Enhanced Plasminogen Activator Research Articles

C-terminal lysine residues enhance plasminogen activation by inducing conformational flexibility and stabilization of activator complex of staphylokinase with plasmin

Staphylokinase (SAK), a potent fibrin-specific plasminogen activator secreted by Staphylococcus aureus, carries a pair of lysine at the carboxy-terminus that play a key role in plasminogen activation. The underlaying mechanism by which C-terminal lysins of SAK modulate its function remains unknown. This study has been undertaken to unravel role of C-terminal lysins of SAK in plasminogen activation. While deletion of C-terminal lysins (Lys135, Lys136) drastically impaired plasminogen activation by SAK, addition of lysins enhanced its catalytic activity 2–2.5-fold. Circular dichroism analysis revealed that C-terminally modified mutants of SAK carry significant changes in their beta sheets and secondary structure. Structure models and RING (residue interaction network generation) studies indicated that the deletion of lysins has conferred extensive topological alterations in SAK, disrupting vital interactions at the interface of SAK.plasmin complex, thereby leading significant impairment in its functional activity. In contrast, addition of lysins at the C-terminus enhanced its conformational flexibility, creating a stronger coupling at the interface of SAK.plasmin complex and making it more efficient for plasminogen activation. Taken together, these studies provided new insights on the role of C-terminal lysins in establishment of precise intermolecular interactions of SAK with the plasmin for the optimal function of activator complex.

Triosephosphate isomerase of Streptococcus pneumoniae is released extracellularly by autolysis and binds to host plasminogen to promote its activation.

Recruitment of plasminogen is an important infection strategy of the human pathogen Streptococcus pneumoniae to invade host tissues. In Streptococcus aureus, triosephosphate isomerase (TPI) has been reported to bind plasminogen. In this study, the TPI of S. pneumoniae (TpiA) was identified through proteomic analysis of bronchoalveolar lavage fluid from a murine pneumococcal pneumonia model. The binding kinetics of recombinant pneumococcal TpiA with plasminogen were characterized using surface plasmon resonance (SPR, Biacore), ligand blot analyses, and enzyme‐linked immunosorbent assay. Enhanced plasminogen activation and subsequent degradation by plasmin were also shown. Release of TpiA into the culture medium was observed to be dependent on autolysin. These findings suggest that S. pneumoniae releases TpiA via autolysis, which then binds to plasminogen and promotes its activation, thereby contributing to tissue invasion via degradation of the extracellular matrix.

Bioresponsive Polyphenol-Based Nanoparticles as Thrombolytic Drug Carriers.

Thrombolytic (clot-busting) therapies with plasminogen activators (PAs) are first-line treatments against acute thrombosis and ischemic stroke. However, limitations such as narrow therapeutic windows, low success rates, and bleeding complications hinder their clinical use. Drug-loaded polyphenol-based nanoparticles (NPs) could address these shortfalls by delivering a more targeted and safer thrombolysis, coupled with advantages such as improved biocompatibility and higher stability in vivo. Herein, a template-mediated polyphenol-based supramolecular assembly strategy is used to prepare nanocarriers of thrombolytic drugs. A thrombin-dependent drug release mechanism is integrated using tannic acid (TA) to cross-link urokinase-type PA (uPA) and a thrombin-cleavable peptide on a sacrificial mesoporous silica template via noncovalent interactions. Following drug loading and template removal, the resulting NPs retain active uPA and demonstrate enhanced plasminogen activation in the presence of thrombin (1.14-fold; p < 0.05). Additionally, they display lower association with macrophage (RAW 264.7) and monocytic (THP-1) cell lines (43 and 7% reduction, respectively), reduced hepatic accumulation, and delayed blood clearance in vivo (90% clearance at 60 min vs 5 min) compared with the template-containing NPs. Our thrombin-responsive, polyphenol-based NPs represent a promising platform for advanced drug delivery applications, with potential to improve thrombolytic therapies.

Schistosoma mansoni glyceraldehyde-3-phosphate dehydrogenase enhances formation of the blood-clot lysis protein plasmin.

