Monovalent cation dependence of tissue plasminogen activator synthesis by HeLa cells.

HeLa cells synthesize and secrete increased levels of tissue plasminogen activator (tPA) when incubated for 18 h with 10-20 nM phorbol myristate acetate. This response was inhibited by a number of conditions which affect intracellular Na+ and K+ concentrations. Removing extracellular Na+, while maintaining isotonicity with choline+, reduced the secretion of both functional and antigenic tPA in a linear fashion. A series of cardiac glycosides and related compounds strongly inhibited tPA secretion with the following rank order of potency: digitoxin = ouabain greater than digoxin greater than digitoxigenin greater than digoxigenin greater than digitoxose greater than digitonin. These compounds also inhibited cellular Na+/K+-ATPase activity over an identical concentration range. Two compounds which selectively increase cellular permeability to K+, valinomycin, and nigericin, strongly inhibited tPA secretion, with IC50 values of approximately 50 nM. In contrast, monensin, which selectively increases cellular permeability to Na+, was much less active. Valinomycin, but not nigericin, also inhibited cellular Na+/K+-ATPase activity. Phorbol myristate acetate, 5-20 nM, increased Na+/K+-ATPase activity up to 2-fold and tPA secretion up to 15-fold. We conclude that the secretion of tPA by HeLa cells treated with phorbol myristate acetate proceeds via a mechanism which requires extracellular Na+ and a functional Na+/K+-ATPase ("sodium pump") enzyme.

Calcium regulation of tissue plasminogen activator and prostaglandin biosynthesis in HeLa cells.

Phorbol 12-myristate 13-acetate, 1-20 nM, induced the synthesis in HeLa cells of a 65 200 Mr tissue-type plasminogen activator, and of prostaglandin E2. Omission of Ca2+ from the incubation medium inhibited the induction of plasminogen activator synthesis by 40-60% and abolished the induction of prostaglandin E2 synthesis. Maximal plasminogen activator synthesis could be maintained at extracellular Ca2+ concentrations of approx. 0.1 mM, while maximal prostaglandin synthesis required at least 0.45-0.9 mM Ca2+. The induction of each factor was inhibited by 10-100 microM 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), an inhibitor of intracellular C2+ mobilization. Prostaglandin synthesis, but not plasminogen activator synthesis, was also inhibited by 10-100 microM verapamil and nifedipine, which inhibit intracellular Ca2+ uptake via the so-called 'slow-channels' and by 0.5-10 microM trifluoperazine, an inhibitor of calmodulin. Neither plasminogen activator synthesis nor prostaglandin synthesis were stimulated by 5-50 microM 1-oleoyl-2-acetylglycerol or 1-250 microM 1,2-dioctanoylglycerol, alone and in combination with 50 nM-1 microM ionophore A23187. These results indicate that the synthesis of plasminogen activator and prostaglandins in HeLa cells is Ca2+-dependent, and that the Ca2+ requirements for each process are not identical. Thus, Ca2+ regulation of the production of tissue plasminogen activator and prostaglandin E2 occurs at multiple points in their biosynthetic pathways.

Alterations in the apparent tissue factor (thromboplastin) expression in HeLa cells by a cellular factor Xa inhibitor.

HeLa cells have undetectable tissue factor (thromboplastin) activity when measured by a one-stage coagulation assay. In contrast, these cells accelerated the factor VII-catalyzed cleavage of factor X. The two assays gave similar results after either heating the samples to 100 degrees C for 2 min or exposure to thrombin. Neither of these treatments altered the tissue factor activity of human foreskin fibroblasts, a cell type with high tissue factor activity. HeLa cells contain an inhibitor(s) directed against factor Xa but not thrombin. The inhibitor(s) was inactivated by exposure to thrombin or by heat treatment. Inhibition of factor Xa-catalyzed cleavage of a synthetic peptide was blocked by ethyleneglycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) so the inhibition was apparently dependent on divalent cations. Inhibition was not accelerated by heparin. The inhibitor(s) was not protein C or other serine proteases since it was not inactivated by diisopropylfluorophosphate. The factor Xa inhibitor(s) has been isolated from HeLa cells with an approximate 500-fold increase in specific activity. After SDS-polyacrylamide gel electrophoresis factor Xa-inhibitory activity was recovered from a region corresponding to the major Coomassie-staining band at 43 kDa and in lesser amounts from regions corresponding to 26 and 17 kDa. Cellular inhibitors of coagulation may partially explain the low apparent tissue factor observed in some in vitro cells and may serve a regulatory role in limiting the expression of tissue factor.

