Plasminogen Receptors and Fibrinolysis.

The ability of cells to promote plasminogen activation on their surfaces is now well recognized, and several distinct cell surface proteins have been demonstrated to function as plasminogen receptors. Here, we review studies demonstrating that plasminogen bound to cells, in addition to plasminogen directly bound to fibrin, plays a major role in regulating fibrin surveillance. We focus on the ability of specific plasminogen receptors on eukaryotic cells to promote fibrinolysis in the in vivo setting by reviewing data obtained predominantly in murine models. Roles for distinct plasminogen receptors in fibrin surveillance in intravascular fibrinolysis, immune cell recruitment in the inflammatory response, wound healing, and lactational development are discussed.

Vimentin coordinates fibroblast proliferation and keratinocyte differentiation in wound healing via TGF-ß-Slug signaling.

Vimentin has been shown to be involved in wound healing, but its functional contribution to this process is poorly understood. Here we describe a previously unrecognized function of vimentin in coordinating fibroblast proliferation and keratinocyte differentiation during wound healing. Loss of vimentin led to a severe deficiency in fibroblast growth, which in turn inhibited the activation of two major initiators of epithelial-mesenchymal transition (EMT), TGF-ß1 signaling and the Zinc finger transcriptional repressor protein Slug, in vimentin-deficient (VIM(-/-)) wounds. Correspondingly, VIM(-/-) wounds exhibited loss of EMT-like keratinocyte activation, limited keratinization, and slow reepithelialization. Furthermore, the fibroblast deficiency abolished collagen accumulation in the VIM(-/-) wounds. Vimentin reconstitution in VIM(-/-) fibroblasts restored both their proliferation and TGF-ß1 production. Similarly, restoring paracrine TGF-ß-Slug-EMT signaling reactivated the transdifferentiation of keratinocytes, reviving their migratory properties, a critical feature for efficient healing. Our results demonstrate that vimentin orchestrates the healing by controlling fibroblast proliferation, TGF-ß1-Slug signaling, collagen accumulation, and EMT processing, all of which in turn govern the required keratinocyte activation.

Novel diagnostic ELISA test for discrimination between infections with Yersinia enterocolitica and Yersinia pseudotuberculosis.

Yersiniosis is a foodborne infection caused by Yersinia enterocolitica or Yersinia pseudotuberculosis. Although yersiniosis is most often self-limiting, some patients develop chronic infections, such as reactive arthritis, glomerulonephritis, or myocarditis, which require an antibiotic treatment. Whereas early infections can be diagnosed by direct detection of bacteria, chronic infections can only be identified by serological tests. At this point, a serological method for differentiation between infections with the two Yersinia species is important since antibiotic susceptibility of these bacteria is different. Traditional immunoassays do not distinguish between infections with Y. enterocolitica and Y. pseudotuberculosis. The only test that allows for this differentiation is Mikrogen's strip test where discrimination between the two types of infection is based on two recombinant bacterial proteins, MyfA and PsaA (specific for Y. enterocolitica and Y. pseudotuberculosis, respectively). Here, we show that Y. enterocolitica and Y. pseudotuberculosis, cultured under the conditions that mimic the natural rout of infection, express surface antigens different from MyfA and PsaA that can also be used in a discrimination test. Further, we describe a new ELISA that is based on the whole bacteria and recombinant MyfA and PsaA as antigens, and that allows the differentiation between infections with Y. enterocolitica and Y. pseudotuberculosis and simultaneous detection of yersiniosis.

Plasminogen initiates and potentiates the healing of acute and chronic tympanic membrane perforations in mice.

