Interaction of single-chain urokinase-type plasminogen activator with human endothelial cells

The interaction of urokinase-type plasminogen activators with receptors on the surface of endothelial cells may play an important role in the regulation of fibrinolysis and cell migration. Therefore, we investigated whether human umbilical vein endothelial cells (HUVEC) express receptors for single-chain urokinase (scu-PA) on the cell surface and examined the effect of such binding on plasminogen activator activity. Binding of 125I-labeled scu-PA to HUVEC, performed at 4 degrees C, was saturable, reversible, and specific (k+1 4 +/- 1 X 10(6) min-1 M-1, k-1 6.2 +/- 1.4 X 10(-3) min-1, Kd 2.8 +/- 0.1 nM; Bmax 2.2 +/- 0.1 X 10(5) sites/cell; mean +/- S.E.). Binding of radiolabeled scu-PA was inhibited by both natural and recombinant wild-type scu-PA, high molecular weight two-chain u-PA (tcu-PA), catalytic site-inactivated tcu-PA, an amino-terminal fragment of u-PA (amino acids 1-143), and a smaller peptide (amino acids 4-42) corresponding primarily to the epidermal growth factor-like domain. Binding was not inhibited by low molecular weight urokinase or by a recombinant scu-PA missing amino acids 9-45. Cell-bound scu-PA migrated at its native molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of plasminogen, scu-PA bound to endothelial cells generated greater plasmin activity than did scu-PA in the absence of cells. In contrast, when tcu-PA was added directly to HUVEC, sodium dodecyl sulfate-stable complexes formed with cell or matrix-associated plasminogen activator inhibitors with a loss of plasminogen activator activity. These studies suggest that endothelial cells in culture express high affinity binding sites for the epidermal growth factor domain of scu-PA. Interaction of scu-PA with these receptors may permit plasminogen activator activity to be expressed at discrete sites on the endothelial cell membrane.     

Ligand specificity of human thrombomodulin. Equilibrium binding of human thrombin, meizothrombin, and factor Xa to recombinant thrombomodulin.

Thrombomodulin is an endothelial glycoprotein that serves as a cofactor for protein C activation. To examine the ligand specificity of human thrombomodulin, we performed equilibrium binding assays with human thrombin, thrombin S205A (wherein the active site serine is replaced by alanine), meizothrombin S205A, and human factor Xa. In competition binding assays with CV-1(18A) cells expressing cell surface recombinant human thrombomodulin, recombinant wild type thrombin and thrombin S205A inhibited 125I-diisopropyl fluorophosphate-thrombin binding with similar affinity (Kd = 6.4 +/- 0.5 and 5.3 +/- 0.3 nM, respectively). However, no binding inhibition was detected for meizothrombin S205A or human factor Xa (Kd greater than 500 nM). In direct binding assays, 125I-labeled plasma thrombin and thrombin S205A bound to thrombomodulin with Kd values of 4.0 +/- 1.9 and 6.9 +/- 1.2 nM, respectively. 125I-Labeled meizothrombin S205A and human factor Xa did not bind to thrombomodulin (Kd greater than 500 nM). We also compared the ability of thrombin and factor Xa to activate human recombinant protein C. The activation of recombinant protein C by thrombin was greatly enhanced in the presence of thrombomodulin, whereas no significant activation by factor Xa was detected with or without thrombomodulin. Similar results were obtained with thrombin and factor Xa when human umbilical vein endothelial cells were used as the source of thrombomodulin. These results suggest that human meizothrombin and factor Xa are unlikely to be important thrombomodulin-dependent protein C activators and that thrombin is the physiological ligand for human endothelial cell thrombomodulin.     

