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PubMed2006

Cell-surface actin binds plasminogen and modulates neurotransmitter release from catecholaminergic cells.

An emerging area of research has documented a novel role for the plasminogen activation system in the regulation of neurotransmitter release. Prohormones, secreted by cells within the sympathoadrenal system, are processed by plasmin to bioactive peptides that feed back to inhibit secretagogue-stimulated release. Catecholaminergic cells of the sympathoadrenal system are prototypic prohormone-secreting cells. Processing of prohormones by plasmin is enhanced in the presence of catecholaminergic cells, and the enhancement requires binding of plasmin(ogen) to cellular receptors. Consequently, modulation of the local cellular fibrinolytic system of catecholaminergic cells results in substantial changes in catecholamine release. However, mechanisms for enhancing prohormone processing and cell-surface molecules mediating the enhancement on catecholaminergic cells have not been investigated. Here we show that plasminogen activation was enhanced >6.5-fold on catecholaminergic cells. Carboxypeptidase B treatment decreased cell-dependent plasminogen activation by approximately 90%, suggesting that the binding of plasminogen to proteins exposing C-terminal lysines on the cell surface is required to promote plasminogen activation. We identified catecholaminergic plasminogen receptors required for enhancing plasminogen activation, using a novel strategy combining targeted specific proteolysis using carboxypeptidase B with a proteomics approach using two-dimensional gel electrophoresis, radioligand blotting, and tandem mass spectrometry. Two major plasminogen-binding proteins that exposed C-terminal lysines on the cell surface contained amino acid sequences corresponding to beta/gamma-actin. An anti-actin monoclonal antibody inhibited cell-dependent plasminogen activation and also enhanced nicotine-dependent catecholamine release. Our results suggest that cell-surface-expressed forms of actin bind plasminogen, thereby promoting plasminogen activation and increased prohormone processing leading to inhibition of neurotransmitter release.

Actins↗
PubMed1988

Complementary modes of action of tissue-type plasminogen activator and pro-urokinase by which their synergistic effect on clot lysis may be explained.

Tissue plasminogen activator (t-PA) and/or pro-urokinase (pro-UK) induced lysis of standard 125I-fibrin clots suspended in plasma was studied. Doses were kept below the concentration at which a nonspecific effect was seen, i.e., where fibrinogenolysis and major plasminogen consumption were observed. Small amounts of t-PA potentiated clot lysis by pro-UK by attenuating the lag phase characteristic of pro-UK, and causing a much earlier transition to the rapid phase of lysis. Similar promotion of the fibrinolytic effect of pro-UK was obtained when clots were pretreated with UK or with a little plasmin (less than 1% clot lysis). Promotion by plasmin was nullified by a subsequent treatment of the clot with carboxypeptidase B, indicating that the plasmin effect was related to the exposure of carboxy terminal lysine residues on fibrin. These lysine termini, absent in undegraded fibrin, are known to be essential for the high affinity binding of plasminogen to fibrin. In contrast, clot lysis by t-PA was unaffected by plasmin pretreatment and little affected by carboxypeptidase B treatment of the fibrin substrate. Therefore, plasminogen bound to lysine termini on fibrin, although found to be essential for pro-UK, did not appear to serve as a substrate for t-PA. Selective activation of fibrin bound plasminogen has been attributed to the conformational change in Glu-plasminogen that occurs as a result of binding. The present findings suggest that this conformational change occurs when plasminogen is bound to a terminal lysine but not to an internal lysine. Plasminogen bound to the latter site on fibrin was activated by t-PA and therefore is involved in the ternary complex. This initiates lysis of the undegraded clot and exposes the plasminogen binding sites required by pro-UK. By their complementary activation of fibrin bound plasminogen, t-PA followed by pro-UK induces efficient and synergistic fibrinolysis, whereas each is relatively inefficient when used alone.

Carboxypeptidase B↗
PubMed1992

Effects of tiaprofenic acid on plasminogen activators and inhibitors in human OA and RA synovium.

