Von Willebrand Factor Deposition Research Articles

Von Willebrand factor: from figurant to main character in the scene of inflammation

von Willebrand factor: from figurant to main character in the scene of inflammation

Anti‐inflammatory protective effect of ADAMTS‐13 in murine arthritis models

BackgroundPatients with rheumatoid arthritis (RA) have frequent thrombotic events with endothelial dysfunction. Von Willebrand factor (VWF) has been shown to bind neutrophil extracellular traps (NETs) and NETs are part of RA etiology. ObjectivesThis study aims to elucidate whether this prothrombotic status exacerbates inflammation in arthritis. Here we focus on the involvement of A Disintegrin And Metalloprotease with ThromboSpondin type 1 motif, member 13 (ADAMTS‐13), an enzyme cleaving VWF and its effect on NET deposition and RA development. MethodsWe evaluated the influence of the Adamts13 gene and recombinant human ADAMTS‐13 (rhADAMTS‐13) on arthritis in the mouse models of collagen‐induced arthritis (CIA). We also assessed VWF and NETs in synovial tissue. ResultsSeveral Adamts13−/− mice developed arthritis, while Adamts13+/+ siblings did not. Synovial tissue from Adamts13−/− showed accumulation of NETs. Treatment of DBA/1 J mice, an arthritis‐susceptible strain, with well‐tolerated doses of rhADAMT13 reduced arthritis incidence and alleviated the severity of arthritis. Mice treated with rhADAMT13 presented less serum interleukin 6 and less bone erosion determined by micro‐computed tomography. The effects on arthritis severity were observed both when administering rhADAMTS‐13 prophylactically and also when given after arthritis has developed. In both conditions, rhADAMTS‐13 reduced VWF and NET deposition on proliferated synovial tissue evaluated by immunoblotting. ConclusionsOur results demonstrate the inhibitory role of Adamts13 in murine arthritis and the effectiveness of rhADAMTS‐13 treatment. Additionally, this study suggests that deposition of VWF in the synovium and subsequent pathogenic NET retention promotes arthritis. Treatment with rhADAMTS‐13 provides a potential therapeutic approach targeting inflammation and pro‐thrombotic state in arthritis.

Ultra-Low Von Willebrand Factor (VWF) Adsorption Under Shear with a Polymer Coating for Blood-Contacting Devices

Introduction. Many human diseases are treated with blood-contacting medical devices. However, they are often compromised because of blood proteins that accumulate on their surfaces. Such accumulation activates both platelets and blood coagulation, and patients supported by blood-contacting devices are at high risk of both bleeding and clotting. Blood passing through medical devices is often subjected to high shear stresses (especially near surfaces) and flow acceleration, conditions that we have shown promote rapid deposition of the large plasma glycoprotein von Willebrand factor (VWF) onto uncoated surfaces. Additionally, acquired Von Willebrand syndrome (AVWS) - a bleeding disorder caused by deficiency of high molecular weight VWF multimers - has been described in patients supported with ECMOs (extracorporeal membrane oxygenators) and LVADs (left ventricular assist devices). Blood in these patients experiences high shear stress when passing through the blood pump in ECMOs and LVADs. Thus, the adsorption of VWF to artificial surfaces under shear is likely to initiate platelet binding and the adsorption of other plasma proteins, including clotting proteins. Here, we show that polymers generated from the sulfobetaine methacrylate (SBMA) when coated onto surfaces are able to render these surfaces resistant to VWF binding (< 5 ng/cm2) under both static conditions and under shear stress.Preliminary results. We synthesized SBMA polymers (pSBMA) from SBMA according to methods described in the literature. Previously, we tested various coating conditions for pSBMA to achieve the lowest VWF adsorption. Under optimal conditions, VWF adsorption to pSBMA-coated polypropylene tubes was 4% of that to uncoated tubes (< 2.6 ng/cm2) after the tubes were exposed to a VWF solution (5 µg/mL) under shear applied with a vortexer (3000 rpm at RT for 90 min). In this work, we prepared a microfluidic device of polydimethylsiloxane that contained a tortuous flow channel to mimic shear stress conditions of blood vessels and tested the ability of pSBMA to prevent VWF deposition and platelet binding under different shear stress conditions.Methods. Purified plasma VWF (2.5 µg/mL) was perfused through the uncoated and pSBMA-coated devices for 1 h with a gravity-driven pump. To image the VWF structure, 5% BSA was perfused through the system to block nonspecific protein adsorption. Then FITC-labeled anti-human VWF antibody was perfused into the chamber system. After incubation at room temperature for 1 h, PBS was introduced to wash the system. The devices were fixed with 3.7% formaldehyde, incubated, and washed with PBS. We also perfused 1 mL of citrated human whole blood containing platelets stained with calcein AM through the devices at 0.05 mL/min with syringe pump to image platelet binding.Results. Under both conditions, VWF and platelet binding to the pSBMA-coated device was much lower than to the uncoated device. On the uncoated device, VWF formed aggregates which could not be washed away. By quantifying fluorescence intensity, we found that most VWF adsorbed along curves compared to the straight regions, the inlet, or the outlet of the chamber, consistent with shear stress simulation results that indicate that VWF deposits in the regions of highest shear and elongational flow. When whole blood was perfused through the chamber, the platelets bound to the entire device surface, with the inlet and outlet having relatively higher levels. In contrast, in the pSBMA-coated device, VWF bound loosely and could be removed almost completely with washing. We then perfused whole blood through uncoated or pSBMA-coated chambers without VWF pre-perfusion. Unlike the findings with VWF pre-perfusion, there was no apparent platelet binding in either device, indicating that the high level of platelet binding on the uncoated devices required time for VWF to deposit on the surface.Conclusions. PSBMA effectively prevented VWF adsorption under shear in two types of devices, and was stable during storage. We expect this coating, or a variation of it to significantly enhance the lifetime of blood-contacting devices and reduce both costs and medical complications of their use. DisclosuresNo relevant conflicts of interest to declare.

