The sequential,complementary and synergistic activation of fibrin-bound plasminogen by tissue plasminogen activator and pro-urokinase

Tissue plasminogen activator (t-PA) and pro-urokinase (pro-UK) are the two principal activators of the fibrinolytic system. They share a relative fibrin specificity related to their selective activation of fibrin-bound plasminogen, but achieve this by very different mechanisms. Some of the properties of pro-UK, which contrast with those of t-PA, are reviewed. These include zymogenicity and stability in plasma, lack of significant fibrin affinity and requirement for a certain conformational change in Glu-plasminogen. This conformation appears to be induced principally when Glu-plasminogen is bound to carboxyterminal lysine residues which interact with the high affinity lysine binding sites. These residues become available on fibrin only after degradation has been initiated. By contrast, in a plasma milieu, fibrin-bound t-PA was found to activate plasminogen on intact fibrin. This plasminogen binding involves internal lysine residues which interact with the weak lysine binding site on Kringle 5. The two activators are therefore sequential and complementary in their activation of fibrin-bound plasminogen. This complementariness helps to explain their synergistic fibrinolytic effects when combined. The findings also explain the lag phase characteristic of pro-UK but not t-PA-induced clot lysis. They also help account for the unusually high dose requirements for t-PA and pro-UK in therapeutic thrombolysis when each is used alone. A rationale for combination therapy is provided which may mimic the natural design for fibrinolysis.     

Diversity in catalytic properties of single chain and two chain tissue-type plasminogen activator

Like many other serine proteases, human tissue-type plasminogen activator (t-PA) is synthesised and released from cells in a single chain (sc-) form. This may later be converted proteolytically to a two chain (tc-) form, in which the chains are held together by a disulfide bridge. With most serine proteases, the sc-form is a genuine zymogen with an activity several orders of magnitude smaller than that of the tc-form. But in the case of t-PA, the relative activity of the two forms has been a controversial issue. We review here old and new investigations of the problem, emphasizing recent experiments with genetically engineered t-PA mutants that cannot be cleaved to the tc-form. Provided steps are taken to prevent plasmin-catalysed conversion of sc- to tc-t-PA during the assays, the results consistently point to a plasminogen activation activity of sc-t-PA that is at least one order of magnitude smaller than that of tc-t-PA. Fibrin stimulates the activity of sc-t-PA strongly and to a higher extent than tc-t-PA, resulting in equal activities of the two forms in the presence of fibrin. On the other hand, there is no doubt that sc-t-PA as such possesses a distinct activity even in the absence of fibrin. It reacts with synthetic substrates, with reactive site reagents and with its natural inhibitors. These properties have important implications as to the way t-PA may fulfil its physiological function in fibrinolysis: sc-t-PA is not likely to circulate as a reservoir of potential plasminogen activation activity. Rather, plasmin generation and subsequent fibrinolysis is likely to be initiated by release of sc-t-PA from the endothelium and to depend on the presence of polymerised fibrin as a cofactor. Other mechanisms of t-PA activation may be involved in non-fibrinolytic functions of the activator.     

Plasmin-catalysed cleavage of single chain tissue-type plasminogen activator in fibrin clots

The activity of single chain t-PA in the presence of fibrin was investigated and compared to that of its twochain counterpart. A plasmin resistant t-PA analogue in which Arg-275 is replaced by Gly was included in this study in order to avoid the complications caused by concomitant two chain generation during plasminogen activation experiments. Substantial plasminogen activation of single chain t-PA was observed during fibrin clot dissolution, still the clot lysis activity of two chain t-PA was found to be about 20% higher than that of the single chain form. Plasmin-catalysed cleavage of single chain t 25I-t-PA was studied in the presence and absence of fibrin. This reaction was not enhanced by fibrin, rather a small inhibition was observed. In addition to the primary cleavage site at Arg-275 Ile-276 secondary plasmin-catalysed cleavage resulting in a 30 000 Da fragment was observed. Appearance of this fragment took place roughly at the same time as a decline in clot lysis activity (but not in amidolytic activity) was observed. Secondary plasmin cleavage was not observed when fibrin was present.     

