A standard nomenclature for von Willebrand factor gene mutations and polymorphisms. On behalf of the ISTH SSC Subcommittee on von Willebrand factor

Examination of the entire von Willebrand factor (VWF) gene for mutations, particularly in types 1 and 3 von Willebrand disease (VWD) is becoming more widely practised. The sequence of the entire VWF gene will soon be compiled as a single sequence. For these reasons, a clearly defined nomenclature to use for numbering the VWF nucleotide and amino acid sequence is required. The following recommendations are made for VWF numbering. VWF cDNA nucleotide sequence should be numbered from the A of the initiator ATG as the +1 position. Genomic DNA should be prefixed with a "g" and also numbered from this position. Amino acid (aa) numbering should be from the initiator methionine as the +1 position with sequential numbering of aa throughout VWF. To avoid confusion with previously used numbering schemes for mature VWF, which started from serine 764 of pre-pro VWF, the use of the single letter amino acid code is recommended.     

The anti-von Willebrand factor aptamer ARC1779 increases von Willebrand factor levels and platelet counts in patients with type 2B von Willebrand disease

Blockade of hyperactive von Willebrand factor (VWF) by ARC1779 blunted the platelet drop induced by desmopressin in patients with type 2B von Willebrand disease (VWD). Thus, we hypothesised that ARC1779 may increase VWF levels and correct thrombocytopenia. Three thrombocytopenic patients suffering from type 2B VWD received a loading dose of 0.23 mg/kg ARC1779 followed by 4 μg/kg/min intravenously for 72 hours in a prospective clinical trial. ARC1779 was well tolerated and safe. Plasma concentrations of ARC1779 increased to 76 μg/ml (59-130) leading to an immediate decrease of free VWF A1 domains. VWF/FVIII levels increased as early as 12 h after start of infusion, peaked near the end of infusion, and returned to baseline at follow-up. VWF ristocetin cofactor activity (VWF:RCo) showed a median 10-fold increase 8 hours after end of infusion, while the median VWF-antigen and FVIII increase was less (5-fold and 4-fold, respectively). Most importantly inhibition of hyperactive VWF rapidly increased platelet counts from 40 x 10(9)/l (38-58 x 10(9)//l) to a maximum of 146 x 10(9)//l (107-248 x 10(9)//l). In conclusion, ARC1779 markedly increases VWF/FVIII levels and most importantly improves or even corrects thrombocytopenia in VWD type 2B patients. This underscores the in vivo potency of ARC1779.     

Acute effects of hyperglycaemia on plasma concentration of soluble P-selectin and von Willebrand factor in healthy volunteers -a prospective randomised double blind controlled study

This study set out to clarify if isolated hyperglycaemia may directly induce acute endothelial and/or platelet activation as diabetes mellitus is a major risk factor for cardio- and cerebrovascular diseases with thromboembolic complications. 12 healthy volunteers were investigated in a prospective randomised, double blind, three-way cross-over trial with a wash-out period of 21 days. Normoglycemic (4.72 mmol/L), moderate (11.1 mmol/L) or high grade (16.7 mmol/L) glucose clamps were maintained for 6 hours by infusion of glucose, insulin and somatostatin. Volunteers were observed for 24 hours. An increase in soluble (s)P-selectin and von Willebrand Factor (VWF) concentrations, of 26 ± 15% and 21 ± 7%, respectively was observed 24 hours after euglycaemic treatment, of 20 ± 7% and 19 ± 5%, respectively after moderate hyperglycaemia and of 22 ± 13% and 18 ± 7%, respectively after high grade hyperglycaemia at 24 hours (p < 0.6 and p < 0.004 in all periods and p = 0.2-0.9 between periods). In conclusion, acute hyperglycaemia for 6 hours does not increase the platelet and endothelial activation markers sP-selectin and VWF in healthy volunteers.     

Investigation of Thr715Pro P-selectin gene polymorphism and soluble P-selectin levels in type 2 diabetes mellitus

Increased levels of soluble P-selectin (sP-selectin) have been shown in a number of different disorders, e.g. diabetes mellitus (DM) and cardiovascular disease (CVD). Several studies have attempted to demonstrate the association of the most intensively examined variant of P-selectin gene polymorphism (Thr715Pro) with sP-selectin levels in healthy subjects and in CVD, but contradictory data have been reported. To clarify the effect of Pro715 allele on the sP-selectin levels in type 2 DM, we analysed this polymorphism in diabetic patients and compared these data with sP-selectin levels. Type 2 DM patients (n = 119), 48 BMImatched non diabetic individuals - consisting mostly of overweight subjects - and 57 healthy volunteers were included in the study. TheThr715Pro polymorphism was analysed by PCR-RFLP, while sP-selectin levels were measured by ELISA. Significantly elevated sP-selectin levels were found in both DM and in overweight subjects compared to healthy controls. We confirmed previous reports that in healthy Pro715 allele carriers lower sPselectin levels could be measured; however, this difference was only significant in case of lean subjects. No significant difference was detected in sP-selectin level among DM and overweight individuals according to this genotype. However, significant difference was observed in sP-selectin levels in older DM patients compared to younger ones, but these levels were not accounted for by the Thr715Pro polymorphism. We suggest that in type 2 DM individuals, the significantly elevated sP-selectin levels are not due to the Thr715Pro P-selectin gene polymorphism.     

