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PubMed2004

Characterization of plasminogen variants in healthy subjects and plasminogen mutants in patients with inherited plasminogen deficiency by isoelectric focusing gel electrophoresis.

Plasmin(ogen) plays an important role in fibrinolysis and wound healing. Severe hypoplasminogenemia has recently been linked to ligneous conjunctivitis. Plasminogen (plg) is known as a polymorphic protein and most of these variants have been identified using isoelectric focusing (IEF) gel electrophoresis. Here, we studied common plg variants from healthy subjects and plg mutants from three patients with hypoplasminogenemia and three subjects with dysplasminogenemia by molecular genetic analysis and IEF. Analysis of 24 healthy subjects showed that subjects with the most common IEF plg phenotype A (n=12) were homozygous for aspartate at position 453 (453D), while both subjects with IEF plg phenotype B were homozygous for asparagine at this position (453N). Subjects with IEF plg phenotype AB (n=10) were compound-heterozygous for 453D/453N. Three patients with severe hypoplasminogenemia and different plg gene mutations exhibited characteristic "abnormal" IEF band patterns when compared with IEF plg phenotypes A and B. In all heterozygous family members the observed IEF plg phenotype was derived from the wild type plg molecule only, probably due to low concentration of the mutant plg molecule in plasma. In contrast, in three unrelated subjects with heterozygous dysplasminogenemia an equal "mixture" of wild type and mutant plg was found by IEF analysis. In conclusion, plg phenotyping by IEF in combination with molecular analysis of the plg gene seems to be a useful method for characterization of plg variants and mutants.

Asparagine↗
PubMedDate not supplied

Temporal expression of urokinase plasminogen activator, plasminogen activator inhibitor and gelatinase-B in chronic wound fluid switches from a chronic to acute wound profile with progression to healing.

The plasminogen activator/plasmin system is known to initiate a proteolytic cascade resulting in the activation of matrix metalloproteinases in vitro leading to the degradation of extracellular matrix. To investigate whether or not this cascade is present during delayed wound healing and contributes to the pathophysiological basis of impaired healing we examined the temporal expression of urokinase plasminogen activator, plasminogen activator inhibitor-1 and gelatinase-B in fluid collected from chronic venous leg ulcers compared to acute surgical mastectomy wounds. Using a chromogenic substrate assay, levels of active urokinase plasminogen activator in chronic wounds were found to be about five fold higher compared to sera and two fold higher compared to mastectomy wounds. Levels of active plasminogen activator inhibitor-1 in chronic wounds were four times higher than those found in sera and two times higher than those found in mastectomy wound fluid. Using a fibrin overlay system and reverse zymography, we found that when the wound was not healing, the expression of urokinase plasminogen activator in chronic wound fluid was initially detected in the active forms (50 and 33 kDa), but that as the wound healed and decreased in size, was detected as an inhibitor- bound urokinase plasminogen activator-plasminogen activator inhibitor-1 complex ( congruent with 80-116 kDa). When the expression of active urokinase plasminogen activator was highest, no plasminogen activator inhibitor-1 was detectable. In contrast, urokinase plasminogen activator was always detected in the inhibitor bound form as a urokinase plasminogen activator-plasminogen activator inhibitor-1 complex in blood- and plasma-derived serum and mastectomy wound fluid. Plasminogen activator inhibitor-1 was detected in blood-derived serum and mastectomy wound fluid but not in plasma derived serum. Expression of matrix metalloproteinase-9 in chronic wound fluids, analyzed by gelatin zymography, showed that when urokinase plasminogen activator was detected in the active forms, matrix metalloproteinase-9 was overexpressed by approximately twice that found in mastectomy wounds and approximately 30 times that detected in blood-derived sera. When urokinase plasminogen activator appeared almost entirely as an enzyme- inhibitor complex, the level of expression of matrix metalloproteinase-9 was similar to that seen in mastectomy wound fluid. We conclude that the switch in urokinase plasminogen activator expression from an active to inhibitor bound form correlates with the decrease seen in matrix metalloproteinase-9 expression suggesting the presence of a proteolytic cascade initiated by the plasminogen activator/plasmin system during wound healing leading to the activation of matrix metalloproteinase-9. In addition, expression of urokinase plasminogen activator and matrix metalloproteinase-9 appear to be useful biomarkers to determine clinical wound healing status.

