Regulation of plasminogen receptor expression on human monocytes and monocytoid cell lines.

The capacity of human monocytoid cell lines and peripheral blood monocytes to modulate their expression of plasminogen receptors has been assessed. After PMA stimulation, THP-1 or U937 monocytoid cells were separated into adherent and nonadherent populations. Plasminogen bound to adherent cells with similar capacity and affinity as to nonstimulated cells. In contrast, the nonadherent cells bound plasminogen with 5-17-fold higher capacity (without a change in affinity). This increase was selective as urokinase bound with similar affinity and capacity to the adherent and nonadherent populations. Upregulation of plasminogen receptors on the nonadherent monocytoid cells was rapid, detectable within 30 min, and reversible, adhesion of the nonadherent cells resulted in a sixfold decrease in plasminogen binding within 90 min. The increase in plasminogen binding to the nonadherent cells was associated with a marked increase in their capacity to generate plasmin activity from cell-bound plasminogen. PMA stimulation of human peripheral blood monocytes increased their expression of plasminogen receptors by two- to fourfold. This increase was observed in both adherent and nonadherent monocytes. Freshly isolated monocytes maximally bound 5.0 x 10(5) plasminogen molecules per cell, whereas monocytes cultured for 18 h or more maximally bound 1.7 x 10(7) molecules per cell, a 30-fold difference in receptor number. These results indicate that both monocytes and monocytoid cell lines can rapidly and markedly regulate their expression of plasminogen binding sites. As enhanced plasminogen binding is correlated with an increased capacity to generate plasmin, an enzyme with broad substrate recognition, modulation of plasminogen receptors may have profound functional consequences.

UV irradiation induces the murine urokinase-type plasminogen activator gene via the c-Jun N-terminal kinase signaling pathway: requirement of an AP1 enhancer element.

UV irradiation leads to severe damage, such as cutaneous inflammation, immunosuppression, and cancer, but it also results in a gene induction protective response termed the UV response. The signal triggering the UV response was thought to originate from DNA damage; recent findings, however, have shown that it is initiated at or near the cell membrane and transmitted via cytoplasmic kinase cascades to induce gene transcription. Urokinase-type plasminogen activator (uPA) was the first protein shown to be UV inducible in xeroderma pigmentosum DNA repair-deficient human cells. However, the underlying molecular mechanisms responsible for the induction were not elucidated. We have found that the endogenous murine uPA gene product is transcriptionally upregulated by UV in NIH 3T3 fibroblast and F9 teratocarcinoma cells. This induction required an activator protein 1 (AP1) enhancer element located at -2.4 kb, since deletion of this site abrogated the induction. We analyzed the contribution of the three different types of UV-inducible mitogen-activated protein (MAP) kinases (ERK, JNK/SAPK, and p38) to the activation of the murine uPA promoter by UV. MEKK1, a specific JNK activator, induced transcription from the uPA promoter in the absence of UV treatment, whereas coexpression of catalytically inactive MEKK1(K432M) and of cytoplasmic JNK inhibitor JIP-1 inhibited UV-induced uPA transcriptional activity. In contrast, neither dominant negative MKK6 (or SB203580) nor PD98059, which specifically inhibit p38 and ERK MAP kinase pathways, respectively, could abrogate the UV-induced effect. Moreover, our results indicated that wild-type N-terminal c-Jun, but not mutated c-Jun (Ala-63/73), was able to mediate UV-induced uPA transcriptional activity. Taken together, we show for the first time that kinases of the JNK family can activate the uPA promoter. This activation links external UV stimulation and AP1-dependent uPA transcription, providing a transcription-coupled signal transduction pathway for the induction of the murine uPA gene by UV.

Cellular regulation of fibrinolysis.

