 4 families were homozygous for R91Q, six patients from 3 families were compound heterozygous for R91Q and a silent yet unidentified allele. The VWD 2N phenotype of father, daughter, and son in one family, was based on 2 different genotypes, compound heterozygosity for R91Q and VWD type 1 in the father and the daughter, and homozygosity for R91Q in the son. Two patients from one family were compound heterozygous for T28M and delta C2680-2685 in exon 18, and one patient was compound heterozygous for a novel candidate missense mutation in exon 18(E24K) and delta C2680-2685 in exon 18 which is regarded the most common defect causing severe VWD type 3. FVIII:C values of 0.07 and 0.14 IU/ml were observed in patients with the genotype T28M/delta C2680-2685 whereas in patients being homozygous or compound heterozygous for R91Q, FVIII:C was 0.19 +/- 0.071 IU/ml. In contrast to the other genotypes, E24K/delta C2680-2685 is correlated with a more severe haemophilic phenotype with a FVIII residual activity of only 0.01-0.02 IU/ml. This emphasizes the need for inclusion of the factor VIII binding assay in the diagnostic workup of suspected haemophilia A.

Adult↗

PubMed1995

Acquired von Willebrand disease in malignant peripheral neuroectodermal tumor (PNET).

In week 12 of the EICESS 92 protocol a 12-year-old boy with pelvic PNET developed acquired von Willebrand disease: bleeding time was prolonged (> 15 min) and von Willebrand factor antigen (54%) and ristocetin cofactor activity (50) were reduced. Platelet aggregations with thrombin and collagen and the number of major platelet glycoproteins/per platelet did not differ from the controls. After Haemate P (Behring Werke, Marburg, Germany) bleeding time normalized and surgery could be performed without occurrence of bleeding episodes. Three weeks after surgery bleeding time, ristocetin cofactor activity, and von Willebrand factor antigen increased to normal paediatric values. Without occurrence of further bleeding episodes the patient received another 10 courses of polychemotherapy (EICESS 92).

Bone Neoplasms↗

PubMed1995

Identification of a candidate missense mutation in a family with von Willebrand disease type IIC.

A screening project to identify candidate molecular defects causing von Willebrand disease type IIC (VWD IIC) in a German family was carried out using polymerase chain reaction (PCR) amplification of all 52 exons of the von Willebrand factor (VWF) gene, subsequent electrophoresis of single and double stranded DNA and direct sequencing of PCR products with aberrant electrophoretic patterns. Only one candidate mutation, G550R, caused by a G-->A transition, was detected in exon 14 of the pro-VWF gene sequence. This mutation was not found on 200 chromosomes of normal individuals. The propositus was homozygous for the mutation and for an extended intragenic haplotype, composed of eight polymorphic markers. Further family members were heterozygous for the mutation and were phenotypically normal or only mildly affected, in accordance with the recessive pattern of inheritance for VWD type IIC. The mutation could influence one of the presumed active centers for the suspected multimerizing enzymatic activity of pro-VWF localized in the D1 and D2 domain, which corresponds to exon 5 and exon 14 of the VWF gene.

Amino Acid Sequence↗

PubMed1995

The Thrombostat 4000. A sensitive screening test for von Willebrand's disease.

The determination of the bleeding time (BT) is an essential diagnostic tool for von Willebrand's disease (vWD). However, the standardized Simplate BT still displays many variables and disadvantages. The present study reports on the sensitivity of the in vitro bleeding test (IVBT) in 51 vWD cases of different types and severity in comparison to the Simplate BT, and the correlation of both to each other as well as to von Willebrand factor (vWF:RCo) activity. The IVBT was performed in two modifications (2 mmol/L CaCl2 and 4 mmol/L ADP) on the Thrombostat 4000. The IVBT, particularly with CaCl2, showed clearly higher sensitivity than the BT (CaCl2: 84.3%, ADP: 61.7%, CaCl2 + ADP: 86.3, BT: 52.9%). The BT even failed in one patient with severe (type 2B) and in three with moderate vWD. The IVBT only failed in very mild forms of the disease (vW-F:RCo > 25%). In addition, the IVBT with ADP showed a close correlation to the vWF:RCo activity (r2 = 0.73). The significantly lower correlation of the BT with vWF:RCo (r2 = 0.49) was particularly due to the poor results in vWD of type 2 (type 2:r2 = 0.29; types 1 and 3:r2 = 0.61). Finally, BT and IVBT-ADP correlated with each other (r2 = 0.53), a rather good correlation considering that both are complex functional tests. It can be concluded from our study that the IVBT not only may replace the BT for most applications, but is clearly superior to BT for the screening (IVBT-CaCl2) and control of therapy in vWD.