Adult↗
PubMed2003

Endogenous plasmin converts Glu-plasminogen to Lys-plasminogen on the monocytoid cell surface.

Recently, we showed that localization of Glu-plasminogen on cell surfaces enhances its conversion to Lys-plasminogen by exogenous plasmin. This leads to stimulation of plasminogen activation because Lys-plasminogen is the preferred substrate on cell surfaces. Here, we show that Glu-plasminogen was converted to Lys-plasminogen on monocytoid cells in the absence of exogenous plasmin. Culture of cells under serum-free conditions did not affect this conversion, suggesting that the enzymatic activity was cell-derived. Therefore, we tested whether endogenous monocytoid plasminogen could provide a source of plasmin to convert cell-associated Glu-plasminogen to Lys-plasminogen because plasmin is the only enzyme known to effect this reaction. We used a recombinant human plasminogen mutant, [D(646)E]Pg, which can be cleaved by plasminogen activators, but cannot catalyze the generation of Lys-plasminogen. Upon incubation with either THP-1 or U937 monocytoid cells, 35 and 38%, respectively, of the cell-bound ligand was converted to Lys-[D(646)E]Pg. Trasylol, alpha2-antiplasmin, and an anticatalytic antiplasminogen monoclonal antibody decreased Lys-[D(646)E]Pg formation to < 5% on monocytoid cells, consistent with a plasmin-dependent mechanism. Plasminogen was detected in these cells by Northern blotting and RT-PCR. Our results suggest that plasmin converts cell-bound Glu-plasminogen to Lys-plasminogen and that this enzyme is produced by activation of monocytoid plasminogen by endogenous monocytoid plasminogen activators to enhance plasminogen activation on the monocytoid cell surface.

Cell Line↗
PubMed2000

Discriminating between cell surface and intracellular plasminogen-binding proteins: heterogeneity in profibrinolytic plasminogen-binding proteins on monocytoid cells.

When plasminogen binds to cell surfaces, its activation is markedly enhanced compared to soluble plasminogen. Although several distinct molecules may contribute to plasminogen binding to a given cell type, the subset of plasminogen receptors responsible for enhancing plasminogen activation expose a carboxyl-terminal lysine on the cell surface and are sensitive to proteolysis by carboxypeptidase B (CpB). To distinguish this subset of plasminogen receptors from plasminogen-binding proteins that are not profibrinolytic, we treated intact U937 monocytoid cells and peripheral blood monocytes with CpB to remove exposed carboxyl-terminal lysines, and subjected the membrane proteins to two-dimensional gel electrophoresis followed by ligand blotting with 125I-plasminogen. Western blotting was performed with antibodies against previously characterized candidate plasminogen receptors to identify plasminogen-binding proteins on the two-dimensional ligand blots. Densitometry of autoradiograms of the 125I-plasminogen ligand blots of U937 cell membranes revealed that membrane-associated alpha-enolase, actin and annexin II showed minimal changes in 125I-plasminogen binding following CpB treatment of intact cells, suggesting that these proteins are not accessible to CpB on the U937 cell surface and most likely do not serve as profibrinolytic plasminogen receptors on U937 cells. In contrast, densitometry of autoradiograms of 125I-plasminogen ligand blots of monocyte membranes revealed that 125I-plasminogen binding to alpha-enolase was reduced 71% by treatment of intact cells with CpB, while binding to annexin II was reduced 14%. Thus, a portion of membrane-associated alpha-enolase and annexin II expose carboxyl terminal lysines that are accessible to CpB on the peripheral blood monocyte surface, suggesting that these molecules may serve as profibrinolytic plasminogen receptors on monocytes. Our data suggest that U937 cells and peripheral blood monocytes have distinct sets of molecules that constitute the population of cell surface profibrinolytic plasminogen-binding proteins. Furthermore, our data suggest that while several plasminogen-binding proteins with carboxyl terminal lysines are associated with cell membranes, only a small subset of these proteins expose a carboxyl terminal lysine that is accessible to CpB on the cell surface.

