Antithrombin III activity, von Willebrand factor antigen and platelet function in young diabetic patients treated with multiple insulin injections versus insulin pump treatment

In a prospective study with cross-over design 20 patients with insulin-dependent diabetes mellitus of more than 2 years duration were treated for 6 months with continuous subcutaneous insulin infusion (CSII) and multiple insulin injections (MII). Metabolic control, platelet aggregability, thromboxane B2 levels in serum and plasma as well as antithrombin III (ATIII) activity and von Willebrand factor antigen were evaluated. A good metabolic control was obtained by both intensified regimens. No difference could be demonstrated between either platelet function tests or serum level of von Willebrand factor antigen during treatment with CSII and MII. However, the plasma level of ATIII activity was significantly higher (P less than 0.01) during MII treatment as compared to CSII treatment. There was no correlation between ATIII activity and daily insulin requirement or serum fructosamine. In conclusion, long-term metabolic control with MII has a favourable effect on ATIII activity in plasma. This may be important for a delay in onset and progression of diabetic vascular complications.     

Time course of changes in in vitro platelet function and plasma von willebrand factor activity (VIIIR:WF) and factor VIII-related antigen (VIIIR:Ag) in the diabetic rat

The relationship between platelet abnormalities and vessel wall changes in diabetes is not known. We have examined the time course of alterations in in vitro platelet function and endothelial damage, as assessed by measurement of plasma levels of von Willebrand factor (VIIIR:WF) and factor VIII-related antigen (VIIIR:Ag), in streptozotocin-induced diabetic rats. Platelet aggregation and the platelet release reaction in response to ADP, thrombin, and collagen were measured in suspensions of washed platelets prepared from rats 3, 7, 14, or 28 days after induction of diabetes and in control animals. Platelets from diabetic animals showed enhanced aggregation response to ADP as early as 3 days after induction of diabetes and became hyperresponsive to thrombin after 7 days, compared to control platelets. Thrombin-induced release of serotonin was greater in platelets from diabetic animals at 14 days. Collagen-induced responses were not different at any time studied. VIIIR:WF was determined by ristocetin-induced platelet agglutination time in gel-filtered platelets, and VIIIR:Ag was determined by immunoelectrophoretic technique. VIIIR:WF and VIIIR:Ag were significantly enhanced in plasma from rats at 28 days after induction of diabetes and VIIIR:Ag was enhanced in plasma from rats at 14 days after induction of diabetes, but at the earlier times studied, neither were different from values in plasma from control-treated rats. Changes in VIIIR:WF and VIIIR:Ag therefore occurred later than the changes in platelet function. Plasma cholesterol concentrations were not significantly different at any of the times studied, but plasma triglyceride concentrations were significantly increased at 3 days and remained increased with further durations of diabetes. This may have contributed to the observed platelet and vessel wall changes. If these in vitro alterations reflect in vivo behavior, then platelet alterations occur before vessel wall changes and therefore do not appear to be a consequence of such changes in experimental diabetes mellitus.     

Subcellular platelet factor VIII antigen and von Willebrand factor

Subcellular membrane and granule fractions derived from human platelets contain factor VIIII antigen and von Willebrand factor activity but not factor VII procoagulant activity. Circulating platelets constitute a significant reservoir of plasma factor VIII antigen, containing approximately 15% of the amount of factor VIII antigen present in platelet-poor plasma. The antibiotic ristocetin, which aggregates human platelets in the presence of von Willebrand factor, nonspecifically precipitates platelet membrane factor VIII antigen. Thus normal platelets contain surface-bound as well as internally stored von Willebrand factor, a protein synthesized by endothelial cells which is necessary for normal platelet function in vivo.     

von Willebrand factor propeptide to antigen ratio identifies platelet activation and reduced von Willebrand factor survival phenotype in mice

Essentials

• Reduced survival of von Willebrand factor (VWF) in plasma causes type 1C von Willebrand disease.
• Blood was collected from mouse strains by various methods and VWF propeptide and antigen assayed.
• VWF propeptide to antigen ratio identifies a reduced VWF survival phenotype in mice.
• This ratio validates the acceptability of murine blood samples for coagulation studies.

