Human Plg Research Articles

Proteoform-Resolved Profiling of Plasminogen Activation Reveals Novel Abundant Phosphorylation Site and Primary N-Terminal Cleavage Site

Plasminogen (Plg), the zymogen of plasmin (Plm), is a glycoprotein involved in fibrinolysis and a wide variety of other physiological processes. Plg dysregulation has been implicated in a range of diseases. Classically, human Plg is categorized into two types, supposedly having different functional features, based on the presence (type I) or absence (type II) of a single N-linked glycan. Using high-resolution native mass spectrometry, we uncovered that the proteoform profiles of human Plg (and Plm) are substantially more extensive than this simple binary classification. In samples derived from human plasma, we identified up to 14 distinct proteoforms of Plg, including a novel highly stoichiometric phosphorylation site at Ser339. To elucidate the potential functional effects of these post-translational modifications, we performed proteoform-resolved kinetic analyses of the Plg-to-Plm conversion using several canonical activators. This conversion is thought to involve at least two independent cleavage events: one to remove the N-terminal peptide and another to release the active catalytic site. Our analyses reveal that these processes are not independent but are instead tightly regulated and occur in a step-wise manner. Notably, N-terminal cleavage at the canonical site (Lys77) does not occur directly from intact Plg. Instead, an activation intermediate corresponding to cleavage at Arg68 is initially produced, which only then is further processed to the canonical Lys77 product. Based on our results, we propose a refined categorization for human Plg proteoforms. In addition, we reveal that the proteoform profile of human Plg is more extensive than that of rat Plg, which lacks, for instance, the here-described phosphorylation at Ser339.

Abstract 3613: Effect of thrombomodulin and plasminogen interaction on melanoma spheroid invasion

Abstract Background: Thrombomodulin (TM), a transmembrane glycoprotein that acts as a plasminogen (Plg) receptor, has been found as a potential molecular and prognostic marker that impacts the spread of various cancers, including malignant melanoma. A previous study showed that TM plays a vital function in angiogenesis activity [1] but its role in the malignant melanoma invasion is unclear. Here, we investigate the role of TM-Plg by using melanoma spheroid (MS) embedded in an extracellular matrix (ECM) modeled to understand how TM-Plg affects the progression of MS invasion. Methods: In this work, A2058 (A2058-GFP and A2058-TMGFP) and MeWo (siC and siTM) MS with different TM expressions were formed, harvested, and embedded in type I collagen gel for invasion assay. TM and Plg expression were determined by western blot. The surface of A2058 MS was observed by SEM imaging. Human Plg, MMPs, and Plg inhibitors, tranexamic acid (TXA) and epsilon-aminocaproic acid (ε-ACA), were utilized to investigate the effect of TM on the plasmin system. Immunofluorescence of TM and F-actin were validated. Results: When the two cell lines expressed with TM, the formed MS invaded to the surrounding matrix more distinctively than that of the TM-silencing ones, with a 2.5-fold difference in the ratio invasive area change. Upon placing the two A2058-TMGFP MS in the vicinity of each other within the ECM, they showed a strong interaction with an observed distance between the two MS from 50 μm to 280 μm. Immunofluorescence of TMGFP assessed the role of TM in MS invasion and interaction in ECM. Microvilli structures were found on the surface of A2058-TMGFP cells that may help the cell have more cellular processes such as adhesion, signal transduction, and metabolic activities. Furthermore, TXA, ε-ACA, and MMP inhibitors inhibited the invasion of A2058-TMGFP MSs in a dose-dependent manner. Human Plg stimulated invasion in TM-expressing MSs while no effect on non-TM MSs. The results suggested that the human Plg only significantly increased the MS invasion with the presence of TM. Conclusion: We concluded that with the presence of TM, the MS invasion and interaction become stronger than MS without TM expression. Moreover, TM enhances MS invasion in the 3D model via interacting Plg. This study provides evidence for the development of TM antagonists in the future to analyze the drug's potential as an anti-metastasis treatment.[1] P. K. Chen et al., “Thrombomodulin functions as a plasminogen receptor to modulate angiogenesis,” FASEB J., vol. 27, no. 11, pp. 4520-4531, 2013, doi: 10.1096/fj.13-227561. Citation Format: Thi Kim Ngan Ngo, Hua-Lin Wu, Cheng-Hsiang Kuo, Ting-Yuan Tu. Effect of thrombomodulin and plasminogen interaction on melanoma spheroid invasion. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3613.