Assessment of platelet function utilizing viscoelastic testing.

Assessment of platelet function utilizing viscoelastic testing.

Von Willebrand factor delays liver repair after acetaminophen-induced acute liver injury in mice

Acetaminophen (APAP)-induced acute liver failure is associated with substantial alterations in the hemostatic system. In mice, platelets accumulate in the liver after APAP overdose and appear to promote liver injury. Interestingly, patients with acute liver injury have highly elevated levels of the platelet-adhesive protein von Willebrand factor (VWF), but a mechanistic connection between VWF and progression of liver injury has not been established. We tested the hypothesis that VWF contributes directly to experimental APAP-induced acute liver injury. Wild-type mice and VWF-deficient (Vwf-/-) mice were given a hepatotoxic dose of APAP (300 mg/kg, i.p.) or vehicle (saline). VWF plasma levels were measured by ELISA, and liver necrosis or hepatocyte proliferation was measured by immunohistochemistry. Platelet and VWF deposition were measured by immunofluorescence. In wild-type mice, VWF plasma levels, high molecular weight (HMW) VWF multimers, and VWF activity decreased 24 h after APAP challenge. These changes coupled to robust hepatic VWF and platelet deposition, although VWF deficiency had minimal effect on peak hepatic platelet accumulation or liver injury. VWF plasma levels were elevated 48 h after APAP challenge, but with relative reductions in HMW multimers and VWF activity. Whereas hepatic platelet aggregates persisted in livers of APAP-challenged wild-type mice, platelets were nearly absent in Vwf-/- mice 48 h after APAP challenge. The absence of platelet aggregates was linked to dramatically accelerated repair of the injured liver. Complementing observations in Vwf-/- mice, blocking VWF or the platelet integrin αIIbβ3 during development of injury significantly reduced hepatic platelet aggregation and accelerated liver repair in APAP-challenged wild-type mice. These studies are the first to suggest a mechanistic link between VWF, hepatic platelet accumulation, and liver repair. Targeting VWF might provide a novel therapeutic approach to improve repair of the APAP-injured liver. Patients with acute liver injury due to acetaminophen overdose have highly elevated levels of the platelet-adhesive protein von Willebrand factor. It is not known whether von Willebrand factor plays a direct role in the progression of acute liver injury. We discovered that von Willebrand factor delays repair of the acetaminophen-injured liver in mice and that targeting von Willebrand factor, even in mice with established liver injury, accelerates liver repair.

Vimentin knockout results in increased expression of sub-endothelial basement membrane components and carotid stiffness in mice

Intermediate filaments are involved in stress-related cell mechanical properties and in plasticity via the regulation of focal adhesions (FAs) and the actomyosin network. We investigated whether vimentin regulates endothelial cells (ECs) and vascular smooth muscle cells (SMCs) and thereby influences vasomotor tone and arterial stiffness. Vimentin knockout mice (Vim−/−) exhibited increased expression of laminin, fibronectin, perlecan, collagen IV and VE-cadherin as well as von Willebrand factor deposition in the subendothelial basement membrane. Smooth muscle (SM) myosin heavy chain, α-SM actin and smoothelin were decreased in Vim−/− mice. Electron microscopy revealed a denser endothelial basement membrane and increased SM cell-matrix interactions. Integrin αv, talin and vinculin present in FAs were increased in Vim−/− mice. Phosphorylated FA kinase and its targets Src and ERK1/2 were elevated in Vim−/− mice. Knockout of vimentin, but not of synemin, resulted in increased carotid stiffness and contractility and endothelial dysfunction, independently of blood pressure and the collagen/elastin ratio. The increase in arterial stiffness in Vim−/− mice likely involves vasomotor tone and endothelial basement membrane organization changes. At the tissue level, the results show the implication of FAs both in ECs and vascular SMCs in the role of vimentin in arterial stiffening.

