Antonella Bertomoro

A shorter von Willebrand factor survival in O blood group subjects explains how ABO determinants influence plasma von Willebrand factor

ABO blood groups greatly influence circulating von Willebrand factor (VWF) levels, and O group subjects have lower VWF values. In this study, we investigated whether ABO groups affect VWF survival by monitoring the post-DDAVP (1-desamino-8-d arginine vasopressin) time courses of VWF antigen (VWF:Ag), VWF collagen binding (VWF:CB), and factor VIII (FVIII) in 47 healthy subjects (28 O and 19 non-O blood groups). The elimination half-life (T1/2el) of VWF was found significantly shorter in O than in non-O subjects (10.0+/-0.8 hours vs 25.5+/-5.3 hours, respectively; P<.01), as was the T1/2el of VWF:CB (7.9+/-0.5 hours vs 20.9+/-4.5 hours; P<.01). A direct linear correlation was found between basal VWF:Ag and T1/2el, subjects with higher VWF levels having longer-surviving VWF. ABO blood groups appeared to strongly influence VWF clearance, but not its synthesis or release from endothelial cells. The VWF propeptide to VWF:Ag ratio, useful for predicting an increased VWF clearance, was found significantly higher in O than in non-O individuals (1.6+/-0.1 vs 1.2+/-0.5, P<.001), with values that correlated inversely with T1/2el (P<.001). Based on these findings, we conclude that the lower VWF values in O group individuals is attributable to a shorter VWF survival and circulating VWF values are strongly influenced by its half-life.

Do hemophilia A and von Willebrand disease protect against carotid atherosclerosis? A comparative study between coagulopathics and normal subjects by means of carotid echo-color Doppler scan.

Atherosclerosis is a multifactorial disease caused by genetic and environmental factors with important clinical sequelae. The aim of this study was to evaluate the degree of carotid atherosclerosis by echo-color Doppler scan in a group of patients affected by hemophilia A and von Willebrand dis ease versus a group of normal subjects apparently free of ath erosclerotic risk factors. All coagulopathics and normal patients who came to our Internal Medicine Department (Padua Hospi tal) underwent physical exam, blood analysis, standard electro cardiogram, chest x-ray, echo-color Doppler scan, and a thor ough history. We examined 156 subjects, 76 coagulopathics (46 men, 30 women) and 77 normals (37 men, 40 women). Coagulopathics were affected by hypertension in 28.9% of cases, diabetes mellitus in 6.5%, dislipidemia in 17.1%, smoke in 39.4%, and obesity in 36.8% (p < .05). Echo-color Doppler scan revealed carotid plaques in 27.2% of control patients ver sus 13.1% of coagulopathics (p < .05). Hemophilics and sub jects with von Willebrand disease with a more serious illness had fewer plaques than those with lighter defects. Coagulopath ics showed 23.6% of the plaques we revealed on the whole, versus 76.3% of control subjects (p < .01), with a lighter degree of stenosis (p < .01). Our data demonstrate that patients with hemophilia A and von Willebrand disease have fewer carotid plaques and a smaller degree of carotid stenosis than normal subjects of the same sex and age. These data seem to strengthen the hypothesis that blood coagulation defects may allow pro tection against carotid atherosclerosis and its sequelae.

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.

Reduced survival of type 2B von Willebrand factor, irrespective of large multimer representation or thrombocytopenia

