Binding of vitronectin-thrombin-antithrombin III complex to human endothelial cells is mediated by the heparin binding site of vitronectin.

The interaction of vitronectin-thrombin-antithrombin III (VN.TAT) complex with endothelial cells (EC) was investigated. Binding was specific and time- and concentration-dependent. Kinetics revealed an apparent dissociation constant of 16 nM and 1.7 x 10(5) binding sites/endothelial cell. The binding determinant of the ternary complex was located on the VN moiety. Since the association of VN to TAT adds its specific properties to the VN.TAT complex, the involvement of the heparin binding domain and the cell attachment site of VN was investigated. Neither addition of RGD peptide nor blocking of the vitronectin receptor with a monoclonal antibody interfered with VN.TAT binding to EC. Addition of heparin, a VN-derived peptide comprising two heparin binding consensus sequences or a monoclonal antibody directed against the heparin binding domain on VN, completely inhibited VN.TAT binding to EC. These results indicate that the interaction is mediated through the heparin binding domain of VN. Digestion of heparan sulfate proteoglycans resulted in a decrease of VN.TAT binding to EC, indicating the involvement of heparin-like structures on the EC surface. Our findings point to an unrecognized mechanism by which VN may act as scavenger in order to enhance the clearance of end products of the clotting system via binding of the ternary VN.TAT complex to the luminal surface of EC.     

Orientation of heparin-binding sites in native vitronectin. Analyses of ligand binding to the primary glycosaminoglycan-binding site indicate that putative secondary sites are not functional

A primary heparin-binding site in vitronectin has been localized to a cluster of cationic residues near the C terminus of the protein. More recently, secondary binding sites have been proposed. In order to investigate whether the binding site originally identified on vitronectin functions as an exclusive and independent heparin-binding domain, solution binding methods have been used in combination with NMR and recombinant approaches to evaluate ligand binding to the primary site. Evaluation of the ionic strength dependence of heparin binding to vitronectin according to classical linkage theory indicates that a single ionic bond is prominent. It had been previously shown that chemical modification of vitronectin using an arginine-reactive probe results in a significant reduction in heparin binding (Gibson, A., Baburaj, K., Day, D. E., Verhamme, I. , Shore, J. D., and Peterson, C. B. (1997) J. Biol. Chem. 272, 5112-5121). The label has now been localized to arginine residues within the cyanogen bromide fragment-(341-380) that contains the primary heparin-binding site on vitronectin. One- and two-dimensional NMR on model peptides based on this primary heparin-binding site indicate that an arginine residue participates in the ionic interaction and that other nonionic interactions may be involved in forming a complex with heparin. A recombinant polypeptide corresponding to the C-terminal 129 amino acids of vitronectin exhibits heparin-binding affinity that is comparable to that of full-length vitronectin and is equally effective at neutralizing heparin anticoagulant activity. Results from this broad experimental approach argue that the behavior of the primary site is sufficient to account for the heparin binding activity of vitronectin and support an exposed orientation for the site in the structure of the native protein.     

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.     

Kinetic analysis of the interaction between vitronectin and the urokinase receptor.

Although the urokinase receptor (uPAR) binds to vitronectin (VN) and promotes the adhesion of cells to this matrix protein, the biochemical details of this interaction remain unclear. VN variants were employed in BIAcore experiments to examine the uPAR-VN interaction in detail and to compare it to the interaction of VN with other ligands. Heparin and plasminogen bound to VN fragments containing the heparin-binding domain, indicating that this domain was functionally active in the recombinant peptides. However, no significant binding was detected when uPAR was incubated with this domain, and neither heparin nor plasminogen competed with it for binding to VN. In fact, uPAR only bound to fragments containing the somatomedin B (SMB) domain, and monoclonal antibodies (mAbs) that bind to this domain competed with uPAR for binding to VN. Monoclonal antibody 8E6 also inhibited uPAR binding to VN, and this mAb was shown to recognize sulfated tyrosine residues 56 and 59 in the region adjacent to the SMB domain. Destruction of this site by acid treatment eliminated mAb 8E6 binding but had no effect on uPAR binding. Thus, there appears to be a single binding site for uPAR in VN, and it is located in the SMB domain and is distinct from the epitope recognized by mAb 8E6. Inhibition of uPAR binding to VN by mAb 8E6 probably results from steric hindrance.     