Pursuing Ultra-Low VWF Adsorption for Blood-Contacting Devices Under Shear

Many human diseases are treated with blood-contacting medical devices. These devices, which include artificial lung and heart machines and extracorporeal membrane oxygenators (ECMO), save or extend many lives every year. However, the devices are often compromised because of blood proteins that accumulate on their surfaces. Such accumulation activates both platelet activation and blood coagulation, and patients supported by blood-contacting devices are at high risk of both bleeding and clotting. Attempts have been made to create nonfouling surfaces, and many have in fact have been produced that resist fouling by plasma when tested under non-flow conditions. However, blood passing through medical devices is often subjected to high shear stresses (especially near surfaces) and flow acceleration, conditions that we have shown promote rapid deposition of the large plasma glycoprotein von Willebrand factor (VWF) onto uncoated plastic surfaces. VWF normally functions to initiate platelet attachment at sites of vessel injury. Thus, its adsorption to artificial surfaces under shear is likely to initiate platelet binding and the adsorption of other plasma proteins, including clotting proteins. We sought to create a surface that achieves ultra-low (< 5 ng/cm2) VWF adsorption under shear, which we anticipate will prevent further protein adsorption and solve the problem of blood and platelet deposition at its initiation.To achieve this objective, we synthesized polymers from the zwitterion sulfobetaine methacrylate (polySBMA) based on published methods. Dopamine was used as the linker between polySBMA and the commonly used materials for devices, resulting in the direct attachment of the polymer to the surface in one step. A more uniform coating of the polymer on certain hydrophobic surfaces was achieved using a mixed solvent of methanol and water. Various coating conditions for polySBMA were tested, including variations in buffer type, salt concentration, polymer concentration, polymer/dopamine ratio, and methanol/water ratio. Eppendorf polypropylene tubes were incubated overnight with polySBMA solution, washed, and then exposed to a VWF solution under shear in a vortexer (3000 rpm, at RT for 90 min). We compared VWF adsorption in the polySBMA-coated PP tubes to adsorption in uncoated tubes. VWF adsorption to tubes coated under optimal conditions was 4% of the adsorption to uncoated tubes, with a VWF surface density below 2 ng/cm2. This coating condition was used for subsequent studies.We next tested the stability of the polySBMA coating after long-term storage. Coated tubes were stored in PBS at 4°C for 1, 4, 7, 28, and 42 days and the integrity of the surface-bound polymers was then examined by evaluating the ability of the stored tubes to resist VWF deposition and comparing their performance to that of freshly coated tubes. Coated tubes stored up to 6 weeks retained their resistance to VWF adsorption under shear (less than 5 ng/cm2), indicating the long-term resilience of the polySBMA coating.To mimic shear stress conditions of blood vessels in capillary, venous, and arterial flow, we used a perfusion system in which a pump (ibidi) generates different flow patterns (continuous unidirectional, oscillating, or pulsatile flow) and shear stresses. Surfaces within the perfusion system were coated with polySBMA to minimize VWF loss. Additional polypropylene test tubing (50 cm long), uncoated or polySBMA-coated, was connected to the perfusion system between two connecters. Purified VWF (2.5 µg/mL in PBS) was perfused through this system for 1 hr. The VWF remaining in the solution was measured by ELISA after the prescribed perfusion period, and multimer composition was compared before and after perfusion. The VWF concentration decreased by 74.1% with uncoated tubing after 1 hr at 20 dyn/cm2, with selective depletion of large VWF multimers. By contrast, in the polySBMA-coated tubing the concentration and multimer composition of VWF was unchanged under identical conditions.In summary, polySBMA effectively prevented VWF adsorption under shear in two types of experiments, and was stable during storage. We expect this coating to significantly enhance the lifetime of blood-contacting devices and reduce both costs and medical complications. DisclosuresNo relevant conflicts of interest to declare.

Von Willebrand Factor Interacts with Surface-Bound C1q and Induces Platelet Rolling.

