Von Willebrand factor propeptide makes it easy to identify the shorter Von Willebrand factor survival in patients with type 1 and type Vicenza von Willebrand disease.

Reduced von Willebrand factor (VWF) half-life has been suggested as a new pathogenic mechanism in von Willebrand disease (VWD). The usefulness of VWF propeptide (VWFpp) in exploring VWF half-life was assessed in 22 type 1 and 14 type Vicenza VWD patients, and in 30 normal subjects, by comparing the findings on post-Desmopressin (DDAVP) VWF t(1/2) elimination (t(1/2el)). The VWFpp/VWF antigen ratio (VWFpp ratio) was dramatically increased in type Vicenza VWD (13.02 +/- 0.49) when compared to normal subjects (1.45 +/- 0.06), whereas it appeared to be normal in all type 1 VWD patients (1.56 +/- 0.7), except for the four carrying the C1130F mutation (4.69 +/- 0.67). A very short VWF t(1/2el) was found in type Vicenza VWD (1.3 +/- 0.2 h), while all type 1 VWD patients had a t(1/2el) similar to that of the controls (11.6 +/- 1.4 and 15.4 +/- 2.5 h respectively), except for the four patients carrying the C1130F mutation, who had a significantly shorter VWF survival (4.1 +/- 0.2 h). A significant inverse correlation emerged between VWFpp ratio and VWF t(1/2el) in both VWD patients and normal subjects. The VWFpp ratio thus seemed very useful for distinguishing between type 1 VWD cases with a normal and a reduced VWF survival, as well as for identifying type Vicenza VWD.

Identifying carriers of type 2N von Willebrand disease: procedures and significance.

The defective FVIII carrier function of von Willebrand factor (VWF) identifies type 2N von Willebrand disease (VWD), a variant with a pattern resembling hemophilia A. Type 2N characterization is based on the evaluation of the capacity of VWF to bind exogenous FVIII (VWF:FVIIIB). Here we report on a retrospective evaluation of hemostatic laboratory parameters most useful in detecting type 2N carriers. The diagnostic capacity of aPTT, FVIII, VWF:Ag, FVIII/VWF:Ag ratio, VWF:FVIIIB and VWF:FVIIIB/VWF:Ag ratio was evaluated in 21 type 2N VWD carriers. Twenty subjects were heterozygous for the R854Q mutation, one was heterozygous for the R760C missense mutation, which interferes with cleavage of the VWF propeptide. We found that prolongation of aPTT and decrease in FVIII and FVIII/VWF:Ag ratio were not frequent findings in type 2N carriers. The same was true for VWF:FVIIIB which was not always abnormal. On the contrary, VWF:FVIIIB/VWF:Ag ratio was always defective and its values were not related with FVIII and FVIII/VWF:Ag ratio or influenced by plasma VWF concentration. Given these results, we attribute the greatest significance to VWF:FVIIIB/VWF:Ag ratio in the diagnosis of type 2N defects, and only search for type 2N mutations, to validate the diagnosis, if the ratio proves abnormal.

Plasma and platelet von Willebrand factor abnormalities in patients with uremia: lack of correlation with uremic bleeding.

Chronic renal failure often is associated with abnormal bleeding that may represent an important complication of this disorder. The hemorrhagic tendency currently is attributed to altered primary hemostasis, mainly platelet dysfunction. However, von Willebrand factor (vWF) also seems to be involved, even though the nature of its abnormalities is still controversial. To gain insight into the role of vWF in determining uremic bleeding, we studied 11 patients with stable, chronic renal failure. We found a significant increase in plasma factor VIII (FVIII), vWF:antigen (Ag), and vWF:ristocetin cofactor (Rco) levels, associated with a mean decrease in platelet vWF:Ag. Plasma vWF multimer pattern was characterized by increased representation of all oligomers in all patients, but five patients also showed a slight decrease in large vWF multimers. In addition, platelet vWF multimer pattern displayed a decrease in all components, especially those with high molecular weight. Despite normal bleeding time, collagen-induced platelet aggregation was defective in almost all patients, whereas vWF collagen binding capacity was normal. The levels of glycocalicin, the circulating fragment of glycoprotein Ib-IX, the major platelet vWF receptor, were also normal. In six patients who also were studied after initiation of dialysis, collagen-induced platelet aggregation was impaired further. Moreover, plasma vWF, and especially FVIII levels, were increased additionally, in association with a normalized platelet vWF content and an improved vWF multimer pattern. The results suggest that vWF abnormalities are present in uremia. Moreover, thrombopathy caused by impaired collagen-induced platelet aggregation is constantly present and apparently not improved by dialytic treatment.

