Binding of tissue plasminogen activator to human monocytes and monocytoid cells

Monocytes and monocytoid cell lines previously have been shown to express receptors for plasminogen and urokinase (u-PA). In the present study, the monocytoid cell lines, U937 and THP-1, are shown to bind tissue plasminogen activator (t-PA) in a specific, saturable, and reversible manner. These cells bound t-PA with low affinity (kd = 0.67 to 0.97 mumol/L) and high capacity (0.71 to 3.3 x 10(6) receptors/cell). Human peripheral blood monocytes bound t-PA with a kd (0.9 mumol/L) similar to that of the monocytoid cells but with a lower capacity (0.17 x 10(6) sites/cell). These binding parameters also were similar to the low-affinity interaction of t-PA with endothelial cells as measured with the cells in suspension (kd = 0.73 mumol/L and 1.1 x 10(6) sites/cell). Lysine analogues and active or diisopropylfluorophosphate-inactivated u-PA inhibited t-PA binding to monocytes, monocytoid cells, and endothelial cells with similar IC50 (concentration producing 50% inhibition) values, suggesting that the same recognition specificity mediates t-PA binding to all of these cell types. The existence of a high-affinity binding site for t-PA on monocytoid cells was also explored in detail. Unlike endothelial cells where plasminogen activator inhibitor-1 has been implicated in mediating a high-affinity interaction of t-PA with the cells, no evidence for a role of this inhibitor in ligand binding to the monocytoid cells was found. Furthermore, using both high and low 125I-t-PA concentrations, competition analyses with lysine analogues or u-PA, or treatment of the cells with carboxypeptidase B, failed to indicate the presence of distinguishable classes of t-PA binding sites. In sum, low-affinity receptors for t-PA are expressed at high density on monocytes and monocytoid cells, identifying a new element in the fibrinolytic arsenal of these cells.     

Competition between plasminogen and tissue plasminogen activator for cellular binding sites

Cellular receptors for plasminogen and tissue plasminogen activator (t- PA) regulate plasminogen activation and cell-associated proteolytic activity. The characteristics of the interactions of both ligands with monocytes and monocytoid cell lines bear certain similarities, including affinity (kd approximately 1 mumol/L) capacity and susceptibility to carboxypeptidase treatment. Therefore, we have undertaken the present study to determine directly whether t-PA and plasminogen share common binding sites on cells. We found that recombinant human single-chain t-PA (rt-PA) could inhibit the binding of 125I-plasminogen to the cells and, conversely, plasminogen could inhibit 125I-rt-PA binding. This relationship was observed with 9 cell types, including both adherent cells and cells in suspension. In addition, under several conditions of cell treatment, plasminogen and t- PA receptor expression was modulated in parallel. Furthermore, molecules that have been implicated as candidate plasminogen receptors, gangliosides, and an alpha-enolase--related molecule, also interacted with t-PA. These results suggest that at least a component of the binding sites for plasminogen is shared with t-PA. Occupancy of these sites by either or both ligand(s) should result in arming the cells with the proteolytic activity of plasmin.     

Low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor is an hepatic receptor for tissue-type plasminogen activator.

Tissue-type plasminogen activator (t-PA), a serine protease that catalyzes the initial and rate-limiting step in the fibrinolytic cascade, is cleared rapidly in vivo by the liver. Using chemical crosslinking, we have recently identified a plasminogen-activator inhibitor type 1 (PAI-1)-independent t-PA clearance receptor on rat hepatoma MH1C1 cells with a relative molecular mass of approximately 500 kDa. Another recently identified membrane receptor, low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP/alpha 2MR), was also detected on MH1C1 hepatoma cells by using immunoprecipitation with anti-LRP/alpha 2MR antibody. When analyzed by SDS/PAGE, we found the t-PA receptor identified on MH1C1 cells comigrated with the large subunit of LRP/alpha 2MR. The t-PA receptor was immunoprecipitated by an anti-LRP/alpha 2MR antibody after chemical crosslinking of specifically bound 125I-labeled t-PA to its receptor. Through chemical crosslinking studies, we found that t-PA and methylamine-activated alpha 2-macroglobulin could bind to LRP/alpha 2MR simultaneously without competing with one another for binding, suggesting that the two ligands bound to two independent sites on the LRP/alpha 2MR molecule. Furthermore, a 39-kDa protein, which modulates ligand binding to LRP/alpha 2MR, was also found to inhibit t-PA binding to its receptor. These data thus show that the t-PA clearance receptor identified on MH1C1 hepatoma cells is LRP/alpha 2MR.     

