Localization Of Von Willebrand Factor Research Articles

Heterogeneity Of Type 3 Von Willebrand Disease (VWD): Evidence From Patient-Derived Blood Outgrowth Endothelial Cells (BOEC)

BackgroundType 3 von Willebrand Disease (VWD) is the rarest and most severe form of the disease. Patients exhibit plasma von Willebrand factor (VWF) levels of<5% for both VWF:Ag and VWF:RCo and have low FVIII levels (<10%). Patient-derived blood outgrowth endothelial cells (BOEC) provide a useful cellular model for studying VWD instead of using heterologous cell systems, which do not completely mimic the native environment. Until recently, we believed the lack of endogenous VWF in patients with type 3 VWD might pose as a challenge for isolating and culturing type 3 VWD BOEC. The objectives of the current study were to isolate BOEC from type 3 VWD patients with different genotypes and to compare the cellular presence, if any, and localization of VWF in the patient-derived BOEC using confocal immunofluorescence microscopy. Patients and MethodsPeripheral blood samples were obtained from seven type 3 VWD index cases (IC) (from six different families) in order to isolate and culture BOEC. These patients were originally enrolled in the Canadian type 3 VWD study as IC or siblings of IC. Research Ethics Board approval was obtained, as well as informed consent from all participants. Endothelial cell phenotype of isolated cells was confirmed using flow cytometry for cell surface markers; positive for CD31, CD144 and/or CD146, and negative for CD14 and CD45. Confocal immunofluorescence microscopy was used to observe intracellular VWF. ResultsWe were able to successfully isolate and culture BOEC from seven type 3 VWD patients with different genotypes. All cells were confirmed as endothelial cells as per the markers listed in the methods. Table 1 shows the patient characteristics, and corresponding VWF phenotype and genotype.Table 1Type 3 VWD Index Case Phenotype and GenotypePatient IDGender/age (M/F year)Bleeding ScoreVWF:Ag (IU/ml)VWF:RCo (IU/ml)FVIII:C (IU/ml)Nucleotide Change, HGVSAmino Acid Change, HGVSExonGenotypeT001M/42190.010.020.02c.221-977_532+7059delp.Asp75_Gly178del4-5HomozygousT050F/21240.010.000.01c.876delC, c.1255C>Tp.Ser293fs, p.Gln419*8/11Compound heterozygousT076M/17230.010.030.01c.1897T>Cp.Cys633Arg15HomozygousT076-SF/1190.010.030.01c.1897T>Cp.Cys633Arg15HomozygousT105F/62180.020.000.03c.8419_8422dupTCCCp.Pro2808Leufs*2452HomozygousT151M/27290.020.040.01c.3939G>A, c.5842+1 G>Cp.Trp1313*, -28/34Compound heterozygousT154M/16240.010.030.01c. 1657dupT, c.8419_8422dupTCCCp.Trp553Leufs*97, p.Pro2808Leufs*2414/52Compound heterozygousDespite a virtual lack of VWF in the plasma of the seven type 3 VWD patients, confocal immunofluorescence microscopy studies showed different patterns of intracellular VWF. Qualitative and/or quantitative defects in Weibel-Palade bodies (WPB) were observed. T001, T076, and T076-S, IC whom are homozygous for VWF propeptide mutations, show a complete lack of WPB formation, with only diffuse staining of VWF, indicative of retention in the endoplasmic reticulum (ER). This was confirmed by co-localization of the diffuse VWF with the ER marker calnexin. T105 and T154, who have in common a frameshift mutation in the CK domain of VWF showed similar VWF staining patterns with diffuse staining and a limited number of rounded WPBs. T151, a compound heterozygote for a nonsense mutation in exon 28 and a splice site mutation in intron 34, showed both diffuse VWF staining, as well as a number of rounded WPBs. The latter three cases indicate abnormalities in WPB biogenesis which would impair secretion of VWF. T050, who is compound heterozygous for two different VWF propeptide mutations, showed a complete absence of VWF, presenting a true VWF “null” endothelial cell. DiscussionWe have shown that BOEC can be successfully isolated from type 3 VWD patients with different VWF genotypes. As well, we have further confirmed the heterogeneity in type 3 VWD as seen in the variety of endothelial cellular phenotypes associated with the different VWF genotypes. Patient-derived BOEC are a useful cellular model for investigating the pathogenic nature of VWF mutations reflective of the native vascular environment. As well, they may be a useful tool to study the effect of novel treatments for VWD, leading the way for personalized treatment of VWD. Disclosures:James:CSL Behring: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; Baxter: Honoraria; Bayer: Honoraria.

