血管性血友病因子对结直肠癌细胞生物学特性的影响

目的 探讨血管性血友病因子（vWF）对人结直肠癌细胞生长、黏附和迁移能力的影响.方法 体外培养人结直肠癌细胞株SW480,采用免疫细胞化学方法检测SW480细胞中vWF的表达情况.用vWF抗体对其进行处理,采用倒置显微镜观察细胞形态学变化;MTT法检测细胞增殖活性和对细胞外基质成分--Ⅳ型胶原黏附能力的变化;Transwell法检测细胞迁移能力的改变.结果 SW480细胞能够表达vWF,染色部位主要位于细胞核,细胞质亦有轻微表达.vWF抗体能够显著抑制SW480细胞的生长,该抑制作用呈剂量及时间依赖性（P〈0.05）;20 ms/L vWF抗体处理48 h后,SW480细胞的黏附力明显下降（P〈0.05）;迁移能力明显减弱[穿膜细胞数（54.60±11.01）比（97.27±10.01）,P〈0.01].结论 人结直肠癌细胞能够表达vWF;vWF对人结直肠细胞的生长、黏附、迁移过程中起重要促进作用.     

vWF及GPIb在慢性肾脏疾病中变化及作用

目的研究假性血管血友病因子（vWF）及血小板膜糖蛋白Ib（GPIb）在慢性肾脏疾病（CKD）不同阶段的变化及意义。方法87例患者按病因及CKD不同阶段分为5组,A组：CKD1期,肾小球肾炎组,17例;B组：CKD1期,原发性肾病综合征组,14例;C组：CKD1期,继发性肾病组,18例;D组：CKD2～4期,慢性肾衰组,18例;E组：CKD5期,慢性肾衰透析组,20例。酶联免疫吸附检测法测定GPIb、双抗体夹心固相酶免疫测定血浆vWF。结果vWF、GPIb在A、B、C、D、E组均明显增高,以B、C组尤为突出;与凝血状况正相关。结论vWF水平可同时反应内皮细胞受损及凝血状态,在肾小球疾病发展中可能有重要意义。     

Endothelial dysfunction marker von Willebrand factor antigen in haemodialysis patients: associations with pre-dialysis blood pressure and the acute phase response.

BACKGROUND: Increased plasma soluble von Willebrand factor antigen (vWF : Ag) level, a marker of vascular endothelial cell dysfunction, is a strong predictor of atherosclerotic cardiovascular disease (CVD) in the general population. We studied cross-sectional associations between vWF : Ag level, prevalence of CVD, and related factors including pre-dialysis arterial blood pressure (BP) and some markers of inflammation in maintenance haemodialysis (HD) patients. Methods and results. Plasma vWF : Ag level measured by an enzyme-linked immunosorbent assay (ELISA) was higher in 110 HD patients than in 20 controls. On bivariate regression analysis, vWF : Ag level was directly associated with the presence of CVD, age, fibrinogen and the use of enoxaparin (vs unfractionated heparin) during HD procedures, and inversely with albumin and pre-dialysis BP. The patients with prevalent CVD were older, had higher vWF : Ag, white blood cell and platelet counts, fibrinogen and triglycerides, lower albumin levels, and were less frequently on combination antihypertensive therapy. Multivariable analyses identified low pre-dialysis BP, hypoalbuminaemia and hyperfibrinogenaemia (in descending order of significance) as independent predictors of high vWF : Ag level. There were no associations between vWF : Ag levels and gender, ABO blood type, smoking, body mass index, renal failure cause, duration of HD therapy, K(t)/V, normalized protein catabolic rate, dialysate buffers, dialysers, viral hepatitis, erythropoietin treatment, specific antihypertensive drugs, haemoglobin, white blood cell and platelet counts, liver enzymes, phosphorous, total cholesterol, and triglycerides. CONCLUSION: Elevated plasma levels of endothelial dysfunction marker vWF : Ag in maintenance HD patients are associated with established cardiovascular mortality risk factors such as low pre-dialysis blood pressure and the activated acute phase response.     

