Generation of a novel proteolysis resistant vascular endothelial growth factor165 variant by a site-directed mutation at the plasmin sensitive cleavage site

Vascular endothelial growth factor (VEGF) is a potent angiogenic mediator in tissue repair. In non-healing human wounds plasmin cleaves and inactivates VEGF165. In the present study, we generated recombinant VEGF165 mutants resistant to plasmin proteolysis. Substitution of Arg110 with Ala110 or Gln110, and Ala111 with Pro111 yielded plasmin-resistant and biologically active VEGF165 mutants. In addition, substitution of Ala111 with Pro111 resulted in a substantial degree of stabilization when incubated in wound fluid obtained from non-healing wounds. These results suggest that the plasmin cleavage site Arg110/Ala111 and the carboxyl-terminal domain play an important role in the mitogenic activity of VEGF165.     

Plasmin degradation of cartilage proteoglycan

Employing agarose gel electrophoresis, physiological concentrations of plasmin have been shown to degrade purified proteoglycan monomers and aggregates isolated from bovine articular cartilage. Proteoglycan degradation was (1) proportional to plasmin concentration, (2) dependent on the conversion of plasminogen to plasmin by plasminogen activator, (3) not displayed by plasminogen or plasminogen activator alone, and (4) inhibited by a serine proteinase inhibitor. These results, coupled with other findings, provide further support for a possible role of plasmin/plasminogen activator in cartilage destruction associated with rheumatoid arthritis.     

Effects of metal ions on activity of plasmin

The effects of some metal ions on amidolytic and fibrinogenolytic activities of highly purified human plasmin were investigated in vitro. In the presence of Zn²⁺, Cu²⁺, Cd²⁺, and Au⁺ in the incubation mixture at the concentrations of 1×10⁻⁵−1×10⁻³ M, the anidolytic plasmin activity was strongly inhibited, whereas Ca²⁺ and Mg²⁺ at the same concentrations were not effective. The analysis of the kinetic study has shown that Zn²⁺ or Cu²⁺ acts as mixed-type inhibitors of plasmin activity. The inhibition of amidolytic plasmin activity by Zn²⁺ and Cu²⁺ was reduced in the presence of EDTA, histidine, or albumin. Incubation of plasmin with Zn²⁺ or Cu²⁺ (at the concentration of 5×10⁻⁴ M) resulted in complete loss of its proteolytic action on fibrinogen, whereas Cd²⁺ and Au⁺ under the same conditions only partially inhibited this process.     

Bacterial plasminogen activators and receptors

Invasive bacterial pathogens intervene at various stages and by various mechanisms with the mammalian plasminogen/plasmin system. A vast number of pathogens express plasmin(ogen) receptors that immobilize plasmin(ogen) on the bacterial surface, an event that enhances activation of plasminogen by mammalian plasminogen activators. Bacteria also influence secretion of plasminogen activators and their inhibitors from mammalian cells. The prokaryotic plasminogen activators streptokinase and staphylokinase form a complex with plasmin(ogen) and thus enhance plasminogen activation. The Pla surface protease of Yersinia pestis resembles mammalian activators in function and converts plasminogen to plasmin by limited proteolysis. In essence, plasminogen receptors and activators turn bacteria into proteolytic organisms using a host-derived system. In Gram-negative bacteria, the filamentous surface appendages fimbriae and flagella form a major group of plasminogen receptors. In Gram-positive bacteria, surface-bound enzyme molecules as well as M-protein-related structures have been identified as plasminogen receptors, the former receptor type also occurs on mammalian cells. Plasmin is a broad-spectrum serine protease that degrades fibrin and noncollagenous proteins of extracellular matrices and activates latent procollagenases. Consequently, plasmin generated on or activated by Haemophilus influenzae, Salmonella typhimurium, Streptococcus pneumoniae, Y. pestis, and Borrelia burgdorferi has been shown to degrade mammalian extracellular matrices. In a few instances plasminogen activation has been shown to enhance bacterial metastasis in vitro through reconstituted basement membrane or epithelial cell monolayers. In vivo evidence for a role of plasminogen activation in pathogenesis is limited to Y. pestis, Borrelia, and group A streptococci. Bacterial proteases may also directly activate latent procollagenases or inactivate protease inhibitors of human plasma, and thus contribute to tissue damage and bacterial spread across tissue barriers.     

