Heavy Chain Of Plasmin Research Articles

P093 PLASMA PROTEINS PROFILE AND THERAPEUTIC RESPONSIVENESS IN PATIENTS WITH PREECLAMPSIA

Preeclampsia (PE) is a hypertensive syndrome of pregnancy, characterized by target organ damage, related to a high incidence of cardiovascular sequelae. PE requires antihypertensive drug therapy, however, about 40% of patients do not respond to this treatment, leading to worse clinical outcomes. Through the proteomic strategy, we 1) compared the profile of circulating proteins expressed in 10 responsive (R-PE) and 10 non-responsive to antihypertensive therapy (NR-PE), besides 10 healthy pregnant (HP); 2) investigated which proteins are differentially expressed between the groups and 3) found out which signaling pathways are altered between them. The study followed the Declaration of Helsinki and was approved by the Research Ethics Committee of the Ribeirao Preto Medical School, University of Sao Paulo (FMRP-USP, protocol code CAAE-37738620.0.0000.5440). We performed plasma protein quantification using mass spectrometry, the obtained data underwent bioinformatics analyses based on Uniprot, PatternLab for Proteomics, String and MetaboAnalyst softwares. Considering a fold change of 1.5, three proteins showed as significantly differentially expressed between HP and R-PE, one downregulated (transtyretin) and two upregulated (apolipoprotein C1 and hemoglobin subunit beta). Between HP and NR-PE, there were six, two downregulated (clusterin and plasmin heavy chain A) and four upregulated (apolipoprotein A-IV; heparin cofactor II; complement C4B and haptoglobin related protein). Between R-PE and NR-PE there were four, one downregulated (fibronectin) and three upregulated (pregnancy-specific beta-1-glycoprotein 1 (PSG1); complement C4B (C4B) and complement C4A (C4A). Regarding PE, C4A and C4B intensity peaks were correlated with blood pressure and creatinine levels. Both, together with PSG1, showed significant correlations with the newborn's weight and gestational week (all p< 0.05). These findings help to understand the pathophysiological mechanisms between the two subgroups of the disease, since the under regulated circulating proteins act in the blood pressure modulation and the upregulated may occur as compensatory mechanisms due to an inflammatory scenario. These data give insights to explain the complexity and clinical consequences of this disorder.

Different Proteomic Profiles Regarding Antihypertensive Therapy in Preeclampsia Pregnant.

Preeclampsia (PE) is a hypertensive pregnancy syndrome associated with target organ damage and increased cardiovascular risks, necessitating antihypertensive therapy. However, approximately 40% of patients are nonresponsive to treatment, which results in worse clinical outcomes. This study aimed to compare circulating proteomic profiles and identify differentially expressed proteins among 10 responsive (R-PE), 10 nonresponsive (NR-PE) patients, and 10 healthy pregnant controls (HP). We also explored correlations between these proteins and clinical data. Plasma protein relative quantification was performed using mass spectrometry, followed by bioinformatics analyses with the UniProt database, PatternLab for Proteomics 4.0, and MetaboAnalyst software (version 6.0). Considering a fold change of 1.5, four proteins were differentially expressed between NR-PE and R-PE: one upregulated (fibronectin) and three downregulated (pregnancy-specific beta-1-glycoprotein 1, complement C4B, and complement C4A). Between NR-PE and HP, six proteins were differentially expressed: two upregulated (clusterin and plasmin heavy chain A) and four downregulated (apolipoprotein L1, heparin cofactor II, complement C4B, and haptoglobin-related protein). Three proteins were differentially expressed between R-PE and HP: one downregulated (transthyretin) and two upregulated (apolipoprotein C1 and hemoglobin subunit beta). These findings suggest a complex interplay of these proteins involved in inflammatory, immune, and metabolic processes with antihypertensive therapy responsiveness and PE pathophysiology.

