A framework for the optimal design of a minimum set of clinical trials to characterize von Willebrand disease.

BACKGROUND AND OBJECTIVE: Von Willebrand disease (VWD) is one of the most severe inherited bleeding disorder in humans, and it is associated with a qualitative and/or quantitative deficiency of von Willebrand factor, a multimeric glycoprotein fundamental in the coagulation process. At present, the diagnosis of VWD is extremely challenging and mostly based on clinical experience. Kinetic models have been recently proposed and applied to help in the diagnosis and characterization of VWD, but the complexity of these models is such that they requires long and stressful clinical tests, such as the desmopressin response test (DDAVP), to achieve a satisfactory estimation of the individual haemostatic parameters. The goal of this paper is to design a minimal set of clinical tests for the identification of akinetic model to decrease the required time and effort for the characterization and diagnosis of VWD. METHODS: A model proposed in the literature is used as a building block to develop a new model, where response surface methodologies have been applied to determine a set of explicit correlations linkingkinetic model parameters to basal clinical trials data. Model-based design of experiments techniques are then used to devise optimally informative tests for model validation which are shorter and easier to implement. RESULTS: Results show an excellent agreement between the original model for VWD and the new proposed model on representing healthy and VWD subjects. The application of experimental design techniques for model validation shows the possibility to drastically reduce the duration of DDAVP tests from 24 h-3 h by exploiting complementary information from basal clinical tests. CONCLUSIONS: Basal clinical tests can be used alongside a time-reduced DDAVP test to validate pharmacokinetic models for a quantitative characterisation of subjects affected by VWD and for a quicker and easier diagnosis of the disease.

Type 2N von Willebrand disease: Characterization and diagnostic difficulties.

INTRODUCTION: An abnormal factor VIII (FVIII) binding capacity of von Willebrand factor (VWF) identifies type 2N von Willebrand disease (VWD). Type 2N VWD patients are identified by means of the VWF FVIII binding (VWF:FVIIIB) assay, and especially their VWF:FVIIIB/VWF:Ag ratio (VWF:FVIIIB ratio). AIM: We report on our 15-year experience of diagnosing type 2N VWD. METHODS: We have performed 2178 VWF:FVIIIB assays in bleeders and normal subjects. RESULTS: von Willebrand factor (VWF):FVIIIB was reduced in 682, but only 60 had low VWF:FVIIIB ratios (<0.74). Among nine patients who had a VWF:FVIIIB ratio below 0.3, four had normal VWF levels and were homozygotes for the p.R854Q mutation; the other five had low VWF levels due to a quantitative VWF mutation combined with p.R854Q. The VWF:FVIIIB ratio ranged between 0.3 and 0.73 in 51 subjects; 34 of them were heterozygotes for the p.R854Q mutation, while one carried the p.R760C. The heterozygotes for type 2N included subjects with or without bleeding symptoms, the former with significantly lower mean VWF levels than the latter. Among the 116 normal subjects tested, six were heterozygotes for the p.R854Q mutation (all asymptomatic). CONCLUSIONS: The prevalence of type 2N in our VWD cohort was 2.5%, and 5.2% of the general population in Northeast Italy was found heterozygous for the p.R854Q mutation. It might be difficult to reveal a type 2N defect using routine tests alone, especially when it is combined with a quantitative VWF mutation. Accordingly, we always recommend VWF:FVIIIB assay in the diagnostic workup of VWD.

Usefulness of the Total Thrombus-Formation Analysis System (T-TAS) in the diagnosis and characterization of von Willebrand disease.

INTRODUCTION: The heterogeneity of von Willebrand disease (VWD) makes its diagnosis a difficult task. METHODS: We report here on the usefulness of a microchip-based flow-chamber system, the total thrombus-formation analysis system (T-TAS), in the identification and characterization of VWD. Thirty VWD patients and 20 healthy subjects were studied with the T-TAS platelet (PL) and atherome (AR) microchips developed for the in vitro assessment of platelet thrombus formation and fibrin-rich platelet thrombus formation respectively. RESULTS: Samples from severe type 1 VWD, characterized by von Willebrand factor (VWF) levels below 10 U dL-1 , failed to occlude either the PL or the AR chip capillaries, while the occlusion times were normal in patients with mild type 1 VWD (VWF above 25 U dL-1 ). PL and/or AR chip occlusion occurred, but took longer than normal, for samples from type Vicenza and type 1 VWD patients, whose VWF levels ranged between 10 and 25 U dL-1 . No PL or AR chip capillary occlusion was seen for samples from patients with type 2A or 2B VWD featuring the absence of large VWF multimers, whereas no abnormalities emerged for type 2B patients with normal multimer patterns. CONCLUSION: The T-TAS appears to be sensitive mainly to plasma VWF concentration and the presence of large multimers. Failure of the PL and AR chips to become occluded points to a lack of large VWF multimers, or type 1 VWD with VWF levels below 10 U dL-1 . Although the T-TAS does not assure a precise VWD diagnosis, it does point us in the right direction, and thus seems a useful global preliminary test.

