Type 2 von Willebrand disease causing defective von Willebrand factor-dependent platelet function.

Type 2 von Willebrand disease causing defective von Willebrand factor-dependent platelet function comprises mainly subtypes 2A, 2B and 2M. The diagnosis of type 2 von Willebrand disease may be guided by the observation of a disproportionately low level of ristocetin cofactor activity or collagen-binding activity relative to the von Willebrand factor antigen level. The decreased platelet-dependent function is often associated with an absence of high molecular weight multimers (types 2A and 2B), but the high molecular weight multimers may also be present (type 2M and some type 2B), and supranormal multimers may exist (as in the Vicenza variant). Today, the identification of mutations in particular domains of the pre-provon Willebrand factor is helpful to classify these variants and to provide further insight into the structure-function relationship and the biosynthesis of von Willebrand factor. Thus, mutations in the D2 domain, involved in the multimerization process, are found in patients with type 2A, formerly named IIC von Willebrand disease. Mutations in the D3 domain characterize the Vicenza variant, or type IIE patients. Mutations in the A1 domain may modify the binding of von Willebrand factor multimers to platelets, either increasing (type 2B) or decreasing (types 2M and 2A/2M) the affinity of von Willebrand factor for platelets. In type 2A disease, molecular abnormalities identified in the A2 domain, which contains a specific proteolytic site, are associated with alterations in folding that impair the secretion of von Willebrand factor or increase its susceptibility to proteolysis. Finally, a mutation localized in the C terminus cysteine knot domain, which is crucial for the dimerization of von Willebrand factor subunit, has been identified in a rare subtype 2A, formerly named IID.     

Expression and characterization of von Willebrand factor dimerization defects in different types of von Willebrand disease

Dimerization defects of von Willebrand factor (vWF) protomers underlie von Willebrand disease (vWD) type 2A, subtype IID (vWD 2A/IID), and corresponding mutations have been identified at the 3' end of the vWF gene in exon 52. This study identified and expressed 2 additional mutations in this region, a homozygous defect in a patient with vWD type 3 (C2754W) and a heterozygous frameshift mutation (8566delC) in a patient with vWD type 2A, subtype IIE. Both mutations involve cysteine residues that we propose are possibly essential for dimerization. To prove this hypothesis, transient recombinant expression of each of the 2 mutations introduced in the carboxy-terminal vWF fragment II and in the complete vWF complementary DNA, respectively, were carried out in COS-7 cells and compared with expression of vWD 2A/IID mutation C2773R and the wild-type (WT) sequence in COS-7 cells. Recombinant WT vWF fragment II assembled correctly into a dimer, whereas recombinant mutant fragments were monomeric. Homozygous expression of recombinant mutant full-length vWF resulted in additional dimers, probably through disulfide bonding at the amino-terminal multimerization site, whereas recombinant WT vWF correctly assembled into multimers. Coexpression of recombinant mutant and recombinant WT vWF reproduced the multimer patterns observed in heterozygous individuals. Our results suggest that a common defect of vWF biosynthesis--lack of vWF dimerization--may cause diverse types and subtypes of vWD. We also confirmed previous studies that found that disulfide bonding at the vWF amino-terminal is independent of dimerization at the vWF carboxy-terminal. (Blood. 2001;97:2059-2066)     

Type 2N von Willebrand disease

Type 2N von Willebrand disease (VWD) refers to patients with a factor VIII (FVIII) deficiency caused by a markedly decreased affinity of von Willebrand factor (VWF) for FVIII. It is inherited as an autosomal recessive trait but is clinically similar to mild hemophilia. The differential biologic diagnosis, which is of major importance for providing relevant genetic counseling and optimal treatment, is based on the measurement of plasma VWF capacity to bind FVIII. Molecular biology techniques have allowed the identification of 20 missense mutations in the VWF gene that cause type 2N VWD. All of them induce changes in amino acid residues located in the N-terminal part of mature VWF, which contains the FVIII binding site. Their identification may provide a genetic diagnosis. Theoretically, patients with type 2N VWD should be treated with products containing VWF that is able to stabilize their endogenous normal FVIII.     