Differential localization of P-selectin and von Willebrand factor during megakaryocyte maturation

An important step in megakaryocyte maturation is the appropriate assembly of at least two distinct subsets of α-granules. The mechanism that sorts the α-granule components into distinct structures and mediates their release in response to specific stimuli is now emerging. P-selectin and von Willebrand factor are two proteins present in the α-granules that recognize P-selectin glycoprotein ligand on neutrophils and collagen in the subendothelial matrix. These proteins may play an important role in determining the differential release of the α-granule contents in response to external stimuli. If P-selectin and von Willebrand factor are localized in the same or different α-granules is not known. To clarify this question, we analyzed by immunoelectron microscopy the localization of von Willebrand factor and P-selectin during the maturation of wild-type and Gata1low megakaryocytes induced in vivo by treating animals with thrombopoietin. Gata1low is a hypomorphic mutation that blocks megakaryocyte maturation, reduces the levels of von Willebrand factor expression and displaces P-selectin on the demarcation membrane system. The maturation block induced by this mutation is partially rescued by treatment in vivo with thrombopoietin. In immature megakaryocytes, both wild-type and Gata1low, the two receptors were co-localized in the same cytoplasmic structures. By contrast, the two proteins were segregated to separate α-granule subsets as the megakaryocytes matured. These observations support the hypothesis that P-selectin and von Willebrand factor may ensure differential release of the α-granule content in response to external stimuli.

Changes in the Localization of Immunoreactive von Willebrand Factor in Microvascular Network of Bovine Ovarian Follicles during Atresia

Changes in the localization of von Willebrand factor (vWF) in the follicular microvasculature were examined to determine whether functional modulation of microvasculature occurred in the atretic follicle in cows. Paraffin sections of healthy follicles and various stages of atretic follicles were immunostained with rabbit monoclonal antibody to vWF. The vWF-positive cells were counted in 4 different regions of a follicle from the apical to the basal side. In all types of follicles, immunoreactions for vWF were observed in the endothelial cells of capillaries as well as veins and arteries in the theca interna and externa. In the theca interna, the vWF-positive area was significantly (P<0.05) greater in advanced and late atretic follicles than in healthy and early atretic follicles at all regions except for the apical region of advanced atretic follicles. In the theca externa, vWF-positive area significantly (P<0.05) increased with a progress of atresia only at the apical region of follicles. However, in the late atretic follicles, the theca tissues and blood vessels did not show obvious histological disintegration. These results suggest that the increase of the vWF expression in the endothelial cells occurs before morphological changes of microvasculature appear in the atretic follicles. The functional modulation of microvasculature that is associated with the expression of vWF may be involved in the events of atresia.

Heterogeneous distribution of thrombomodulin and von Willebrand factor in endothelial cells in the human pulmonary microvessels.

Laser scanning confocal fluorescence microscopy techniques were used to study the localization of von Willebrand factor (vWf; Factor VIII-related antigen) and thrombomodulin (transmembrane receptor for thrombin) in the microvascular endothelial cells in the normal human lung. Tissues were obtained from lobectomy specimens resected for solitary nodules (7 adenocarcinomas and 4 hamartomas) from 11 patients. The plasma membranes of the capillary endothelial cells in the alveolar zones (A-zones) showed red linear fluorescence for thrombomodulin. However, their cytoplasm was mostly unreactive for vWf. The microvessels which were located in the connective tissue (C-zones), including peribronchial, and subpleural areas and large vascular walls, consistently demonstrated band-like green fluorescence for vWf in their cytoplasm, and their plasma membranes usually lacked reactivity for thrombomodulin. Only a limited number of peribronchial capillaries measuring <10 microm in diameter showed a mosaic-like appearance, in which red fluorescence along the plasma membranes was found together with green fluorescence in the subjacent cytoplasm. In the juxtaalveolar (J-zones) microvessels located along the borders between A- and C-zones, and measuring up to 40 microm in diameter, the endothelial cells showed a mosaic-like pattern of distribution of the two antigens. However, the localization of thrombomodulin in the J-zone microvessels was separate and independent from that of vWf. The thrombomodulin-reactive cells were directly connected to the alveolar capillary endothelial cells. Heterogeneous patterns of distribution of thrombomodulin and vWf suggest that topographic differences of endothelial function occur to maintain a balance of coagulation and anticoagulation in the normal human lung.