BACKGROUND: Most tympanic membrane (TM) perforations heal spontaneously, but approximately 10-20% remain open as chronic TM perforations. Chronic perforations can lead to an impaired hearing ability and recurrent middle ear infections. Traditionally, these perforations must be surgically closed, which is costly and time consuming. Therefore, there is a need for simpler therapeutic strategies. Previous studies by us have shown that plasminogen (plg) is a potent pro-inflammatory regulator that accelerates cutaneous wound healing in mice. We have also shown that the healing of TM perforations is completely arrested in plg-deficient (plg(-/-)) mice and that these mice develop chronic TM perforations. In the present study, we investigated the therapeutic potential of local plg injection in acute and chronic TM perforation mice models. METHODS: Plg(-/-) mice and wild-type mice were subjected to standardized TM perforations followed by local injection of plg into the soft tissue surrounding the TM. TM perforations with chronic characteristics were induced by leaving TM perforations in plg(-/-) mice untreated for 9 days before treatment. The healing process was observed through otomicroscope and finally confirmed by immunostaining. The quality of TM healing was evaluated based on the morphology of the TM. RESULT: Daily local injections of plg into the soft tissue surrounding the TM restored the ability to heal TM perforations in plg-/- mice in a dose-dependent manner, and potentiated the healing rate and quality in wild-type mice. A single local injection of plg initiated the healing of the chronic-like TM perforations in these mice, resulting in a closed TM with a continuous but rather thick outer keratinocyte layer. However, three plg injections led to a completely healed TM with a thin keratinizing squamous epithelium covering a connective tissue layer. CONCLUSION: Our data suggests that plg is a promising drug candidate for the treatment of chronic TM perforations in humans.

Plasminogen is a key proinflammatory regulator that accelerates the healing of acute and diabetic wounds.

Despite decades of research on wound healing, effective biologic agents for the treatment of chronic wounds, especially diabetic wounds, are still lacking. In the present study, we report that the inert plasma protein plasminogen (plg) acts as a key regulatory molecule that potentiates wound healing in mice. Early in the healing process, plg bound to inflammatory cells is transported to the wound area, where the level of plg is increased locally, leading to the induction of cytokines and intracellular signaling events and to a potentiation of the early inflammatory response. Systemic administration of additional plg not only accelerates the healing of acute burn wounds in wild-type mice, but also improves the healing of chronic diabetic wounds in a mouse model of diabetes. Our results suggest that the administration of plg may be a novel therapeutic strategy to treat many different types of wounds, especially chronic wounds such as those caused by diabetes.

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.

Plasminogen is a master regulator and a potential drug candidate for the healing of radiation wounds.

Around 95% of cancer patients undergoing radiotherapy experience cutaneous side effects, and some develop radiation wounds or fibrosis. Currently, there is no effective treatment for these indications. We show here that plasminogen administration enhanced the healing of radiation wounds via pleiotropic effects on gene expression. Using RNA sequencing, we found that plasminogen downregulated the expression of genes in the TLR, TNF, WNT, MAPK, and TGF-ß signaling pathways, and enhanced the anti-inflammatory effect of arachidonic acid, leading to significantly decreased inflammation and improved remodeling of granulation tissue compared with placebo treatment. In addition, plasminogen induced metabolic changes, including decreased glycolysis. Importantly, many of the factors downregulated by plasminogen are pro-fibrotic. Therefore, in radiation wounds with excessive inflammation, plasminogen is able to enhance and redirect the healing process, such that it more closely resembles physiological healing with significantly reduced risk for developing fibrosis. This makes plasminogen an attractive drug candidate for the treatment of radiation wounds in cancer patients.

The plasminogen receptor, Plg-RKT, plays a role in inflammation and fibrinolysis during cutaneous wound healing in mice.

Wound healing is a complex physiologic process that proceeds in overlapping, sequential steps. Plasminogen promotes fibrinolysis and potentiates the inflammatory response during wound healing. We have tested the hypothesis that the novel plasminogen receptor, Plg-RKT, regulates key steps in wound healing. Standardized burn wounds were induced in mice and time dependence of wound closure was quantified. Healing in Plg-RKT-/- mice was significantly delayed during the proliferation phase. Expression of inflammatory cytokines was dysregulated in Plg-RKT-/- wound tissue. Consistent with dysregulated cytokine expression, a significant delay in wound healing during the proliferation phase was observed in mice in which Plg-RKT was specifically deleted in myeloid cells. Following wound closure, the epidermal thickness was less in Plg-RKT-/- wound tissue. Paradoxically, deletion of Plg-RKT, specifically in keratinocytes, significantly accelerated the rate of healing during the proliferation phase. Mechanistically, only two genes were upregulated in Plg-RKT-/- compared with Plg-RKT+/+ wound tissue, filaggrin, and caspase 14. Both filaggrin and caspase 14 promote epidermal differentiation and decrease proliferation, consistent with more rapid wound closure and decreased epidermal thickness during the remodeling phase. Fibrin clearance was significantly impaired in Plg-RKT-/- wound tissue. Genetic reduction of fibrinogen levels to 50% completely abrogated the effect of Plg-RKT deletion on the healing of burn wounds. Remarkably, the effects of Plg-RKT deletion on cytokine expression were modulated by reducing fibrinogen levels. In summary, Plg-RKT is a new regulator participating in different phases of cutaneous burn wound healing, which coordinately plays a role in the interrelated responses of inflammation, keratinocyte migration, and fibrinolysis.