Fibrinolysis↗
PubMed1997

Acceleration of plasminogen activation by tissue plasminogen activator on surface-bound histidine-proline-rich glycoprotein.

Histidine-proline-rich glycoprotein (HPRG), also known as histidine-rich glycoprotein, is a major plasminogen-binding protein. In this work we characterized extensively the circumstances under which HPRG accelerates plasminogen activation and the specificity of this effect. Soluble HPRG did not significantly influence plasminogen activation. In contrast, native HPRG bound to hydrazide or nickel chelate surfaces strongly stimulated the activation of plasminogen by tissue plasminogen activator, but not by urokinase or streptokinase. The efficiency of activation on surface-bound HPRG was increased for Glu-plasminogen (41-fold), Lys-plasminogen (17-fold), and cross-linked Glu-plasminogen (11-fold) but not for mini-plasminogen, and was mainly due to a decrease in the apparent Km. A reduced susceptibility to inhibition by chloride ions contributed to the higher activation rate of Glu-plasminogen on an HPRG surface. The immobilized N- and C-terminal domains, but not the histidine-proline-rich domain of HPRG, also bound plasminogen and stimulated its activation. HPRG-enhanced plasminogen activation was proportional to the quantity of HPRG immobilized and was abolished by anti-HPRG antiserum, by low concentrations of epsilon-aminocaproic acid, by methylation of lysine residues in HPRG, and by treatment of HPRG with carboxypeptidase B. Soluble HPRG and a plasminogen fragment, kringle 1-2-3, acted as competitive inhibitors by binding to plasminogen and immobilized HPRG, respectively. The interaction of the conserved C-terminal lysine of HPRG with the high affinity lysine binding site of plasminogen is necessary and sufficient to accelerate plasminogen activation. Unlike other stimulators of plasminogen activation, the effect of HPRG on fibrinolysis is modulated by factors that influence the equilibrium between solution and surface-bound HPRG.

Animals↗
PubMed2000

The potential mechanism for the effect of heparin on tissue plasminogen activator-mediated plasminogen activation.

The effects and possible role of heparin on tissue plasminogen activator-mediated plasminogen activation was thoroughly investigated. Direct analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that heparin increased the conversion of plasminogen to plasmin. Experiments by fluorescence quenching suggested that the stimulation of tissue plasminogen activator activity probably was due to a direct binding of heparin to tissue plasminogen activator, causing a conformational change of tissue plasminogen activator and rendering it more accessible to plasminogen interaction. The absence of additive stimulation effects on tissue plasminogen activator-mediated plasminogen activation when both heparin and fibrinogen were present also implied that both compounds interacted with tissue plasminogen activator via the same domain; it appeared to be most likely via the kringle-2 domain in tissue plasminogen activator based on studies using epsilon-aminocaproic acid as an inhibitor. Unlike heparin-induced stimulation of antithrombin-thrombin interaction, the heparin-induced stimulation of tissue plasminogen activator did not seem to follow a template model. Only in the presence of a high plasminogen or a low tissue plasminogen activator concentration, massive stimulation of tissue plasminogen activator activity was observed via a pseudotemplate model. The results suggest that precautions concerning high heparin dose should be given during its conjunctive clinical use with tissue plasminogen activator in thrombolytic therapy to reduce the risk of hemorrhage.

Binding Sites↗
PubMed1992

Plasminogen binding by alpha 2-antiplasmin and histidine-rich glycoprotein does not inhibit plasminogen activation at the surface of fibrin.