Purification and properties of a single-chain urokinase-type plasminogen activator form produced by subcultured human umbilical vein endothelial cells

Single-chain Mr 54,000 u-PA (scu-PA) was isolated, in the presence of aprotinin, from 3-liter batches of 60-h serum-free conditioned media obtained from subcultured (4-6th passage) human umbilical vein endothelial cells (HUVECs, approximately 1.8 x 10(9) cells). In the presence of heparin and endothelial cell growth factor, subcultured human umbilical vein endothelial cells produced u-PA proteins consisting of about 85-90% Mr 54,000 scu-PA and 10-15% two-chain Mr 54,000. The major scu-PA form was purified to homogeneity by ion-exchange chromatography on CM-Sephadex C-50, immunoadsorption on purified anti-u-PA IgG-Sepharose and affinity chromatography on p-amino-benzamidine-Agarose. Typically, about 8-10 micrograms of purified scu-PA protein (antigen/protein ratio = 1) was isolated from 3-liter batches of heparin-containing serum-free conditioned media with a yield of about 41% of the total starting u-PA antigen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this purified u-PA protein showed a single Ag-stained band (nonreduced and reduced), with an estimated molecular weight of about 54,000, which exhibited very low fibrinolytic activity. Purified HUVEC-derived scu-PA did not incorporate 3H-labeled diisopropyl fluorophosphate. This protein did, however, exhibit very low amidolytic activity (approximately 5,000 IU/mg) on the u-PA-specific synthetic substrate pyroglu-Gly-Arg-p-nitroanilide, very low plasminogen-dependent fibrinolytic activity on 125I-labeled fibrin coated plates, and directly activated 125I-labeled plasminogen following Michaelis-Menten kinetics with high affinity, Km = 0.72 microM and low turnover number, kcat = 0.0005 s-1. Treatment with plasmin rapidly converted the HUVEC-derived scu-PA to the active two-chain Mr 54,000 u-PA form (approximately 90,000 IU/mg). Binding to fibrin clots, using antigen quantitation, indicated about 20, 10, and 90% binding for equimolar amounts of HUVEC-derived scu-PA, two-chain u-PA, and tissue plasminogen activator standards, respectively. These results indicate that subcultured HUVECs synthesize and secrete their u-PA protein as a single-chain molecule with low intrinsic amidolytic and fibrinolytic activity, high affinity for plasminogen and no specific affinity for fibrin. The role of scu-PA in endothelial cell-mediated vascular function has yet to be clearly defined.     

ACTH receptors in nervous tissue. High affinity binding-sequestration of [125I]Phe2,Nle4]ACTH 1-24 in homogenates and slices from rat brain

We have demonstrated specific, high affinity binding of a biologically active Tyr23-monoiodinated derivative of ACTH, [125I][Phe2,Nle4]ACTH 1-24, in rat brain homogenates. Similarly, in metabolically inhibited and noninhibited rat whole brain slices there is a specific "binding-sequestration" process that is dependent on time, protein concentration, and pH. In homogenates, binding curves were best described by a two-site model and provided the following parameters: Kd1 = 0.65 +/- 0.47 nM, Bmax1 = 21 +/- 41 fmol/mg protein; Kd2 = 97 +/- 48 nM, Bmax2 = 3.5 +/- 1.8 pmol/mg protein. In metabolically viable brain slices, concentration-competition curves of [125I][Phe2,Nle4]ACTH 1-24 binding-sequestration can be described by three components (Kd1 = 14 +/- 24 nM, Bmax1 = 50 +/- 95 fmol/mg protein; Kd2 = 2.4 +/- 1.9 microM, Bmax2 = 44 +/- 49 pmol/mg protein; Kd3 = 0.16 +/- 1.0 mM, Bmax3 = 5.3 +/- 54 nmol/mg protein). Metabolic inhibition, by removal of glucose and addition of 100 microM ouabain, abolishes the lowest affinity, highest capacity binding-sequestrian component only (Kd1 = 7.1 +/- 14 nM, Bmax1 = 8.7 +/- 16 fmol/mg protein; Kd2 = 7.4 +/- 4.49 microM, Bmax2 = 37 +/- 27 pmol/mg protein). The two binding-sequestration parameter estimates obtained from metabolically inhibited tissue slices are not significantly different from those of the two higher affinity components obtained with noninhibited tissue. Thus, metabolic inhibition permits demonstration of ACTH receptor binding only, unconfounded by sequestration or internalization of ligand:receptor complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photoaffinity labeling of the K+-channel-associated apamin-binding molecule in smooth muscle, liver and heart membranes