The effect of therapeutic and pharmacological concentrations of tiaprofenic acid, a non-steroidal anti-inflammatory drug (NSAID), on the synthesis of the plasminogen activators, urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA), and the plasminogen activator inhibitors 1 and 2 (PAI-1 and PAI-2), by human synovial membranes isolated from osteoarthritis (OA) and rheumatoid arthritis (RA) sufferers was evaluated. Both forms of plasminogen activator (PA) and PA inhibitor (PAI) were synthesized by the arthritic synovium. PAI-1 and PAI-2 were both synthesized in greater amounts than the plasminogen activators. Tiaprofenic acid induced a dose-dependent decrease in uPA synthesis in both OA and RA, particularly in OA synovium, but had no true effect on tPA. Tiaprofenic acid also exerted a suppressive effect on the synthesis of PAI-1 in both OA and RA synovial membranes, and on the release of PAI-2 in RA synovium. The results of this study indicate that a decrease in uPA synthesis may be one of the mechanisms by which tiaprofenic acid could exert its effects on the arthritic process. The suppressive action of tiaprofenic acid on PAI is not likely to have a significant impact on the balance of plasminogen activators and plasminogen activator inhibitors, as plasminogen activator inhibitors are synthesized in greater amounts than plasminogen activators.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1996

Plasminogen activator inhibitor-1 overexpression in nonproliferative diabetic retinopathy.

Plasminogen activator inhibitor-1 is secreted bidirectionally by endothelial cells, acts as the primary regulator of fibrinolysis and as a key modulator of extracellular matrix proteolysis. Elevated serum levels of plasminogen activator inhibitor-1 are observed in serum of diabetic individuals. We investigated whether plasminogen activator inhibitor-1 is overexpressed in capillaries of diabetic donors with non-proliferative retinopathy compared to non-diabetic donors. We also assessed plasminogen activator inhibitor-1 expression in an animal model of retinopathy induced by exposing rabbit retinas to insulin-like growth factor-I. Colloidal gold immunocytochemistry was used to quantify plasminogen activator-1 antigen in donor retinas from diabetic subjects (n = 10) and control subjects (n = 10). This technique was also used to examine expression of plasminogen activator inhibitor-1 for correlation with retinal changes in the insulin-like growth factor-I-induced retinopathy model (n = 14). Plasminogen activator inhibitor-1 immunoreactivity was significantly increased in the retinas of all diabetic subjects as compared to controls. In the rabbit model, the expression of plasminogen activator inhibitor-1 immunoreactivity correlated with pathological retinal changes. In both the diabetic human and insulin-like growth factor-I-injected rabbit, overproduction of plasminogen activator inhibitor-1 was seen within the lumen of capillaries, within the cytoplasm of endothelial cells and in the basement membrane and extracellular matrix surrounding these capillaries. Minimal plasminogen activator inhibitor-1 was detected in the retinas of non-diabetics and in control rabbits injected with either heat-inactivated insulin-like growth factor-I or balanced salt solution. These studies support the conclusion that plasminogen activator inhibitor-1 is overexpressed in the retinal capillaries of diabetics with non-proliferative diabetic retinopathy and in rabbits with insulin-like growth factor-I-induced retinopathy.

Adult↗
PubMed1990

The role of carbohydrate in the function of human plasminogen: comparison of the protein obtained from molecular cloning and expression in Escherichia coli and COS cells.

A cDNA library was constructed in the phage lambda gt11 from human liver mRNA enriched for plasminogen mRNA by chromatography on Sepharose 4B. A full-length cDNA clone of human plasminogen was isolated. The 2.7 kb cDNA encoded the entire plasminogen molecule, a signal peptide sequence and two start codons with a 5'-untranslated region of about 80 base pairs. In the 3'-non coding region of 280 base pairs a consensus signal AATAAA was found at a distance of 46 base pairs upstream of the poly(A) tail. The plasminogen cDNA was subcloned in the eukaryotic expression vector p91023 (B), and human plasminogen was expressed in monkey kidney (COS m6) cells and in Escherichia coli. The recombinant molecule obtained from COS cells has physicochemical and biological properties similar to native human plasminogen I, indicating that it has folded in a manner similar to plasminogen synthesized by liver. By contrast, plasminogen expressed in E. coli could not be activated and showed biological properties which are very different from glycosylated forms of plasminogen. However, the non-glycosylated plasminogen was bound by lysine-Sepharose and reacted with a conformation dependent monoclonal antibody to kringles 1 to 3. These data suggest that the protein has properly folded kringle domains. Our studies suggest that the carbohydrate domains may play an important role in the function of the plasminogen molecule.