血管性血友病因子对冠心病发生影响的研究进展

von Willebrand因子又称血管性血友病因子,通过特异的血小板膜受体（糖蛋白Ιb-IX复合物）介导血小板与内皮下层的黏附,是一个反应内皮损伤的标志物,其增高有利于血小板的黏附。vWF与冠心病的发生相关,是反映内皮细胞受损的可靠指标之一。本研究通过对vWF生物学特点、生理功能、作用机理及对冠心病发生影响的相关论文的研究整理,为临床探讨冠心病影响因素及治疗提供参考依据。     

Elevated levels of plasma von Willebrand factor and the risk of macro- and microvascular disease in type 2 diabetic patients with microalbuminuria.

BACKGROUND: The purpose of this study was to examine the concept suggesting that microalbuminuria in combination with high levels of plasma von Willebrand factor is a stronger predictor for cardiovascular disease and microvascular complications than microalbuminuria alone in type 2 diabetic patients. METHODS: One hundred and sixty patients with type 2 diabetes mellitus and persistent microalbuminuria were followed for an average of 3.8 (SD 0.3) years. 70% of the patients were treated with angiotensin converting enzyme (ACE)-inhibitors. Patients in this subanalysis were divided into two groups according to baseline plasma von Willebrand factor levels below or above the median. The main outcome was cardiovascular disease (cardiovascular mortality, non-fatal stroke, non-fatal myocardial infarction, coronary artery bypass graft and revascularization or amputation of legs), progression to diabetic nephropathy or progression in diabetic retinopathy. RESULTS: At baseline the two groups were comparable for HbA(1c), fasting levels of s-total-cholesterol, s-HDL-cholesterol and s-triglycerides, systolic and diastolic blood pressure, gender, known diabetes duration, smoking habits, previous cardiovascular disease and antihypertensive therapy as well as retinopathy. Odds ratio for cardiovascular disease was 1.11 (95% CI 0.45-2.73, P=0.82) (multiple logistic regression), odds ratio for progression to nephropathy was 1.08 (0.41-2.85, P=0.87) and odds ratio for progression in retinopathy was 0.96 (0.46-2.00, P=0.92), all with plasma von Willebrand factor levels above the median. CONCLUSIONS: Our results do not support the suggestion that the combination of high plasma levels of von Willebrand factor and microalbuminuria is a stronger predictor for cardiovascular disease, progression to diabetic nephropathy or progression in diabetic retinopathy than microalbuminuria alone in patients with type 2 diabetes and persistent microalbuminuria.     

Von Willebrand factor and factor XIII in children with Henoch-Schonlein purpura

Levels of von Willebrand factor antigen (vWf: Ag) and factor XIII activity (F XIII) were studied in relation to the severity of clinical symptoms (scored from 0 to 3) and to immunological parameters [IgA, C3, C4, and circulating immune complexes (CIC) in 16 children (7 males, 9 females, aged 3–11 years) with Henoch-Schonlein purpura (HSP) at presentation. vWf: Ag was increased in 7 patients, F XIII activity was decreased in 6. In all children we found high levels of IgA, while C3 and C4 levels were normal; CIC were elevated in 11. vWf: Ag correlated with clinical score and with IgA and CIC, probably as a result of immune-mediated endothelial cell damage. The haemostatic alterations observed in HSP are important for understanding the pathophysiology of the disease.     

Correlation between von Willebrand factor levels and early graft function in clinical liver transplantation

Cold preservation/reperfusion leads to sinusoidal endothelial cell (SEC) activation and damage in nearly every liver transplantation; the extent of these changes influences early graft function. Upon reperfusion, activated SEC show increased expression of adhesion molecules, including von Willebrand factor (vWF) which is released into the circulation. This study was designed to evaluate the levels of vWF measured in the caval effluent and correlate these findings with known markers of SEC damage and early graft function. Data were obtained from 35 patients undergoing orthotopic liver transplantation (LTx). Two samples were taken from each patient for measurement of vWF: a) from the portal vein immediately prior to reperfusion; and b) from the first 50 ml of the caval effluent. Commercial assays were used to measure vWF, as well as hyaluronic acid (HA), thrombomodulin (TM), IL-1β, IL-6, IL-8 and TNF-α. Patients were divided into two groups based on early graft function. Poor early graft function (PEGF) was defined as a peak aspartate transaminase (AST) or alanine transaminase (ALT) level> 2500 U/L during the first three postoperative days (POD) and a prothrombin time (PT)> 16 s on POD 2 (n=8). The remaining 27 patients had good early graft function (GEGF). In patients with GEGF, vWF levels dropped significantly between the two time points. This change was not observed in those with PEGF. A positive linear correlation was observed in the PEGF group between vWF and HA and IL-6. The different pattern of change in vWF between the two groups, as well as the positive correlation between HA, IL-6 and vWF in PEGF, suggest that vWF may be a useful marker of early graft function.     