High-affinity binding sites for mono[125I]iodoapamin were detected in membranes (Kd = 59 pM, Bmax = 24 fmol/mg protein) and cultured cells (Kd = 69 pM, Bmax = 2.8 fmol/mg protein) from rat heart and in membranes from guinea-pig ileum (Kd = 67 pM, Bmax 42 fmol/mg protein) and liver (Kd = 15 pM, Bmax = 43 fmol/mg protein). Binding was stimulated by K+ ions (K0.5 = 0.3-0.5 mM). Covalent labeling with arylazide [125I]iodoapamin derivatives showed that smooth muscle, liver and heart binding molecules are associated with a 85-87-kDa polypeptide. A second strongly labeled 57-kDa component was identified in liver membranes only.     

Urokinase mutant with better fibrin-specificity

A 150 -156 amino acids-deleted single-chain urokinase-type plasminogen activator (dscu-PA) and its rccombinant wild-type counterpart (rscu-PA) were both expressed in Escherichia coli. After denaturation and renaturation in nitro, the expressed products were both purified lo a single silver-stained band by means of IgG affinity chromatography. After activation by plasrran, similar enzymatic constants based on the hydrolysis of synthetic substrate S2444 by the two-chain molecular forms of dscu-PA and rscu-PA, or native tcu-PA were observed, suggesting that no impairment had been exerted on the catalytic active site of dtcu-PA by the 150-156 amino acids deletion. In both in vitro fibrin-clot and 125I-fibrin sepharose lysis tests, dtcu-PA showed a significantly higher fibrinolytic activity than rtcu-PA or rscu-PA. Hardly any effect on the concentration of fibrinogen in plasma was found in dtcu-PA. It was concluded that dtcu-PA had a higher fibrin specificity and that tcu-PA could be provided with better fib     

Characterization of prostaglandin (PG)-binding sites expressed on human basophils. Evidence for a prostaglandin E1, I2, and a D2 receptor.

Recent data suggest that prostaglandins (PGs) are involved in the regulation of basophil activation. The aim of this study was to characterize the basophil PG-binding sites by means of radioreceptor assays using 3H-labeled PGs. Scatchard analysis for pure (greater than 95%) chronic myeloid leukemia (CML) basophils revealed two classes of PGE1-binding sites differing in their affinity for the natural ligand (Bmax1 = 217 +/- 65 fmol/10(8) cells; Kd1 = 0.5 +/- 0.2 nM; Bmax2 = 2462 +/- 381 fmol/10(8) cells; Kd2 = 47 +/- 20 nM; IC50 = PGE1 less than PGI2 less than PGD2 less than PGE2 less than PGF2 alpha) as well as two classes of PGI2 (iloprost)-binding sites (Bmax1 = 324 +/- 145 fmol/10(8) cells; Kd1 = 0.5 +/- 0.3 nM; Bmax2 = 2541 +/- 381; Kd2 = 27 +/- 6 nM; IC50 = PGI2 less than PGE1 less than PGD2 less than PGE2 less than PGF2 alpha. In addition, CML basophils exhibited a single class of PGD2-binding sites (Bmax = 378 +/- 98 fmol/10(8) cells; Kd = 13 +/- 4 nM; IC50: PGD2 less than PGI2 less than PGE1 less than PGE2 less than PGF2 alpha). In contrast, we were unable to detect specific saturable PGE2-binding sites. Primary and immortalized (KU812) CML basophils revealed an identical pattern of PG receptor expression. Basophils (KU812) expressed significantly (p less than 0.001) lower number of PGE1 (PGI2)-binding sites (Bmax1: 9% (20%) of control; Bmax2: 36% (50%) of control) when cultured with recombinant interleukin 3 (rhIL-3), a basophil-activating cytokine, whereas rhIL-2 had no effect on PG receptor expression. Functional significance of binding of PGs to basophils was provided by the demonstration of a dose-dependent increase in cellular cAMP upon agonist activation, with PGE1 (ED50 = 1.7 +/- 1.1 nM) and PGI2 (ED50 = 2.8 +/- 2.3 nM) being the most potent compounds. These findings suggest that human basophils express specific receptors for PGE1, PGI2 as well as for PGD2.     