Differing roles for urokinase and tissue-type plasminogen activator in collagen-induced arthritis

The plasminogen activators, urokinase PA (u-PA) and tissue-type PA (t-PA), are believed to play important roles in inflammatory cell infiltration, fibrin deposition, and joint destruction associated with rheumatoid arthritis; however, their precise roles in such processes, particularly u-PA, have yet to be defined. Using gene-deficient mice we examined the relative contribution of the PAs to the chronic systemic collagen-induced arthritis model. Based on clinical and histological assessments, u-PA-/- mice developed significantly milder disease and t-PA-/- mice more severe disease compared with the relevant wild-type mice. Fibrin deposition within joints paralleled disease severity and was particularly pronounced in t-PA-/- mice. Likewise, cytokine levels in the synovium reflected the severity of disease, with interleukin-1beta levels in particular being lower in u-PA-/- mice and increased in t-PA-/- mice. The antibody response to type II collagen was normal in both knockouts; however, T cells from u-PA-/- mice had a reduced proliferative response and produced less interferon-gamma on antigen stimulation in vitro. These results indicate that the major effect of u-PA in the collagen-induced arthritis model is deleterious, whereas that of t-PA is protective. Our data highlight the complexities of PA function, and suggest that approaches either to target u-PA or to enhance local t-PA activity in joints may be of therapeutic benefit in rheumatoid arthritis.     

Amyloid-beta-stimulated plasminogen activation by tissue-type plasminogen activator results in processing of neuroendocrine factors

Alzheimer's disease brain is characterized by the abundant presence of amyloid deposits. Accumulation of the major constituent of these deposits, amyloid-beta (Abeta), has been associated with decreased neurotransmission, increased neuronal cell death, and with cognitive decline. The mechanisms underlying these phenomena have not yet been fully elucidated. We have previously shown that amyloid peptides like Abeta bind tissue-type plasminogen activator (tPA) and cause enhanced plasmin production. Here we describe the identification of five major neuronal cell-produced Abeta-associated proteins and how Abeta-stimulated plasmin formation affects their processing. These five proteins are all neuroendocrine factors (NEFs): chromogranins A, B and C; truncated chromogranin B; and VGF. Plasminogen caused processing of Abeta-bound (but not soluble) tPA, chromogranin B and VGF and the degradation products were released from Abeta. Processing of the neuroendocrine factors was dependent on tPA as it was largely abrogated in tPA-/- cells or in the presence of a specific tPA-inhibitor. If plasmin indeed produces NEF-derived peptides in vivo, some of these peptides may have biological activity, for instance in regulating neurotransmitter release that may affect the pathology of Alzheimer's disease.     

尿激酶型纤溶酶原激活物受体及可溶性尿激酶型纤溶酶原激活物受体与炎症相关的研究进展

尿激酶型纤溶酶原激活物受体（urokinase plasminogen activator receptor,uPAR）与尿激酶型纤溶酶原激活物（urokinase plasminogen activator,uPA）同为纤溶酶原激活系统的主要成员,是一种协调多种信号转导途径的多功能分子,可溶性尿激酶型纤溶酶原激活物受体（soluble urokinase plasminogen activator receptor,suPAR）是其可溶形式。除凝血-纤溶以外,uPAR参与了肿瘤侵袭及炎症等多种疾病过程,而suPAR可能是一种良好的炎性标志物。本文就uPAR及suPAR在炎症中的作用进行简要综述。     

Influence of heparin and various glycosaminoglycans on activation of single chain urokinase-type plasminogen activator (scu-PA) by plasmin

Increase in the rate of activation of single chain urokinase-type plasminogen activator (scu-PA) to two chain urokinase (tcu-PA) by plasmin caused by heparin was observed to be molecular mass dependent. A heparin fraction of 30 kDa increased the catalytic efficiency of activation by 3.7-fold as opposed to 1.4-fold by heparin of average molecular weight. All of a range of glycosaminoglycans tested were observed to cause some increase in the rate of activation through chondroitin sulphate K (Glc UA-3-SO₄- Gal-NAc-4-SO₄) was twice as potent as unfractionated heparin. Increase in rate was due to variation in kcat and Km. This effect was concentration-dependent such that Km was decreased at low heparin concentration, but at high concentration of heparin increased to beyond that in its absence. A progressive increase in kcat was observed with heparin concentration. Heparin also caused increase in catalytic efficiency of K5 plasmin suggesting that the observed effects may be due to interaction with scu-PA rather than plasmin. Heparin had no influence on kallikrein activation of scu-PA. The influence of glycosaminoglycans upon this reaction may be of importance in controlling the physiological function of scu-PA.     