Oligosaccharide structures of von Willebrand factor and their potential role in von Willebrand disease

Oligosaccharides make up approximately 20% of the mass of VWF and although their structures are well established, their functional role remains unclear. Modification of the VWF oligosaccharide structures has been shown to result in increased plasma clearance of the protein. A mutation which alters cell type-specific expression of the Galgt2 glycosyltransferase gene in the RIIIS/J mouse results in an autosomal dominant partial quantitative deficiency of VWF. Increased plasma clearance of VWF has been demonstrated in some individuals with a partial quantitative deficiency of the protein and it is possible that variation in VWF glycosylation may contribute towards this. ABH antigens occur within the oligosaccharide component of VWF and may account for the variation in plasma VWF:Ag levels observed between individuals of different ABO blood groups. The structures and functional roles of the oligosaccharide side chains of VWF and possible pathogenetic mechanisms by which they may contribute towards VWD are reviewed in this article.     

Thrombomodulin and von Willebrand factor: relation to endothelial dysfunction and disease outcome in children with acute lymphoblastic leukemia

The severe endothelial dysfunction in children with acute lymphoblastic leukemia (ALL) can result from the disease itself, from treatment, or from other conditions (e.g. sepsis). The aim of this study was to determine the levels of markers of endothelial activation in children with ALL and to assess their potential prognostic value. Fifty-two children with ALL, 19 children with ALL 1-10 years after the completion of therapy, and 28 healthy children were studied. In children with ALL, there was a significant increase in thrombomodulin (TM) and von Willebrand factor (vWF) levels during the acute phase of the disease and during treatment. Children with an unfavorable outcome had higher levels of TM. In conclusion, severe endothelial dysfunction is present during the acute phase of ALL and during treatment and appears to result from the disease itself. Serum TM and vWF levels might represent additional, but not independent, prognostic markers in childhood ALL.     

Current perspective. Diabetic vascular disease: from endothelial dysfunction to atherosclerosis.

Cardiovascular complications are the leading cause of death in diabetes. Over the past decade a number of studies have addressed the underlying mechanisms. Derangements of endothelial function, also referred to as endothelial dysfunction, have emerged to be the crucial early step in the development of atherosclerosis and are also involved in plaque progression and clinical emergence. Endothelial dysfunction is a condition of impaired endothelium-dependent vasodilation and most important of "endothelial activation", characterized by a proinflammatory, proliferative, and procoagulatory milieu that promotes initiation and complications of atherogenesis. A synergistic cross-talk among the conventional cardiovascular risk factors associated with diabetes contributes to disruption of endothelial integrity and accelerated atherosclerosis. This review will focus on the multifactorial nature of endothelial dysfunction in diabetes, the relationship between endothelial dysfunction, conventional cardiovascular risk factors and atherosclerosis, and the therapeutic options to improve endothelial function.     

Primary endothelial dysfunction: atherosclerosis

The endothelium synthesizes and releases several vasodilating factors, including nitric oxide, endothelium-derived hyperpolarizing factor, and prostacyclin. Under certain conditions, it also liberates vasocontracting factors. Thus, the endothelium plays an important role in regulating vascular homeostasis. Several intracellular mechanisms are involved in the synthesis of nitric oxide, including receptor-coupled G proteins, the availability of L-arginine, cofactors for endothelial nitric oxide synthase and the expression of the enzyme. Endothelial dysfunction by aging, menopause and hypercholesterolemia is involved in the development of atherosclerotic vascular lesions, and predisposes the blood vessel to several vascular disorders, such as vasospasm and thrombosis. Multiple mechanisms are apparently involved in the pathogenesis of the endothelial dysfunction in atherosclerosis. The reduced production of nitric oxide by the endothelium is caused by abnormalities in endothelial signal transduction, availability of L-arginine, cofactors for endothelial nitric oxide synthase and expression of the enzyme. Other mechanisms may also be involved in the impaired endothelium-dependent relaxations in atherosclerosis, including increased destruction of nitric oxide by superoxide anion, altered responsiveness of vascular smooth muscle, and concomitant release of vasocontracting factors. In addition to the treatment of the underlying risk factors, several pharmacological agents can improve endothelial dysfunction in atherosclerosis. Thus, the endothelium is a novel therapeutic target for the treatment of atherosclerotic cardiovascular disease.     