Mechanism of the inhibitory effect of angiostatin on plasminogen activation by its physiologic activators

The influence of angiostatin K1-4.5--a fragment of the heavy chain of plasmin and a powerful inhibitor of angiogenesis--on kinetic parameters (k(Pg) and K(Pg)) of human Glu-plasminogen activation under the action of urokinase (uPA) not having affinity for fibrin and fibrin-specific tissue plasminogen activator (tPA) was investigated. Angiostatin does not affect the k(Pg) value, but increases the value K(Pg) urokinase plasminogen activation. A decrease in the k(Pg) value and an increase in the K(Pg) value were found for fibrin-stimulated plasminogen activation by tPA with increasing concentrations of angiostatin. The obtained results show that angiostatin is competitive inhibitor of the uPA activator activity, while it inhibits the activator activity of tPA by mixed type. Such an influence ofangiostatin on the kinetic constants ofthe urokinase plasminogen activation suggests that angiostatin dose dependent manner replaces plasminogen in the binary enzyme-substrate complex uPA-Pg. In case of fibrin-stimulated plasminogen activation by tPA, both zymogen and tPA are bound to fibrin with formation of the effective triple tPA-Pg-fibrin complex. Angiostatin replaces plasminogen both from the fibrin surface and from the enzyme-substrate tPA-Pg complex that leads to a decrease in k(Pg) and an increase in K(Pg) of plasminogen activation. Inhibition constants by angioststin (Ki) of plasminogen-activator activities of uPA and tPA determined by Dixon method were found to be 0.59 +/- 0.04 and 0.12 +/- 0.05 microM, respectively.

Structural/Functional Characterization of the α2-Plasmin Inhibitor C-Terminal Peptide

The alpha(2)-plasmin inhibitor (A2PI) is a main physiological regulator of the trypsin-like serine proteinase plasmin. It is composed of an N-terminal 15 amino acid fibrin cross-linking polypeptide, a 382-residue serpin domain, and a flexible C-terminal segment. The latter, peptide Asn(398)-Lys(452), and its Lys452Ala mutant were expressed as recombinant proteins in Escherichia coli (r-A2PIC and r-A2PICmut, respectively). CD and NMR analyses indicate that r-A2PIC is flexible, loosely folded, and with low content of regular secondary structure. Functional characterization via intrinsic fluorescence ligand titrations shows that r-A2PIC interacts with the isolated plasminogen kringle 1 (r-K1) (K(a) approximately 69.9 mM(-)(1)), K4 (K(a) approximately 45.7 mM(-)(1)), K5 (K(a) approximately 4.3 mM(-)(1)), and r-K2 (K(a) approximately 3.2 mM(-)(1)), all of which are known to exhibit lysine-binding capability. The affinities of these kringles for r-A2PIC are consistently larger than those reported for the ligand N(alpha)-acetyllysine, a mimic of a C-terminal Lys residue. The r-A2PICmut, with a C-terminal Ala residue, also interacts with r-K1 and K4, although with approximately 5-fold lesser affinities relative to r-A2PIC, demonstrating that while Lys(452) plays a major role in the binding, internal residues in r-A2PIC tether the kringles. (1)H NMR spectroscopy shows that key aromatic residues within the K4 lysine-binding site (LBS), namely, Trp(25), Trp(62), Phe(64), Trp(72), and Tyr(74), selectively respond to the addition of r-A2PIC and r-A2PICmut, indicating that these interactions proceed via the kringles' canonical LBS. We conclude that r-A2PIC docks to kringles primarily through lysine side chains and that Lys(452) most definitely enhances the binding. This suggests that multiple Lys residues within A2PI could contribute, perhaps in a zipper-like fashion, to its binding to the in-tandem, multikringle array that configures the plasmin heavy chain.

Stopped-flow fluorescence kinetics of bovine alpha 2-antiplasmin inhibition of bovine midiplasmin.