Levels of tissue-type plasminogen activator and plasminogen activator inhibitor 1 in disseminated intravascular coagulation syndrome

Since disseminated intravascular coagulation (DIC) may directly reflect the abnormal regulation of the fibrinolytic system by endothelial cells, we have measured the levels of tissue-type plasminogen activator (t-PA), type 1 PA inhibitor (PAI-1) and t-PA . PAI-1 complex which is formed as a result of interaction on the two factors, in the plasma of patients with DIC (n = 51) and healthy controls (n = 42). Antigens of t-PA, PAI-1 and t-PA . PAI-1 complex were significantly increased in the DIC plasma (36.4 +/- 25.1, 106.8 +/- 54.7 and 46.6 +/- 34.5 ng/ml, respectively) compared with those in normal plasma (8.5 +/- 4.3, 54.4 +/- 21.2 and 8.6 +/- 3.5 ng/ml, respectively). The molar ratio of t-PA to PAI-1 was much higher in the DIC plasma (1:3) than in normal plasma (1:6), which caused enhancement of the whole fibrinolytic activity in the DIC plasma. These changes resulted in significant consumption of plasminogen, alpha 2-plasmin inhibitor (alpha 2-PI) and a significant increase of plasmin . alpha 2-PI complex (PPI) and D-dimer. These results suggest that t-PA and its specific inhibitor PAI-1 both of which are secreted from endothelial cells into blood, play an important role on the progress of DIC.     

Secretion of tissue-type plasminogen activator and plasminogen activator inhibitor by Rickettsia conorii- and Rickettsia rickettsii-infected cultured endothelial cells.

Hemostasis abnormalities have been described in patients with Mediterranean spotted fever and Rocky Mountain spotted fever. Evidence of the activation of the fibrinolytic system has been obtained in both diseases. After experimental Rocky Mountain spotted fever, an elevated level of fibrinogen was found in parallel with the activation of the fibrinolytic system and transient elevation of the tissue-type plasminogen activator. Later protein is mainly synthesized by endothelial cells. The ability to culture human endothelial cells in vitro provides a unique system to study the secretion of tissue-type plasminogen activator and of plasminogen activator inhibitor after rickettsial infection. Human vascular endothelial cells derived from the umbilical vein, when infected with Rickettsia conorii or Rickettsia rickettsii, secreted as much tissue-type plasminogen activator as control cells. The activity of plasminogen activator inhibitor however, was higher in the supernatants of infected cells than in those of control cells. This rickettsia-induced imbalance of the tissue-type plasminogen activator-inhibitor pair was a very early event after in vitro infection. The involvement of this system during Mediterranean spotted fever and Rocky Mountain spotted fever remains to be demonstrated.     

Refolding and purification of a urokinase plasminogen activator fragment by chromatography

A fragment of recombinant urokinase plasminogen activator (u-PA), was expressed in E. coli in the form of inclusion bodies. Purification and renaturation was achieved in a three-stage process. Capture of the inclusion bodies was achieved by coupling wash steps in Triton X-100 and urea with centrifugation. Solubilised inclusion bodies were then renatured by buffer exchange performed by size-exclusion chromatography (SEPROS). Use of size-exclusion media with higher fractionation ranges resulted in an increase in the recovery of u-PA activity, to a maximum fractionation range of Mr 10000-1500000 after which recovery is reduced, due to a low resolution between the refolded u-PA and denaturant. Fractions of refolded u-PA were concentrated using cation ion-exchange chromatography, which selectively binds correctly folded u-PA. The result is concentrated, active, homogeneous u-PA.     