ABSTRACTSchistosomes are intravascular blood flukes that cause the parasitic disease schistosomiasis. In agreement with Schistosoma mansoni (Sm) proteomic analysis, we show here that the normally intracellular glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is also found at the parasite surface; live worms from all intravascular life stages display GAPDH activity. Suppressing GAPDH gene expression using RNA interference significantly lowers this live worm surface activity. Medium in which the worms are cultured overnight displays essentially no activity, showing that the enzyme is not shed or excreted but remains associated with the worm surface. Immunolocalization experiments confirm that the enzyme is highly expressed in the parasite tegument (skin). Surface activity in schistosomula amounts to ∼8% of that displayed by equivalent parasite lysates. To address the functional role of SmGAPDH, we purified the protein following its expression in Escherichia coli strain DS113. The recombinant protein displays optimal enzymatic activity at pH 9.2, shows robust activity at the temperature of the parasite's hosts, and has a Michaelis–Menten constant for glyceraldehyde-3-phosphate (GAP) of 1.4 mM±0.24. We show that recombinant SmGAPDH binds plasminogen (PLMG) and promotes PLMG conversion to its active form (plasmin) in a dose response in the presence of tissue plasminogen activator. Since plasmin is a key mediator of thrombolysis, our results support the hypothesis that SmGAPDH, a host-interactive tegumental protein that can enhance PLMG activation, could help degrade blood clots around the worms in the vascular microenvironment and thus promote parasite survival in vivo.This article has an associated First Person interview with the first author of the paper.

Stimulation of plasminogen activation by recombinant cellular prion protein is conserved in the NH2-terminal fragment PrP23-110.

The cellular prion protein (PrP(c)), tissue-type plasminogen activator (t-PA) and plasminogen are expressed in synaptic membranes in vivo. In the central nervous system the fibrinolytic system is associated with excitotoxin-mediated neurotoxicity and Alzheimer's disease. Recently binding of the disease associated isoform of the prion protein (PrP(Sc)) to plasminogen and stimulation of t-PA activity have been reported. In this study the interaction of PrP(c) and plasminogen was investigated using chromogenic assays in vitro. We found that plasmin is able to cleave recombinant PrP(c) at lysine residue 110 generating an NH(2)-terminal truncated molecule that has previously been described as a major product of PrP(c) metabolism. We further characterized the proteolytic fragments with respect to their ability to stimulate plasminogen activation in vitro. Our results show that the NH(2)-terminal part of PrP(c) spanning amino acids 23-110 (PrP23-110) together with low molecular weight heparin stimulates t-PA mediated plasminogen activation in vitro. The apparent rate constant was increased 57 fold in the presence of 800 nM PrP23-110. Furthermore, we compared the stimulation of t-PA activity by PrP(c) and beta-amyloid peptide (1-42). While the activity of the beta-amyloid was independent of low molecular weight heparin, PrP23-110 was approximately 4- and 37 fold more active than beta-amyloid in the absence or presence of low molecular weight heparin. In summary, plasmin cleaves PrP(c) in vitro and the liberated NH(2)-terminal fragment accelerates plasminogen activation. Cleavage of PrP c has previously been reported. Thus cleavage of PrP(c) enhancing plasminogen activation at the cell surface could constitute a regulatory mechanism of pericellular proteolysis.

Cation‐independent Mannose 6‐Phosphate Receptor Interacts with Several Components of the Plasminogen Activation System