Von Willebrand factor collagen binding activity in the diagnosis of von Willebrand disease: an alternative to ristocetin co-factor activity?

The capability of von Willebrand factor (VWF) to bind platelet glycoprotein Ib (GPIb) and promote platelet plug formation is currently evaluated in vitro using the ristocetin co-factor activity (VWF:RCo) assay. The replacement of this cumbersome and not always reproducible test with the collagen binding activity of VWF (VWF:CBA) has been attempted with controversial results. To evaluate the capacity of VWF:CBA to identify classic and variant von Willebrand disease (VWD) compared with VWF:RCo, we studied 10 type 2A and 12 type 2B VWD patients, together with 30 type 1 VWD patients with reduced platelet VWF content. In both 2A and 2B VWD, VWF:CBA and VWF:RCo were decreased, but that of VWF:CBA was more consistent. The difference was more evident when values were expressed as a ratio, obtained by normalizing VWF:CBA and VWF:RCo with the VWF antigen value; the ratio for VWF:CBA was always below 0.2, while that for VWF:RCo was greater than 0.4, and in no patient was the VWF:CBA value higher than VWF:RCo. In contrast, in type 1 VWD, the decrease in VWF:CBA was similar to that seen in VWF:RCo with the ratios always within the normal range. To better investigate the relationship between VWF:CBA and VWF:RCo, and the representation of large/intermediate VWF multimers, to which both tests are sensitive, 1-deamino-cys-8-D-arginine-vasopressin (DDAVP) was infused in type 2A and 2B VWD patients. The differences between the two tests were even more evident after DDAVP, and in type 2A, even though large multimers were persistently decreased, VWF:RCo was normalized, while VWF:CBA remained defective. These findings clearly indicate that VWF:CBA detects the absence of large and intermediate VWF multimers better than VWF:RCo. Hence, we suggest adding VWF:CBA to the panel of tests employed in the diagnosis of VWD. Moreover, owing to the difficulty in performing VWF:RCo and its low reproducibility, we suggest that, when necessary, VWF:CBA may be substituted for VWF:RCo.

Type 2M von Willebrand disease variant characterized by abnormal von willebrand factor multimerization.

We describe a von Willebrand disease (VWD) variant characterized by low plasma and platelet von Willebrand factor (VWF), impaired ristocetin-induced VWF binding to platelet glycoprotein Ib (GPIb), and abnormal VWF multimer pattern not associated with the absence of large forms. A C-to-T transition at nucleotide 4120 in exon 28 of the VWF gene was found; this mutation introduces a cysteine at the codon for Arg 611 of mature VWF. In addition to the decreased factor VIII (FVIII) and VWF levels, ristocetin-induced platelet aggregation (RIPA) was almost absent, and VWF ristocetin cofactor activity (VWF:RCo) was significantly more decreased than VWF antigen. The patients (mother and son) also showed a defect in VWF collagen-binding activity. Plasma VWF multimers were decreased, with no limit in the size of large forms, and the normal discontinuous multimer organization was replaced by a diffuse smear, especially detectable in the large forms. This picture was emphasized by 1-deamino-8-D -arginine vasopressin (DDAVP) infusion, so that the abnormal VWF multimers appeared to have a molecular weight higher than those present in, or released by, human umbilical vein endothelial cells. DDAVP also increased FVIII and VWF levels but did not normalize the GPIb-dependent VWF functions expressed as RIPA and VWF:RCo. We include this variant in type 2M VWD, focusing on the abnormality in GPIb-dependent VWF function. We advance that this defect depends on the mutation in the GPIb binding domain of VWF rather than the abnormal VWF multimer pattern.