Premature atherosclerosis and thrombotic complications are major causes of morbidity and mortality in patients with systemic lupus erythematosus (SLE). However, the high incidence of these complications cannot be explained by traditional risk factors alone, suggesting direct effects of an activated immune system on hemostasis. The unexpected nucleotide sequence homology between SLE patient-derived autoantibodies against complement C1q (Fab anti-C1q) and von Willebrand factor (VWF) led us to investigate a potential interaction between the complement and hemostatic systems on the level of initiating molecules. VWF was found to bind to surface-bound C1q under static conditions. The binding could specifically be inhibited by Fab anti-C1q and C1q-derived peptides. Under shear stress the C1q-VWF interaction was enhanced, resembling the binding of VWF to collagen I. Additionally, we could show that C1q-VWF complexes induced platelet rolling and firm adhesion. Furthermore, we observed VWF binding to C1q-positive apoptotic microparticles and cholesterol crystals, as well as increased VWF deposition in C1q-positive glomeruli of SLE patients compared with control nephropathy. We show, to our knowledge for the first time, binding of VWF to C1q and thus a direct interaction between starter molecules of hemostasis and the classical pathway of complement. This direct interaction might contribute to the pathogenic mechanisms in complement-mediated, inflammatory diseases.

Abstract 202: The Role of Neutrophil Extracellular Traps in the Pathogenesis of Pulmonary Hypertension.

Rationale: Inflammation contributes to vasoconstriction and remodeling in pulmonary hypertension (PH), but the mechanisms are not fully understood. Recent data link neutrophil extracellular traps (NETs) and NET components with endothelial dysfunction, thrombosis, and lung injury. NETs, produced as a result of neutrophil cell death induced by inflammation or cytotoxic stress, comprise chromatin fibers associated with a number of proteins including myeloperoxidase and neutrophil elastase. We hypothesized that NETs contribute to pulmonary vascular remodeling in pulmonary hypertension. The aim of the present study was to measure NET markers in plasma of PH patients and to analyze their effects on human pulmonary artery endothelial cell (HPAEC) function in vitro. Methods and Results: Patients with prevalent chronic thromboembolic pulmonary hypertension (CTEPH) (n=26) and idiopathic pulmonary arterial hypertension (IPAH) (n=52) show increased plasma levels of NET components including myeloperoxidase, neutrophil elastase and circulating DNA, compared with plasma from sex- and age-matched healthy volunteers (n=24). Endarterectomy specimens from CTEPH patients show accumulation of NET-forming peptidylarginine deiminase 4+ (PAD4+) neutrophils. PAD4+ neutrophils are also abundant in and around plexiform lesions in IPAH lungs, compared to corresponding uninvolved regions of lung tissues from cancer patients. Incubation of NETs with HPAECs in vitro activates the transcription factor nuclear factor-kB and increases expression of pro-inflammatory cytokines and leukocyte-binding adhesion molecules. NETs also increase endothelial angiogenesis in vitro and induce plateletet aggregation, possibly due to increased expression and surface deposition of von Willebrand factor and P-selectin. Conclusion: CTEPH and IPAH patients show elevated NET markers in plasma and increased numbers of NET-forming neutrophils in the remodelled lung. NETs induce endothelial dysfunction in vitro and are likely to contribute to vascular remodelling in vivo. Inhibition of NET components or NET formation in vivo may represent a new therapeutic approach in the treatment of PH.

Postoperative thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening disease characterized by acute episodes of thrombocytopenia and microangiopathic hemolytic anemia occurring due to platelet and von Willebrand factor deposition and hyaline thrombi formation in arterioles and capillaries throughout the body, which results in organ ischemia. TTP can be idiopathic or secondary, and there are several causes of secondary TTP. There is a clinical syndrome resembling TTP that occurs after surgical procedures, so-called "postoperative TTP" (pTTP). In this review, the differential diagnosis, pathogenesis and clinical and laboratory features of pTTP, together with the treatment modalities and outcomes of the patients, are discussed. The pTTP is a diagnosis of exclusion, and disseminated intravascular coagulation, heparin-induced thrombocytopenia and medication-induced effects should be ruled out. As in classical TTP, patients with pTTP should be diagnosed and treated with therapeutic plasma exchange (TPE) as early as possible to reduce their morbidity and mortality. Although rarely seen, surgeons and physicians of all specialties should be alert to the possibility of pTTP, and since pTTP is a life-threatening event that usually can be treated successfully with TPE, especially when diagnosed early in its course, it is critical to recognize and treat pTTP promptly.

