Influence Of Von Willebrand Factor Research Articles

Influence of von Willebrand factor on common pathophysiological mechanisms of cerebrovascular diseases and multiple sclerosis

Endothelial dysfunction is a universal pathological mechanism underlying or contributing to the development/progression of many diseases, including cerebrovascular disease and multiple sclerosis. Von Willebrand factor is a multimeric glycoprotein synthesized by endothelial cells and megakaryocytes that participates in a range of physiological and pathological processes, including primary hemostasis and coagulation. It also regulates secretion and transport of a variety of molecules, exerts a proinflammatory effect, modulates angiogenesis and smooth muscle mitotic activity, influences atherogenesis. In this review, we discuss the synthesis, secretion, and regulation of the von Willebrand factor within the context of endothelial dysfunction and other common mechanisms that play a significant role in brain tissue damage in cerebrovascular diseases and multiple sclerosis.

A Factor VIII Fusion with an Inactivated FIX Generates a Biologically Active Molecule with Delayed Clearance in a FVIII/VWF Knockout Mouse Model

Background: Developing a delayed clearance factor VIII for the treatment of haemophilia A is of importance due to the cost and inconvenience associated with current FVIII products which require several intravenous injections each week to maintain therapeutic FVIII levels. While there are a number of extended half-life rFVIII products in development, they only have a ~1.8-fold extension of half-life due to the dominant influence of von Willebrand factor (VWF).Objective: To generate a long-acting rFVIII through fusion to inactive FIX allowing intermolecular binding of FVIII and FIX as well as a dynamic equilibrium with FVIII being free to bind endogenous FIXa. We hypothesized that the fusion to inactive FIX will protect FVIII from proteolytic inactivation, create steric hindrance for binding to VWF and FVIII clearance receptors and thereby delay the in vivo clearance of FVIII.Methods: We cloned a novel rFVIII fusion molecule linked through its C-terminus to an inactivated factor IX designated 13A1FVIII. 13A1FVIII was expressed in mammalian cells (CHO) and protein expression confirmed by western blotting. Biological activity was tested using a chromogenic assay and thrombin generation test and clearance examined in VWF/FVIII double knock out mice.Results: 13A1FVIII protein purification was achieved by affinity based chromatography with >95% purity as judged by silver staining. Purified 13A1FVIII bioactivity was confirmed in vitro using a chromogenic FVIII assay and an activated partial thromboplastin time (aPTT) test. In a PK study using VWF/FVIII double knock out mice, 13A1FVIII exhibited a reduced plasma clearance over Advate rFVIII with terminal half-life of 32hrs vs 12hrs, respectively. The observed 2.7-fold increase in half-life was achieved independent of VWF.Conclusions: A fusion of FVIII to inactive FIX retains biological activity in two in vitro tests of haemostasis. The fusion protein also demonstrates delayed plasma clearance and an increase in circulating half-life in a haemophiliac mouse model. The results suggest it is possible to delay FVIII clearance independent of VWF using this fusion protein approach. DisclosuresNo relevant conflicts of interest to declare.

Pre-surgical level of von Willebrand factor as an evident indicator of breast cancer recurrence.

Endothelial and platelet activation as well as a disruption of haemostatic balance are crucial in cancer-dependent venous thromboembolism development. The aim of this study was to investigate the influence of von Willebrand factor (VWF), sE-selectin, sP-selectin as well as VWF/sE-selectin and sP-selectin/sE-selectin ratios on the probability of disease relapse in invasive breast carcinoma (IBrC) cases. Eighty-four patients with IA-IIB stage of IBrC who passed a comprehensive clinicopathologic evaluation were included in the study. Follow-up was completed in all patients with a 15.48 % recurrence rate. An immunoassay of VWF antigen, sE-selectin, sP-selectin, as well as an immunohistochemistry of oestrogen and progesterone receptors, human epidermal growth factor receptor 2 (HER2) and Ki67 was performed in all cases. The VWF/sE-selectin ratio was significantly higher in patients with poorly differentiated tumours than in those with high-differentiated tumours. A positive correlation between VWF concentration and tumour grade was noted. Eleven of 13 events happened in patients with VWF value below 600 mU/mL with recurrence rate of 25%, but only two events occurred in subject with VWF values above the 600 mU/mL (5%; P= 0.0028). Our study show that VWF could be considered as a suitable biomarker of breast cancer relapse.

