Tissue plasminogen activator and urokinase enhance the binding of plasminogen to thrombospondin

Thrombospondin (TSP) is a multifunctional platelet alpha-granule and extracellular matrix glycoprotein that binds specifically to plasminogen (Plg) via that protein's lysine-binding site and modulates activation by tissue activator (TPA). In this study we report that the plasminogen activators, TPA and urokinase, greatly influence the binding of Plg to TSP. Using an enzyme-linked immunosorbent assay and a TSP-Sepharose affinity bead-binding assay we have found that Plg-TSP complex formation was markedly enhanced (up to 5-fold) when catalytic concentrations of Plg activators were included in the reaction mixtures. The enhancement was dependent upon the generation of small amounts of active plasmin and was duplicated by pretreatment of the immobilized TSP with plasmin prior to addition of the Plg. The enhancement effect was associated with selective proteolysis of the immobilized TSP. Purified Lys-Plg (the plasmin modified form of native Glu-Plg) bound to TSP to a greater extent than Glu-Plg, and binding of both forms was augmented by Plg activators. The apparent KD values of complex formation were unchanged in the presence of Plg activators suggesting that the enhancement effect was due to the generation of additional binding sites. The increased amount of bound Plg was demonstrated to result in a similar increase in the amount of plasmin generated from the complexes by TPA. Plg activators did not influence binding of Plg to histidine-rich glycoprotein or of histidine-rich glycoprotein to TSP, demonstrating specificity. In addition when TSP was treated with other proteases (human thrombin or human leukocyte elastase) no augmentation of Plg binding was seen. Thus, the initial production of small amounts of plasmin from Plg immobilized on TSP in fibrin-free microenvironments could generate a positive feedback loop by enzymatically modifying both TSP and Plg, resulting in an increase in TSP-Plg complex formation leading to the localized production of substantially more plasmin.     

Thrombospondin forms complexes with single-chain and two-chain forms of urokinase

Thrombospondin (TSP), an adhesive glycoprotein found in platelets and extracellular matrix, has been shown previously to interact with plasminogen and tissue plasminogen activator, resulting in efficient plasmin generation. We now demonstrate specific complex formation of TSP with both the single-chain and two-chain forms of urokinase (scuPA and uPA). Binding of uPA and scuPA to immobilized TSP was detected and quantified using colorimetric immunoassays and a functional amidolytic assay. Binding was time and concentration dependent with apparent affinity constants of 40-50 nM. Binding was not affected by serine protease inhibitors, EDTA, or epsilon-aminocaproic acid. scUPA and uPA bound to TSP retained functional activity. Using a sensitive amidolytic assay we found that TSP. scuPA complexes were efficiently converted to TSP. uPA by catalytic plasmin concentrations. Additionally, TSP.uPA complexes were found to have plasminogen-activating activity equivalent to fluid-phase uPA and to be protected from inhibition by plasminogen activator inhibitor type 1, the major plasma and matrix plasminogen activator inhibitor. Using immunohistochemical techniques, we also demonstrated co-distribution of TSP and uPA in normal and malignant breast tissue. Complex formation of TSP with uPA may serve to localize, concentrate, and protect these enzymes on cell surfaces and within the extracellular matrix, thereby providing a reservoir of plasminogen activator activity.     

Plasminogen binding by alpha 2-antiplasmin and histidine-rich glycoprotein does not inhibit plasminogen activation at the surface of fibrin.

