Distribution Of Von Willebrand Factor Research Articles

Platelet factor 4 inhibits ADAMTS13 activity and regulates the multimeric distribution of von Willebrand factor.

The efficiency of von Willebrand factor (VWF) in thrombus formation is related to its multimeric size, which is controlled by the protease ADAMTS13. However, it is not clear what regulates ADAMTS13 activity. In this study, we investigated whether PF4 could bind to VWFand inhibit ADAMTS13 activity. We found that PF4 binds to VWF and protects against ADAMTS13 activity. We also found that VWF-PF4 complexes circulate in patients with thrombotic thrombocytopenic purpura (TTP). Our data provides the first evidence that PF4 may have a novel role in regulating VWF multimers during primary haemostasis and thrombosis.

Immunohistochemical Analysis of the Lacrimal Sac Mucopeptide Concretions.

To investigate the presence and distribution patterns of proteins in the lacrimal sac mucopeptide concretions. The study was performed on 10 mucopeptide concretions obtained from the lacrimal sac during an endoscopic dacryocystorhinostomy. Immunohistochemical labeling was performed for assessing the presence and distribution of von Willebrand factor, fibronectin, collagen I, collagen III, human beta-defensin 2, beta-defensin 3, lysozyme, prolactin, cytokeratin (CK)-CK4, CK7, CK18, immunoglobulin M (IgM), immunoglobulin G (IgG), psoriasin (S100-A7), and migration inhibitory factor-related protein (MRP14/S100-A9). The results were then scored as positive or negative and the distribution pattern, if any, within the sectioned concretions was assessed. Immunohistochemical labeling was strongly positive for von Willebrand factor and fibronectin in the core areas of the concretions. Collagen I was widely dispersed both in the peripheral and core areas whereas collagen III was mostly limited to only few areas of the core. Strong immunoreactivity was noted for lysozyme, which was abundantly distributed, whereas prolactin receptors were positive on the surface and also the dispersed cells within the concretion matrices. Very few peripheral areas of concretions showed MRP14 or S100-A9 positivity in a noncontiguous fashion. Immunoreactivity was uniformly negative for CK-18, IgM, IgG, and psoriasin. The presence of fibronectin and von Willebrand factor in the core of the concretions strengthens the earlier hypothesis that blood components may act as a nidus for subsequent mucopeptide deposition. The study also provides possible clues to the lacrimal defenses mounted during dacryolithiasis.Fibronectin and vWF are present in the core of the dacryoliths. Blood components may act as a nidus in the evolution of lacrimal sac mucopeptide concretions.

Immunohistochemistry of von Willebrand factor in the lungs with regard to the cause of death in forensic autopsy

Pulmonary microvascular injury is involved in severe trauma or disease. The present study investigated the immunohistochemical distribution of von Willebrand factor (vWF) and platelet CD61 factor in forensic autopsy cases ( n = 157, >18 years of age, within 48 h postmortem), which comprised fatalities from blunt and sharp instrument injuries, strangulation, fire fatality and acute cardiac death (ACD). vWF immunoreactivity was clearly detected in the endothelia of large vessels (LV, ϕ > 200 μm), small vessels (SV, ϕ 40–200 μm) and capillaries (Cap, ϕ < 40 μm). Cap-vWF positivity was also detected in microthrombi with CD61 immunopositivity. The vWF positivity was higher in non-edema areas than in the edema area in the lungs. For acute deaths, Cap-vWF positivity of non-edema areas was frequently detected for strangulation ( n = 8/13, 61.5%), fire fatality ( n = 11/26, 42.3%) and ACD ( n = 8/15, 53.3%), but was infrequent for blunt and sharp instrument injuries ( n = 6/27, 22.5%, and n = 2/15, 13.3%, respectively), irrespective of the complication of chest injury. However, for non-acute deaths, Cap-vWF positivity was more frequent for non-chest blunt injury ( n = 12/27, 44.4%) than for blunt chest injury ( n = 3/13, 23.1%) and sharp instrument injury ( n = 0/10, 0%). For fire fatality, Cap-vWF positivity was relatively frequent in cases with a lower blood carboxyhemoglobin (COHb) level of <60% ( n = 6/14, 42.8%) than in cases with a higher COHb level of >60% ( n = 3/12, 25.0%). These findings suggest that Cap-vWF positivity is closely related to the death process involving pulmonary microvascular injury.

