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The binding and assembly of the coagulation proteases on the endothelial cell surface are important steps not only in the generation of thrombin and thrombogenesis, but also in vascular cell signaling. Effector cell protease receptor (EPR-1) was identified as a novel leukocyte cell surface receptor recognizing the coagulation serine protease Factor Xa but not the precursor Factor X. We now demonstrate that EPR-1 is expressed on vascular endothelial cells and smooth muscle cells. Northern blots of endothelial and smooth muscle cells demonstrated three abundant mRNA bands of 3.0, 1.8, and 1.3 kDa. 125I-Labeled Factor Xa bound to endothelial cells in a dose-dependent saturable manner, and the binding was inhibited by antibody to EPR-1. No specific binding was observed with a recombinant mutant Factor X in which the activation site was substituted by Arg196 --> Gln to prevent the proteolytic conversion to Xa. EPR-1 was identified immunohistochemically on microvascular endothelial and smooth muscle cells. Functionally, exposure of smooth muscle cells or endothelial cells to Factor Xa induced a 3-fold and a 2-fold increase in [3H]thymidine uptake, respectively. However, receptor occupancy alone is insufficient for mitogenic signaling because the active site of the enzyme is required for mitogenesis. Thus, EPR-1 represents a site of specific protease-receptor complex assembly, which during local initiation of the coagulation cascade could mediate cellular signaling and responses of the vessel wall.

Lipoprotein (a) (Lp(a)) is a low density lipoprotein-like particle which contains the plasminogen-like apolipoprotein a. Lp(a) levels are elevated in patients with atherosclerotic coronary artery disease. Recent studies suggest that Lp(a) competitively inhibits plasminogen binding to the endothelial cell and interferes with surface-associated plasmin generation. In this study, we present evidence for the presence of Lp(a) in the microvasculature of inflamed tissue. In addition, we demonstrate that Lp(a) regulates endothelial cell synthesis of a major fibrinolytic protein, plasminogen activator inhibitor-1 (PAI-1). In cultured human endothelial cells, Lp(a) enhanced PAI-1 antigen, activity, and steady-state mRNA levels without altering tissue plasminogen activator activity or mRNA transcript levels. This effect was cell-specific. Although other lipoproteins did not coordinately raise PAI-1 mRNA levels in endothelial cells, low density lipoprotein treatment selectively raised the level of the 3.4-kilobase mRNA species of PAI-1 without a concomitant increase in PAI-1 activity or antigen. Endothelial cell exposure to Lp(a) did not cause generalized endothelial cell activation since the functional activity and mRNA levels for tissue factor, platelet-derived growth factor and interleukin-6 were not elevated following Lp(a) exposure. These data suggest a molecular mechanism whereby Lp(a) may support a specific prothrombotic endothelial cell phenotype, namely by increasing PAI-1 expression.

Tumor cell attachment to thrombospondin (TSP) in the extracellular matrix may be of critical importance in the processes of invasion and hematogenous dissemination. To determine the specific receptor systems that mediate the interaction of tumor cells with insoluble TSP, the attachment of HT1080 fibrosarcoma and C32 and G361 melanoma cells to TSP-coated discs was studied in the presence of heparin, Arg-Gly-Asp-Ser, or antibodies to glycoprotein (GP) IV (CD36, GPIIIb), a TSP receptor. HT1080 and C32 cell attachment to TSP was inhibited by the combination of heparin and a monoclonal (or polyclonal) antibody to GPIV but not by either alone. Heparin alone inhibited cell spreading. Neither control monoclonal antibodies nor the cell attachment peptide Arg-Gly-Asp-Ser inhibited tumor cell attachment to TSP, alone or in the presence of heparin. HT1080 cells attached equally as well to a 140-kDa proteolytic TSP fragment lacking the heparin-binding domain as to intact TSP. A monoclonal antibody to GPIV alone inhibited tumor cell attachment to the heparin-domainless 140-kDa TSP fragment. No attachment to the heparin-binding fragment was observed, but the addition of the heparin fragment to 140-kDa heparin-domainless TSP restored the heparin sensitivity of binding. G361 cells that lack GPIV attached well to TSP but were not inhibited by heparin or anti-GPIV alone or in combination. The combination of heparin and Arg-Gly-Asp-Ser inhibited G361 attachment to TSP. These studies suggest that tumor cells may utilize separate receptor systems in a cooperative manner to adhere to TSP. HT1080 fibrosarcoma and C32 melanoma cells utilize GPIV in concert with a heparin-modulated binding systems to attach and spread on TSP. G361 cells, which lack GPIV expression, attach and spread on TSP using an integrin system as well as a heparin-modulated system.

