Thrombospondin-plasminogen interactions: modulation of plasmin generation.
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Fibrinolysin↗
Biomedical subjects
R L Nachman
Publications and source records attributed to R L Nachman.
Thrombospondin binds to monocytes-macrophages and mediates platelet-monocyte adhesion.
Thrombospondin (TSP) is a multifunctional platelet glycoprotein synthesized by a variety of cells in culture including monocytes and macrophages. We now report that 125I-TSP binds specifically, saturably, and reversibly to mouse peritoneal macrophages and to cells of the monocyte-like human cell line U937 with dissociation constants of 6.7-14.5 X 10(-8) M and 3-4 X 10(5) binding sites per cell. TSP mediates an adhesive interaction between thrombin-stimulated platelets and both U937 cells and human blood monocytes. Using a sensitive rosetting assay, we found that monocytes were not rosetted by resting platelets whereas greater than 90% were rosetted by thrombin-stimulated platelets. Monoclonal and polyclonal anti-TSP antibodies markedly inhibited rosetting as did TSP itself. Neither control antibodies nor heparin, fibronectin, fibrinogen, nor the fibronectin adhesion tetrapeptide Arg-Gly-Asp-Ser inhibited rosetting. TSP may thus serve as a molecular bridge linking activated platelets with monocytes at sites of early vascular injury. Such interaction may be of critical importance in the regulation of thrombosis and the initiation of atherosclerosis.
Animals↗
Isolation of the thrombospondin membrane receptor.
Thrombospondin (TSP), a 450-kD multifunctional glycoprotein with a broad tissue distribution, is secreted upon platelet stimulation, binds to the activated platelet surface, and supports platelet aggregation. We have identified and isolated an 88-kd membrane glycoprotein present in platelets, endothelial cells, monocytes, and a variety of human tumor cell lines that is the membrane binding site for TSP. Endogenous platelet TSP binding to thrombin- and ionophore-stimulated human platelets was inhibited in the presence of the monoclonal antibody OKM5. TSP binding to C32 melanoma cells and HT1080 fibrosarcoma cells was specific and also inhibitable with OKM5 Mab. Cell labeling followed by specific immunoprecipitation demonstrated biosynthesis of a single 88-kD glycoprotein. Binding of TSP to the isolated membrane protein was specific and saturable. These studies identify an 88-kD membrane glycoprotein that reacts with the monoclonal antibody, OKM5, and may function as the cellular TSP receptor.
Antibodies, Monoclonal↗
Plasminogen activator inhibitor is associated with the extracellular matrix of cultured bovine smooth muscle cells.
The extracellular matrix secreted by cultured bovine smooth muscle cells (BSMC) contains an endothelial type plasminogen activator (PA) inhibitor. When PA is incubated with the matrix, a high molecular weight complex containing a truncated PA inhibitor is released into the supernatant. The inhibitor also dissociates from the matrix by treatment with glycine, pH 2.7, in its intact, functionally active, 45-kD form, whereas treatment of the matrix with thrombin results in the release of a cleaved, inactive, 41 kD PA inhibitor. Bowes melanoma cells but not smooth muscle cells cultured on BSMC matrices decrease available matrix associated PA inhibitor. PA inhibitor incorporated into the extracellular matrix may serve an important role in the regulation of plasminogen activator mediated matrix degradation.
Animals↗
Binding of tissue plasminogen activator to cultured human endothelial cells.
Tissue plasminogen activator (t-PA) and urokinase (u-PA), the major activators of plasminogen, are synthesized and released from endothelial cells. We previously demonstrated specific and functional binding of plasminogen to cultured human umbilical vein endothelial cells (HUVEC). In the present study we found that t-PA could bind to HUVEC. Binding of t-PA to HUVEC was specific, saturable, plasminogen-independent, and did not require lysine binding sites. The t-PA bound in a rapid and reversible manner, involving binding sites of both high (Kd, 28.7 +/- 10.8 pM; Bmax, 3,700 +/- 300) and low (Kd, 18.1 +/- 3.8 nM; Bmax 815,000 +/- 146,000) affinity. t-PA binding was 70% inhibited by a 100-fold molar excess of u-PA. When t-PA was bound to HUVEC, its apparent catalytic efficiency increased by three- or fourfold as measured by plasminogen activation. HUVEC-bound t-PA was active site-protected from its rapidly acting inhibitor: plasminogen activator inhibitor. These results demonstrate that t-PA specifically binds to HUVEC and that such binding preserves catalytic efficiency with respect to plasminogen activation. Therefore, endothelial cells can modulate hemostatic and thrombotic events at the cell surface by providing specific binding sites for activation of plasminogen.
Binding Sites↗
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.
Animals↗
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.