By virtue of their capacity to bind plasminogen activators and plasminogen, to accelerate plasminogen activation and to protect bound plasmin from inactivation by alpha 2 antiplasmin, cells can harness the broad proteolytic activity of plasmin to their surface. Most cells bind plasminogen with a very high capacity, a relatively low affinity (Kd approximately 1 microM) and recognize the lysine binding sites of the molecule. Gangliosides serve as non-protein plasminogen binding sites, and a subset of membrane proteins with carboxy-terminal lysine residues also serve as receptors. The alpha isoform of enolase possesses a carboxy-terminal lysine and is a prominent plasminogen binding protein of cells. Cells of the monocytoid lineage, including peripheral blood monocytes, can markedly upregulate their expression of plasminogen receptors. The capacity to modulate expression of receptors for fibrinolytic components establishes an additional mechanism by which the cell-surface regulates the function of the plasminogen system.

Inhibition of urokinase-type plasminogen activator (uPA) abrogates myogenesis in vitro.

Urokinase-type plasminogen activator (uPA) is one of the components of blood's fibrinolytic cascade. uPA acts as a broad spectrum proteolytic enzyme involved in different physio-pathological processes including cellular fibrinolysis, adhesion, migration, invasion and remodeling. Here, we present evidence that uPA participates in myogenesis, a process which requires drastic cell membrane reorganization, leading to the plurinucleated myotube from the progenitor myoblast. We have dissected the expression of uPA throughout the different myogenic compartments and found an increase in uPA enzymatic activity associated with myotube formation in C2C12 myoblast cells, with uPA mRNA increasing prior the onset of fusion and differentiation. When both fusion and differentiation were blocked by specific inhibitors (DMSO, cytochalasin B) the levels of uPA were strongly downregulated. This process was reversible and specific: the removal of the inhibitors immediately restored the levels of uPA mRNA while the specific inhibition of uPA enzymatic activity by an anti-uPA antibody resulted in a 50% reduction of the extent of fusion and in the abrogation of muscle-specific gene products, such as alpha-actin and MyoD. Moreover, the conversion of fibroblasts to muscle-like cells upon acquisition of MyoD resulted in a dramatic increase of uPA mRNA, which was partially due to transcriptional activation of the uPA gene. These results indicate that the increase in uPA expression prior to fusion and differentiation occurs via a MyoD-mediated mechanism whereas the normal MyoD expression requires the plasminogen activation-dependent activity of this protease. Therefore, these studies extend the sphere of influence of myogenic factors to fibrinolysis, an intrinsic component of the hematological system. Taken together, one mechanism used by the myoblast cell to become a differentiated myotube, involving the inductive extracellular proteolysis of urokinase, is proposed.

AT III Barcelona: a familial quantitative-qualitative AT III deficiency.

A quantitative and qualitative deficiency of antithrombin III (ATIII) was found in four members of a Spanish family with thrombotic tendency. In all affected members, levels of ATIII antigen and activity (heparin cofactor activity) were reduced to 50% of the normal range. When crossed immunoelectrophoresis (CIE) was performed in the presence of heparin, an abnormal slow-moving peak was found. Crossed immunoelectrofocusing (CIEF) from normal and affected individuals showed that normal ATIII migrated between pH 4.9-5.3 while the ATIII under study was asymmetrically distributed between two pH ranges: 4.9-5.3 and 4.6-4.8. Affinity adsorption of affected members' plasma to heparin-sepharose beads revealed one population of ATIII in the supernatant corresponding to the abnormal ATIII, devoid of heparin cofactor activity and showing a peak between pH range: 4.6-4.8 in CIEF. Our data supports the view that a quantitative-qualitative deficiency was present in the heterozygous state in all the affected family members. Both normal and abnormal ATIII were present in plasma of the affected individuals. This abnormal ATIII was characterized by a lack of affinity for heparin. This familial ATIII deficiency was named ATIII Barcelona.

Plasminogen binding properties of macrophage inflammatory protein (MIP)-2alpha.

