Nontoxic irreversible inhibitors of factors IXa, Xa, and thrombin: potential therapeutic agents for the in vivo regulation of thrombin generation and activity.
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Animals↗
Biomedical subjects
S V Pizzo
Publications and source records attributed to S V Pizzo.
Specificity of alpha 2-macroglobulin covalent cross-linking for the active domain of proteinases.
The reaction of alpha 2-macroglobulin (alpha 2M) with the two-chain enzyme plasma kallikrein results in covalent bond formation between the catalytic subunit and the inhibitor. We have recently published a model of alpha 2M which suggests that this phenomenon may be a general mechanism when multisubunit proteinases are inactivated by alpha 2M. In order to test this hypothesis, we studied the reactions of factor Xa, plasmin, streptokinase-plasmin and alpha-thrombin with alpha 2M. In the case of factor Xa the catalytic heavy chain demonstrated greater than 99% covalent incorporation while over 97% of the light chain failed to crosslink to the inhibitor. Preferential binding of the catalytic light chains of plasmin (70% covalent incorporation) and plasmin in complex with streptokinase (79% covalent incorporation) was also observed. Finally, 82% covalent incorporation of the catalytic heavy chain of alpha-thrombin was found. These studies demonstrate that in the case of multisubunit proteinases, the chain containing the active site demonstrates preferential binding as predicted by the model supporting placement of the site of covalent binding close to the "bait region" of alpha 2M.
Binding Sites↗
Release of tissue plasminogen activator and its fast-acting inhibitor in defective fibrinolysis.
Releasable tissue plasminogen activator (t-PA) and the fast inhibitor of t-PA were measured in 18 controls and a pedigree with venous thrombosis. The functional assay was performed by a technique that destroys the t-PA inhibitor when blood is drawn. It was found that activator and inhibitor levels varied widely in the control group. One patient demonstrated inhibitor levels, on two different occasions, of 2.82 and 3.54 IU of t-PA per milliliter of plasma, as compared with a releasable activator level of 1.87 IU/mL. The t-PA antigen levels of this patient and the remainder of the pedigree were essentially normal for all seven subjects. Thus, it is suggested that the previously reported fibrinolytic disorder in this pedigree represents an imbalance between activator and inhibitor levels rather than an actual deficiency of t-PA.
Antigens↗
Characterization of murine peritoneal macrophage receptors for fibrin(ogen) degradation products.
The binding of human fibrinogen degradation fragments D1, E, X, and Y, as well as fibrin fragment D1 dimer, to mouse peritoneal macrophages was examined. A Scatchard plot of fragment D1 binding was biphasic, suggesting two classes of receptors. Fragments D1, D1 dimer, X, and Y in low concentrations bound to macrophages with high affinity (Kd = 23 to 73 X 10(-11) mol/L). Fragment E bound specifically but at a much lower level than the other fragments. Fragment D1 was able to compete for the binding of radiolabeled fragments X and Y but not radiolabeled fragment E. These studies indicate that fragments D and E are recognized by separate receptor systems but that all of the fibrinogen degradation products that contain the D domain are recognized by the same receptor system.
Animals↗
Atrophie blanche. A disorder associated with defective release of tissue plasminogen activator.
Atrophie blanche is a skin disorder characterized by recurrent ulceration and fibrosis. It is one of a group of cutaneous vasculitides associated with decreased blood fibrinolytic activity. We examined pre- and post-venous occlusion plasma from eight patients with atrophie blanche for release of vascular (tissue-type) plasminogen activator (t-PA). The average plasma level of releasable t-PA was only 0.03 IU/mL, compared with 0.70 IU/mL for 118 healthy controls. Therefore, the fibrinolytic disorder in atrophie blanche is associated with defective release of t-PA from blood vessel walls.
Atrophy↗
Oral contraceptives and thromboembolic disease.
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Contraceptives, Oral↗
Binding and endocytosis of alpha 2-macroglobulin-plasmin complexes.