Bleeding Time↗

PubMed1994

Genetic heterogeneity of severe von Willebrand disease type III in the German population.

The genetic heterogeneity of severe von Willebrand disease (vWd) type III was estimated by analysing extended haplotypes of eleven intragenic restriction fragment length polymorphisms and one variable number of tandem repeat polymorphism in 32 patients from 28 families from Germany or of German origin. All patients were screened for gross deletions and for mutations at potential "hot spot" regions of the von Willebrand factor (vWf) gene. Disease-associated haplotypes were established in 24 families. Only a few, apparently unrelated families shared common haplotypes suggesting a considerable genetic heterogeneity in the German population of vWd type III patients. Defects causing vWd type III were identified on 14 out of 56 chromosomes (25%). Gross deletions were detected in two families. A complete homozygous deletion of the vWf gene was displayed in one patient. Another patient was compound heterozygous for a large deletion of at least 100 kb of the vWf gene with an additional, as yet unidentified, defect. One homozygous missense mutation was detected in exon 10, and two nonsense mutations were detected in exon 8 and exon 45 of the vWf gene, respectively. A frameshift mutation (delta C) in exon 18 was identified in five families and an additional frameshift mutation (delta G) was found in exon 28 in one family. It appears that delta C is the most common molecular defect in German patients with vWd type III. Its association with a number of different haplotypes suggests repeated de novo mutations at a mutation "hot spot". Evidence is presented that particular molecular defects causing vWd type III are associated with different patterns of inheritance, depending on their location within the vWf gene. Complete deletions of the gene and nonsense mutations in the pro-sequence are correlated with recessive inheritance, whereas frameshift and nonsense mutations in the gene sequence corresponding to the mature vWf subunit tend to be inherited in a dominant fashion.

Adolescent↗

PubMed1994

Von Willebrand factor multimers in virus-inactivated plasmas and F VIII concentrates.

The von Willebrand factor (vWf) stabilizes F VIII in plasma and is involved in platelet adhesion at high shear rates. While the first function is independent of the grade of multimerization, the latter needs the full range of large vWf multimers. Recently new virus-inactivated plasmas and F VIII concentrates, double-inactivated by heat and chemical treatment, became available. To evaluate whether these treatments have detrimental effects on the structure of the vWf protein we compared some of these products with fresh frozen plasma (FFP), and the Biotest F VIII concentrate with two F VIII concentrates inactivated by pasteurization or solvent/detergent (SD) treatment. The large vWf multimeres were preserved in single-donor plasmas and laboratory plasma pools. All commercial pool products showed a loss of the largest vWf multimers. While SD-treated plasma and the F VIII concentrates from Behring and Intersero contained enough large vWf multimers to allow treatment of patients with von Willebrand disease (vWd), the Biotest F VIII concentrate showed a loss of large and some intermediate multimers and is less suitable for the treatment of patients with vWd.

Electrophoresis, Agar Gel↗

PubMed1993

Elevated platelet count as a cause of abnormal von Willebrand factor multimer distribution in plasma.