The chemokine macrophage inflammatory protein (MIP)-2alpha was identified as a plasminogen binding protein by phage display analysis. MIP-2alpha and a truncated form lacking 5 lysine residues in the COOH-terminal region (mut-MIP-2alpha) were expressed in E. coli and purified to apparent homogeneity. Purified MIP-2alpha but not mut-MIP-2alpha bound specifically to plasminogen, with K(A) of 3.7 X 10(5) M(-1) for the interaction of plasminogen with surface-bound MIP-2alpha. Binding and competition experiments indicated that the interaction involves the region comprising the first 3 kringles of plasminogen and the COOH-terminal lysine-rich domain of MIP-2alpha. Activation of plasminogen bound to surface-associated MIP-2alpha by two-chain urokinase-type plasminogen activator (tcu-PA) was about 2.5-fold more efficient than in solution (catalytic efficiency k(cat)K(M) of 0.1 microM(-1)s(-1), as compared to 0.04 microM(-1)s(-1). In contrast, binding of plasminogen to MIP-2alpha in solution was very weak, as evidenced by the absence of competition of MIP-2alpha with lysine-Sepharose or with human THP-1 cells for binding of plasminogen. In agreement with this finding, addition of excess MIP-2alpha did not affect the main functional properties of plasmin(ogen) in solution, as indicated by unaltered activation rates of plasminogen by tcu-PA or tissue-type plasminogen activator (t-PA), t-PA-mediated fibrinolysis, and inhibition rate of plasmin by alpha2-antiplasmin. Thus, association of MIP-2alpha with surfaces exposes its COOH-terminal plasminogen-binding site, and may result in enhanced local plasmin generation.

Procoagulant activity of T lymphoblastoid cells due to exposure of negatively charged phospholipid.

We have characterised the Procoagulant activity (PCA) of six well-established cell lines by assays of tissue factor (TF), thrombin and FXa generation, flow cytometry using Annexin V, and by binding studies with Factors VIII and IXa. The monocytic (THP-1 & U937) and promyelocytic (NB4) cells expressed high concentrations of TF antigen and activity, whereas TF in the lymphocytic cells (Molt 4, Jurkat & Nalm 6) was very low or absent. However the T-lymphoblastoid cells (Molt 4 & Jurkat) promoted the generation of large amounts of thrombin despite their low TF content, and these cells were also the most active in supporting Factor Xa generation. Molt 4 cells bound Factors VIII and IXa with high capacity and their activity was inhibited by Annexin V. These results indicate that the PCA of T-lymphoblastoid lines is due to expression of negatively charged phospholipids. Flow cytometry studies showed Annexin V binding to the major population of nonapoptotic Molt 4 cells and the PCA of Molt 4 was not increased when apoptosis was induced by staurosporine, indicating that PCA is independent of apoptotic status.

Identification of an epitope of alpha-enolase (a candidate plasminogen receptor) by phage display.

Alpha-enolase is an ubiquitous cytoplasmic glycolytic enzyme which also exhibits cell surface mediated functions and a structural role in the lens of some species. An alpha-enolase related molecule (alpha-ERM) is present on the surfaces of neutrophils, monocytes and monocytoid cells and has the capacity to specifically bind plasminogen, suggesting that alpha-ERM may function as a plasminogen receptor. We have generated a monoclonal antibody (mAB), 9C12, against alpha-ERM. This mAB reacted with both alpha-ERM and purified human alpha-enolase in Western blotting and in enzyme linked immunosorbent assays (ELISA). mAB 9C12 detected a cell surface associated molecule on human peripheral blood neutrophils and on U937 human monocytoid cells as assessed by fluorescence activated cell sorting (FACS) analyses. In addition, mAB 9C12 recognized an intracellular pool of alpha-enolase/alpha-ERM in permeabilized U937 cells. A phage display approach was employed to identify the alpha-enolase epitope recognized by mAB 9C12. Random fragments of 100-300 base pairs (bp), obtained from the full length human alpha-enolase cDNA, were cloned into the filamentous phage vector pComb3B, to generate a phage-displayed peptide library. Recombinant phages binding to mAB 9C12 were selected and their DNA inserts characterized by direct sequencing. All of the fragments which bound to mAB 9C12 encoded the common sequence DLDFKSPDDPSRYISP, spanning amino acids 257-272 of human alpha-enolase. This sequence is located within an external loop of the molecule. These data indicate that this sequence contains the epitope recognized by mAB 9C12 and is, therefore, exposed on the cell surface, further suggesting that alpha-enolase and alpha-ERM share common amino acid sequences.

