Enzymatic modifications of human plasma fibronectin in relation to opsonizing activity.

Plasma fibronectin is one of the largest plasma proteins (Mr approximately 440 000), comprising two approximately equal polypeptide chains which are held together by a disulfide linkage near the C-terminal end of the molecule. The binding of gelatinized latex beads to liver slices as well as the internalization of these particles by macrophages, in the presence of heparin, is greatly enhanced by fibronectin. The question as to whether the entire covalent structure of fibronectin was necessary for opsonizing activity was approached by limited proteolytic degradations of the molecule. Patterns of controlled digestion with trypsin, cathepsin D, Staphylococcus aureus protease, and plasmin all indicate that the minimal unit necessary for retention of opsonic activity is some large (Mr 200 000 and 190 000) single-chain entity. Treatment with plasmin proved to be the most reliable procedure for generating the active split product which could be readily separated from the inactive, disulfide-containing C-terminal fragment. Incorporation of dansylcadaverine into plasma fibronectin (3.5 mol/mol of protein) by fibronoligase (coagulation factor XIIIa) did not affect the opsonic activity of the protein.

Coagulation testing with the Du Pont aca discrete clinical analyzer.

Methods for the measurement of the functional concentration of AT III, PLG, and FBG have been developed for the Du Pont aca discrete clinical analyzer. These methods are fully automated, provide excellent reproducibility, and compare favorably with existing methodologies. The availability of these and future coagulation assays on this automated analyzer will provide the hospital laboratory with a reliable means for evaluating hemostatic function. In addition, excellent method reproducibility and use of defined reference standards should significantly contribute to the standardization of coagulation methods.

New colored and fluorescent amine substrates for activated fibrin stabilizing factor (Factor XIIIa) and for transglutaminase.

Two fluorescent (FITC and 6-chloro-2-methoxyacridine) and an intensely colored (dabsyl) derivative of cadaverine were synthesized, following earlier work from this laboratory with dansyl-cadaverine, in order to enlarge the scope of possibilities for labeling some gamma-glutamine sites in proteins. Enzyme affinities of the amine substrates for human Factor XIIIa and for guinea pig liver transglutaminase were measured. The utility of dabsylcadaverine was further demonstrated by activity staining of these enzymes, following electrophoresis in agarose, and by measuring the Factor XIII zymogen of human plasma colorimetrically.

Development and analytical performance of automated tests for antithrombin III and plasminogen on the Du Pont aca analyzer.

We describe assays for functional antithrombin III (AT III) and plasminogen in plasma with the Du Pont aca discrete clinical analyzer. Both are two-stage kinetic assays, based on synthetic substrate methodologies, and require 20-microL sample volumes. In the AT III assay the sample is incubated with excess thrombin and heparin to form the functionally inactive AT III-thrombin complex. Residual thrombin is measured through its rate of hydrolysis of a lysine thioester and is inversely related to analyte concentration. In the plasminogen assay excess streptokinase is reacted with the sample to form an enzymatically active complex. The substrate hydrolysis rate of this complex is measured, which is linearly related to the concentration of plasminogen in the sample. Reaction conditions for both assays were optimized by univariate and response surface techniques. The assay for AT III has a range of 0 to 150% of the value for normal human plasma (% NHP) with a CV of 3% at 80% NHP. The plasminogen assay is linear from 25 to 200% NHP with a CV of less than 2% at 80% NHP. No significant interferences with either method by common blood components or drugs were found.

Degradation of transmembrane proteins in Ca2+-enriched human erythrocytes. An immunochemical study.

Apart from causing the formation of gamma-glutamyl-epsilon-lysine cross-linked polymers, exposure of human erythrocytes to Ca2+ and ionophore A23187 leads to a breakdown of the two major transmembrane proteins, i.e. the anion-transporting band 3 and glycophorin. This apparently proteolytic phenomenon was examined by crossed immunoelectrophoretic techniques. The main product of the cleavage of band 3 had a chain weight of about 55,000 and showed good precipitation with the antibody raised against the intact protein. The degradation of glycophorin was more rapid and, when complete, gave rise to small fragments which were barely precipitated with antiglycophorin antibody. Incubation of the cells with pepstatin or N-ethylmaleimide prior to and during Ca2+ loading prevented the breakdown of both transmembrane proteins. Histamine, a competitive inhibitor of the transglutaminase-catalyzed formation of gamma-glutamyl-epsilon-lysine cross-links in Ca2+-enriched erythrocytes, also provided some protection, suggesting that the breakdown of the two transmembrane proteins might in some manner be related to the transglutaminase-dependent polymerization process. Pathophysiological implications of the proteolytic event, which would distort the normal interaction of membrane proteins with the cytoskeleton, are discussed.

Development and analytical performance of a functional assay for fibrinogen on the Du Pont aca analyzer.