Characterization of von Willebrand factor gene defects in two unrelated patients with type IIC von Willebrand disease

Genetic studies were performed in two unrelated patients with the IIC phenotype of von Willebrand disease (vWD) characterized by the increased concentration of the protomeric form of von Willebrand factor (vWF). In patient B, the sequencing of both exons 15 and 16 of the vWF gene showed two sequence alterations: a 3-bp insertion in exon 15 resulting in the insertion of a Glycine at position 625 (625insGly) and a 2-bp deletion in exon 16 leading to a premature translational stop at codon 711 (711 ter), at the heterozygote state. Patient A was found homozygous for a single point mutation also localized in exon 15 and responsible for the substitution Cys623Trp. These candidate mutations were not found in a panel of 96 normal chromosomes, suggesting a causal relationship with IIC vWD phenotypic expression. The composite heterozygote or homozygote state of both patients supports the recessive mode of inheritance already described for this phenotype. Furthermore, the localization of these gene defects in the D2 domain of vWF propeptide, known to play an important role in vWF multimerization, provides another argument in favor of their causative effect regarding the peculiar multimeric pattern of vWF in these patients.     

Glycosaminoglycan metabolism defects and atherosclerosis: frequent association of endothelial dysfunction in patients with Mucopolysaccharidosis

Cardiovascular lesions, including coronary artery stenosis, are frequently associated and can cause sudden death in patients with genetic defects of glycosaminoglycan (GAG) metabolism. Early diagnosis of coronary artery lesions is difficult, although potentially lifesaving. Histopathological similarities between atherosclerotic changes in adults and in patients with genetic GAG metabolism defects have been known. Atherosclerosis is the result of a complex process involving metabolism of GAGs and proteoglycans preceded by endothelial dysfunction as a key event. Decreased nitric oxide (NO) bioavailability is considered the hallmark of endothelial dysfunction. Reduced NO synthase (NOS) has been reported in atherosclerotic arteries. Impairment in reactive hyperemia-digital peripheral arterial tonometry (RH-PAT) with EndoPAT has been validated to correlate coronary microvascular function in patients with atherosclerosis. RH-PAT is thought to reflect endothelial NO production. Immunohistological staining of endothelial NOS was performed in the stenotic lesions in the coronary artery of a 3-year-old patient with Mucopolysaccharidosis-I, showing decreased activities. This prompted a study to measure endothelial function in patients with GAG metabolism defects for early diagnosis of endothelial dysfunction in the coronary arteries as an early sign of coronary artery changes. Evaluation by RH-PAT in 30 patients with variable genetic defects in GAG metabolism revealed significantly decreased Reactive Hyperemia Indexes compared with 12 controls. Evaluation of endothelial function with RH-PAT in patients with GAG metabolism defects may detect coronary artery lesions that can be underdiagnosed by the other measures such as coronary angiography. Use of this method may prove vital in the management of patients with GAG metabolism defects.     

Expression of abnormal von Willebrand factor by endothelial cells from a patient with type IIA von Willebrand disease.

Studies were conducted to characterize the biosynthesis of von Willebrand factor (vWf) by cultured endothelial cells (EC) derived from the umbilical vein of a patient with type IIA von Willebrand disease. The patient's EC, compared with those from normal individuals, produced vWf that had decreased amounts of large multimers and an increase in rapidly migrating satellite species, features characteristic of plasma vWf from patients with type IIA von Willebrand disease. The type IIA EC did produce a full spectrum of vWf multimers in both cell lysates and postculture medium, although the relative amounts of the largest species were decreased. The large multimers were degraded in conjunction with the appearance of rapidly migrating satellites that contained approximately equal to 170-kDa proteolytic fragments, suggesting that this patient's functional defect is due to abnormal proteolysis and not to a primary failure of vWf subunit oligomerization. Moreover, the observed degradation appears to result from an abnormal vWf molecule and not elevated protease levels. These results suggest that this patient's von Willebrand disease phenotype is caused by increased proteolytic sensitivity of his vWf protein.     

Endothelial cell senescence in human atherosclerosis: role of telomeres in endothelial dysfunction