Summary

Background

Reduced plasma survival of von Willebrand factor (VWF) is characteristic of patients with type 1C von Willebrand disease (VWD). These subjects can be identified by an increased steady‐state ratio of plasma VWF propeptide (VWFpp) to VWF antigen (VWF:Ag). A similar phenotype occurs in mice with the Mvwf1 allele.

Objectives

To (i) determine if the VWFpp/VWF:Ag ratio can be used to identify a ‘type 1C’ phenotype in mice, (ii) determine the most reliable method for murine blood sampling, and (iii) identify the source of VWF released during problematic blood collection.

Methods

‘Platelet‐VWF’ and ‘endothelial‐VWF’ mice were generated by bone marrow transplantation between C57BL/6J and VWF‐/‐ mice. Several blood sampling methods were used and murine VWFpp and VWF:Ag levels determined. Plasma and platelet VWF:Ag and VWFpp, VWF multimers and VWF half‐life were examined in mouse strains with and without Mvwf1.

Results

A single retro‐orbital bleed and vena cava collection were found to be the optimal methods of blood collection. Problematic collection resulted in release of VWF from platelets and endothelium. The VWFpp/VWF:Ag ratio identified strains of mice with reduced VWF survival.

Conclusion

Assay of murine VWFpp and VWF:Ag has utility in determining the acceptability of murine blood samples for coagulation testing and in identification of a reduced VWF survival phenotype in mice.

     

Endothelial cell synthesis of von Willebrand antigen II, von Willebrand factor, and von Willebrand factor/von Willebrand antigen II complex.

von Willebrand antigen II (vW AgII) and von Willebrand factor (vWf) are immunochemically distinct proteins that are deficient in the plasma and platelets of patients with severe von Willebrand's disease. Normal human umbilical vein endothelial cells were cultured in the presence of [35S]methionine. Crossed immunoelectrophoresis of endothelial cell supernates and detergent-solubilized endothelial cells demonstrated specific incorporation of the [35S]methionine into vW AgII. Furthermore, when endothelial cells were lysed in the presence of proteolytic inhibitors, a second, less anodal peak was identified on crossed immunoelectrophoresis. This peak represented a complex of vW AgII and vWf and demonstrated a reaction of complete identity with the vW AgII immunoprecipitate. When plasma, serum, or platelets were evaluated by crossed immunoelectrophoresis, this "complex" peak was not present. When antibodies to vWf, fibronectin, or fibrinogen were present in the first dimension of crossed immunoelectrophoresis, only the antibodies to vWf removed the complex. Radioiodinated polyclonal and monoclonal antibodies to vWf also localized vWf to this complex. Under reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled immunoprecipitates indicated that the molecular weight of vW AgII is 98,000 and that vWf was present as two species of 220,000 and 260,000 mol wt, respectively. Immunofluorescent microscopy of endothelial cells demonstrated colocalization of vW AgII and vWf in endothelial cells with intense immunostaining of the same subcellular granules.     

Effect of calcium on the availability of platelet von Willebrand factor

The binding of a polyclonal immune-purified rabbit anti-human von Willebrand factor (VWF) antibody to activated and nonactivated normal human platelets was studied in the presence and absence of extracellular calcium. Extracellular Ca++ in the incubation buffer results in no increase in the amount of antibody bound to the surface of unstimulated platelets when compared with that bound in the absence of Ca++. Alpha thrombin stimulation of the platelets increases antibody binding to the platelets in both the presence and absence of extracellular Ca++; however, the increase is approximately four times as great in the presence of extracellular Ca++. The amount of VWF detected in the incubation supernatants of unstimulated platelets is similar in either the presence or absence of Ca++, and this amount is increased without demonstrating a Ca++ dependency after thrombin stimulation. Measurement of thrombin-induced beta thromboglobulin (beta TG) release reveals some parallel between anti-VWF antibody binding and beta TG release. However, the amount of beta TG release after thrombin stimulation is independent of extracellular Ca++, whereas the amount of bound anti-VWF is markedly increased in the presence of Ca++. These studies demonstrate that thrombin induces an increase in the surface exposure of platelet VWF even in the absence of extracellular Ca++. The data also suggest that there may be a quantity of platelet VWF that can become exposed on the platelet surface by an action of thrombin that is independent of the Ca++-dependent granule release reaction induced by thrombin.     