Animals↗
PubMed1992

On the molecular interactions between plasminogen-staphylokinase, alpha 2-antiplasmin and fibrin.

The molecular interactions between the plasminogen-staphylokinase complex, alpha 2-antiplasmin and fibrin were studied by measuring the effect of CNBr-digested fibrinogen on the inhibition rate of the plasminogen-staphylokinase complex by alpha 2-antiplasmin. The second-order rate constant for the inhibition of plasminogen-staphylokinase by alpha 2-antiplasmin was 2.7 +/- 0.3.10(6) M-1 s-1 (mean +/- S.D.; n = 7). Addition of CNBr-digested fibrinogen, but not of fibrinogen, resulted in a concentration-dependent reduction of the apparent inhibition rate constant, with a 50 percent reduction at a concentration of 5 nM CNBr-digested fibrinogen. The second-order rate constant for the inhibition of the low-Mr plasminogen-staphylokinase complex (plasminogen lacking the kringle structures comprising the lysine-binding sites) by alpha 2-antiplasmin was about 30-fold lower (9.3 +/- 0.7.10(4) M-1 s-1, mean +/- S.D.; n = 4) than that of plasminogen-staphylokinase and was not affected by addition of CNBr-digested fibrinogen. Inhibition of the plasminogen-staphylokinase complex by the chloromethylketone D-Val-Phe-Lys-Ch2Cl is 9-fold less efficient than that of plasmin (k2/Ki of 700 M-1 s-1 versus 6300 M-1 s-1). Our results confirm and establish that rapid inhibition of plasminogen-staphylokinase by alpha 2-antiplasmin requires the availability of the lysine-binding sites in the plasminogen moiety of the complex. Fibrin, but not fibrinogen, reduces the inhibition rate by alpha 2-antiplasmin by competition for interaction with the lysine-binding site. Protection of the plasminogen-staphylokinase complex bound to fibrin from rapid inhibition by alpha 2-antiplasmin thus appears to contribute to the fibrin-specificity of clot lysis with staphylokinase in a plasma milieu, by allowing preferential plasminogen activation at the fibrin surface, while the free complex is rapidly inhibited in plasma.

Amino Acid Chloromethyl Ketones↗
PubMed1995

Effect of staphylokinase concentration of plasminogen activation.

Activation of Glu- and Lys-plasminogen by various concentrations of recombinant staphylokinase (SAK) were studied by the generation of amidolytic activity from the chromogenic substrate S-2251(H-D-Val-Leu-Lys-pNA) and by SDS-PAGE analysis. Surprisingly, excess SAK decreased and fixed the rate of S-2251 hydrolysis in a mixture of Lys-plasminogen and SAK. Since the effect of SAK on S-2251 hydrolysis by plasma was similar, the hydrolysis kinetics by free plasmin and plasmin-SAK complex were studied. Hydrolysis by either enzyme form followed Michaelis-Menten kinetics with a Km of 0.38 mM for plasma and 3.74 mM for SAK-plasmin complex. The catalytic rate constant was 22.7 s-1 for plasmin and 21.0 s-1 for the SAK-plasmin complex. With excess SAK and vigorous removal of plasmin activity from plasminogen, the pre-activation lag period differed greatly between Glu- and Lys-plasminogen. Based on the different substrate specificity of plasmin and plasmin-SAK complex, we analyzed the Glu-plasminogen activation with either catalytic or excess SAK. With excess SAK, almost no Lys-plasminogen was detectable and whole Glu-plasminogen was converted directly to Glu-plasmin, then gradually to Lys-plasmin. In contrast, Lys-plaminogen appeared rapidly with catalytic amount of SAK. These results suggest that inhibition of Glu-plasminogen to Lys-plasminogen to Lys-plasminogen conversion in the plasminogen-SAK complex in the presence of excess SAK prolonged the initial lag phase of activation.