Increased clearance explains lower plasma levels of tissue-type plasminogen activator by estradiol: evidence for potently enhanced mannose receptor expression in mice.

Several clinical studies have demonstrated an inverse relationship between circulating levels of estrogen and tissue-type plasminogen activator (t-PA). The present study was designed to test the hypothesis that estrogens lower plasma levels of t-PA by increasing its clearance from the bloodstream. 17alpha-Ethinyl estradiol (EE) treatment resulted in a significant increase in the clearance rate of recombinant human t-PA in mice (0.46 mL/min in treated mice v 0. 32 mL/min in controls; P <.01). The clearance of endogenous, bradykinin-released t-PA in rats was also significantly increased after EE treatment (area under the curve [AUC], 24.9 ng/mL. min in treated animals v 31.9 ng/mL. min in controls; P <.05). Two distinct t-PA clearance systems exist in vivo: the low-density lipoprotein receptor-related protein (LRP) on liver parenchymal cells and the mannose receptor on mainly liver endothelial cells. Inhibition of LRP by intravenous injection of receptor-associated protein (RAP) as a recombinant fusion protein with Salmonella japonicum glutathione S-transferase (GST) significantly retarded t-PA clearance in control mice (from 0.41 to 0.25 mL/min; n = 5, P <.001) and EE-treated mice (from 0.66 to 0.35 mL/min; n = 5, P <.005), but did not eliminate the difference in clearance capacity between the 2 experimental groups. Similar results were obtained in mice in which LRP was inhibited via overexpression of the RAP gene in liver by adenoviral gene transduction. In contrast, administration of mannan, a mannose receptor antagonist, resulted in identical clearances (0.22 mL/min in controls and 0.24 mL/min in EE-treated mice). Northern blot analysis showed a 6-fold increase in mannose receptor mRNA expression in the nonparenchymal liver cells of EE-treated mice, whereas the parenchymal LRP mRNA levels remained unchanged. These findings were confirmed at the protein level by ligand blotting and Western blotting analysis. Our results demonstrate that EE treatment results in increased plasma clearance rate of t-PA via induction of the mannose receptor and could explain for the inverse relationship between estrogen status and plasma t-PA concentrations as observed in humans.     

Annexin II mediates plasminogen-dependent matrix invasion by human monocytes: enhanced expression by macrophages

Monocytes and macrophages participate in a wide variety of host defense mechanisms. Annexin II, a fibrinolytic receptor, binds plasminogen and tissue plasminogen activator (t-PA) independently at the cell surface, thereby enhancing the catalytic efficiency of plasmin production. We demonstrated previously that annexin II on the surface of both cultured monocytoid cells and monocyte-derived macrophages promotes their ability to remodel extracellular matrix. Here, we demonstrate that human peripheral blood monocytes represent the major circulating annexin II-expressing cell. Annexin II supported t-PA-dependent generation of cell surface plasmin and the matrix-penetrating activity of human monocytes. Compared to polymorphonuclear leukocytes, monocytes supported a 12.9-fold greater rate of plasmin generation in the presence of exogenous t-PA, and this activity was largely attributable to annexin II. Likewise, anti-annexin II IgG directed against the t-PA-binding tail domain inhibited plasminogen-dependent, cytokine-directed monocyte migration through extracellular matrix. On differentiation of monocytes to macrophages, there was a 2.4-fold increase in annexin II-specific mRNA, and a 7.9-fold increase in surface annexin II. Thioglycolate-elicited peritoneal macrophages, furthermore, displayed an additional 3.8-fold increase in annexin II surface expression compared with resident cells. Thus, annexin II-mediated assembly of plasminogen and t-PA on monocyte/macrophages contributes to plasmin generation, matrix remodeling, and directed migration.     