Cultured murine cerebral microvascular endothelial cells contain von Willebrand factor-positive Weibel-Palade bodies and support rapid cytokine-induced neutrophil adhesion.

To obtain cultures of rodent brain microvascular endothelium (BMEC) that retain endothelial cell-specific markers and functions for two purposes: investigating whether these cultures contain endothelial cell-specific storage granules or Weibel-Palade bodies and have the ability to rapidly bind neutrophils upon cytokine induction; and setting the groundwork for future studies examining endothelium derived from mice strains with targeted deficiencies in endothelial adhesion molecules. Capillaries were obtained by collagenase/dispase digestion and subsequent density centrifugation of either rodent brain or meninges. The yield was then plated onto fibronectin-coated dishes. For some studies, pure murine endothelial cultures were obtained by flow-cytometric sorting, using uptake of fluorescently labeled diI-acetylated low-density lipoprotein as a marker for endothelium. Endothelial cell-specific markers were analyzed via immunofluorescence, immunoprecipitation and light microscopy. Cytokine-induced neutrophil adhesion and associated upregulation of leukocyte adhesion molecules were measured as described previously for human umbilical vein endothelial cells. BMEC possess numerous von Willebrand factor-containing Weibel-Palade bodies and synthesize and secrete all multimeric forms of von Willebrand factor. They take up diI-acetylated low-density lipoproteins, contain platelet-endothelial cell adhesion molecules and form capillary-like structures on three-dimensional extracellular matrix substrates. Sorted murine brain microvascular endothelial cells treated with IL-1 beta or TNF-alpha for 4 h show an increase in surface expression of the cytokine-inducible leukocyte adhesion molecules E-selectin, VCAM-1, and ICAM-1, and they support rapid neutrophil adhesion, which is, on average, three times greater than that of nonstimulated cells. The brain microvascular endothelial cultures described here exhibit many of the markers of endothelial cells including the presence of Weibel-Palade bodies. The localization of von Willebrand factor almost exclusively to Weibel-Palade bodies indicates that murine cerebral endothelium has evolved an efficient mechanism for storage of this platelet adhesion protein, which plays an important role in hemostasis. In addition, this is the first demonstration of rapid neutrophil adhesion to murine brain microvascular endothelial cells. Finally, the reproducible culture and the characterization of murine BMEC makes feasible future studies on endothelium isolated from gene-targeted mice.

Differential localization of von Willebrand factor, fibronectin and 13-HODE in human endothelial cell cultures

Von Willebrand factor (vWF), fibronectin (FN) and 13-hydroxy-octadecadienoic acid (13-HODE) are known to influence the regulation of the adhesive properties of vascular surfaces. In the present study vWF, FN and 13-HODE were comparatively localized in endothelial cells (EC) and in the extracellular matrix (ECM) produced by EC. An indirect immunofluorescent technique was applied to coverslips containing human EC cultures previously fixed and permeabilized following different procedures: A. Alcohol/acetone; B. Paraformaldehyde alone and C. Paraformaldehyde followed by Triton X-100. vWF was observed inside EC (A), on the ECM produced by EC (B) or in EC and ECM (C) depending on the fixation procedures used. FN was mainly localized in the ECM despite the fixation procedures employed. FN was only seen in relation to cell bodies after strong permeabilization (A). Under our experimental conditions 13-HODE was never found in ECM. This latter antigen was observed randomly dispersed in those preparations fixed with alcohol/acetone, indicating that it is probably extracted by this fixative. 13-HODE was detected in granular shaped structures in EC after permeabilization with detergent (C). These results suggest that the cellular localization of vWF and FN is compatible with an adhesive role related to the abluminal side of ECs. 13-HODE was readily observed after mild permeabilization. This finding would be morphologically consistent with its contribution to the regulation of the vessel wall thromboresistance.