Disturbed Laminar Blood Flow Causes Impaired Fibrinolysis and Endothelial Fibrin Deposition In Vivo.

Endothelial expression of tissue-type plasminogen activator (t-PA) is crucial for maintaining an adequate endogenous fibrinolysis. It is unknown how endothelial t-PA expression and fibrinolysis are affected by blood flow in vivo. In this study, we investigated the impact of different blood flow profiles on endothelial t-PA expression and fibrinolysis in the arterial vasculature. Induction of disturbed laminar blood flow (D-flow) in the mouse carotid artery potently reduced endothelial t-PA messenger ribonucleic acid and protein expression, and caused fibrin deposition. En face immunohistochemistry demonstrated that arterial areas naturally exposed to D-flow had markedly lower endothelial t-PA levels than areas with sustained laminar blood flow (S-flow), and displayed pronounced fibrin deposition despite an intact endothelium. In t-PA and plasminogen-deficient mice, fibrin deposition did not extend into S-flow areas, indicating that areas of D-flow and S-flow differ, not only in fibrinolytic capacity, but also in coagulation. Furthermore, plasminogen accumulation was found at D-flow areas, and infusion of recombinant t-PA activated fibrinolysis and significantly reduced the fibrin deposits. In conclusion, D-flow potently impairs the fibrinolytic capacity and causes endothelial fibrin deposition in vivo. Our data also indicate that t-PA is the limiting factor for efficient fibrinolysis at the thrombosis-prone D-flow areas in the arterial vasculature.

Expression and localization of the serine proteases high-temperature requirement factor A1, serine protease 23, and serine protease 35 in the mouse ovary.

Proteolytic degradation of extracellular matrix components has been suggested to play an essential role in the occurrence of ovulation. Recent studies in our laboratory have indicated that the plasminogen activator and matrix metalloproteinase systems, which were previously believed to be crucial for ovulation, are not required in this process. In this study we have used a microarray approach to identify new proteases that are involved in ovulation. We found three serine proteases that were relatively highly expressed during ovulation: high-temperature requirement factor A1 (HtrA1), which was not regulated much during ovulation; serine protease 23 (PRSS23), which was down-regulated by gonadotropins; and serine protease 35 (PRSS35), which was up-regulated by gonadotropins. We have further investigated the expression patterns of these proteases during gonadotropin-induced ovulation in immature mice and in the corpus luteum (CL) of pseudopregnant mice. We found that HtrA1 was highly expressed in granulosa cells throughout follicular development and ovulation, as well as in the forming and regressing CL. PRSS23 was highly expressed in atretic follicles, and it was expressed in the ovarian stroma and theca tissues just before ovulation. PRSS35 was expressed in the theca layers of developing follicles. It was also highly induced in granulosa cells of preovulatory follicles. PRSS35 was also expressed in the forming and regressing CL. These data suggest that HtrA1 and PRSS35 may be involved in ovulation and CL formation and regression, and that PRSS23 may play a role in follicular atresia.

Expression patterns of neurotrophins and neurotrophin receptors in articular chondrocytes and inflammatory infiltrates in knee joint arthritis.

BACKGROUND: It is likely that neurotrophins (NTs) are of great importance for the articular cartilage and the inflammation process in arthritis. METHODS: The immunohistochemical expression of the NTs nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and the associated receptors p75, TrkA and TrkB was examined in the knee joint of arthritic and healthy mice. RESULTS: Immunoreactions for NGF and BDNF were detected in cells and nerve fiber varicosities in the inflammatory infiltrates of the synovial tissue of arthritic joints but not in synovial tissue of controls. p75-immunoreactive nerve fiber-like strands were detected in inflammatory infiltrates. Immunostaining for NGF, BDNF, p75, TrkA and TrkB was noted in articular chondrocytes. There was a statistically significant decrease in reactions for NGF (p < 0.001), TrkA (p = 0.001) and p75 (p < 0.001) in articular chondrocytes in joints exhibiting severe arthritis. CONCLUSION: The findings show that an NT system develops in inflammatory infiltrates of the synovial tissue. Furthermore, most interestingly, autocrine/paracrine effects appear to exist concerning NTs for the articular chondrocytes. The downregulated expression of NGF and NT receptors in articular chondrocytes in arthritis is a new aspect concerning the involvement of NTs in cartilage.