Binding of fibrin fragments to one-chain and two-chain tissue-type plasminogen activator.

To explore whether fibrin fragments have binding affinity for the tissue-type plasminogen activator (t-PA) molecule, the interactions were studied of (DD)E complex and fragments DD, E1, and E3 with one-chain and two-chain t-PA. For this purpose, a solid-phase binding assay was developed using microtiter plates with nitrocellulose filters. It was found that (DD)E complex and fragments DD and E3 retained the t-PA binding function of the parent fibrin molecule, thus demonstrating that t-PA binds to both the D and E domains of fibrin. Unexpectedly, fragment E1 did not bind t-PA. Fibrin fragments had different binding properties for one-chain and two-chain t-PA. (DD)E complex had the highest and fragment E3 the lowest affinity for one-chain t-PA, both binding curves being consistent with one class of binding sites. However, binding of the fragments with two-chain t-PA was distinguished by more than one class of binding sites, with fragment E3 having the highest affinity for this form of the activator. epsilon-Aminocaproic acid, even at 50 mmol/L concentration, had only minimal effect on binding of (DD)E complex or fragment DD to either one-chain or two-chain t-PA. The potentiating effect of fibrin fragments on plasminogen activation by t-PA was measured by a chromogenic substrate assay. Fragment DD was the most effective stimulator of plasminogen activation by t-PA. In conclusion, (DD)E complex and fragment DD retained most of the regulatory functions of fibrin, which included t-PA binding and t-PA-mediated acceleration of plasminogen activation to plasmin.     

Isolation and characterization of the mannose receptor from human liver potentially involved in the plasma clearance of tissue-type plasminogen activator.

Various studies have shown that mannose receptors rapidly eliminate glycoproteins and microorganisms bearing high mannose-type carbohydrate chains from the blood circulation. The purpose of this study was to characterize the mannose receptor in the liver, which in vivo is involved in the rapid clearance of tissue-type plasminogen activator from the circulation. Human liver membranes were solubilized in Triton X-100, and the solution was applied to a tissue-type plasminogen activator Sepharose column. Bound proteins were eluted with ethylenediaminetetraacetate (10 mmol/L). A second, similar purification step rendered a single liver protein of 175,000 daltons. A combination of ligand blotting and a chromogenic assay for tissue-type plasminogen activator demonstrated that the identified liver protein is a mannose receptor because it bound tissue-type plasminogen activator, this tissue-type plasminogen activator binding being fully inhibited by 0.2 mol/L D-mannose. Western-blot analysis revealed that the isolated liver protein is immunologically identical to the human mannose receptor from placenta. Treatment of the liver protein and the placenta mannose receptor with trypsin yielded the same pattern of proteolytic degradation products as identified on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that the physiologically relevant mannose receptor for tissue-type plasminogen activator clearance isolated from human liver is immunologically and structurally similar to or identical with the human mannose receptor isolated from placenta.     

Differences between binding of one-chain and two-chain tissue plasminogen activators to non-cross-linked and cross-linked fibrin clots