Absence of tubular structures and immunolabeling for von Willebrand factor in the platelet alpha-granules from porcine von Willebrand disease [published erratum appears in Blood 1987 Feb;69(2):707

The electron microscopic localization of von Willebrand factor (vWF) was studied in platelets from normal and von Willebrand disease (vWD) pigs. In normal pig platelets, immunolabeling for vWF was far more intense and extensive than in human platelets and was either localized at one pole of the alpha-granule or all along its periphery or long axis. As in human platelets, this immunolabeling coincided with the presence of tubules about 200 nm in diameter. These structures were more numerous than in human platelets, with up to 30 tubules per alpha- granule. They were easily identified either in transverse sections, usually grouped in a less electron-dense part of the matrix at one pole of the alpha-granule, or in longitudinal sections parallel to the long axis of the elongated granules, or coiled around the alpha-granule core. They closely resemble those structures found in Weibel-Palade bodies. In platelets from pigs with severe vWD, these structures were absent, as was the immunolabeling for vWF; however, cytoplasmic microtubules were normally present in these platelets. Thus, the granule-associated tubules can be distinguished from the microtubules, which are larger in diameter (250 nm), are present in both normal and vWD platelets, and do not stain for vWF. These results strongly suggest that the tubular structures present in the alpha-granules of normal porcine platelets correspond to the vWF molecule itself.

Absence of Tubular Structures and Immunolabeling for von Willebrand Factor in the Platelet a-Granules From Porcine von Willebrand Disease

The electron microscopic localization of von Willebrand factor (vWF) was studied in platelets from normal and von Willebrand disease (vWD) pigs. In normal pig platelets, immunolabeling for vWF was far more intense and extensive than in human platelets and was either localized at one pole of the alpha-granule or all along its periphery or long axis. As in human platelets, this immunolabeling coincided with the presence of tubules about 200 nm in diameter. These structures were more numerous than in human platelets, with up to 30 tubules per alpha-granule. They were easily identified either in transverse sections, usually grouped in a less electron-dense part of the matrix at one pole of the alpha-granule, or in longitudinal sections parallel to the long axis of the elongated granules, or coiled around the alpha-granule core. They closely resemble those structures found in Weibel-Palade bodies. In platelets from pigs with severe vWD, these structures were absent, as was the immunolabeling for vWF; however, cytoplasmic microtubules were normally present in these platelets. Thus, the granule-associated tubules can be distinguished from the microtubules, which are larger in diameter (250 nm), are present in both normal and vWD platelets, and do not stain for vWF. These results strongly suggest that the tubular structures present in the alpha-granules of normal porcine platelets correspond to the vWF molecule itself.

Gray platelet syndrome: immunoelectron microscopic localization of fibrinogen and von Willebrand factor in platelets and megakaryocytes

An immunogold method was used for investigating the subcellular localization of von Willebrand factor (vWF) and fibrinogen (Fg) in platelets and cultured megakaryocytes from normal subjects and from three patients with the gray platelet syndrome (GPS), a rare congenital disorder characterized by the absence of alpha-granules. In normal platelets at rest, vWF was detected exclusively in alpha-granules, with a characteristic distribution: gold particles were localized at one pole of each labeled granule, outlining the inner face of its membrane. vWF was distributed similarly in the alpha-granules of megakaryocytes at day 12 of culture, where it was also found in small vesicles near the Golgi complex. In contrast, Fg was observed in the whole matrix of all platelet alpha-granules but not in the nucleoids. In platelets from three patients with GPS, vWF and Fg were distributed homogeneously in the rare normal alpha-granules, which could be recognized by their size, and also in small granules identified as abnormal alpha-granules, which were similar in size to the small, possibly immature granules present in normal megakaryocytes. In addition, in some unstimulated platelets, Fg labeling was associated with dense material in the lumen of the surface-connected canalicular system (SCCS). At day 12 of culture, megakaryocytes from the patients with GPS contained some small alpha-granules labeled for Fg and vWF identical to those found in mature platelets. The majority of alpha-granules of normal size appeared partially or completely empty. Thus, we conclude that vWF is distributed differently from Fg in normal alpha-granules, and that unstimulated platelets from patients with GPS contain Fg and vWF in a population of small granules identifiable as abnormal alpha-granules only by immunoelectron microscopy. In addition, the presence of Fg in the SCCS of gray platelets suggests a spontaneous release of the alpha- granule content.