The clearance of 125I-labeled alpha 2-macroglobulin-plasmin complexes (125I-alpha 2M-PM) from mouse circulation is slower than that of 125I-labeled alpha 2M-methylamine complexes (125I-alpha 2M-CH3NH2). In addition, clearance of 125I-alpha 2M-PM is biphasic, but that of 125I-alpha 2M-CH3NH2 follows simple first-order kinetics. Treatment of alpha 2M-PM with trypsin yields a complex that clears like alpha 2M-CH3NH2. Complexes of alpha 2M with Val442-plasmin (alpha 2M-Val442-PM) were prepared; alpha 2M-Val442-PM has a stoichiometry of 2 mol of Val442-PM to 1 mol of alpha 2M and also clears like alpha 2M-CH3NH2. In vitro 4 degrees C binding inhibition studies with mouse peritoneal macrophages show that alpha 2M-CH3NH2, alpha 2M-PM, trypsin-treated alpha 2M-PM, and alpha 2M-Val442-PM bind with the same affinity, apparent Kd = 0.4 nM. The binding isotherms at 4 degrees C are the same for 125I-alpha 2M-CH3NH2, 125I-alpha 2M-PM, and 125I-trypsin-treated alpha 2M-PM in both mouse peritoneal macrophages and 3T3-L1 fibroblasts. The Scatchard plots for the binding isotherms in macrophages were curved; those in 3T3-L1 fibroblasts were linear with an apparent Kd of 0.48 nM and a receptor activity of 140 fmol/mg of cell protein for alpha 2M-CH3NH2, an apparent Kd of 0.29 nM and a receptor activity of 110 fmol/mg of cell protein for alpha 2M-PM, and an apparent Kd of 0.35 nM and a receptor activity of 210 fmol/mg of cell protein for trypsin-treated alpha 2M-PM.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Precipitation of fibrinogen, fibrinogen degradation products and fibrin monomer by histone H3.
Incubation of histone H3 with normal citrated plasma resulted in the formation of insoluble aggregates, as determined by turbidity measurements. The precipitate was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, confirming that fibrinogen was a major component. Purified fibrinogen precipitated rapidly as determined with turbidity experiments and experiments with radioiodinated protein. The amount of fibrinogen precipitation was strongly dependent on H3 concentration. Variation of ionic strength (0.2-0.84) and pH (5.3-7.4), however, had little or no effect on the reaction. Fibrinogen subjected to gelatin-Sepharose chromatography or dialysis against 3.3M urea reacted equivalently with H3. Precipitation of 125I-fibrinogen by H3 was strongly favored by increasing temperature (4 degrees-45 degrees C). Precipitation of fibrinogen by protamine was maximized by decreasing the temperature. In addition, formation of insoluble fibrinogen-protamine aggregates was highly dependent on ionic strength and pH, suggesting that different types of protein-interaction are involved in the two studied precipitation reactions. Of the fibrinogen degradation products, only fragment X precipitated significantly when incubated with H3. Radioiodinated fibrin monomer also precipitated when incubated with H3 in solutions of sufficient ionic strength to prevent spontaneous polymerization. The extent of precipitation was equivalent for fibrin monomer and fibrinogen. Fragment D inhibited the precipitation of fibrinogen by H3 or protamine. These studies indicate that the proteins termed "paracoagulants" are not all equivalent and that the hydrophobic domain of H3 plays a critical role in fibrinogen precipitation.
Chemical Precipitation↗
Purification and characterization of frog alpha-macroglobulin: receptor recognition of an amphibian glycoprotein.