alpha 2-antiplasmin (alpha 2-AP) exerts its inhibitory effect on fibrinolysis by rapidly inhibiting the plasmin evolved; in addition, it has been suggested that interference with the binding of plasminogen to fibrin, a function shared with histidine-rich glycoprotein (HRGP), may also be significant in inhibition of fibrinolysis. To elucidate if plasminogen binding by these two alpha 2-globulins may decrease the generation of plasmin by tissue-type plasminogen activator (t-PA) at the surface of fibrin, a system mimicking the fibrin/plasma interface was used. Attempts were made to differentiate the plasminogen binding from the plasmin inhibitory function of alpha 2-AP. The activation of human Glu-plasminogen (native plasminogen with NH2-terminal glutamic acid) by fibrin-bound t-PA was performed in a plasma environment using either normal plasma, alpha 2-AP- or HRGP-depleted plasmas supplemented with increasing amounts of the lacking protein, or in a reconstituted system with purified plasminogen and various concentrations of alpha 2-AP and HRGP. The activation of Glu-plasminogen in alpha 2-AP-depleted plasma containing a normal concentration of HRGP produced a time-dependent increase in the generation of plasmin. The addition of 1 microM-alpha 2-AP to this plasma prevented the formation of Lys-derivatives and produced a marked decrease (42%) in the number of plasminogen-binding sites. In contrast, the addition of 1.5 microM-HRGP to HRGP-depleted plasma containing a normal amount of alpha 2-AP produced only a modest (17%) decrease in the amount of plasmin(ogen) bound. Moreover, in a purified system the amount of plasminogen-binding sites and thereby of plasmin generated at the surface of fibrin in the presence of both alpha-2 globulins was similar to the amount generated in the presence of alpha 2-AP alone. These results indicate clearly that the formation of reversible complexes between plasminogen and alpha 2-AP does not interfere with the binding and activation of plasminogen at the fibrin surface. In contrast, the inhibition of plasmin by alpha 2-AP decreases importantly the number of plasminogen-binding sites (carboxyl-terminal lysines) and inhibits thereby the accelerated phase of fibrinolysis. It can be concluded that interference of the binding of plasminogen to fibrin by alpha 2-AP during plasminogen activation, does not play a significant role in inhibition of fibrinolysis, and that the plasminogen-binding effect of HRGP, if any, is obscured by the important inhibitory effect of alpha 2-AP.     

Kinetic analysis of the effects of heparin and lipoproteins on tissue plasminogen activator mediated plasminogen activation

Heparin sulfate and the less sulfated glycosaminoglycan heparan sulfate enhance human plasminogen (Pg) conversion to plasmin by tissue-type plasminogen activator (t-PA). Kinetic studies indicate that both heparin and heparan increase the kcat of t-PA-mediated Pg activation by 25- and 3.5-fold, respectively. The Km of plasmin formation is unaltered by the presence of either heparin or heparan. Both heparin and heparan stimulate the activity of t-PA by interacting with the finger domain of t-PA, with association constants of 1 microM and 200 nM, respectively. Additionally, the lipoproteins lipoprotein(a) [Lp(a)] and low-density lipoprotein (LDL) inhibit the heparin enhancement of Pg activation. Lp(a) is a competitive inhibitor and LDL is a mixed inhibitor of t-PA-mediated Pg activation, with inhibition constants of 30 and 70 nM, respectively. The inhibition constants correspond to physiologic concentrations of these lipoproteins. These data suggest that heparin, heparan, and lipoproteins may play an important in vivo role in regulating cell surface associated activation of the fibrinolytic system.     

Plasminogen activation by receptor-bound urokinase. A kinetic study with both cell-associated and isolated receptor

The specific cellular receptor for urokinase-type plasminogen activator (uPA) is found on a variety of cell types and has been postulated to play a central role in the mediation of pericellular proteolytic activity. We have studied the kinetics of plasminogen (Plg) activation catalyzed by uPA specifically bound to its receptor on the human monocytoid cell-line U937 and demonstrate this process to have properties differing widely from those observed for uPA in solution. The solution-phase reaction was characterized by a Km of 25 microM and for the cell-associated reaction this fell 40-fold to 0.67 microM, below the physiological Plg concentration of 2 microM. A concomitant 6-fold reduction in kcat resulted in an increase in the overall catalytic efficiency, kcat/Km, of 5.7-fold. This high affinity Plg activation was abolished in the presence of a Plg-binding antagonist. In contrast to intact cells, purified uPA receptor (isolated from phorbol 12-myristate 13-acetate-stimulated U937 cells) was observed to partially inhibit uPA-catalyzed Plg activation, although activity against low molecular weight substrates was retained. Therefore, the cellular binding of Plg appears to be of critical importance for the efficient activation of Plg by receptor-bound uPA. Plasmin generated in the cell-surface Plg activation system described here was also observed to be protected from its principal physiological inhibitor alpha-2-antiplasmin. Together, these data demonstrate that the cell surface constitutes the preferential site for Plg activation when uPA is bound to its specific cellular receptor, which therefore has the necessary characteristics to play an efficient role in the generation of pericellular proteolytic activity.     