Cell-binding domain of endothelial cell thrombospondin: localization to the 70-kDa core fragment and determination of binding characteristics.

Endothelial and other cell types synthesize thrombospondin (TSP), secrete it into their culture medium, and incorporate it into their extracellular matrix. TSP is a large multifunctional protein capable of specific interactions with other matrix components, as well as with cell surfaces, and can modulate cell adhesion to the extracellular matrix. With the aim of understanding the mechanism by which TSP exerts its effect on cell adhesion, we studied the interaction of endothelial cell TSP (EC-TSP) with three different cell types: endothelial cells, granulosa cells, and myoblasts. We find that endothelial cells specifically bind radiolabeled EC-TSP with a Kd of 25 nM, and the number of binding sites is 2.6 X 10(6)/cell. Binding is not inhibitable by the cell-adhesion peptide GRGDS, indicating that the cell-binding site of EC-TSP is not in the RGD-containing domain. Localization of the cell-binding site was achieved by testing two chymotryptic fragments representing different regions of the TSP molecule, the 70-kDa core fragment and the 27-kDa N-terminal fragment, for their ability to bind to the cells. Cell-binding capacity was demonstrated by the 70-kDa fragment but not by the 27-kDa fragment. Binding of both intact [125I]EC-TSP and of the 125I-labeled 70-kDa fragment was inhibited by unlabeled TSP, heparin, fibronectin (FN), monoclonal anti-TSP antibody directed against the 70-kDa fragment (B7-3), and by full serum, but not by heparin-absorbed serum or the cell-adhesion peptide GRGDS. The 70-kDa fragment binds to endothelial cells with a Kd of 47 nM, and the number of binding sites is 5.0 x 10(6)/cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of binding and cyclic GMP accumulation by atrial natriuretic peptides in endothelial cells

Rat 125I-labeled atrial natriuretic factor (ANF (8-33)) was used to identify ANF receptors on cultured bovine aortic endothelial cells. Specific binding of 125I-ANF at 37 degrees C to confluent endothelial cells was saturable and of high affinity. Scatchard analysis of the equilibrium binding data indicated that endothelial cells contain a single class of binding sites with a Kd of 0.1 +/- 0.01 nM. This particular clone of endothelial cells had 16000 +/- 1300 receptors per cell. The order of potency for competing with 125I-ANF binding was human atrial natriuretic peptide (hANP) = atrial natriuretic factor (ANF (8-33)) greater than atriopeptin II greater than atriopeptin III greater than atriopeptin. The weakest competitor, atriopeptin I, had a K1 of 0.45 nM, which was only 6-fold higher than the K1 for hANP and ANF (8-33). ANF (8-33) and hANP in the presence of 0.5 mM isobutylmethyl-xanthine produced a 15-20-fold increase in cyclic GMP content at 10 pM and a maximal 500-fold elevation of cyclic GMP at 10 nM. The concentrations required to elicit a half-maximal increase in cyclic GMP for hANP, ANF (8-33), atriopeptin I, atriopeptin II and atriopeptin III were 0.30, 0.35, greater than 500, 4.0 and 5.0 nM, respectively. Although atriopeptin I acted as a partial agonist, it was unable to antagonize the effect of ANF (8-33) on cyclic GMP formation. These findings suggest that endothelial cells have multiple and functionally distinct ANF-binding sites.     

