Rabbit Plasminogen Research Articles

The mechanism of activation of rabbit plasminogen by urokinase.

The data presented in this paper show that when rabbit plasminogen is activated to plasmin by urokinase at least two peptide bonds are cleaved in the process. Urokinase first cleaves an internal peptide bond in plasminogen, leading to two-chain disulfide-linked plasmin molecule. The plasmin heavy chain of molecular weight 66,000 to 69,000 possesses an NH2-terminal amino acid sequence identical with the original plasminogen (molecular weight 88,000 to 92,000). The plasmin light chain of molecular weight 24,000 to 26,000 is known to be derived from the COOH-terminal portion of plasminogen. The plasmin generated during the activation of plasminogen is capable, by a feedback process, of cleaving a peptide of molecular weight 6,000 to 8,000 from the NH2 terminus of the heavy chain, producing a proteolytically modified heavy chain of molecular weight 58,000 to 62,000. Plasmin also can cleave this same peptide from the original plasminogen, yielding an altered plasminogen of molecular weight 82,000 to 86,000. This plasmin-altered plasminogen and the plasmin heavy chain derived from it by urokinase activation process NH2-terminal amino acid sequences which are identical with each other and with the plasminolytic product of the original plasmin heavy chain. These studies support a mechanism of activation of plasminogen by urokinase which involves loss of a peptide located on the NH2 terminus of plasminogen. However, these same results show that this NH2-terminal peptide need not be released from rabbit plasminogen prior to the cleavage of the internal peptide bond which leads to the two-chain plasmin molecule. Furthermore, these studies show that urokinase cannot remove this peptide from either the original rabbit plasminogen molecule or from the heavy chain of the initial plasmin formed.

The effect of heparin on the affinity chromatography of plasminogen Demonstration of heparin-plasminogen interaction

Evidence is presented that heparin binds rabbit plasminogen types I and II under affinity chromatographic conditions using the single stage technique earlier described (Hatton, M. W. C. and Regoeczi, E. (1974) Biochim. Biophys. Acta 359, 55–65). Thus, the affinity of types I and II for Sepharose-lysine is markedly increased in the presence of heparin and elution by ε-aminohexanoic acid requires a steeper gradient to recover the plasminogen types. Furthermore by adding sufficient ε-aminohexanoic acid to non-heparinised plasma to suppress plasminogen affinity, the presence of heparin is shown to encourage binding of plasminogen (type II more so than type I) to the gel. However, the heparin effect is quickly reversed by washing the column with 0.5 M NaCl prior to elution by ε-aminohexanoic acid. No evidence of a stable plasminogen-heparin complex has been found from gel filtration studies and any interaction between plasminogen and heparin probably only takes place when heparin is bound to an affinity site. Studies with 35S-labelled heparin have shown the mucopolysaccharide to bind to the free amino group of Sepharose-lysine and Sepharose-cadaverine and to be displaced by 0.5 M NaCl elution but not by 0.1 M ε-aminohexanoic acid. The plasminogen types produced from heparinised plasma are free from heparin and closely resemble preparations from non-heparinised plasma when compared by polyacrylamide gel electrophoresis, Sephadex gel filtration and arginine esterase activity after urokinase activation.

The relevance of the structure of lysine bound to Sepharose for the affinity of rabbit plasminogen

The features of the structure of lysine, linked to Sepharose by the α-amino group, which are important for affinity chromatography of rabbit plasminogen were studied. Nine lysine and lysine-like conjugates, including ε-aminohexanoic acid, dl-norleucine, dl-α-aminoadipic acid, dl-α-ε-diaminopimelic acid, cadaverine, lornithine, l-arginine and d-lysine, were prepared. Using labelled rabbit plasminogen added to plasma, the ability of each conjugate to absorb plasminogen and separate the allomeric forms, type I and type II, during ε-aminohexanoic acid gradient elution was compared to Sepharose- l-lysine. Plasminogen had no affinity for Sepharose-ε-aminohexanoic acid, and was only weakly attracted by Sepharose-norleucine, Sepharose-cadaverine and others. Sepharose-ornithine held a greater attraction to the protein but the strongest binding was obtained with Sepharose-arginine. The affinity of plasminogen type I was always less than type II for the Sepharose-lysine analogues and the recovery of type II greater than type I from Sepharose-ornithine and Sepharose-arginine. Plasminogen affinity was in the order of Sepharose-arginine > Sepharose-lysine > Sepharose-ornithine. However, because of the present difficulty in recovering plasminogen from Sepharose-arginine the use of Sepharoselysine in the affinity chromatography of rabbit plasminogen remains unchallenged. It is concluded that binding of rabbit plasminogen to conjugates of lysine and its analogues is determined by the presence of both a free carboxyl and a free amino group and that the distance between these groups is critical.