Distribution of von Willebrand factor levels in young women with and without bleeding symptoms. Influence of ABO blood group and promoter haplotypes

The normal distribution of von Willebrand factor (VWF) levels is wide. Low levels are associated with bleeding symptoms and von Willebrand disease (VWD). We have recently described a high prevalence of bleeding symptoms in a whole age group of young females (n = 1,019) from Malmo, Sweden. It was the objective of the present study to evaluate the distribution of VWF levels in young females with or without bleeding symptoms in this population, and the influence of ABO blood group and promoter haplotypes on VWF levels and to identify a possible increased prevalence of VWD in females with bleeding symptoms. A random selection of the female age group (n = 246), into a study group (n = 176) with, and a control group (n = 70) without bleeding symptoms, was evaluated. Eighteen girls had VWF:RCo below the reference range, of which 17 belonged to the study group (17/176, 9.7%), and one to the control group (1/70, 1.4%) (p = 0.017). Blood group O was found in 14/18 girls with low VWF:RCo. There was a highly significant correlation between VWF:RCo and blood group O and non-O genotypes. Two common VWF promoter haplotypes did not contribute to the VWF:RCo variation. VWF levels did not correlate with time during menstrual cycle, or the use of oral contraceptives. No case fulfilled the diagnostic criteria for VWD. In conclusion, low VWF:RCo was significantly more frequent in females with bleeding symptoms. However, we found no case fulfilling strict diagnostic criteria for VWD. The ABO blood group was a strong modifier, but VWF promoter haplotypes had no association to VWF levels in this population.

Deletion of Pro1648-Leu1650 in VWF73, the Conservative Substrate for ADAMTS13, Causes von Willebrand Disease.

The multimer distribution of Von Willebrand factor (VWF) in plasma is regulated by the specific VWF cleaving protease ADAMTS13, which cleaves at the Y1605-M1606 bond in the A2 domain of VWF under the shear stress, plays paramount roles in mediating platelet adhesion to the subendothelium during vascular damage. Quantitative deficiency or qualitative abnormity in VWF caused by the mutations in the VWF gene leads to von Willebrand disease (vWD). There exist three types of vWD. Type 1 vWD is characterized by the partial quantitative deficiency of VWF and normal multimers. Type 3 refers to complete deficiency of VWF. Type 2 vWD refers to the qualitative deficiency of VWF and is subdivided into types of 2A, 2B, 2M, 2N. Meanwhile, the subtype of 2A vWD is also subdivided into two groups regarding ADAMTS13-dependent proteolysis of VWF. Group I includes the mutations G1505R, S1506L, L1540P, V1607D, which hinder the multimer assembly and diminish the secretion of VWF while group II includes R1597W, R1597Q, G1505E, I1628T, E1628K, which make VWF more susceptible to ADAMTS13 -dependent proteolysis. All these published point mutations cluster in the A2 domain of VWF and the corresponding mutation mechanism upon VWF has been elucidated. We have identified a patient with bleeding symptoms and reduced plasma VWF antigen, factor VIII and ristocetin cofactor activity, compatible with clinical von Willebrand disease. Analysis of proband's plasma VWF multimers in low resolution agarose gels demonstrated similar results compared to the healthy. The patient carried a heterozygous deletion mutation from position 1648 to 1650 resulting in loss of three consecutive amino acids (ProIleLeu) in the pre-pro-VWF. It has been demonstrated that the minimal substrate for ADAMTS13 is intact VWF73, a region from Asp1596 to Arg1668 of von Willebrand factor. The novel deletion mutation in this patient occurred in the intact VWF73 and its mutated effect upon cleavage by ADAMTS13 could be clarified by further experiments such as in vitro recombinant expression of mutated VWF and might strengthen our understanding of the interaction between VWF and ADAMTS13.