Annexin II-mediated plasmin generation activates TGF-beta3 during epithelial-mesenchymal transformation in the developing avian heart

Epithelial-mesenchymal transformation (EMT), the process by which epithelial cells are converted into motile, invasive mesenchymal cells, is critical to valvulogenesis. Transforming growth factor-beta3 (TGF-beta3), an established mediator of avian atrioventricular (AV) canal EMT, is secreted as a latent complex. In vitro, plasmin-mediated proteolysis has been shown to release active TGF-betas from the latent complex. Annexin II, a co-receptor for tissue plasminogen activator (tPA) and plasminogen, promotes cell-surface generation of the serine protease plasmin. Here, we show that annexin II-mediated plasmin activity regulates release of active TGF-beta3 during chick AV canal EMT. Primary embryonic endocardial-derived cells express annexin II which promotes plasminogen activation in vitro. Incubation of heart explant cultures with either alpha(2)antiplasmin (alpha(2)AP), a major physiological plasmin inhibitor, or anti-annexin II IgG, blocked EMT by approximately 80%, and 50%, respectively. Anti-annexin II IgG-mediated inhibition of EMT was overcome by the addition of recombinant TGF-beta3. Upon treatment with anti-annexin II IgG or alpha(2)AP, conditioned medium from heart explant cultures showed absence of the active fragment of TGF-beta3 by Western blot analysis and a approximately 50% decrease in TGF-beta specific bioactivity. Our results suggest that annexin II-mediated plasmin activity regulates the release of active TGF-beta during cardiac valve development in the avian heart.     

Electrochemical assay of plasmin activity and its kinetic analysis

A sensitive and convenient electrochemical assay of plasmin activity and its kinetic analysis are described. Thus, a ferrocenyl peptide substrate (FcPS) having a plasmin-specific substrate sequence, Lys-Thr-Phe-Lys, and a Cys residue was prepared and immobilized on a gold electrode through the sulfur-gold linkage. The obtained electrode showed a redox signal based on the ferrocene moiety, suggesting the immobilization of FcPS on the electrode. After treatment of this electrode with plasmin, its electrochemical signal was decreased in proportion to an increase of the amount of plasmin. The detection limit for plasmin in this assay system was as low as 50 ng/ml or 0.15 mU/ml. Real-time monitoring of plasmin reaction on the electrode could also be achieved, and the kinetic parameters of this enzymatic reaction could be determined; for example, the k_cat/K_m value was 0.063 μM⁻¹ s⁻¹. Furthermore, a quantitative assay for streptokinase as a plasminogen activator was also demonstrated by using this system.     

Plasmin reduces fibronectin deposition by mesangial cells in a protease-activated receptor-1 independent manner

BackgroundProtease-activated receptor-1 (PAR-1) potentiates diabetic nephropathy (DN) as evident from reduced kidney injury in diabetic PAR-1 deficient mice. Although thrombin is the prototypical PAR-1 agonist, anticoagulant treatment does not limit DN in experimental animal models suggesting that thrombin is not the endogenous PAR-1 agonist driving DN.ObjectivesTo identify the endogenous PAR-1 agonist potentiating diabetes-induced nephropathy.MethodsUnbiased protease expression profiling in glomeruli from human kidneys with DN was performed using publically available microarray data. The identified prime candidate PAR-1 agonist was subsequently analysed for PAR-1-dependent induction of fibrosis in vitro.ResultsOf the 553 proteases expressed in the human genome, 247 qualified as potential PAR-1 agonists of which 71 were significantly expressed above background in diabetic glomeruli. The recently identified PAR-1 agonist plasmin(ogen), together with its physiological activator tissue plasminogen activator, were among the highest expressed proteases. Plasmin did however not induce mesangial proliferation and/or fibronectin deposition in vitro. In a PAR-1 independent manner, plasmin even reduced fibronectin deposition.ConclusionExpression profiling identified plasmin as potential endogenous PAR-1 agonist driving DN. Instead of inducing fibronectin expression, plasmin however reduced mesangial fibronectin deposition in vitro. Therefore we conclude that plasmin may not be the endogenous PAR-1 agonist potentiating DN.     