Heparin-binding properties of vitronectin are linked to complex formation as illustrated by in vitro polymerization and binding to the terminal complement complex.

Vitronectin (VN, complement S-protein) is a multifunctional protein which participates in cell adhesion, coagulation, fibrinolysis, and protection against complement lysis. VN is incorporated into several complexes, such as the terminal complement complex and thrombin-antithrombin III, and is bound to plasminogen activator inhibitor 1. The present study showed that purified VN spontaneously forms polymers of approximately 1000 kDa with a Stokes radius of 10 nm. The polymers are to a varying extent stabilized by disulfide bonds, but are quite stable even after reduction and alkylation, indicating the importance of noncovalent bonds. Plasma VN circulates mainly as a 65/75-kDa monomer containing a cryptic heparin-binding site which is exposed upon a conformational change induced by different stimuli, such as coagulation, heating, adsorption to surfaces, or exposure to acids, urea, or other denaturating agents. In the present study, VN was demonstrated to expose its heparin-binding site and its conformationally dependent 8E6 epitope when incorporated into the terminal complement complex. We suggest that exposure of the heparin-binding site and a putative hydrophobic binding site of VN are linked events dependent upon the same conformational change. In vivo, complex formation probably induces the heparin-binding site. Such a link might also explain why purified heparin-binding VN spontaneously forms polymers. The heparin-binding site may be involved in the elimination of multimolecular complexes containing VN.     

Modulation of integrin antagonist signaling by ligand binding of the heparin-binding domain of vitronectin to the alphaVbeta3 integrin

The interaction between the arginine glycine and aspartic acid motif (RGD) of integrin ligands such as vitronectin and the integrin receptor alphaVbeta3 in mediating cell attachment has been well described. Similarly, the ability of disintegrins, small RGD containing peptides, to inhibit cell attachment and other cellular processes has also been studied extensively. Recently, we characterized a second site of interaction between vitronectin and its integrin partner. We determined that amino acids within the heparin-binding domain of vitronectin bind to a cysteine loop (C-loop) region of beta3 and that this interaction is required for the positive effects of alphaVbeta3 ligand occupancy on IGF-I signaling in smooth muscle cells. In this study we examine the signaling events activated following ligand binding of disintegrins to the alphaVbeta3 and the ability of these signals to be regulated by binding of the heparin-binding domain of vitronectin. We demonstrate that disintegrin ligand binding activates a series of events including the sequential activation of the tyrosine kinases c-Src and Syk. This leads to the activation of calpain and the cleavage of the beta3 cytoplasmic tail. Addition of vitronectin or a peptide homologous to the heparin-binding domain inhibited activation of this pathway. Our results suggest that the signaling events that occur following ligand binding to the alphaVbeta3 integrin reflects a balance between the effects mediated through the RGD binding site interaction and the effects mediated by the heparin binding site interaction and that for intact vitronectin the effect of the heparin-binding domain predominates.     

Vitronectin's basic domain is a syndecan ligand which functions in trans to regulate vitronectin turnover

During the process of tissue remodeling, vitronectin (Vn) is deposited in the extracellular matrix where it plays a key role in the regulation of pericellular proteolysis and cell motility. In previous studies we have shown that extracellular levels of vitronectin are controlled by receptor-mediated endocytosis and that this process is dependent upon vitronectin binding to sulfated proteoglycans. We have now identified vitronectin's 12 amino acid “basic domain” which is contained within the larger 40 amino acid heparin binding domain, as a syndecan binding site. Recombinant vitronectins representing wild type vitronectin (rVn) and vitronectin with the basic domain deleted (rVnΔ347-358) were prepared in a baculoviral expression system. The rVn as well as a glutathione S-transferase (GST) fusion protein, consisting of vitronectin's 40 amino acid heparin binding domain (GST-VnHBD), exhibited dose dependent binding to HT-1080 cell surfaces, which was attenuated following deletion of the basic domain. In addition, GST-VnHBD supported both HT-1080 and dermal fibroblast cell adhesion, which was also dependent upon the basic domain. Similarly, ARH-77 cells transfected with syndecans -1, -2, or -4, but not Glypican-1, adhered to GST-VnHBD coated wells, while adhesion of these same cells was lost following deletion of the basic domain. HT-1080 cells were unable to degrade rVnΔ347-358. Degradation of rVnΔ347-358 was completely recovered in the presence of GST-VnHBD but not in the presence of GST-VnHBDΔ347-358. These results indicate that turnover of soluble vitronectin requires ligation of vitronectin's basic domain and that this binding event can work in trans to regulate vitronectin degradation.     