Cells, Cultured↗
Blockade of clearance of immune complexes by an anti-Fc gamma receptor monoclonal antibody.
Clearance of immune complexes by the mononuclear phagocyte system is important for maintaining normal host defenses against bacterial and viral assault (1), but also contributes to the pathogenesis of a variety of immune- mediated diseases . For example, removal from the circulation of IgG-coated erythrocytes and platelets by the MPS is the sine qua non of immune-mediated cytopenias (2, 3). On the other hand, abnormally decreased removal by the MPS of smaller, soluble immune complexes may play a role in the pathogenesis of immune complex-mediated tissue damage found in such autoimmune diseases as SLE (4). Although the physicochemical nature and the size of immune complexes can influence rates of clearance and sites of deposition (reviewed in 5), interactions between immune complexes and the MPS in vivo are poorly understood. The inability to directly measure binding or internalization of immune complexes by cells in the liver and spleen has made the analysis of the molecular basis of immune complex clearance very difficult . Receptors for the Fc portion of IgG (FcgammaR) and for complement (CR) undoubtedly play a role in the removal of immune complexes, but the relative importance of these receptors is not known.
Animals↗
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.
Enzyme-Linked Immunosorbent Assay↗
Interleukin 1 induces endothelial cell synthesis of plasminogen activator inhibitor.
Human endothelial cells activated with IL-1 express a surface membrane-oriented procoagulant generating system characterized by increased tissue factor synthesis and decreased thrombomodulin activity. We now report that IL-1 also stimulates endothelial cell synthesis of plasminogen activator inhibitor. This array of IL-1-induced activities shifts the balance at the endothelial cell surface to a prothrombotic influence and may reflect an early response of the blood vessel wall to injury.
Cells, Cultured↗
Treatment of refractory immune thrombocytopenic purpura with an anti-Fc gamma-receptor antibody.
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Adult↗
Human brain glial cells synthesize thrombospondin.
Thrombospondin, a 450-kDa multinodular glycoprotein with lectin-type activity, is found in human platelets, endothelial cells, fibroblasts, smooth muscle cells, monocytes, and granular pneumocytes. Thrombospondin interacts with heparin, fibrinogen, fibronectin, collagen, histidine-rich glycoprotein, and plasminogen. Recently, thrombospondin synthesis by smooth muscle cells has been reported to be augmented by platelet-derived growth factor. We present evidence that thrombospondin is present within and synthesized by astrocytic neuroglial cells. Heparin-Sepharose affinity chromatography of material derived from a human brain homogenate yielded a protein that, when reduced, had an apparent size of 180 kDa and comigrated with reduced platelet thrombospondin on NaDodSO4/PAGE. Immunoblot analysis with monospecific anti-thrombospondin confirmed the presence of immunoreactive thrombospondin. Indirect immunofluorescence of cultured human glial cells indicated the presence of thrombospondin. Metabolic labeling of glial cell cultures with [35S]methionine followed by immunoprecipitation with monospecific anti-thrombospondin revealed synthesis of a 180-kDa polypeptide that comigrated with platelet thrombospondin on NaDodSO4/PAGE. Cultured human glial cells were incubated for 48 hr in serum-free medium with purified platelet-derived growth factor at concentrations up to 50 ng/ml. Aliquots taken at intervals were analyzed by a quantitative double-antibody ELISA. The growth factor stimulated the release of thrombospondin into the culture medium by as much as 10-fold over control cultures. The presence of thrombospondin within glial cells of the central nervous system and the augmentation of its synthesis by platelet-derived growth factor suggest that thrombospondin may play an important role in regulating cell-cell and cell-matrix interactions during periods of cell division and growth.
Brain↗
Activation of immobilized plasminogen by tissue activator. Multimolecular complex formation.
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.
Enzyme Activation↗
Platelet thrombospondin forms a trimolecular complex with plasminogen and histidine-rich glycoprotein.