Characterization of cellular binding sites and interactive regions within reactants required for enhancement of plasminogen activation by tPA on the surface of leukocytic cells.

Plasminogen and tPA bind to a common set of binding sites on nucleated cells. To assess the functional consequences of cellular binding, we have measured the kinetic changes induced by plasminogen activation by tPA on cell surfaces. These studies were carried out with U937 and THP-1 monocytoid cells, with Raji, Nalm6 and Molt4 lymphoid cells and with peripheral blood monocytes and neutrophils. The interactions of plasminogen and tPA with cells induced an increase in the rate of plasmin generation which depended upon the cell concentration. With saturating amounts of U937 monocytoid cells (1.25 x 10(5)/ml) the rate of plasmin generation was 0.39 nM.s-1 versus 0.07 and 0.09 nM.s-1 without cells or without tPA, respectively. The catalytic efficiency of Glu- or Lys-plasminogen activation by tPA increased by 7.2- and 24.2-fold, respectively. These changes were induced by a 72-242-fold reduction in the Km of these interactions which was in the range of 0.3-0.9 microM. These values are below the plasminogen concentration in plasma (1-2 microM). Moreover, we provide new data indicating that 1) only a specific subset of plasminogen binding sites, i.e. molecules exposing carboxyl terminal lysines on the cell surface, promotes plasminogen activation on cells; 2) the first four kringles of plasminogen and the finger of tPA are critical for enhanced plasmin generation on cell surfaces; 3) the simultaneous co-localization of tPA with plasminogen on cell surfaces is required for enhanced plasminogen activation; 4) modulation of plasminogen/tPA receptor expression induces concomitant modulation of the stimulatory effects of cells on plasminogen activation and 5) in a direct comparison, the mechanism by which cells and fibrin fragments accelerate plasminogen activation are similar but not identical. These data suggest that modulation of plasminogen/tPA binding sites permits local and efficient generation of plasmin on cell surfaces.

Dysfunctional activated protein C (PC Cádiz) in a patient with thrombotic disease.

The partial characterization of a dysfunctional protein C (PC), provisionally named "PC Cádiz", in a 45-year-old male patient suffering from recurrent venous thrombosis is described. The only defect found in laboratory assays for haemostasis and hepatic function was a half normal level of both amidolytic and anticoagulant protein C activity, measured by different functional assays that use thrombin-thrombomodulin complex and a snake venom to activate protein C. Protein C antigen was always found to be within normal levels. Two young daughters of the propositus were found to have the same defect. Double-crossed immunoelectrophoresis, performed in the presence and absence of Ca2+ in the first dimension, showed no clear differences between patient and control PC. PC adsorption to barium salts was also found to be normal. Measurement of the PC activation peptide in the barium citrate eluates after PC activation showed no significant differences between patient and 10 normal controls, the concentration of this peptide being very similar to that of PC zymogen in the same eluates before PC activation. These results indicate that this abnormal PC is able to be normally activated by thrombin-thrombomodulin complex but does not exhibit serine protease activity, probably due to a defect in the PC molecule near the active site center.

Plasma and urinary heparin cofactor II levels in patients with nephrotic syndrome.

Heparin cofactor II (HC II) levels were measured by electroimmunoassay in plasmas and urines from 68 patients with nephrotic syndrome. In addition, antithrombin III (AT III) and protein C (PC) activities and antigens were measured also in the same group of patients. Seven of these patients had histories of thrombosis. Plasma HC II levels (mean +/- SD 105 +/- 43) were not different from levels in healthy subjects (94 +/- 17). Only 5 patients had low plasma levels of HC II. None of the patients with thrombosis had low HC II levels. Even though measurable amounts of HC II were found in 25 urines from 50 patients. There was a relationship in the urinary excretion between HC II and AT III and their urinary clearances were quite similar. However, no correlation was found between plasma HC II and AT III levels, and levels of AT III activity and antigen were significantly lower than in healthy subjects. Three patients with histories of thrombosis had low AT III levels. Most patients (including those with thrombosis histories) had high plasma PC levels and increased urinary loss. It is suggested that HC II does not play an important role in the pathogenesis of thrombosis in nephrotic syndrome.