Hyperstimulation and a Gonadotropin-Releasing Hormone Agonist Modulate Ovarian Vascular Permeability by Altering Expression of the Tight Junction Protein Claudin-5

We investigated the mechanism by which a GnRH agonist (GnRHa) affects ovarian vascularity, vascular permeability, and expression of the tight junction protein claudin-5 in a rat model of ovarian hyperstimulation syndrome (OHSS). Hyperstimulated rats received excessive doses of pregnant mare serum gonadotropin (PMSG; 50 IU/d) for 4 consecutive days, from d 25 to 28 of life, followed by 25 IU human chorionic gonadotropin (hCG) on d 29. Control rats received 10 IU PMSG on d 27 of life, followed by 10 IU hCG on d 29. GnRHa (leuprolide 100 microg/kg.d) was administered to some hyperstimulated rats either on d 29 and 30 (short-term GnRHa treatment) or from d 25 to 30 (long-term GnRHa treatment). Ovarian vascular density (vessels per 10 mm(2)) and vessel endothelial area (percent) were assessed by immunohistochemical analysis of the distribution of von Willebrand factor, whereas vascular permeability was evaluated based on leakage of Evans blue. High doses of PMSG and hCG significantly increased ovarian weight, vascular permeability, vascular density, and the vessel endothelial area and significantly reduced expression of claudin-5 protein and mRNA. All of these effects were significantly and dose-dependently inhibited by administration of GnRHa. This suggests that reduced expression of claudin-5 plays a crucial role in the increased ovarian vascular permeability seen in OHSS and that its expression can be modulated by GnRHa treatment. Indeed, preventing redistribution of tight junction proteins in endothelial cells and the resultant loss of endothelial barrier architecture might be the key to protecting patients against massive extravascular fluid accumulation in cases of OHSS.

Histomorphometric analysis of aortic medial structural elements in cases with an apparently normal aortic wall

Pulmonary microvascular injury is involved in severe trauma or disease. The present study investigated the immunohistochemical distribution of von Willebrand factor (vWF) and platelet CD61 factor in forensic autopsy cases (n = 157, >18 years of age, within 48 h postmortem), which comprised fatalities from blunt and sharp instrument injuries, strangulation, fire fatality and acute cardiac death (ACD). vWF immunoreactivity was clearly detected in the endothelia of large vessels (LV, ϕ > 200 μm), small vessels (SV, ϕ 40–200 μm) and capillaries (Cap, ϕ < 40 μm). Cap-vWF positivity was also detected in microthrombi with CD61 immunopositivity. The vWF positivity was higher in non-edema areas than in the edema area in the lungs. For acute deaths, Cap-vWF positivity of non-edema areas was frequently detected for strangulation (n = 8/13, 61.5%), fire fatality (n = 11/26, 42.3%) and ACD (n = 8/15, 53.3%), but was infrequent for blunt and sharp instrument injuries (n = 6/27, 22.5%, and n = 2/15, 13.3%, respectively), irrespective of the complication of chest injury. However, for non-acute deaths, Cap-vWF positivity was more frequent for non-chest blunt injury (n = 12/27, 44.4%) than for blunt chest injury (n = 3/13, 23.1%) and sharp instrument injury (n = 0/10, 0%). For fire fatality, Cap-vWF positivity was relatively frequent in cases with a lower blood carboxyhemoglobin (COHb) level of <60% (n = 6/14, 42.8%) than in cases with a higher COHb level of >60% (n = 3/12, 25.0%). These findings suggest that Cap-vWF positivity is closely related to the death process involving pulmonary microvascular injury.