Identification and characterization of Taenia solium enolase as a plasminogen-binding protein

The larval stage of Taenia solium (cysticerci) is the causal agent of human and swine cysticercosis. When ingested by the host, T. solium eggs are activated and hatch in the intestine, releasing oncospheres that migrate to various tissues and evolve into cysticerci. Plasminogen (Plg) receptor proteins have been reported to play a role in migration processes for several pathogens. This work is aimed to identify Plg-binding proteins in T. solium cysticerci and determine whether T. solium recombinant enolase (rTsEnoA) is capable of specifically binding and activating human Plg. To identify Plg-binding proteins, a 2D-SDS-PAGE ligand blotting was performed, and recognized spots were identified by MS/MS. Seven proteins from T. solium cysticerci were found capable of binding Plg: fascicilin-1, fasciclin-2, enolase, MAPK, annexin, actin, and cytosolic malate dehydrogenase. To determine whether rTsEnoA binds human Plg, a ligand blotting was performed and the results were confirmed by ELISA both in the presence and absence of εACA, a competitive Plg inhibitor. Finally, rTsEnoA-bound Plg was activated to plasmin in the presence of tPA. To better understand the evolution of enolase isoforms in T. solium, a phylogenetic inference analysis including 75 enolase amino acid sequences was conducted. The origin of flatworm enolase isoforms, except for Eno4, is independent of their vertebrate counterparts. Therefore, herein we propose to designate tapeworm protein isoforms as A, B, C, and 4. In conclusion, recombinant enolase showed a strong plasminogen binding and activating activity in vitro. T. solium enolase could play a role in parasite invasion along with other plasminogen-binding proteins.

Plasminogen Tochigi mice exhibit phenotypes similar to wild-type mice under experimental thrombotic conditions.

Plasminogen (Plg) is a precursor of plasmin that degrades fibrin. A race-specific A620T mutation in Plg, also known as Plg-Tochigi, originally identified in a patient with recurrent venous thromboembolism, causes dysplasminogenemia with reduced plasmin activity. The Plg-A620T mutation is present in 3–4% of individuals in East Asian populations, and as many as 50,000 Japanese are estimated to be homozygous for the mutant 620T allele. In the present study, to understand the changes of thrombotic phenotypes in individuals with the mutant 620T allele, we generated knock-in mice carrying the homozygous Plg-A622T mutation (PlgT/T), an equivalent to the A620T mutation in human Plg. PlgT/T mice grew normally but showed severely reduced plasmin activity activated by urokinase, equivalent to ~8% of that in wild-type mice. In vitro fibrin clot lysis in plasma was significantly slower in PlgT/T mice than in wild-type mice. However, all experimental models of electrolytic deep vein thrombosis, tissue factor-induced pulmonary embolism, transient focal brain ischaemic stroke, or skin-wound healing showed largely similar phenotypes between PlgT/T mice and wild-type mice. Protein S-K196E mutation (Pros1E/E) is a race-specific genetic risk factor for venous thromboembolism. Coexistence in mice of PlgT/T and Pros1E/E did not affect pulmonary embolism symptoms, compared with those in Pros1E/E mice. Hence, the present study showed that the Plg-A622T mutation, which confers ~8% plasmin activity, does not increase the risk of thrombotic diseases in mice under experimental thrombotic conditions and does not modify the thrombotic phenotype observed in Pros1E/E mice. PlgT/T mice can be used to investigate the potential pathophysiological impact of the Plg-A620T mutation.