Plasminogen activation is required for the development of radiation-induced dermatitis.

Skin damage caused by radiation therapy (radiodermatitis) is a severe side effect of radiotherapy in cancer patients, and there is currently a lack of effective strategies to prevent or treat such skin damage. In this work, we show with several lines of evidence that plasminogen, a pro-inflammatory factor, is key for the development of radiodermatitis. After skin irradiation in wild-type (plg+/+) mice, the plasminogen level increased in the irradiated area, leading to severe skin damage such as ulcer formation. However, plasminogen-deficient (plg-/-) mice and mice lacking plasminogen activators were mostly resistant to radiodermatitis. Moreover, treatment with a plasminogen inhibitor, tranexamic acid, decreased radiodermatitis in plg+/+ mice and prevented radiodermatitis in plg+/- mice. Together with studies at the molecular level, we report that plasmin is required for the induction of inflammation after irradiation that leads to radiodermatitis, and we propose that inhibition of plasminogen activation can be a novel treatment strategy to reduce and prevent the occurrence of radiodermatitis in patients.

Functional corpora lutea are formed in matrix metalloproteinase inhibitor-treated plasminogen-deficient mice.

Corpus luteum (CL) formation involves dramatic tissue remodeling and angiogenesis. To determine the functional roles of the plasminogen activator and matrix metalloproteinase (MMP) systems in these processes, we have studied CL formation and function in plasminogen (plg)-deficient mice, with or without treatment with the broad-spectrum synthetic MMP inhibitor galardin. Both the adult pseudopregnant CL model and the gonadotropin-primed immature mouse model were used. We found that CL formed normally not only in plasminogen-deficient mice and in galardin-treated wild-type mice, but also in galardin-treated plg-deficient mice, suggesting that neither of the plasminogen activator and MMP systems is essential for CL formation. Nevertheless, in plg-deficient mice, serum progesterone levels were reduced by approximately 50%, and the progesterone levels were not reduced further by galardin treatment. When CL from plg-deficient mice were stained for several molecular markers for CL development and regression, they appeared healthy and vascularized, and were indistinguishable from CL from wild-type mice. This implies that the reduced progesterone levels were not caused by impaired CL formation. Taken together, our data suggest that neither plasmin nor MMPs, alone or in combination, are required for CL formation. Therefore, the tissue remodeling and angiogenesis processes during CL formation may be mediated by redundant protease systems. However, the reduced serum progesterone levels in plg-deficient mice suggest that plasmin, but not MMPs, plays a role in maintenance of luteal function. This role may be performed through proteolytic activation of growth factors and other paracrine factors.

Plasmin/plasminogen is essential for the healing of tympanic membrane perforations.

Plasminogen has been proposed to play an important role in different tissue remodeling processes such as wound healing and tissue regeneration after injuries. The healing of tympanic membrane perforations is a well-organized chain of inflammatory events, with an initial invasion of inflammatory cells followed by reparative and restoration phases. Here we show that the healing of tympanic membrane perforations is completely arrested in plasminogen-deficient mice, with no signs of any healing even 143 days after perforation. Inflammatory cells were recruited to the wounded area, but there were no signs of tissue debridement. In addition, removal of fibrin, keratinocyte migration and in-growth of connective tissue were impaired. This contrasts with skin wound healing, where studies have shown that, although the healing process is delayed, it reaches completion in all plasminogen-deficient mice. Our finding that keratinocyte migration and re-epithelialization were completely arrested in plasminogen-deficient mice indicates that plasminogen/plasmin plays a more profound role in the healing of tympanic membrane perforations than in the healing of other epithelial wounds.

Plasminogen is a critical regulator of cutaneous wound healing.