The 300 kDa cation‐independent mannose 6‐phosphate receptor (CI‐MPR) functions in the biogenesis of lysosomes by trafficking newly synthesized lysosomal enzymes with mannose 6‐phosphate (Man‐6‐P)‐containing glycans to the late endosome. In addition, CI‐MPR binds a number of proteins at the cell surface in a glycan‐independent manner, including plasminogen, urokinase‐type plasminogen activator receptor, and insulin‐like growth factor 2. The ability of CI‐MPR to interact with these different ligands is facilitated by its large extracellular region that is composed of 15 contiguous domains. Previous studies have shown that plasminogen binds to CI‐MPR through one or more of its lysine‐binding kringle domains. We report here that tissue‐type plasminogen activator (tPA), which contains two kringle domains, also binds to CI‐MPR. Using surface plasmon resonance, we demonstrate that tPA binds with high affinity to the N‐terminus of CI‐MPR and that CI‐MPR domain 1 appears to be sufficient for binding. The interaction of tPA with CI‐MPR can be partially inhibited by the lysine analog 6‐aminohexanoic acid and by Man‐6‐P, however, the exact nature of the protein interaction is unknown. Addition of CI‐MPR to tPA and plasminogen enhances the rate of plasmin formation. This rate enhancement shows saturable kinetics for receptor with a K½ of approximately 120 nM. The rate of enhancement is dependent on more than one domain of the receptor as CI‐MPR domain 1 by itself is not sufficient to enhance tPA‐dependent plasminogen activation. We propose that CI‐MPR acts as a scaffold to bring tPA and plasminogen together and augments conversion of plasminogen to plasmin by tPA. Angiostatin K1‐4 protein, which includes the pan domain and kringles 1‐4 of plasminogen, is able to inhibit the tPA‐dependent activation of plasminogen in the presence of CI‐MPR. Addition of a lysosomal enzyme also inhibits the CI‐MPR enhancement of plasminogen activation by tPA in a concentration‐dependent manner. Characterization of the mechanisms of these inhibitors will help in understanding the interaction of CI‐MPR with tPA and plasminogen along with providing a better understanding of CI‐MPR's ability to interact with multiple ligands.Support or Funding InformationFunding: NIH R01DK042667

Isoprene Side-chain of SMTP is Essential for Soluble Epoxide Hydrolase Inhibition and Cellular Localization

SMTPs, a family of natural small molecules that effectively treat ischemic stroke, are subject to clinical development. SMTPs enhance plasminogen activation and inhibit soluble epoxide hydrolase (sEH), leading to promotion of endogenous thrombolysis and anti-inflammation. The SMTP molecule consists of atricyclic γ-lactam moiety, an isoprene side-chain, and an N-linked side-chain. Here, we investigate the yet-to-be-characterized function of the isoprene side- chain of SMTPs in sEH inhibition and cellular distribution. The results demonstrated that oxidative modification as well as truncation of the side-chain abolished epoxide hydrolase inhibition. The introduction of a terminal hydroxy group exceptionally unaffected epoxide hydrolase, but led to impaired cellular localization, resulting in diminution of cellular epoxide hydrolase inhibition. Thus, the isoprene side-chain of SMTP is an important pharmacophore for epoxide hydrolase inhibition and cellular localization.

Enolase, a plasminogen receptor isolated from salivary gland transcriptome of the ixodid tick Haemaphysalis flava

Enolase, a multifunctional protein, is shown to act as a plasminogen receptor that contributes to fibrinolysis, which plays an important role in preventing the formation of blood clots during tick feeding. The study of enolase genes provides opportunities to develop a potential antigen target for tick control. So far, enolase has been identified in only a few species of ticks. Knowledge of the exact mechanisms of plasminogen activation and fibrinolysis by enolase as a plasminogen receptor is limited. Here, we cloned the enolase full-length complementary DNA (cDNA) from the salivary glands of Haemaphysalis flava, expressed it, and analyzed the function of the recombinant H. flava enolase. The enolase cDNA was 1988bp in length and encoded 433 amino acid residues. It contained two domains and some highly conserved functional motifs including an assumed membrane re-association region "AAVPSGASTGI." The enolase exhibited 83.3% amino acid similarity to that of the putative enolase of Ixodes ricinus, and 85% to that of Ornithodoros moubata enolase. After eukaryotic expression in insect cells, Western blot analysis showed that the mouse antiserum against the hexahistidine-tagged recombinant enolase protein recognized a band of approximately 48kDa. The recombinant enolase bound human plasminogen in a dose-dependent manner and enhanced plasminogen activation in the presence of host tissue plasminogen activator (t-PA), most probably to promote fibrinolysis and maintain blood flow at the host-tick interface. Real-time quantitative polymerase chain reaction (qPCR) analysis showed that the expression level of enolase in salivary glands was significantly higher than in other tested tissues. Although the enolase was expressed in all developmental stages, it had the highest expression in the rapid blood feeding period of ticks. These findings indicate that the enolase might play an important role in blood feeding of H. flava.