早期胰岛素治疗对糖尿病大鼠心肌功能的干预作用

目的 探讨速度向量成像(VVI)技术结合负荷超声心动图评价早期应用胰岛素对糖尿病(DM)大鼠心肌功能的干预作用.方法 30只大鼠中20只腹腔注射链脲佐菌素复制糖尿病模型,其余10只作为对照组.糖尿病大鼠成模后又随机分为DM组(10只)和治疗组(10只),治疗组每天给予皮下注射胰岛素.每只大鼠分别进行基础和潘生丁负荷VVI检查.结果 静息状态下DM组和治疗组各项VVI指标均低于对照组,治疗组的收缩期最大速度、切向应变及收缩与舒张期最大切向应变率均较DM组明显提高;各项VVI心肌功能储备指标三组间比较差异均有统计学意义,并且治疗组的各项储备指标与DM组比较均显著增加.结论 胰岛素的早期应用可以改善糖尿病大鼠心肌功能.

Abstract：

Objective To assess the myocardial function with early insulin treatment by velocity vector imaging (VVI) combined with dipyridamole stress in diabetic rats.Methods The 30 rats comprised 20 diabetic rats and 10 normal rats in the study.The 20 diabetic rats were divided into the diabetes mellitus(DM) group and the treatment group.Every rat in the treatment group was hypodermic injected with protamime biosynthetic human insulin after the diabetic model established.The control group was including 10 normal rats of the same age.After 8 weeks,each rat was performed with VVI at baseline and after dipyridamole stress.Results At baseline VVI,the parameters in the DM and the treatment groups were reduced compared with those in the control group.The systolic velocity,circumferential strain,systolic and diastolic circumferential strain rate in the treatment group were increased significantly compared with those in the DM group.After stress,the VVI reserve parameters in the treatment group were higher than those in the DM group.Conclusions The myocardial function are improved with early insulin treatment in the diabetic rats.     

Heparin inhibition of von Willebrand factor-dependent platelet function in vitro and in vivo.

The intravenous administration of heparin to patients before open heart surgery reduced ristocetin cofactor activity by 58% (P less than 0.01, t test), and this impairment of von Willebrand factor-dependent platelet function was closely related to plasma heparin levels (r2 = 0.9), but not to plasma von Willebrand factor (vWF) levels. We hypothesized that heparin may inhibit vWF-dependent platelet hemostatic functions by directly binding vWF in solution and interfering with vWF-GpIb binding. Using the in vitro techniques of ristocetin-induced platelet agglutination, fluorescent flow cytometric measurement of vWF-platelet binding, and conventional radioligand binding assays we observed that heparin inhibited both vWF-dependent platelet function and vWF-platelet binding in a parallel and dose-dependent manner. Heparin also inhibited platelet agglutination induced by bovine vWF and inhibited the binding of human asialo-vWF to platelets in ristocetin-free systems. The inhibitory potency of heparin was not dependent upon its affinity for antithrombin III, but was molecular weight dependent: homogeneous preparations of lower molecular weight were less inhibitory. Heparin impairment of vWF function may explain why some hemorrhagic complications of heparin therapy are not predictable based on techniques for monitoring the conventional anticoagulant effects of heparin.     