A method for determination of functional fibrinogen has been developed for the Du Pont aca discrete clinical analyzer. This fully automated assay is based on the rate of fibrin turbidity formation when thrombin is added to the test sample. The rate of this reaction is enhanced by added calcium chloride and dextran. The assay range is from 0.50 to 8.00 g/L. Within-run reproducibility studies at the medical decision level of 1.0 g/L showed a CV of 2.5%. There was no interference by heparin up to 10 USP units per milliliter of plasma.

Lens transglutaminase and cataract formation.

A protein polymer characteristically present in human cataract was shown to contain significant amounts of gamma-glutamyl-epsilon-lysine isopeptides. It is proposed that these crosslinks are produced by the action of transglutaminase (R-glutaminyl-peptide:amine-gamma-glutamyl-yltransferase, EC 2.3.2.13), which is all the more plausible because lens contains the enzyme and endogenous protein substrates for it. The enzyme is similar to that obtained from liver and is Ca2+ dependent. Highest apparent activity is found in lens cortex. When cortex homogenate from the rabbit was incubated in the presence of Ca2+ with either [14C]putrescine or with dansylcadaverine, a a selective incorporation of the radioactive or fluorescent amine into the heavier subunits (Mr approximately 26,000 and 30,000) of beta-crystallins could be demonstrated. Possible modes of regulating the crosslinking activity of this enzyme in lens are discussed.

Effects of the calcium-mediated enzymatic cross-linking of membrane proteins on cellular deformability.

Excess calcium binding affects the shape and dynamics of cellular deformation of human erythrocytes. It may be hypothesized that incorporation of calcium may modify cellular deformability by processes which include specific cross-linking of membrane proteins with resultant changes in cell shape and deformability. Since previous studies indicate that accumulation of calcium ions causes development of gamma-glutamyl-epsilon-lysine bridges in membrane proteins, under control of a membrane transamidating enzyme which specifically requires calcium ions for activation, experiments were devised to examine the relationship between cross-linking and deformability and to determine the effects of specific inhibitor of membrane protein cross-linking on the calcium-dependent modification of erythrocyte to the echinocytic shape. The elastic shear modulus of the membrane was not significantly affected by calcium-induced cross-linking, indicating that induced shape change, not altered elasticity, causes the observed reduction in cellular deformability. These findings support the interpretation that Ca++-induced and transamidase-catalyzed cross-linking of membrane proteins contributes to fixation of altered cellular shape and decreased cellular deformability.

Fibronectin-mediated uptake of gelatin-coated latex particles by peritoneal macrophages.

The present study demonstrates the ability of plasma fibronectin or cold-insoluble globulin (Clg) to promote the uptake of 125I-labeled, gelatin-coated latex beads (g-Ltx*) by monolayers of peritoneal macrophages (PM). The uptake of g-Ltx* by PM was enhanced by Clg in a concentration-dependent fashion and required the presence of heparin (10 U/ml) as an obligatory cofactor for maximal particle uptake. Treatment of PM monolayers with trypsin (1 mg/ml) for 15 min at 37 degrees C after particle uptake removed less than 15% of the radioactivity incorporated by the monolayers. However, a similar trypsin treatment of the monolayers before the addition of latex particles depressed Clg-dependent uptake by greater than 75%. Pretreatment of PM monolayers with inhibitors of glycolysis effectively reduced the Clg-dependent uptake of latex. Similarly, pretreatment of monolayers with either inhibitors of protein synthesis or agents that disrupt cytoskeletal elements also significantly depressed Clg-dependent particle uptake. Phagocytosis of g-Ltx* by PM in the presence of Clg and heparin was confirmed by electron microscopy. Finally, g-Ltx* could also be effectively opsonized with Clg at 37 degrees C before their addition to the monolayers. These studies suggest that the recognition of g-Ltx* in the presence of Clg required cell surface protein(s) and that subsequent phagocytosis of these particles by PM was energy dependent and required intact intracellular cytoskeleton elements. Thus, PM monolayers provide a suitable system for further studies on the function of Clg in the recognition and phagocytosis of gelatin-coated particles by phagocytic cells.

Enzymatic basis for the Ca2+-induced cross-linking of membrane proteins in intact human erythrocytes.

The accumulation of Ca2+ ions in intact human erythrocytes leads to the production of membrane protein polymers larger than spectrin. The polymer has a heterogeneous size distribution and is rich in gamma-glutamyl-epsilon-lysine cross-links. Isolation of this isodipeptide, in amounts as high as 6 mol/10(5) g of protein, confirms the idea [Lorand L., Weissmann, L.B., Epel, D.L., and Bruner-Lorand, J. (1976), Proc. Natl. Acad. Sci. U.S.A. 73, 4479] that the Ca2+-induced membrane protein polymerization is mediated by transglutaminase. Formation of the polymer in the intact cells is inhibited by the addition of small, water-soluble primary amines. Inasmuch as these amines are known to prevent the Ca2+-dependent loss of deformability of the membrane, it is suggested that transglutaminase-catalyzed cross-linking may be a biochemical cause of irreversible membrane stiffening.