Schistosomes Enhance Plasminogen Activation: The Role of Tegumental Enolase.

Schistosoma mansoni is a blood fluke parasite that causes schistosomiasis, a debilitating disease of global public health importance. These relatively large parasites are able to survive prolonged periods in the human vasculature without inducing stable blood clots around them. We show here that the intravascular life stages (schistosomula and adult males and females) can all promote significant plasminogen (PLMG) activation in the presence of tissue plasminogen activator (tPA). This results in the generation of the potent fibrinolytic agent plasmin which could degrade blood clots forming around the worms in vivo. We demonstrate that S. mansoni enolase (SmEno) is a host-interactive tegumental enzyme that, in recombinant form, can bind PLMG and promote its activation. Like classical members of the enolase protein family, SmEno can catalyze the interconversion of 2-phospho-D-glycerate (2-PGA) and phosphoenolpyruvate (PEP). The enzyme has maximal activity at pH 7.5, requires Mg2+ for optimal activity and can be inhibited by NaF but not mefloquin. Suppressing expression of the SmEno gene significantly diminishes enolase mRNA levels, protein levels and surface enzyme activity but, surprisingly, does not affect the ability of the worms to promote PLMG activation. Thus, while SmEno can enhance PLMG activation, our analysis suggests that it is not the only contributor to the parasite’s ability to perform this function. We show that the worms possess several other PLMG-binding proteins in addition to SmEno and these may have a greater importance in schistosome-driven PLMG activation.

A Novel Interaction between Complement Inhibitor C4b-binding Protein and Plasminogen That Enhances Plasminogen Activation

The complement, coagulation, and fibrinolytic systems are crucial for the maintenance of tissue homeostasis. To date numerous interactions and cross-talks have been identified between these cascades. In line with this, here we propose a novel, hitherto unknown interaction between the complement inhibitor C4b-binding protein (C4BP) and plasminogen of the fibrinolytic pathway. Binding of C4BP to Streptococcus pneumoniae is a known virulence mechanism of this pathogen and it was increased in the presence of plasminogen. Interestingly, the acute phase variant of C4BP lacking the β-chain and protein S binds plasminogen much stronger than the main isoform containing the β-chain and protein S. Indeed, the complement control protein (CCP) 8 domain of C4BP, which would otherwise be sterically hindered by the β-chain, primarily mediates this interaction. Moreover, the lysine-binding sites in plasminogen kringle domains facilitate the C4BP-plasminogen interaction. Furthermore, C4BP readily forms complexes with plasminogen in fluid phase and such complexes are present in human serum and plasma. Importantly, whereas the presence of plasminogen did not affect the factor I cofactor activity of C4BP, the activation of plasminogen by urokinase-type plasminogen activator to active plasmin was significantly augmented in the presence of C4BP. Taken together, our data demonstrate a novel interaction between two proteins of the complement and fibrinolytic system. Most complexes might be formed during the acute phase of inflammation and have an effect on the homeostasis at the site of injury or acute inflammation.

Structure-activity relationships of the plasminogen modulator SMTP with respect to the inhibition of soluble epoxide hydrolase.

A family of fungal metabolites, SMTP, is a small-molecule plasminogen modulator that enhances plasminogen activation, leading to thrombolysis. We recently demonstrated that SMTP-7 effectively treats ischemic stroke due to its thrombolytic activity as well as anti-inflammatory action, which is attributable to soluble epoxide hydrolase (sEH) inhibition. In this paper, we studied detailed structure-activity relationships of plasminogen modulation and sEH inhibition using 25 SMTP congeners including six newly synthesized ones. The results clearly demonstrate that the structure of the N-linked side chain of SMTP congeners markedly affect their activities toward plasminogen modulation and inhibitions of the two activities of sEH (C-terminal epoxide hydrolase and N-terminal phosphatase). A slight change in the N-linked side chain results in affording selectivity of SMTP congeners. Many congeners, which lacked plasminogen modulation activity, differently inhibited the two sEH activities depending on the structures of the N-linked side chain. Some congeners were active in plasminogen modulation and inhibition of both activities of sEH. These results help comprehensive understanding of ideal design of a drug useful for ischemic diseases that are associated with inflammation, such as stroke.