罗格列酮对糖尿病大鼠肺炎性病变的影响

目的 现察过氧化物酶体增殖体活化受体-r(PPAR-r)激动剂罗格列酮对链脲菌素诱导的糖尿病大鼠肺部炎性病变的影响.方法 30只10周龄SD大鼠随机分为正常对照组(C组,雌雄各5只)、糖尿病模型组(D组,雌雄各5只)、糖尿病罗格列酮处理组(DR组,雌雄各5只),采用链脲菌素诱导建立10周糖尿病大鼠模型.其中糖尿病罗格列酮处理组用马来酸罗格列酮1mg.kg-1.d-1灌胃治疗,对照组及糖尿病模型组用同体积0.9%氯化钠注射液代替.模型建立10周后,全部大鼠于氯胺酮35mg/kg和苯巴比妥50mg/kg腹腔内注射麻醉处死,并取肺组织.光学显微镜下现察肺组织炎症程度、检测杯状细胞的分布,免疫组织化学方法检测肺组织TNF-α表达的阳性面积百分比与平均积分光密度值(I0D).结果 ①肺部炎症观察:大多数正常对照组大鼠肺组织内未见炎症细胞.糖尿病模型组可见片状炎症细胞聚集,局部肺泡结构消失.罗格列酮处理组糖尿病大鼠肺组织炎症明显比模型组轻;②杯状细胞观察:在AB/PAS染色下,正常对照组大鼠各级气管上皮内均未见杯状细胞出现,糖尿病模型组在一级或二级支气管内出现较多的杯状细胞.罗格列酮处理组糖尿病大鼠在一级或二级支气管内也出现杯状细胞,但数量明显少于糖尿病模型组;③TNF-α的表达TNF-α主要表达于气管、血管周围的成纤维细胞内,糖尿病模型组、罗格列酮处理组还可见巨噬细胞胞浆内阳性表达.正常对照组肺组织阳性表达较其余两组弱,罗格列酮处理组阳性面积百分比及平均积分光密度值介于正常对照组和糖尿病模型组之间[其中阳性面积百分比(%)C:D:DR组为9.07±4.17vs23.75±5.66vs12.21±1.50(F=54.5,P<0.05;平均光密度C:D:DR组为0.60±0.03vs0.73±0.08vs0.66±0.04(F=22.73.P<0.05)],差异有统计学意义.结论 高血糖能引起肺部炎症及组织结构的破坏.罗格列酮能够保护糖尿病大鼠肺组织,该作用独立于降血糖之外. 内阳性表达.正常对照组肺组织阳性表达较其余两组弱,罗格列酮处理组阳性面积百分比及平均积分光密度值介于正常对照组和糖尿病模型组之间[其中阳性面积百分比(%)C:D:DR组为9.07±4.17vs23.75±5.66vs12.21±1.50(F=54.5,P<0.05;平均光密度C:D:DR组为0.60±0.03vs0.73±0.08vs0.66±0.04( =22.73.P<0.05)],差异有统计学意义.结论 高血糖能引起肺部炎症及组织结构的破坏.罗格列酮能够保护糖尿病大鼠肺组织,该作用独立于降血糖之外. 内阳性表达.正常对照组肺组织阳性表达较其余两组弱,罗格列酮处理组阳性面积百分比及平均积分光密度值介于正常对照组和糖尿病模型组之间[其中阳性面积百分比(%)C:D:DR组为9.07±4.17vs23.75±5.66vs12.21±1.50(F=54.5,P<0.05;平均光密度C:D:DR组为0.60±0.03vs0.73±0.08vs0.66±0.04( =22.73.P<0.05)],差异有统计学意义.结论 高血糖能引起肺部炎症及组织结构的破坏.罗格列酮能够保护糖尿病大鼠肺组织,该作用独立于降血糖之外. 内阳性表达.正常对照组肺组织阳性表达较其余两组弱,罗格列酮处理组阳性面积百分比及平均积分光密度值介于正常对照组和糖尿病模型组之间[其中阳性面积百分比(%)C:D:DR组为9.07±4.17vs23.75±5.     

Role of von Willebrand factor in platelet thrombus formation

Haemostasis is the process that arrests bleeding from wounds, preventing blood from flowing outside of the vascular bed. Thrombosis is an abnormal condition in which the vascular lumen becomes occluded by a mass constituted primarily of aggregated platelets and polymerized fibrin. These thrombi impede the normal flow of circulating blood, becoming the acute cause of diseases that represent a great health concern in the developed world. Haemostasis and thrombosis are two aspects of the same function, ie the biological response to vascular injury leading to formation of a thrombus, which is platelet-rich specifically in the arteries. In either situation, von Willebrand factor is a crucial participant in the process as its main biological activity is to support platelet adhesion and aggregation in vessels where rapid blood flow challenges the firm attachment of thrombi to the vascular wall or exposed extravascular tissues. Advances in understanding the structure and function of von Willebrand factor indicate that this protein, for its unique biomechanical properties, may be a potentially useful target of antithrombotic drugs.     