The Role of Sialic Acid in the Determination of Distinct Properties of the Isozymes of Rabbit Plasminogen

Abstract Previous studies from our laboratory have resulted in the separation of two major forms of plasminogen from rabbit plasma. These two forms differ in their affinity characteristics for antifibrinolytic amino acids, metabolic survival times in the circulation, charge characteristics of the subforms resolved from each major form, and sialic acid content. In order to investigate the role of sialic acid in determining these distinct properties of the plasminogen forms, we have studied these same properties of the asialo rabbit plasminogen forms. We find that the charge differences between the two forms are essentially abolished upon removal of the sialic acid. The number of subforms resolved from each major form is decreased as an effect of removal of the sialic acid, but the remaining subforms possess greatly increased isoelectric points. Contrarily, the binding of each plasminogen to antifibrinolytic amino acids appears no different from normal. The metabolic survival times of the asialoplasminogen forms are not greatly different from each other but are significantly lower than normal, a phenomena also found for many other circulating proteins. These studies suggest that the charge differences between the two forms of rabbit plasminogen are incidental to their functional differences.

The mechanism of activation of rabbit plasminogen by urokinase. Lack of a preactivation peptide

We have obtained direct evidence which we interpret to prove that an amino terminal peptide need not be released from rabbit plasminogen prior to its conversion to plasmin by urokinase. The single chain plasminogen molecule possesses an amino terminal amino acid sequence of NH 2-glu-pro-leu-asp-asp. When this plasminogen is activated to plasmin by urokinase in the presence of the Kunitz bovine trypsin-plasmin-kallikrein inhibitor (BTI), a two chain disulfide linked molecule of plasmin is obtained. The heavy chain of this plasmin is directly derived from the original amino terminus of plasminogen since it possesses the identical amino terminal sequence as does native plasminogen. When the same plasminogen activation is carried out in the absence of BTI, the heavy chain of the plasmin obtained has a molecular weight of 6,000–8,000 less than the heavy chain of the plasmin obtained in the presence of this inhibitor. In addition, the heavy chain of this latter plasmin has an amino terminal sequence which differs from the original native plasminogen. These data show, in agreement with others, that the activation of plasminogen by urokinase is accompanied by the loss of an amino terminal peptide from plasminogen but also show, in contrast to the human plasminogen system, that cleavage of the internal peptide bond, leading to plasmin formation, can occur without cleavage of the amino terminal peptide.

The Interaction of Streptokinase with Human, Cat, Dog, and Rabbit Plasminogens: THE FRAGMENTATION OF STREPTOKINASE IN THE EQUIMOLAR PLASMINOGEN-STREPTOKINASE COMPLEXES

Abstract Highly purified human (Lys-forms), cat, dog, and rabbit plasminogens showed significantly different sensitivities to activation by highly purified streptokinase. The most sensitive plasminogen is the human zymogen, followed by the cat, dog, and rabbit zymogens, respectively. These human, cat, dog, and rabbit zymogens reacted with streptokinase to form homogeneous equimolar plasminogen-streptokinase complexes. The electrophoretic mobilities of the human and cat complexes on cellulose acetate were different from their respective zymogens, but the electrophoretic mobilities of the dog and rabbit complexes were similar to their respective zymogens. The human, cat, and dog complexes showed different mobilities from their respective zymogens in acrylamide gel electrophoretic systems and appeared to show multiple electrophoretic forms. With these species, all of the multiple isoelectric forms of the zymogens reacted with streptokinase. Analyses of the human (Glu- and Lys-forms), cat, dog, and rabbit equimolar plasminogen-streptokinase complexes, incubated for various time intervals, in an acrylamide gel-dodecyl sulfate-urea electrophoretic system, showed the gradual conversion, or transformation, of the plasminogen-streptokinase complexes into plasmin-streptokinase complexes. These transformations occurred at different rates in each of the mammalian plasminogen-streptokinase complexes. This acrylamide gel system also permitted a comparison between the plasmin-derived carboxymethyl heavy (A) and carboxymethyl light (B) chains produced from these mammalian zymogens by two different methods of activation, namely with equal molar ratios of streptokinase to zymogen and with low molar ratios of urokinase to zymogen (1:1000). The carboxymethyl heavy (A* and A') chains produced by streptokinase activation of the human (Glu-forms), cat, and rabbit plasminogens in the complex appeared to have higher molecular weights than the major carboxymethyl heavy (A and A1) chains derived from each of these zymogens by urokinase activation; the human (Lys-forms) and dog carboxymethyl heavy (A and A') chains appeared to be similar in both activation systems. The carboxymethyl heavy (A* and A') chains of the human (Glu- and Lys-forms), cat, dog, and rabbit plasmins found in the complexes were similar but not identical with each other in molecular weight. The plasmin-derived carboxymethyl light (B') chains produced by streptokinase activation of the human (Glu- and Lys-forms), cat, dog, and rabbit plasminogens in the complex appeared to have molecular weights similar to the carboxymethyl light (B) chains derived from each of these plasminogens by urokinase activation. But, the plasmin-derived carboxymethyl light (B') chains of the four species obtained from the equimolar complexes appeared to have somewhat different molecular weights. Streptokinase fragmentation within the complexes occurs within a few seconds after the complexes are formed, and intact streptokinase disappears after several minutes of incubation. Four major streptokinase fragments (SK1, SK2, SK3, and SK4), having molecular weights between 47,600 and 25,700, are produced in each of the equimolar human (Glu- and Lys-forms), cat, dog, and rabbit complexes. They appear to be the same fragments in each of the mammalian plasminogen-streptokinase and plasmin-streptokinase complexes; each of the complexes contains varying amounts of streptokinase fragments, SK1, SK2, SK3, and SK4. But, the rate of degradation of streptokinase into these major fragments differed with each species. Plasmin and streptokinase moieties prepared by dissociation of an equimolar plasminogen (Lys-forms)-streptokinase complex at pH 3.0 (Summaria, L., Robbins, K. C., and Barlow, G. H. (1971) J. Biol. Chem. 246, 2136–2142) were also analyzed in the acrylamide gel-dodecyl sulfateurea electrophoretic system. The isolated plasmin moiety gave carboxymethyl heavy and carboxymethyl light chains, both of which were of lower molecular weight than the plasmin-derived carboxymethyl heavy (A) and carboxymethyl light (B) chains produced by urokinase activation. The isolated streptokinase moiety contains approximately equal amounts of streptokinase fragments SK2 and SK4, and a third smaller fragment.

