Comparison of secretion and subcellular localization of von Willebrand protein with that of thrombospondin and fibronectin in cultured human vascular endothelial cells

Reinders, Jan Hendrik; Groot, P. G. De; Dawes, J.; Hunter, Nikolas R.; Heugten, Han A. A. Van; Zandbergen, J.; Gonsalves, Mervin D.; Mourik, Jan A. van

doi:10.1016/0167-4889(85)90131-4

Cited by 69 publications

(29 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Before seeding the cells, gelatin was fixated with 0.5% glutardialdehyde. 35 Cell monolayers grown to confluence in 5-7 days were used (50,000 cells/cm 2 ). At confluence, the cells were stimulated for 18 hours by addition of 20 ng/ml 4£-phorbol-12-myristate-13-acetate (Sigma Chemical Co., St. Louis, Mo.).…”

mentioning

confidence: 99%

Defects in platelet adhesion and aggregate formation in uremic bleeding disorder can be attributed to factors in plasma.

Zwaginga¹,

Ijsseldijk²,

Groot³

et al. 1991

Arterioscler Thromb

View full text Add to dashboard Cite

Uremia is associated with bleeding diathesis. Platelet adhesion to the subendothelium is inhibited by a factor in uremic plasma that may play a role in the disturbed hemostasis of uremic patients. In the formation of the hemostatic plug, platelet adherence is followed by stimulus-induced platelet aggregation and reinforcement by thrombin-generated fibrin. To study these processes in uremic blood, a newly developed thrombosis model was used. Perfusates anticoagulated with low-molecular-weight heparin were circulated over a matrix of stimulated cultured endothelial cells. By stimulation of the endothelial cells, tissue factor was synthesized and deposited in the matrix. When this tissue factor rich-matrix was exposed to flowing blood, local thrombin was formed via activation of the extrinsic coagulation pathway. With this system, platelet adhesion, thrombin-dependent platelet activation, and fibrin formation can all be studied at the same surface. In addition to an adhesion defect, decreased aggregate formation was also found in uremic perfusates. Normal platelets in uremic plasma showed similar results, which indicates that a factor in uremic plasma caused this adhesion and aggregation defect. Platelet aggregation in the system was dependent on endogeneously formed thrombin. Fibrinopeptide A generation, however, was normal in uremic perfusates; therefore, uremic plasma has a normal capacity to form thrombin. Resuspension of washed uremic platelets in control plasma did not reverse the aggregation defect in perfusions. In contrast, aggregometer studies with isolated uremic platelets could not detect an abnormal response to threshold concentrations of exogenous thrombin. Thus, uremic toxin (s) cause defective aggregate formation in flow, but not necessarily in the aggregometer. This apparent discrepancy may be due to the higher shear forces in the flow system, which may prevent aggregate formation that is allowed in the aggregometer. Another explanation, that uremic platelets are less responsive to locally formed thrombin than they are to exogenously added thrombin, seems less likely. [Arteriosclerosis and Thrombosis 1991;ll:733-744)

show abstract

mentioning

confidence: 99%

Defects in platelet adhesion and aggregate formation in uremic bleeding disorder can be attributed to factors in plasma.

Zwaginga¹,

Ijsseldijk²,

Groot³

et al. 1991

Arterioscler Thromb

View full text Add to dashboard Cite

show abstract

“…Before seeding the cells, gelatin was coated on the surfaces and was fixed with 0.5% glutardialdehyde. 14 For counting, confluent cells (9.5 cm 2 ) were removed with 0.5 ml of 0.075% trypsin (GIBCO) and 0.01% ethylenediaminetetraacetate (EDTA) in PBS and were counted in a hemocytometer. To isolate the extracellular matrix, the cells were removed by exposure to 0.1 M NH 4 OH at room temperature for 30 minutes and gentle shaking.…”

mentioning

confidence: 99%

Thrombogenicity of vascular cells. Comparison between endothelial cells isolated from different sources and smooth muscle cells and fibroblasts.