alpha 2-antiplasmin (alpha 2-AP) exerts its inhibitory effect on fibrinolysis by rapidly inhibiting the plasmin evolved; in addition, it has been suggested that interference with the binding of plasminogen to fibrin, a function shared with histidine-rich glycoprotein (HRGP), may also be significant in inhibition of fibrinolysis. To elucidate if plasminogen binding by these two alpha 2-globulins may decrease the generation of plasmin by tissue-type plasminogen activator (t-PA) at the surface of fibrin, a system mimicking the fibrin/plasma interface was used. Attempts were made to differentiate the plasminogen binding from the plasmin inhibitory function of alpha 2-AP. The activation of human Glu-plasminogen (native plasminogen with NH2-terminal glutamic acid) by fibrin-bound t-PA was performed in a plasma environment using either normal plasma, alpha 2-AP- or HRGP-depleted plasmas supplemented with increasing amounts of the lacking protein, or in a reconstituted system with purified plasminogen and various concentrations of alpha 2-AP and HRGP. The activation of Glu-plasminogen in alpha 2-AP-depleted plasma containing a normal concentration of HRGP produced a time-dependent increase in the generation of plasmin. The addition of 1 microM-alpha 2-AP to this plasma prevented the formation of Lys-derivatives and produced a marked decrease (42%) in the number of plasminogen-binding sites. In contrast, the addition of 1.5 microM-HRGP to HRGP-depleted plasma containing a normal amount of alpha 2-AP produced only a modest (17%) decrease in the amount of plasmin(ogen) bound. Moreover, in a purified system the amount of plasminogen-binding sites and thereby of plasmin generated at the surface of fibrin in the presence of both alpha-2 globulins was similar to the amount generated in the presence of alpha 2-AP alone. These results indicate clearly that the formation of reversible complexes between plasminogen and alpha 2-AP does not interfere with the binding and activation of plasminogen at the fibrin surface. In contrast, the inhibition of plasmin by alpha 2-AP decreases importantly the number of plasminogen-binding sites (carboxyl-terminal lysines) and inhibits thereby the accelerated phase of fibrinolysis. It can be concluded that interference of the binding of plasminogen to fibrin by alpha 2-AP during plasminogen activation, does not play a significant role in inhibition of fibrinolysis, and that the plasminogen-binding effect of HRGP, if any, is obscured by the important inhibitory effect of alpha 2-AP.

Biotransformation↗
PubMed1985

A monoclonal antibody specific for Lys-plasminogen. Application to the study of the activation pathways of plasminogen in vivo.

Human plasminogen, a glycoprotein with NH2-terminal Glu, is rapidly converted by traces of plasmin to proteolytic derivatives with NH2-terminal Met 68, Lys 77, or Val 78 ("Lys-plasminogen"), which are much more readily activated to plasmin than is Glu-plasminogen. It has, therefore, been proposed that physiological activation of Glu-plasminogen occurs mainly via Lys-plasminogen intermediates (Wiman, B., and Wallén, P. (1973) Eur. J. Biochem. 36, 25-31). In the present study we have characterized a murine monoclonal antibody (LPm1) directed against an epitope exposed in Lys-plasminogen but not in Glu-plasminogen. The antibody was secreted by a hybridoma obtained by fusion of mouse myeloma cells (P3X63-Ag8-6.5.3) with spleen cells of a mouse immunized with purified Lys-plasmin-alpha 2-antiplasmin complex. Coupling of the alpha-amino groups of Lys-plasminogen with phenylisothiocyanate resulted in complete loss of immunoreactivity for LPm1, which was, however, fully restored by cleavage of the derivatized NH2-terminal amino acid. After a second cycle, immunoreactivity was not restored, indicating that the LPm1 antibody-binding site depends on the presence of Lys 77 and/or Val 78 as NH2-terminal amino acids. The immunoreactivity of Lys-plasminogen with LPm1 is abolished by reduction of the protein, suggesting that conversion of Glu-plasminogen to Lys-plasminogen is associated with a conformational alteration exposing the epitope for the LPm1 monoclonal antibody. In order to investigate the pathways of plasminogen activation in vivo, total plasmin-alpha 2-antiplasmin and Lys-plasmin-alpha 2-antiplasmin complexes were measured with sandwich-type micro enzyme-linked immunosorbent assays. Therefore, microtiter plates were coated with monoclonal antibodies against alpha 2-antiplasmin, and bound antigen was quantitated with horseradish peroxidase-conjugated LPm1 or a monoclonal antibody reacting equally well with Glu-plasmin as with Lys-plasmin. In 25 healthy subjects the plasmin-alpha 2-antiplasmin levels in plasma were undetectable (less than 0.1 nM). Infusion of tissue-type plasminogen activator in patients with thromboembolic disease resulted in generation of high concentrations of Glu-plasmin-alpha 2-antiplasmin complex (620 +/- 150 nM, n = 7) whereas neither Lys-plasmin-alpha 2-antiplasmin complex nor Lys-plasminogen were consistently detected. It is, therefore, concluded that activation of the fibrinolytic system in vivo occurs by direct cleavage of the Arg 560-Val 561 bond in Glu-plasminogen and not via formation of the Lys-plasminogen intermediates.