儿童血管性血友病因子抗原和活性水平参考区间建立

目的 观察性别、年龄和ABO血型对儿童血管性血友病因子(vWF)检测的影响并建立参考区间。方法 随机选取2022年1月至9月苏州大学附属儿童医院无出凝血临床表现且常规凝血检查结果均正常的857例儿童，检测其血浆vWF抗原和活性水平与ABO血型。结果 ≤6岁儿童vWF抗原和活性水平低于>6岁且≤18岁儿童(P<0.05);在不同年龄分组中O型血儿童vWF水平均低于非O型血儿童(P<0.001)。对于≤6岁儿童，O型血儿童vWF抗原和活性参考区间分别为44.4%～130.6%和43.0%～194.7%;非O型血儿童vWF抗原和活性参考区间分别66.3%～190.4%和71.3%～282.3%。对于>6岁儿童，O型血儿童vWF抗原和活性参考区间分别为50.4%～145.4%和51.7%～221.4%;非O型血儿童vWF抗原和活性参考区间分别为75.4%～195.6%和84.0%～296.5%。结论 儿童血浆vWF抗原和活性水平受年龄和ABO血型影响，实验室积极建立针对儿童vWF的特异性参考区间有利于临床全面评估患儿的出血与血栓风险。     

von Willebrand factor and endothelial abnormalities in diabetic microangiopathy

We briefly summarize current knowledge on 1) the abnormalities of von Willebrand factor (vWF) as an indicator of endothelial cell (EC) dysfunction in diabetes and 2) the modifications induced in the growth of cultured ECs by high glucose in the incubation media. A MEDLINE search (1986 through Sept. 1989) was performed to update previous relevant references on vWF and ECs in healthy and diabetic subjects. Main data in the literature and personal contributions were scrutinized. Study quality, information, and relevance to the subject were assessed. vWF is synthesized and stored mainly in ECs. Its plasma levels are increased in diabetic microangiopathy but are not influenced by circulating glucose, insulin, or growth hormone, nor do they acutely affect platelet function in diabetes. Supraphysiological concentrations of glucose inhibit the replication of cultured ECs from large vessels via different possible mechanisms but appear to stimulate pathways involved in the activation of capillary ECs. vWF is a possible marker of EC damage in diabetes, and prospective studies will ascertain its role as a predictor for the development of microangiopathy. The possible dichotomy in the response of cultured ECs from large and small vessels to high glucose in the culture media may help explain some of the lesions observed in the walls of arteries and capillaries in diabetes.     

杏丁治疗对糖尿病肾病患者血浆血栓调节蛋白和血管性血友病因子的影响

目的：观察杏丁治疗对糖尿病肾病（DN）患者血浆血栓调节蛋白（TM）和血管性血友病因子（vWF）的影响。方法：60例DN患者，随机分为常规治疗组和杏丁治疗组。所有患者均于治疗前后抽血测TM、vWF、纤维蛋白原（FIB）、活化部分凝血活酶时间（APT）及血黏度。结果：杏丁治疗组患者治疗后。TM、vWF、FIB及血浆黏度均较治疗前显著下降（P〈0．01），全血黏度下降（P〈0．05），APTT显著延长（P〈0．01），且与常规治疗组相比较，差异有显著性（P〈0．05）。结论：DN患者体内存在内皮的损伤，高凝倾向。对DN患者早期使用杏丁治疗，可以有效地保护其血管内皮系统，改善其体内的高凝状态，对延缓疾病的进展有着重要意义。     