Calcium channel binding in nerves and muscle of canine small intestine

Using [3H]-nitrendipine (Nit) and [125I]-omega conotoxin (w-CTX), the cellular and subcellular distribution of calcium channel subtypes in the homogenates of canine small intestinal circular muscle was studied. Nit. bound to the membranes from the circular smooth muscle cells (PM) and to the synaptosomal membranes from the deep muscular plexus (DMP); the Kd and Bmax values of Nit binding from these two sources were similar (Kd 0.4 +/- 0.16 nM and 0.77 +/- 0.24 nM; Bmax 206 +/- 22 and 192 +/- 39 fmol/mg of protein in DMP and PM respectively). w-CTX, however, bound only to the DMP (Kd 18.41 +/- 7.5 pM, Bmax 265 +/- 36 fmol/mg of protein). In DMP, nifedipine (10(-6) M) failed to interact with the binding of w-CTX; similarly, no modulation of Nit binding with unlabelled w-CTX (10(-7) M) could be detected. Therefore w-CTX and Nit binding sites represent two distinct, non-interactive and differentially distributed binding sites in canine small intestine.     

Assembly and expression of an intrinsic factor IX activator complex on the surface of cultured human endothelial cells.

Endothelial cells expose specific receptors for blood clotting factors and, upon perturbation, can initiate and propagate the reactions of the extrinsic pathway of blood coagulation leading to fibrin formation on the cell surface. The existence of an intrinsic mechanism of Factor IX activation on cultured human umbilical vein cells (HUVECs) was investigated by studies of the interaction between HUVECs and two proteins of the contact activation system, the cofactor high molecular weight kininogen (H-kininogen) and the zymogen Factor XI. In the presence of zinc ions (10-300 microM), 125I-labeled H-kininogen bound to HUVECs in a time-dependent, reversible, and saturable manner, with calcium ions exerting an inhibitory effect on the zinc-dependent binding. Analysis of the binding data by the LIGAND computer program indicated that HUVECs, in the presence of 2 mM CaCl2 and 100 microM ZnCl2 at 37 degrees C, bound 1.14 x 10(7) H-kininogen molecules per cell with an apparent dissociation constant of 55 nM. HUVEC-bound H-kininogen functions as the cell surface receptor for both 125I-labeled Factor XI and 125I-labeled Factor XIa, since HUVECs cultured in contact factor-depleted serum do not detectably bind either the zymogen or the enzyme in the absence of H-kininogen and zinc ions. In the presence of saturating concentrations of H-kininogen, 2 mM CaCl2 and 100 microM ZnCl2, the binding of 125I-labeled Factor XI and Factor XIa to HUVECs was time-dependent, reversible, and saturable, with apparent dissociation constants of 4.5 and 1.5 nM, respectively. HUVEC-bound complexes of H-kininogen and Factor XI generated Factor XIa activity only after the addition of purified Factor XIIa, and cell-bound Factor XIa in turn activated Factor IX, as documented by a 3H-labeled activation peptide release assay for 3H-Factor IX activation. The results indicate that cultured HUVECs provide a surface for the assembly and expression of an intrinsic Factor IX activator complex that may participate in the initiation of blood coagulation at sites of vascular injury.     

Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in syrian hamster hypothalamus

1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, [125I]iodomelatonin, was examined using an incubation temperature (30 degrees C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing [125I]iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax = 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus.     

Characterization of gonadotropin-releasing hormone (GnRH) binding sites in the pituitary and testis of the frog, Rana esculenta