Effects of Trypsin and Plasmin Treatment of Myelin on the Myelin-Associated Glycoprotein and Basic Protein

Human and rat myelin preparations were incubated with varying concentrations of trypsin and plasmin to determine the effects of these proteolytic enzymes on myelin-associated glycoprotein (MAG), basic protein, and other myelin proteins and to compare the effects with those of the neutral protease that was reported to be endogenous in myelin. Basic protein was most susceptible to degradation by both trypsin and plasmin, whereas MAG was relatively resistant to their actions. Under the assay conditions used, the highest concentrations of trypsin and plasmin degraded greater than 80% of the basic protein but less than 30% of the MAG, and lower concentrations caused significant loss of basic protein without appreciably affecting MAG. Neither trypsin nor plasmin caused a specific cleavage of MAG to a derivative of MAG (dMAG) in a manner analogous to the endogenous neutral protease. Thus the endogenous protease appears unique in converting human MAG to dMAG much more rapidly than it degrades basic protein. MAG is slowly degraded along with other proteins when myelin is treated with trypsin or plasmin, but it is less susceptible to their action than is basic protein.     

Contribution of conserved lysine residues in the alpha2-antiplasmin C terminus to plasmin binding and inhibition

α(2)-Antiplasmin is the physiological inhibitor of plasmin and is unique in the serpin family due to N- and C-terminal extensions beyond its core domain. The C-terminal extension comprises 55 amino acids from Asn-410 to Lys-464, and the lysine residues (Lys-418, Lys-427, Lys-434, Lys-441, Lys-448, and Lys-464) within this region are important in mediating the initial interaction with kringle domains of plasmin. To understand the role of lysine residues within the C terminus of α(2)-antiplasmin, we systematically and sequentially mutated the C-terminal lysines, studied the effects on the rate of plasmin inhibition, and measured the binding affinity for plasmin via surface plasmon resonance. We determined that the C-terminal lysine (Lys-464) is individually most important in initiating binding to plasmin. Using two independent methods, we also showed that the conserved internal lysine residues play a major role mediating binding of the C terminus of α(2)-antiplasmin to kringle domains of plasmin and in accelerating the rate of interaction between α(2)-antiplasmin and plasmin. When the C terminus of α(2)-antiplasmin was removed, the binding affinity for active site-blocked plasmin remained high, suggesting additional exosite interactions between the serpin core and plasmin.     

Structure-activity studies of cyclic ketone inhibitors of the serine protease plasmin: design, synthesis, and biological activity

Three series of cyclic ketone inhibitors were synthesized and evaluated against the serine protease plasmin. Peptide inhibitors that incorporated 3-oxotetrahydrofuran and 3-oxotetrahydrothiophene 1,1-dioxide groups had the highest activities. Alkylamino substituents, which were designed to bind in the S1 subsite of plasmin, were attached to the inhibitors. Compounds 5c and 5g, which incorporated 6-aminohexyl substituents, were found to be optimal and demonstrated IC₅₀ values in the low micromolar range. Incorporating conformationally constrained peptide segments into the inhibitors did not improve their activities.     

Pyrrolopyrimidine-inhibitors with hydantoin moiety as spacer can explore P4/S4 interaction on plasmin

In the development of plasmin inhibitors, a novel chemotype, pyrrolopyrimidine scaffold possessing two motifs, a hydantoin-containing P4 moiety and a warhead-containing P1 moiety, is uncovered. A unique feature of the new line of the plasmin inhibitors is that the interaction between the plasmin inhibitors and key subsites in plasmin can be controlled by a spacer like hydantoin. The application of the novel chemotype is demonstrated by 1n and provides further evidence on the importance of hydantoin as the spacer.     