Hormonal regulation of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 in cultured monkey Sertoli cells

Sertoli cells play a central role in the control and maintenanceof spermatogenesis. Isolated Sertoli cells of mouse and rattestes have been shown to secrete plasminogen activator (PA)and a plasminogen activator inhibitor type-1 (PAI-1) in culture.In this study, we have investigated the hormonal regulationof PA and PAI-1 activities in cultured monkey Sertoli cells.Sertoli cells (5x10⁵ cells/well) isolated from infant rhesusmonkey testes were preincubated at 35°C for 16 h in 24-wellplates precoated with poly(D-lysine) (5 µg/cm²) in 0.5ml McCoy's 5a medium containing 5% of fetal calf serum and furtherincubated for 48 h in 0.5 ml serum-free medium with or withoutvarious hormones or other compounds. PA as well as PAI-1 activitiesin the conditioned media were assayed by fibrin overlay andreverse fibrin autography techniques respectively. The Sertolicells in vitro secreted only tissue-type PA (tPA), no detectableamount of urokinase-type PA (uPA) could be observed. MonkeySertoli cells were also capable of secreting PAI-1. Immunocytochemicalstudies indicated that both tPA and PAI-1 positive staininglocalized in the Sertoli cells, spermatids and residual bodiesof the seminiferous epithelium; Northern blot analysis furtherconfirmed the presence of both tPA and PAI-1 mRNA in monkeySertoli cells. Addition of follicle-stimulating hormone (FSH)or cyclic adenosine monophosphate (cAMP) derivatives or cAMP-generatingagents and gonadotrophin-releasing hormone (GnRH) agonist orphorbol ester (PMA) to the cell culture significantly increasedtPA activity. PAI-1 activity in the culture was also enhancedby these reagents except 8-bromo-dibutyryl-cAMP, forskolin and3-isobutyl-1-methylxanthin (MIX) which greatly stimulated tPAactivity, whereas decreased PAI-1 activity, implying that neutralizationof PAI-1 activity by the high level of tPA in the conditionedmedia may occur. These data suggest that increased intracellularsignals which activate protein kinase A (PKA), or protein kinaseC (PKC) can modulate Sertoli cell tPA and PAI-1 activities.The concomitant induction of PA and PAI-1 by the same reagentsin the Sertoli cells may reflect a finely tuned regulatory mechanismin which PAI-1 could limit the excession of the proteolysis. plasminogen activator inhibitor type-1/Rhesus monkey/Sertoli cells/tissue-type plasminogen activator     

Interaction of tissue-type plasminogen activator with fibronectin and fibronectin fragments

In this paper we present data on the interaction, in solution, of the tissue-type plasminogen activator (t-PA) with fibronectin (FN) and its degradation products (FNdp). A cross radial caseinolytic assay (CRACA) was developed for the evaluation of the effect of the FN and/or FNdp on t-PA and urokinase plasminogen activator (u-PA) activity. A directional caseinolysis was observed when t-PA or u-PA were tested in the proximity of FNdp; no directionality was observed when intact FN or BSA were used. After incubation of t-PA, but not u-PA, with FNdp, PA activity at 170, 150, 100 and 30 kDa was detected by SDS-PAGE followed by zymography. The incubation of intact FN with t-PA gives rise to two forms of 500 and 150 kDa after prolonged incubation of the zymograms; no higher MW forms appear when u-PA substitutes t-PA.The immunoblotting analysis of the mixtures of t-PA and FN or FNdp with anti-t-PA or anti-FN sera showed that intact FN and some of its fragments interact with t-PA, giving rise to complexes recognized by both antisera and resistant to SDS-PAGE. Similar complexes are also evident, in vivo, in biological fluids like human plasma cryoprecipitates (cryos).     