Lipoprotein(a) Binds to Recombinant Nontypeable <i>Haemophilus influenzae</i> Aspartase

The respiratory pathogen nontypeable <i>Haemophilus influenza</i> (NTHi) can recruit plasminogen (Plg) on the cell surface by its Plg receptor aspartase (ASP) and utilize host Plg and fibrinolytic system to achieve its adherence and immune invasion. Lipoprotein(a) [Lp(a)] consists of one molecule low-density lipoprotein (LDL) and one molecule apolipoprotein(a) [Apo(a)]. Apo(a) shares a high degree of homology with the human Plg, and both of them contain lysine-binding sites (LBS), which enables them to interact with various cell-surface receptors or fibrin(ogen). However, the definite physiological function of Lp(a) remains vague. Here, we present evidence that Lp(a) via its Apo(a) may bind to thePlg receptor ASP. Recombinant aspartase (rASP) and C-terminal lysine-deleted variant of ASP (rASPΔK) were used in the current study. The rASP specifically bound to Lp(a), but rASPΔK did not, indicating that C-terminal lysine residue of rASP was responsible for the interaction. In addition, rASP interacted with Lp(a), but not with LDL, revealing that LBS of Apo(a) was involved in the binding. Our results also showed that Lp(a) could inhibit the binding of Plg to rASP. Plasma Lp(a) might play a role in anti-NTHi infection by binding to its Plg receptor ASP.

Glycolytic and Non-glycolytic Functions of Mycobacterium tuberculosis Fructose-1,6-bisphosphate Aldolase, an Essential Enzyme Produced by Replicating and Non-replicating Bacilli

The search for antituberculosis drugs active against persistent bacilli has led to our interest in metallodependent class II fructose-1,6-bisphosphate aldolase (FBA-tb), a key enzyme of gluconeogenesis absent from mammalian cells. Knock-out experiments at the fba-tb locus indicated that this gene is required for the growth of Mycobacterium tuberculosis on gluconeogenetic substrates and in glucose-containing medium. Surface labeling and enzymatic activity measurements revealed that this enzyme was exported to the cell surface of M. tuberculosis and produced under various axenic growth conditions including oxygen depletion and hence by non-replicating bacilli. Importantly, FBA-tb was also produced in vivo in the lungs of infected guinea pigs and mice. FBA-tb bound human plasmin(ogen) and protected FBA-tb-bound plasmin from regulation by α(2)-antiplasmin, suggestive of an involvement of this enzyme in host/pathogen interactions. The crystal structures of FBA-tb in the native form and in complex with a hydroxamate substrate analog were determined to 2.35- and 1.9-Å resolution, respectively. Whereas inhibitor attachment had no effect on the plasminogen binding activity of FBA-tb, it competed with the natural substrate of the enzyme, fructose 1,6-bisphosphate, and substantiated a previously unknown reaction mechanism associated with metallodependent aldolases involving recruitment of the catalytic zinc ion by the substrate upon active site binding. Altogether, our results highlight the potential of FBA-tb as a novel therapeutic target against both replicating and non-replicating bacilli.

A pilot study on purification of human plasminogen from cord blood plasma and inducing posterior vitreous detachment

Background Many studies and clinical trials of pharmacologic vitreolysis are already under way to try to improve vitreo-retinal surgery and to liquefy and detach the vitreous from the retina ultimately, including chondroitinase,hyaluronidase,dispase and plasmin. However, there has not been any report on purification of human plasminogen from cord blood plasma and inducing posterior vitreous detachment of the animal eye at present.Objective This study was designed to isolate and purify the production of human plasminogen (Plg) from cord blood plasma with ethanol precipitation and evaluate the efficacy of Plg in inducing posterior vitreous detachment (PVD).Methods Human Plg was Separated and purified from cord blood plasma by ethanol precipitation method. The protein band corresponding to Plg with molecular mass of 92 000 was revealed in SDS-PAGE and confirmed by MALDI-TOF and Mascot database. Anion-exchange chromatography and plasminogen activity assay kit were used to obtain purified Plg with biological activity. Twenty-five fresh pig eyes were enucleated and assigned to 5 groups and 5 eyes for each group. The normal eyes were used as control group. Balanced salt solution(BSS)of 0.1 ml was intravitreally group and standard substance group. All of the eyes were then incubatedfor 60 minutes under the 37 ℃. Retinal histopathology and ultrastructure were examined under the light microscopy, scanning electron microscopy ( SEM ) and transmission electron microscopy (TEM). Results The Plg with potential fibrinolytic activity was successfully extracted and purified from cord blood plasma by ethanol precipitation method. No posterior vitreous detachment (PVD) was seen in normal control group, BSS group and r-SK group following the intravitreal injection under the sem. However,PVD was demonstrated in r-SK+ Plg group and standard substance group under the SEM. The inner limiting membrane ( ILM ) and the retina were well preserved in all of the experimental eyes. No retinal morphology and ultrastructural abnormality were found under the light and SEM and TEM. Conclusion Ethanol precipitation is a feasible way to isolate and purify Plg from human cord blood plasma. Extracted Plg shows potential fibrinolytic intravitreal injection of Plg. Key words: Human plasminogen; Recombinant streptokinase; Cord blood; Posterior vitreous detachment