Platelet glycoprotein Ib is a major platelet membrane protein composed of two disulfide-linked chains, termed the alpha and beta chains. The larger alpha chain (GpIb alpha), a platelet receptor for von Willebrand factor, plays a major role in mediating platelet adhesion to the subendothelium. Our laboratories have previously reported synthesis of a protein in human endothelial cells that is immunoprecipitated with polyclonal and monoclonal antibodies to platelet GpIb alpha. Lopez et al. (Lopez, J. A., Chung, D. W., Fujikawa, K., Hagan, F. S., Papayannopoulou, T., and Roth, G. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5615-5619) have reported the cloning of GpIb alpha from a human erythroleukemia (HEL) cell cDNA library. Using this clone as probe, we have isolated two partial GpIb alpha clones from a human umbilical vein endothelial cell lambda gt11 cDNA library. These clones were localized within HEL-derived GpIb alpha cDNA by sequence and restriction enzyme analysis. Additionally, they detected the same message species in HEL and tonsilar RNA that was detected with the HEL GpIb alpha cDNA. Low level GpIb alpha mRNA expression was detected in cultured human umbilical vein endothelial cells, which was increased by treatment of the cells with tumor necrosis factor-alpha. This effect was enhanced by pretreatment with interferon-gamma. Additionally, localization of GpIb alpha in endothelium of fresh tonsilar tissue was demonstrated by immunohistochemistry and in situ hybridization. GpIb alpha may play a role in mediating platelet or other effector cell adhesion to activated endothelium.

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.

Glycoprotein IIb (GPIIb) and glycoprotein IIIa (GPIIIa) form a macromolecular complex on the activated platelet surface which contains the fibrinogen-binding site necessary for normal platelet aggregation. To identify the specific region of the fibrinogen molecule responsible for its interaction with the GPIIb-GPIIIa complex, purified fragment D1 (Mr = 100,000) and fragment E (Mr = 50,000) were prepared from plasmin digests of purified human fibrinogen. In addition, the polypeptide chain subunits A alpha, B beta, and gamma of fibrinogen were prepared. Using an enzyme-linked immunosorbent assay we have demonstrated that isolated fragment D1 in a solid phase system forms a complex with a mixture of GPIIb and GPIIIa. The binding of the GPIIb-GPIIIa mixture to fragment D1-coated plates reached saturation at 8 nM and to fibrinogen-coated plates at 24 nM. Isolated A alpha, B beta, and gamma chains were not reactive with added glycoproteins. Fragment E coated directly on plastic plates or immobilized on antibody-coated plastic plates did not form a complex with GPIIb-GPIIIa. Only fluid phase fibrinogen and fragment D1 but not fragment E were inhibitory toward formation of a complex between solid phase fibrinogen and GPIIb-GPIIIa. Isolated A alpha, B beta, and gamma chains at concentrations equivalent to fluid phase fibrinogen were inactive. Binding of fragment D1 but not fragment E to the GPIIb-GPIIIa complex was also demonstrated by rocket immunoelectrophoresis of the membrane glycoprotein mixture through a gel containing the individual fragments and subsequent autoradiography of the complex following exposure to 125I-anti-fibrinogen. These observations with isolated platelet membrane glycoproteins provide strong evidence that each of the D domains of the fibrinogen molecule interacts directly with the GPIIb-GPIIIa complex on the activated platelet surface, thus allowing formation of a tertiary molecular "bridge" across the surface of two adjacent activated platelets.

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.

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.

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.