Gray Platelet Syndrome: Immunoelectron Microscopic Localization of Fibrinogen and von Willebrand Factor in Platelets and Megakaryocytes

An immunogold method was used for investigating the subcellular localization of von Willebrand factor (vWF) and fibrinogen (Fg) in platelets and cultured megakaryocytes from normal subjects and from three patients with the gray platelet syndrome (GPS), a rare congenital disorder characterized by the absence of alpha-granules. In normal platelets at rest, vWF was detected exclusively in alpha-granules, with a characteristic distribution: gold particles were localized at one pole of each labeled granule, outlining the inner face of its membrane. vWF was distributed similarly in the alpha-granules of megakaryocytes at day 12 of culture, where it was also found in small vesicles near the Golgi complex. In contrast, Fg was observed in the whole matrix of all platelet alpha-granules but not in the nucleoids. In platelets from three patients with GPS, vWF and Fg were distributed homogeneously in the rare normal alpha-granules, which could be recognized by their size, and also in small granules identified as abnormal alpha-granules, which were similar in size to the small, possibly immature granules present in normal megakaryocytes. In addition, in some unstimulated platelets, Fg labeling was associated with dense material in the lumen of the surface-connected canalicular system (SCCS). At day 12 of culture, megakaryocytes from the patients with GPS contained some small alpha-granules labeled for Fg and vWF identical to those found in mature platelets. The majority of alpha-granules of normal size appeared partially or completely empty. Thus, we conclude that vWF is distributed differently from Fg in normal alpha-granules, and that unstimulated platelets from patients with GPS contain Fg and vWF in a population of small granules identifiable as abnormal alpha-granules only by immunoelectron microscopy. In addition, the presence of Fg in the SCCS of gray platelets suggests a spontaneous release of the alpha-granule content.

Eccentric localization of von Willebrand factor in an internal structure of platelet alpha-granule resembling that of Weibel-Palade bodies

Immunogold staining was used to study the ultrastructural distribution of von Willebrand factor (vWF) in unstimulated platelets. vWF was detected in the alpha-granules with a specific eccentric distribution pattern opposite the nucleoids. Similar findings were obtained with a polyclonal antibody or a pool of monoclonal antibodies to human vWF. This labeling coincided with the presence of tubular structures located at the periphery of the alpha-granules. These structures were better visualized on platelets treated for standard electron microscopy: they formed a group of one to four tubules ranging from 200 A to 250 A in diameter. They closely resembled the internal tubular structures found in Weibel-Palade bodies, which are the storage organelles of vWF in endothelial cells.

Use of monoclonal antibody and colloidal gold in E.M. localization of von Willebrand Factor in megakaryocytes and platelets

The subcellular localization of Factor VIII/von Willebrand protein (VIII R:Ag) is studied with monoclonal antibody and gold immunocytochemical technique. Monoclonal antibody against purified VIII R:Ag is brightly fluorescent on megakaryocytes and platelets. In E.M., gold immunolabeling is performed on thin cell sections of human megakaryocytes and platelets. Different embedding materials are used to preserve the antigenicity: Epon embedded megakaryocytes show a high concentration of VIII R:Ag in α-granules using 4F9 monoclonal antibody. In comparison, lowicryl K4M embedded material does not improve the same specificity, only a few platelets granules were stained. This subcellular localization, in full agreement with biochemical results appears visualized for the the first time in E.M.