Severe, recessive type 1 is a discrete form of von Willebrand disease: the lesson learned from the c.1534-3C>A von Willebrand factor mutation.

Type 1 von Willebrand disease (VWD) is transmitted mainly as a dominant trait - especially in forms involving von Willebrand factor (VWF) levels below 20 U/dL - and less frequently as a recessive trait. In the latter case, mutations at heterozygous level may be associated with type 3 carrier status, while mutations at homozygous or compound heterozygous level often coincide with type 3 VWD. Here we present a recessive, severe type 1 form as a distinct type of VWD. Eight patients with severe type 1 VWD belonging to 7 unrelated families were studied. They had VWF levels below 10 U/dL, FVIII higher than 10 U/dL, and a significantly lower than normal platelet VWF content. All patients were homozygous or compound heterozygous for the c.1534-3C>A VWF mutation, that simultaneously induces the skipping of exon 14, the activation of a cryptic splice site, and a normal VWF gene transcription. This means that one of the three different mRNA generated assures the synthesis of normal VWF. The probands' relatives who were heterozygous for the c.1534-3C>A mutation always had low platelet VWF levels, sometimes with circulating VWF levels within normal range. This finding confirms the utility of measuring platelet VWF content to identify an abnormal VWF synthesis. Because the c.1534-3C>A mutation impairs, but does not abolish normal mRNA processing, it may never cause type 3 VWD. We propose a model of severe recessive type 1 VWF defect associated with mutations that sporadically go undetected by the cells' molecular machinery, as the c.1534-3C>A VWF mutation. BULLET POINTS: What is known about this topic? - Type 1 VWD is transmitted mainly as a dominant trait. - Recessive type 1 mutations at homozygous or compound heterozygous level are often associated with type 3 VWD, and at heterozygous level with type 3 VWD carrier status. What does this paper add? - There are quantitative VWF mutations, such as c.1534-3C>A, that impair, but do not abolish normal mRNA processing. - The c.1534-3C>A VWF mutation simultaneously induces the skipping of exon 14, the activation of a cryptic splice site, and a normal VWF gene transcription. - The c.1534-3C>A mutation is the archetype of mutations that cause severe recessive type 1 VWD, but never type 3 VWD. - Recessive, severe type 1 appears to be a distinct form of VWD.

Loss of cysteine 584 impairs the storage and release, but not the synthesis of von Willebrand factor.

Cysteines play a key part in von Willebrand factor (VWF) dimerisation and polymerisation, and their loss may severely affect VWF structure and function. We report on three patients with type 3 von Willebrand disease carrying the new c.1751G>T missense mutation that induces the substitution of cysteine 584 by phenylalanine (C584F), and the deletion of seven nucleotides in exon 7 (c.729_735del), producing a premature stop codon at position 454 (E244Lfs*211). VWF was almost undetectable in the patients' plasma and platelets, while a single, poorly represented, oligomer emerged on plasma VWF multimer analysis. No post-DDAVP increase in VWF and factor VIII was observed. Expressing human recombinant C584F-VWF in HEK293T cells showed that C584F-VWF was synthesised and multimerised but not secreted - apart from the first oligomer, which was slightly represented in the conditioned medium, with a pattern similar to the patients' plasma VWF. The in vitro expression of the E244Lfs*211-VWF revealed a defective synthesis of the mutated VWF, with a behavior typical of loss of function mutations. Cellular trafficking, investigated in HEK293 cells, indicated a normal C584F-VWF content in the endoplasmic reticulum and Golgi apparatus, confirming the synthesis and multimerisation of C584F-VWF. No pseudo-Weibel Palade bodies were demonstrable, however, suggesting that C584F mutation impairs the storage of C584F-VWF. These findings point to cysteine 584 having a role in the release of VWF and its targeting to pseudo-Weibel Palade bodies in vitro, as well as in its storage and release by endothelial cells in vivo.