Plasmin Is Essential in Preventing Periodontitis in Mice

Periodontitis involves bacterial infection, inflammation of the periodontium, degradation of gum tissue, and alveolar bone resorption, which eventually leads to loss of teeth. To study the role of the broad-spectrum protease plasmin in periodontitis, we examined the oral health of plasminogen (Plg)-deficient mice. In wild-type mice, the periodontium was unaffected at all time points studied; in Plg-deficient mice, periodontitis progressed rapidly, within 20 weeks. Morphological study results of Plg-deficient mice revealed detachment of gingival tissue, resorption of the cementum layer, formation of necrotic tissue, and severe alveolar bone degradation. IHC staining showed massive infiltration of neutrophils in the periodontal tissues. Interestingly, doubly deficient mice, lacking both tissue- and urokinase-type plasminogen activators, developed periodontal disease similar to that in Plg-deficient mice; however, mice lacking only tissue- or urokinase-type plasminogen activator remained healthy. Supplementation by injection of Plg-deficient mice with human plasminogen for 10 days led to necrotic tissue absorption, inflammation subsidence, and full regeneration of gum tissues. Notably, there was also partial regrowth of degraded alveolar bone. Taken together, our results show that plasminogen is essential for the maintenance of a healthy periodontium and plays an important role in combating the spontaneous development of chronic periodontitis. Moreover, reversal to healthy status after supplementation of Plg-deficient mice with plasminogen suggests the possibility of using plasminogen for therapy of periodontal diseases.

Activation of Plasminogen by Staphylokinase Reduces the Severity ofStaphylococcus aureusSystemic Infection

Staphylokinase (SAK) is produced by the majority of Staphylococcus aureus strains. It is an extracellular protein that activates the conversion of human plasminogen (plg) to plasmin. The role played by SAK in staphylococcal infection is unclear. Wild-type S. aureus strain LS-1, which lacks the ability to produce SAK, was modified by an insertion of the sak gene into its chromosome. The sak gene was integrated in 2 forms--(1) linked to its own promoter and (2) fused to the promoter of the protein A gene--which resulted in the overexpression of SAK. SAK is highly specific for human plg and exhibits almost no activity toward murine plg. To investigate the role played by SAK in a murine infection model, human plg transgenic mice and their wild-type counterparts were inoculated intravenously with congenic S. aureus strains differing in SAK production. Human plg transgenic mice inoculated with SAK-expressing strains displayed significantly reduced mortality, less weight loss, and lower bacterial loads in kidneys than did the wild-type mice. No difference in the severity of sepsis was observed between transgenic and wild-type mice infected with a SAK-deficient strain. The results suggest that expression of SAK followed by activation of plg alleviates the course of S. aureus sepsis.

Comparison of the effect of homocysteine and its thiolactone on the fibrinolytic system using human plasma and purified plasminogen