Amino Acids↗
PubMed1985

Tissue plasminogen activator and urokinase mediate the binding of Glu-plasminogen to plasma fibrin I. Evidence for new binding sites in plasmin-degraded fibrin I.

The effect of tissue plasminogen activator (TPA) or urokinase on the specific binding of human Glu-plasminogen to fibrin I formed in plasma by clotting with Reptilase was studied using 125I-plasminogen and 131I-fibrinogen. In the absence of TPA, small amounts of plasminogen were bound to fibrin I. TPA induced binding of plasminogen to plasma fibrin I that was dependent upon the concentrations of TPA and plasminogen as well as upon the time of incubation. Plasminogen binding occurred in association with fibrin clot lysis and the formation in the clot supernatant of alpha 2-plasmin inhibitor-plasmin complexes. Urokinase also induced binding of plasminogen to plasma fibrin I that was concentration- and time-dependent. The molecular form of plasminogen bound to the fibrin I plasma clot was identified as Glu-plasminogen by dodecyl sulfate-polyacrylamide gel electrophoresis and by fast performance liquid chromatography. Further studies demonstrated that fibrin I formed from fibrinogen that had been progressively degraded by plasmin-bound Glu-plasminogen. The mole ratio of plasminogen bound increased with the time of plasmin digestion. Glu-plasminogen did not bind to fibrin I formed from fibrinogen progressively digested by human leukocyte elastase, thereby demonstrating the specificity of plasmin. These studies demonstrate that plasminogen activators regulate the binding of Glu-plasminogen to fibrin I by catalyzing plasmin-mediated modifications in the fibrin substrate.

Densitometry↗
PubMed1987

Localization of the binding sites of porcine tissue-type plasminogen activator and plasminogen to heparin.

To localize the binding region of porcine tissue-type plasminogen activator (EC 3.4.21.31) (t-plasminogen activator) to heparin, functionally active A and B chains (molecular mass of each 33 kDa) were separated from the two-chain t-plasminogen activator after mild reduction and alkylation. The A chain bound to fibrin-Sepharose, but not to heparin-Sepharose. In contrast, the B chain showed amidase activity toward HD-Ile-Pro-Arg-p-nitroanilide (S-2288) and a high affinity for heparin-Sepharose, but no affinity for fibrin-Sepharose. Plasminogen activator activity of the B chain was stimulated by heparin (about 3-fold), but not by fibrin. On the other hand, the elastase digestion fragments of plasminogen, kringle 1-3 and kringle 4, had no affinity for a heparin-Sepharose column, whereas the other fragment, Val442-plasminogen, efficiently bound to the column and was eluted with 1.6 M KSCN-containing buffer. The stimulatory effect of fibrin on two-chain t-plasminogen activator-catalyzed Val442-plasminogen activation was clearly diminished by heparin. These results suggest that heparin can form a complex with both t-plasminogen activator and plasminogen molecules through their catalytic regions located in each B chain, and that the heparin connection between t-plasminogen activator and plasminogen may improve the plasminogen activation kinetics by making a situation in which t-plasminogen activator is easily approachable to plasminogen.

Animals↗
PubMed2004

Regulation of expression of tissue plasminogen activator and plasminogen activator inhibitor-1 by dichloroacetic acid in human fibroblasts from normal peritoneum and adhesions.