Frog, Rana esculenta, pituitary and testis gonadotropin-releasing hormone (GnRH) receptors were characterized by using 125I-chicken IIGnRH (cIIGnRH) as radiolabeled ligand. At 4 C equilibrium binding of 125I-cIIGnRH to pituitary homogenates was achieved after 90 min of incubation; binding of 125I-cIIGnRH to testis membrane fractions reached its maximum at 60 min of incubation. Binding of the radioligand was a function of tissue concentration, with a positive correlation over the range 0.5-2 tissue equivalents per tube. One pituitary and one testis per tube were used as standard experimental condition. Incubation of the pituitary homogenate with increasing concentrations of 125I-cIIGnRH indicated saturable binding at radioligand concentrations of 1 nM and above while for the testis membrane preparation saturation was achieved using 5 nM 125I-cIIGnRH. The binding of 125I-cIIGnRH was found to be reversible after addition of the cold analog and the displacement curves could be resolved into one linear component for both tissues. Scatchard analysis suggested the presence of one class of binding sites for both pituitary and testis (Pituitary: Kd = 1.25 +/- 0.14 nM and Bmax = 8.55 +/- 2.72 fmol/mg protein; testis: Kd = 2.23 +/- 0.89 nM and Bmax = 26.48 +/- 7.39 fmol/mg protein). Buserelin displaced the labeled 125I-cIIGnRH with a lower IC50 as compared with cIIGnRH cold standard, while Arg-vasopressin (AVP) was completely ineffective, confirming the specificity of binding.     

Motilin receptors in rabbit stomach and small intestine

Motilin receptors in rabbit antral and duodenal smooth muscle tissue were characterized by direct binding technique using 125I-labeled porcine motilin as a tracer ligand. Binding at 30 degrees C was maximal at 90 min, was saturable and partially reversible. Displacement studies with natural porcine motilin, synthetic leucine-motilin or norleucine-motilin indicated a dissociation constant (Kd) of 1.1 +/- 0.3 nM and a maximal binding capacity (Bmax) of 42 +/- 10 fmol/mg protein. Binding was unaffected by glucagon, pancreatic polypeptide and somatostatin, but substance P interfered via an unknown mechanism. By density gradient centrifugation motilin receptors were shown to be present in plasma membranes. Binding could only be demonstrated in preparations from antrum and upper duodenum. These observations provide evidence for a localized target region for motilin in the gastrointestinal tract, and for a direct interaction of motilin with gastrointestinal smooth muscle tissue.     

Demonstration of specific binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) in rat astrocytes

The high and low affinity binding sites for PACAP were identified in rat astrocytes using [125I]PACAP27 as the labeled ligand. Scatchard analysis of displacement of the bound tracer by unlabeled PACAP27 indicated the existence of two classes of binding sites, with the dissociation constant (Kd) = 1.22 +/- 0.4 nM, the binding maximal capacity (Bmax) = 821 +/- 218 fmols/mg protein for the high affinity binding site, and Kd = 0.59 +/- 0.06 microM, Bmax = 563 +/- 12 pmols/mg protein for the low affinity binding site, respectively. The specificity of [125I]PACAP27 binding was tested using PACAP38 and peptides structurally related to PACAP, such as VIP, GHRF, PHI, secretin and glucagon. PACAP38 completely displaced the binding of [125I]PACAP27 and Scatchard analysis also indicated the presence of two classes of binding sites with similar Kd and Bmax to those for PACAP27. VIP and GHRF competed with [125I]PACAP27, but to a much lesser extent than unlabeled PACAP27 in binding. Other peptides tested did not displace the binding of [125I]PACAP27 at 10(-6) M.     

Biochemical characterization of single-chain chimeric plasminogen activators consisting of a single-chain Fv fragment of a fibrin-specific antibody and single-chain urokinase.