Cell invasion is affected by differential expression of the urokinase plasminogen activator/urokinase plasminogen activator receptor system in muscle satellite cells from normal and dystrophic patients

The aim of this study was to evaluate the differential expression and the function in cell movement and proliferation of the urokinase plasminogen activator (u-PA) system in muscle satellite cells (MSC) of normal individuals and patients with Duchenne muscular dystrophy (DMD). By immunoenzymatic, zymographic, and radioligand binding methods and by quantitative polymerase chain reaction of the specific mRNA we have shown that both normal and DMD MSC produce u-PA and the plasminogen activator inhibitor-1 and express u-PA receptors (u-PAR). During the proliferation phase of their growth-differentiation program, MSC from DMD patients show more u-PAR than their normal counterpart, produce more plasminogen activator inhibitor-1, and release low amounts of u-PA into the culture medium. By Boyden chamber Matrigel invasion assays we have shown that normal MSC are more prone than DMD cells to spontaneous invasion but, when subjected to a chemotactic gradient of u-PA, DMD MSC sense the ligand much better and to a greater extent than normal MSC. u-PA also stimulates proliferation of MSC, but no difference is observable between normal and DMD patients. Antagonization of u-PA/u-PAR interaction with specific anti-u-PA and anti-u-PAR monoclonal antibodies and with antisense oligonucleotides inhibiting u-PAR expression indicates that u-PA/u-PAR interaction is required in spontaneous and u-PA-induced invasion, as well as in u-PA-induced proliferation.     

The possible role of tissue-type plasminogen activator and the plasminogen system in the pathogenesis of major depression

Major depressive disorder (MDD) is one of the most common psychiatric illnesses with an unknown etiology. Evidence from animal and human studies has suggested that brain-derived neurotrophic factor (BDNF) function may be implicated in the pathogenesis of MDD. Tissue-type plasminogen activator (tPA) is a highly specific serine proteinase that catalyses the generation of zymogen plasminogen from the proteinase plasmin. Recent studies have found that the proteolytic cleavage of proBDNF, a BDNF precursor, to BDNF by the plasmin represents a mechanism by which the direction of BDNF action is controlled. Furthermore, studies using mice deficient in tPA has demonstrated that tPA is important for the stress reaction, a common precipitating factor for MDD. A study of the serum levels of the plasminogen activator inhibitor-1 (PAI-1), the major inhibitor of tPA, found that women with MDD had a higher PAI-1 concentration than normal controls. From these findings, it is proposed that the tPA/plasminogen system may play a role in the pathogenesis of MDD. Attempts to confirm the tPA/plasminogen hypothesis may lead to new directions in the study of the pathogenesis of MDD and the development of a novel intervention of this disorder.     

Invasion by esophageal cancer cells: functional contribution of the urokinase plasminogen activation system, and inhibition by antisense oligonucleotides to urokinase or urokinase receptor

Early metastasis contributes to the very poor prognosis of esophageal carcinoma. The recent immunohistochemical finding that invasive esophageal carcinomas express elevated levels of urokinase (uPA) and urokinase receptor (uPA-R) in vivo suggest that the plasminogen activation system may contribute to metastasis in esophageal cancer. The aim of our study was to functionally investigate, at the molecular level, the relative contribution of uPA and uPA-R to the invasiveness of esophageal cancer cells in vitro. The three esophageal cancer cell lines, OC1-3, generated in our laboratory, were analyzed for uPA and uPA-R expression by RT-PCR, immunoenzymatic staining, and quantitative ELISA. Invasiveness of all cell lines was quantified as percentage cellular invasiveness in a standardized Matrigel in vitro assay. OC1 and OC3, which were found to coexpress both uPA and uPA-R, displayed stronger invasiveness (44% and 32.5% respectively) relative to OC2 (19%) which expressed uPA-R but was negative for uPA. Transfection of OC2 cells with the uPA cDNA resulted in two variants, OC2.uPA1 and OC2.uPA2, stably expressing functional uPA. Both transfectants exhibited enhanced invasiveness (60% and 50% respectively) relative to the parent uPA-negative OC2 cells (19%). Antisense oligonucleotide inhibition of either uPA or uPA-R expression resulted in a similar, marked reduction in invasiveness of esophageal tumor cells which normally coexpress both molecules (OC1, OC3 and the uPA-expressing OC2-transfectant clones). Neither antisense treatment altered the basal invasiveness of OC2, which expresses uPA-R but not uPA. In conclusion, coexpression of uPA with its receptor, uPA-R, is required for functional involvement of the urokinase system in invasion by esophageal carcinoma cells. Our results suggest that these synergistic mediators of invasiveness are quantitatively major contributors to the invasiveness of esophageal carcinoma.