Mechanism of the action of SMTP-7, a novel small-molecule modulator of plasminogen activation.

SMTP-7 is a small molecule that promotes the proteolytic activation of plasminogen by relaxing its conformation. SMTP-7 has excellent therapeutic activities against thrombotic stroke in several rodent models. The objective of this study was to elucidate detailed mechanism of the action of SMTP-7 in vitro. We report here that the action of SMTP-7 requires a cofactor with a long-chain alkyl or alkenyl group, and that the fifth kringle domain (kringle 5) of plasminogen is involved in the SMTP-7 action. In this study, we found that the SMTP-7 action to enhance plasminogen activation depended on the presence of a certain type of surfactant, and we screened biologically relevant molecules for their cofactor activity for the SMTP action. As a result, phospholipids, sphingolipids, and oleic acid were found to be active in assisting the SMTP-7 action. On the contrary, stearic acid and bile acids were inactive. Thus, a certain structural element, not only the surface-activating potential, is required for a compound to act as a cofactor for the SMTP-7 action. The plasminogen molecule consists of a PAN domain, five kringle domains, and a serine protease domain. The cofactor-dependent effects of SMTP-7 was observed with plasminogen species including kringle 5 such as intact plasminogen (Glu-plasminogen), des-PAN plasminogen (Lys-plasminogen), and des-[PAN - (kringles 1-4)] plasminogen (mini-plasminogen). However, SMTP-7 effect was not observed with the smallest plasminogen species des-[PAN - (kringles 1-4) and a half of kringle 5)] plasminogen (micro-plasminogen). Thus, kringle 5 is crucial for the action of SMTP-7.

Soluble Epoxide Hydrolase As An Anti-Inflammatory Target Of The Thrombolytic Stroke Drug Candidate Smtp-7