Frog alpha-macroglobulin was purified to apparent homogeneity by Ni2+ chelate affinity chromatography. Frog alpha-macroglobulin migrated as an alpha 1-globulin in cellulose acetate electrophoresis. A molecular weight of 730 000 was obtained by equilibrium sedimentation, and in sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), the protein migrated as a single band of Mr approximately 360 000 before reduction and Mr approximately 180 000 after reduction. Treatment with trypsin resulted in subunit cleavage to yield a fragment of Mr approximately 90 000. After being heated, the protein fragmented, migrating in SDS-PAGE as two bands of Mr approximately 120 000 and 60 000. This fragmentation was inhibited by prior reaction of the protein with methylamine. In native pore-limit electrophoresis the protein exhibited the characteristic "slow" to "fast" conformational change of protease-treated alpha-macroglobulins. In contrast, typical "slow" to "fast" conformational change was not observed in native PAGE with this preparation. Moreover, the protein incorporated approximately 2 mol of [14C]methylamine/mol of inhibitor without demonstrating a change in mobility in native PAGE. In circular dichroism studies, the protein exhibited a spectrum similar to that of human alpha 2M. Reaction with trypsin resulted in a broadening and decrease in the magnitude of the spectrum. Reaction with methylamine resulted in similar changes, but of smaller magnitude. The inhibitor bound approximately 0.7 mol of trypsin in both radiolabeled protease binding and amidolytic titration studies. 125I-Labeled native frog alpha 1M was removed slowly from the circulation of mice with a t1/2 greater than 2h.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acids↗
Binding of alpha 2-macroglobulin to hepatocytes: mechanism of in vivo clearance.
The binding of 125I-labeled human alpha 2-macroglobulin-methylamine to adult rat hepatocytes in primary culture was studied at 4 degrees C. Cells which had been in culture for 4 hours exhibited steady state ligand binding after 1 hour, a receptor number of 22,400 receptors per cell, and a dissociation constant of 0.6 nM. Adult rat hepatocytes exhibited a significant decrease in receptor number with increased time in primary culture with less than 10% of the initial number of receptors remaining after 2 days (p less than 0.01). In autopsy studies of mice injected intravenously with 125I-labeled alpha 2-macroglobulin-methylamine, greater than 90% of the cleared ligand was found in the liver. Autoradiography of the liver demonstrated that 80% of the ligand was cleared by hepatocytes. It is concluded that the hepatocytes are the primary pathway for clearance from the circulation of receptor recognized alpha 2-macroglobulin.
Animals↗
In vitro effects of acetaminophen and its analogues on human platelet aggregation and thromboxane B2 synthesis.
We examined the effect of acetaminophen and the structural analogues 2,6-dimethylacetaminophen, 3,5-dimethylacetaminophen, and N-acetyl-p-benzoquinone imine on human platelet aggregation, 14C-serotonin secretion, and thromboxane B2 synthesis. Preincubation with 1 mM acetaminophen for 2 min completely inhibited arachidonic acid- and collagen-stimulated platelet aggregation. Thromboxane B2 production and 14C-serotonin secretion by arachidonic acid-stimulated platelets also were completely inhibited. Preincubation of platelets with 1 mM 3,5-dimethylacetaminophen inhibited collagen and arachidonic acid-induced aggregation and arachidonic acid-stimulated thromboxane B2 synthesis, while treatment with 2,6-dimethylacetaminophen did not inhibit aggregation and blocked thromboxane B2 formation to a much lesser degree. Preincubation with 1 mM N-acetyl-p-benzoquinone imine inhibited arachidonic acid-induced aggregation and 14C-serotonin secretion but had no effect on arachidonic acid-induced thromboxane B2 formation and collagen-induced platelet aggregation.
Acetaminophen↗
Immunochemical and biochemical characterization of a glioma-associated extracellular matrix glycoprotein.
A novel human glioma-associated extracellular matrix (ECM) glycoprotein has been identified by murine monoclonal antibody 81C6. The glycoprotein, designated GMEM, is expressed in the ECM of glioma and mesenchymal cell cultures, in the perivascular matrix of endothelial proliferations of human gliomas, and in the stroma of human glioma xenografts in athymic mice, where it has been used as a target antigen for monoclonal antibody tumor localization and radioimaging. We report here on the immunochemical and biochemical characterization of GMEM. Polyacrylamide gel analysis of immunoprecipitated [3H]-leucine- and [3H]-glucosamine-labeled ECM from the human glioma cell line U-251MG has shown that GMEM is a high-molecular-weight macromolecule (Mr approximately 1,000,000) composed of Mr approximately 230,000 disulfide-bonded glycoprotein subunits. Immunoprecipitation, immunoblot, and one-dimensional peptide map analysis have shown that GMEM is distinct from human fibroblast and plasma fibronectin. These results support previous immunohistology and absorption analysis findings, indicating that GMEM is distinct from fibronectin, laminin, and glycosaminoglycans secreted by U-251MG.