Venous thromboembolism in patients with Cushing's syndrome: need of a careful investigation of the prothrombotic risk profile.

A high incidence of venous thromboembolic (VTE) complications has been reported in Cushing's syndrome (CS), mostly post-operatively and attributable to hypercoagulability. The prevalence of symptomatic VTE was investigated retrospectively in 58 consecutive CS patients in relation to acquired and genetic thrombotic risk factors. Eight CS patients (14 %) developed VTE (group A), 3 of them related and 5 unrelated to surgery. These patients had higher urinary free cortisol (p = 0.01) and VWF levels (p = 0.02) than the 50 patients without VTE (group B), as well an increase in the hemostatically more efficient, high-molecular-weight VWF multimers (p = 0.002). Factor V Leiden and the prothrombin gene 20210A variants (the most common inherited thrombophilic defects) were more represented in group A than in group B, as was the genotype GCAG/GCAG of the VWF gene promoter, known to hyperinduce VWF upregulation under cortisol excess. All but one of the patients with VTE unrelated to surgery had at least four acquired and at least one inherited risk factor. Severe hypercortisolism and VWF levels with increased haemostatic activity are strongly associated with VTE in CS. VTE episodes unrelated to surgery are attributable to the synergistic action of acquired and inherited thrombotic risk factors. Based on these observations, we believe that severely affected CS patients should be screened for coagulation disorders and receive antithrombotic prophylaxis whenever they have concomitant prothrombotic risk factors.

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.

Diagnosis and follow-up of thrombotic thrombocytopenic purpura by means of von Willebrand factor collagen binding assay.

Thrombotic thrombocytopenic purpura (TTP) is characterized by intravascular thrombosis leading to consumption of large or unusually large von Willebrand factor (VWF) multimers. The usefulness of VWF collagen binding (VWF:CB) assay was assessed in detecting the decrease/absence of large VWF multimers or the presence of abnormally large forms in patients with TTP. Nine patients with TTP were studied during the acute phase of the disorder and the absence of large VWF multimers was demonstrated by means of the VWF:CB assay. These findings were confirmed by VWF multimer pattern analysis; VWF:CB deficiency appeared to correlate with abnormalities in large VWF multimers. The diagnostic potency of VWF:CB was especially evident when the values were expressed as VWF:CB/VWF:Ag ratio. VWF:CB was also used during the follow-up of the disorder to document improvement or restoration of large VWF multimers. VWF:CB was able to detect the absence or decrease of large VWF multimers better than VWF ristocetin cofactor (VWF:RCo); in fact, VWF:CB was defective when large VWF multimers persisted to be decreased, in contrast with what observed with VWF:RCo. In conclusion, VWF:CB is a simple test that appears to be useful, together with clinical symptoms and reduced platelet count, for the diagnosis and follow-up of TTP.

Arterial and venous thrombosis in patients with von Willebrand's disease: a critical review of the literature.

All patients with von Willebrand's disease (vWD) who showed an arterial or venous thrombosis and were reported in the literature have been evaluated. 11 patients had arterial thrombosis while 19 had venous thrombosis for a total of 30 cases. 9 out the 11 cases with arterial thrombosis had myocardial infarction. Two had cerebral thrombosis. Associated risk factors for arterial thrombosis were available only for three patients who showed, respectively, smoking and dyslipidemia (2 cases) and smoking and intravenous desmopressin infusion (1 case). The majority of patients with venous thrombosis showed DVT with or without PE. Four patients presented with apparently isolated PE. In two instances thrombosis occurred in unusual sites (central retinal vein and portal vein, respectively). Several associated risk factors were present, mainly: infusion of FVIII or FVIII + vWF concentrates in 7 cases; surgery in 8 cases, pregnancy in 1, desmopressin infusion in 1, variable coagulation defects or polymorphisms in 5. More than one of these associated conditions were present in a few patients. The majority of vWD patients who showed thrombotic phenomena were type I patient, but in 6 cases were also type 3. The type of defect was not reported in 6 patients. As a conclusion of this review it seems safe to assume that both arterial and venous thrombosis appear rare in vWD. This is confirmed by the fact that arterial or venous thrombosis appears slightly more frequent in hemophilia A and B.

Biochemical markers of endothelial activation in primary hyperparathyroidism.