Enhanced fibrinolysis by proteolysed coagulation factor Xa

We previously showed that coagulation factor Xa (FXa) enhances activation of the fibrinolysis zymogen plasminogen to plasmin by tissue plasminogen activator (tPA). Implying that proteolytic modulation occurs in situ, intact FXa (FXaα) must be sequentially cleaved by plasmin or autoproteolysis, producing FXaβ and Xa33/13, which acquire necessary plasminogen binding sites. The implicit function of Xa33/13 in plasmin generation has not been demonstrated, nor has FXaα/β or Xa33/13 been studied in clot lysis experiments. We now report that purified Xa33/13 increases tPA-dependent plasmin generation by at least 10-fold. Western blots confirmed that in situ conversion of FXaα/β to Xa33/13 correlated to enhanced plasmin generation. Chemical modification of the FXaα active site resulted in the proteolytic generation of a product distinct from Xa33/13 and inhibited the enhancement of plasminogen activation. Identical modification of Xa33/13 had no effect on tPA cofactor function. Due to its overwhelming concentration in the clot, fibrin is the accepted tPA cofactor. Nevertheless, at the functional level of tPA that circulates in plasma, FXaα/β or Xa33/13 greatly reduced purified fibrin lysis times by as much as 7-fold. This effect was attenuated at high levels of tPA, suggesting a role when intrinsic plasmin generation is relatively low. FXaα/β or Xa33/13 did not alter the apparent size of fibrin degradation products, but accelerated the initial cleavage of fibrin to fragment X, which is known to optimize the tPA cofactor activity of fibrin. Thus, coagulation FXaα undergoes proteolytic modulation to enhance fibrinolysis, possibly by priming the tPA cofactor function of fibrin.     

A Bacterial Pathogen Co-opts Host Plasmin to Resist Killing by Cathelicidin Antimicrobial Peptides

The bacterial pathogen Group A Streptococcus (GAS) colonizes epithelial and mucosal surfaces and can cause a broad spectrum of human disease. Through the secreted plasminogen activator streptokinase (Ska), GAS activates human plasminogen into plasmin and binds it to the bacterial surface. The resulting surface plasmin protease activity has been proposed to play a role in disrupting tissue barriers, promoting invasive spread of the bacterium. We investigated whether this surface protease activity could aid the immune evasion role through degradation of the key innate antimicrobial peptide LL-37, the human cathelicidin. Cleavage products of plasmin-degraded LL-37 were analyzed by matrix-assisted laser desorption ionization mass spectrometry. Ska-deficient GAS strains were generated by targeted allelic exchange mutagenesis and confirmed to lack surface plasmin activity after growth in human plasma or media supplemented with plasminogen and fibrinogen. Loss of surface plasmin activity left GAS unable to efficiently degrade LL-37 and increased bacterial susceptibility to killing by the antimicrobial peptide. When mice infected with GAS were simultaneously treated with the plasmin inhibitor aprotinin, a significant reduction in the size of necrotic skin lesions was observed. Together these data reveal a novel immune evasion strategy of the human pathogen: co-opting the activity of a host protease to evade peptide-based innate host defenses.     

FDP相关肽修饰物合成与抗栓活性研究

采用多肽固相合成法合成了ＦＤＰ相关肽的修饰产物ＨＰＡ,经ＨＰＬＣ鉴定,其纯度为８５．５％,氨基酸组成分析及质谱法测定分子量结果均与理论值相符合。生物活性研究表明,在体外,该肽能有效地抑制ＡＴＰ引起的人血小板聚集作用．在体内,该肽能显著增强家兔血纤溶酶的活性,并能有效抑制家兔实验性血栓的形成。     

Plasminogen/plasmin modulates bone metabolism by regulating the osteoblast and osteoclast function

The contribution of plasminogen (Plg)/plasmin, which have claimed to be the main fibrinolytic regulators in the bone metabolism, remains unclear. This study evaluated how the absence of Plg affects the function of osteoblast (OB) and osteoclast (OC). There was a larger population of pre-OCs in bone marrow-derived cells from the Plg(-/-) mice than the population of that from the WT mice. In addition, the absence of Plg suppressed the expression of osteoprotegerin in OBs. Moreover, an exogenous plasmin clearly induced the osteoprotegerin expression in Plg(-/-) OBs. The osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells in co-culture with OBs from the Plg(-/-) mice was significantly accelerated in comparison with that in co-culture with OBs from the WT mice. Intriguingly, the accelerated OC differentiation of RAW264.7 cells co-cultured with Plg(-/-) OBs was clearly suppressed by the treatment of an exogenous plasmin. Consequently, Plg(-/-) mice display decreased bone mineral density. These findings could eventually lead to the development of new clinical therapies for bone disease caused by a disorder of the fibrinolytic system.     