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

Journal Website (http: www.nms.ac.jp jnms ) Confocal laser scanning microscopy revealed the unique distribution of von Willebrand factor (vWf)and thrombomodulin(TM)in microvascular endothelial cells of the human lung. In dual immunofluorescence technique, vWf and TMwere labeled by FITC(green), and Texas red, respectively. Cell nucleus was stained by TOTO 3(blue). ( See page 118―125 of this journal ) 64 J Nippon Med Sch 2000;67(2)

Ultrastructural analysis of the distribution of von Willebrand factor and fibrinogen in platelet aggregates formed in the PFA-100

The PFA-100 is a new apparatus used to detect platelet dysfunction in vitro . Anticoagulated blood flows under constant pressure through a capillary, and across an aperture that pierces a membrane coated with collagen and either epinephrine or ADP. Through their ability to adhere and aggregate, platelets occlude the orifice and the closure time is a test of platelet function. Using electron microscopy and immunogold staining, we have analyzed the ultrastructure of platelet aggregates formed within the aperture and that are responsible for the occlusion. Standard electron microscopy showed that the aggregates formed on both collagen-epinephrine and collagenADP cartridges presented the same morphological features. The aggregates were exclusively composed of platelets, some of which were degranulated. Degranulation was particularly intense at the periphery of the aggregate where platelets were often totally devoid of secretory organelles. Immunogold staining on ultrathin frozen sections with polyclonal antibodies, allowed us to evaluate the distribution of adhesive proteins such as fibrinogen and von Willebrand factor (vWF) within the aggregate. The latter was found to be abundant in the intercellular spaces between adjoining platelets. Although fibrinogen was also present, its labeling was less intense suggesting that vWF is the major protein implicated in the platelet-platelet interactions in the aggregates formed in the PFA-100 system. This may be because of the high shear rate that occurs across the aperture which suggests that the PFA-100 is particularly sensitive for detecting abnormalities of vWFplatelet interactions.

Vascular origin determines angiotensin I-converting enzyme expression in endothelial cells.

Previous observations on the heterogeneous distribution of von Willebrand factor in the vascular endothelium led us to examine the expression of angiotensin I-converting enzyme (ACE) in function of the vascular origin of endothelial cells (EC). EC from pig thoracic aorta, pulmonary artery, inferior vena cava and brain capillaries were cultured and assayed for ACE by enzymatic radiochemical determination and by western-blot and immunofluorescence using an antiACE polyclonal antibody. EC from the various vascular levels secreted ACE in the culture medium; western-blot analysis showed its presence at cellular level and immunofluorescence confirmed its location on the plasma membrane. But quantification revealed that EC from pulmonary artery contain more ACE than EC from the other vessels, especially from brain capillaries; immunofluorescence correlated well with the functional data. In contrast, secretion of ACE by brain capillaries EC was faster than that of arteries and of vena cava, the latter being the less effective. This differential ACE expression along the vascular tree could have a pharmacological implication since ACE inhibitors, used in the treatment of arterial hypertension, may act more at the vascular level than on the plasma renin-angiotensin system. On the other hand, endothelial distribution of ACE was different from that of von Willebrand factor; in particular we showed that EC cultured from vessels of pigs homozygous for the von Willebrand disease, in which von Willebrand factor synthesis was completely abolished, normally express ACE.

Distribution of von Willebrand factor in capillary endothelial cells of rat lungs with pulmonary fibrosis

To determine the value of von Willebrand factor (vWF) antigen as a marker of endothelial injury in radiation-induced fibrosis of rat lungs, we studied endothelial immunoreactivity to antibodies against vWF using the indirect immunoperoxidase technique combined with morphometric analysis. Using immunoelectron microscopy of LR White embedded lung samples to detect vWF, immunogold-labelled Weibel Palade bodies were found in endothelial cells of capillary endothelium. The irradiated lungs showed a statistically significant elevation of vWF expression, ie, vWF positive endothelia per unit area, and a significant increase of vWF expression per unit of parenchyma as well. The results suggest that vWF antigen expression and the number of vWF positive structures is modulated under condition of injury in radiation-induced fibrosis.