Dose-Response Relationship, Drug↗
PubMed1998

Selective plasminogen binding: cysteinyl-lysine-dextran protein interactions.

The dipeptide, L-cysteine-L-lysine, when coupled to a dextran matrix demonstrates a much higher binding of Glu-plasminogen than dextran alone. Plasminogen binds to the dipeptide coupled matrix with a high affinity and binding capacity and is retained by the surface for long periods. In comparison, other proteins such as bovine serum albumin, alpha2-antiplasmin, tPA, fibrinogen, and plasmin are not retained by the material demonstrating that the L-cysteine-L-lysine-dextran is highly selective for Glu-plasminogen. While fibrinogen alone does not bind to the surface, fibrinogen on surfaces to which plasminogen has been pre-adsorbed does demonstrate adsorption interactions. The nature of the interactions depend on the amount of plasminogen bound. Fibrinogen in contact with high concentrations of adsorbed plasminogen causes an increase in the desorption rate of the surface bound protein; if low amounts of plasminogen are initially bound, fibrinogen exposure causes an increase in total protein adsorbed by the surface. A higher amount of protein adsorbs from a mixed plasma solution. This does not appear to inhibit the binding of plasminogen, but rather may actually facilitate more plasminogen binding than is the case of plasminogen alone. The dipeptide coupled to the dextran matrix appears to be a preferred substrate for plasminogen binding compared to free lysine and epsilon-amino caproic acid.

Animals↗
PubMed1984

In vitro biosynthesis of plasminogen in a cell-free system directed by mRNA fractions isolated from monkey liver.

mRNA was isolated from total RNA of monkey liver by oligo(dT)-cellulose chromatography and was translated in a rabbit reticulocyte cell-free system. Analysis of the translation products immunoprecipitated with specific antibodies to monkey plasma plasminogen revealed a molecule with characteristics similar to those of native plasminogen. The purification of the mRNA by centrifugation on sucrose gradients indicated the presence of plasminogen mRNAs in both the 23S and 18S RNA fractions. Both plasminogen mRNAs can be further purified by chromatography on Sepharose 4B. Affinity chromatography of the proteins synthesized in vitro by total mRNA from liver, as well as by the purified mRNAs, on L-lysine-substituted Sepharose revealed that both major plasma plasminogen forms (1 and 2) are synthesized, as precursors, in the system. The in vitro synthesized plasminogen is similar in its physical and chemical properties to native plasma plasminogen as determined by its ability to bind to L-lysine-substituted Sepharose and its molecular interaction with streptokinase. The purified mRNAs were also translated in the presence of dog pancreas microsomal membranes, and and fractionated on concanavalin A-Sepharose. The 23S mRNA directed the synthesis of a plasminogen molecule similar to the circulating plasma plasminogen form 1, whereas the 18S mRNA directed the synthesis of a molecule similar to the circulating plasma plasminogen form 2. Our evidence indicates that the synthesis of the two major circulating plasma plasminogen forms is directed in the liver by separate mRNAs.

Animals↗
PubMed1985

Quantitative characterization of the binding of plasminogen to intact fibrin clots, lysine-sepharose, and fibrin cleaved by plasmin.

The binding of human Glu- and Lys-plasminogens to intact fibrin clots, to lysine-Sepharose, and to fibrin cleaved by plasmin was quantitatively characterized. On intact fibrin clots, there was one strong binding site for Glu-plasminogen with a dissociation constant, Kd, of 25 microM and one strong binding site for Lys-plasminogen with a Kd of 7.9 microM. In both cases, the number of plasminogen binding sites per fibrin monomer was 1. Also, a much weaker binding site for Glu-plasminogen was observed with a Kd of about 350 microM. Limited digestion of fibrin by plasmin created additional binding sites for plasminogen with Kd values similar to the binding of plasminogen to lysine-Sepharose. This was predictable given the observations that plasminogen binds to lysine-Sepharose and can be eluted with epsilon-aminocaproic acid [Deutsch, D.G., & Mertz, E.T. (1970) Science (Washington, D.C.) 170, 1095-1096] and that plasmin preferentially cleaves fibrin at the carboxy side of lysyl residues [Weinstein, M.J., & Doolittle, R.F. (1972) Biochim. Biophys. Acta 258, 577-590], because the structures of the lysyl moiety in lysine-Sepharose and of epsilon-aminocaproic acid are identical with the structure of a COOH-terminal lysyl residue created by plasmin cleavage of fibrin. The Kd for the binding of Glu-plasminogen to lysine-Sepharose was 43 microM and for fibrin partially cleaved by plasmin 48 microM. The Kd for the binding of Lys-plasminogen to lysine-Sepharose was 30 microM. With fibrin partially cleaved by plasmin, there were two types of binding sites for Lys-plasminogen, one with a Kd of 7.6 microM and the other with a Kd of 44 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1995