K12G0S32 is a 57-kDa recombinant single-chain chimeric plasminogen activator consisting of scFv-K12Go, a single-chain variable-region antigen-binding fragment (Fv) of the monoclonal antibody MA-15C5, which is specific for fragment D-dimer of human cross-linked fibrin, and a low-molecular-mass (33 kDa) urokinase-type plasminogen activator (u-PA-33k) containing amino acids Ala132-Leu411 (Holvoet, P., Laroche, Y., Lijnen, H. R., Van Cauwenberghe, R., Demarsin, E., Brouwers, E., Matthyssens, G. & Collen D. (1991) J. Biol. Chem. 266, 19717-19724). In addition, the Arg156-Phe157 thrombin-cleavage site in the u-PA moiety of K12G0S32 is removed by substitution of Phe157 with Asp. In the present study, the fibrinolytic potency of K12G0S32, determined in a system composed of a 125I-fibrin-labeled human plasma clot submerged in citrated plasma, was found to be only twofold higher than that of intact single-chain u-Pa (rscu-PA), but 17-fold higher than that of rscu-PA(M), a variant of rscu-PA in which the thrombin-cleavage site was removed by substitution of Phe157 with Asp. The fibrinolytic potency of K12G0S32T, with an intact thrombin-cleavage site, was 6-15-fold higher than that of rscu-PA. Conversion of 1 microM single-chain K12G0S32 or rscu-PA(M) into their two-chain derivatives with plasmin occurred at a rate of 1.0 +/- 0.15 nmol.min-1.nmol plasmin-1 and 0.85 +/- 0.074 nmol.min-1.nmol plasmin-1, compared to 14 +/- 2.3 nmol.min-1.nmol plasmin-1 and 18 +/- 2.6 nM.min-1.nmol plasmin-1 for K12G0S32T and rscu-PA, respectively. Purified fragment D-dimer of human cross-linked fibrin inhibited the fibrinolytic potency of single-chain K12G0S32T, but not of two-chain K12G0S32T, in a dose-dependent manner. Furthermore, the fibrinolytic potencies of two-chain K12G0S32 and K12G0S32T were not significantly higher than those of recombinant two-chain u-PA (rtcu-PA) or of rtcu-PA(M). These findings suggest that the 59-fold increase in fibrinolytic potency of K12G0S32T, relative to that of rscu-PA(M), is due both to targeting of the activator to the clot via the single-chain Fv fragment (sixfold increase) and to a more efficient conversion of single-chain K12G0S32T to its two-chain derivative (eightfold increase). Thus, targeting to clots by means of fibrin-specific antibodies results in a significant increase of the fibrinolytic potency of single-chain but not of two-chain u-PA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distinct atrial natriuretic factor receptor sites on cultured bovine aortic smooth muscle and endothelial cells

Cultured bovine aortic smooth muscle and endothelial cells each display distinct specific binding sites for radiolabeled atrial natriuretic peptide (ANF). 125I-pro-rANF (103-126)I binding to both cell types is rapid, reversible and competitive. Scatchard plots of the binding data show Bmax values of 5.5 and 0.1 - 2.1 X 10(5) sites/cell and Kd values of 2.1 and 0.3 nM for smooth muscle and endothelial cells, respectively. In addition, ANF elevates levels of cGMP substantially in both cell types at concentrations of ANF close to its Kd and Ki for binding. Sodium nitroprusside, however, has essentially no effect on cGMP levels in either cell type. These results show that distinct functionally active receptor sites for ANF exist on both vascular smooth muscle and endothelial cells.     

Plasminogen activation with single-chain urokinase-type plasminogen activator (scu-PA). Studies with active site mutagenized plasminogen (Ser740----Ala) and plasmin-resistant scu-PA (Lys158----Glu)

The mechanism of the activation of plasminogen by single-chain urokinase-type plasminogen activator (single-chain u-PA, scu-PA) was studied using rscu-PA-Glu158, a recombinant plasmin-resistant mutant of human scu-PA obtained by site-specific mutagenesis of Lys158 to Glu, and rPlg-Ala740, a recombinant human plasminogen in which the catalytic site is destroyed by mutagenesis of the active-site Ser740 to Ala. Conversion of 125I-labeled single-chain plasminogen to two-chain plasmin was quantitated on reduced sodium dodecyl sulfate-gel electrophoresis combined with autoradiography and radioisotope counting of gels bands. The efficiencies of both rscu-PA-Glu158 and rscu-PA for the activation of rPlg-Ala740 and of natural plasminogen were comparable and were 250-500-fold lower than that of recombinant two-chain u-PA (rtcu-PA) for rscu-PA-Glu158 and 100-200-fold lower for rscu-PA. Pretreatment of rscu-PA-Glu158 or rscu-PA with excess alpha 2-antiplasmin, which efficiently neutralizes all contaminating rtcu-PA, did not significantly reduce the catalytic efficiency of these single-chain moieties, indicating that they have a low but significant intrinsic plasminogen activating potential. The low intrinsic catalytic efficiency of rscu-PA for the conversion of plasminogen to plasmin may be sufficient to generate trace amounts of plasmin, which may regulate plasminogen activation by converting poorly active rscu-PA to very active rtcu-PA.     