In the conversion of bovine plasminogen to bovine plasmin not only the expected urokinase-catalysed cleavage of Arg-557-Val-558, and the following autocatalytic cleavage separating the N-terminal peptide 1-77 from the heavy chain of plasmin, but also a cleavage at Arg-342-Met-343 between kringles 3 and 4 is seen. Here, kinetic studies of the interaction of bovine alpha 2-antiplasmin with bovine plasmin were performed on isolated bovine midiplasmin (lacking kringles 1-3) and on bovine plasmin containing all of the activation products from the bovine plasminogen. A series of experiments using stopped-flow fluorescence fast kinetics as well as conventional techniques suggests a reaction model in accordance with the one known for the human system. First, a tight complex (K1 in the nanomolar range) is formed in a fast reaction step; and second, a tightening of this complex occurs in a slow reaction step. The final complex is indeed so tight (Ki < or = pM), that the reaction for many practical purposes is legitimately considered irreversible. The stopped-flow method allows for the determination of reliable values of the second-order rate constant for the fast association step. At pH 7.4 and 25 degrees C, k+1 = 1.7 x 10(6) M-1 s-1 was obtained in the absence and k+1 = 0.9 x 10(6) M-1.s-1 in the presence of the kringles 1-3 domain of bovine plasmin. In contrast to this, substantial reductions of k+1 were seen in the presence of concentrations of 6-amino-hexanoic acid corresponding to lysine-binding-site interactions and far too low to be attributed to active-site interactions with the bovine plasmins (for each, Ki = 42 mM). All in all, the data indicated that the lysine-binding site(s) not of kringle 1, but of midiplasmin (those of kringles 4 and 5) are regulating the inhibition reaction.

Localization of epsilon-lysyl-gamma-glutamyl cross-links in alpha 2-macroglobulin-plasmin complex.

The major epsilon-lysyl-gamma-glutamyl cross-links present in the largely covalent 1:1 alpha 2-macroglobulin-(alpha 2M)-plasmin complex have been localized. Cross-linking engaged Lys607, -708, and -750 of the plasmin light chain, Lys550, -556, and -557 of the heavy chain-light chain connecting strand, Lys258 and -298 of the kringle 3 region, and Lys473 of the kringle 5 region of the plasmin heavy chain. Lys607, -708, and -750 accounted for 75% of all cross-linking, Lys550, -556, and -557 accounted for 20%, and Lys258, -298, and -473 accounted for 5%. Hence, cross-linking engaged only nine of the 41 Lys residues of plasmin, showing that, probably due to their large size, individual plasmin molecules become deposited in the large elongated binding cavity in alpha 2M in a relatively uniform way. The cross-linking of Lys residues in the heavy chain-light chain connecting strand to alpha 2M explains earlier findings that a substantial portion of the heavy chain is cross-linked to alpha 2M [Pizzo et al. (1986) Biol. Chem. Hoppe-Seyler 367, 1177-1182]. Although located in the heavy chain, Lys550, -556 and -557 should be considered part of the serine proteinase domain. That part of plasmin must be deeply buried in the alpha 2M structure in close vicinity to the four thiol esters, while most of the elongated heavy chain is protruding from the binding cavity. The pattern of cross-linked species seen in sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that individual plasmin molecules are bound to alpha 2M through one or two cross-links. Bivalent cross-linking can take place within or between 360-kDa alpha 2M dimers, pointing to the tetrameric alpha 2M structure as the functional proteinase binding unit.

Analysis of thiolester bond cleavage-dependent conformational changes in binary α 2-macroglobulin-proteinase complexes