ADAMTS13 activity is decreased in a septic porcine model

During sepsis, the balance between abundantly secreted von Willebrand factor (VWF) and the activity of its size regulating protease ADAMTS13 is assumed to be involved in coagulation abnormalities. We aimed to establish a porcine model with haemorrhagic shock with consecutive sepsis and hypothesised that a decreased ADAMTS13-activity as well as an altered VWF multimer pattern is associated with renal failure. Animals (n=21) were subjected to haemorrhagic shock. After volume replacement, intraperitoneal Escherichia coli sepsis was induced. Blood samples were drawn at baseline, after haemorrhage and sepsis induction. Directly postmortem we examined renal tissue by JONES-silver, CD61, VWF and fibrin staining for characterisation of thrombi. Renal failure was analysed by scoring PAS-stained sections for acute tubular damage. Glomerular microthrombi were observed in six of 21 septic animals. Porcine ADAMTS13 activity declined significantly during sepsis, accompanied by a drop-off in platelet count. At 12 hours after sepsis induction, ADAMTS13 activity was significantly diminished compared to sham controls, and an elevated acute tubular damage score was associated with an increased proportion of high-molecular-weight VWF multimers. Compared to baseline the proportion of high-molecular-weight VWF multimers increased significantly in septic animals. Similar to human sepsis, diminished ADAMTS13 activity was observed in a septic porcine model associated with a shift to rather thrombogenic VWF multimers and deposition of microthrombi. Therefore, this porcine model seems to be appropriate for performing functional and therapeutic studies in sepsis-associated ADAMTS13 deficiency.

Rituximab for the Treatment of Recurrent Thrombotic Thrombocytopenic Purpura.

Background: Thrombotic thrombocytopenic purpura (TTP) is a rare and severe disease, characterized by thrombocytopenia, microangiopathic anaemia, fever, renal failure, and neurological manifestation caused by deposition of von Willebrand factor - platelet rich hyaline thrombi in the arterioles and capillaries. Plasma exchange has decreased the mortality in TTP from almost 100% to 10–30%. However, relapses occur in more than one-third of patients, a subset of whom develop multiple relapses or chronic disease requiring numerous sessions of plasma exchange. This treatment is costly and associated with many adverse reactions. CaseReport: We describe a female patient - diagnosed with TTP in 1996 - who did not achieve a sustained remission with plasma exchange, steroids and vincristine, but who remained in remission after a splenectomy performed in 1997. The patient underwent relapse in 2003 and restarted plasma exchange, with progressive improvement of cytopenias. Plasma exchange was reduced in frequency from daily sessions to every other day. There was a lowering of platelet levels and an increase in LDH levels - steroids and vincristine were re-initiated, but without response. The patient was treated with four, weekly doses (375 mg/m2) of rituximab. Plasma exchange was only required during the first 2 weeks of rituximab treatment, and was discontinued after the second dose of rituximab. The patient remained in complete remission for 9 months, but was then admitted to hospital with femoral thrombosis, and normal LDH and platelet levels. After 40 days of thrombosis therapy, the patient had a further relapse and refused plasma exchange. Treatment with rituximab was restarted as previously, and the patient achieved a complete remission within 2 weeks - now sustained for 11 months.Conclusion: In comparing this patient with eight previous cases not treated with rituximab, we conclude that rituximab is a very effective treatment for TTP, which is not associated with clinically significant toxicity. Randomized clinical trials are required to confirm these findings.

Gender-Dependent Up-Regulation of the VWF-Cleaving metalloprotease ADAMTS-13 in Mice with Obesity and Hypercholesterolemia.

The development of atherothrombosis is associated with hypercholesterolemia and systemic inflammation. Recent studies have shown that adipose tissue may directly promote inflammation. Since one of the hallmark events of atherosclerosis is the deposition of von Willebrand factor (VWF) on vascular endothelial cells, the release and proteolysis of ultra-large (UL) and hyperreactive VWF may play an important role in the development of thrombotic diseases. We therefore investigated how the synthesis of ADAMTS-13, the newly characterized metalloprotease that cleaves the hyperreactive ULVWF to smaller and less active form, may be regulated in mice with obesity and hypercholesterolemia. Wild-type C57BL/6 and Apo E −/− C57Bl/6 mice (18 in each group with equal gender distribution) were fed with regular Chow or western-type high fat diet for 6 months and then analyzed for ADAMTS-13 mRNA in the liver (by quantitative PCR), the primary site of ADAMTS-13 synthesis, and ADAMTS-13 activity under flow condition. We found that ADAMTS-13 mRNA was more than doubled for wild-type mice on high fat diet than those on regular diet (4.14±0.46 vs. 1.72±0.57 units, respectively, p < 0.01). The increase was 47.9% for Apo E−/− mice on high fat diet compared to mice on regular diet (2.61±0.31 vs. 3.86±0.72 units, respectively, p = 0.05). When the data were stratified for gender difference, the increase was primarily observed in male mice. For male mice, ADAMTS-13 mRNA on high fat diet was more than four and two folds of those on regular diet for wild-type and Apo E−/− mice, respectively. , For female mice, however, the increase was less than 40% and only detected in wild-type mice fed with high fat diet. Consistent with this observation, ADAMTS-13 activity in general was lower in female (61±12%) than males (81±10%, p < 0.05). For both wild-type and Apo E−/− mice, we detected no difference in ADAMTS-13 activity in males, but a significantly lower activity in the female Apo E−/− mice on high fat diet (19.6±10.5% vs. 49.1±9.2% for high fat and regular diets, respectively, p < 0.05). These results demonstrate that the synthesis of ADAMTS-13 is up-regulated in obese mice (wild-type on high fat diet) and mice with hypercholesterolemia (Apo E−/− mice on high fat diet). The increase may serve as a protective measure to encounter the likely increase in ULVWF release due to systemic inflammation and endothelial cell stimulation seen in these mice. Interestingly, male mice showed a much stronger up-regulation than females. The low rate of ADAMTS-13 synthesis may contribute to the lower ADAMTS-13 activity found in female mice, suggesting that the machinery of ULVWF proteolysis in female mice may be less responsive to obesity/hypercholesterolemia and systemic inflammation.