Interaction of tissue plasminogen activator (t-PA) with fibrin plays a key role in regulation of plasminogen activation and clot dissolution. Previous investigations of t-PA-fibrin interaction, using incorporation of t-PA into polymerizing fibrin clots, have suggested that no significant differences exist in the binding of one-chain or two-chain t-PA to non-cross-linked or cross-linked fibrin. In the present study, binding of 125I-labeled and affinity-purified one-chain and two-chain forms of t-PA to preformed non-cross-linked or cross-linked, sonicated suspension of fibrin was investigated. Interaction of one-chain t-PA with cross-linked fibrin involved a single type of binding site with dissociation constant (kd) of 0.58 mumol/L and a stoichiometry (n) of 1.5. Interaction of one-chain t-PA with non-cross-linked fibrin, however, involved two classes of binding sites with dissociation constants of 0.32 and 1.5 mumol/L and corresponding number of binding sites equal to 0.57 and 2.0, respectively. In contrast to the binding of one-chain t-PA to cross-linked fibrin by a limited number of sites, two-chain t-PA appeared to involve a considerably greater number of sites (minimum six) whose dissociation constant was 3.2 mumol/L. Interaction of two-chain t-PA with non-cross-linked fibrin also showed the presence of many binding sites (minimum seven) with approximate dissociation constant of 6.4 mumol/L, as well as a few (n = 0.012) high-affinity sites with a kd of 0.011 mumol/L epsilon-Aminocaproic acid did not completely reverse the binding of either one-chain t-PA or two-chain t-PA to fibrin. The present findings suggest that the fibrin-binding properties of t-PA undergo considerable changes on proteolytic conversion from one-chain to two-chain t-PA, catalyzed under physiologic conditions by plasmin. The cleavage of one-chain t-PA to two-chain t-PA allows to bind to a large number of low-affinity binding sites on fibrin. Cross-linking of fibrin by factor XIIIa results in masking of high-affinity binding sites that are present in non-cross-linked fibrin. We propose that both plasmin and factor XIIIa play an important regulatory role in dissolution of blood clots by modulating t-PA-fibrin interaction.     

The binding of monomeric IgG to human blood monocytes and alveolar macrophages

Macrophage receptor sites for IgG are important in the immune clearance of particles both from the blood and lung. We studied the number, affinity, and density of binding sites for monomeric IgG on human blood monocytes and alveolar macrophages. Monocytes and alveolar macrophages had a similar affinity for monomeric IgG at 37 degrees and 4 degrees C. The half-time for dissociation of the IgG-receptor complex was also similar for both cells. However, alveolar macrophages expressed approximately 5-fold more IgG binding sites than monocytes at both 37 degrees and 4 degrees C. Nevertheless, when cell surface area was estimated, these cells expressed a similar density of IgG binding sites (monocytes = 110 +/- 14.8 IgG binding sites/square micron; pulmonary macrophages = 138 +/- 46.9 IgG binding sites/square micron; p greater than 0.50). Gamma interferon increased the number and density of monocyte binding sites for monomeric IgG by 162 +/- 89%. Furthermore, patients with sarcoidosis, a disorder in which gamma interferon is spontaneously elaborated, expressed a similar increase in the number of IgG binding sites per monocyte, from 24,968 +/- 1,361 for normal subjects to 44,860 +/- 6,652 for patients with sarcoidosis. However, alveolar macrophages from 5 patients with sarcoidosis expressed a normal number of IgG binding sites. These data suggest that there is no major alteration in the Fc(IgG) receptor as monocytes differentiate into alveolar macrophages. Both gamma interferon treatment and sarcoidosis are associated with enhanced expression of the Fc(IgG) receptor on monocytes.     

A monoclonal antibody preventing binding of tissue-type plasminogen activator to fibrin: useful to monitor fibrinogen breakdown during t-PA infusion

One (MA-1C8) of 36 monoclonal antibodies obtained by fusion of P3X63- Ag8-6.5.3 myeloma cells with spleen cells of mice immunized with purified human tissue-type plasminogen activator (t-PA) blocked the activity of t-PA on fibrin plates but not on chromogenic substrates. MA- 1C8 at a concentration of 200 micrograms/mL inhibited plasma clot lysis and binding of t-PA to the clot. MA-1C8 had no influence on the activation of plasminogen by t-PA, which obeys Michaelis-Menten kinetics with Km = 105 mumol/L and kcat = 0.05 s-1; however, it abolished the influence of CNBr-digested fibrinogen on Km. These findings confirm that the stimulatory effect of fibrin on the activation of plasminogen by t-PA is mediated by binding of t-PA to fibrin and provide additional support for the kinetic model. Addition of t-PA to pooled fresh human plasma to a concentration of 5 micrograms/mL resulted in extensive fibrinogen breakdown after incubation for one hour at 37 degrees C or during storage at -20 degrees C for one day. In both instances, fibrinogen degradation was completely prevented by addition of MA-1C8 to a concentration of 200 micrograms/mL of plasma. MA-1C8 also effectively prevented in vitro fibrinogen degradation and in vitro plasminogen activation in plasma samples obtained during infusion of recombinant t-PA in patients with thromboembolic disease. Thus, MA-1C8 is a useful tool for discriminating between in vivo and in vitro fibrinolysis during thrombolytic therapy with t-PA.     