BACKGROUND: The functional changes associated with cellular senescence may be involved in human aging and age-related vascular disorders. We have shown the important role of telomeres and telomerase in vascular cell senescence in vitro. Progressive telomere shortening in vivo has been observed in the regions susceptible to atherosclerosis, implicating its contributions to atherogenesis. However, whether senescent vascular cells are present in the vascularture and contribute to the pathogenesis of atherosclerosis remains unclear. METHODS AND RESULTS: Senescence-associated beta-galactosidase (beta-gal) activity was examined in the coronary arteries and the internal mammary arteries retrieved from autopsied individuals who had ischemic heart diseases. Strong beta-gal staining was observed in atherosclerotic lesions of the coronary arteries but not in the internal mammary arteries. An immunohistochemical analysis using anti-factor VIII antibody demonstrated that beta-gal stained cells are vascular endothelial cells. To determine whether endothelial cell senescence causes endothelial dysfunction, we induced senescence in human aortic endothelial cells (HAECs) by inhibiting telomere function and examined the expression of intercellular adhesion molecule (ICAM)-1 and endothelial nitric oxide synthase (NOS) activity. Senescent HAECs exhibited increased ICAM-1 expression and decreased eNOS activity, both of which are alterations implicated in atherogenesis. In contrast, introduction of telomerase catalytic component significantly extended the life span and inhibited the functional alterations associated with senescence in HAECs. CONCLUSIONS: Vascular endothelial cells with senescence-associated phenotypes are present in human atherosclerotic lesions, and endothelial cell senescence induced by telomere shortening may contribute to atherogenesis.     

Type I von Willebrand disease, subtype 'platelet low': decreased platelet adhesion can be explained by low synthesis of von Willebrand factor in endothelial cells

Summary Endotheial cells (EC) were isolated from the umbilical vein of a newborn girl with type I 'platelet low' von Willebrand disease (I vWD) and endothelial localization and release and the ability of subendothelial von Willebrand factor (vWF) to support platelet adhesion were compared with those of normal EC. vWF was detectable by immunofluorescence in Weibel-Palade bodies, but the number of Weibel-Palade bodies positive for vWF was lower than in control EC. Patient EC released into the medium significantly smaller amount of vWF, both constitutively and after their stimulation. The vWF content of the extracellular matrix of patient EC was 38% that of control EC matrix. Platelet adhesion studies were performed under flow conditions with umbilical arteries and EC matrices of cultured EC. Using normal citrated whole blood as perfusate, platelet adhesion was lower in the umbilical artery of the patient (9 ± 1% v 35 ± 4% for the control) and in her EC matrix (7 ± 1% v 21 ± 2% of control). When patient EC matrix was perfused with vWF-deficient reconstituted blood, adhesion was 17 ± 3% v 32 ± 3% for control EC matrix; preincubation of patient EC matrix with 1 U/ml vWF increased the adhesion to 30 ± 6%. These data establish that low contents of vWF in EC and subendothelium are important characteristics of type I vWD 'platelet low', and that such characteristics correlate with low platelet adhesion to the subendothelium.     

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.     

Reduced von Willebrand factor survival in type Vicenza von Willebrand disease.

Type Vicenza variant of von Willebrand disease (VWD) is characterized by a low plasma von Willebrand factor (VWF) level and supranormal VWF multimers. Two candidate mutations, G2470A and G3864A at exons 17 and 27, respectively, of the VWF gene were recently reported to be present in this disorder. Four additional families, originating from northeast Italy, with both mutations of type Vicenza VWD are now described. Like the original type Vicenza subjects, they showed a mild bleeding tendency and a significant decrease in plasma VWF antigen level and ristocetin cofactor activity but normal platelet VWF content. Unlike the original patients, ristocetin-induced platelet aggregation was found to be normal. Larger than normal VWF multimers were also demonstrated in the plasma. Desmopressin (DDAVP) administration increased factor VIII (FVIII) and VWF plasma levels, with the appearance of even larger multimers. However, these forms, and all VWF oligomers, disappeared rapidly from the circulation. The half-life of VWF antigen release and of elimination was significantly shorter than that in healthy counterparts, so that at 4 hours after DDAVP administration, VWF antigen levels were close to baseline. Similar behavior was demonstrated by VWF ristocetin cofactor activity and FVIII. According to these findings, it is presumed that the low plasma VWF levels of type Vicenza VWD are mainly attributed to reduced survival of the VWF molecule, which, on the other hand, is normally synthesized. In addition, because normal VWF-platelet GPIb interaction was observed before or after DDAVP administration, it is proposed that type Vicenza VWD not be considered a 2M subtype.     

血管内皮功能障碍与动脉粥样硬化

血管内皮不仅是血液与内皮下组织的屏障,还具有内分泌功能.当血管内皮功能障碍时,会引起一系列的病理生理反应,导致动脉粥样硬化.在内皮功能障碍向动脉粥样硬化演变的过程中,血管紧张素Ⅱ和氧化型低密度脂蛋白起重要作用.内皮细胞损伤时,机体自身的修复机制发挥作用,相关药物可改善内皮功能,稳定动脉粥样硬化,改善预后.内皮细胞功能的...