L-Homocysteine (Hcys) occurs in human blood in free (i.e. as reduced form—about 100 nM) and protein bound form. Increased concentration of Hcys in the blood is called hyperhomocysteinemia (about [15 lM). Mechanisms involved in the relationship between hyperhomocysteinemia and haemostatic process are still unclear and sometimes controversial. In the literature there are few papers describing studies on the effects of Hcys on proteins that participate in blood coagulation and fibrinolysis in human. The most reactive form of Hcys is its cyclic thioester— homocysteine thiolactone (HTL; formed through editing mechanisms with methionyl t-RNA synthetase), which represents up to 0.29% of plasma total homocysteine, and the Hcysor tiolactone-induced modifications of haemostatic proteins (N-homocysteinylation or S-homocysteinylation) seem to be the main mechanism of biotoxicity of these compounds. Fibrinogen, plasminogen (Plg) and other haemostatic proteins can be also covalently modified by Hcys and HTL [1, 2]. The aim of our study was to establish and compare the effect of a reduced form of L-Hcys (C95%, Sigma Chemical Company, St. Louis, MO, USA) and its cyclic thioester (C99%, Sigma Chemical Company, St. Louis, MO, USA) on the fibrinolytic system (using human plasma or purified plasminogen). Blood samples were taken from 12 healthy volunteers (aged between 26 and 32 years; mean 29.7 ± 2.5) without cardiovascular disorders, allergy and lipid or carbohydrate metabolism disorders, untreated with drugs. Healthy subjects did not use addictive substances and antioxidant supplementation, their diet was balanced (meat and vegetables), lived in similar socio-economic conditions. Subjects with significant medical illness were excluded. They were no smokers. Human blood was collected at 3.2% citrate (used to 12 mM final concentration) and immediately centrifuged (20009g, 15 min) to get plasma. Plg was isolated by affinity chromatography on Lysine–Sepharose [3]. The natural concentration of total Hcys in plasma was 10.6 ± 3.8 lM. The endogenous concentration of reduced form of Hcys and HTL was about 100 ± 11.2 and 0–35 nM, respectively. The classical technique HPLC has been used to analysis Hcys or HTL in human plasma. The HPLC analysis was performed with a Hewlett-Packard 1100 Series system according to Glowacki et al. [4] and Bald et al. [5]. The concentration of Plg in plasma was 2 ± 0.3 lM. Samples of human Plg (2 lM) were exposed to the reduced form of L-homocysteine (at a final concentration between 10 and 100 lM); or to homocysteine thiolactone (at a final concentration between 0.1 and 1 lM) in the presence of 100 mM potassium phosphate buffer, pH 7.4, for 30 min at 37 C. Human plasma was exposed also to the reduced form of L-homocysteine (at a final concentration between 10 and 100 lM); or to homocysteine thiolactone (at a final concentration between 0.1 and 1 lM) for 30 min at 37 C. The tested concentrations of Hcys or HTL correspond to levels found in human plasma during hyperhomocysteinemia in vivo. Plasmin activity and Plg activation were estimated by the hydrolysis of chromogenic substrate by streptokinase (SK) or by tissue plasminogen activator (tPA); assays were performed at room temperature in 96well polystyrene flat-bottom plates. The absorbance measurements were performed in a microplate reader (Bio-Rad Microplate Reader, model 550) at 415 nm. No activity of generated plasmin was detected in the absence of SK or tPA. We also studied changes of proteolytic activities of J. Kolodziejczyk J. Malinowska P. Nowak B. Olas (&) Department of General Biochemistry, Institute of Biochemistry, University of Łodź, Banacha 12/16, 90-237 Lodz, Poland e-mail: olasb@biol.uni.lodz.pl

Human PLG Antibodies as a HNSCC Therapeutic Benefit

Free accessAbstractFirst published online August 2010Human PLG Antibodies as a HNSCC Therapeutic BenefitIsaac Dingle, MBA, presenter, James Byrd, MD, […], Brian Hoel, PhD, M. Boyd Gillespie, MSc, MD, and Natalie Sutkowski, PhD, +2 -2Volume 143, Issue 2_supplhttps://doi.org/10.1016/j.otohns.2010.06.346

Structural Consideration of the Formation of the Activation Complex between the Staphylokinase-Like Streptococcal Plasminogen Activator PadA and Bovine Plasminogen

The characteristics of a streptococcal plasminogen activator (PA) displaying specificity for ruminant plasminogen (Plg) were defined using molecular approaches. The 16-kDa secreted protein PadA was found to be prevalent in Streptococcus dysgalactiae subspecies dysgalactiae isolated from cases of bovine mastitis and septic arthritis in lambs. PadA was able to activate bovine, ovine and caprine Plg, but not human Plg. Amino acid sequence analysis identified a limited level of homology to other streptococcal PAs, including streptokinase; however, PadA was found to align well with and match in size the staphylococcal PA, staphylokinase. Recombinant PadA was used to investigate interaction with bovine Plg, leading to formation of an activator complex that was capable of recruiting and converting further substrate Plg into plasmin. Individual non-overlapping peptides of PadA or bovine microplasminogen were found to block the interaction between PadA and bovine Plg, preventing the formation of the activation complex. Homology modelling based upon structures of staphylokinase complexed with human microplasminogen supported these findings by placing critical residues in close proximity to the plasmin component of the activation complex.