Background Type 2B von Willebrand factor (VWF) is characterized by gain of function mutations in the A1 domain inducing a greater affinity for platelet GPIb, possibly associated with the disappearance of large VWF multimers and thrombocytopenia. Design and Methods VWF survival was explored using 1-desamino-8-D-arginine vasopressin (DDAVP) in 18 patients with type 2B von Willebrand disease (VWD) and compared with their platelet count and large VWF multimer representation. Results A similarly significant shorter VWF survival, expressed as T1/2elimination (T1/2el), was observed in patients lacking large VWF multimers (type 2B) and in those with a normal multimer pattern (atypical type 2B) (4.47±0.41 h and 4.87±0.9 h, respectively, vs. normal 15.53±2.17 h) due mainly to a greater VWF clearance. The half-life of large VWF multimers, explored by VWF collagen binding (VWF:CB) activity, was likewise reduced. The similarly reduced VWF half-life was also confirmed by the increase in the VWF propeptide ratio (a useful tool for exploring VWF survival) which was found to be the same in type 2B and atypical type 2B patients. The post-DDAVP drop in platelet count occurred in all patients lacking large multimers but not in those with a normal multimer pattern. A correlation was always found between pre- and/or post-DDAVP thrombocytopenia and the lack of large VWF multimers in type 2B VWD while these were unrelated to the reduced VWF half-life. Conclusions In addition to demonstrating that a shorter VWF survival contributes to the type 2B and atypical type 2B VWD phenotype, our findings suggest that VWF clearance and proteolysis are independent phenomena.

Abnormal collagen binding activity of 2A von Willebrand factor: evidence that the defect depends only on the lack of large multimers.

It is well established that the large von Willebrand factor (vWf) multimers bind with high affinity to the extracellular matrix. To explore the different roles of intermediate and large vWf multimers, we studied the collagen-binding activity (vWf:CBA) of 2A vWf under nonflowing conditions in relation to the multimer organization of the molecule. Regardless of the anticoagulant used for blood collection, vWf:CBA was significantly decreased, in 4 patients with 2A von Willebrands disease (vWd), in accordance with the lack of high and intermediate vWf multimers. After 1-deamino-8-D-arginine vasopressin (DDAVP) infusion, the appearance of circulating large and unusually large vWf multimers, in samples collected in the presence of protease inhibitors, induced a complete normalization of vWf:CBA. The peak was observed 15 minutes after DDAVP, when large and unusually large multimers were maximally represented. These effects were transient because vWf:CBA decreased after 60 minutes, even though values were still significantly higher than pre-DDAVP figures; at the same time, large vWf multimers appeared to be decreased. In contrast, samples anticoagulated with sodium citrate after DDAVP did not show a normalized vWf multimer pattern and were characterized by a persistently decreased vWf:CBA. Moreover, in all of the patients studied, platelet vWf presented normal vWf:CBA values in accordance with the normal levels and multimer organization of the vWf molecule. Our findings indicate that the collagen-binding defect displayed in vitro by type 2A vWf depends only on the lack of circulating large vWf multimers. Moreover, the observation of normal platelet vWf:CBA seems to indicate a primary role of plasma rather than platelet vWf in assuring platelet plug formation.

A novel von Willebrand factor mutation (I1372S) associated with type 2B-like von Willebrand disease: An elusive phenotype and a difficult diagnosis

Mutations in the A1 domain of von Willebrand factor (VWF) may be associated with gain of function in the VWF-platelet GPIb interaction and consumption of large VWF multimers, as seen in type 2B von Willebrand disease (VWD). We report a new VWF abnormality associated with greater VWF-GPIb interaction in the presence of all VWF multimers. The index case is a woman with a lifelong history of bleeding, found hyperresponsive to ristocetin with spontaneous platelet aggregation (SPA). She had normal factor VIII, VWF:Ag, VWF:RCo and VWF:CB levels, normal VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag ratios, and a full panel of plasma and platelet VWF multimers. A missense mutation (4115T>G) was found in exon 28 of the VWF gene, which replaced a isoleucine with a serine at position 1372 of pre-pro-VWF (I1372S) at heterozygous level. Recombinant VWF carrying the I1372S mutation and showing a normal VWF multimer organisation was capable of inducing SPA on normal platelet-rich plasma (unlike wild-type VWF), as well as a hyper-response to ristocetin in the same platelets (0.6 mg/ml ristocetin vs. 1.2 of wild-type VWF). The new I1372S VWF mutation, characterized by SPA and hyper-responsiveness to ristocetin thus has some of the features of type 2B VWD, but not the lack of large VWF multimers, so we defined this variant as type 2B-like VWD. Why I1372S VWF is associated with bleeding symptoms, despite normal VWF levels and multimer organisation, remains to be seen.