The activation-resistant conformation of recombinant human plasminogen is stabilized by basic residues in the amino-terminal hinge region.

Fully activable recombinant human plasminogen (rPlg) was expressed in mammalian cells employing either recombinant vaccinia virus or stable lines coexpressing alpha 2-plasmin inhibitor. A panel of eight variants of rPlg was constructed, in which progressively up to 6 basic amino acid residues in the hinge region of rPlg between the NH2-terminal acidic domain ("proactivation peptide") and kringle 1 were substituted by neutral residues. Analysis of the cleavage rates of these variants by plasmin revealed that the peptide bond at Arg68 is most susceptible, followed by Lys62 and Lys77. A variant with all 6 basic residues substituted was cleaved at Lys20. Three of these variants, PlgB (R68A, R70A), PlgF (R68A, R70A, K77H, K78H), and PlgG (R61A, K62A, R68A, R70A, K77H, K78H), as well as rPlg, were analyzed in more detail. The conformation of these plasminogens was analyzed by monitoring the change in intrinsic fluorescence upon binding of lysine analogs. This revealed that rPlg exhibits the native tight Glu1-plasminogen conformation, whereas PlgB, PlgF, and Plg G display an open conformation similar to Lys78-plasminogen, leading to an increased affinity for lysine analogs. This allowed a direct study of the impact of the activation-resistant conformation on the properties of Glu1-plasminogen. The open conformation of rPlg variants leads to an increased rate of activation by urokinase-type plasminogen activator and streptokinase and increased binding to a fibrin clot. Fibrin clot lysis mediated by tissue-type plasminogen activator was accelerated for the variants as a result of a lower Km for tissue-type plasminogen activator-mediated plasminogen activation, resulting from the increased affinity of rPlg (variants) for intact fibrin. We conclude that the basic residues in the extremely plasmin susceptible hinge region of plasminogen are directly involved in maintaining the activation resistant Glu1-plasminogen conformation.

Base Sequence↗
PubMed1972

The fibrinolytic pathway of human plasma. Isolation and characterization of the plasminogen proactivator.

The conversion of the plasminogen proactivator to plasminogen activator by activated Hageman factor or its fragments has been recognized as an essential step in the conversion of plasminogen to plasmin. The plasminogen proactivator has been completely separated from prekallikrein and pre-PTA, two other proenzyme substrates of activated Hageman factor or its fragments. Plasminogen proactivator, free of any contaminating proteins as assessed by disc gel electrophoresis or isoelectric focusing, revealed a single band with an isoelectric point of 8.9 corresponding in position to the Hageman factor activatable material eluted from replicate unstained gels. After conversion of plasminogen proactivator by Hageman factor fragments to the plasminogen activator, the active site of the plasminogen activator is not inhibited by C1INH and is thus readily distinguished from that of kallikrein or PTA. The plasminogen activator is susceptible to inactivation by DFP while the plasminogen proactivator is not, as has been the case for esterases having a serine in the active site. Its interaction with plasminogen is inhibited by epsilon-aminocaproic acid.

Aminocaproates↗
PubMed1990

Binding and activation of plasminogen at the surface of Staphylococcus aureus. Increase in affinity after conversion to the Lys form of the ligand.