Subcellular membrane and granule fractions derived from human platelets contain immunologically identifiable alpha2-macroglobulin and alpha1-antitrypsin. These platelet-derived inhibitors show a reaction of immunologic identity when compared to alpha2-macroglobulin and alpha1-antitrypsin purified from human plasma. Further, the platelet protease inhibitors possessed a similar subunit polypeptide chain structure to their plasma counterparts as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. Studies of the binding of radiolabeled trypsin to the various solubilized platelet subcellular fractions suggest that the granule-associated alpha2-macroglobulin and alpha1-antitrypsin, as well as membrane-associated alpha2-macroglobulin were functionally active. Quantitatively, circulating platelets contain relatively small concentrations of these inhibitors as compared to platelet-associated fibrinogen and factor VIIIAGN. Platelet protease inhibitors may modulate the protease-mediated events involved in the formation of hemostatic plugs and thrombi.

Ternary complex formation of tissue plasminogen activator (TPA) and plasminogen (Plg) with thrombospondin (TSP) or histidine-rich glycoprotein (HRGP) has been demonstrated using an enzyme-linked immunosorbent assay, an affinity bead assay, and a rocket immunoelectrophoresis assay. The formation of these complexes was specific, concentration dependent, saturable, lysine binding site-dependent, and inhibitable by fluid phase plasminogen. Apparent Kd values were approximately 12-36 nM for the interaction of TPA with TSP-Plg complexes and 15-31 nM with HRGP-Plg complexes. At saturation the relative molar stoichiometry of Plg:TPA was 3:1 within the TSP-containing complexes and 1:1 within HRGP-containing complexes. The activation of Plg to plasmin by TPA on TSP- and HRGP-coated surfaces was studied using a synthetic fluorometric plasmin substrate (D-Val-Leu-Lys-7-amino-4-trifluoromethyl coumarin). Kinetic analysis demonstrated a marked increase in the affinity of TPA for plasminogen in the presence of surface-associated TSP or HRGP. Compared to fluid phase activation or activation on fibronectin- or Factor VIII-related antigen-coated surfaces there was a 35-fold increase in efficiency of plasmin generation. A substantial amount (up to 71%) of the plasmin formed remained surface-associated and was found to be protected from inhibition by alpha 2-plasmin inhibitor. Greater than 200-fold increase in inhibitor concentration was required to effect 50% inhibition. Complex formation of locally released tissue plasminogen activator with Plg immobilized on TSP or HRGP surfaces may thus play an important role in effecting proteolytic events in nonfibrin-containing microenvironments.

We have previously demonstrated that plasminogen activator inhibitor (PAI-1) is associated with the extracellular matrix of cultured bovine smooth muscle cells (Knudsen, B.S., Harpel, P.C., Nachman, R.L. (1987) J. Clin. Invest. 80, 1082-1089). In this report we describe the physiologic role of PAI-1 during the interaction of the tissue plasminogen activator (t-PA) secreting Bowes human melanoma cell line with endothelial extracellular matrices. In addition we have characterized the t-PA.PAI complexes formed during this interaction in the presence and absence of plasminogen. In the absence of plasminogen, a 104-kDa complex between Bowes t-PA and PAI-1 appears in the supernatant. In the presence of plasminogen, PAI initially prevents plasmin formation on the matrix and protects the matrix from degradation by plasmin. The 104-kDa t-PA.PAI complex is degraded into a 68 and a 47-kDa complex by small amounts of plasmin generated from secreted Bowes t-PA and plasminogen. Analysis of these complexes revealed that t-PA is rapidly cleaved by plasmin within the complex whereas complexed PAI-1 is not further degraded. Matrix-associated PAI-1 may play an important role in the protection of extracellular matrices from remodeling and degradation by cellular t-PA and plasminogen.

Platelet membrane glycoproteins IIb and IIIa were isolated and purified from human platelet membranes using lentil lectin affinity chromatography and electrophoretic elution from sodium dodecyl sulfate-polyacrylamide gels. Two-dimensional immunoelectrophoresis of a mixture of the purified proteins against monospecific antisera showed antigenic uniqueness of the separate polypeptides. Computerized analysis of autoradiographs of two-dimensional tryptic 125I peptide maps revealed that the two glycoproteins had completely different structures. Monospecific anti-glycoproteins IIb and IIIa Fab'2 fragments, either singly or in combination, induced platelet agglutination but did not inhibit or alter the platelet aggregation response to physiologic stimuli. The results demonstrate that human platelet membrane glycoproteins IIb and IIIa are separate molecular entities. In the native state, the membrane macromolecular IIb.IIIa complex may play an important role in mediating platelet-platelet interactions.