Elevation of serum von Willebrand factor and anti-endothelial cell antibodies in patients with immunoglobulin A nephropathy are associated with intrarenal arterial lesions

Aim: Recent studies suggest that intrarenal arterial lesions are frequently observed in patients with immunoglobulin A nephropathy (IgAN). However, the mechanisms of the injury have not been elucidated. The level of serum von Willebrand factor (vWF) and the prevalence of anti-endothelial cell antibodies (AECA) were investigated in patients with IgAN with different intrarenal arterial lesions. Methods: Sera from 28 patients with mild intrarenal arterial lesions (group 1) and 36 patients with severe intrarenal arterial lesions (group 2) were collected. Sera from 20 patients with idiopathic membranous nephropathy (group 3) and 50 healthy volunteers were also obtained as disease and normal controls, respectively. Serum vWF and AECA of both IgG and IgA isotype were detected. Results: In comparison with normal controls, serum vWF was significantly higher in group 2 and group 3. Serum vWF was also significantly higher in group 2 than in group 1. Both IgG-AECA and IgA-AECA could be detected in three groups of patients. The prevalence of anti-87 kD IgG-AECA was greatest in patients in group 2. IgAN patients, especially those in group 2 with IgG-AECA or anti-87 kD IgG-AECA, had significantly higher serum creatinine and lower creatinine clearance than those without. No significant difference could be found for IgA-AECA. The level of serum vWF was higher in IgAN patients with IgG-AECA than that in patients without. Conclusion: Intrarenal arterial lesions are associated with endothelial cell damage in IgAN, and vWF is a useful serological biomarker of severe intrarenal arterial lesions. AECA, especially IgG-AECA, may play an important role in the pathogenesis of intrarenal arterial damage in IgAN.     

Severe thrombotic and bleeding complications in a baby with heterozygous factor V Leiden and acquired von Willebrand disease on ECMO

We aim to present the case of a 5-week-old girl with severe respiratory failure placed on veno-venous extracorporeal membrane oxygenation (ECMO) that was then switched to veno-arterial ECMO. She required up to 60 units/kg/hr of heparin to keep her heparin level within the target range at .3-.7 units/mL. During the ECMO course, substantial thrombus formation was observed within the venous site of the ECMO cannula, which led to two circuit changes on ECMO day 9 and day 20. On ECMO day 15, she was noticed to have purpuric lesions on her chest and her right hand with no obvious arterial or venous clot detected by Doppler ultrasound. She was also noted to have remarkable hemolysis as the plasma free hemoglobin levels were substantially elevated up to 700 mg/dL. She was noted to have continuous oozing from the catheter insertion sites despite adequate underlying coagulation status. Her subsequent platelet function analysis, the thromboelastography, and thromboelastography platelet mapping suggested substantial platelet dysfunction. Her von Willebrand panel revealed absence of high molecular weight multimers. Further coagulation workup was prompted which revealed heterozygosity for factor V Leiden. The patient developed severe pulmonary hemorrhages and ECMO was discontinued on day 40.     

The effect of vibration on back discomfort and serum levels of von Willebrand factor antigen: A preliminary communication

Summary The von Willebrand factor (vWf) is a complex protein whose release is a marker for endothelial damage; serum levels of its antigen (vWFAg) can be used as a marker for such changes. We measured the levels of back discomfort and vWFAg in 11 subjects following 25-min periods of (1) lying down, (2) sitting upright, (3) vibrating whilst sitting and (4) sitting upright. Back discomfort appeared and vWf levels were significantly increased following sitting upright, compared with lying flat, and increased further following vibration. They fell thereafter with a period of sitting still upright. These results demonstrate that vibration has a significant effect in increasing back discomfort and the serum levels of vWFAg, and it is possible that vibration may induce vascular damage within the spine.     