Receptor recognition specificity of plasminogen for the novel plasminogen receptor, Plg‐RKT

Plasminogen (Plg) receptors promote Plg activation to arm cells with plasmin to promote both thrombolysis and cell migration. Plg‐RKT is a new type II integral membrane Plg receptor on leukocytes and other tissues. Our objective was to define the recognition specificity of Plg for Plg‐RKT. The C‐terminal peptide of Plg‐RKT was coupled to microtiter wells. Biotinylated Plg was incubated with the immobilized peptide and detected with streptavidin‐HRP. Plg bound to the peptide in a concentration‐dependent manner (IC50=5 nM). In specificity controls, Plg binding was competed by unlabeled Plg and no binding to the reverse peptide was detected. Plg binding was inhibited in a dose‐dependent manner by the soluble peptide, but was not affected by a mutant peptide with the C‐terminal K replaced with A. A peptide with the proximal upstream R replaced with K was a more effective competitor than the wild type peptide. Human Plg also recognized the C‐terminal peptides of murine, rat, and bovine Plg‐RKT's that contain the same R6K substitution. Plg domains containing disulfide‐bonded kringles (K) inhibited the interaction with the following order of effectiveness: Plg&gt;K1‐3&gt;K5&gt;K4. In conclusion, the interaction of Plg‐RKT with Plg requires the presence of a C‐terminal lysine and can be modulated by a proximal upstream basic residue. Furthermore, kringle domains within the Plg molecule interact with the C‐terminus of Plg‐RKT.

Extracellular proteins of Lactobacillus crispatus enhance activation of human plasminogen

The abundant proteolytic plasminogen (Plg)/plasmin system is important in several physiological functions in mammals and also engaged by a number of pathogenic microbial species to increase tissue invasiveness or to obtain nutrients. This paper reports that a commensal bacterium, Lactobacillus crispatus, interacts with the Plg system. Strain ST1 of L. crispatus enhanced activation of human Plg by the tissue-type Plg activator (tPA), whereas enhancement of the urokinase-mediated Plg activation was lower. ST1 cells bound Plg, plasmin and tPA only poorly, and the Plg-binding and activation-enhancing capacities were associated with extracellular material released from the bacteria into buffer. The extracellular proteome of L. crispatus ST1 contained enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as major components. The enolase and the GAPDH genes of ST1 were cloned, sequenced and expressed in recombinant Escherichia coli as His(6)-fusion proteins, which bound Plg and enhanced its activation by tPA. Variable levels of secretion of enolase and GAPDH proteins as well as of the Plg activation cofactor function were detected in strains representing major taxonomic groups of the genus Lactobacillus. So far, interference with the Plg system has been addressed with pathogenic microbes. The results reported here demonstrate a novel interaction between a member of the microbiota and a major proteolytic system in humans.

Plasminogen enhances virulence of group A streptococci by streptokinase-dependent and streptokinase-independent mechanisms.

Interactions between host plasminogen (Plg) and streptokinase (SK) secreted by group A streptococci (GAS) have been hypothesized to promote bacterial invasion of tissues. The virulence of GAS strain UMAA2616, after being subcutaneously inoculated into mice, was studied. Skin lesions and mortality were observed after inoculation of 7x106 cfu. Coadministration of human Plg with UMAA2616 markedly increased virulence. SK-deficient UMAA2616 (UMAA2616-SK(-)) was generated. Mean skin-lesion area and mortality, after bacterial inoculation (3x105 cfu), were significantly greater with UMAA2616 in the presence of human Plg than with UMAA2616-SK(-) in the presence of human Plg (P=.0001). Human Plg also enhanced UMAA2616-SK(-) virulence. Exogenous human Plg enhanced the virulence of MGAS166, a human clinical isolate. These findings suggest that SK-Plg interactions are an important determinant of GAS invasiveness in vivo and that both SK and host Plg activators appear to promote virulence of GAS by catalyzing plasmin formation.

Domain interactions between streptokinase and human plasminogen.

