Biomedical subjects

Triantafyllos Chavakis

Publications and source records attributed to Triantafyllos Chavakis.

40 records · Page 3Linked to original sources

PubMed2002

A novel antithrombotic role for high molecular weight kininogen as inhibitor of plasminogen activator inhibitor-1 function.

The adhesive glycoprotein vitronectin (VN) forms a function-stabilizing complex with plasminogen activator inhibitor-1 (PAI-1), the major fibrinolysis inhibitor in both plasma and vessel wall connective tissue. VN also interacts with two-chain high molecular weight kininogen (HKa), particularly its His-Gly-Lys-rich domain 5, and both HKa and PAI-1 are antiadhesive factors that have been shown to compete for binding to VN. In this study the influence of HKa and domain 5 on the antifibrinolytic function of PAI-1 was investigated. In a purified system, HKa and particularly domain 5 inhibited the binding of PAI-1 to VN and promoted PAI-1 displacement from both isolated VN as well as subendothelial extracellular matrix-associated VN. The sequence Gly(486)-Lys(502) of HKa domain 5 was identified as responsible for this inhibition. Although having no direct effect on PAI-1 activity itself, HKa domain 5 or the peptide Gly(486)-Lys(502) markedly destabilized the VN.PAI-1 complex interaction, resulting in a significant reduction of PAI-1 inhibitory function on plasminogen activators, resembling the effect of VN antibodies that prevent stabilization of PAI-1. Furthermore, high affinity fibrin binding of PAI-1 in the presence of VN as well as the VN-dependent fibrin clot stabilization by the inhibitor were abrogated in the presence of the kininogen forms mentioned. Taken together, our data indicate that the peptide Gly(486)-Lys(502) derived from domain 5 of HKa serves to interfere with PAI-1 function. Based on these observations potential low molecular weight PAI-1 inhibitors could be designed for the use in therapeutic interventions against thromboembolic complications.

Binding, Competitive↗

PubMed2002

Inhibition of platelet adhesion and aggregation by a defined region (Gly-486-Lys-502) of high molecular weight kininogen.

Proteolytic cleavage of single chain high molecular weight kininogen (HK) by kallikrein releases the short-lived vasodilator bradykinin and leaves behind two-chain high molecular weight kininogen (HKa). HKa and particularly its His-Gly-Lys-rich domain 5 have been previously reported to exert anti-adhesive properties by binding to the extracellular matrix protein vitronectin (VN). In this study the ability of HKa and domain 5 to interfere with platelet adhesion and aggregation was investigated. In a purified system HKa and particularly domain 5 but not HK inhibited the binding of VN to the alpha(IIb)beta(3) integrin, whereas the binding of fibrinogen to this integrin was not affected. The region Gly-486-Lys-502 from the carboxyl terminus of the domain 5 was identified as responsible for inhibition of the VN-alpha(IIb)beta(3)-integrin interaction, as this portion was also found to mediate kininogen binding to VN. Through these interactions, HKa, the isolated domain 5, and the peptide Gly-486-Lys-502 abrogated the alpha(IIb)beta(3)-integrin-dependent adhesion of human platelets to VN but not to fibrinogen. The codistribution of VN and HKa at sites of ex vivo platelet aggregation was demonstrated by transmission immune electron microscopy, indicating that the described interaction is likely to take place in vivo. Moreover, domain 5 and the peptide Gly-486-Lys-502 dose-dependently blocked platelet aggregation, resembling the inhibitory effect of monoclonal antibody 13H1 against multimeric VN. Finally, treatment of mice with isolated domain 5 resulted in a significantly prolonged tail bleeding time. Taken together, our data emphasize the inhibitory role of HK domain 5 on platelet adhesion and aggregation; new anti-thrombotic compounds may become available on the basis of peptide Gly-486-Lys-502 of HK domain 5.

Amino Acid Sequence↗

PubMed2002

Molecular interactions and functional interference between vitronectin and transforming growth factor-beta.

Different extracellular matrix proteins have been described as binding proteins for growth factors, influencing their storage or presentation towards cellular receptors. The multifunctional adhesive glycoprotein vitronectin (VN), which is found in the circulation and widely distributed throughout different tissues, has been implicated in the regulation of vascular cell functions, and these activities could be related to interactions with various growth factors. In vitro, soluble VN interfered with transforming growth factor-beta (TGF-beta) binding to isolated extracellular matrix and was found to associate with TGF-beta1 and TGF-beta2 as well as with other growth factors such as vascular endothelial growth factor, epidermal growth factor, or basic fibroblast growth factor in a saturable manner. In particular, binding of TGF-beta was maximal for the heparin-binding multimeric isoform of VN, whereas VN in a ternary complex with thrombin and antithrombin or plasma VN exhibited weaker binding. Plasminogen activator inhibitor-1 (PAI-1) or heparin, but not desulfated glycosaminoglycans, interfered with binding of VN to TGF-beta, and soluble PAI-1 was able to dissociate VN-bound TGF-beta. Upon limited plasmin proteolysis of VN, only the fragments comprising the intact aminoterminal portion of VN bound to TGF-beta as did a synthetic peptide (amino acids 43 to 62), indicating that TGF-beta and PAI-1 share common binding site(s) on VN. Although VN did not influence TGF-beta bioactivity for mink lung epithelial cells, TGF-beta dose dependently inhibited both urokinase-receptor as well as alpha(v)-integrin-dependent adhesion to VN. This activity of TGF-beta was reminiscent of the antiadhesive function of PAI-1. In atherosclerotic tissue sections, staining patterns of VN and TGF-beta indicated their colocalization. These findings describe VN as a new binding protein for TGF-beta, whereby specific functions of both factors become modulated by this interaction.

Animals↗

PubMedDate not supplied

Variability in the expression of urokinase receptor (CD87) mutants on cells: relevance to cell adhesion.

The urokinase-type plasminogen activator receptor (uPAR, CD87) is a glycosylphosphatidylinositol (GPI)-anchored protein, containing three homologous Ly-6 domains, that mediates integrin-independent cell adhesion by directly binding to extracellular matrix protein vitronectin (VN). To elucidate the structural requirements for the uPAR-dependent cell adhesion on VN, several glycolipid-anchored variants of uPAR were expressed in BAF3 cells, (mouse pre B-lymphocytes) followed by functional analysis. The individual domains of uPAR were expressed at very low levels, the two domain mutants were expressed to a higher level and the wild type uPAR was expressed highly. Point mutations in domain 2 of uPAR have been shown to diminish cellular binding of the ligand urokinase and we observed a lack of VN binding to this mutant. Flow cytometry with a number of monoclonal antibodies indicated that the domain-specific antigenic determinants in these mutants were well preserved. Only the cells expressing the intact uPAR with all three domains adhered strongly to a VN substrate, whereas none of the other transfected cells showed significant cell adhesion. Hence, any alterations in the domain structure of uPAR reduce its expression and only the intact receptor can sustain the direct cell adhesion on VN-rich matrices found at sites of inflammation and injury.

Amino Acid Substitution↗