Characterization of a Porcine Model for Von Willebrand Disease Type 1 and 3 Regarding Expression of Angiogenic Mediators in the Nonpregnant Female Reproductive Tract.

Von Willebrand disease (VWD), a blood coagulation disorder, is also known to cause angiodysplasia. Hitherto, no animal model has been found with angiodysplasia that can be studied in vivo. In addition, VWD patients tend to have a higher incidence of miscarriages for reasons unknown. Thus, we aimed to examine the influence of von Willebrand factor (VWF) on the female reproductive tract histology and the expression and distribution of angiogenic factors in a porcine model for VWD types 1 and 3. The disease-causing tandem duplication within the VWF gene occurred naturally in these pigs, making them a rare and valuable model. Reproductive organs of 6 animals (2 of each mutant genotype and 2 wildtype (WT) animals) were harvested. Genotype plus phenotype were confirmed. Several angiogenic factors were chosen for possible connections to VWF and analyzed alongside VWF by immunohistochemistry and quantitative gene expression studies. VWD type 3 animals showed angiodysplasia in the uterus and shifting of integrin αVβ₃ from the apical membrane of uterine epithelium to the cytoplasm accompanied by increased vascular endothelial growth factor (VEGF) expression. Varying staining patterns for angiopoietin (Ang)-2 were observed among the genotypes. As compared with WT, the ovaries of the VWD type 3 animals showed decreased gene expression of ANG2 and increased gene expression of TIE (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains) 2, with some differences in the ANG/TIE-system among the mutant genotypes. In conclusion, severely reduced VWF seems to evoke angiodysplasia in the porcine uterus. Varying distribution and expression of angiogenic factors suggest that this large animal model is promising for investigation of influence of VWF on angiogenesis in larger groups.

Expression Profile of VEGF and VEGFR2 in the Female Reproductive Tract at the Time of Placentation in a Porcine Model of Von Willebrand Disease Type 1

Objectives: Clinical studies showed that women affected by von Willebrand disease (VWD) trend towards higher rates of miscarriages, but the underlying pathomechanisms remain unclear. Several in vitro studies demonstrated an influence of von Willebrand factor (VWF) on angiogenesis, which seems to be partly mediated via VEGF. Since angiogenesis in the reproductive organs is essential to establish and maintain pregnancy, we aimed to investigate gene expression of VWF, VEGF, and its receptor VEGFR2 in non-pregnant and pregnant individuals, using a porcine model of VWD type 1.Methods: Tissue samples of uterus, oviduct and ovary were harvested from eight female pigs of which four were pregnant on day 30 (time of placentation) and four were non-pregnant and in estrus. Of each group, two were affected by VWD type 1 and two were wildtype (WT) individuals. The gene expression of VWF, VEGF, and VEGFR2 was measured by qRT-PCR and relatively quantified against the endothelial specific housekeeping genes PROCR and CD31 using the ΔΔCT method and calculating the respective x-fold changes. The gene expression differences were compared between both genotypes as well as between both reproductive states Mean differences were taken into account, if the divergences were consistent in both individuals of each group and not within the range of the group they were compared to.Results: Regarding the non-pregnant sows, VWF expression was lower in uterus and ovary of the VWD type 1 animals. This difference was not seen comparing the pregnant animals of each genotype, but the VWD type 1 animals showed higher VEGF expression in oviduct and higher VEGF and VEGFR2 expression in the ovary. The expression of VEGFR2 was reduced in the uterus. Comparing the non-pregnant with the pregnant animals within each genotype, the following results were found: the pregnant WT pigs showed increased expression of VEGFR2 in uterus and ovary, but decreased expression of VEGF in the uterus. For the pregnant VWD type 1 animals increased expression was found for VWF and VEGFR2 in the ovary, and decreased expression for VEGF in uterus and oviduct and VEGFR2 in the uterus.Discussion and Conclusion: Comparison of the different groups revealed differences of gene expression in the female reproductive tract during early pregnancy. The expectedly lower expression of VWF in the VWD type 1 animals was not found for the pregnant animals. Apparently, there is an increase of VWF levels during pregnancy as seen in women. While VEGFR2 expression in the uterus increases during placentation in the WT animals, it decreases in the VWD type 1 animals. This suggests altered regulation of early angiogenesis, which is essential during placentation. Expression of VEGF and VEGFR2 was increased in the ovaries of the VWD type 1 animals. This points to an enhanced involvement of this pathway during conversion of the ruptured follicles to sufficient corpora lutea graviditates, which may affect this process. Enhanced angiogenesis via the VEGF/VEGFR2-pathway due to lack of VWF was already shown in vitro. Our study shows that the expression of VEGF and VEGFR2 differs during early pregnancy in VWD type 1 compared to wildtype sows. Therefore, this pathway may influence angiogenesis in the reproductive tract. DisclosuresNo relevant conflicts of interest to declare.