Overexpression of von Willebrand factor is an independent risk factor for pathogenesis of intimal hyperplasia: Preliminary studies

Deposition of von Willebrand factor (vWF) is increased in hyperplastic intima of grafts, vWF levels are elevated in patients with cardiovascular diseases, and there is resistance to progression of atherosclerosis in pigs with von Willebrand disease. We hypothesize that increased expression of endothelial vWF has mitogenic effects on smooth muscle cell (SMC) proliferation. In an in vitro study, mouse aortic smooth muscle cells (SMC) were exposed to vWF in various concentrations (0, 5, 20, 100, 500, and 1000 ng/mL). DNA synthesis of SMC was measured with (3)H-thymidine incorporation. In an in vivo study, 108 mice from inbred strains of C57BL/6J (control) and RIIIS/J (characteristic of low plasma vWF) underwent carotid artery ligation (flow cessation model) and were divided into three groups: C57BL/6J, RIIIS/J, and RIIIS/J treated with desmopressin (DDAVP; intraperitoneal injection at 3 micro g/kg/d). At 2 and 4 weeks, carotid arteries were harvested for analysis with immunohistochemical analysis, morphometric studies, and reverse transcriptase polymerase chain reaction; plasma vWF was measured with an enzyme-linked immunosorbent assay. In vitro SMC proliferation showed a positive dose-response curve with vWF stimulation. Intimal hyperplasia (IH) in carotid arteries was prominent in C57BL/6J mice, absent in RIIIS/J mice, and moderate in RIIIS/J treated with DDAVP (intima-media ratio, 71% +/- 18%, 0, and 32% +/- 12%, respectively; P <.01). vWF deposition occurred in all hyperplastic intima subjacent to intact endothelium. Plasma vWF correlated with degree of IH (110% +/- 10%, 21% +/- 7%, and 45% +/- 8%, respectively; P <.05). vWF-messenger RNA was 9 times higher in carotid arteries of C57BL/6J mice and 4 times higher in RIIIS/J with DDAVP, compared with RIIIS/J. vWF directly stimulates SMC proliferation in vitro via a direct dose-response effect. In vivo low shear stress accelerates IH proportional to vWF expression. This could occur under intact endothelium without platelet activation and platelet-derived growth factor release. In effect, control of IH may entail modulation of vWF expression.

Redistribution of von Willebrand factor in porcine carotid arteries after balloon angioplasty.

von Willebrand factor (VWF) is a well-characterized multimeric glycoprotein present in platelets and plasma and synthesized by vascular endothelial cells and megakaryocytes. Its role in platelet-vessel wall interactions has been studied extensively, but its involvement in intravascular events after balloon angioplasty has not been clarified. VWF antigen is not present in porcine arterial endothelium (except for the pulmonary artery) but is readily detected in porcine venous endothelial cells. We have examined the localization of VWF in porcine vessel walls during neointima formation after bilateral carotid balloon-angioplasty. Endothelium was denuded by balloon injury but regenerated by 7 days and was fully confluent by 42 days. VWF was detected at the site of injury in localized, adherent platelet aggregates at 10 minutes after angioplasty that were not present at later time points. A well-demarcated homogeneous layer of VWF was observed on the luminal surface from 30 minutes to day 7, but there was a progressive shift of positive staining from the lumen to the outer media from days 1 to 7. VWF was also strongly detected at sites proximal and distal to the balloon injury from 30 minutes to day 7, although endothelial disruption was minimal and the monolayer remained substantially intact at these sites. Regrowing endothelial cells appeared to contain granular VWF from days 12 to 21, but this was not readily evident at later time points. The results suggest that balloon injury is associated with deposition and medial absorption of plasma or platelet VWF in this porcine model over a time period that precedes and overlaps vascular smooth muscle proliferation and endothelial recoverage. The findings provide evidence to support the concept of a wider role for VWF in tissue injury responses.