Zwaginga¹,

Boer²,

Ijsseldijk³

et al. 1990

Arteriosclerosis

View full text Add to dashboard Cite

When the endothelial cell layer Is damaged, a thrombotic reaction starts on the cells' subendothelium and on the connective tissue deposited by smooth muscle cells in the deeper layers. When more severe vascular damage occurs, hemostasis will Involve the vessel adventitia In which fibroblasts are found. In this article, the influence of in vitro cultured endothelial cells, smooth muscle cells, and fibroblasts on the hemostatic balance was studied. To do so, perfusions were performed with low molecular weight heparln antlcoagulated blood over the extracellular matrix of the cells. This method allowed the study of tissue factor-dependent thrombln generation and its influence on formation of fibrin and platelet aggregates. The experiments described in this article show that endothelial cells isolated from different human organs Interfere differently In the hemostatic response. Endothelial cells Isolated from umbilical veins are nonthrombogenic; they do not synthesize tissue factor under unstlmulated conditions. On their extracellular matrix, only adherent platelets are found, but no aggregates and no fibrin. Endothelial cells Isolated from omentum and atrium contain tissue factor activity under unstimulated conditions. As a consequence, thrombln Is generated on their surfaces, and platelet aggregates and fibrin deposition are found on the extracellular matrices after perfusions with whole blood. The matrix of smooth muscle cells and fibroblasts behaved similarly. Increase In shear rate and perfuslon time resulted In an Increase in platelet aggregate formation. Polymerized fibrin deposition decreased when perfusions were performed at higher shear. Both platelet aggregation and fibrin deposition were tissue factor dependent and could be blocked more than 70% by an antibody against tissue factor. Based on these results, we conclude that endothelial cells isolated from umbilical veins form the best nonthrombogenic surface In vitro. Moreover, coagulation-dependent hemostasis should be Included when thrombogenicity of subendothelium Is discussed, especially when it concerns matrix derived from cells present In the deeper layer of the vessel wall.

show abstract

“…The release into the medium of von Willebrand factor by cultured endothelial cells was not altered by incubating the cells with cigarette smoke condensate (Figure 5). Also the PMA-stimulated release of von Willebrand factor 26 was not hampered by cigarette smoke condensate. The von Willebrand protein levels in the 35 cigarette smoke condensate did not seriously alter the subcellular localization of von Willebrand factor and the secretion of this protein from these organelles, following incubation of the cells with PMA ( Figure 6).…”

mentioning

confidence: 90%

“…18 Immunofluorescence staining for von Willebrand factor in endothelial cells was performed as described elsewhere 26 with use of monoclonal antibody CLB-RAg-1 as indicator antibody.…”

Section: Enzymes and Von Wlllebrand Factormentioning

confidence: 99%

Cigarette smoke impairs endothelial cell prostacyclin production.

Reinders¹,

Brinkman²,

Mourik³

et al. 1986

Arteriosclerosis

View full text Add to dashboard Cite

Production of prostacyclin by endothelial cells is considered to be important in rendering the vessel wall nonthrombogenic. Cigarette smoking is an important risk factor in the pathogenesis of atherosclerosis. Here we show that the incubation of cultured human endothelial cells with a cigarette smoke condensate impaired the basal prostacyclin release. Also, the enhanced release of prostacyclin provoked by phorbol myristate acetate was inhibited by cigarette smoke condensate. Furthermore, cigarette smoke condensate impaired the thrombin-induced prostacyclin production. The production of prostacyclin from exogenous arachidonate was not affected by cigarette smoke condensate, indicating that cigarette smoke condensate constituents exert their inhibitory properties on the level of arachidonate mobilization from cellular phospholipids, rather than on cyclooxygenase or prostaglandin synthetase. The effects noted for cigarette smoke condensate could not be attributed to the cigarette smoke constituents nicotine and cadmium. While inhibiting the endothelial cell prostacyclin production significantly, cigarette smoke condensate did not cause cell death or impairment of secretory function, as measured by the release of von Willebrand factor. This in vitro study shows that impairment of an endothelial cell function is related to a risk factor for atherosclerosis.

show abstract

Comparison of secretion and subcellular localization of von Willebrand protein with that of thrombospondin and fibronectin in cultured human vascular endothelial cells

Cited by 69 publications

References 32 publications

Defects in platelet adhesion and aggregate formation in uremic bleeding disorder can be attributed to factors in plasma.

Defects in platelet adhesion and aggregate formation in uremic bleeding disorder can be attributed to factors in plasma.

Thrombogenicity of vascular cells. Comparison between endothelial cells isolated from different sources and smooth muscle cells and fibroblasts.

Cigarette smoke impairs endothelial cell prostacyclin production.

Contact Info

Product

Resources

About