Untreated Staphylococcus aureus cells, strain Cowan I, specifically bound 125I-Glu-plasminogen. The binding was inhibited by both unlabeled Glu-plasminogen and Glu-plasmin. The Lys form of plasminogen, which lacks the 8-kDa amino-terminal activation peptide, was approximately 100-fold more effective than the Glu form in competing with the binding of 125I-labeled Glu-plasminogen. This suggests an increase in binding affinity upon removal of the activation peptide. Fibronectin, fibrinogen and IgG, plasma components known to bind to the staphylococcal surface, did not significantly interfere with the binding. The competing activity in plasma was abolished by specifically absorbing plasminogen from the plasma sample. L-Lysine and a fragment of plasminogen containing three of the first five protein attachment domains present in the molecule (kringle structures) also competed with plasminogen for binding suggesting that the lysine-binding sites of plasminogen were involved in its interaction with staphylococci. Scatchard analysis revealed high- and low-affinity binding sites. Kd and the number of high-affinity binding sites were 1.7 nM and 780 binding sites/bacterial cell, respectively. 125I-Glu-plasminogen bound to staphylococcal surface was converted to plasmin by tissue-type plasminogen activator. The conversion took place also in the presence of plasma. If the conversion was carried out in the absence of low-molecular-mass plasmin inhibitors such as aprotinin, the bound Glu-plasmin was further converted to Lys-plasmin. The surface-bound plasmin was enzymically active, as judged by digestion of the synthetic substrate, S-2251. The plasminogen conversion shown by the present experiments not only leads to the surface-bound plasmin but seems to considerably increase the affinity of plasmin for its binding site. This may represent a physiologically relevant method for a bacterial cell to retain surface-bound active plasmin which is also protected from its soluble plasma inhibitors. This novel mechanism for staphylococci to adopt surface-bound proteolytic activity, without the interference of plasma components, may have some role in the tissue penetration and invasion of microbes during infection.

Aprotinin↗
PubMed2006

Induction of catalytic activity of plasminogen by monoclonal antibody IV-Ic in the presence of divalent metal cations and alpha2-antiplasmin.

Investigation of the influence of divalent metal cations on the induction of plasminogen catalytic activity by monoclonal antibody IV-Ic showed that the presence of metal cations in the reaction medium changes the induction by slowing down or accelerating the process. Ions of Zn(2+), Mn(2+), and Cu(2+) completely inhibit activation. Ions of Co(2+) and Ni(2+) decrease the rate of the first and second phases of the reaction more than 2 times. Ca(2+) ions do not have any effect on the activation rate. Ions of Mg(2+), Ba(2+), and Sr(2+) increase the rate of the first phase of the reaction by 1.5, 2.0, and 2.0 times and the rate of the second phase by 2.0, 3.8, and 4.7 times, correspondingly. Sr(2+) ions have the strongest stimulating effect on plasminogen activation by monoclonal antibody IV-Ic. Investigation of the dose dependent effect of Sr(2+) on the rate of plasminogen activation by monoclonal antibody IV-Ic showed stimulating effect of Sr(2+) at concentrations from 0.1 to 1.0 mM with half maximum at 0.6 mM. However, Sr(2+) ions do not affect amidolytic activity of plasmin and activation of plasminogen by streptokinase. Sr(2+) ions also do not affect monoclonal antibody IV-Ic binding to plasminogen. The effect of Sr(2+) is specific and mediated by the IV-Ic component. The presence of metal cations affects conformational changes in the process of active site formation. Metal cations also affect structure of the plasminogen molecule active site in the complex with monoclonal antibody IV-Ic and enzyme-substrate interaction. The effect of alpha(2)-antiplasmin on the induction of plasminogen catalytic activity by monoclonal antibody IV-Ic in range of concentrations from 5 to 30 nM has been studied. alpha(2)-Antiplasmin at concentration 30 nM almost completely inhibits induction of plasminogen catalytic activity by monoclonal antibody IV-Ic at the ratio plasminogen/alpha(2)-antiplasmin of 3 : 1. This can be explained by competition of alpha(2)-antiplasmin and monoclonal antibody IV-Ic for the lysine-binding sites of plasminogen and inhibition of the active center in activated complex plasminogen*-mAB IV-Ic. Divalent metal cations and alpha(2)-antiplasmin are important factors in induction of plasminogen catalytic activity by monoclonal antibody IV-Ic.