Gene disruption and basic phenotypic analysis of nine novel yeast genes from chromosome XIV.

In this work, we describe the disruption of nine ORFs of S. cerevisiae (YNL123w, YNL119w, YNL115c, YNL108c, YNL110c, YNL124w, YNL233w, YNL232w and YNL231c) in two genetic backgrounds: FY1679 and CEN.PK2. For the construction of the deletant strains, we used the strategy of short flanking homology (SFH) PCR. The SFH-deletion cassette was made by PCR amplification of the KanMX4 module with primers containing a 5' region of 40 bases homologous to the target yeast gene and with a 3' region of 20 bases homologous to pFA6a-KanMX4 MCS. Sporulation and tetrad analysis of heterozygous deletants revealed that YNL110c, YNL124w and YNL232w are essential genes. The subcellular localization of the protein encoded by the essential gene YNL110c was investigated using the green fluorescent protein (GFP) approach, revealing a nuclear pattern. Basic phenotypic analysis of the non-essential genes revealed that the growth of ynl119w delta haploid cells was severely affected at 37 degrees C in N3 medium, indicating that this gene is required at high temperatures with glycerol as a non-fermentable substrate. The ynl233w delta haploid cells also showed a particular phenotype under light microscopy and were studied in detail in a separate work.

Abnormalities of von Willebrand factor are also part of the prothrombotic state of Cushing's syndrome.

Glucocorticoids are known to increase plasma concentrations of factor VIII (FVIII) and von Willebrand factor (vWF), and their administration is associated with an increased incidence of thrombotic complications. Because Cushing's syndrome is characterized by an endogenous increase in glucocorticoids, we studied levels of FVIII and vWF in 20 patients with Cushing's syndrome. Plasma levels of FVIII and vWF were found to be markedly increased. Moreover, the molecular organization of plasma vWF appeared to have been altered by the presence of unusually large multimers, normally present only in the cellular compartments. Spontaneous platelet aggregation and hyperresponsiveness to ristocetin were also observed. All patients underwent therapeutic surgery. Within 1 month of the intervention, regardless of its efficacy as evaluated by the assay of plasma and urinary cortisol, an additional significant increase in levels of FVIII and vWF was observed, with a concomitant more pronounced representation of abnormally large vWF multimers in circulation. In the cured patients, a progressive decrease in the levels of FVIII and vWF was observed, beginning in the third month after surgery, with complete normalization of the pattern within 12 months of surgery; a concomitant improvement in the plasma vWF multimer pattern was also observed. In contrast, no significant changes in FVIII or vWF were found in patients with persistent Cushing's syndrome. Our findings emphasize that vWF abnormalities are also part of the prothrombotic state of Cushing's syndrome. Moreover, this study also identified a period of additional thrombotic risk immediately after surgery, as a result of the worsening of the hemostatic pattern.

The DNA sequence of cosmid 14-13b from chromosome XIV of Saccharomyces cerevisiae reveals an unusually high number of overlapping open reading frames.

This work is part of the effort for sequencing chromosome XIV of Saccharomyces cerevisiae. Cosmid 14-13b contains a 37.8 kb insert derived from a partial Sau3A digestion of the genome, cloned into the BamHI site of the vector Pou6. The strategy used for sequencing is based on the fragmentation of the whole cosmid by sonication, followed by shotgun sequencing on an Applied Biosystem DNA sequencer. The clones with inserts corresponding to the vector were identified by dot-blot hybridization, without the need of sequencing. The analysis of the DNA sequence reveals 29 open reading frames (ORFs) longer than 300 bases. Nine ORFs are internal to some other ORFs. Similarity searches against DNA and protein data banks show that six ORFs correspond to already known yeast genes (OMP1, PSU1, MLS1, RPC19, DBP2, CYB5) and one ORF matches the sequence of a putative yeast gene (ESBP6).