Mechanism of activation of human plasminogen by the activator complex, streptokinase-plasmin.

The object of this investigation was to distinguish between two potential mechanisms of activation of human plasminogen (HPg) to plasmin (HPm) by catalytic levels of the activator complex, streptokinase.plasmin (SK.HPm). One mechanism, which is widely supported, postulates an enzymatic role for SK.HPm in the conversion of molar excesses of plasminogen to plasmin. A more recently described kinetic mechanism involves a direct conversion of HPg to HPm by streptokinase (SK). Here, it is believed that displacement of HPm from SK.HPm by excess HPg is the major source of free HPm in the activation process. The present paper shows that SK is not capable of undergoing rapid exchange from SK.HPm to other HPg or HPm molecules, thus precluding the possibility of direct activation of HPg by SK. Our evidence supports a mechanism involving an enzymatic role for SK.HPm as the major means of converting free HPg to HPm.

Formation of gamma-glutamyl-epsilon-lysine bridges between membrane proteins by a Ca2+-regulated enzyme in intact erythrocytes.

A rise in the intracellular concentration of Ca2+-ions in human erythrocytes causes the formation of high-molecular-weight membrane protein polymers, cross-linked by gamma-glutamyl-epsilon-lysine side chain bridges. Cross-linking involves proteins at the cytoplasmic side of the membrane (band 4.1, spectrin, and band 3 materials) and the reaction is catalyzed by the intrinsic transglutaminase. This enzyme is regulated by Ca2+-ions and it exits in a latent form in normal cells. The protein polymer, isolated from the membranes of Ca2+-loaded intact human red cells, is heterogeneous in size and may contain as many as 6 moles of gamma-glutamyl-epsilon-lysine cross-links per 100,000 gm of protein. Synthetic compounds, which either compete against the epsilon-lysine cross-linking functionalities of the protein substrates (eg, histamine, aminoacetonitrile, cystamine) or directly inactivate the transamidase (eg, cystamine), inhibit the membrane polymerization reaction in intact human erythrocytes. They also interfere with the Ca2+-induced irreversible shape change from discocyte to echinocyte and inhibit the irreversible loss of membrane deformability. Thus, the transamidase-catalyzed production of gamma-glutamyl-epsilon-lysine cross-links in the membrane may be a common denominator in these cellular manifestations.

Interaction of streptokinase with plasminogen. Isolation and characterization of a streptokinase degradation product.

When streptokinase is incubated with human or rabbit plasminogen, one event which occurs is a specific fragmentation of streptokinase. At least five major identifiable streptokinase fragments appear with time, and they possess molecular weights of approximately 40,000 (SK 1), 36,000 (SK 2), 31,000 (SK 3), 26,000 (SK 4), and 10,000 (SK 5) under denaturing conditions, as observed on calibrated sodium dodecyl sulfate-polyacrylamide gels, compared to native streptokinase of molecular weight 45,000. The amount of each of the fragments generated at given times of incubation of plasminogen and streptokinase depends upon the species of plasminogen employed. Utilizing rabbit plasminogen and streptokinase, the SK 4 fragment was purified. This fragment arises by proteolysis at both the NH2 and COOH regions of native streptokinase. However, when isolated utilizing dilute aqueous buffers, the SK 4 fragment contained a portion of the original NH2 terminus of native streptokinase noncovalently bound to the molecule (SK 4'). SK 4' is capable of activating human plasminogen to plasmin, albeit more slowly than native streptokinase. However, the SK 4'-human plasmin complex possess only very weak plasminogen-activating activity toward sheep plasminogen. Upon removal of the noncovalently bound small NH2-terminal peptide of native streptokinase from SK 4', SK 4 is formed. This particular fragment possesses practically no human plasminogen-activating activity and cannot be used as an activator of sheep plasminogen, even with added human plasminogen.

De novo biosynthesis of plasminogen in the anephric rat.

Two major forms of plasminogen exist in the plasma of many different species. Each form is present in normal amounts in anephric patients and each form is synthesized de novo in bilaterally nephrectomized rats. Although the rate of synthesis of each form of plasminogen is lower than normal immediately after nephrectomy, the rate of plasma protein synthesis is also apparently lower than normal in these cases. This is thought to reflect the fact that the nephrectomized rat is in a state of circulatory distress immediately after kidney ligation. It is concluded that the kidney is not responsible for de novo biosynthesis of plasminogen. This point has been the subject of some confusion.