Patients with primary hyperparathyroidism (PHPT) have impaired vasodilation both dependent and independent of endothelium. The aims of our study were to measure three different biochemical markers of endothelial activation, i. e., plasma thrombomodulin, soluble(s) E-selectin, and von Willebrand factor, in PHPT patients before and one year after successful parathyroidectomy, and to distinguish the potential effect of hypercalcemia and/or high parathyroid hormone from that of major cardiovascular risk factors (diabetes mellitus, hyperlipidemia, hypertension, obesity, smoking habit) on endothelial function. Twenty consecutive patients with PHPT subdivided into two groups according to the absence (n = 8) or presence (n = 12) of one or more risk factors, and fifteen healthy normocalcemic subjects were studied. Baseline thrombomodulin levels were similar in the groups with and without risk factors, and in controls. In contrast, sE-selectin and von Willebrand factor were higher in PHPT patients with risk factors than in those without risk factors (p < 0.05 and p < 0.01, respectively) and controls (p < 0.01). Neither thrombomodulin nor sE-selectin changed after parathyroidectomy in either PHPT group. Plasma von Willebrand factor decreased (p < 0.01) in patients without risk factors, while persisting at high levels in patients with risk factors. In conclusion, in spite of a limitation due to the small number of patients, our study suggests that classic cardiovascular risk factors seem to be the main determinants for the high plasma levels of sE-selectin and vWF in PHPT. Together with unaltered thrombomodulin and sE-selectin levels, a plasma vWF decrease after parathyroidectomy might reflect a specific mechanism of its endothelial calcium- and/or PTH-stimulated secretion in some PHPT patients without risk factors. Whether a vWF reduction after parathyroidectomy may be used as a biochemical index for improved endothelial function in PHPT patients without risk factors has yet to be demonstrated in larger studies.

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

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 patient's 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.

Early transfusion of factor VIII/von Willebrand factor concentrates seems to be effective in the treatment of gastrointestinal bleeding in patients with von Willebrand type III disease.

The association between gastrointestinal angiodysplasia and von Willebrand disease was reported 30 years ago. The clinical course of patients with von Willebrand disease and angiodysplasia is characterized by numerous admissions to hospital for gastrointestinal bleeding necessitating transfusion with packed red cells, factor VIII and plasma. The management of these patients is problematic. Numerous treatments for the gastrointestinal bleeding have been proposed: surgery, electrocoagulation, laser photocoagulation, sclerotherapy, arteriography with embolization, immunoglobulins, oestrogens, and octreotide, but no treatment modality has been successful in all cases. We report a 66-year-old-female with small bowel angiodysplasia and von Willebrand type III disease in whom prompt administration of factor VIII/vWF concentrates was effective. Education of patients to recognize minimal gastrointestinal bleeding manifestations, periodical clinical visits and early infusion of factor VIII/vWF seems to be fundamental for the success of this therapy. A longer follow-up and the study of other patients are needed to confirm our observation.

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.

Adrenaline potentiates type 2B von Willebrand factor-induced activation of human platelets by enhancing both the formation and action of thromboxanes.

Von Willebrand factor (vWF) is a large plasma glycoprotein that mediates platelet adhesion at sites of vascular injury. We have previously reported that the pathological type 2B (formerly named type IIB) variant of vWF promotes platelet activation through phospholipase A(2)-mediated release of arachidonic acid. The present report shows that adrenaline (1 microM) potentiates type 2B vWF-induced platelet aggregation, serotonin secretion, rise in cytosolic Ca(2+) concentration, and pleckstrin phosphorylation, as well as thromboxane B(2) production. The hormone also increases the partially inhibited release of serotonin observed in platelets pretreated with the anti-GPIIb-IIIa antibody LJCP8 but does remove the total inhibition on the secretion caused by the anti-GPIb antibody LJIB1. Adrenaline also increases type 2B vWF-elicited tyrosine phosphorylation of proteins with apparent molecular masses of 60 and 80 kDa. Furthermore, adrenaline potentiates the rise in cytosolic Ca(2+) and the release of thromboxane B(2) in platelets stimulated with arachidonic acid (2 microM) as well as the increase in Ca(2+) induced by the thromboxane mimetic U46619 (0.3 microM). Platelet pretreatment with yohimbine or 13-azaprostanoic acid, which are antagonists of the alpha(2)-adrenergic and thromboxane receptors, respectively, or with acetylsalicylate and indomethacin, both of which act as inhibitors of thromboxane formation, abolishes the potentiating effect of adrenaline. These observations lead to the conclusion that the potentiating action of adrenaline on type 2B vWF-promoted platelet responses is due to an increase in both the formation and activating action of thromboxanes.