New Horizons for Approach of ST-Segment Elevation Myocardial Infarction

In the past decades, there was a reduction in mortality from ST segment elevation acute myocardial infarction (STEMI) associated with a set of actions combining technological advances and public policies. However, its characteristic of a time-dependent disease is still responsible for a high number of cases of sudden death and the consequences of late or inefficient reperfusion are related to heart failure and increased morbidity and mortality. From this point of view, three different aspects were reviewed: the impact of ventricular arrhythmias in prehospital care; the influence of Von Willebrand factor and the role of microparticles in the diagnosis of the disease.

The influence of von Willebrand factor on factor VIII activity measurements.

It has been reported by multiple laboratories that the quantitation of factor (F)VIII by activity-based assays is influenced by the method, procedure and the quality of reagents used in the assays. To evaluate the influence of von Willebrand factor (VWF) on FVIII activity in vitro. The activated partial thromboplastin time (APTT) and synthetic coagulation proteome assays were used. Citrated FVIII/VWF-depleted substrate plasma (SP) (both antigens < 0.5%) was used in all APTT assays. The concentration of FVIII antigen in pooled plasma from healthy donors [normal plasma (NP)] was 1.5 nm. The SP reconstituted with 1.5 nm recombinant (r)FVIII clotted in 23.8 +/- 0.2 s (standard deviation). The addition of 10 microg mL(-1) VWF to the SP increased the clotting time to 28.7 +/- 0.1 s; that is, the activity of rFVIII (FVIIIc) decreased to 50%. This inhibitory effect of VWF decreased with decreasing rFVIII concentration in SP, and became negligible at rFVIII <or= 10% (150 pm). The FVIIIc of 1.5 nm FVIII in two products formulated with VWF (Immunate and Hemofil M) was not affected by the addition of exogenous VWF to the SP, whereas the FVIIIc of the B-domainless rFVIII product ReFacto was decreased to 50% by the addition of VWF. In the synthetic coagulation proteome triggered with 5 pm tissue factor, VWF had no effect on thrombin generation. VWF has an inhibitory effect on the measurement of FVIII clotting activity. This effect depends upon the structure and formulation of the FVIII product.