Conversion of low-affinity platelet factor 4 to β-thromboglobulin by plasmin and trypsin

Low-affinity platelet factor 4 and β-thromboglobulin are platelet-secreted proteins that bind with low affinity to heparin. They show extensive immunological cross-reactivity and appear to differ in amino acid sequence only by an amino-terminal peptide unique to low-affinity platelet factor 4. The possibility that β-thromboglobulin is derived from low-affinity platelet factor 4 by proteolysis was investigated by exposing this protein to the action of plasmin, thrombin and trypsin. While thrombin had no effect, plasmin and trypsin converted low-affinity platelet factor 4 to a species with the same electrophoretic mobility and isoelectric point as β-thromboglobulin. We conclude that β-thromboglobulin is a breakdown product of low-affinity platelet factor 4.     

Evaluation of phage display system and leech-derived tryptase inhibitor as a tool for understanding the serine proteinase specificities

A small combinatorial library of LDTI mutants (5.2 x 10(4)) restricted to the P1-P4' positions of the reactive site was displayed on the pCANTAB 5E phagemid, and LDTI fusion phages were produced and selected for potent neutrophil elastase and plasmin inhibitors. Strong fusion phage binders were analyzed by ELISA on enzyme-coated microtiter plates and the positive phages had their DNA sequenced. The LDTI variants: 29E (K8A, I9A, L10F, and K11F) and 19E (K8A, K11Q, and P12Y) for elastase and 2Pl (K11W and P12N), 8Pl (I9V, K11W, and P12E), and 10Pl (I9T, K11L, and P12L) for plasmin were produced with a Saccharomyces cerevisiae expression system. New strong elastase and plasmin inhibitors were 29E and 2Pl, respectively. LDTI-29E was a potent and specific neutrophil elastase inhibitor K(i) =0.5 nM), affecting no other tested enzymes. LDTI-2Pl was the strongest plasmin inhibitor ( K(i) =1.7nM) in the LDTI mutant library. This approach allowed selection of new specific serine proteinase inhibitors for neutrophil elastase and plasmin (a thrombin inhibitor variant was previously described), from a unique template molecule, LDTI, a Kazal type one domain inhibitor, by only 2-4 amino acid replacements. Our data validate this small LDTI combinatorial library as a tool to generate specific serine proteinase inhibitors suitable for drug design and enzyme-inhibitor interaction studies.     

Effects of extracellular DNA on plasminogen activation and fibrinolysis

The increased levels of extracellular DNA found in a number of disorders involving dysregulation of the fibrinolytic system may affect interactions between fibrinolytic enzymes and inhibitors. Double-stranded (ds) DNA and oligonucleotides bind tissue-(tPA) and urokinase (uPA)-type plasminogen activators, plasmin, and plasminogen with submicromolar affinity. The binding of enzymes to DNA was detected by EMSA, steady-state, and stopped-flow fluorimetry. The interaction of dsDNA/oligonucleotides with tPA and uPA includes a fast bimolecular step, followed by two monomolecular steps, likely indicating slow conformational changes in the enzyme. DNA (0.1-5.0 μg/ml), but not RNA, potentiates the activation of Glu- and Lys-plasminogen by tPA and uPA by 480- and 70-fold and 10.7- and 17-fold, respectively, via a template mechanism similar to that known for fibrin. However, unlike fibrin, dsDNA/oligonucleotides moderately affect the reaction between plasmin and α(2)-antiplasmin and accelerate the inactivation of tPA and two chain uPA by plasminogen activator inhibitor-1 (PAI-1), which is potentiated by vitronectin. dsDNA (0.1-1.0 μg/ml) does not affect the rate of fibrinolysis by plasmin but increases by 4-5-fold the rate of fibrinolysis by Glu-plasminogen/plasminogen activator. The presence of α(2)-antiplasmin abolishes the potentiation of fibrinolysis by dsDNA. At higher concentrations (1.0-20 μg/ml), dsDNA competes for plasmin with fibrin and decreases the rate of fibrinolysis. dsDNA/oligonucleotides incorporated into a fibrin film also inhibit fibrinolysis. Thus, extracellular DNA at physiological concentrations may potentiate fibrinolysis by stimulating fibrin-independent plasminogen activation. Conversely, DNA could inhibit fibrinolysis by increasing the susceptibility of fibrinolytic enzymes to serpins.     