Signal transduction pathways underlying the expression of tissue factor and thrombomodulin in promyelocytic cells induced to differentiate by retinoid acid and dibutyryl cAMP.

Acute promyelocytic leukaemia (APL) may be associated with disseminated intravascular coagulation, as a result of increased tissue factor (TF) expression and reduced thrombomodulin (TM) expression by APL blast cells. During retinoid acid (RA)- and dibutyryl cAMP (dbcAMP)-induced differentiation of the APL cells, there is a marked up-modulation of both the protein kinase A (PKA) and C (PKC) activities. In order to further assess whether these kinases are intimately associated with both the differentiation process and the regulation of TF and TM expression, we have correlated the modulation of their respective pathways with the extent of differentiation and modulation of these cellular receptors. NB4 cells were incubated with all-trans-RA (ATRA) or dbcAMP for up to 48 h. The contribution of phospholipase C (PLC), inositol phosphate (IP), PKC and PKA in the expression of CD11b, TF and TM was studied by the use of specific inhibitors. Myo-inositol uptake and PKC activity increased in cells induced to differentiate by ATRA but the retinoid did not affect cAMP levels or PKA activity. Under treatment with dbcAMP, PKA activity was increased while inositol uptake and PKC activity remained unchanged. Our results show that the effects of ATRA and dbcAMP on promyelocytic cells are closely related, respectively, to the PLC/IP/PKC and the cAMP/PKA pathways. In cells induced to differentiate by ATRA, CD11b expression seems more closely related to inositol uptake than to PKC activity while the expression of TF and TM show the opposite pattern, which suggests cellular events regulated at a different level within a common signal transduction pathway.

Tissue factor (TF) and urokinase plasminogen activator receptor (uPAR) and bleeding complications in leukemic patients.

Tissue factor (TF) and urokinase receptor (uPAR) are key cellular receptors triggering, respectively, coagulation and fibrinolysis. Bleeding complications among leukemic patients have been related to an abnormal expression of TF by blast cells and/or to an abnormal fibrinolytic response. In this study the expression of TF and uPAR has been assessed in 18 acute non-lymphoblastic and 8 lymphoblastic leukemic blast cells using several methodological approaches. TF mRNA was evaluated by in situ hybridization and TF and uPAR antigen were evaluated immunologically in cell lysates and on the cell surface by flow cytometry. In addition, TF-procoagulant activity was measured in coagulation-based assays. The reliability of these methods was corroborated in six leukemic cell lines of different lineages and states of maturation. Disseminated intravascular coagulation was detected in two M3 leukemia patients whose blast cells expressed high amounts of TF. Hyperfibrinolysis was detected in one M1 and two M2 patients, whose blast cells displayed a high content of uPAR antigen, but no TF. Furthermore, M5 leukemia blast cells expressed both TF and uPAR, although no hemostatic defects or bleeding complications were detected in these patients. Taken together, although a limited number of patients was included in this study, these data suggest that in leukemia patients exhibiting bleeding, either TF or uPAR are expressed by their blast cells. However, the presence of these receptors does not necessarily imply the existence of a hemostatic disorder.

Distinct patterns of urokinase receptor (uPAR) expression by leukemic cells and peripheral blood cells.