Evidence for a novel binding protein to urokinase-type plasminogen activator in platelet membranes

Endogenous urokinase-type plasminogen activator (u-PA) has been identified in platelet membrane, and platelets have been shown to take up exogenous high molecular weight u-PA from the ambient medium. In this report, the mechanism of the association of u-PA with platelets was investigated using recombinant, single chain u-PA. When gel filtered human platelets were incubated with radiolabeled u-PA, the u- PA was found to specifically and saturably bind to the resting platelets in a dose-dependent manner. Unlabeled u-PA and the amino terminal fragment of u-PA inhibited 125I-u-PA binding to platelets with a mean IC50 of 65 and 58 nmol/L, respectively. A single saturable binding site in intact resting platelets was found with a mean kd of 43 +/- 25 nmol/L and 2263 +/- 809 sites per platelet. In contrast to resting platelets, 125I-u-PA did not bind to thrombin-induced platelets. Western blotting studies, using a monoclonal or a polyclonal antibody specific for the u-PA cell-surface receptor (u- PAR), failed to show evidence of u-PAR in resting platelets, whereas, u-PAR was found at approximately 54 and approximately 48 kD on U937 monocytes, which served as a positive control. Ligand blotting of platelet membrane and of U937 cell proteins with 125I-u-PA revealed a u-PA binding protein of approximately 70 kD in the platelets and one of approximately 54 kD in the U937 cells. Complexion of u-PA with a platelet membrane protein was also shown by gel filtration of a mixture of u-PA and platelet membrane proteins. A u-PA complex was further shown by enzyme-linked immunosorbent assay when microtiter plates were coated with platelet membrane proteins, and this complex formation was shown to be dose-dependent and saturable with an apparent kd of 17 nmol/L. It was concluded that platelet membrane contains a specific, high affinity u-PA-binding protein that is distinct from u-PAR.     

The low density lipoprotein receptor-related protein mediates the cellular degradation of tissue factor pathway inhibitor.

The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) is a cell-surface glycoprotein of 4525 amino acids that functions as a hepatic endocytosis receptor for several plasma proteins. These include alpha 2-macroglobulin-protease complexes, free plasminogen activators as well as plasminogen activators complexed with their inhibitors, and beta-migrating very low density lipoproteins complexed with either apolipoprotein E or lipoprotein lipase. In the current study we used human and rat hepatoma cell lines to demonstrate that LRP can mediate the degradation of tissue factor pathway inhibitor (TFPI), a Kunitz-type plasma serine protease inhibitor that regulates tissue factor-induced blood coagulation. The cellular degradation of 125I-labeled TFPI (125I-TFPI) was inhibited more than 80% both by antibodies directed against LRP and by the LRP-associated 39-kDa protein, a protein that inhibits the binding and/or cell-mediated degradation of all ligands by LRP. Using rat hepatoma cells, we report that at 4 degrees C, 125I-TFPI binds to approximately 2 x 10(6) sites per cell with an equilibrium dissociation constant of approximately 30 nM. 125I-TFPI binding to the cell surface is not inhibited by the 39-kDa protein. Taken together, our results suggest that TFPI binds to an as-yet-unidentified cell surface molecule. After binding, LRP mediates the cellular degradation of TFPI.     