Some observations on the affinity chromatography of rabbit plasminogen

The feasibility of isolating plasminogen allomers from plasma by affinity chromatography in a single stage and the specificity of Sepharose-lysine for plasminogen are examined. It is shown that the method of Castellino can readily be adapted by relatively minor modifications as a one-stage procedure without endangering the quality of the product. The optimal degree of lysine conjugation with Sepharose is defined for this process. Experiments with labelled allomers are described showing the competitive nature of the capture of plasminogen by Sepharose-lysine. The affinity behaviour of plasmin and of complexes of plasmin with serum protease inhibitors are compared to plasminogen. Several other plasma proteins which also exhibit affinity to lysyl groups are identified. Plasminogen obtained in the present study compares well with previously reported analyses for molecular weight, N-terminal amino acid and sialic acid content, but not for neutral carbohydrates.

Metabolic Turnover Studies on the Two Major Forms of Rat and Rabbit Plasminogen

Abstract Two major forms of plasminogen can be isolated from the plasmas of several species by affinity chromatography methods. These two forms are easily isolated in high purity from a single small animal, a situation which is particularly suitable for biological investigations. This paper shows that the two forms of rat and rabbit plasminogen must exist as such in the circulating plasma, are not interconvertible, and appear in the plasma at different rates following a single injection of l-[4,5-3H]leucine. Studies on the biological turnover characteristics of each major plasminogen form under homologous and heterologous conditions demonstrate that plasminogen Form 2 from affinity chromatography columns possesses a circulatory half-life which is slightly lower than plasminogen Form 1.

Corrections - Interaction of Streptokinase and Rabbit Plasminogen

Corrections - Interaction of Streptokinase and Rabbit Plasminogen

NH2-Terminal Sequences of Mammalian Plasminogens and Plasmin S-Carboxymethyl Heavy (A) and Light (B) Chain Derivatives: A RE-EVALUATION OF THE MECHANISM OF ACTIVATION OF PLASMINOGEN