OBJECTIVE: As part of our ongoing studies to understand the biologic mechanisms of wound repair that lead to postoperative adhesions, we have identified characteristics of an adhesion phenotype that differs between fibroblasts that are obtained from human normal peritoneum and adhesions. In this study, we sought to examine whether stimulation of aerobic metabolism would alter differential expression of tissue plasminogen activator and plasminogen activator inhibitor-1, thereby creating a milieu likely to be less favorable to postoperative adhesion development. To examine this issue, we used a compound, dichloroacetic acid, that stimulates the pyruvate dehydrogenase complex, which causes pyruvate to be metabolized in the Kreb's cycle rather than being converted into lactate, thereby switching anaerobic to aerobic metabolism. STUDY DESIGN: Human fibroblasts from normal peritoneum and adhesions were cultured in the absence or presence of dichloroacetic acid (100 microg/mL) for 24 hours, under normal and hypoxic (2% O(2)) conditions. Real-time reverse transcriptase-polymerase chain reaction of tissue plasminogen activator, plasminogen activator inhibitor-1, and a housekeeping gene beta-actin was performed with messenger RNA that was extracted from all treatment points. RESULTS: Dichloroacetic acid stimulated normal peritoneal fibroblast tissue plasminogen activator messenger RNA expression under hypoxic conditions. In adhesion fibroblasts, dichloroacetic acid treatment enhanced tissue plasminogen activator messenger RNA expression under both normoxic and hypoxic conditions. Plasminogen activator inhibitor-1 messenger RNA expression was unaltered by dichloroacetic acid in normoxic normal peritoneal fibroblasts; but during culture under hypoxic conditions, dichloroacetic acid reduced plasminogen activator inhibitor-1 messenger RNA expression. Similarly, in adhesion fibroblasts, dichloroacetic acid reduced plasminogen activator inhibitor-1 messenger RNA expression under both normoxic and hypoxic conditions. As a result, in normal peritoneal fibroblasts under hypoxic conditions and in adhesion fibroblasts under normoxic and hypoxic conditions, dichloroacetic acid greatly increased the tissue plasminogen activator/plasminogen activator inhibitor-1 ratios. CONCLUSION: These findings confirm that fibroblasts from adhesions are characterized by reduced tissue plasminogen activator and increased plasminogen activator inhibitor-1 production. These observations are extended to show the stimulation of oxidative metabolism by dichloroacetic acid increases tissue plasminogen activator expression under hypoxic conditions. Dichloroacetic acid reduces plasminogen activator inhibitor-1 production by hypoxic normal peritoneal fibroblasts and adhesion fibroblasts under hypoxic conditions. The resultant increases in the tissue plasminogen activator/plasminogen activator inhibitor-1 ratios would favor the development of a fibrinolytic milieu, which would be expected potentially to limit postoperative adhesion development. Thus, regulation of metabolic activity of peritoneal cells may provide a target for future interventions for the reduction of the development of postoperative adhesions, particularly as intervention relates to the healing of peritoneal sites that previously had adhesions. (eg, sites of potential adhesion reformation).

Cell Hypoxia↗
PubMed1995

Identification of a plasminogen binding region in streptokinase that is necessary for the creation of a functional streptokinase-plasminogen activator complex.

Streptokinase is a plasminogen activator widely used to treat patients with myocardial infarction. However, streptokinase is not a protease, and must first bind and interact with plasminogen to form an enzymatic complex. By measuring the binding of recombinant streptokinase fragments to plasminogen, we have sought, first, to identify a plasminogen binding region in streptokinase and, second, to explore the relation between binding (via this region) and the generation of a functional streptokinase--plasminogen activator complex. Recombinant streptokinase bound in a saturable and specific manner to human Glu-plasminogen with a dissociation constant of 4.2 x 10(-10) M. Recombinant streptokinase fragments spanning amino acids 1-127 and 1-253 could not be shown to bind to Glu-plasminogen, whereas fragments spanning amino acids 1-352, 120-352, and 244-414 bound tightly to plasminogen and each fragment completely inhibited the binding of full-length streptokinase to plasminogen. Although these latter streptokinase fragments formed a complex with plasminogen, enzymatic assays indicated that none of them was capable of generating an active site. When the streptokinase region shared by these three fragments, spanning residues 244-352, was expressed, it also bound plasminogen and competitively inhibited the formation of a functional plasminogen activator complex by full-length streptokinase. Taken together, these data indicate that streptokinase binds to plasminogen with high affinity, that a primary binding region for plasminogen is located within amino acids 244-352, and that binding via this region is necessary for the generation of a functional plasminogen activator complex.

