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Fibrin/agar films were prepared and used to detect plasminogen activators produced by cultured bovine aortic endothelial cells (fibrin autography). One preparation of fibrin underwent spontaneous lysis upon incubation at 37 degrees C. This lysis was prevented by antibodies to tissue-type plasminogen activator but not by antibodies to urokinase. Conditioned medium from the confluent endothelial cells was fractionated by polyacrylamide gel electrophoresis in the presence of NaDodSO4. The gels were analyzed on indicator films prepared with the spontaneously lysing fibrin (reverse fibrin autography). Unexpectedly, as the opaque fibrin film cleared, a distinct lysis-resistant zone appeared in the indicator gel at a region corresponding to Mr 55,000. Experiments were devised to determine whether the lysis-resistant zone in the indicator film reflected the presence of a cellular inhibitor in the polyacrylamide gel. The corresponding region was excised from a polyacrylamide gel, extracted with buffer, and tested directly for antifibrinolytic activity by the 125I-labeled fibrin plate method. Urokinase-mediated fibrinolytic activity was inhibited by the gel extract in a dose-dependent manner indicating the presence of such an inhibitor. Inhibitor activity was detected in Triton X-100 extracts of washed monolayers and in conditioned medium, where it accumulated with time. The endothelial cell inhibitor not only survived exposure to NaDodSO4 but also was active after incubation at pH 12 or treatment with 5% (vol/vol) 2-mercaptoethanol, 6 M urea, 4 M guanidine hydrochloride, or 1 M acetic acid. Considerable activity also remained after heating at 100 degrees C for 30 min. These results indicate that cultured bovine aortic endothelial cells synthesize and secrete a previously undetected, unusually stable fibrinolytic inhibitor of Mr 55,000. Reverse fibrin autography offers a convenient approach for studying such molecules.

Glanzmann's thrombasthenia is a bleeding disorder, inherited in an autosomal recessive way and characterized by an absence or deficiency of the platelet glycoprotein (GP) IIb/IIIa complex. Recently, we and others demonstrated that cultured human umbilical vein endothelial cells synthesized a membrane protein complex similar to the platelet GP IIb/IIIa complex. In this article, we demonstrate that endothelial cells isolated from the umbilical vein of a newborn with Glanzmann's thrombasthenia, as compared with normal endothelial cells, show no difference in their ability to synthesize and express this GP IIb/IIIa complex. Our results indicate that Glanzmann's thrombasthenia is not accompanied by an "endotheliopathy."

A human endothelial cDNA expression library, based on the Escherichia coli plasmid pUC9, was screened with a heterologous antibody raised against purified bovine aortic endothelial plasminogen activator inhibitor (PAI). A synthetic oligonucleotide, derived from a partial PAI cDNA expression clone, was used to select a full-length PAI cDNA, the size of which coincides with the length of PAI mRNA (approximately 2350 nucleotides) as determined by Northern blot analysis. The authenticity of full-length PAI cDNA is demonstrated by the expression of biologically active PAI both in lysates of transformed E. coli cells and in conditioned media of mouse Ltk- cells, transfected with PAI cDNA inserted into vector pSV2. Analysis of the de novo synthesized anti-plasminogen activator activity, employing reverse fibrin autography, shows that transfected mouse Ltk- cells synthesize a polypeptide with a mol. wt identical to that of the native PAI glycoprotein (Mr 52,000), whereas in E. coli an unglycosylated, active product with a mol. wt of 43,000 is made. The amino acid sequence, derived from the determined nucleotide sequence, shows that pre-PAI consists of 402 amino acids. It is proposed that the mature PAI is preceded by a signal peptide of 23 amino acid residues. The amino acid sequence of mature PAI includes three potential asparagine-linked glycosylation sites and lacks cysteine residues. The predicted amino acid sequence reveals significant homology with members of the serine protease inhibitor (Serpin) family, e.g. alpha 1-proteinase inhibitor and antithrombin III.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Endothelial cells were cultured from various human arteries and veins, obtained from adult individuals and from umbilical cords. We compared the storage and secretion of von Willebrand factor by endothelial cells from umbilical veins with that of endothelial cells cultured from a number of adult vessels, including aorta, arteria iliaca, vena saphena magna and vena cava. There were no differences in the way the cultured endothelial cells handled the von Willebrand factor they synthesized. Endothelial cells from the various vessels responded to stimuli in secreting stored von Willebrand factor. The cells also responded to thrombin and ionophore A23187 in producing enhanced amounts of prostacyclin. Thus, cultured umbilical vein endothelial cells have properties that are very similar to those of cultured endothelial cells of various other origins. It is concluded that foetal venous cells provide a representative model for studies of endothelial cell von Willebrand factor biosynthesis and prostacyclin production.