Vitronectin's Basic Domain is a Syndecan Ligand which Functions in transto Regulate Vitronectin Turnover

During the process of tissue remodeling, vitronectin (Vn) is deposited in the extracellular matrix where it plays a key role in the regulation of pericellular proteolysis and cell motility. In previous studies we have shown that extracellular levels of vitronectin are controlled by receptor-mediated endocytosis and that this process is dependent upon vitronectin binding to sulfated proteoglycans. We have now identified vitronectin's 12 amino acid “basic domain” which is contained within the larger 40 amino acid heparin binding domain, as a syndecan binding site. Recombinant vitronectins representing wild type vitronectin (rVn) and vitronectin with the basic domain deleted (rVnΔ347–358) were prepared in a baculoviral expression system. The rVn as well as a glutathione S-transferase (GST) fusion protein, consisting of vitronectin's 40 amino acid heparin binding domain (GST-VnHBD), exhibited dose dependent binding to HT-1080 cell surfaces, which was attenuated following deletion of the basic domain. In addition, GST-VnHBD supported both HT-1080 and dermal fibroblast cell adhesion, which was also dependent upon the basic domain. Similarly, ARH-77 cells transfected with syndecans -1, -2, or -4, but not Glypican-1, adhered to GST-VnHBD coated wells, while adhesion of these same cells was lost following deletion of the basic domain. HT-1080 cells were unable to degrade rVnΔ347–358. Degradation of rVnΔ347–358 was completely recovered in the presence of GST-VnHBD but not in the presence of GST-VnHBDΔ347–358. These results indicate that turnover of soluble vitronectin requires ligation of vitronectin's basic domain and that this binding event can work in transto regulate vitronectin degradation.     

Functional interaction of plasminogen activator inhibitor type 1 (PAI-1) and heparin.

Plasminogen activator inhibitor type 1 (PAI-1), the fast-acting inhibitor of tissue-type plasminogen activator (t-PA) and urokinase (u-PA), is a member of the serpin superfamily of proteins. Both in plasma and in the growth substratum of cultured endothelial cells, PAI-1 is associated with its binding protein vitronectin, resulting in a stabilization of active PAI-1. Recently, it has been demonstrated that the PAI-1-binding site on vitronectin is adjacent to a heparin-binding site (Preissner et al., 1990). Furthermore, it can be deduced that the amino acid residues, proposed to mediate heparin binding in the serpins antithrombin III and heparin cofactor II, are conserved in PAI-1. Consequently, here we have investigated whether PAI-1 also interacts with heparin. At pH 7.4, PAI-1 quantitatively binds to heparin-Sepharose and can be eluted with increasing [NaCl]. Binding of PAI-1 to heparin-Sepharose can be efficiently competed with heparin in solution (IC50, 7 microM). In the presence of heparin, the protease specificity of PAI-1 toward thrombin is substantially increased. This is shown by (i) quenching of thrombin activity of PAI-1 in the presence of heparin and (ii) induction of the formation of SDS-stable complexes between thrombin and PAI-1 by heparin. In a dose response curve, both effects reached a maximum at approximately 1 unit/mL and then diminished again upon further increasing the heparin concentration, strongly suggesting a template mechanism as an explanation for the observed effect. In contrast to vitronectin, heparin does not stabilize the active conformation of PAI-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Heparin-Binding Activity of Secreted Modular Calcium-Binding Protein 1 (SMOC-1) Modulates Its Cell Adhesion Properties