Changes in the pattern of distribution of von Willebrand factor in rat aortic endothelial cells following thrombin generation in vivo

The pattern of distribution of von Willebrand factor (VWF) in relatively large sheets of rat aortic endothelial cells (EC) obtained by the Häutchen technique were analysed by immunocytochemistry and light microscopy. EC were examined pre and post administration of a procoagulant mixture of factor Xa (F.Xa) and phosphotidylcholine/phosphotidylserine (PCPS) vesicles which was demonstrated to result in the selective loss of high molecular weight multimers (HMWM) of plasma VWF in the rat. In placebo animals the pattern was heterogenous both in overall distribution and in individual cells which showed both a diffuse and granular pattern. Groups of intensely stained EC were oriented parallel to the longitudinal axis of the aorta and staining was particularly prominent around the orifices of the intercostal arteries, implicating shear-stress as a possible factor in VWF expression by EC. Changes in the pattern of distribution of staining were observed at various time points post-infusion of F.Xa/PCPS, suggesting the immediate release of VWF from EC stores followed by the recruitment of EC to synthesize and store VWF. These changes are consistent with the decrease in EC Weibel-Palade Body (WPB) content observed by EM in previously reported studies using this model.

Evidence for a factor promoting the conversion of VWF from low and intermediate to high molecular mass polymers on the platelet membrane

On the membrane surface of resting platelets exists a factor capable of promoting the conversion of plasma von Willebrand factor from low and intermediate molecular mass to high molecular mass polymers. The process is accompanied by a parallel increase in the von Willebrand factor activity. This could be a regulatory system for the molecular mass distribution of von Willebrand factor during its lifetime in plasma. Glycoprotein Ib of the platelet membrane appears not to be involved in the process.

Stimulated secretion of endothelial von Willebrand factor is accompanied by rapid redistribution to the cell surface of the intracellular granule membrane protein GMP-140

We have examined the cell activation-dependent redistribution of the intracellular granule membrane protein GMP-140 of human endothelial cells. By dual-label immunofluorescence, the distribution of GMP-140 within cultured human umbilical vein endothelial cells was found to coincide with the distribution of von Willebrand factor (vWF), suggesting that GMP-140 is located in the membranes of vWF-containing storage granules. Stimulation of vWF secretion resulted in an increase in GMP-140 on the cell surface, as detected by increased binding of the monoclonal antibody S12 which recognizes the extracytoplasmic domain of GMP-140. For each agonist tested (histamine, thrombin, phorbol 12-myristate 13-acetate, and the calcium ionophore A23187) a dose-dependent redistribution of GMP-140 to the endothelial surface was observed which closely paralleled the dose-dependent secretion of vWF into the cell supernatant. When cells were maximally stimulated by histamine in the presence of antibody S12, a 4-fold increase in S12 uptake by the cells was observed. This increase occurred rapidly and reached a plateau by 10 min. In contrast, when histamine-stimulated cells were first fixed with paraformaldehyde or chilled to 4 degrees C before addition of antibody S12, only a transient increase in cell surface GMP-140 was detected. Under these conditions of arrested membrane turnover during antibody binding, cell surface GMP-140 was maximal 3 min after histamine stimulation and then declined to control levels by 20 min. These data suggest that stimulated secretion of vWF from endothelial cells entails fusion of vWF-containing storage granules with the plasma membrane. Once inserted into the plasma membrane, GMP-140 is subsequently removed from the endothelial surface, most likely by an endocytic mechanism.