Effects of endothelin-1 and phenylephrine on plasma levels of von Willebrand factor and protein S

Endothelial dysfunction is considered a major factor in the pathogenesis of a wide array of diseases and often leads to increased production of, or increased responsiveness to endogenous vasoconstrictive mediators, such as endothelin-1 (ET-1) or adrenergic agonists. Based on previous studies in animals and in-vitro, we hypothesized that ET-1 and alpha adrenergic stimulation by phenylephrine (PE) may alter plasma levels of von Willebrand factor-Ag (vWF) and protein S in humans.Ten healthy men were studied in a prospective randomized double blind trial: we investigated the effects of infusions of ET-1 and PE on plasma levels of vWF-Ag and protein S. Aditionally, we examined the effects of these vasoconstrictors on thrombomodulin, tissue plasminogen activator and on protein C.ET-1 increased plasma levels of vWF-Ag by 19% (CI: 9-36%; p = 0.008) and increased plasma levels of protein-C by 7% (CI: 0.2 -12 %; p = 0.028), even at doses which produced no increase in blood pressure. Plasma levels of protein S and TPA were not significantly affected by ET-1. After PE-infusion we observed a relative increase in plasma levels of protein S by 20% (CI: 5-28%; p = 0.036) and of TPA by 14 % (CI: 2-39%; p = 0.036). Also, platelet counts increased by 36% (CI: 12-53%; p = 0.028) which correlated (r² = 0.86; p = 0.014) with an increase in leukocytes by 49 % (CI: 18-84%; p = 0.028). Plasma levels of vWF and protein C were not affected by PE and neither drug had a significant effect on plasma levels of thrombomodulin.In conclusion, our results support the study hypothesis, that ET-1 and PE increase the plasma levels of vWF and protein S, respectively. ET-1 may induce a procoagulant status in various disease states by increasing vWF-Ag levels.     

Increased levels of plasma von Willebrand factor in migraine crisis

Introduction – To evaluate the participation of the vessel wall in the pathogenesis of migraine attack, we measured the plasma levels of von Willebrand factor (vWF), a protein secreted from the endothelial cells. Material & methods – 17 patients suffering from migraine without aura and 25 healthy volunteers were studied. von Willebrand factor and platelet aggregation tests were studied by conventional methods. Results – The levels of vWF:antigen increased from 72.4 ± 29 U/dl in the intercrisis to 130.2 ± 75 U/dl during the attack (p < 0.01). We did not detect difference in the platelet aggregability in both phases. Plasma vWF activity measured as ristocetin cofactor (vWF:RCo) was similar in intercrisis and crisis (100.6 ± 31 U/dl vs 94.5 ± 44 U/dl). Conclusions – There is a plasma release of vWF molecules during the migraine crisis. This feature is not platelet dependent and is probably a consequence of endothelial stress.     

Sequential changes in von Willebrand factor and soluble thrombomodulin in acute ischemic stroke

The purposes of the present study were to investigate sequential changes in plasma von Willebrand factor (vWf) activities and serum soluble thrombomodulin (sTM) concentrations, compared with white blood cell (WBC) counts, and to disclose the different roles of vWf and sTM in acute ischemic stroke. Forty-three acute ischemic stroke patients admitted to our hospital within 48 hours from onset were enrolled. Plasma vWf activities, serum sTM concentrations, and WBC counts were measured at the acute stage and 1 and 6 months after admission. The time course study revealed that vWf activities increased more markedly 1 month after admission than at the acute stage. However, sTM concentrations were low at the acute stage and increased sequentially at 1 month (not significantly) and 6 months (significantly) after admission. In contrast, elevated WBC counts at the acute stage decreased significantly at 1 and 6 months after admission. Raised vWf activities 1 month after admission were suggested to occur through continuous endothelial dysfunction or repair and platelet activation, compared with the acute stage, and decreased sTM at the acute stage through down-regulation of sTM synthesis by acute inflammatory response after acute ischemic stroke, compared with the chronic stage.     

Three distinct candidate point mutations of the von Willebrand factor gene in four patients with type IIA von Willebrand disease.