Antibodies, Monoclonal↗
PubMed1995

Comparative metabolism of plasminogen glycoforms I and II in the alloxan-diabetic rabbit.

The metabolism of plasminogen glycoforms I and II was measured in alloxan-induced diabetic and in age-matched control rabbits. Radiolabeled plasminogen I and II were degraded significantly more slowly in diabetic compared with control rabbits; plasminogen II [half-time (T1/2), 1.31 days] was degraded faster than plasminogen I (T1/2), 1.86 days) in diabetic rabbits and in control rabbits (T1/2, 1.18 and 1.58 days, respectively). From the catabolic rates and relative quantities in plasma, we calculated that approximately four molecules of plasminogen II were degraded for one molecule of plasminogen I in the diabetic and control rabbits. To verify this later observation, plasminogen I and II production by diabetic rabbit livers was compared with that by the control livers in vitro. During perfusion with [3H]leucine, 3H-labeled protein was released more slowly from diabetic than from control livers, but no quantitative difference in total plasminogen yield between diabetic and control livers was found. Nevertheless, plasminogen II was produced 0.7 +/- 0.4 and 4.3 +/- 0.3 times faster than plasminogen I by diabetic and control livers, respectively. Plasminogen metabolism in the diabetic rabbit did not differ qualitatively from that in the control rabbit except that catabolism was slowed.

Animals↗
PubMed1988

Plasminogen: a brief introduction into its biochemistry and function.

Human plasminogen is a beta-globulin (2% carbohydrate, molecular weight 90 KD), which in its native form has NH2-terminal glutamic acid (Glu-plasminogen) whose primary structure is known (31, 37, 38). From human plasma plasminogen can easily be isolated by affinity chromatography techniques (10, 25, and Table 1). Plasminogen is synthesized in many organs. The production site of the zymogen may be the liver (21), the eosinophiles (3) or the kidney (15). The plasma-plasminogen level is low in newborns (22) and even lower in the premature infant (2). In healthy adults it is found in plasma or serum in a concentration of 200 mg/l (= 2 microM, 22, 39). The half-life of the native (Glu-) plasminogen is 2.24 +/- 0.29 days (6). Two types of Glu-plasminogen occur in human plasma, which differ in their carbohydrate composition as well as in their content of sialic acid. Genetic variants (see Mayr, 3.1.); of plasminogen have been reported (16) after isoelectric focusing of human plasma in polyacrylamide gels. Three patterns were found, two completely different and the third most likely a mixture of the other two. Characteristical functional properties of plasminogen are related to its molecular structure, e.g. its in vivo specificity for fibrin in contrast to the fairly unspecific in vitro activity of plasmin. Glu-plasminogen is easily converted by limited plasmic digestion to modified forms with NH2-terminal lysine, valine or methionine, which are commonly designated "Lys-plasminogen" displaying a plasma half-life time of 0.8 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrate Conformation↗
PubMed1988

Physiology of plasminogen: with special reference to activation and degradation.

Plasminogen is said to be synthesized in many organs, but the major source of its production is considered to be the liver. The production of plasminogen is observed in fetal life, but its plasma level is low in new born infants, rising rapidly to attain adult levels in about 13 weeks. The plasma levels do not change much with age. Although the fibrinolytic enzyme system is influenced by many factors such as hormones, exercise, emotion, age, sex, nutritional states etc., the plasma levels of plasminogen are relatively stable. In this presentation we would like to discuss three important subjects related to the physiology of plasminogen. The first subject is the activation pathway of the native form of plasminogen (Glu-plg) by various activators in the plasma or clotted plasma. Glu-plg was not easily activated by activators such as urokinase (UK) or tissue plasminogen activator (t-PA), but activated very easily in the presence of fibrin clot. In the presence of purified clot, Glu-plg was partly activated by activators to Glu-plasmin, but also converted to Lys-plg by preformed plasmin, subsequently being activated to plasmin. Glu-plg I (containing two carbohydrate chains) changed conformation more easily upon interaction with fibrin than Glu-plg II (containing one carbohydrate chain) and Glu-plg I was also more easily activated by activators than Glu-plg II. Although Glu-plg was hardly activated by activators in the plasma, Glu-plg was activated easily in the presence of plasma clot. Results of immunoblotting experiments indicated that Glu-plg was mainly activated by activators directly to plasmin, not via Lys-plg in contrast to purified systems. The second subject is the degradation of plasminogen by elastase. Plasminogen may be degraded by proteolytic enzymes such as cathepsin or elastase. The degradation of plasminogen by elastase is shown to give rise to K1 to K3, K4, and mini-plg (containing K5). We have shown that the degradation rate to Glu-plg by elastase increased in the presence of tranexamic acid, indicating that the conformational change of Glu-plg in the presence of tranexamic acid resulted in the exposure of the hydrophobic regions connecting K3, K4 and K5, thus making them accessible to elastase. Consequently, Lys-plg or conformationally altered Glu-plg (possibly bound to fibrin or FDP) is more easily degraded in vivo. The last subject is relationship between plasma plasminogen levels and fibrinolytic activity in various ages. Although plasma plasminogen levels do not change with age, the fibrinolytic activity lowers.(ABSTRACT TRUNCATED AT 400 WORDS)