Plasminogen-mediated matrix invasion and degradation by macrophages is dependent on surface expression of annexin II

Genetic evidence demonstrates the importance of plasminogen activation in the migration of macrophages to sites of injury and inflammation, their removal of necrotic debris, and their clearance of fibrin. These studies identified the plasminogen binding protein annexin II on the surface of macrophages and determined its role in their ability to degrade and migrate through extracellular matrices. Calcium-dependent binding of annexin II to RAW264.7 macrophages was shown using flow cytometry and Western blot analysis of EGTA eluates. Ligand blots demonstrated that annexin II comigrates with one of several proteins in lysates and membranes derived from RAW264.7 macrophages that bind plasminogen. Preincubation of RAW264.7 macrophages with monoclonal anti-annexin II IgG inhibited (35%) their binding of 125I-Lys-plasminogen. Likewise, plasmin binding to human monocyte-derived macrophages and THP-1 monocytes was inhibited (50% and 35%, respectively) when cells were preincubated with anti-annexin II IgG. Inhibition of plasminogen binding to annexin II on RAW264.7 macrophages significantly impaired their ability to activate plasminogen and degrade [3H]-glucosamine-labeled extracellular matrices. The migration of THP-1 monocytes through a porous membrane, in response to monocyte chemotactic protein-1, was blocked when the membranes were coated with extracellular matrix. The addition of plasminogen to the monocytes restored their ability to migrate through the matrix-coated membrane. Preincubation of THP-1 monocytes with anti-annexin II IgG inhibited (60%) their plasminogen-dependent chemotaxis through the extracellular matrix. These studies identify annexin II as a plasminogen binding site on macrophages and indicate an important role for annexin II in their invasive and degradative phenotype.     

Common binding structure for granulocyte macrophage colony-stimulating factor and interleukin-3 on human acute myeloid leukemia cells and monocytes

Granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) control the proliferation of human acute myeloid leukemia (AML) cells in vitro. Previously, we have shown that receptors for GM-CSF and IL-3 are often coexpressed on AML cells. Here we present experiments with purified AML blasts, normal monocytes, and granulocytes that were conducted to analyze the properties of GM-CSF and IL-3 binding proteins in more detail. On AML cells from eight cases we demonstrate two types of GM-CSF receptors: one with low affinity (dissociation constant [kd] 5.1 to 24.8 nmol/L) and one with a high affinity (kd 31 to 104 pmol/L). These AML cells also expressed high affinity receptors for IL-3 (kd 24 to 104 pmol/L). Cross-competition experiments showed that an excess concentration of nonlabeled IL-3 completely prevented the high affinity binding of radiolabled GM-CSF. This competition occurred at 37 degrees C as well as 4 degrees C. Low affinity GM-CSF binding was not affected by IL-3. Binding of radiolabeled IL-3 could be prevented by nonlabeled GM-CSF. In certain cases, this competition was complete, whereas in others only partial (49% to 77%) reduction of the radiolabeled IL-3 binding was seen. On the basis of these ligand binding features, we propose the existence of three receptor types on AML cells: (1) low affinity GM-CSF receptors that do not bind IL-3, (2) dual high affinity GM-CSF/IL-3 receptors, and (3) high affinity IL-3 receptors that do not bind GM-CSF. We could also demonstrate these receptor types on normal monocytes. In addition, a fourth type of receptor was apparent on normal granulocytes (4), incapable of binding IL-3 and with an intermediate affinity for GM-CSF (approximately 400 pmol/L). Chemical crosslinking showed that GM-CSF and IL-3 both bind to proteins with molecular weight values of 130, 105, and 75, which provides additional evidence for the existence of a common GM-CSF/IL-3 receptor complex.     

Antithrombotic regulation in human endothelial cells by fibrinolytic factors

Vascular endothelial cells (ECs) modulate the blood fibrinolytic system by secreting tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), and their inhibitor, type-1 plasminogen activator inhibitor (PAI-1). ECs also express t-PA receptors (t-PAR) and u-PA receptors (u-PAR) on their cell surfaces, assembling both enzymes to regulate the cellular fibrinolytic activity. In addition, ECs modulate these factors in response to several stimuli. Fibrin clots on ECs induce the up- and downregulation of t-PA and PAI-1 production, respectively, thus causing an effective lysis of the fibrin clot. Heat shock (43 degrees C) increases the expression of u-PA, t-PA, PAI-1, and u-PAR by which ECs become more fibrinolytic around the cells. Furthermore, because ECs possess t-PAR and u-PAR on their cell surfaces, the binding of t-PA and u-PA is a critical event, which affords ECs the localized and condensed fibrinolytic potential. Therefore, ECs play a central role in antithrombotic activity by regulating the levels of these fibrinolytic factors.     