The structures of the two proteinase-binding sites in human α 2-macroglobulin (α 2M) were probed by treatment of α 2M with the serine proteinases thrombin and plasmin. Each proteinase forms an equimolar complex with α 2M (a binary α 2M-proteinase complex) which results in the activation and cleavage of two internal thiolester bonds in α 2M. Binary α 2M-proteinase complexes demonstrated an incomplete conformational change as determined by nondenaturing polyacrylamide gel electrophoresis and incomplete receptor recognition site exposure as determined by in vivo plasma elimination studies. Treatment of binary α 2M-proteinase complexes with CH 3NH 2, trypsin, or elastase resulted in cleavage of an additional one or two thiolester bonds in α 2M and complete receptor recognition site exposure, demonstrating that a limited conformational change had occurred. Treatment of the α 2M-thrombin complex with elastase resulted in the incorporation of approximately 0.5 mol proteinase/mol α 2M and completion of the conformational change in the complex. Similar treatment of the α 2M-plasmin complex resulted in the incorporation of less than 0.1 mol proteinase/mol α 2M. Unlike the α 2M-thrombin complex, the α 2M-plasmin complex did not undergo a complete conformational change following treatment with CH 3NH 2 or trypsin. Incubation of this complex with elastase resulted in proteolysis of the kringle 1–4 region of the α 2M-bound plasmin heavy chain, and following this treatment the α 2M-plasmin complex underwent a complete conformational change. The results of this investigation demonstrate that binary α 2M-proteinase complexes retain a relatively intact proteinase-binding site. In the case of the α 2M-plasmin complex, however, the heavy chain of α 2M-bound plasmin protrudes from the proteinase-binding site and prevents a complete conformational change in the complex despite additional thiolester bond cleavage.

Isolation, purification and 1H-NMR characterization of a kringle 5 domain fragment from human plasminogen

A scheme is proposed for generating the intact Val-448-Phe-545 polypeptide of human plasminogen which contains the fifth kringle domain of the plasmin heavy chain. The procedure is based on a pepsin fragmentation of miniplasminogen and involves the purification of the kringle 5-containing fragment by gel filtration and ion-exchange chromatography. The final product is characterized by amino acid analysis, N- and C-terminal analyses, and high-resolution 1H-NMR spectroscopy at both 300 MHz and 611 MHz. We detect a (40:60%) Asp / Asn heterogeneity at site 452 of the Glu-plasminogen molecule. In the conventional kringle numbering system, the kringle 5 domain extends from Cys-1 to Cys-80, which corresponds to Cys-461 to Cys-540 in plasminogen. A preliminary 1H-NMR characterization of kringle 5 focuses on the global conformational features of the polypeptide. Assignments are given for a number of resonances, including the Tyr-72, the His imidazoles' and the Trp indoles' spin systems. Comparison with human plasminogen kringles 1 and 4 shows that the kringle 5 conformation is highly structured and very similar to that of the homologous domains. This conservancy is particularly striking in the environment surrounding Leu-46 and in the overall features of the aromatic spectrum. There are some differences, particularly in the buried His-33 imidazole group, whose H2 resonance is shifted to 9.67 ppm. A preliminary study of benzamidine-binding shows that the ligand interacts weakly ( K a ≈ 1.7 mM 1) mainly through the amidino functional group. Trp-62 and Tyr-72 are significantly perturbed by benzamidine, suggesting that these residues are part of the ligand-binding site.

Topography of the high-affinity lysine binding site of plasminogen as defined with a specific antibody probe.

An antibody population that reacted with the high-affinity lysine binding site of human plasminogen was elicited by immunizing rabbits with an elastase degradation product containing kringles 1-3 (EDP I). This antibody was immunopurified by affinity chromatography on plasminogen-Sepharose and elution with 0.2 M 6-aminohexanoic acid. The eluted antibodies bound [125I]EDP I, [125I]Glu-plasminogen, and [125I]Lys-plasminogen in radioimmunoassays, and binding of each ligand was at least 99% inhibited by 0.2 M 6-aminohexanoic acid. The concentrations for 50% inhibition of [125I]EDP I binding by tranexamic acid, 6-aminohexanoic acid, and lysine were 2.6, 46, and 1730 microM, respectively. Similar values were obtained with plasminogen and suggested that an unoccupied high-affinity lysine binding site was required for antibody recognition. The antiserum reacted exclusively with plasminogen derivatives containing the EDP I region (EDP I, Glu-plasminogen, Lys-plasminogen, and the plasmin heavy chain) and did not react with those lacking an EDP I region [miniplasminogen, the plasmin light chain or EDP II (kringle 4)] or with tissue plasminogen activator or prothrombin, which also contain kringles. By immunoblotting analyses, a chymotryptic degradation product of Mr 20,000 was derived from EDP I that retained reactivity with the antibody. The high-affinity lysine binding site was equally available to the antibody probe in Glu- and Lys-plasminogen and also appeared to be unoccupied in the plasmin-alpha 2-antiplasmin complex. alpha 2-Antiplasmin inhibited the binding of radiolabeled EDP I, Glu-plasminogen, or Lys-plasminogen by the antiserum, suggesting that the recognized site is involved in the noncovalent interaction of the inhibitor with plasminogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of molecular forms of plasminogen and plasmin-inhibitor complexes in urokinase-activated human plasma.