Structural consideration of the formation of the activation complex between the staphylokinase-like streptococcal plasminogen activator PadA and bovine plasminogen

The characteristics of a streptococcal plasminogen activator (PA) displaying specificity for ruminant plasminogen (Plg) were defined using molecular approaches. The 16-kDa secreted protein PadA was found to be prevalent in Streptococcus dysgalactiae subspecies dysgalactiae isolated from cases of bovine mastitis and septic arthritis in lambs. PadA was able to activate bovine, ovine and caprine Plg, but not human Plg. Amino acid sequence analysis identified a limited level of homology to other streptococcal PAs, including streptokinase; however, PadA was found to align well with and match in size the staphylococcal PA, staphylokinase. Recombinant PadA was used to investigate interaction with bovine Plg, leading to formation of an activator complex that was capable of recruiting and converting further substrate Plg into plasmin. Individual non-overlapping peptides of PadA or bovine microplasminogen were found to block the interaction between PadA and bovine Plg, preventing the formation of the activation complex. Homology modelling based upon structures of staphylokinase complexed with human microplasminogen supported these findings by placing critical residues in close proximity to the plasmin component of the activation complex.     

The interaction of streptokinase.plasminogen activator complex, tissue-type plasminogen activator, urokinase and their acylated derivatives with fibrin and cyanogen bromide digest of fibrinogen. Relationship to fibrinolytic potency in vitro.

The effects of purified soluble fibrin and of fibrinogen fragments (fibrin mimic) on the activation of Lys-plasminogen (i.e. plasminogen residues 77-790) to plasmin by streptokinase.plasminogen activator complex and by tissue-type plasminogen activator were studied. Dissociation constants of both activators were estimated to lie in the range 90-160 nM (fibrin) and 16-60 nM (CNBr-cleavage fragments of fibrinogen). The kinetic mechanism for both types of activator comprised non-essential enzyme activation via a Rapid Equilibrium Ordered Bireactant sequence. In order to relate the fibrin affinity of plasminogen activators to their fibrinolytic potency, the rate of lysis of supported human plasma clots formed in the presence of unmodified or active-centre-acylated precursors of plasminogen activators was studied as a function of the concentration of enzyme derivative. The concentrations of unmodified enzyme giving 50% lysis/h in this assay were 0.9, 2.0 and 11.0 nM for tissue-type plasminogen activator, streptokinase.plasmin(ogen) and urokinase respectively. However, the potencies of active-centre-acylated derivatives of these enzymes suggested that acylated-tissue plasminogen activator and streptokinase.plasminogen complexes of comparable hydrolytic stability were of comparable potency. Both types of acyl-enzyme were significantly more potent than acyl-urokinases.     

Mapping of binding sites for heparin, plasminogen activator inhibitor-1, and plasminogen to vitronectin's heparin-binding region reveals a novel vitronectin-dependent feedback mechanism for the control of plasmin formation.

Vitronectin (VN) has been implicated as a major matrix-associated regulator component of plasminogen activation by serving as a potent stabilizing cofactor of plasminogen activator inhibitor-1 (PAI-1). The direct binding of heparin, plasminogen as well as PAI-1 in its latent and active form to immobilized VN was studied in the absence or presence of competitors. Monoclonal antibodies against the carboxyl-terminal portion of VN inhibited both PAI-1 and plasminogen binding, whereas heparin, heparan sulfate with a high degree of sulfation, or dextran sulfate interfered with PAI-1 binding (KD = 20 nM) only. Utilizing synthetic peptides encompassing overlapping sequences of the heparin-binding domain of VN, adjacent heparin and PAI-1-binding sites were localized within the sequence 348-370 of VN. Although a number of other serine protease inhibitors which do not form binary complexes with VN contain a reactive-site Ser at their P1'-position, a reactive-site P1' mutant of PAI-1 (Met----Ser) showed comparable if not increased binding to VN. Binding of Lys-plasminogen and active-site-blocked plasmin was at least 10-fold higher in affinity (KD = 85-100 nM) compared to Glu-plasminogen (KD approximately 1 microM) and could be inhibited by lysine analogs but not by glycosaminoglycans or PAI-1, indicating that heteropolar plasmin(ogen) binding of VN occurs to an adjacent segment upstream to the heparin and PAI-1-binding sites. This contention was further supported in binding studies with plasmin-modified VN which lost both heparin and PAI-1 binding but exhibited 2-3-fold higher capacity to bind plasminogen. The essential plasmin(ogen)-binding site was mapped by ligand blot analysis to the carboxyl-terminal portion of proteolytically trimmed VN (M(r) = 61,000). Moreover, treatment of the extracellular matrix of human umbilical vein endothelial cells with plasmin resulted in partial degradation of matrix-associated VN and concomitant release of PAI-1, but increased the ability of the matrix by about 2-fold to bind plasminogen. These results are indicative of differential interactions of VN with components of the plasminogen activation system, whereby plasmin itself may provoke the switch of VN from an anti-fibrinolytic into a pro-fibrinolytic cofactor. This process reflects a novel role for the adhesive protein and its degradation product(s) in the possible feedback regulation of localized plasmin formation at extracellular sites.     