Antibodies, Monoclonal↗
Releasable vascular plasminogen activator and thrombotic strokes.
Releasable vascular plasminogen activator was measured in 28 patients (14 males and 14 females) with a history of thrombotic strokes documented by computed tomographic scanning. Levels were compared with those in a control population of 126 healthy subjects with no history of thromboembolic disease. The patient population tended to have higher levels of activator than the control population, 0.53 Committee on Thrombolytic Agents (CTA) units/ml of plasma for patients versus 0.21 CTA units/ml for control subjects; however, there was a wide distribution of values, as reported in all previous populations. Since plasminogen activator levels distribute in a non-Gaussian manner, patient values and control values were stratified into deciles. By this approach, the distribution among the patient was not significantly different from that among the control subjects except in females, who demonstrated skewing to higher deciles (p = 0.019). It is concluded that thrombotic strokes are not associated with low levels of releasable vascular plasminogen activator, and in fact, these patients may present with levels considerably above the mean for normal subjects.
Adolescent↗
Trypanosoma brucei gambiense: growth in vitro of bloodstream forms inhibited by dichlorodiammineplatinum (II) compounds and hypolipidemic drugs.
Infectious bloodstream forms of Trypanosoma brucei gambiense were grown in microcultures of murine bone marrow cells in 96-well tissue culture plates. Limiting dilution studies showed that fewer than 10 cultured trypanosomes developed into populations of about 5 X 10(4) parasites per well in a week. Bloodstream parasites were reisolated with high efficiency from mice infected with cultured parasites; fewer than 10 bloodstream parasites successfully established a trypanosome population in a microculture. Both the cis and trans isomers of dichlorodiammineplatinum (II) (cisplatin and transplatin) and a hypolipidemic agent, Wy 14643, were found to have activity against T. b. gambiense growing in microcultures. The minimum concentration of drug necessary to completely eliminate parasites from microcultures was 4 microM for cisplatin, 40 microM for transplatin, and 500 microM for Wy 14643. A preformed complex of cisplatin and bovine serum albumin and another hypolipidemic agent, chlofibric acid, were inactive. This culture system should be useful for rapid screening of large numbers of compounds for trypanocidal activity.
Animals↗
Model of alpha 2-macroglobulin structure and function.
A model of alpha 2-macroglobulin is presented that is compatible with previous structural, functional, and phylogenetic studies of the protein. The model of the molecule resembles a hollow cylinder and is comprised of two identical functional halves with three C2 axes of symmetry and no mirror planes. The "trap mechanism" of this proteinase inhibitor is effected by slight movement of two trap arms per "half-molecule." Evidence for this model is obtained from the study of the structure and proteinase binding of the molecule. By using this model, predictions are made concerning proteinase binding ratios, receptor recognition, and "slow-to-fast" conformational change of the molecule.
Animals↗
A nonantigenic covalent streptokinase-polyethylene glycol complex with plasminogen activator function.
A series of new, covalent polyethylene glycol (PEG)-streptokinase adducts were synthesized and characterized. PEGs with average molecular weights of 2,000, 4,000, and 5,000 were activated with carbonyldiimidazole and coupled to the protein under standardized reaction conditions. Steady-state kinetic analysis demonstrated comparable Km values for the activation of plasminogen by streptokinase, PEG-2-streptokinase, and PEG-4-streptokinase. The kcat values were somewhat decreased when PEG-2 or PEG-4 was coupled to the streptokinase. Activation by the PEG-5 adduct did not follow Michaelis-Menten kinetics under the conditions employed in this study. Plasmin activity obtained by incubating streptokinase derivatives with plasminogen also was studied as a function of time with each of the PEG-streptokinase derivatives. By this assay, incubations containing PEG-5-streptokinase and unmodified streptokinase demonstrated comparable activity while reaction mixtures containing PEG-2-streptokinase and PEG-4-streptokinase were slightly more active. Streptokinase incubated with plasminogen at a 1:1 molar ratio was extensively degraded after 30 min whereas PEG-2-streptokinase was resistant to plasmin cleavage. The derivatized proteins were radioiodinated and incubated in plastic microtiter plates that were coated with an immunoglobulin fraction containing antibodies to streptokinase. Binding of the PEG-streptokinase adducts was decreased by greater than 95% compared with unmodified streptokinase. Plasminogen activator complexes were formed by reacting the streptokinases with human plasminogen in vitro and the clearance studied in mice. Radioiodinated plasmin in complex with the PEG-streptokinase adducts cleared at a slower rate than did plasmin complexed with unmodified streptokinase. Catabolism of the protease still occurred by a mechanism that involved reaction with alpha 2-macroglobulin as has been described for nonderivatized streptokinase-plasminogen complex (Gonias, S. L., M. Einarsson, and S. V. Pizzo, 1982, J. Clin. Invest., 70:412-423). When more extensive derivatization procedures were utilized, PEG-2-streptokinase preparations were obtained that further prolonged the clearance of complexed 125I-plasmin; however, these adducts did not uniformly retain comparable activity. It is suggested that PEG-streptokinase complexes with greatly reduced antigenicity may be useful in the treatment of thrombotic disorders.