Spinal anesthesia for a cesarean delivery in a woman with type-2M von Willebrand disease: case report and mini-review

Von Willebrand disease is the most common inherited bleeding disorder. No consensus exists about the use of neuraxial analgesia or anesthesia in patients with von Willebrand disease. We report on a 38-year-old multiparous woman who presented at 36 weeks’ of gestation with spontaneous rupture of membranes for urgent cesarean delivery. Preoperative coagulation tests were normal except for prolonged platelet adhesion and aggregation tests. The cesarean delivery was performed under spinal anesthesia with hyperbaric bupivacaine, fentanyl and morphine sulfate. Desmopressin was administered immediately after delivery. No perioperative complications were observed.     

Prognostic implications of retinopathy and a high plasma von Willebrand factor concentration in type 2 diabetic subjects with microalbuminuria.

BACKGROUND: Microalbuminuria in subjects with type 2 diabetes may be heterogeneous with respect to clinical features, renal histology, and prognosis. There may be at least two types of microalbuminuria in diabetes, namely with and without generalized endothelial dysfunction. We investigated whether, among microalbuminuric subjects with type 2 diabetes, the presence of generalized endothelial dysfunction, as indicated by the presence of retinopathy or a high plasma von Willebrand factor (vWf) level, has prognostic implications. METHODS: In 173 type 2 diabetic subjects of a population-based cohort, we assessed the urinary albumin-to-creatinine ratio, the plasma vWf level, and the presence of retinopathy. The main outcome was cardiovascular mortality. RESULTS: The absolute difference in 7 years' cardiovascular mortality between microalbuminuric (albumin-to-creatinine ratio 2.0-30.0 mg/mmol) and normoalbuminuric subjects was higher in the presence as compared to the absence of retinopathy (55.6 vs 11.1%). The age- and sex-adjusted relative risk (95% confidence interval) of cardiovascular mortality, as compared to normoalbuminuric subjects without retinopathy, was 1.1 (0.1-9.2) for normoalbuminuric subjects with retinopathy, 1.8 (0.5-6.7) for microalbuminuric subjects without retinopathy, and 9.8 (3.1-30.9) for microalbuminuric subjects with retinopathy. The absolute difference in risk of 7 years' cardiovascular mortality between microalbuminuric and normoalbuminuric subjects was higher in the presence as compared to the absence of a high (>1.89 IU/ml) vWf level (49.8 vs 16.4%). The age- and sex-adjusted relative risk of cardiovascular mortality, as compared to normoalbuminuric subjects without a high vWf level, was 1.5 (0.4-5.5) for normoalbuminuric subjects with a high vWf level, 2.6 (0.7-9.6) for microalbuminuric subjects without a high vWf level, and 12.0 (2.9-49.5) for microalbuminuric subjects with a high vWf level. These differences in risk of cardiovascular mortality did not change materially after further adjustment for known duration of diabetes, hypertension, creatinine clearance, level of glycated haemoglobin and high-density lipoprotein cholesterol, and presence of cardiovascular disease. Analysis of all-cause instead of cardiovascular mortality showed a similar difference in risk of mortality between microalbuminuric subjects with or without retinopathy or a high vWf level. CONCLUSIONS: Among type 2 diabetic subjects with microalbuminuria, the presence of retinopathy or a high plasma vWf level affects the risk of cardiovascular death. Although larger studies are necessary, these findings support the concept that microalbuminuria in type 2 diabetes can occur in the absence or the presence of generalized endothelial dysfunction, and that the latter is a much more 'malignant' condition than the former.     