Urokinase-activated human plasma was analysed by acetic acid/urea/polyacrylamide-gel electrophoresis. The bands representing plasminogen, the plasmin-alpha 2-plasmin inhibitor and plasmin-alpha 2-macroglobulin complexes were identified by immunoprecipitation with specific antibodies and by comparison with purified components. Plasminogen and the plasmin-inhibitor complexes were isolated from plasma or thrombin-clotted plasma containing 125I-labelled Glu-plasminogen (residues 1-790) and urokinase. The plasma was kept at 37 degrees C for 0.5 and 10 times the lysis time of the clotted plasma, the clotted plasma until lysis. The plasmin heavy chain from the plasmin-inhibitor complexes was subsequently prepared. Only in one case could a low-grade proteolytic conversion of Glu- forms into Lys/Met/Val-forms (residues 77-790, 68-790 and 78-790 respectively) during the preparations be detected. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and N-terminal sequence analysis of the purified plasminogen and plasmin heavy chain showed the following. The plasminogen in plasma was on the Glu- form. Glu-plasmin constituted 0.74 and 0.58 of the plasmin bound to the alpha 2-plasmin inhibitor in plasma after brief and prolonged activation respectively. The rest was Lys/Met/Val-plasmin. The clotted plasma contained about equal amounts of Glu-plasminogen and Lys/Met/Val-plasminogen, and predominantly Lys/Met/Val-plasmin complexed to alpha 2-plasmin inhibitor and alpha 2-macroglobulin. The results of the analysis of the purified material substantiated the identity of radioactive protein bands in the gel after acetic acid/urea/polyacrylamide-gel electrophoresis.

Plasmin regulating system from embryonal carcinoma F9 cells: Plasminases A,B and embrinogen

Two plasmin inactivators, plasminase A and B, and their inhibitor embrinogen were isolated from embryonal carcinoma F9 cells by preparative two-dimensional electrophoresis. Plasminases A and B have molecular weights of 160,000 and 82,000, respectively. Both are serine proteinases which digest the light chain of plasmin in a time dependent inactivation process. The heavy chain of plasmin is not affected by this action. Plasminases A and B show similar specificity towards synthetic and natural polypeptide inhibitors. The interaction of the two enzymes leads to their inhibition. Embrinogen (m.w. 84,000) inhibits both plasminases A and B as well as urokinase and plasmin. Its activation by trypsin creates embrin, a proteinase directed against plasmin heavy chain.

Activation pathway of glu-plasminogen to lys-plasmin by urokinase

When Glu-plasminogen (Glu-plg) was incubated with plasmin for various time intervals, and the mixture was activated by urokinase (UK), the activation rate increased gradually as incubation time increased. The presence of fibrin not only enhanced the activation rate of Glu-plg but also that of proteolytically modified form to some extent. The results of SDS-PAGE indicate that the release of N-terminal peptides from Glu-plg or Glu-plasmin takes place gradually when the concentration of plg was about 1 μM, and that Glu-plasmin I of larger molecular weight is more slowly converted to Lys-plasmin than Glu-plasmin II of smaller molecular weight. The amounts of carbohydrate moieties on the heavy chain of plasmin may influence the release of N-terminal peptide from Glu-plasmin. Kinetic studies indicate that Lys-plasmin has smaller Km than Glu-plasmin, thus the former being better enzymatically than the latter.