Persistent factor VIII-dependent factor X activation on endothelial cells is independent of von Willebrand factor

Endothelial cells are able to support the activation of coagulation factor X by activated factor IX in the presence of its cofactor, factor VIII. We have previously reported that this reaction is persistent on endothelial cells, but transient on activated platelets and phospholipid vesicles when activated factor X (Xa) is used as activator of factor VIII. Aim of the present study was to explore the influence of von Willebrand factor and that of the factor VIII activator, either factor Xa or thrombin, on the decay of factor X activation on the endothelial cell surface. Kinetics of factor X activation on human umbilical vein endothelial cells was compared with that on phospholipid vesicles employing purified coagulation factors from plasma as well as recombinant factor VIII variants. Employing factor Xa as factor VIII activator, rate constants for decay of membrane-bound factor X activation were consistently low on endothelial cells (0.02 min) as compared with phospholipid vesicles (0.2 min). Activation of factor VIII by thrombin resulted in two-fold increased decay rates. In the presence of excess of von Willebrand factor over factor VIII, decay rates were not significantly changed. Factor VIII variants with and without a Tyr to Phe substitution, which abolishes high-affinity binding to von Willebrand factor, displayed the same factor X activation decay kinetics. Although previous studies have shown that von Willebrand factor modulates factor VIII activation and stabilisation, this apparently does not affect the progression of factor X activation at the endothelium.

Adenovirus-induced thrombocytopenia: the role of von Willebrand factor and P-selectin in mediating accelerated platelet clearance

AbstractThrombocytopenia has been consistently reported following the administration of adenoviral gene transfer vectors. The mechanism underlying this phenomenon is currently unknown. In this study, we have assessed the influence of von Willebrand Factor (VWF) and P-selectin on the clearance of platelets following adenovirus administration. In mice, thrombocytopenia occurs between 5 and 24 hours after adenovirus delivery. The virus activates platelets and induces platelet-leukocyte aggregate formation. There is an associated increase in platelet and leukocyte-derived microparticles. Adenovirus-induced endothelial cell activation was shown by VCAM-1 expression on virus-treated, cultured endothelial cells and by the release of ultra-large molecular weight multimers of VWF within 1 to 2 hours of virus administration with an accompanying elevation of endothelial microparticles. In contrast, VWF knockout (KO) mice did not show significant thrombocytopenia after adenovirus administration. We have also shown that adenovirus interferes with adhesion of platelets to a fibronectin-coated surface and flow cytometry revealed the presence of the Coxsackie adenovirus receptor on the platelet surface. We conclude that VWF and P-selectin are critically involved in a complex platelet-leukocyte-endothelial interplay, resulting in platelet activation and accelerated platelet clearance following adenovirus administration.

Immune Tolerance Induction (ITI) in Hemophilia A and Inhibitors using VWF Containing Factor VIII Concentrates.

Immune tolerance induction using the Bonn protocol (ITI) is the most successful approach to eliminate inhibitors in hemophilia A patients. The influence of the type of concentrate, particularly the content of von-Willebrand-Factor (VWF) used for ITI is under discussion and has never been investigated comparatively.A longitudinal study at the Frankfurt center on the influence of VWF on ITI using the Bonn protocol (low responder: 50–100 IU FVIII/kg bw daily or every other day; high responder: 100–150 IU FVIII/kg bw every 12 hours; according to the bleeding tendency concomitant treatment with FEIBA®/Baxter) showed a significantly decreased success rate since the introduction of high purity plasma derived (pd) and recombinant (rec) F VIII products (success rate with pd VWF-F VIII 91% vs ultrapure F VIII 29%). Similar observations have been reported by the Bonn and Bremen centers (success rate with pd VWF-F VIII 87% vs ultrapure F VIII 54%). A meta-analysis of different ITI studies revealed a higher success rate with the use of VWF-FVIII concentrates (88% using VWF-FVIII and 63% using rec and pd/monoclonal purified F VIII).The change to VWF-FVIII concentrates during ITI in inhibitor patients who showed an unsatisfactory treatment course with ultrapure F VIII concentrates (n=12 high responders) led to success in 10 out of 12 patients after a median treatment period of 17 months (5–36 months). Successful IT after changing to VWF-F VIII concentrates was evaluated by a questionnaire in another 10 high responders who had unsatisfactory treatment courses with ultrapure F VIII concentrates.These observations indicate that VWF has a major impact on the success of ITI.