Enzyme Activation↗
PubMed1983

Thrombolysis with human extrinsic (tissue-type) plasminogen activator in rabbits with experimental jugular vein thrombosis. Effect of molecular form and dose of activator, age of the thrombus, and route of administration.

A simple venous thrombosis model in rabbits was used for the quantitative evaluation of the thrombolytic effect of human extrinsic (tissue-type) plasminogen activator as compared with urokinase.A thrombus was formed in an isolated segment of the jugular vein from a mixture of (125)I-labeled fibrinogen, whole rabbit blood, and thrombin. In order to immobilize the thrombus during lysis, it was formed around a woolen thread introduced longitudinally in the lumen of the vein. Thrombotic extension of the clot was prevented by subcutaneous injection of heparin. The extent of thrombolysis was measured as the difference between the radioactivity introduced in the clot and that recovered in the vein segment at the end of the experiment. In control animals the extent of thrombolysis was 5.6+/-1.4% (n = 5) after 6 h, 14.5+/-1.7% (n = 10) after 30 h, 16.0+/-1.5% (n = 11) after 78 h, and 48.1+/-2.7% (n = 10) after 174 h (mean+/-SEM). Extrinsic (tissue-type) plasminogen activator, highly purified from the culture fluid of a human melanoma cell line, was administered systemically or locally over a time period of 4 h and the percent thrombolysis measured 2 h after the end of the infusion. One- and two-chain extrinsic plasminogen activator had very similar thrombolytic potency. Systemic infusion resulted in a dose-dependent degree of thrombolysis. The activator-induced thrombolysis, after infusion of 100,000 IU ( congruent with1 mg protein), was approximately 75% for fresh clots, 35% for 1-d-old clots, 30% for 3-d-old clots, and 50% for 7-d-old clots. The thrombolytic activity of urokinase was more than five times lower than that of extrinsic plasminogen activator: Infusion of 500,000 IU resulted in approximately 40% lysis of fresh clots and 25% of 1-3-d-old clots, while 7-d-old clots appeared to have become resistent to urokinase. Local infusion resulted in a 5-10 times higher thrombolytic effect of both extrinsic plasminogen activator and urokinase. Thrombolysis with extrinsic plasminogen activator was not associated with systemic activation of the fibrinolytic system as evidenced by unaltered plasma levels of fibrinogen, plasminogen, and alpha(2)-antiplasmin. Systemic infusion of urokinase resulted in significant thrombolysis only at doses that were associated with disseminated plasminogen activation. Local infusion of urokinase required a 5-10-fold higher dose than extrinsic plasminogen activator to obtain a similar degree of thrombolysis, which also occurred in the absence of systemic activation of the fibrinolytic system. It is concluded that the extent of thrombolysis by extrinsic plasminogen activator is mainly determined by the dose of activator and its delivery in the vicinity of the thrombus and much less by the age of the thrombus or the molecular form of the activator. Extrinsic plasminogen activator appears to be superior to urokinase because of its higher (5-10-fold) specific thrombolytic activity and the absence of systemic activation of the fibrinolytic system, which results in defibrinogenation and a bleeding tendency.

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