Does apolipoprotein(a) heterogeneity influence lipoprotein(a) effects on fibrinolysis?

High plasma levels of lipoprotein(a) [Lp(a)] are considered to be an independent risk factor for premature cardiovascular disease and are inversely associated with apolipoprotein(a) [apo(a)] isoform sizes. The contribution of apo(a) polymorphism to the inhibition of fibrinolysis, a mechanism that may favor thrombus development, was therefore evaluated by measuring the ability of Lp(a) particles of distinct apo(a) isoform content to compete with plasminogen for fibrin binding during plasminogen activation by fibrin-bound tissue-type plasminogen activator. The rate of plasmin generation was most efficiently inhibited by an isoform with a molecular weight (M(r)) of approximately 540 Kd. An isoform with M(r) approximately 590 Kd produced a less pronounced effect, whereas the isoform with M(r) approximately 610 Kd failed to inhibit plasminogen activation. These effects were directly proportional to the amount of Lp(a) bound to the carboxy-terminal lysine residues of degraded fibrin. The relative affinity of the binding (kd range, 16 to 180 nmol/L) reflected the ability of individual Lp(a) isoforms to inhibit the binding of plasminogen (kd, 600 nmol/L). The question of the influence of kringle sequence variability on the binding to fibrin was not addressed by the present work. These data suggest that apo(a) isoform types with high affinity for fibrin may influence the ability of Lp(a) to interfere with fibrinolysis and contribute thereby to the association of elevated levels of Lp(a) with atherosclerotic and thrombotic risks.     

Formation, inhibition and clearance of plasmin in vivo

A 5 microg/kg bolus of tissue plasminogen activator (t-PA) was infused into 11 healthy subjects followed by measurement of t-PA activity and antigen, PAI-1 activity and antigen, t-PA/PAI-1 complex, plasmin/antiplasmin (PAP) complex and D-dimer over 4 h. Infusion of t-PA resulted in a rise in PAP levels in all subjects from a baseline of 2.4 +/- 1.1 nmol/l to a peak of 5.1 +/- 2.3 nmol/l, but had no effect on plasminogen, antiplasmin or D-dimer levels. Using a kinetic model of plasminogen activation in vivo, the second-order rate constant for t-PA binding to plasminogen was estimated to be 3,100 +/- 1,300 mol(-1)l x s(-1), 200-500 times slower than t-PA accelerated by fibrin. The half-life of PAP was 4.5 +/- 1.6 h. In healthy subjects, the PAP level in plasma represents the average rate of plasminogen activation over the last 2-8 h.     

Urokinase, tissue-type plasminogen activator and plasminogen activator inhibitor-1 expression in severely stenosed and occluded vein grafts with thrombosis.

Intimal hyperplasia and subsequent thrombotic occlusions limit the success of vascular reconstructive procedures. Plasminogen activation in situ may be an important factor affecting re-stenosis of the graft. Tissue specimens from eight patients with failing or failed infra-inguinal vein bypasses and three specimens from normal veins were harvested to study urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) by in situ hybridization and immunohistochemistry. The possible presence of thrombi was monitored by platelet and fibrin-specific stainings. In occluded grafts, platelet endothelial cell adhesion molecule (PECAM-1) antibody stained the thrombi but not the endothelial area, indicating the absence of endothelium. Platelet glycoprotein (GP) IIb/IIIa co-localized with PECAM-1 and, furthermore, GP IIb/IIIa staining was positive on the vein walls with thrombi and to some extent in the grafts without thrombi. PAI-1 and u-PA were uniformly upregulated in intimal thickening in grafts without thrombus. In organized thrombi, enhanced u-PA, t-PA and PAI-1 reactivity was detected in the ingrowing subendothelium. In non-occluded grafts with small thrombi, u-PA expression was enriched beneath microthrombi co-localizing with the graft wall injury, while PAI-1 was scattered in the (sub)endothelium. We conclude that fibrinolytic system is upregulated at sites of graft stenosis, and local proteolytic degradation of the graft wall associates with thrombus formation.     

Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells

The vascular endothelial growth factor (VEGF) family has recently expanded by the identification and cloning of three additional members, namely VEGF-B, VEGF-C, and VEGF-D. In this study we demonstrate that VEGF-B binds selectively to VEGF receptor-1/Flt-1. This binding can be blocked by excess VEGF, indicating that the interaction sites on the receptor are at least partially overlapping. Mutating the putative VEGF receptor-1/Flt-1 binding determinants Asp⁶³, Asp⁶⁴, and Glu⁶⁷ to alanine residues in VEGF-B reduced the affinity to VEGF receptor-1 but did not abolish binding. Mutational analysis of conserved cysteines contributing to VEGF-B dimer formation suggest a structural conservation with VEGF and platelet-derived growth factor. Proteolytic processing of the 60-kDa VEGF-B₁₈₆ dimer results in a 34-kDa dimer containing the receptor-binding epitopes. The binding of VEGF-B to its receptor on endothelial cells leads to increased expression and activity of urokinase type plasminogen activator and plasminogen activator inhibitor 1, suggesting a role for VEGF-B in the regulation of extracellular matrix degradation, cell adhesion, and migration.     

Fibrin(ogen) is internalized and degraded by activated human monocytoid cells via Mac-1 (CD11b/CD18): a nonplasmin fibrinolytic pathway

Fibrin(ogen) (FGN) is important for hemostasis and wound healing and is cleared from sites of injury primarily by the plasminogen activator system. However, there is emerging evidence in plasminogen activator- deficient transgenic mice that nonplasmin pathways may be important in fibrin(ogen)olysis, as well. Given the proximity of FGN and monocytes within the occlusive thrombus at sites of vascular injury, we considered the possibility that monocytes may play an ancillary role in the degradation and clearance of fibrin. We found that monocytes possess an alternative fibrinolytic pathway that uses the integrin Mac- 1, which directly binds and internalizes FGN, resulting in its lysosomal degradation. At 4 degrees C, FGN binds to U937 monocytoid cells in a specific and saturable manner with a kd of 1.8 mumol/L. Binding requires adenosine diphosphate stimulation and is calcium- dependent. At 37 degrees C, FGN and fibrin monomer (FM) are internalized and degraded at rates of 0.37 +/- 0.13 and 0.55 +/- 0.03 microgram/10(6) cells/h by U937 cells, 1.38 +/- 0.02 and 1.20 +/- 0.30 microgram/10(6) cells/h by THP-1 cells, and 2.10 +/- 0.20 and 2.52 +/- 0.18 micrograms/10(6) cells/h by human peripheral blood mononuclear cells, respectively. The serine protease inhibitors, PPACK and aprotinin, and the specific elastase inhibitor, AAPVCK, do not significantly inhibit degradation. However, degradation is inhibited by chloroquine, suggesting that a lysosomal pathway is involved. Factor X, a competitive ligand with FGN for the Mac-1 receptor, also blocks degradation, as does a monoclonal antibody to the alpha-subunit of Mac- 1. Autoradiography of radioiodinated, internalized FGN shows that FGN proteolysis by the pathway produces a unique degradation pattern distinct from that observed with plasmin. In a fibrin clot lysis assay, Mac-1-mediated fibrinolysis contributed significantly to total fibrinolysis. In summary, FGN is internalized and degraded by activated human monocytoid cells via Mac-1 in the absence of plasmin, thereby providing an alternative fibrinolytic pathway. Thus, in addition to the function of cell adhesion, integrins may also act as receptors that mediate the internalization and degradation of bound ligands.