Influence of von Willebrand factor on the reactivity of human factor VIII inhibitors with factor VIII

In order to determine the difference in reactivity of factor (F) VIII inhibitors against the FVIII/von Willebrand factor (vWF) complex and against vWF‐deficient FVIII, we investigated a panel of 10 antibodies to FVIII from multitransfused individuals with severe haemophilia A and other pathologies. Immunoblotting of purified FVIII and purified thrombin‐cleaved FVIII revealed that in all cases inhibitor epitopes could be localized in the heavy chain (A2 subunit) while in four cases they were also present in the light chain. One of the FVIII inhibitors remained unclassified. The effect on FVIII:C of purified IgG from inhibitor plasmas was tested against a high purity FVIII/vWF concentrate and a monoclonally purified FVIII concentrate with only trace contents of vWF, by two different functional assays. Our results suggest that for those inhibitors showing A2 plus light chain (LC) reactivity, the IgG concentration required to inhibit 50% of FVIII activity in vitro is higher for the FVIII/vWF complex than for the vWF‐deficient FVIII. We conclude that there might be a protective role of vWF (at least in vitro) against FVIII inhibitors with A2 and LC subunit specificity.

Influence of von Willebrand factor on the reactivity of human factor VIII inhibitors with factor VIII.

In order to determine the difference in reactivity of factor (F) VIII inhibitors against the FVIII/von Willebrand factor (vWF) complex and against vWF-deficient FVIII, we investigated a panel of 10 antibodies to FVIII from multitransfused individuals with severe haemophilia A and other pathologies. Immunoblotting of purified FVIII and purified thrombin-cleaved FVIII revealed that in all cases inhibitor epitopes could be localized in the heavy chain (A2 subunit) while in four cases they were also present in the light chain. One of the FVIII inhibitors remained unclassified. The effect on FVIII:C of purified IgG from inhibitor plasmas was tested against a high purity FVIII/vWF concentrate and a monoclonally purified FVIII concentrate with only trace contents of vWF, by two different functional assays. Our results suggest that for those inhibitors showing A2 plus light chain (LC) reactivity, the IgG concentration required to inhibit 50% of FVIII activity in vitro is higher for the FVIII/vWF complex than for the vWF-deficient FVIII. We conclude that there might be a protective role of vWF (at least in vitro) against FVIII inhibitors with A2 and LC subunit specificity.

Development of coronary atherosclerosis in swine with severe hypercholesterolemia. Lack of influence of von Willebrand factor or acute intimal injury.

The development of coronary atherosclerosis in response to acute intimal injury and severe hypercholesterolemia was studied in 18 swine, nine normal and nine with von Willebrand's disease, an inherited disorder affecting platelet-vessel wall interactions. The left anterior descending coronary artery was denuded of endothelium by balloon catheterization, while the circumflex and right coronary arteries served as nonballooned controls. All swine were maintained on a 2% cholesterol diet for 4 months. The extent of atherosclerotic involvement was evaluated from four indices: percent intimal area, percent luminal narrowing, ratio of intimal to medial area, and luminal form. No differences in coronary atherosclerosis were observed between phenotypes in either ballooned or nonballooned vessels, nor were there any differences between ballooned and nonballooned vessels within either phenotype (p greater than 0.05). The major variable affecting coronary atherosclerosis was serum cholesterol. There was a significant positive relationship between serum cholesterol concentration and the extent of intimal lesions (r = 0.62, p = 0.006) that was independent of plasma von Willebrand factor concentration. These findings suggest that severe hypercholesterolemia promotes the development of porcine coronary atherosclerosis through a mechanism(s) that is independent of acute intimal injury or von Willebrand factor-mediated platelet response to injury.