Partial purification and characterization of a new fast-acting plasmin inhibitor from human platelets. Evidence for non-identity with the known plasma proteinase inhibitors.

An inhibitor of the plasma proteinase plasmin (EC 3.4.21.7) was partially purified from washed and lysed human blood platelets by (NH4)2SO4 fractionation and affinity chromatrography on Sepharose-linked purified plasminogen. The material contained none of the known plasma proteinase inhibitors when studied by crossed-immunoelectrophoresis and electroimmunoassay, but inhibited a clot-lysis-time assay and an esterolytic assay that used the synthetic substrate S-2251 (D-Val-Leu-Lys-p-nitroanilide). The inhibitory activity had the same mobility as the alpha 2-plasma proteins on preparative agarose-gel electrophoresis. Titration of the inhibitor preparation by active-site-titrated plasmin demonstrated a dissociation constant of approx. 0.1 nM. The inhibition was complete within 1 min. The inhibitor increased the mobility in agarose-gel electrophoresis of purified activator-free plasmin or 125I-labelled plasmin, as demonstrated by crossed-immunoelectrophoresis against specific immunoglobulins against plasminogen or by radioautography. The results strongly suggest the presence in platelets of a plasmin inhibitor different from the known plasma proteinase inhibitors.     

Stimulation of tPA-dependent provisional extracellular fibrin matrix degradation by human recombinant soluble melanotransferrin

Tissue-type plasminogen activator (tPA) and its substrate plasminogen (Plg) are key components in the fibrinolytic system. We have recently demonstrated, that truncated human recombinant soluble melanotransferrin (sMTf) could stimulate the activation of Plg by urokinase plasminogen activator and inhibit angiogenesis. Since various angiogenesis inhibitors were shown to stimulate tPA-mediated plasminogen activation, we examined the effects of sMTf on tPA-dependent fibrinolysis. This study demonstrated that sMTf enhanced tPA-activation of Plg by 6-fold. sMTf also increased the release of [125I]-fibrin fragments by tPA-activated plasmin. Moreover, we observed that the interaction of sMTf with Plg provoked a change in the fibrin clot structure by cleaving the fibrin alpha and beta chains. Overall, the present study shows that sMTf modulates tPA-dependent fibrinolysis by modifying the clot structure. These results also suggest that sMTf properties could involve enhanced dissolution of the provisional extracellular fibrin matrix.     

The plasminogen system and cell surfaces: evidence for plasminogen and urokinase receptors on the same cell type

The capacity of cells to interact with the plasminogen activator, urokinase, and the zymogen, plasminogen, was assessed using the promyeloid leukemic U937 cell line and the diploid fetal lung GM1380 fibroblast cell line. Urokinase bound to both cell lines in a time- dependent, specific, and saturable manner (Kd = 0.8-2.0 nM). An active catalytic site was not required for urokinase binding to the cells, and 55,000-mol-wt urokinase was selectively recognized. Plasminogen also bound to the two cell lines in a specific and saturable manner. This interaction occurred with a Kd of 0.8-0.9 microM and was of very high capacity (1.6-3.1 X 10(7) molecules bound/cell). The interaction of plasminogen with both cell types was partially sensitive to trypsinization of the cells and required an unoccupied high affinity lysine-binding site in the ligand. When plasminogen was added to the GM1380 cells, a line with high intrinsic plasminogen activator activity, the bound ligand was comprised of both plasminogen and plasmin. Urokinase, in catalytically active or inactive form, enhanced plasminogen binding to the two cell lines by 1.4-3.3-fold. Plasmin was the predominant form of the bound ligand when active urokinase was added, and preformed plasmin can also bind directly to the cells. Plasmin on the cell surface was also protected from its primary inhibitor, alpha 2-antiplasmin. These results indicate that the two cell lines possess specific binding sites for plasminogen and urokinase, and a family of widely distributed cellular receptors for these components may be considered. Endogenous or exogenous plasminogen activators can generate plasmin on cell surfaces, and such activation may provide a mechanism for arming cell surfaces with the broad proteolytic activity of this enzyme.     