Wound healing is a complicated biological process that consist of partially overlapping inflammatory, proliferation and tissue remodelling phases. A successful wound healing depends on a proper activation and subsequent termination of the inflammatory phase. The failure to terminate the inflammation halts the completion of wound healing and is a known reason for formation of chronic wounds. Previous studies have shown that wound closure is delayed in plasminogen-deficient mice, and a role for plasminogen in dissection of extracellular matrix was suggested. However, our finding that plasminogen is transported to the wound by inflammatory cells early during the healing process, where it potentiates inflammation, indicates that plasminogen may also have other roles in the wound healing process. Here we report that plasminogen-deficient mice have extensive fibrin and neutrophil depositions in the wounded area long after re-epithelialisation, indicating inefficient debridement and chronic inflammation. Delayed formation of granulation tissue suggests that fibroblast function is impaired in the absence of plasminogen. Therefore, in addition to its role in the activation of inflammation, plasminogen is also crucial for subsequent steps, including resolution of inflammation and activation of the proliferation phase. Importantly, supplementation of plasminogen-deficient mice with human plasminogen leads to a restored healing process that is comparable to that in wild-type mice. Besides of being an activator of the inflammatory phase during wound healing, plasminogen is also required for the subsequent termination of inflammation. Based on these results, we propose that plasminogen may be an important future therapeutic agent for wound treatment.

Structural Insights into Polymorphic ABO Glycan Binding by Helicobacter pylori.

The Helicobacter pylori adhesin BabA binds mucosal ABO/Le(b) blood group (bg) carbohydrates. BabA facilitates bacterial attachment to gastric surfaces, increasing strain virulence and forming a recognized risk factor for peptic ulcers and gastric cancer. High sequence variation causes BabA functional diversity, but the underlying structural-molecular determinants are unknown. We generated X-ray structures of representative BabA isoforms that reveal a polymorphic, three-pronged Le(b) binding site. Two diversity loops, DL1 and DL2, provide adaptive control to binding affinity, notably ABO versus O bg preference. H. pylori strains can switch bg preference with single DL1 amino acid substitutions, and can coexpress functionally divergent BabA isoforms. The anchor point for receptor binding is the embrace of an ABO fucose residue by a disulfide-clasped loop, which is inactivated by reduction. Treatment with the redox-active pharmaceutic N-acetylcysteine lowers gastric mucosal neutrophil infiltration in H. pylori-infected Le(b)-expressing mice, providing perspectives on possible H. pylori eradication therapies.

Structural bases of the redox-dependent conformational switch in the serpin PAI-2.

Depending on the redox-status, the serpin plasminogen activator inhibitor type 2 (PAI-2) can exist in either a stable monomeric or polymerogenic form. The latter form, which spontaneously forms loop-sheet polymers, has an open beta-sheet A and is stabilized by a disulfide bond between C79 (in the CD-loop) and C161 (at the bottom of PAI-2). Reduction of this bond results in a closing of the beta-sheet A and converts PAI-2 to a stable monomeric form. Here we show that the stable monomeric and polymerogenic forms of PAI-2 are fully interconvertible, depending on redox-status of the environment. Our intramolecular distance measurements indicate that the CD-loop folds mainly on one side of the stable monomeric form of the inhibitor. However, the loop can translocate about 54A to the bottom of PAI-2 so that the C79-C161 disulfide bond can form under oxidizing conditions. We show also that the redox-active C79 can form a disulfide-link to the matrix protein vitronectin, suggesting that vitronectin can stabilize active PAI-2 in extracellular compartments. PAI-2 is therefore a rare example of a redox-sensitive protein for which the activity and polymerization ability are regulated by reversible disulfide bond formation leading to major translocation of a loop and significant conformational changes in the molecule.

The reactive-center loop of active PAI-1 is folded close to the protein core and can be partially inserted.

Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of plasminogen activators and plays an important role in many pathophysiological processes. Like other members of the serpin family, PAI-1 has a reactive center consisting of a mobile loop (RCL) with P1 and P1' residues acting as a "bait" for cognate protease. In contrast to the other serpins, PAI-1 loses activity by spontaneous conversion to an inactive latent form. This involves full insertion of the RCL into beta-sheet A. To search for molecular determinants that could be responsible for conversion of PAI-1 to the latent form, we studied the conformation of the RCL in active PAI-1 in solution. Intramolecular distance measurements by donor-donor energy migration and probe quenching methods reveal that the RCL is located much closer to the core of PAI-1 than has been suggested by the recently resolved X-ray structures of stable PAI-1 mutants. Disulfide bonds can be formed in double-cysteine mutants with substitutions at positions P11 or P13 of the RCL and neighboring residues in beta-sheet A. This suggests that the RCL may be preinserted up to residue P13 in active PAI-1, and possibly even to residue P11. We propose that the close proximity of the RCL to the protein core, and the ability of the loop to preinsert into beta-sheet A is a possible reason for PAI-1 being able to convert spontaneously to its latent form.