O-linked glycosylation and functional incompatibility of porcine von Willebrand factor for human platelet GPIb receptors

BACKGROUND: Xenograft rejection is associated with vascular inflammation, thrombocytopenia and the accelerated consumption of coagulation factors. Primary biological incompatibilities of the xenograft in the regulation of clotting appear to amplify pathological processes associated with rejection. The functional incompatibility of porcine von Willebrand factor (vWF) expressed within the xenograft vasculature may heighten interactions with the primate platelet receptor GPIb, hence augmenting formation of platelet microthrombi and vascular injury. Here, we address the functional impact of O-linked glycosylation of the vWF A1 domain on primate platelet activation. METHODS: Recombinant human or porcine vWF A1-domains were transiently over-expressed in COS-7 cells as FLAG-tagged fusion protein, linked to plasma membranes via GPI anchors. O-linked glycosylation was blocked by the addition of phenyl-alpha-GalNAc2 to cultures. Expressed vWF-A1 domains were characterized utilizing cytofluometric- and Western blot analyses. RESULTS: Cytofluometric analysis confirmed equivalent levels of human and porcine vWF A1-domain expression irrespective of the levels of O-linked glycosylation. Differential glycosylation patterns of vWF-A1 under these conditions were confirmed by Western blot analyses. Native porcine vWF A1-domains had enhanced human platelet activation potential when compared with human recombinant vWF A1. However, the loss of O-linked glycosylation abolished differences in aggregatory responses between human and porcine vWF A1 domains. CONCLUSIONS: Various degrees of O-linked glycosylation of vWF-A1-domains modulate levels of functional interaction with platelet receptor GPIb and consequent platelet aggregation responses in vitro. These data may have implications for outcomes of xenotransplantation. We speculate that alterations in glycosylation of vWF and other adhesion proteins associated with the targeting of the alpha1,3-Gal-epitope in mutant swine may have salutatory effects on the primate platelet activation observed in these xenografts.     

The Effect of Shear Stress on the Size,Structure, and Function of Human von Willebrand Factor

Clinical outcomes from ventricular assist devices (VADs) have improved significantly during recent decades, but bleeding episodes remain a common complication of long‐term VAD usage. Greater understanding of the effect of the shear stress in the VAD on platelet aggregation, which is influenced by the functional activity of high molecular weight (HMW) von Willebrand factor (vWF), could provide insight into these bleeding complications. However, because VAD shear rates are difficult to assess, there is a need for a model that enables controlled shear rates to first establish the relationship between shear rates and vWF damage. Secondly, if such a dependency exists, then it is relevant to establish a rapid and quantitative assay that can be used routinely for the safety assessment of new VADs in development. Therefore, the purpose of this study was to exert vWF to controlled levels of shear using a rheometer, and flow cytometry was used to investigate the shear‐dependent effect on the functional activity of vWF. Human platelet‐poor plasma (PPP) was subjected to different shear rate levels ranging from 0 to 8000/s for a period of 6 h using a rheometer. A simple and rapid flow cytometric assay was used to determine platelet aggregation in the presence of ristocetin cofactor as a readout for vWF activity. Platelet aggregates were visualized by confocal microscopy. Multimers of vWF were detected using gel electrophoresis and immunoblotting. The longer PPP was exposed to high shear, the greater the loss of HMW vWF multimers, and the lower the functional activity of vWF for platelet aggregation. Confocal microscopy revealed for the first time that platelet aggregates were smaller and more dispersed in postsheared PPP compared with nonsheared PPP. The loss of HMW vWF in postsheared PPP was demonstrated by immunoblotting. Smaller vWF platelet aggregates formed in response to shear stress might be a cause of bleeding in patients implanted with VADs. The methodological approaches used herein could be useful in the design of safer VADs and other blood handling devices. In particular, we have demonstrated a correlation between the loss of HMW vWF, analyzed by immunoblotting, with platelet aggregation, assessed by flow cytometry. This suggests that flow cytometry could replace conventional immunoblotting as a simple and rapid routine test for HMW vWF loss during in vitro testing of devices.     

Closure of a ventricular septal defect in a patient with von Willebrand disease

Few reports of open heart surgery being performed in patients with von Willebrand disease (vWD) have been documented. We describe, herein the case of a 5-year-old girl with a ventricular septal defect (VSD) and vWD who underwent patch closure under cardiopulmonary bypass (CPB). The hematological parameters relating to vWD, namely, factor VIII, von Willebrand factor, and factor VIII-related antigen were monitored perioperatively. These laboratory findings were elevated after the termination of CPB, and consistently maintained within the normal range for 4 months postoperatively. The operation was successfully completed without the administration of factor VIII concentrates or homologous blood products.