Acquired factor VIII:C inhibitor in a patient with Sjögren's syndrome: successful treatment with steroid and immunosuppressive therapy.

A 57-year-old woman affected with Sjögrens syndrome without bleeding history developed spontaneous hematomas at the arms, the left foot and the thigh, cutaneous hemorrhages and hematuria. Routine coagulation tests showed a prolongation of activated partial thromboplastin time associated with a marked reduction of factor VIII activity (VIII: C 5%). Other deficiencies of blood coagulation factors, especially von Willebrand factor, were excluded. Measurement of factor VIII inhibitor revealed an activity of 26.4 Bethesda units/ml. These findings were consistent with the diagnosis of acquired hemophilia A due to the presence of a factor VIII inhibitor. The patient was treated with a combination of prednisone and azathioprine. The therapy led, in a few months, to a significant reduction of factor VIII: C inhibitor and she did not require replacement therapy. Furthermore, there was a complete remission of the bleeding tendency. Long-term therapy for about 3 years induced the complete disappearance of the inhibitor and a full normalization of coagulation tests.

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.

Combined partial exon skipping and cryptic splice site activation as a new molecular mechanism for recessive type 1 von Willebrand disease

We describe the complex picture associated with a mutated splice junction in intron 13 of von Willebrand factor (VWF) gene. The proband, characterized by a marked decrease in plasma and platelet VWF and near normal multimer organization, was classified as recessive type 1 von Willebrand disease (VWD). Genetic analysis demonstrated that he was homozygous for the 1534-3C > A mutation in the consensus sequence of the acceptor splicing site of intron 13 of the VWF gene. Platelet mRNA analysis documented three VWF transcripts: a wild type generated by the correct recognition of the mutated splice site, a smaller transcript not containing exon 14, and a longer one that, in addition to exons 13 and 14, included a 62bp fragment corresponding to the end of intron 13. The small transcript derives from the skipping of exon 14, the long one from the activation of a cryptic splice site in intron 13; both show a premature stop codon in VWF propeptide, so the proband VWF derives entirely from the correct splice site recognition. Combined incomplete exon skipping and cryptic splice site activation are first recognized in VWD. Since the 1534-3C > A mutation does not abolish the normal processing of mRNA, it is unlikely to be found in type 3 VWD. This mutation therefore appears to be peculiar to type 1 VWD.

Lack of multimer organization of von Willebrand factor in an acquired von Willebrand syndrome

Summary. We report a case of acquired von Willebrand syndrome (AVWS) in a 20‐year‐old‐woman with systemic lupus erythematosus, in whom severe bleeding complications followed kidney biopsy. Coagulation studies demonstrated undetectable levels of ristocetin‐induced platelet aggregation (RIPA), von Willebrand factor antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo), associated with significantly prolonged bleeding time; unlike type 3 von Willebrand disease (VWD), platelet VWF was reduced but not undetectable. The plasma VWF multimer pattern was characterized by the presence of only two bands, one of low molecular weight (MW) running as the protomer of plasma VWF in normals, the other of abnormally high MW without detectable intermediate multimers; this pattern resembles that of VWF present in endothelial cells. A search for an anti‐VWF antibody demonstrated the presence of an inhibitor at high titre. This anti‐VWF antibody did not interfere in the interaction of VWF with platelet glycoprotein (GP) Ib through the A1 domain, and did not react with the A2 domain of VWF; instead, it seemed to modify the relative representation of high and low MW VWF multimers released by normal human umbilical vein endothelial cells (HUVEC). After Azathioprine and corticosteroid treatment, the anti‐VWF antibody disappeared and the patients haemostatic profile normalized, except for the platelet VWF content which still remained decreased. We suggest that the anti‐VWF antibody present in the AVWS described compromised both circulating VWF levels and their multimeric organization, inducing the maintenance of the multimer structure that VWF normally has before or in the early phase after secretion from endothelial cells.