Animals↗
In vitro preparation of nonenzymatically glucosylated human transferrin, alpha 2-macroglobulin, and fibrinogen with preservation of function.
Human transferrin, alpha 2-macroglobulin, and fibrinogen were incubated with [3H]-glucose. After a 7-day, 37 degrees C incubation at 20 mM glucose, transferrin incorporated 1.1 mol of glucose/mol protein; alpha 2-macroglobulin, 10 mol of glucose/mol; and fibrinogen, 3.8 of glucose/mol, or approximately 14 mumol of glucose/g for each protein. These results were the same for glucose labeled in the 1 or 6 position. No radiolabel was incorporated into the proteins during incubations with glucose labeled in the 2 position. The rate and extent of iron binding were identical for both glucosylated and nonglucosylated transferrin. Glucosylated transferrin bound to Wil-2 human lymphoblast cells with a Kd = 33 nM and receptor number of 3.4 X 10(5) receptors/cell; nonglucosylated transferrin with a Kd = 31 nM and receptor number of 3.9 X 10(5) receptors/cell. Glucosylated and nonglucosylated alpha 2-macroglobulin showed the same conformational change as determined on native PAGE after reaction with trypsin, plasmin, or methylamine and had the same activity in the Ganrot assay after reaction with trypsin or plasmin. The clearance of 125I-labeled, methylamine-treated alpha 2-macroglobulin from the mouse circulation was identical for both glucosylated and nonglucosylated alpha 2-macroglobulin, t1/2 = 3 min. alpha 2-Macroglobulin that was first glucosylated then reacted with methylamine bound to mouse peritoneal macrophages with a Kd of 2.5 nM and receptor activity of 370 fmol/mg cell protein. alpha 2-Macroglobulin that was first reacted with methylamine and then glucosylated bound with a Kd of 3 nM and receptor activity of 320 fmol/mg cell protein. Glucosylated fibrinogen had the same clotting time as control fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Catabolism of human tissue plasminogen activator in mice.
The catabolism of human tissue plasminogen activator (t-PA) was studied in mice. The clearance of t-PA labeled with iodine 125 was rapid (t1/2). The clearance of phenylmethylsulfonyl-125I-t-PA, which is active site-inhibited, was identical to the active enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the vast majority of 125I-t-PA injected into the circulation was present as free enzyme and not in a complex with inhibitors. The clearance of 125I-t-PA was unaltered by large molar excesses of several ligands of known clearance specificities, including macroalbumin, asialoorosomucoid, and diisopropylphosphorylthrombin and was also not altered in the presence of a 1,000-fold molar excess of unlabeled t-PA. Organ distribution studies demonstrated that the early rapid clearance of 125I-t-PA occurred in hepatocytes, followed by a later renal phase of clearance. The clearance of 125I-urokinase (UK) also was studied and was very similar in all aspects to the clearance of 125I-t-PA. These results suggest that both t-PA and UK are cleared from the circulation by unique nonsaturable processes localized in the liver that are independent of the proteinase active site.
Albumins↗