Base Sequence↗
PubMed1994

Catabolism of plasminogen glycoforms I and II in rabbits: relationship to plasminogen synthesis by the rabbit liver in vitro.

The metabolisms of the two glycoforms of rabbit plasminogen have been compared in rabbits. Plasminogen I and II (ratio in plasma, 1:2.2) differ only in glycan content: plasminogen I probably possesses one N-glycan and one O-glycan, and plasminogen II only one O-glycan. New Zealand White (NZW) rabbits were injected intravenously with 125I-plasminogen I and 131I-plasminogen II, and blood samples were taken at regular intervals over 5 days. Kinetic behaviors were determined from protein-bound radioactivities using a three-compartment model. Fractional catabolic rates for plasminogen II in the vascular space (2.42 d-1) and the total body (0.56 d-1) were significantly greater than those measured for plasminogen I (1.12 and 0.45 d-1); half-lives were 1.53 and 1.23 days for plasminogen I and II, respectively (P < .01). Fractional distributions among the vascular, noncirculating vascular, and extravascular compartments were 0.41, 0.13, and 0.46 for plasminogen I, and 0.23, 0.11, and 0.65 for plasminogen II. From these data, we determined that plasminogen II was catabolized 4.8 times more rapidly than plasminogen I and was quantitatively contained largely in the extravascular space. By comparison, perfusion of rabbit livers ex corpora showed that plasminogen II was synthesized and released 5.0 times faster than plasminogen I over a 5-hour period. The possible roles for these glycoforms in vivo with respect to their different turnover rates and compartmental distributions are discussed.

Animals↗
PubMed1992

Aggregation of washed platelets by plasminogen and plasminogen activators is mediated by plasmin and is inhibited by a synthetic peptide disulfide.

Plasmin is known to activate platelets. However, it is not clear whether plasminogen activators as used in thrombolytic therapy can aggregate platelets and how this relates to the ability of each activator to convert plasminogen to plasmin. Urokinase (UK) and streptokinase (SK) activated purified plasminogen (2 microM) in a concentration-dependent manner. The rates of aggregation of washed platelets by the above plasminogen activators and plasminogen were similar to the extent of activation of plasminogen to plasmin in the absence of platelets. UK or SK (0.2 microM) and plasminogen (2 microM) aggregated platelets modified by an ADP affinity analog, 5'-p-fluorosulfonylbenzoyladenosine (FSBA), and cleaved aggregin, a putative ADP receptor, in [3H]FSBA-modified platelets. These results suggest that the effect was independent of ADP. In contrast, incubation mixtures containing only plasminogen (2 microM) and single chain tissue plasminogen activator (sc-tPA) (less than or equal to 0.12 microM) neither activated the zymogen to an appreciable extent nor aggregated platelets. But, in the presence of fibrin(ogen) fragments (tPA-stimulator), a mixture of plasminogen and sc-tPA aggregated unmodified and FSBA-modified platelets, and cleaved aggregin. The results imply that platelets, in the presence of t-PA stimulator, potentiate activation of plasminogen to plasmin by t-PA, as previously reported. P1, Phe-Gln-Val-Val-Cys-(NpyS)-Gly-NH2, (NpyS = 3-nitro-2-thiopyridine), a synthetic hexapeptide capable of binding to and inhibiting calpain, has been shown to inhibit platelet aggregation induced by purified plasmin. P1 inhibited platelet aggregation by plasminogen and any of the three plasminogen activators. Our results show that at plasma concentrations of plasminogen and at levels of UK and SK attained after infusion of these agents during thrombolysis, these mixtures can cause maximum aggregation which may contribute to reocclusion and stenosis following infarct therapy. P1 can effectively inhibit platelet aggregation under such conditions.

Amino Acid Sequence↗
PubMed1984

Sequence of formation of molecular forms of plasminogen and plasmin-inhibitor complexes in plasma activated by urokinase or tissue-type plasminogen activator.