Storage and Secretion of von Willebrand Factor by Endothelial Cells

Endothelial cells synthesize and store von Willebrand factor. We have studied the storage and secretion of von Willebrand factor in cultured human umbilical vein endothelial cells. In particular, we were interested in the nature of the storage compartment and the effects of perturbation on the storage and secretion processes. The storage compartment for von Willebrand factor was isolated from homogenates of endothelial cells. By an immunostaining technique the isolated vesicles stained for von Willebrand factor. The staining pattern was similar to that of Weibel-Palade bodies in intact endothelial cells. We concluded that the storage compartment containing von Willebrand factor is identical to the Weibel-Palade body. The von Willebrand factor of the isolated storage vesicles is predominantly constructed of polypeptide chains with a Mr of 220 kD. On the other hand, von Willebrand factor continuously secreted by endothelial cells is constructed of both a 220 kD and a larger precursor (apparent Mr of 275 kD) subunit. The storage vesicles contain von Willebrand factor that supports ristocetin-induced platelet aggregation. Thus, endothelial cells store fully processed, biologically active von Willebrand factor within Weibel-Palade bodies. Short-term (less than 1 h) treatment of endothelial cells with the perturbing phorbol ester 4 beta-phorbol-12-myristate-13-acetate (PMA) results in release of cellular stored von Willebrand factor. 24-48 h after exposure to PMA the endothelial cell distribution of von Willebrand factor is changed distinctly. While the contents of the von Willebrand factor storage sites in the cells are gradually restored within 48 h, enhanced amounts of von Willebrand factor are secreted into the medium. The number as well as the size of von Willebrand factor storage granules in the endothelial cells increase after exposure to phorbol ester, as determined by immunofluorescence microscopy. Phorbol ester treated cells release stored von Willebrand factor 48 h after they have been stimulated. PMA decreases the von Willebrand factor contents of the extracellular matrix; the deposition of von Willebrand factor in the subendothelium is blocked by PMA, whereas the degradation of matrix von Willebrand factor is not affected. Thus, perturbation of endothelial cells changes the cellular distribution of von Willebrand factor.

Perturbation of cultured human vascular endothelial cells by phorbol ester or thrombin alters the cellular von Willebrand factor distribution.

We have studied the influence of perturbation of cultured human umbilical vein endothelial cells on the distribution of the von Willebrand factor. As shown previously, short-term (less than 1 hr) treatment of endothelial cells with the phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) or thrombin resulted in the release of cellular stored von Willebrand factor. Long-term treatment with PMA or thrombin evoked a distinct change in the endothelial cell distribution of von Willebrand factor, evident 24 to 48 hrs after exposure. Whereas the contents of the von Willebrand factor storage sites in the cells were gradually restored within 48 hrs, enhanced amounts of von Willebrand factor were secreted into the medium. However, PMA did not increase the endothelial cell contents of mRNA encoding for von Willebrand factor. The number as well as the size of von Willebrand factor storage granules in the endothelial cells increased after exposure to the phorbol ester, as determined by immunofluorescence microscopy. A second treatment with PMA or thrombin, 48 hrs after cells had been stimulated with these agents, resulted again in the instantaneous release of von Willebrand factor. PMA and thrombin caused a decrease in the von Willebrand factor contents of the extracellular matrix. Pulse-chase experiments revealed that PMA blocked the deposition of von Willebrand factor in the subendothelium, whereas PMA did not affect the degradation of matrix von Willebrand factor. Thus, perturbation of endothelial cells changes the cellular distribution of von Willebrand factor.

Distribution of von Willebrand factor in porcine intima varies with blood vessel type and location.

The von Willebrand factor (vWF) has been generally accepted as a marker for endothelial cells. In a systematic immunolocalization study of porcine blood vessels that used indirect immunofluorescence with a monospecific polyclonal anti-vWF and two monoclonal anti-vWFs, we observed that vWF is not universally distributed in intact, fresh endothelia. vWF is consistently localized in veins, with the exception of the pulmonic vein. In arteries, vWF is generally absent except for areas of the distal abdominal aorta, the vaso vasorum of the thoracic aorta, and the pulmonic artery. We conclude that there are regional differences in the distribution of vWF in the various endothelial beds of pigs.

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.

Eccentric Localization of von Willebrand Factor in an Internal Structure of Platelet α-Granule Resembling That of Weibel-Palade Bodies

AbstractImmunogold staining was used to study the ultrastructural distribution of von Willebrand factor (vWF) in unstimulated platelets. vWF was detected in the α-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 α-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 Å to 250 Å in diameter. They closely resembled the internal tubular structures found in Weibel-Palade bodies, which are the storage organelles of vWF in endothelial cells.