Fibrin(ogen) and von Willebrand factor deposition are associated with intimal thickening after balloon angioplasty of the rabbit carotid artery.

The aim of the study was to assess the contribution of thrombus incorporation into neointimal thickening in the rabbit carotid artery after deep vascular injury induced by balloon angioplasty compared with superficial vascular injury induced by a perivascular collar. Besides CD 31 (PECAM 1), vimentin, alpha-smooth muscle actin, rabbit anti-macrophage monoclonal antibody and proliferating cell nuclear antigen, fibrin(ogen) and von Willebrand factor (vWF) deposition was assessed immunohistochemically. Angioplasty was performed in 47 rabbits and evaluated immediately (n = 7), after 6 hours (n = 4), and after 1 (n = 7), 2 (n = 9), or 3 (n = 20) weeks. A collar was placed in 29 rabbits and evaluated immediately (n = 5), after 6 hours (n = 5), and after 1 (n = 7), 2 (n = 10), or 3 (n = 2) weeks. After dilatation, the arteries were extensively denuded of endothelium, the internal elastic membrane was ruptured and blood-filled clefts were present in the media, pointing to deep vascular (type III) injury. Six hours later, mural fibrin(ogen) thrombi were formed, specially at sites with severe damage. This fibrin(ogen) matrix became infiltrated by phagocytes and smooth muscle cells. A luminal cap covered by regenerating endothelium was formed, demonstrating increased immunoreactivity to vWF. vWF was deposited in the extracellular neointimal spaces. Fibrin(ogen) thrombus deposition and incorporation appeared to be protracted phenomena for at least 2 weeks. After collar placement, minimal endothelial denudation was documented, pointing to focal superficial (type I) vascular injury. In subsequent weeks, neointimal thickening was associated with vWF deposition but not with fibrin(ogen) thrombus incorporation. In conclusion, mural fibrin(ogen) thrombus formation and incorporation contribute to neointima formation after deep vascular injury and seem to occur for several weeks after the initial insult.

Cultured Endothelial Cells Regulate Platelet Adhesion to their Extracellular Matrix by Regulating its von Willebrand Factor Content

Endothelial cells and their extracellular matrix formed in vitro are often used as a model for subendothelium in studies on platelet-vessel wall interaction. We have characterized the influence of culture conditions of endothelial cells on the formation of extracellular matrix and on the interaction of the matrix with platelets. Passage number, time of confluence, serum concentration and the addition of heparin, growth factors and antibiotics to the culture medium were varied and the extracellular matrices were isolated. The amount of fibronectin and von Willebrand factor present in the matrix were measured and the number of platelets adhering to these matrices after perfusion with citrated whole blood at a shear rate of 1000 s-1 was determined. A three times increase of the amount of von Willebrand factor in the matrix was found when the serum concentration was increased from 2.5% to 30%. When the passage number of the cells was increased or the period during which the cells were at confluence was extended, the amount of von Willebrand factor in the matrix was decreased up to 50%. Addition of heparin or ECGS (endothelial cell growth supplement) decreased the von Willebrand factor content in the matrix. Addition of penicillin or streptomycin to the culture medium had no influence on the amount of von Willebrand factor deposited in the matrix or secreted into the medium, however, other antibiotics such as gentamycin and neomycin decrease the amount of von Willebrand factor in the matrix. No influence on the amount of fibronectin in the matrix was found under all conditions tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical trials of restenosis after coronary angioplasty.