Secreted modular calcium-binding proteins 1 and 2 (SMOC-1 and SMOC-1) are extracellular calcium- binding proteins belonging to the BM-40 family of proteins. In this work we have identified a highly basic region in the extracellular calcium-binding (EC) domain of the SMOC-1 similar to other known glycosaminoglycan-binding motifs. Size-exclusion chromatography shows that full length SMOC-1 as well as its C-terminal EC domain alone bind heparin and heparan sulfate, but not the related chondroitin sulfate or dermatan sulfate glycosaminoglycans. Intrinsic tryptophan fluorescence measurements were used to quantify the binding of heparin to full length SMOC-1 and the EC domain alone. The calculated equilibrium dissociation constants were in the lower micromolar range. The binding site consists of two antiparallel alpha helices and mutagenesis experiments have shown that heparin-binding residues in both helices must be replaced in order to abolish heparin binding. Furthermore, we show that the SMOC-1 EC domain, like the SMOC-2 EC domain, supports the adhesion of epithelial HaCaT cells. Heparin-binding impaired mutants failed to support S1EC-mediated cell adhesion and together with the observation that S1EC in complex with soluble heparin attenuated cell adhesion we conclude that a functional and accessible S1EC heparin-binding site mediates adhesion of epithelial cells to SMOC-1.     

A novel synthetic peptide from the B1 chain of laminin with heparin- binding and cell adhesion-promoting activities

Recent studies using solid-phase-binding assays and electron microscopy suggested the presence of a heparin-binding domain between the inner globule of a lateral short arm and the cross region of laminin. Using the information from the amino acid sequence of the B1 chain of laminin, several peptides were synthesized from areas with a low hydropathy index and a high density of lysines and/or arginines. One of these, peptide F-9 (RYVVLPRPVCFEKGMNYTVR), which is derived from the inner globular domain of the lateral short arm, demonstrated specific binding to heparin. This was tested in direct solid-phase binding assays by coating the peptide either on nitrocellulose or on polystyrene and in indirect competition assays where the peptide was in solution and either laminin or heparin was immobilized on a solid support. The binding of [3H]heparin to peptide F-9 was dramatically reduced when heparin but not other glycosaminoglycans other than heparin (dextran sulfate, dermatan sulfate) were used in competition assays. Modification of the free amino groups of peptide F-9 by acetylation abolished its ability to inhibit the binding of [3H]heparin to laminin on polystyrene surfaces. Peptide F-9 promoted the adhesion of various cell lines (melanoma, fibrosarcoma, glioma, pheochromocytoma) and of aortic endothelial cells. Furthermore, when peptide F-9 was present in solution, it inhibited the adhesion of melanoma cells to laminin-coated substrates. These findings suggest that peptide F-9 defines a novel heparin-binding and cell adhesion-promoting site on laminin.     

Highly sulfated glycosaminoglycans augment the cross-linking of vitronectin by guinea pig liver transglutaminase. Functional studies of the cross-linked vitronectin multimers.

Vitronectin (VN) is an adhesive glycoprotein with roles in the complement, coagulation, and immune systems. Many of the functions of VN are mediated by a glycosaminoglycan binding site, near its carboxyl-terminal end. In this paper, we show that the highly sulfated glycosaminoglycans (GAGs), dextran sulfate, pentosan polysulfate, and fucoidan effectively augment [14C]putrescine incorporation into VN and cross-linking of VN into high molecular multimers by guinea pig liver transglutaminase (TG). Other GAGs including heparin, low molecular weight heparin, dermatan sulfate, keratan sulfate, and the nonsulfated dextrans were ineffective in accelerating these reactions. Dextran sulfate of average molecular mass 500 kDa was more effective than dextran sulfate of average molecular mass 5 kDa, supporting a template mechanism of action of the GAGs, in which VN molecules align on the GAG in a conformation suitable for cross-linking. The VN multimers catalyzed by TG retained functional activity in binding [3H]heparin, platelets, and plasminogen activator inhibitor type-1 (PAI-1). [3H]Heparin bound selectively to the 65-kDa monomeric band of VN and to the multimers derived from this band. PAI-1, however, bound equally to both the 75- and 65-kDa monomeric forms of VN, suggesting that the PAI-1 binding site on VN is distinct from the GAG binding site. The interaction of GAGs with the TG-catalyzed cross-linking of VN may facilitate studies of VN structure-function relationships.     

Structural requirements for the extracellular interaction of plasminogen activator inhibitor 1 with endothelial cell matrix-associated vitronectin.