Expression and characterization of erythropoietin receptors on normal human bone marrow cells

We studied the specific binding of 125I-labeled bioactive recombinant human erythropoietin (Epo) to human bone marrow mononuclear cells (BMNC) obtained from normal subjects. The 125I-labeled Epo bound specifically to the BMNC. Scatchard analysis of the data showed two classes of binding sites; one high affinity (Kd 0.07 nM) and the other low affinity (Kd 0.38 nM). The number of Epo binding sites per BMNC was 46 +/- 16 high-affinity receptors and 91 +/- 51 low-affinity receptors. The specific binding was displaced by unlabeled Epo, but not by other growth factors. Receptor internalization was observed significantly at 37 degrees C, but was prevented by the presence of 0.2% sodium azide. These findings indicate that human BMNC possess two classes of specific Epo receptors with characteristics of a hormone-receptor association.     

Intracellular receptors for inositol 1,4,5-trisphosphate in angiotensin II target tissues

Many cells (including angiotensin II target cells) respond to external stimuli with accelerated hydrolysis of phosphatidylinositol 4,5-bisphosphate, generating 1,2-diacylglycerol and inositol 1,4,5-trisphosphate, a rapidly diffusible and potent Ca2+-mobilizing factor. Following its production at the plasma membrane level, inositol 1,4,5-trisphosphate is believed to interact with specific sites in the endoplasmic reticulum and triggers the release of stored Ca2+. Specific receptor sites for inositol 1,4,5-trisphosphate were recently identified in the bovine adrenal cortex (Baukal, A. J., Guillemette, G., Rubin, R., Sp?t, A., and Catt, K. J. (1985) Biochem. Biophys. Res. Commun. 133, 532-538) and have been further characterized in the adrenal cortex and other target tissues. The inositol 1,4,5-trisphosphate-binding sites are saturable and present in low concentration (104 +/- 48 fmol/mg protein) and exhibit high affinity for inositol 1,4,5-trisphosphate (Kd 1.7 +/- 0.6 nM). Their ligand specificity is illustrated by their low affinity for inositol 1,4-bisphosphate (Kd approximately 10(-7) M), inositol 1-phosphate and phytic acid (Kd approximately 10(-4) M), fructose 1,6-bisphosphate and 2,3-bisphosphoglycerate (Kd approximately 10(-3) M), with no detectable affinity for inositol 1-phosphate and myo-inositol. These binding sites are distinct from the degradative enzyme, inositol trisphosphate phosphatase, which has a much lower affinity for inositol trisphosphate (Km = 17 microM). Furthermore, submicromolar concentrations of inositol 1,4,5-trisphosphate evoked a rapid release of Ca2+ from nonmitochondrial ATP-dependent storage sites in the adrenal cortex. Specific and saturable binding sites for inositol 1,4,5-trisphosphate were also observed in the anterior pituitary (Kd = 0.87 +/- 0.31 nM, Bmax = 14.8 +/- 9.0 fmol/mg protein) and in the liver (Kd = 1.66 +/- 0.7 nM, Bmax = 147 +/- 24 fmol/mg protein). These data suggest that the binding sites described in this study are specific receptors through which inositol 1,4,5-trisphosphate mobilizes Ca2+ in target tissues for angiotensin II and other calcium-dependent hormones.