Thrombospondin (TSP), a multifunctional alpha-granule glycoprotein of human platelets binds fibrinogen, fibronectin, heparin, histidine-rich glycoprotein (HRGP), and plasminogen (Plg), and thus, may play an important role in regulating thrombotic influences at vessel surfaces. In this study we have demonstrated that purified human platelet TSP formed a trimolecular complex with human Plg and HRGP. Complex formation was detected by a specific binding enzyme-linked immunosorbent assay (ELISA) which demonstrated simultaneous binding of fluid-phase Plg and HRGP to TSP adsorbed to microtitration wells. While neither ligand inhibited complex formation of the other with TSP, 10 mM epsilon-amino-n-caproic acid selectively blocked incorporation of Plg into the complex, suggesting that TSP contains independent binding sites for Plg and HRGP. Comparable extent of trimolecular complex formation was also detected when TSP monomer was substituted for whole TSP in the ELISA. HRGP covalently cross-linked to Sepharose 4B simultaneously bound both 125I-TSP and 131I-Plg, confirming trimolecular complex formation. Rocket immunoelectrophoresis of mixtures of the purified radiolabeled proteins into anti-Plg containing agarose also confirmed trimolecular complex formation. The TSP-HRGP-Plg complex bound a similar amount of heparin as the TSP-HRGP complex, demonstrating that the HRGP within the trimolecular complex maintained functional capability. Similarly, using a fluorometric plasmin substrate, the trimolecular complex was shown to be an effective substrate for tissue plasminogen activator. Significant amounts of plasmin were generated from the TSP-HRGP-Plg complex (equivalent to that from the TSP-Plg complex), but the rate of plasmin generation from the trimolecular complex was greater than from the bimolecular complex, suggesting an important interaction of HRGP with Plg when both are complexed to TSP. The macromolecular assembly of these three proteins on cellular surfaces, such as the platelet, may serve important regulatory functions, both prothrombotic at sites of active fibrin deposition and proteolytic in nonfibrin-containing microenvironments.
Blood Platelets↗
Platelet membrane topography: colocalization of thrombospondin and fibrinogen with the glycoprotein IIb-IIIa complex.
The distribution of platelet thrombospondin (TSP), fibrinogen, and glycoproteins IIb-IIIa (GPIIb-IIIa) and GPIb were studied in resting and activated human platelets using frozen thin-section immunoelectron microscopy. In resting platelets, TSP and fibrinogen were found within alpha granules and not on the platelet surface. In unstimulated platelets, GPIIb-IIIa and GPIb were distributed diffusely over the platelet membrane as well as within the body of the platelets. Upon thrombin or A23187 stimulation, TSP, fibrinogen, and GPIIb-IIIa colocalized on the platelet membrane and the canalicular system as well as on pseudopodia and between adherent platelets. GPIb distribution was unchanged by platelet activation. The findings support the hypothesis that a macromolecular complex of TSP-fibrinogen and GPIIb-IIIa forms on the activated platelet membrane.
Blood Platelets↗
Complex formation of platelet membrane glycoproteins IIb and IIIa with the fibrinogen D domain.
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.
Blood Platelets↗
Plasma and platelet fibrinogen differ in gamma chain content.
The polypeptide chain composition of fibrinogen and crosslinked fibrin from normal platelets and plasma has been compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fibrinogen was prepared from a lysate of normal human platelets by ethanol precipitation and by antifibrinogen immunoaffinity chromatography. While the A alpha chains of platelet fibrinogen appeared to be somewhat degraded, the electrophoretic mobilities of purified platelet and plasma fibrinogen B beta and gamma chains were the same. Crosslinked fibrin was prepared from platelet and plasma fibrinogen and gamma chain dimers identified by their electrophoretic mobility, incorporation of the lysine analog dansyl cadaverine during crosslinking, and by reaction with antifibrinogen antibody in Western blots. Platelet crosslinked fibrin had a different polypeptide chain composition than that of plasma crosslinked fibrin with absence of the gamma 50-gamma 57.5 dimer in the platelet fibrin. This finding indicates that the gamma 57.5 chain, which constitutes 5% of total normal plasma fibrinogen gamma chains, is either absent or present in markedly reduced amounts in platelet fibrinogen.
Blood Platelets↗
Complex formation of platelet thrombospondin with histidine-rich glycoprotein.
Thrombospondin and histidine-rich glycoprotein are two proteins with diverse biological activities which have been associated with human platelets and other cell systems. Using an enzyme-linked immunosorbent assay, we have demonstrated that purified human platelet thrombospondin formed a complex with purified human plasma histidine-rich glycoprotein. The formation of the thrombospondin-histidine-rich glycoprotein complex was specific, concentration dependent, and saturable. Significant binding was detected when histidine-rich glycoprotein was incubated with thrombospondin immobilized on anti-thrombospondin IgG-coated plates, indicating that the observed complex formation was not due to a thrombospondin interaction with the plastic surface. Sucrose-density-gradient ultracentrifugation of a mixture of thrombospondin and histidine-rich glycoprotein also revealed the formation of fluid-phase complexes, with an estimated stoichiometry of 1 thrombospondin: 3.5 histidine-rich glycoprotein. Fibrinogen, which has been previously shown to bind to absorbed thrombospondin, did not inhibit the formation of the thrombospondin-histidine-rich glycoprotein complex. Histidine-rich glycoprotein complexed with thrombospondin was capable of binding heparin and neutralizing the anticoagulant activity of heparin in plasma. Specific complex formation between thrombospondin and histidine-rich glycoprotein may play a significant role in influencing platelet blood vessel wall interactions as well as modulating the association of various cells with the extracellular matrix.
Binding Sites↗