The interaction of plasminogen activator inhibitor-1 (PAI-1) with its binding protein vitronectin (VN) (Declerck, P. J., De Mol, M., Alessi, M.-C., Baudner, S., Paques, E.-P., Preissner, K. T., Müller-Berghaus, G., and Collen, D. (1988) J. Biol. Chem. 263, 15454-15461) in the extracellular matrix (ECM) of cultured human endothelial cells (HUVEC) was studied. Like PAI-1, VN was found associated with the ECM as evidenced by direct antibody binding, by Western blot analysis as well as by diffuse immunofluorescence staining in permeabilized HUVEC. The specific interaction of VN with confluent monolayers of HUVEC was found to be saturable within 2-4 h at 37 degrees C only with respect to binding to the cells, while no saturable binding to the underlying ECM was observed, indicating that the majority if not all ECM-associated VN was derived from the culture medium. In contrast to PAI-1, ECM-associated VN was resistant toward glycine (pH 2.3), guanidine or urokinase treatment, suggesting that VN was tightly associated with the ECM network. Binding of recombinant PAI-1 (rPAI-1) was largely blocked by anti-VN IgG and only partly by anti-collagen IgG but not by antibodies against other ECM components, indicating that VN constitutes the primary binding protein for ECM-associated PAI-1. This contention was supported by ligand blotting experiments in which rPAI-1 was reacted with nitrocellulose replicas of electrophoretically separated ECM components. Protein band(s) (Mr = 63,000-67,000), comigrating with bovine VN (i.e. medium-derived VN) rather than with human VN were identified as major binding component(s). Moreover, binding studies with purified components revealed that PAI-1 did not show any affinity for collagen (type I/III) alone, whereas VN collagen coating was a much better template for PAI-1 binding than VN alone and that conformationally extended VN provides maximal PAI-1 binding capacity. Binding of rPAI-1 to surface-coated VN was saturable and revealed that (unlike urokinase) heparin or the synthetic peptide Gly-Arg-Gly-Asp-Ser did not inhibit PAI-1 binding. Ligand binding of rPAI-1 to nitrocellulose replicas from sodium dodecyl sulfate-polyacrylamide gels containing electrophoretically separated peptides from VN digests documented the association of PAI-1 with Mr = 10,000-20,000 fragments originating from the heparin-binding domain of VN. These results indicate that the exposure of the glycosaminoglycan-binding domain in VN may allow the concomitant binding of PAI-1 and heparin-like molecules to this region of the VN molecule.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sialylation of vitronectin regulates stress fiber formation and cell spreading of dermal fibroblasts via a heparin-binding site

Vitronectin (VN) plays an important role in tissue regeneration. We previously reported that VN from partial hepatectomized (PH) rats results in a decrease of sialylation of VN and de-sialylation of VN decreases the cell spreading of hepatic stellate cells. In this study, we analyzed the mechanism how sialylation of VN regulates the properties of mouse primary cultured dermal fibroblasts (MDF) and a dermal fibroblast cell line, Swiss 3T3 cells. At first, we confirmed that VN from PH rats or de-sialylated VN also decreased cell spreading in MDF and Swiss 3T3 cells. The de-sialylation suppressed stress fiber formation in Swiss 3T3 cells. Next, we analyzed the effect of the de-sialylation of VN on stress fiber formation in Swiss 3T3 cells. RGD peptide, an inhibitor for a cell binding site of VN, did not affect the cell attachment of Swiss 3T3 cells on untreated VN but significantly decreased it on de-sialylated VN, suggesting that the de-sialylation attenuates the binding activity of an RGD-independent binding site in VN. To analyze a candidate RGD-independent binding site, an inhibition experiment of stress fiber formation for a heparin binding site was performed. The addition of heparin and treatment of cells with heparinase decreased stress fiber formation in Swiss 3T3 cells. Furthermore, de-sialylation increased the binding activity of VN to heparin, as detected by surface plasmon resonance (SPR). These results demonstrate that sialylation of VN glycans regulates stress fiber formation and cell spreading of dermal fibroblast cells via a heparin binding site.     

Solution structure of midkine, a new heparin-binding growth factor.