Abstract The NH2-terminal 12-residue sequences of human and rabbit plasminogens were determined to be Glu-Pro-Leu-Asp-Asp-Tyr-Val-Asn-X-Gln-Gly-Ala-. The cat plasminogen 12-residue sequence differed only in the 1st residue, in which aspartic acid replaced glutamic acid. The bovine plasminogen NH2-terminal 7-residue sequence was determined to be Asp-Leu-Leu-Asp-Asp-Tyr-Val-. The NH2-terminal residue of dog plasminogen is blocked since the protein could not be sequenced. The NH2-terminal 5-residue sequences of the isolated S-carboxymethyl heavy (A) chain derivatives of cat, dog, rabbit, and bovine plasmins (urokinase-activated) were determined and found to be homologous with each other and with the human S-carboxymethyl heavy (A) chain 5-residue sequence. These NH2-terminal sequences were different from those found in the five mammalian zymogens studied, indicating a specific cleavage at the NH2-terminal end of each zymogen during activation at apparently the same position in the peptide chain. These sequences are also homologous with the human Lys-plasminogen NH2-terminal sequence. The NH2-terminal 21-residue sequences of the isolated S-carboxymethyl light (B) chain derivatives of cat, dog, rabbit, and bovine plasmins (urokinase-activated) were determined and found to be homologous with each other and with a previously reported human S-carboxymethyl light (B) chain 20-residue sequence (Robbins, K. C., Bernabe, P., Arzadon, L., and Summaria, L. (1972) J. Biol. Chem. 247, 6757–6762). The human NH2-terminal 21-residue sequence was determined to be Val-Val-Gly-Gly-Cys-Val-Ala-His-Pro-His-Ser-Trp-Pro-Trp-Gln-Val-Val-Leu-Leu-Arg-Arg-. The 5th residue previously reported as glutamine is incorrect and is cysteine in all the sequences. This cysteine is part of the hinge disulfide bond connecting the heavy (A) and light (B) chains of human plasmin. The evolutionary changes in this portion of the molecule appear to be in the pairs of basic residues at positions 8 and 10, and 20 and 21, and in the pairs of neutral residues at positions 16 and 17. The replacement of serine at the 17th residue of bovine light (B) chain is a rather significant evolutionary change which could be related to the activator binding site in bovine plasminogen for streptokinase. The mechanism of activation of plasminogen to plasmin by urokinase involves two specific peptide bond cleavages instead of one previously reported by us for the activation of human Lys-plasminogen (Robbins, K. C., Summaria, L., Hsieh, B., and Shah, R. J. (1967) J. Biol. Chem. 242, 2333–2342). The specific cleavage of the sensitive arginyl-valine peptide bond in the COOH-terminal portion of the molecule to give the two-chain plasmin molecule and the specific cleavage of an X-arginyl, or X-lysyl, peptide bond in the NH2-terminal portion of the molecule are the two principal events which occur during activation of the zymogen. The latter event apparently results in the release of a peptide.

Amino terminal amino acid sequences and carbohydrate of the two major forms of rabbit plasminogen

Two major forms of plasminogen exist in the plasma of many animal species and are distinguished by their affinities for certain antifibrinolytic amino acids. Quantitative end group analysis demonstrated that each isolated form of rabbit plasminogen possessed a single amino terminal residue of glutamic acid. Amino acid sequence analysis indicated that at least the first twelve amino terminal amino acids were identical in the two forms. The unique amino terminal sequence obtained for each form was NH 2-glu-pro-leu-asp-asp-tyr-val-asn-thr-gln-gly-ala-. Analysis of the carbohydrate content of each major plasminogen form revealed some striking differences. The first major form of rabbit plasminogen isolated from affinity chromatography columns contained 1.5–1.7 percent neutral carbohydrate and 3.0–3.3 moles of sialic acid per mole of protein. The second major form of rabbit plasminogen isolated from affinity chromatography columns contained 0.6–0.8 percent neutral carbohydrate and 1.8–2.2 moles of sialic acid per mole of protein.

ACTIVATION OF RABBIT PLASMINOGEN BY STREPTOKINASE.

When streptokinase was injected into whole rabbits the fibrinolytic and proteolytic activity of the plasma increased and antifibrinolytic and antiproteolytic activity and fibrinogen level fell. In vitro treatment of rabbit plasma with streptokinase produced strong fibrinolytic activity, but proteolytic activity was not observed unless trace amounts of human plasma were added. However, the euglobulin fraction of rabbit plasma also became proteolytic after streptokinase had been added to the system. A trace amount of human plasma was required for complete activation of rabbit plasminogen by streptokinase. Addition of rabbit plasma to streptokinase-bovine euglobulin mixture produced a strong fibrinolytic activity but failed to produce any proteolytic activity. This indicates the presence of proactivator in rabbit plasma. Rabbit plasma and the euglobulin supernatant fraction contain an inhibitor of proteolytic activity.

Studies on specificity of streptokinase.

Summary1. Ability of SK to activate the fibrinolytic system in plasma or euglobulin fractions of various animal species has been studied. 2. Human, cat, dog and rabbit plasminogens were readily activated by SK. Pig, horse and cow plasminogens were much less readily activated. Sheep, guinea pig and goat plasminogens were not activatable by concentrations of SK employed. 3. When SK is able to induce formation of a fibrinolytic system on addition to a given species of plasma or euglobulin fraction, that particular mixture gains ability to activate plasminogen of an animal species not activatable by SK alone. 4. Mixtures of SK and a species of plasminogen in which fibrinolysis or LEEase activity does not occur are incapable of activating plasminogens refractory to SK activation. 5. Addition of plasma from all animal species studied partially inhibits the LEEase activity of a spontaneous plasmin preparation. 6. Possible implications of these findings are briefly discussed.