Purification and characterization of a stimulator of plasmin generation from the antiangiogenic agent Neovastat: identification as immunoglobulin kappa light chain

We have recently shown that Neovastat, an antiangiogenic extract from shark cartilage, stimulates the in vitro activation of plasminogen by facilitating the tissue-type plasminogen activator (tPA)-dependent conversion of plasminogen to plasmin. In this report, we describe the purification and characterization of the stimulatory molecules. Neovastat was subjected to a three-step purification procedure including gel filtration, preparative isoelectric focusing, and preparative SDS-PAGE. Two 28-kDa proteins with pIs of approximately 4.5 and 6.5 were purified to apparent homogeneity and identified as immunoglobulin (Ig) kappa light chains by N-terminal microsequencing. Ig light chains do not directly stimulate the activity of tPA or plasmin, suggesting a mechanism of action involving an interaction with plasminogen. Kinetic analysis showed that both Ig light chains accelerate the in vitro tPA-dependent conversion of plasminogen in plasmin by increasing the affinity of tPA for plasminogen by 32- and 38-fold (Km decrease from 456 nM to 12-14 nM). Shark Ig light chains also stimulated the degradation of fibrin by the tPA/plasminogen system in an in vitro assay. A direct interaction between Ig light chains and plasminogen (KA=4.0-5.5 x 10(7) M(-1); KD=18-25 nM) and with tPA (KA=2.8 x 10(7) M(-1); KD=36 nM) was demonstrated using real time binding measured by surface plasmon resonance. Ig light chain is the first molecule associated with the antiangiogenic activity of Neovastat to be purified and identified.     

Dissecting mannose 6-phosphate-insulin-like growth factor 2 receptor complexes that control activation and uptake of plasminogen in cells

The plasminogen (Plg) activation cascade on the cell surface plays a central role in cell migration and is involved in a plethora of physiological and pathological processes. Its regulation is coordinated by many receptors, in particular the urokinase-type plasminogen activator receptor (uPAR, CD87), receptors that physically interact and functionally cooperate with uPAR, and Plg binding molecules. Here we studied the impact of one of the Plg binding molecules, the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P-IGF2R, CD222), on cellular Plg activation. By developing both in vitro and in vivo Plg activation assays on size-fractionated lysates of M6P-IGF2R-silenced cells, we identified Plg-associated complexes with M6P-IGF2R as the regulatory factor. Using lipid raft preserving versus dissolving detergents, we found lipid dependence of the Plg regulatory function of these complexes. Furthermore, M6P-IGF2R-silencing in uPAR-positive human cell lines reduced internalization of Plg, resulting in elevated Plg activation. In contrast, the expression of human M6P-IGF2R in mouse embryonic fibroblasts derived from M6P-IGF2R knock-out mice enhanced Plg internalization. Finally, peptide 18-36 derived from the Plg-binding site within M6P-IGF2R enhanced Plg uptake. Thus, by targeting Plg to endocytic pathways, M6P-IGF2R appears to control Plg activation within cells that might be important to restrict plasmin activity to specific sites and substrates.     

Binding of plasminogen to extracellular matrix

We have previously demonstrated that plasminogen immobilized on various surfaces forms a substrate for efficient conversion to plasmin by tissue plasminogen activator (t-PA) (Silverstein, R. L., Nachman, R. L., Leung, L. L. K., and Harpel, R. C. (1985) J. Biol. Chem. 260, 10346-10352). We now report the binding of human plasminogen to the extracellular matrix synthesized in vitro by cultured endothelial cell monolayers. The binding was specific, saturable at plasma plasminogen concentrations, reversible, and lysine-binding site-dependent. Functional studies demonstrated that matrix immobilized plasminogen was a much better substrate for t-PA than was fluid phase plasminogen as shown by a 100-fold decrease in Km. Activation of plasminogen by t-PA and urokinase on the matrix was equally efficient. The plasmin generated on the matrix, in marked contrast to fluid phase, was protected from its fast-acting inhibitor, alpha 2-plasmin inhibitor. Matrix-associated plasmin converted bound Glu- into Lys-plasminogen, which in turn is more rapidly activated to plasmin by t-PA. The extracellular matrix not only binds and localizes plasminogen but also improves plasminogen activation kinetics and prolongs plasmin activity in the subendothelial microenvironment.     