Midkine (MK) is a 13 kDa heparin-binding polypeptide which enhances neurite outgrowth, neuronal cell survival and plasminogen activator activity. MK is structurally divided into two domains, and most of the biological activities are located on the C-terminal domain. The solution structures of the two domains were determined by NMR. Both domains consist of three antiparallel beta-strands, but the C-terminal domain has a long flexible hairpin loop where a heparin-binding consensus sequence is located. Basic residues on the beta-sheet of the C-terminal domain form another heparin-binding site. Measurement of NMR signals in the presence of a heparin oligosaccharides verified that multiple amino acids in the two sites participated in heparin binding. The MK dimer has been shown to be the active form, giving signals to endothelial cells and probably to neuronal cells. We present a head-to-head dimer model of MK. The model was supported by the results of cross-linking experiments using transglutaminase. The dimer has a fused heparin-binding site at the dimer interface of the C-terminal domain, and the heparin-binding sites on MK fit the sulfate group clusters on heparin. These features are consistent with the proposed stronger heparin-binding activity and biological activity of the dimer.     

Protein kinase CK2: evidence for a protein kinase CK2beta subunit fraction, devoid of the catalytic CK2alpha subunit, in mouse brain and testicles

Apolipoprotein E (apoE), a key lipid transport protein, displays a heparin-binding property that is critical in several apoE functions. The kinetics of the interaction between apoE isoforms and glycosaminoglycans (GAGs) were studied using surface plasmon resonance. The dissociation constant of equilibrium K(D) for apoE3-heparin interaction was estimated to be 12 nM for apoE3 and three common apoE isoforms revealed similar affinities for heparin. ApoE binds to GAGs in the following order: heparin>heparan sulfate>dermatan sulfate>chondroitin sulfate. The affinity parameter of the binding of low molecular weight heparins to apoE is correlated with the chain length. The effective number Z of electrostatic interactions between plasma apoE3 and heparin was assessed to be three. Metal chelators were able to diminish apoE-binding to heparin, suggesting some stabilizing effect of metal ions while reconstitution with lipids did not affect binding affinities for heparin, suggesting that the N-terminal heparin-binding site is responsible for apoE-containing lipoprotein interactions with heparin.     

Modulation of endothelial cell proliferation,adhesion, and motility by recombinant heparin-binding domain and synthetic peptides from the type I repeats of thrombospondin

Thrombospondin is an inhibitor of angiogenesis that modulates endothelial cell adhesion, proliferation, and motility. Synthetic peptides from the second type I repeat of human thrombospondin containing the consensus sequence -Trp-Ser-Pro-Trp- and a recombinant heparin binding fragment from the amino-terminus of thrombospondin mimic several of the activities of the intact protein. The peptides and heparin-binding domain promote endothelial cell adhesion, inhibit endothelial cell chemotaxis to basic fibroblast growth factor (bFGF), and inhibit mitogenesis and proliferation of aortic and corneal endothelial cells. The peptides also inhibit heparin-dependent binding of bFGF to corneal endothelial cells. The antiproliferative activities of the peptides correlate with their ability to bind to heparin and to inhibit bFGF binding to heparin. Peptides containing amino acid substitutions that eliminate heparin-binding do not alter chemotaxis or proliferation of endothelial cells. Inhibition of proliferation by the peptide is time-dependet and reversible. Thus, the antiproliferative activities of the thrombospondin peptides and recombinant heparin-binding domain result at least in part from competition with heparin-dependent growth factors for binding to endothelial cell proteoglycans. These results suggest that both the Trp-Ser-Xaa-Trp sequences in the type I repeats and the amino-terminal domain play roles in the antiproliferative activity of thrombospondin.     

Role of exosites 1 and 2 in thrombin reaction with plasminogen activator inhibitor-1 in the absence and presence of cofactors.