2型糖尿病患者子女胰岛素抵抗和血浆第八辅助因子水平的变化

目的:检测年龄超过30岁的2型糖尿病患者的子女胰岛素抵抗与血浆第八辅助因子(vonWillebrandfactor,vWF)水平的变化,评价糖尿病子女是否存在胰岛素抵抗及血管内皮损伤。方法:对38例至少双亲之一有2型糖尿病的子女及28例正常人进行了体质量、体质量指数、腰围、腰臀比、血压、空腹血糖、餐后血糖、血脂、空腹胰岛素、胰岛素抵抗稳定模式评估法(homeostasismodelassessment-insulinresistance,HOMA-IR)的测定,同时也观察了代表内皮损伤的vWF水平的变化。结果:糖尿病子女组:体质量(68±14)kg、体质量指数(24.5±4.0)kg/m、腰臀比(0.85)、血压收缩压(117±8)mmHg,舒张压(73±8)mmHg、血糖(5.2±1.0)mmol/L、胆固醇(4.6±1.1)mmol/L、高密度脂蛋白胆固醇(1.5±0.3)mmol/L及低密度脂蛋白胆固醇(2.4±0.8)mmol/L等未发现改变,而空腹胰岛素(13±6)mIU/L、代表胰岛素抵抗的HOMA-IR(3.0±1.5)和代表血管内皮损伤的血浆vWF(112±50)%与对照组比较是升高的,差异具有显著性意义(t=-2.29～2.00,P<0.05)。结论:2型糖尿病的子女当年龄超过30岁以后,在体质量、体脂、血压、血糖尚未发生变化之前,血胰岛素水平已发生变化,胰岛素抵抗及血管内皮损伤已经出现。     

N-acetylcysteine reduces the size and activity of von Willebrand factor in human plasma and mice

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease characterized by systemic microvascular thrombosis caused by adhesion of platelets to ultra-large vWF (ULVWF) multimers. These multimers accumulate because of a deficiency of the processing enzyme ADAMTS13. vWF protein forms long multimers from homodimers that first form through C-terminal disulfide bonds and then join through their N termini by further disulfide bonding. N-acetylcysteine (NAC) is an FDA-approved drug that has long been used to treat chronic obstructive lung disease and acetaminophen toxicity and is known to function in the former disorder by reducing mucin multimers. Here, we examined whether NAC could reduce vWF multimers, which polymerize in a manner similar to mucins. In vitro, NAC reduced soluble plasma-type vWF multimers in a concentration-dependent manner and rapidly degraded ULVWF multimer strings extruded from activated ECs. The effect was preceded by reduction of the intrachain disulfide bond encompassing the platelet-binding A1 domain. NAC also inhibited vWF-dependent platelet aggregation and collagen binding. Injection of NAC into ADAMTS13-deficient mice led to the rapid resolution of thrombi produced by ionophore treatment of the mesenteric venules and reduced plasma vWF multimers. These results suggest that NAC may be a rapid and effective treatment for patients with TTP.     

Fibrinogen and von Willebrand factor-independent platelet aggregation in vitro and in vivo

BACKGROUND: Fibrinogen (Fg) has been considered essential for platelet aggregation. However, we recently demonstrated formation of occlusive thrombi in Fg-deficient mice and in mice doubly deficient for Fg and von Willebrand factor (Fg/VWF(-/-)). METHODS AND RESULTS: Here we studied Fg/VWF-independent platelet aggregation in vitro and found no aggregation in citrated platelet-rich plasma of Fg/VWF(-/-) mice. Surprisingly, in Fg/VWF(-/-) plasma without anticoagulant, adenosine diphosphate induced robust aggregation of Fg/VWF(-/-) platelets but not of beta(3)-integrin-deficient (beta(3) (-/-)) platelets. In addition, beta(3) (-/-) platelets did not significantly incorporate into thrombi in Fg/VWF(-/-) mice. This Fg/VWF-independent aggregation was blocked by thrombin inhibitors (heparin, hirudin, PPACK), and thrombin or thrombin receptor activation peptide (AYPGKF-NH(2)) induced aggregation of gel-filtered Fg/VWF(-/-) platelets in 1 mm Ca(2+) PIPES buffer. Notably, aggregation in PIPES buffer was only 50-60% of that observed in Fg/VWF(-/-) plasma. Consistent with the requirement for thrombin in vitro, hirudin completely inhibited thrombus formation in Fg/VWF(-/-) mice. These data define a novel pathway of platelet aggregation independent of both Fg and VWF. Although this pathway was not detected in the presence of anticoagulants, it was observed under physiological conditions in vivo and in the presence of Ca(2+)in vitro. CONCLUSIONS: beta(3) integrin, thrombin, and Ca(2+) play critical roles in this Fg/VWF-independent aggregation, and both plasma and platelet granule proteins contribute to this process.