Twelve of 19 patients with myeloproliferative disorders showed a decrease of absence of the largest multimers of plasma von Willebrand factor (vWF) that correlated with elevated platelet counts but not with leukocyte counts. This suggested that platelets, rather than leukocytes, may be associated with the pathogenesis of the acquired vWF abnormality seen in these patients. To examine the hypothesis further, we studied 12 patients with reactive thrombocytosis after splenectomy. Increased platelet count (> 5 x 10(11)/L) after splenectomy was associated with vWF abnormalities indistinguishable from those detected in patients with myeloproliferative disorders. Accordingly, there was an inverse correlation between proportion of large vWF multimers and platelet, but not leukocyte, number: normalization of the platelet count was accompanied by restoration of a normal vWF multimeric pattern. These findings suggest that an increase in the number of platelets circulating in blood may favor the adsorption of larger vWF multimers onto the platelet membrane, resulting in their removal from the circulation and subsequent degradation.

Adult↗

PubMed1993

von Willebrand factor mutation enhancing interaction with platelets in patients with normal multimeric structure.

Variant von Willebrand disease designated as type I New York or type Malmö is characterized by enhanced ristocetin-induced platelet agglutination with normal von Willebrand factor multimeric distribution in plasma. We have studied four such patients belonging to three unrelated families and found in all of them a unique cytosine-to-thymine transition changing the codon for Pro503 (CCG) to Leu (CTG). In three patients the mutant allele also had a silent mutation in the codon for Ser500 (TCG-->TCA). Both nucleotide changes are present in the von Willebrand factor pseudogene; however, the characterization of distinctive markers where the gene and pseudogene differ, as well as the examination of amplified cDNA derived from platelet mRNA, confirmed that the abnormality occurs in the von Willebrand factor gene of the patients. Moreover, recombinant expression of the isolated glycoprotein Ib-binding domain of von Willebrand factor provided direct evidence that the Pro503-->Leu mutation is responsible for enhanced platelet reactivity to lower ristocetin concentrations. These results define a new structural element affecting the affinity of von Willebrand factor for glycoprotein Ib and establish the molecular basis of a variant form of von Willebrand disease.

Adult↗

PubMed1991

Luminography--a new, highly sensitive visualization method for electrophoresis.

A highly sensitive method for protein visualization following electrophoresis and protein blotting was developed. The method is based on the light-emitting reaction of luminol and hydrogen peroxide catalyzed by horseradish peroxidase. The luminescent assay can be applied either to the native gel or after protein blotting, and it has a sensitivity two orders of magnitude higher than that achieved with chromogenic detection systems. Analogous to autoradiography the luminescent signal is recorded on an X-ray film with similar sensitivity. We present several examples of application emphasizing the general versatility of this innovative method.

Animals↗

PubMed1990

Luminographic detection of von Willebrand factor multimers in agarose gels and on nitrocellulose membranes.

Two methods for visualization of vWf multimers were compared with respect to sensitivity and detection of normal vWf and vWd variants IIA, IIB, IIC, IID, IIE, and IIF. Autoradiography and luminography after electrotransfer of vWf multimers onto nitrocellulose showed comparable sensitivity with vWf:Ag detectable after 1:500 dilution of normal plasma. The least sensitive method was luminography in agarose gels with vWf:Ag detectable after 1:300 dilution of normal plasma. No difference existed in the banding patterns of plasmas from patients with variant vWd.

Autoradiography↗

PubMed1990

[Hemostatic therapy in a patient with acquired von Willebrand disease and Waldenström disease with plasmapheresis and DDAVP].

A 73-year old patient suffering from IgM-paraproteinemia, plasma hyperviscosity, and acquired v. Willebrand's disease developed two consecutive bleeding episodes. He was treated by double-bag plasmapheresis and double-bag plasmapheresis combined with DDAVP. This combined therapy was superior to plasmapheresis alone, since bleeding ultimately stopped and did not reoccur. Thus, labour and expense of large volume plasma exchange and the costs of F VIII therapy could be prevented.

Aged↗

PubMed1988

Luminography--an alternative assay for detection of von Willebrand factor multimers.

A luminescence assay was adapted for detection of von Willebrand factor multimers subsequent to SDS-agarose gel electrophoresis and electroblotting onto nitrocellulose. The method is as fast as chromogenic detection methods and appears to be as sensitive as autoradiography without the disadvantages of the latter.

Electrophoresis, Agar Gel↗