Human low-molecular-weight urinary urokinase. Partial characterization and preliminary sequence data of the two polypeptide chains.

Low-molecular-weight urokinase (molecular weight 33100) was separated by analytical and preparative isoelectric focusing into five major subforms with isoelectric points between 8.7 and 9.6. These subforms are very similar in molecular weight, specific activity, amino acid composition and content of amino sugar and their N-terminal sequence constellation is identical. Low-molecular-weight urokinase consists of two polypeptide chains connected by a single disulfide bridge. The N-terminal region of the heavy chain (calculated Mr 30700) exhibits homology within the first 46 residues analyzed, with the known primary structure of other serine proteases. The mini chain (Mr 2426), whose complete sequence was determined, consists of 21 residues which show homology with the primary structure of the C-terminal region of the plasmin heavy chain. Based on sequence data and homology criteria with serine proteases a single-chain urokinase precursor is postulated having a peptide bond constellation between heavy and light chain region compatible with the requirements for serine protease activation.

Plasmin: Photoaffinity labeling of a lysine-binding site which regulates clot lysis

Human plasmin has been photoaffinity-labeled by 4-azidobenzoylglycyl-L-lysine. When a solution of plasmin and this peptide is irradiated, the clot-lysis activity is markedly reduced, with only a small loss in activity toward an active-site titrant. Studies with radioactive peptide show that the label is incorporated into the heavy chain of plasmin, with little incorporation into the light chain containing the active site. Plasminogen has also been photoaffinity-labeled by using this reagent. The modified plasminogen can be activated to a plasmin with both active-site titrant and clot-lysis activity. These results establish the presence in plasmin of a lysine-binding site, distinct from that in plasminogen, which regulates clot lysis.

Effects of human granulocyte elastase on fibrinolysis.

The specific limited proteolysis of human plasminogen by human granulocyte elastase results in the cleavage of three peptides from the heavy chain of plasminogen. These three peptides bind to lysine- and fibrin-Sepharose. The remainder of the molecule possesses a molecular weight of 38000 and represents a fully activatable plasminogen lacking the lysine binding site (s) in the heavy chain. Upon treatment of this low molecular weight form of plasminogen with urokinase, esterolytic, fibrinolytic and amidolytic activities appear. The low molecular weight form of plasmin consists of two disulfide-linked polypeptide chains with molecular weights of approximately 25000 and 13000. The kinetic parameters of hydrolysis of synthetic substrates by the low molecular weight form of plasmin and native plasmin, respectively, are similar. Furthermore, the inhibitions of the two enzymes by several inhibitors of the hydrolysis of Blue Dextran-fixed fibrin are identical. The initial rate of activation with urokinase is about two times faster for the low molecular weight form of plasminogen than for native plasminogen, and the activation of native plasminogen by urokinase is inhibited by plasma α2-antiplasmin, whereas the activation of the low molecular weight form of plasminogen is not. Urokinase-induced clot lysis is inhibited efficiently by α2-antiplasmin, but the inhibitory effect of α2-antiplasmin is removed by heavy chain fragments containing lysine binding site (s), which are obtainable from plasminogen by limited elastase digestion. Urokinaseinduced fibrinolytic activity of plasma is promoted by human granulocyte elastase in an apparently time-dependent manner. The findings further indicate that the lysine binding site (s) in the heavy chain of plasmin (ogen) are of great importance in relation to the rate of its reaction with α2-antiplasmin.

The primary inhibitor of plasmin in human plasma.