Type IIA von Willebrand disease (vWD) is the most common type II vWD and is characterized by the selective loss of large and intermediate sized multimers. One explanation for this disorder has been postulated to be a qualitative defect in von Willebrand factor (vWF) which results in increased susceptibility to proteolysis at the bond between residues Tyr842 and Met843. Four missense mutations that may cause type IIA vWD have recently been identified near the cleavage site. We analyzed the molecular basis for type IIA vWD in six patients. A 512 bp DNA sequence spanning the proteolytic cleavage site was targeted for PCR amplification and sequencing. We exploited a difference in restriction sites between the vWF gene and the pseudogene and have designed allele-specific oligomer used with PCR to distinguish these two genes. Three candidate missense mutations; Ser743 (TCG)----Leu (TTG), Leu799 (CTG)----Pro (CCG), and Arg834 (CGG)----Trp (TGG) were identified in 4 out of 6 patients. The amino acid substitution at Arg834 has been reported previously, but the other substitutions at Ser743 and Leu799 are novel candidate mutations locating 99 and 43 amino acids to the N-terminal side of the cleavage site, respectively. Our results indicate that amino acid substitutions located relatively distant from the cleavage site may also be involved in type IIA vWD.     

ABO blood group genotypes, plasma von Willebrand factor levels and loading of von Willebrand factor with A and B antigens

ABO blood group is a genetic determinant of von Willebrand factor (VWF) levels. We investigated the effect of ABO genotypes on VWF and factor VIII (FVIII) levels and on the degree to which VWF is loaded with A- and B-antigens, expressed as normalized ratios, nA-ratio and nB-ratio, respectively, in the Leiden Thrombophilia Study, a large case-control study on venous thrombosis. We found that the ABO locus had an allele-specific, dosage dependent effect on VWF and FVIII levels and on the loading of VWF with A-antigen and B-antigen. The highest mean nA- and nB-ratios were found in A(1)A(1) and BB genotypes, respectively. Four A(1)O carriers had four 43-bp repeats in the minisatellite region of the ABO gene in stead of the expected one repeat. All had a reduced nA-ratio compared to A(1)O carriers with one repeat in their A(1) allele. The amount of A- and B-antigens expressed onVWF (nA-ratio and nB-ratio) explained about 18% (R(2)) of the variation in VWF levels.     

Correlation between plasma levels of growth factors and von Willebrand factor

Intimal proliferation of smooth muscle cells is an important characteristic of developing atherosclerotic lesions and late occlusion of venous bypass grafts. Platelet-derived growth factor, released from aggregating platelets at sites of endothelial injury, has been suggested as a main factor responsible for intimal hyperplasia. The von Willebrand factor is a platelet binding protein secreted by endothelial cells and increased plasma levels of this factor has been identified as a marker of endothelial injury. In the present study we have analysed plasma levels of von Willebrand factor and growth factors in healthy controls, young post-infarction patients and patients with recent coronary bypass surgery. The results demonstrate a significant correlation between plasma growth factor activity and the level of von Willebrand factor (R = 0.52, p less than 0.01) and support the notion of a coupling between endothelial injury and release of platelets mitogens.     

Association between the Thr715Pro P-selectin gene polymorphism and soluble P-selectin levels in a multiethnic population in South London

The aim was to investigate whether the Thr715Pro P-selectin polymorphism is associated with soluble P-selectin (sP-selectin) levels in individuals from different ethnic groups. Plasma sP-selectin and Thr715Pro (A/C) P-selectin gene polymorphism were measured in 237 white (106 females), 177 black African origin (92 females) and 201 South Asian (94 females) individuals living in England. All were free from coronary heart disease (CHD), stroke and other cardiovascular disease, diabetes, drug therapy for hypertension or high lipids, hormone replacement therapy or oral contraceptive pill. The Thr715Pro C allele was rare in blacks (0.8%) and intermediate in South Asians (3.0%) compared to whites (11.2%; p <0.001). sP-selectin levels were significantly lower in the individuals with the AC or CC compared to the AA genotype in both whites (-25% (95% C.I. -33.3 to -16.9); p <0.001) and South Asians (-25.2% (-40.5 to -6.1); p <0.012). There was insufficient power for this analysis in blacks. In conclusion, in whites and South Asians the C allele of the Thr715Pro P-selectin polymorphism is associated with lower sP-selectin levels. Lower levels of sP-selectin were not accounted for by this polymorphism in blacks, in whom the C allele was very rare.     

藻酸双酯钠对急性脑梗死患者血浆血管性假血友病因子和血栓调节蛋白的影响

目的 研究藻酸双酯钠(PSS)对急性脑梗死患者血浆血管性假血友病因子(vWF)和血栓调节蛋白(TM)水平的影响.方法 将急性脑梗死患者64例随机分为PSS治疗组(PSS组,32例)和对照组(32例).PSS组患者用PSS 150mg静脉滴注,每日1次,连续14d;对照组患者每日静脉滴注维脑路通600mg,连续14d.于治疗前、治疗后7d、14d检测两组患者血浆vWF、TM的水平.结果 治疗后第7d、14d PSS组血浆vWF水平明显低于对照组(均P＜0.05),且两组治疗后均低于治疗前(P＜0.05～0.01).治疗后第7d、14d PSS组血浆TM水平明显高于对照组(均P＜0.05),且两组患者治疗后14d均高于治疗前(均P＜0.01).结论 PSS可使急性脑梗死患者血浆vWF水平降低及TM水平升高,故有抗凝和保护血管内皮细胞的作用.