Proteolytic Processing Regulates Placental Growth Factor Activities

Placental growth factor (PlGF) is a critical mediator of blood vessel formation, yet mechanisms of its action and regulation are incompletely understood. Here we demonstrate that proteolytic processing regulates the biological activity of PlGF. Specifically, we show that plasmin processing of PlGF-2 yields a protease-resistant core fragment comprising the vascular endothelial growth factor receptor-1 binding site but lacking the carboxyl-terminal domain encoding the heparin-binding domain and an 8-amino acid peptide encoded by exon 7. We have identified plasmin cleavage sites, generated a truncated PlGF118 isoform mimicking plasmin-processed PlGF, and explored its biological function in comparison with that of PlGF-1 and -2. The angiogenic responses induced by the diverse PlGF forms were distinct. Whereas PlGF-2 increased endothelial cell chemotaxis, vascular sprouting, and granulation tissue formation upon skin injury, these activities were abrogated following plasmin digestion. Investigation of PlGF/Neuropilin-1 binding and function suggests a critical role for heparin-binding domain/Neuropilin-1 interaction and its regulation by plasmin processing. Collectively, here we provide new mechanistic insights into the regulation of PlGF-2/Neuropilin-1-mediated tissue vascularization and growth.     

Osteopontin Is Cleaved at Multiple Sites Close to Its Integrin-binding Motifs in Milk and Is a Novel Substrate for Plasmin and Cathepsin D

Osteopontin (OPN) is a highly modified integrin-binding protein present in most tissues and body fluids where it has been implicated in numerous biological processes. A significant regulation of OPN function is mediated through phosphorylation and proteolytic processing. Proteolytic cleavage by thrombin and matrix metalloproteinases close to the integrin-binding Arg-Gly-Asp sequence modulates the function of OPN and its integrin binding properties. In this study, seven N-terminal OPN fragments originating from proteolytic cleavage have been characterized from human milk. Identification of the cleavage sites revealed that all fragments contained the Arg–Gly–Asp¹⁴⁵ sequence and were generated by cleavage of the Leu¹⁵¹–Arg¹⁵², Arg¹⁵²–Ser¹⁵³, Ser¹⁵³–Lys¹⁵⁴, Lys¹⁵⁴–Ser¹⁵⁵, Ser¹⁵⁵–Lys¹⁵⁶, Lys¹⁵⁶–Lys¹⁵⁷, or Phe¹⁵⁸–Arg¹⁵⁹ peptide bonds. Six cleavages cannot be ascribed to thrombin or matrix metalloproteinase activity, whereas the cleavage at Arg¹⁵²–Ser¹⁵³ matches thrombin specificity for OPN. The principal protease in milk, plasmin, hydrolyzed the same peptide bond as thrombin, but its main cleavage site was identified to be Lys¹⁵⁴–Ser¹⁵⁵. Another endogenous milk protease, cathepsin D, cleaved the Leu¹⁵¹–Arg¹⁵² bond. OPN fragments corresponding to plasmin activity were also identified in urine showing that plasmin cleavage of OPN is not restricted to milk. Plasmin, but not cathepsin D, cleavage of OPN increased cell adhesion mediated by the α_Vβ₃- or α₅β₁-integrins. Similar cellular adhesion was mediated by plasmin and thrombin-cleaved OPN showing that plasmin can be a potent regulator of OPN activity. These data show that OPN is highly susceptible to cleavage near its integrin-binding motifs, and the protein is a novel substrate for plasmin and cathepsin D.