A complex between plasmin and an inhibitor was isolated by affinity chromatography from urokinase-activated human plasma. The complex did not react with antibodies against any of the known proteinase inhibitors in plasma. A rabbit antiserum against the complex was produced. It contained antibodies agianst plasminogen+plasmin and an alpha2 protein. By crossed immunoelectrophoresis the alpha2 protein was shown to form a complex with plasmin, when generated by urokinase in plasma, and with purified plasmin. The alpha2 protein was eluted by Sephadex G-200 gel filtration with KD approx. 0.35, different from the other inhibitors of plasmin in plasma, and corresponding to an apparent relative molecular mass (Mr) of about 75000. By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Mr of the complex was found to be approx. 130000. After reduction of the complex two main bands of protein were observed, with Mr, about 72000 and 66000, probably representing an acyl-enzyme complex of plasmin-light chain and inhibitor-heavy chain, and a plasmin-heavy chain. A weak band with Mr 9000 was possibly an inhibitor-light chain. The inhibitor was partially purified and used to titrate purified plasmin of known active-site concentration. The inhibitor bound plasmin rapidly and strongly. Assuming an equimolar combining ratio, the concentration of active inhibitor in normal human plasma was estimated to be 1.1 mumol/1. A fraction about 0.3 of the antigenic inhibitor protein appeared to be functionally inactive. In plasma, plasmin is primarily bound to the inhibitor. Only after its saturation does lysis of fibrinogen and fibrin occur and a complex between plasmin and alpha2 macroglobulin appear.

Mechanism of the urokinase-catalyzed activation of human plasminogen.

When human plasminogen (Glu-Pga) is activated by urokinase in the presence of pancreatic trypsin inhibitor, the plasmin produced (Glu-Pma) exclusively contains a heavy chain (Glu-Ha) derived intact from the original NH2 terminus of Glu-Pga. Similar activations, utilizing a low molecular weight synthetic plasmin acylating agent, p-nitrophenyl-p-(pyridiniummethyl) benzoate, still result in a plasmin molecule with approximately 50% of the plasmin heavy chain containing the intact NH2 terminus of the original Glu-Pga. Activations performed at high levels of urokinase in the absence of any inhibitors initially produce Glu-Pma. However, the final stable plasmin, Lys-Pmb, which is obtained contains a heavy chain (Lys-Hb) which arises by plasminolysis of a small peptide from the NH2 terminus of Glu-Ha. Alternatively, Lys-Pmb can be formed in a separate series of reactions initially involving plasminolysis of Glu-Pga to yield Lys-Pgb. The peptide removed in this step is identical to the peptide removed in the Glu-Ha to Lys-Hb reaction. Next, urokinase catalyzes the conversion of Lys-Pgb to Lys-Pmb without further loss of peptide material. This latter pathway involving Lys-Pgb is probably the major pathway for human Lys-Pmb generation. These studies support a mechanism of activation of human plasminogen which involves at least two bond cleavages in Glu-Pga. However, these same studies strongly indicate that the Nh2-terminal peptide need not be released from Glu-Pga prior to plasmin formation. Further, we feel that plasmin and not urokinase catalyzes cleavage of the NH2-terminal peptide bond from Glu-Pga and the Glu-Ha heavy chain of Glu-Pma.

Carbohydrate compositions of the rabbit plasminogen isozymes

The carbohydrate compositions of the two affinity-chromatography-resolved isozymes of rabbit plasminogen and plasmin as well as the isoelectric-focusing-resolved subforms of each plasminogen isozyme have been investigated in detail. The first plasminogen isozyme as well as its subforms all possess four to five residues of N-acetylglucosamine, two residues of N-acetylgalactosamine, three residues of mannose and five residues of galactose per molecule of protein. Additionally, we previously reported three residues of sialic acid present on this protein molecule. The corresponding plasmin heavy chain for this isozyme contains essentially all of the carbohydrate, and the plasmin light chain appears devoid of carbohydrate. On the other hand, the second plasminogen isozyme as well as its subforms all possess only trace amounts of N-acetylglucosamine, two residues of N-acetylgalactosamine, less than one residue of mannose and three residues of galactose per molecule of protein. In addition, we have previously reported two residues of sialic acid for this molecule. Here, also, all carbohydrate appears on the heavy chain of the plasmin, which is prepared by activation of this particular plasminogen. Thus, the carbohydrate differences which we reported earlier in rabbit plasminogen isozymes are confirmed and extended.

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.