Binding of plasminogen to cultured human endothelial cells

Endothelial cells are known to release the two major forms of plasminogen activator, tissue plasminogen activator (TPA) and urokinase. We have previously demonstrated that plasminogen (PLG) immobilized on various surfaces forms a substrate for efficient conversion to plasmin by TPA (Silverstein, R. L., Nachman, R. L., Leung, L. L. K., and Harpel, P. C. (1985) J. Biol. Chem. 260, 10346-10352). We now report the binding of human PLG to cultured human umbilical vein endothelial cell (HUVEC) monolayers, utilizing a newly devised cell monolayer enzyme-linked immunosorbent assay system. PLG binding to HUVEC was concentration dependent and saturable at physiologic PLG concentration (2 microM). Binding of PLG was 70-80% inhibited by 10 mM epsilon-aminocaproic acid, suggesting that it is largely mediated by the lysine-binding sites of PLG. PLG bound at an intermediate level to human fibroblasts, poorly to human smooth muscle cells, and not at all to bovine smooth muscle or bovine endothelial cells; unrelated proteins such as human albumin and IgG failed to bind HUVEC. PLG binding to HUVEC was rapid, reaching a steady state within 20 min, and quickly reversible. 125I-PLG bound to HUVEC with an estimated Kd of 310 +/- 235 nM (S.E.); each cell contained 1,400,000 +/- 1,000,000 (S.E.) binding sites. Functional studies demonstrated that HUVEC-bound PLG is activatable by TPA according to Michaelis-Menten kinetics (Km, 5.9 nM). Importantly, surface-bound PLG was activated with a 12.7-fold greater catalytic efficiency than fluid phase PLG. These results indicate that PLG binds to HUVEC in a specific and functional manner. Binding of PLG to endothelial cells may play a pivotal role in modulating thrombotic events at the vessel surface.     

Binding and activation of plasminogen at the surface of Staphylococcus aureus. Increase in affinity after conversion to the Lys form of the ligand.

Untreated Staphylococcus aureus cells, strain Cowan I, specifically bound 125I-Glu-plasminogen. The binding was inhibited by both unlabeled Glu-plasminogen and Glu-plasmin. The Lys form of plasminogen, which lacks the 8-kDa amino-terminal activation peptide, was approximately 100-fold more effective than the Glu form in competing with the binding of 125I-labeled Glu-plasminogen. This suggests an increase in binding affinity upon removal of the activation peptide. Fibronectin, fibrinogen and IgG, plasma components known to bind to the staphylococcal surface, did not significantly interfere with the binding. The competing activity in plasma was abolished by specifically absorbing plasminogen from the plasma sample. L-Lysine and a fragment of plasminogen containing three of the first five protein attachment domains present in the molecule (kringle structures) also competed with plasminogen for binding suggesting that the lysine-binding sites of plasminogen were involved in its interaction with staphylococci. Scatchard analysis revealed high- and low-affinity binding sites. Kd and the number of high-affinity binding sites were 1.7 nM and 780 binding sites/bacterial cell, respectively. 125I-Glu-plasminogen bound to staphylococcal surface was converted to plasmin by tissue-type plasminogen activator. The conversion took place also in the presence of plasma. If the conversion was carried out in the absence of low-molecular-mass plasmin inhibitors such as aprotinin, the bound Glu-plasmin was further converted to Lys-plasmin. The surface-bound plasmin was enzymically active, as judged by digestion of the synthetic substrate, S-2251. The plasminogen conversion shown by the present experiments not only leads to the surface-bound plasmin but seems to considerably increase the affinity of plasmin for its binding site. This may represent a physiologically relevant method for a bacterial cell to retain surface-bound active plasmin which is also protected from its soluble plasma inhibitors. This novel mechanism for staphylococci to adopt surface-bound proteolytic activity, without the interference of plasma components, may have some role in the tissue penetration and invasion of microbes during infection.     