Plasmin (Pm), the main fibrinolytic protease in the plasma, is derived from its zymogen plasminogen (Plg) by cleavage of a peptide bond at Arg(561)-Val(562). Streptokinase (SK), a widely used thrombolytic agent, is an efficient activator of human Plg. Both are multiple-domain proteins that form a tight 1:1 complex. The Plg moiety gains catalytic activity, without peptide bond cleavage, allowing the complex to activate other Plg molecules to Pm by conventional proteolysis. We report here studies on the interactions between individual domains of the two proteins and their roles in Plg activation. Individually, all three SK domains activated native Plg. While the SK alpha domain was the most active, its activity was uniquely dependent on the presence of Pm. The SK gamma domain also induced the formation of an active site in Plg(R561A), a mutant that resists proteolytic activation. The alpha and gamma domains together yielded synergistic activity, both in Plg activation and in Plg(R561A) active site formation. However, the synergistic activity of the latter was dependent on the correct N-terminal isoleucine in the alpha domain. Binding studies using surface plasmon resonance indicated that all three domains of SK interact with the Plg catalytic domain and that the beta domain additionally interacts with Plg kringle 5. These results suggest mechanistic steps in SK-mediated Plg activation. In the case of free Plg, complex formation is initiated by the rapid and obligatory interaction between the SK beta domain and Plg kringle 5. After binding of all SK domains to the catalytic domain of Plg, the SK alpha and gamma domains cooperatively induce the formation of an active site within the Plg moiety of the activator complex. Substrate Plg is then recognized by the activator complex through interactions predominately mediated by the SK alpha domain.

The complete cDNA sequence encoding plasminogen from the European hedgehog ( Erinaceus europaeus)

Using a PCR-based strategy, we have determined the complete cDNA sequence encoding hedgehog plasminogen (Plg). The 2700-nucleotide cDNA (corresponding to a 2.9-kb liver-derived transcript) encodes an open reading frame of 811 amino acids which shares 74–76% identity with Plg characterized from mouse, human and rhesus monkey. Residues corresponding to the catalytic triad, tPA-cleavage site, as well as seven of the eight lysine-binding residues in kringle IV are conserved in the hedgehog. However, potential N-linked glycosylation sites which have been reported in human and rhesus Plg are not present in analogous positions in the hedgehog Plg sequence.

Platelet thrombospondin forms a trimolecular complex with plasminogen and histidine-rich glycoprotein.

Thrombospondin (TSP), a multifunctional alpha-granule glycoprotein of human platelets binds fibrinogen, fibronectin, heparin, histidine-rich glycoprotein (HRGP), and plasminogen (Plg), and thus, may play an important role in regulating thrombotic influences at vessel surfaces. In this study we have demonstrated that purified human platelet TSP formed a trimolecular complex with human Plg and HRGP. Complex formation was detected by a specific binding enzyme-linked immunosorbent assay (ELISA) which demonstrated simultaneous binding of fluid-phase Plg and HRGP to TSP adsorbed to microtitration wells. While neither ligand inhibited complex formation of the other with TSP, 10 mM epsilon-amino-n-caproic acid selectively blocked incorporation of Plg into the complex, suggesting that TSP contains independent binding sites for Plg and HRGP. Comparable extent of trimolecular complex formation was also detected when TSP monomer was substituted for whole TSP in the ELISA. HRGP covalently cross-linked to Sepharose 4B simultaneously bound both 125I-TSP and 131I-Plg, confirming trimolecular complex formation. Rocket immunoelectrophoresis of mixtures of the purified radiolabeled proteins into anti-Plg containing agarose also confirmed trimolecular complex formation. The TSP-HRGP-Plg complex bound a similar amount of heparin as the TSP-HRGP complex, demonstrating that the HRGP within the trimolecular complex maintained functional capability. Similarly, using a fluorometric plasmin substrate, the trimolecular complex was shown to be an effective substrate for tissue plasminogen activator. Significant amounts of plasmin were generated from the TSP-HRGP-Plg complex (equivalent to that from the TSP-Plg complex), but the rate of plasmin generation from the trimolecular complex was greater than from the bimolecular complex, suggesting an important interaction of HRGP with Plg when both are complexed to TSP. The macromolecular assembly of these three proteins on cellular surfaces, such as the platelet, may serve important regulatory functions, both prothrombotic at sites of active fibrin deposition and proteolytic in nonfibrin-containing microenvironments.