During the past decade, the thrombolytic enzyme tissue plasminogen activator (t-PA)-based treatment has been the standard therapy for acute ischemic stroke. However, due to its hemorrhagic risk and narrow therapeutic time window (TTW), only limited patients benefit from t-PA-based therapy, and the development of an alternative therapeutic agent is urgently needed. Reducing inflammation within the infarction area to rescue penumbra is particularly important. SMTP-7 is a small molecule that enhances plasminogen activation by modulating plasminogen conformation. SMTP-7 promotes plasmin formation and clot clearance in vivo and it is effective in treating thrombotic and embolic strokes in experimental models in rodents and a nonhuman primate. Unexpectedly, SMTP-7 reduces hemorrhagic transformation and has extended TTW as compared with t-PA. The distinct effects of SMTP-7 are partly explained by suppression of inflammatory responses following thrombolytic reperfusion, unlike t-PA. Experiments with animal inflammatory disease models (ulcerative colitis, Crohn's disease, and Guillain-Barré syndrome models) suggest that the anti-inflammatory action of SMTP-7 is independent of thrombolytic activity, as a thrombolytically inactive congener, SMTP-44D, exhibits anti-inflammatory action in those models. In this study, we searched for anti-inflammatory target of SMTP and found soluble epoxide hydrolase (sEH) as a possible candidate.We searched for a target protein using an SMTP-conjugated affinity matrix, which was synthesized by coupling SMTP-50, a congener with a primary amino group on the side chain, with gel beads. Mouse liver homogenates were subjected to affinity chromatography on this matrix, and specifically bound proteins were analyzed by peptide mass fingerprint. As a result, 4 major bound proteins were assigned to full length or fragments of soluble epoxide hydrolase (sEH), a hybrid enzyme with epoxide hydrolase activity in the C-terminal domain and lipid phosphatase activity in the N-terminal domain. The sEH hydrolase converts epoxy fatty acids, such as epoxyeicosatrienoic acids (EETs) which are endogenous anti-inflammatory lipid mediators, to less-active diol forms, such as dihydroeicosatrienoic acids (DHETs). The sEH phosphatase is implicated in lipid metabolism and hydrolysis of lysophospatidic acid, whereas its precise biological role is still unclear. SMTP-7 and SMTP-44D inhibited both hydrolase (IC50 20 and 27 µM, respectively) and phosphatase (IC50 6 and 25 µM, respectively) activities of sEH. The simplest congener SMTP-0 (IC50 28 µM for hydrolase and 29 µM for phosphatase), which consists of only the core structure common with all the SMTP congeners, was used to analyze the kinetic mechanism of sEH inhibition. The inhibition of hydrolase by SMTP-0 was competitive with respect to 14,15-EET, and the inhibition of phosphatase is uncompetitive with respect to the synthetic substrate Attophos. The inhibition of phosphatase was unchanged in the presence of a potent competitive inhibitor of hydrolase, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid. Thus, SMTP-0 may bind to two distinct sites in sEH: one is the active site in the hydrolase domain, and the other is an allosteric site that affects the phosphatase domain. Inhibition of sEH hydrolase was also observed in cells in culture. The conversion of 14,15-EET to 14,15-DHET in HepG2 cells was inhibited by SMTP-7, SMTP-44D, and SMTP-0 with IC50 at 4.5, 8.8, and 1.3 mM, respectively. To confirm sEH inhibition in vivo, we traced the fate of intravenously injected EET in the liver. Treatment of wild-type mice with SMTP-7 significantly reduced the 14, 15-DHET level (∼41% reduction, P <0.05), while no significant reduction was observed in sEH KO mice. Thrombolytically inactive SMTP-44D reduced the degree of edema and pro-inflammatory cytokine expression in a mouse embolic stroke model, whereas infarct size and neurological deficits were not ameliorated. Thus, it is possible that both thrombolytic and anti-inflammatory potentials of SMTP are important in its excellent therapeutic activity.Our present study provides evidence that SMTP-7 targets sEH for anti-inflammatory action. The inhibition of sEH and the profibrinolytic action due to plasminogen modulator activity may synergistically contribute to treatment of ischemic stroke. SMTP-7 is thus a promising alternative therapy for ischemic stroke. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Ambivalent roles of carboxypeptidase B in the lytic susceptibility of fibrin

BackgroundRemoval of C-terminal lysine residues that are continuously exposed in lysing fibrin is an established anti-fibrinolytic mechanism dependent on the plasma carboxypeptidase TAFIa, which also removes arginines that are exposed at the time of fibrinogen clotting by thrombin.ObjectiveTo evaluate the impact of alterations in fibrin structure mediated by constitutive carboxypeptidase activity on the function of fibrin as a template for tissue plasminogen activator-(tPA) induced plasminogen activation and its susceptibility to digestion by plasmin.Methods and resultsWe used the stable carboxypeptidase B (CPB), which shows the same substrate specificity as TAFIa. If 1.5 – 6 μM fibrinogen was clotted in the presence of 8 U/mL CPB, a denser fibrin network was formed with thinner fibers (the median fiber diameter decreased from 138 – 144 nm to 89 – 109 nm as established with scanning electron microscopy). If clotting was initiated in the presence of 5 – 10 μM arginine, a similar decrease in fiber diameter (82 -95 nm) was measured. The fine structure of arginine-treated fibrin enhanced plasminogen activation by tPA, but slowed down lysis monitored using fluorescent tPA and confocal laser microscopy. However, if lysis was initiated with plasmin in CPB-treated fibrin, the rate of dissolution increased to a degree corresponding to doubling of the plasmin concentration.ConclusionThe present data evidence that CPB activity generates fine-mesh fibrin which is more difficult to lyse by tPA, but conversely, CPB and plasmin together can stimulate fibrinolysis, possibly by enhancing plasmin diffusion.