Tissue plasminogen activator and urokinase mediate the binding of Glu-plasminogen to plasma fibrin I. Evidence for new binding sites in plasmin-degraded fibrin I

The effect of tissue plasminogen activator (TPA) or urokinase on the specific binding of human Glu-plasminogen to fibrin I formed in plasma by clotting with Reptilase was studied using 125I-plasminogen and 131I-fibrinogen. In the absence of TPA, small amounts of plasminogen were bound to fibrin I. TPA induced binding of plasminogen to plasma fibrin I that was dependent upon the concentrations of TPA and plasminogen as well as upon the time of incubation. Plasminogen binding occurred in association with fibrin clot lysis and the formation in the clot supernatant of alpha 2-plasmin inhibitor-plasmin complexes. Urokinase also induced binding of plasminogen to plasma fibrin I that was concentration- and time-dependent. The molecular form of plasminogen bound to the fibrin I plasma clot was identified as Glu-plasminogen by dodecyl sulfate-polyacrylamide gel electrophoresis and by fast performance liquid chromatography. Further studies demonstrated that fibrin I formed from fibrinogen that had been progressively degraded by plasmin-bound Glu-plasminogen. The mole ratio of plasminogen bound increased with the time of plasmin digestion. Glu-plasminogen did not bind to fibrin I formed from fibrinogen progressively digested by human leukocyte elastase, thereby demonstrating the specificity of plasmin. These studies demonstrate that plasminogen activators regulate the binding of Glu-plasminogen to fibrin I by catalyzing plasmin-mediated modifications in the fibrin substrate.     

Characterization of plasmin-mediated activation of plasma procarboxypeptidase B. Modulation by glycosaminoglycans

Plasma carboxypeptidase B (PCB) is an exopeptidase that exerts an antifibrinolytic effect by releasing C-terminal Lys and Arg residues from partially degraded fibrin. PCB is produced in plasma via limited proteolysis of the zymogen, pro-PCB. In this report, we show that the K(m) (55 nM) for plasmin-catalyzed activation of pro-PCB is similar to the plasma concentration of pro-PCB (50-70 nM), whereas the K(m) for the thrombin- or thrombin:thrombomodulin-catalyzed reaction is 10-40-fold higher than the pro-PCB level in plasma. Additionally, tissue-type plasminogen activator triggers activation of pro-PCB in blood plasma in a reaction that is stimulated by a neutralizing antibody versus alpha(2)-antiplasmin. Together, these results show that plasmin-mediated activation of pro-PCB can occur in blood plasma. Heparin (UH) and other anionic glycosaminoglycans stimulate pro-PCB activation by plasmin but not by thrombin or thrombin:thrombomodulin. Pro-PCB is a more favorable substrate for plasmin in the presence of UH (16-fold increase in k(cat)/K(m)). UH also stabilizes PCB against spontaneous inactivation. The presence of UH in clots prepared with prothrombin-deficient plasma delays tissue-type plasminogen activator-triggered lysis; this effect of UH on clot lysis is blocked by a PCB inhibitor from potato tubers. These results show that UH accelerates plasmin-catalyzed activation of pro-PCB in plasma and PCB, in turn, stabilizes fibrin against fibrinolysis. We propose that glycosaminoglycans in the subendothelial extracellular matrix serve to augment the levels of PCB activity thereby stabilizing blood clots at sites where there is a breach in the integrity of the vasculature.     

Dengue virus-induced autoantibodies bind to plasminogen and enhance its activation

Dengue virus infection can lead to life-threatening dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) in patients. Abnormal activation of the coagulation and fibrinolysis system is one of the hallmarks associated with DHF/DSS patients. However, the mechanisms that cause pathology in DHF/DSS patients are still unclear. Because conversion of plasminogen (Plg) to plasmin (Plm) is the first step in the activation of fibrinolysis, Abs against Plg found in DHF/DSS patients may be important. Therefore, to investigate the specificity, function, and possible origin of these Abs, we generated several Plg cross-reactive mAbs from DENV-immunized mice. An IgG mAb, 6H11, which recognizes an epitope associated with a dengue envelope protein, demonstrated a high level of cross-reactivity with Plg. The 6H11 Ab was further characterized with regard to its effect on Plg activation. Using Plm-specific chromogenic substrate S-2251, we found that mAb 6H11 demonstrated serine protease activity and could convert Plg directly to Plm. The serine protease activity of mAb 6H11 was further confirmed using serine protease chromogenic substrate S-2288. In addition, we found several Plg cross-reactive mAbs that could enhance urokinase-induced Plg activation. Lastly, mAb 6H11 could induce Plm activity and increase the level of D-dimer (a fibrin degradation product) in both human and mouse platelet-poor plasma. Taken together, these data suggest DENV-induced Plg cross-reactive Abs may enhance Plg conversion to Plm, which would be expected to contribute to hyperfibrinolysis in DHF/DSS patients.