HomeCirculationVol. 84, No. 3Clinical trials of restenosis after coronary angioplasty. Free AccessAbstractPDF/EPUBAboutView PDFSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessAbstractPDF/EPUBClinical trials of restenosis after coronary angioplasty. J J Popma, R M Califf and E J Topol J J PopmaJ J Popma Search for more papers by this author , R M CaliffR M Califf Search for more papers by this author and E J TopolE J Topol Search for more papers by this author Originally published1 Sep 1991https://doi.org/10.1161/01.CIR.84.3.1426Circulation. 1991;84:1426–1436 Previous Back to top Next FiguresReferencesRelatedDetailsCited ByBosmans J, Kockx M, Vrints C, Bult H, De Meyer G and Herman A (1997) Fibrin(ogen) and von Willebrand Factor Deposition Are Associated With Intimal Thickening After Balloon Angioplasty of the Rabbit Carotid Artery, Arteriosclerosis, Thrombosis, and Vascular Biology, 17:4, (634-645), Online publication date: 1-Apr-1997.Chen S, Chen Y, Miller D, Li H and Oparil S (1994) Mithramycin inhibits myointimal proliferation after balloon injury of the rat carotid artery in vivo., Circulation, 90:5, (2468-2473), Online publication date: 1-Nov-1994.Cooke T, Sheahan R, Foley D, Reilly M, D'Arcy G, Jauch W, Gibney M, Gearty G, Crean P and Walsh M (1994) Lipoprotein(a) in restenosis after percutaneous transluminal coronary angioplasty and coronary artery disease., Circulation, 89:4, (1593-1598), Online publication date: 1-Apr-1994.Lincoff A, Topol E and Ellis S (1994) Local drug delivery for the prevention of restenosis. Fact, fancy, and future., Circulation, 90:4, (2070-2084), Online publication date: 1-Oct-1994.Serruys P, Klein W, Tijssen J, Rutsch W, Heyndrickx G, Emanuelsson H, Ball S, Decoster O, Schroeder E and Liberman H (1993) Evaluation of ketanserin in the prevention of restenosis after percutaneous transluminal coronary angioplasty. A multicenter randomized double-blind placebo-controlled trial., Circulation, 88:4, (1588-1601), Online publication date: 1-Oct-1993.Soma M, Donetti E, Parolini C, Mazzini G, Ferrari C, Fumagalli R and Paoletti R (1993) HMG CoA reductase inhibitors. In vivo effects on carotid intimal thickening in normocholesterolemic rabbits., Arteriosclerosis and Thrombosis: A Journal of Vascular Biology, 13:4, (571-578), Online publication date: 1-Apr-1993.Santoian E, Schneider J, Gravanis M, Foegh M, Tarazona N, Cipolla G and King S (1993) Angiopeptin inhibits intimal hyperplasia after angioplasty in porcine coronary arteries., Circulation, 88:1, (11-14), Online publication date: 1-Jul-1993.Topol E, Ellis S, Cosgrove D, Bates E, Muller D, Schork N, Schork M and Loop F (1993) Analysis of coronary angioplasty practice in the United States with an insurance-claims data base., Circulation, 87:5, (1489-1497), Online publication date: 1-May-1993.Kuntz R and Baim D (1993) Defining coronary restenosis. Newer clinical and angiographic paradigms., Circulation, 88:3, (1310-1323), Online publication date: 1-Sep-1993.Uprichard A, Texter M and Canter D (1992) Clinical restenosis trials: a perspective from the pharmaceutical industry., Circulation, 85:4, (1636-1637), Online publication date: 1-Apr-1992.Ferrell M, Fuster V, Gold H and Chesebro J (1992) A dilemma for the 1990s. Choosing appropriate experimental animal model for the prevention of restenosis., Circulation, 85:4, (1630-1631), Online publication date: 1-Apr-1992. September 1, 1991Vol 84, Issue 3Article InformationMetrics Copyright © 1991 by American Heart Associationhttps://doi.org/10.1161/01.CIR.84.3.1426 Originally publishedSeptember 1, 1991 PDF download Advertisement

Relationship of Intraglomerular Coagulation and Platelet Aggregation to Glomerular Sclerosis

In order to investigate the relationship between intraglomerular coagulation and glomerular sclerosis, the distribution of fibrin-related antigen (FRA) in glomeruli without extracapillary lesions was examined by immunoperoxidase microscopy in 80 patients with IgA nephropathy (IgA-N). A total of 302 glomeruli were examined, including 20 with global sclerosis, 31 with segmental sclerosis (SS glomeruli), and 251 nonsclerosed glomeruli. In the nonsclerotic areas of SS glomeruli, the deposition of FRA was significantly greater than in the nonsclerosed glomeruli. In the nonsclerosed glomeruli FRA was mainly found in the mesangium, while in the nonsclerotic areas of SS glomeruli FRA was not only present in the mesangium but also in the endothelium of the glomerular capillary loops. FRA-positive microclots were also often observed attached to the endothelium of the capillaries of the nonsclerotic areas of SS glomeruli. Cross-linked FRA was also observed in the endothelium of the same capillaries using the monoclonal antibody DD3B6/22. Deposition of von Willebrand factor (vWF) was greater in the endothelium than in the mesangium in the same areas. Aggregated platelets adhering to the glomerular capillary walls in these areas were frequently detected using the monoclonal antibody P2. Such distribution of platelets and vWF showed that the endothelium of the nonsclerotic areas of SS glomeruli was more severely damaged than that of nonsclerosed glomeruli. These findings suggest that endothelial cell damage might activate the intraglomerular coagulation, which might be one of the factors in the development of global glomerular sclerosis.

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.