Detection of an unusually stable fibrinolytic inhibitor produced by bovine endothelial cells.

Abstract

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 37TC. 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 'lS-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 1000C 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.The production of both urokinase-like and tissue-type plasminogen activators (PAs) by cultured endothelial cells (1) emphasizes the potential role of endothelium in the specific catabolism of locally deposited fibrin and in the general maintenance of blood vessel patency. Expression of these cellular activities should be regulated precisely to ensure that the normal hemostatic role of endothelium (2) is not compromised.Plasma contains a number of molecules that may function in this way by either promoting (3, 4) or inhibiting (5) endothelial cell-mediated fibrinolysis. In addition, cultured rabbit (6, 7) and human (8-11) endothelial cells were found to be associated with an antifibrinolytic activity, suggesting that endothelium itself also may produce molecules that modulate the fibrinolytic activity of the vessel wall. However, no such inhibitor activity has been detected in confluent bovine aortic endothelial cells (BAEs) (9,12).We have used cultured i3AEs as a model to identify factors that influence the overall fibrinolytic activity of endothelium (13). Cellular fibrinolytic activity was found to change with the...

Concomitant secretion of prourokinase and of a plasminogen activator-specific inhibitor by cultured human monocytes-macrophages.

“…Furthermore, these inhibitors appear to differ between different cell types. The secretion of Uk inhibitors by murine macrophages, which also synthesize a Uk-type PA (15,16, manuscript in preparation), as well as by hepatoma tissue culture cells (36), endothelial cells (37)(38)(39), and other cell types (40)(41)(42), further underlines the possible generality of these phenomena.…”

Activated protein C stimulates the fibrinolytic activity of cultured endothelial cells and decreases antiactivator activity.

“…1251-labeled fibrinogen was prepared by using the chloramine-T method (11,19 (20). The cells used for these studies had been passaged [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] …”

“…The interaction of this urokinase with plasminogen caused the indicator film to lyse. The opaque lysis-resistant zones that develop in the otherwise clear indicator film result from the presence of inhibitors in the NaDodSO4/polyacrylamide gel (14,22).…”

“…These cells produce multiple forms of PA (11,12), including both the urokinase-like (u-PA) and tissue-type (t-PA) molecules (13). They also produce an antiactivator that can inhibit the activities of these PAs (14,15). In this report, we show that purified bovine APC can increase the fibrinolytic activity of BAEs in the absence of blood cells or plasma proteins.…”

Plasma Concentration of Coagulation and Fibrinolysis Factors and Platelet Function in Hypertension

“…With regard to the significant enhancement of the mean plasminogen activator inhibitor concentrations, it should be mentioned that it has recently been reported that two pools of plasminogen activator inhibitor-1 exist in blood, which may originate from different cell types. The first one is the free plasma pool, which might be synthesized and secreted by endothelial cells and/or hepatocytes s reported by Loskutoff (28) and Sprengers (39), but this is not unambiguously established. The second pool of plasminogen activator inhibitor has been detennined in the α-granules of the platelets (30) and thus in megakaryocytes.…”