The cofactors heparin, vitronectin (VN), and thrombomodulin (TM) modulate the reactivity of alpha-thrombin with plasminogen activator inhibitor (PAI-1). While heparin and VN accelerate the reaction by approximately 2 orders of magnitude, TM protects alpha-thrombin from rapid inactivation by PAI-1 in the presence of VN. To understand how these cofactors function, we studied the kinetics of PAI-1 inactivation of alpha-thrombin, the exosite 1 variant gamma-thrombin, the exosite 2 mutant R93,97,101A thrombin, and recombinant meizothrombin in both the absence and presence of these cofactors. Heparin and VN accelerated the second-order association rate constant [k(2) = (7.9 +/- 0.5) x 10(2) M(-)(1) s(-)(1)] of alpha-thrombin with PAI-1 approximately 200- and approximately 240-fold, respectively. The k(2) value for gamma-thrombin [(7.9 +/- 0.7) x 10(1) M(-)(1) s(-)(1)] was impaired 10-fold, but was enhanced by heparin and VN approximately 280- and approximately 75-fold, respectively. Similar to inactivation of gamma-thrombin, PAI-1 inactivation of alpha-thrombin in complex with the epidermal growth factor-like domains 4-6 of TM (TM4-6) was impaired approximately 10-fold. The exosite 2 mutant R93,97,101A thrombin, which was previously shown not to bind heparin, and meizothrombin, in which exosite 2 is masked, reacted with PAI-1 at similar rates in both the absence and presence of heparin [k(2) = (1.3-1.5) x 10(3) M(-)(1) s(-)(1) for R93,97,101A thrombin and k(2) = (3.6-5.1) x 10(2) M(-)(1) s(-)(1) for meizothrombin]. Unlike heparin, however, VN enhanced the k(2) of R93,97,101A thrombin and meizothrombin inactivation approximately 80- and approximately 30-fold, respectively. Continuous kinetic analysis as well as competition kinetic studies in the presence of S195A thrombin suggested that the accelerating effect of VN or heparin occurs primarily by lowering the dissociation constant (K(d)) for formation of a noncovalent, Michaelis-type complex. Analysis of these results suggest that (1) heparin binds to exosite 2 of alpha-thrombin to accelerate the reaction by a template mechanism, (2) VN accelerates PAI-1 inactivation of alpha-thrombin by lowering the K(d) for initial complex formation by an unknown mechanism that does not require binding to either exosite 1 or exosite 2 of alpha-thrombin, (3) alpha-thrombin may have a binding site for PAI-1 within or near exosite 1, and (4) TM occupancy of exosite 1 partially accounts for the protection of thrombin from rapid inactivation by PAI-1 in the presence of vitronectin.     

Antimicrobial activities of heparin-binding peptides.

Antimicrobial peptides are effector molecules of the innate immune system. We recently showed that the human antimicrobial peptides alpha-defensin and LL-37 bind to glycosaminoglycans (heparin and dermatan sulphate). Here we demonstrate the obverse, i.e. structural motifs associated with heparin affinity (cationicity, amphipaticity, and consensus regions) may confer antimicrobial properties to a given peptide. Thus, heparin-binding peptides derived from laminin isoforms, von Willebrand factor, vitronectin, protein C inhibitor, and fibronectin, exerted antimicrobial activities against Gram-positive and Gram-negative bacteria. Similar results were obtained using heparin-binding peptides derived from complement factor C3 as well as consensus sequences for heparin-binding (Cardin and Weintraub motifs). These sequence motifs, and additional peptides, also killed the fungus Candida albicans. These data will have implications for the search for novel antimicrobial peptides and utilization of heparin-protein interactions should be helpful in the identification and purification of novel antimicrobial peptides from complex biological mixtures. Finally, consensus regions may serve as templates for de novo synthesis of novel antimicrobial molecules.     

Full-length and truncated forms of vitronectin provide insight into effects of proteolytic processing on function

A genetic polymorphism in the vitronectin allele directs the production of two distinct forms of the 459 amino acid glycoprotein. A methionine present at position 381 favors production of the single-chain form of vitronectin, while threonine at this position increases the susceptibility of vitronectin to cleavage just beyond its heparin-binding domain at residue 379. This reaction gives rise to a disulfide-bonded, two-chain form of vitronectin. In order to investigate the functional significance of the vitronectin polymorphism, the baculovirus system has been used to express recombinant full-length vitronectin and a truncated form of the molecule that represents the 62-kDa fragment of two-chain vitronectin. Both forms of vitronectin bind and neutralize heparin anticoagulant activity. The proteins also bind PAI-1 and stabilize its active conformation. These experiments suggest that the C-terminal 80 amino acids do not confer a functional difference in the two allelic variants. Immunoassays and gel filtration experiments indicate that both full-length and truncated recombinant forms of vitronectin are multimeric. Together with other reports from this laboratory, these results provide information regarding the primary binding sites for two vitronectin ligands and further define regions that may be involved in multimerization of the protein.