Cultured human vascular endothelial cells synthesize von Willebrand protein, thrombospondin and fibronectin. These proteins are secreted in the culture medium and incorporated into the extracellular matrix. We have compared the subcellular localization and the secretion of these proteins in response to stimulants in cultured human umbilical vein endothelial cells. Density gradient centrifugation using colloidal silica showed that the storage and secretion organelle with von Willebrand protein did not contain thrombospondin or fibronectin. Indirect immunofluorescence microscopy indicated that thrombospondin and fibronectin are not located in the rod-shaped organelles containing von Willebrand protein. Thrombin, ionophore A23187 and phorbol myristate acetate did not affect secretion of thrombospondin and fibronectin, while von Willebrand protein secretion was stimulated upon incubation of cells with these agents for 30 min. Prolonged incubation of cultured endothelial cells after a 1-h treatment with phorbol myristate acetate resulted in an increased secretion of von Willebrand protein into the conditioned medium; in contrast, accumulation of thrombospondin and fibronectin in endothelial cell-conditioned medium was decreased. These findings indicate that, unlike in platelets, these major endothelial proteins are not located in the same subcellular compartments. Von Willebrand protein is distinguished from thrombospondin and fibronectin both by its unique subcellular localization and its secretion rate in response to stimuli.

To study the biology of the endothelium under conditions that mimic the architecture of the vessel wall, endothelial cells were grown on a collagen lattice containing a multilayer of smooth muscle cells. Light and electron microscopy of such cultures revealed a confluent monolayer of flattened endothelial cells. In co-culture, endothelial cells tend to elongate, whereas in the absence of smooth muscle cells, the endothelial cells show the polygonal morphology typical for cultures of endothelial cells grown on polystyrene substrates. As conditioned culture media of endothelial cells contain substances that may both promote or inhibit the growth of smooth muscle cells, the availability of this vessel wall model prompted us to examine to what extent endothelial cells regulate the proliferation of smooth muscle cells when these cells are maintained in co-culture. Here we show that endothelial cells suppress the proliferation of co-existing smooth muscle cells. This finding suggests that under physiological conditions the balance of the action of growth-promoting and growth-inhibiting substances produced by endothelial cells is in favour of the latter.

Cultured human endothelial cells derived from the umbilical cord vein are able to release factor VIII-related antigen into the culture medium. The experiments described in this paper show the presence of two pathways for the secretion of factor VIII-related antigen from endothelial cells. There is a basal release of this antigen, independent of the presence of extracellular calcium ions. This release can be inhibited by cycloheximide and is therefore directly related to de novo protein synthesis. Besides this basal release, there is an extra release of factor VIII-related antigen that can be stimulated by thrombin, the Ca2+-ionophore A23187 or phorbol myristate acetate. As demonstrated by immunofluorescence, the stimulus-inducible release originates from storage granules in the cells. This stimulus-inducible release is dependent on extracellular Ca2+ but independent of intracellular cAMP.

Von Willebrand protein was synthesized and secreted by human endothelial cells in culture. Ca2+ ionophore A23187 and phorbol myristate acetate stimulated the release of Von Willebrand protein from the cultured cells. Stimulated release was accompanied by the disappearance of rod-like structures from the cultured endothelial cells immunostained for Von Willebrand protein, suggesting the existence of a storage organelle for Von Willebrand protein in these cells (Loesberg, C., Gonsalves, M.D., Zandbergen, J., Willems, C., Van Aken, W.G., Stel, H.V., Van Mourik, J.A. and De Groot, P.G. (1983) Biochim. Biophys. Acta 763, 160-168). Cultured human endothelial cells were fractionated on a density gradient of colloidal silica. Von Willebrand protein was found in two organelle populations: a buoyant one sedimenting with a variety of cell organelle marker enzymes, including those of the Golgi apparatus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum and plasma membrane fragments (peak density of this fraction: 1.08 g X ml-1), and a dense one with a peak density of 1.12 g X ml-1. The dense organelles containing Von Willebrand protein were apparently free of other organelles. Stimulating Von Willebrand protein release with phorbol myristate acetate or Ca2+ ionophore A23187 resulted in a decrease or even complete disappearance of Von Willebrand protein from the high-density organelle fraction, implying a role of this organelle in the stimulus-induced release of Von Willebrand protein. The Von Willebrand protein content of the buoyant fraction was lowered to some extent or did not change upon incubation of the cells with ionophore A23187 and phorbol myristate acetate. Restoration of Von Willebrand protein content of the dense organelle fraction after stimulation occurred within 2 days; this was accompanied by recurrence of immunostaining of rod-shaped structures in cells and an increase in cellular Von Willebrand protein. The excretion of restored Von Willebrand protein could be stimulated again.