The pathway of plasminogen transformation was studied in plasma, particularly in relation to fibrin formation and the subsequent stimulation of plasminogen activation. Plasminogen was activated by urokinase (low fibrin-affinity) or tissue-type plasminogen activator (high fibrin-affinity). Formation of 125I-labelled free and inhibitor-bound plasminogen derivatives was quantified after their separation by acetic acid/urea/polyacrylamide-gel electrophoresis. In plasma activator converted Glu-plasminogen (residues 1-790) into Glu-plasmin, which was complexed to alpha 2-plasmin inhibitor. When this inhibitor was saturated, Glu-plasmin was autocatalytically converted into Lys-plasmin (residues 77-790). No plasmin-catalysed Lys-plasminogen formation was observed. Upon fibrin formation, activation initially followed the same Glu-plasminogen-into-Glu-plasmin conversion pathway, and stimulation of plasminogen activation was only observed with tissue-type plasminogen activator. In agreement with the emergence of novel effector function, on early plasmin cleavage of fibrin [Suenson, Lützen & Thorsen (1984) Eur. J. Biochem. 140, 513-522] the fibrin-binding of Glu-plasminogen increased when solid-phase fibrin showed evident signs of degradation. This was associated with the formation of considerable amounts of the more easily activatable Lys-plasminogen, most of which was fibrin-bound. At the same time the rate of plasmin formation with urokinase increased over that in unclotted plasma and the rate of plasmin formation with tissue-type plasminogen activator accelerated. Altogether these processes favoured enhanced fibrin degradation. The rates of Lys-plasminogen and plasmin formation abruptly decreased after lysis of fibrin, probably owing to a compromised effector function on further fibrin degradation.

Antifibrinolytic Agents↗
PubMed1989

The mechanism of plasminogen activation and fibrin dissolution by single chain urokinase-type plasminogen activator in a plasma milieu in vitro.

The relative contribution of several mechanisms to plasminogen activation and fibrin dissolution by urokinase-type plasminogen activator (u-PA) in vitro was quantitated. The activation of plasminogen by recombinant single chain u-PA (rscu-PA), by its two chain derivative (rtcu-PA) and by a plasmin-resistant mutant, rscu-PA-Glu158, obeys Michaelis-Menten kinetics with catalytic efficiencies of 0.00064, 0.046, and 0.00005 L/mumol.s for native plasminogen (Glu-plasminogen) and of 0.0061, 1.21, and 0.0004 L/mumol.s for partially degraded plasminogen (Lys-plasminogen). In a purified system consisting of a fibrin clot submerged in a plasminogen solution, the equi-effective doses (50% lysis in one hour) for rscu-PA, rtcu-PA, and rscu-PA-Glu158 were 16, 6.5, and 32,000 ng/mL for Glu-plasminogen and two- to fourfold lower for Lys-plasminogen. In a plasma milieu, 50% lysis in two hours was obtained for a plasma clot with 2.1 micrograms/mL rscu-PA, 0.5 micrograms/mL rtcu-PA, and greater than 200 micrograms/mL rscu-PA-Glu158 and for a purified fibrin clot with 1.3 micrograms/mL rscu-PA and 0.27 microgram/mL rtcu-PA. After predigestion of a purified fibrin clot with plasmin, the apparent potency of rscu-PA and rtcu-PA increased by 40% and 20%, respectively. In conclusion, rscu-PA has an intrinsic plasminogen activating potential that is only about 1% of that of rtcu-PA and that is 13 times higher than that of rscu-PA-Glu158. Conformational transition of Glu-plasminogen to Lys-plasminogen enhances its sensitivity to activation by all u-PA moieties ten- to 20-fold. Predigestion of fibrin clots with associated increased binding of plasminogen results in a minor apparent increase of the fibrinolytic potency of rscu-PA and rtcu-PA. The relative fibrinolytic potency of rtcu-PA is two to three orders of magnitude higher than that of rscu-PA-Glu158 but only two- to five-fold higher than that of rscu-PA, both in purified systems and in a plasma milieu. These results indicate that conversion of rscu-PA to rtcu-PA constitutes the primary mechanism of fibrin dissolution.

Blood Coagulation↗