Delayed fracture healing in tetranectin-deficient mice

Tetranectin is a plasminogen-binding protein that enhances plasminogen activation, which has been suggested to play a role in tissue remodeling. Recently, we showed that tetranectin has a role in the wound-healing process. In this study, we investigated whether tetranectin plays a role in fracture healing. The fracture-healing process was studied using a femoral osteotomy model in tetranectin-null mice, previously generated by the authors. Radiographic imaging, micro-computed tomography (μCT), and histological analysis were used to evaluate osteotomy healing. In wild-type mice, a callus was apparent from 7days, and most samples showed marked callus formation and rebridging of the cortices at the osteotomy site at 21days. In contrast, in the tetranectin-null mice there was no callus formation at 7days and much less callus formation and no bridging of cortices were observed at 21days. At 35days, all osteotomy sites showed clear rebridging, and secondary bone formation was achieved in wild-type mice by 42days. In contrast, no clear rebridging or secondary bone formation was observed at 42days in the tetranectin-null mice. Analysis using μCT at 21days after osteotomy revealed that the callus area in tetranectin-null mice was smaller than that in wild-type mice. Histological analysis also showed that soft tissue and callus formation were smaller in the tetranectin-null mice at the early stage of the healing process after drill-hole injury. These results suggested that tetranectin could have a role in the positive regulation at the early stage of the fracture-healing process, which was reflected in the delayed fracture healing in tetranectin-deficient mice.

A new series of the SMTP plasminogen modulators with a phenylamine-based side chain

SMTPs are a family of small-molecule plasminogen modulators that enhance plasminogen activation. SMTP-7, one of the most potent congeners, is effective in treating thrombotic cerebral infarction. The SMTP molecule consists of a tricyclic γ-lactam moiety, a geranylmethyl group, and an N-linked side chain. The presence of both an aromatic group and a negatively ionizable group in the N-linked side chain is crucial for activity. Investigations of the congeners with a phenylglycine-based side chain suggest that a phenolic hydroxy group affects potency. In this study, we isolate and characterize a series of novel SMTP congeners with a phenylamine-based N-linked side chain. Of the 11 congeners isolated, SMTP-19 (with a 4-phenylcarboxylic acid moiety), SMTP-22 (with a 3-hydroxyphenyl-4-carboxylic acid moiety) and SMTP-25 (with a 2-hydroxyphenyl-3-carboxylic acid moiety) are as potent as SMTP-7 in plasminogen-modulating activity. Their isomers with a carboxylic acid group and/or a phenolic hydroxy group at different positions have <40% of the activity of these congeners. Both SMTP-22 and SMTP-25 have >1.7 times more oxygen radical absorbance capacity as compared with SMTP-7.

Potential Role of Kringle-Integrin Interaction in Plasmin and uPA Actions (A Hypothesis)

We previously showed that the kringle domains of plasmin and angiostatin, the N-terminal four kringles (K1–4) of plasminogen, directly bind to integrins. Angiostatin blocks tumor-mediated angiogenesis and has great therapeutic potential. Angiostatin binding to integrins may be related to the antiinflammatory action of angiostatin. We reported that plasmin induces signals through protease-activated receptor (PAR-1), and plasmin-integrin interaction may be required for enhancing plasmin concentration on the cell surface, and enhances its signaling function. Angiostatin binding to integrin does not seem to induce proliferative signals. One possible mechanism of angiostatin's inhibitory action is that angiostatin suppresses plasmin-induced PAR-1 activation by competing with plasmin for binding to integrins. Interestingly, plasminogen did not interact with αvβ3, suggesting that the αvβ3-binding sites in the kringle domains of plasminogen are cryptic. The kringle domain of urokinase-type plasminogen activator (uPA) also binds to integrins. The uPA-integrin interaction enhances uPA concentrations on the cell surface and enhances plasminogen activation on the cell surface. It is likely that integrins bind to the kringle domain, and uPAR binds to the growth factor-like domain (GFD) of uPA simultaneously, making the uPAR-uPA-integrin ternary complex. We present a docking model of the ternary complex.