This report describes the isolation and culture of venous endothelial cells from the umbilical cord of an obligate heterozygote for homocystinuria. The effect of different sulphur-containing amino acids on the viability and function of these cells was studied and compared with cultured normal endothelial cells. When endothelial cells were cultured in the presence of methionine (10 mmol/l) or homocystine (10 mmol/l), differences occurred between the viability and function of the heterozygote and normal cells in terms of 51Cr release and ability to prevent platelet adherence. The Cr release corrected for spontaneous release increases for the heterozygote cells after incubation/for 21 h in the presence of methionine to 81.3% (control cells, range: 0-23.3%, n = 5) and in the presence of homocystine to 141% (control cells, range: 13.5-55.2%, n = 5). The total number of platelets that adhere to confluent monolayers increases for heterozygote cells cultured in the presence of methionine to 0.98 X 10(7) platelets cm-2 (normal cells, range: 0.56-0.72 X 10(7) platelets cm-2) and in the presence of homocystine to 1.41 X 10(7) platelets cm-2 (normal cells, range: 0.94-1.06 X 10(7) platelets cm-2). Both normal and control cells were sensitive to homocysteine. This study/indicates for the first time what vascular endothelial cells, derived from an obligate heterozygote, are (partly) deficient in cysthathionine synthase and are more susceptible to methionine- and homocystine-mediated injury than normal endothelial cells. Consequently, in homocystinuria, due to dysfunction of the endothelial cells, toxic sulphur-containing amino acids may accumulate in these cells, causing injury of these cells.

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.

Studies on the identification of molecules involved in the interaction of endothelial cells with their environment have led to the discovery of membrane receptors that are similar or identical to previously characterized platelet membrane glycoproteins. Some of these surface molecules belong to a family of widely distributed cell adhesion receptors, termed integrins. One of the integrins, the vitronectin receptor, a platelet glycoprotein (GP) IIb-IIIa related molecule, has now been characterized in some detail. It is a heterodimeric molecule consisting of a subunit similar, but not identical, to GP IIb and a subunit (almost) identical to GP IIIa. Alloantigens (Zwa, Zwb, and Yukb) expressed by platelet GP IIIa are also expressed by endothelial GP IIIa. The vitronectin receptor is involved in the adhesion of endothelial cells to Arg-Gly-Asp-containing immobilized proteins such as vitronectin, fibrinogen, and von Willebrand factor. Endothelial cells also express a molecule indistinguishable from the platelet VLA-2 or GP Ia-IIa complex which is another member of the integrin family. This molecule functions as a platelet collagen receptor, and perhaps it also functions as a collagen receptor on endothelial cells. By mediating cell-matrix contact, the vitronectin receptor, VLA-2 and other similar receptors might be involved in the anchorage of endothelial cells to their extracellular matrix.

We have previously demonstrated that endothelial cells synthesize a plasma membrane protein indistinguishable from platelet glycoprotein (GP) IIa. The present study provides evidence for a further analogy between the platelet and the endothelial cell membrane by showing that cultured endothelial cells also synthesize a membrane protein complex immunologically related to the platelet GP IIb/GP IIIa complex. This evidence is based on the following observations: (1) C17, a murine monoclonal antiplatelet GP IIIa antibody, consistently precipitates two proteins, apparent molecular weights, respectively, 115,000 and 125,000 reduced and 95,000 and 135,000 nonreduced, from metabolically (35S-methionine) as well as surface 125I-labeled cultured human endothelial cells; (2) upon crossed immunoelectrophoresis of solubilized endothelial cells against a polyclonal rabbit antiplatelet antiserum and 125I-labeled C17 IgG, a single precipitate of the protein(s) recognized by C17 is observed. As judged by their mobility in 9% polyacrylamide gels, both endothelial proteins appear to have a somewhat larger molecular weight than their platelet counterparts. Patterns obtained by crossed immunoelectrophoresis are also indicative of a difference in electrophoretic behavior of the platelet GP IIb/IIIa complex and the endothelial cell protein complex.