The urinary type plasminogen activator, urokinase (uPA) is localized on the cell surface through the binding of a specific receptor, the uPA receptor (uPAR). The uPA localization enhances plasmin formation on the cell surface and facilitates cell migration. The cellular and tissue distribution of uPAR is not fully established. We have analyzed uPAR expression in nine leukemic cell lines of distinct lineages and maturational states and correlated this with expression of plasminogen receptors, tissue-type plasminogen activator (tPA) receptors and LDL receptor-related protein (LRP). The most immature and least differentiated cell line (an erythro-myeloid cell line) and cells of lymphoid lineage, did not express uPAR, whereas cells differentiated along the myelo-monocytic pathway displayed this receptor. Plasminogen and tPA receptors were expressed by all leukemic cell lines and by all nucleated peripheral blood cells but B and T lymphocytes were negative for cell surface expression of both uPAR and LRP while monocytes and neutrophils were positive for expression of both uPAR and LRP. PMA stimulation induced surface expression of uPAR in lymphocytes but did not induce expression of LRP by these cells. In contrast, lymphoid cell lines were negative for uPAR expression even after PMA stimulation, indicating differences in regulation of uPAR expression between lymphocytes and lymphoid cell lines. The pattern of uPAR expression on leukemic cell lines was also studied on bone marrow blast cells from leukemic patients. Only the most mature myeloid cells expressed uPAR on their surfaces. In contrast, M3 leukemic cells and other blast cells displaying lymphoid markers such as TdT (+) and/or CD2 (+) did not express intracellular or cell-surface associated uPAR, indicating an heterogeneity among these promyelocytic cells and suggesting that uPAR may be a useful marker for leukemia typing. Myeloid blast cells from some patients contained intracellular pools of uPAR but displayed no receptor on the cell surface, suggesting that translocation may be a mechanism regulating uPAR expression in these cells. The comparison of uPAR expression between these cell lines and peripheral blood cells and it correlation with plasminogen receptors, tPA receptors and LRP expression offers new insights regarding potential mechanisms for regulation of uPA-uPAR-mediated pericellular proteolysis.

Quantitative and qualitative congenital deficiency of antithrombin III: a new molecular variant called ATIII-Barcelona 2.

A Spanish family with a quantitative-qualitative antithrombin III (ATIII) deficiency and thrombotic tendency is reported. The qualitative defect was suggested by the crossed immunoelectrophoresis (CIE) in the presence of heparin in plasma of all those affected. However, the crossed immunoelectrofocusing (CIEF) showed the same ATIII pattern in controls and affected members. Two populations of ATIII were detected by affinity chromatography on heparin-sepharose from affected members' plasma. The ATIII unbound to sepharose beads was devoid of heparin cofactor activity and showed a lack of anodal migration in CIE in the presence of heparin. The ATIII eluted corresponded to normal ATIII. Our data supports the view that an abnormal ATIII molecule is present in all affected family members in the heterozygous state. This is the first reported ATIII variant in which a molecular abnormality produces a lack of affinity for heparin but no changes in its isoelectric point. This familial ATIII deficiency was named ATIII- Barcelona 2.

Massive pulmonary embolism: short-term effects of thrombolytic treatment.

This study assessed the short-term effects of thrombolytic treatment in 38 patients with massive pulmonary embolism. Thirty-two were treated with streptokinase and six with urokinase. Intrapulmonary artery instillation of fibrinolytic agents was utilized except in 3 patients. There was a marked hemodynamic and arteriographic improvement (p less than 0.0005) in 33 patients (86.8%). Four patients (10.5%) died because of treatment failure. In these cases the fibrinogen concentration remained above 1 gr/liter during therapy. Bleeding was detected in 22 patients (57.8%) but was most often related to puncture or cut-down sites, and only 2 patients (5.2%) had major bleeding. One patient (2.6%) had cerebral hemorrhage. It is concluded that "classic" thrombolytic treatment is to be chosen in life-threatening pulmonary embolism. However, the difficulties sometimes encountered in producing an intense lytic effect and its low fibrinolytic specificity for the thrombus do not permit the obtainment of better results.

[Intracranial hemorrhage in patients undergoing anticoagulant treatment with acenocoumarol]. / Hemorragia intracraneal en pacientes sometidos a tratamiento anticoagulante con acenocumarol.

Between 1980 and 1985, 2,012 patients treated with acenocoumarol were followed up. 31 of them had intracranial hemorrhage (ICH), representing an incidence of 1.54%. Its mortality rate was as high as 45%. The most common indication of anticoagulant therapy was cardiac valvular disease. No precipitating factors or concomitant hemorrhages in other sites were detected. Most patients with ICH were, at the time of the accident, within the therapeutic anticoagulant range. The most common localization of ICH was intraparenchymal; most of them developed after one year of anticoagulant therapy. In 32% of instances there was previous hypertension and previous stroke in 26%. ICH had a three times greater incidence in patients over 65 years. The patients who died showed larger hematomas than survivors in computed tomography.