A new series of the SMTP plasminogen modulator with a phenylglycine-based side chain

SMTPs are a family of small-molecule plasminogen modulators that enhance plasminogen activation. SMTP-7, one of the most potent congeners, is effective in treating thrombotic cerebral infarction. The SMTP molecule consists of a tricyclic γ-lactam moiety, a geranylmethyl group, and an N-linked side chain. The presence of both an aromatic group and a negatively ionizable group in the N-linked side chain is crucial for activity. Investigations of the congeners with a phenylglycine-based side chain suggest that a phenolic hydroxy group affects potency. In this study, we isolate and characterize a series of novel SMTP congeners with a phenylamine-based N-linked side chain. Of the 11 congeners isolated, SMTP-19 (with a 4-phenylcarboxylic acid moiety), SMTP-22 (with a 3-hydroxyphenyl-4-carboxylic acid moiety) and SMTP-25 (with a 2-hydroxyphenyl-3-carboxylic acid moiety) are as potent as SMTP-7 in plasminogen-modulating activity. Their isomers with a carboxylic acid group and/or a phenolic hydroxy group at different positions have 1.7 times more oxygen radical absorbance capacity as compared with SMTP-7.

Modifications of the defense and remodeling functionalities of bovine neutrophils inside the mammary gland of milk stasis cows received a commercial dry-cow treatment

An appropriate length of milk stasis between two consecutive lactations of dairy cows is crucial for sustainable milk production. This dry period of cows allows extensive remodeling and sufficient cell renewal in mammary gland. Nevertheless, early dry period is one of the most risky stages in cow lactation cycle to intramammary infection. Dry-cow treatment through teats is, therefore, widely practiced at the commencement of milk stasis. Neutrophils are the most abundant cellular components in cow mammary secretion during early dry period, which in turn attribute to the meanwhile elevation of somatic cell counts and matrix metalloproteinase-9 (MMP-9) level. This study used bovine peripheral neutrophils as a cell model to examine the mode of modifications in their defense and remodeling functionalities after infiltration into mammary gland during early dry period. Results indicate a dose-dependent suppression of phorbol 12-myristate 13-acetate-stimulated free radical production and induction of MMP 9 degranulation in bovine peripheral neutrophils exposure to the d 7-dry secretion of cows received dry cow treatment at d 0 in milk stasis. Meanwhile, an enhancement of plasminogen activation and TNF-α shedding on bovine peripheral neutrophils were also observed. These two cellular events might be involved in the functional modifications on infiltrated neutrophils during early dry period. In conclusion, the opposite trend of modifications in the defense and matrix remodeling functionalities of neutrophils inside the mammary gland of cows at early dry period reflect the collaboration of infiltrated neutrophils for promoting extensive glandular remodeling at minimum compromise of local defense during the acute involution period without apparent disturbance by dry cow treatment.

A synthetic peptide derived from staphylokinase enhances plasminogen activation by tissue‐type plasminogen activator

A synthetic nonadecapeptide (SP; GPYLMVNVTGVDGKGNELL) previously enhanced the activation of plasminogen by the SAK/plasmin complex. To identify the binding site for SP on plasminogen and elucidate the effects of SP on plasminogen activation by the tissue-type plasminogen activator (t-PA). The effects of SP on plasminogen activation were estimated using a chromogenic substrate and from the cleavage of plasmin on SDS-PAGE under reduced conditions. The binding to SP of various peptides derived from the amino acid sequence of plasminogen was analyzed with an IAsys biosensor. The SP-mediated structural change to plasminogen was analyzed by circular dichroism (CD) spectroscopy. The thrombolytic effects of SP were examined using a mouse model of thrombosis. SP enhanced the activation of plasminogen by t-PA. The catalytic efficiency (k(cat)/K(m)) of Glu-plasminogen activation by t-PA was 11.4-fold higher in the presence than absence of SP. The binding of SP to plasminogen was greatly inhibited by a synthetic peptide, FEKDKYILQGVTSWGLG, located close to the C-terminal of the plasminogen B region. Near-ultraviolet CD spectra of the complex between SP and Glu-plasminogen significantly differed from those of Glu-plasminogen. When SP was administered in a mouse model of thrombosis, early recanalization was observed in a dose-dependent manner. However, SP did not cause recanalization in t-PA gene-deficient mice. SP bound to the B region and promoted the activation of plasminogen by t-PA, and then induced effective thrombolysis.