Residues determining the binding specificity of uncompetitive inhibitors to tissue-nonspecific alkaline phosphatase.

UNLABELLED: Recent data have pointed to TNALP as a therapeutic target for soft-tissue ossification abnormalities. Here, we used mutagenesis, kinetic analysis, and computer modeling to identify the residues important for the binding of known ALP inhibitors to the TNALP active site. These data will enable drug design efforts aimed at developing improved specific TNALP inhibitors for therapeutic use. INTRODUCTION: We have shown previously that the genetic ablation of tissue-nonspecific alkaline phosphatase (TNALP) function leads to amelioration of soft-tissue ossification in mouse models of osteoarthritis and ankylosis (i.e., Enpp1-/- and ank/ank mutant mice). We surmise that the pharmacologic inhibition of TNALP activity represents a viable therapeutic approach for these diseases. As a first step toward developing suitable TNALP therapeutics, we have now clarified the residues involved in binding well-known uncompetitive inhibitors to the TNALP active site. MATERIALS AND METHODS: We compared the modeled 3D structure of TNALP with the 3D structure of human placental alkaline phosphatase (PLALP) and identified the residues that differ between these isozymes within a 12 A radius of the active site, because these isozymes differ significantly in inhibitor specificity. We then used site-directed mutagenesis to substitute TNALP residues to their respective homolog in PLALP. In addition, we mutagenized most of these residues in TNALP to Ala and the corresponding residues in PLALP to their TNALP homolog. All mutants were characterized for their sensitivity toward the uncompetitive inhibitors l-homoarginine (L-hArg), levamisole, theophylline, and l-phenylalanine. RESULTS AND CONCLUSIONS: We found that the identity of residue 108 in TNALP largely determines the specificity of inhibition by L-hArg. The conserved Tyr-371 is also necessary for binding of L-hArg. In contrast, the binding of levamisole to TNALP is mostly dependent on His-434 and Tyr-371, but not on residues 108 or 109. The main determinant of sensitivity to theophylline is His-434. Thus, we have clarified the location of the binding sites for all three TNALP inhibitors, and we have also been able to exchange inhibitor specificities between TNALP and PLALP. These data will enable drug design efforts aimed at developing improved, selective, and drug-like TNALP inhibitors for therapeutic use.

Tissue-type plasminogen activator and its inhibitor plasminogen activator inhibitor type 1 are coordinately expressed during ovulation in the rhesus monkey.

Ovulation is a gonadotropin-controlled process that is essential for the propagation of all mammalian species. In the present study, we used a pregnant mare serum gonodotropin/human chorionic gonadotropin (hCG)-induced, synchronized ovulation model in rhesus monkeys and systematically investigated the roles of the plasminogen activator (PA) system in the ovulatory process of the primate. At different follicular developmental stages throughout the periovulatory period, samples of ovaries, granulosa cells, and theca-interstitial cells as well as follicular fluid were collected, and levels of PA and PA inhibitor type-1 (PAI-1) were evaluated by fibrin overlay, reverse fibrin overlay, Northern blot analysis, and in situ hybridization, respectively. We showed that in response to an injection of ovulation-triggering hCG, which mimics the preovulatory surge of LH in the circulation, granulosa cell-derived tissue-type PA (tPA) was substantially elevated in preovulatory follicles and reached its maximum level just before ovulation. Although theca-interstitial cell-derived PAI-1 was also stimulated by pregnant mare serum gonodotropin and hCG treatments, however, the maximum level of PAI-1 appeared 12 h earlier than that of tPA. When ovulation approached, accompanying the highest tPA level in the preovulatory follicles, the follicular PAI-1 level declined dramatically to its minimum value. Moreover, our data on the expression of follicular PA and PAI-1 over the periovulatory period were reinforced by results obtained at the mRNA level. Our data suggest that the coordinated expression of tPA and PAI-1 may be of importance for the follicular rupture process during ovulation in the primate.