Purification of a platelet protein which stimulates fibrinolytic inhibition and tissue factor in human fibroblasts.

Platelets are able to stimulate an increase in two distinct activities, tissue factor (thromboplastin) and fibrinolytic inhibition, in human fibroblasts in vitro. A procedure has been developed which allows the purification of a platelet macromolecule which is able to stimulate both of these changes. Washed human platelets were homogenized, sonicated, and then centrifuged at 90,000 x g for 2 h. The resulting pellet was solubilized in 0.05 M sodium carbonate, pH 10.5, and chromatographed on Sephadex G-200, then on hydroxylapatite, resulting in a 135-fold purification and a 20% yield. When the purified material was further fractionated on sodium dodecyl sulfate-polyacrylamide gels, stimulatory activity for both tissue factor and fibrinolytic inhibition was found only in the 75,000-dalton region. The active material could be inactivated by mercaptoethanol with no change in its apparent molecular weight. It was readily inactivated by trypsin with the concomitant loss of the 75,000-dalton Coomassie-staining band. Assay of the purified material for carbohydrate was negative. After isoelectric focusing, the purified material had a major band at pH 5.8 which stimulated both tissue factor and fibrinolytic inhibition. Subcellular fractionation of platelet homogenates by sucrose density gradient centrifugation resulted in a 2-fold increase in stimulatory material in the granule/mitochondrial fraction. This platelet-derived protein may represent a physiologically important regulator for both cellular procoagulant and the net fibrinolytic activity of systemic cells.

Platelet effects on tissue factor and fibrinolytic inhibition of cultured human fibroblasts and vascular cells.

Platelets stimulate tissue factor, the initiator of the extrinsic coagulation pathway, and increase fibrinolytic inhibition in fibroblasts grown in vitro. Cellular tissue factor increases an average of 2.8-fold over the control levels after a 6-hr incubation with platelets, and no activity is present in the media. Fibrinolytic inhibition is stimulated in both the fibroblasts and their media in the presence of platelets and accumulates throughout a 24-hr incubation. Neither leukocytes nor erythrocytes stimulate these changes. Both tissue factor and fibrinolytic inhibition increases are dependent on platelet concentration and are blocked by inhibitors of RNA or protein synthesis. Control smooth muscle cells have higher tissue factor and fibrinolytic inhibition than fibroblasts, but their response to the presence of platelets is similar. Confluent monolayers of endothelial cells have very low levels of tissue factor that are not altered by the presence of platelets. However, the ability of endothelial cells to inhibit fibrinolysis is enhanced by the presence of platelets. The fraction that stimulates tissue factor and fibrinolytic inhibition is distinct from platelet-derived growth factor and from the fraction that enhances leukocyte tissue factor. It is associated with an insoluble, nonmitogenic fraction that is not inactivated by phospholipase C, or diisopropylfluorophosphate, nor is it chloroform:methanol extractable. Platelets are a physiologic modulator for both cellular tissue factor and the fibrinolytic system in vitro.

Evidence for a carrier-mediated exchange-diffusion entry of calcium into erythrocytes.

The enthalpy of activation of entry of 45Ca into erythrocytes under conditions of zero net transport (19 Kcal/mol), inhibition of entry by N-ethylmaleimide, and transport pH optimum of 7 - 7.5 are consistent with a protein carrier for inward calcium movement. Initial rate analysis shows saturation kinetics and transstimulation, consistent with a carrier-mediated exchange-diffusion mechanism.

Erythrocyte calcium metabolism. Calcium exchange in normal and sickle-cell-anaemia erythrocytes.

Under exchange conditions (no net increase in calcium), erythrocytes incubated in isoosmotic phosphate-buffered saline have an exchangeable calcium pool comprising about 10% of the total erythrocyte calcium. This pool reaches exchange equilibrium, for either inward-directed or outward-directed transfer of the 45Ca-exchange label, with a half-time of about 20 min. The uptake of Ca2+ requires phosphate, even under hypo-osmotic conditions, where the calcium loading expected as the cells swell is obtained only when phosphate is present. The phosphate requirement is not due to Ca2+ transport as a phosphate salt. This exchangeable-calcium pool is also present in sickle-cell-anemia erythrocytes, and comprises a similar proportion of total cellular calcium.