Integrin alphavbeta3-mediated endocytosis of immobilized fibrinogen by A549 lung alveolar epithelial cells.

Fibrinogen (FBG), together with its polymerized form fibrin, modulates cellular responses during wound repair and tissue remodeling. Thus, we sought to determine whether A549 lung epithelial type II-like cells would endocytose insoluble, surface-bound FBG as a potential mechanism of alveolar matrix remodeling. Surface-bound FBG was endocytosed into either lysosomes or late endosomes by A549 cells through arg-gly-asp-dependent binding to alphavbeta3 but not alpha5beta1 integrin receptors. Soluble FBG added to confluent monolayers of A549 cells was not endocytosed. Unlike the uptake of the extracellular matrix glycoproteins vitronectin and thrombospondin by other cell types, endocytosis of FBG by A549 cells was neither inhibited by heparin nor dependent on binding to cell-surface heparan sulfate proteoglycans. FBG did not colocalize with endocytosed transferrin, whereas dextran showed partial colocalization with FBG in endocytic vesicles, suggesting nonclathrin-mediated endocytosis. Inhibition of actin filament polymerization blocked endocytosis of both dextran and FBG but not transferrin, providing further support that FBG is endocytosed via a nonclathrin pathway. Disruption of actin polymerization inhibited integrin-mediated cell spreading, which contributed to an overall reduction in FBG clearance that was most likely due to reduced cell migration and associated pericellular proteolysis. Trasylol inhibition of extracellular plasmin activity did not inhibit endocytosis of FBG. The endocytosed FBG was degraded to trichloroacetic acid-soluble fragments that showed an electrophoretic pattern distinctly different from plasmin-degraded FBG. Together, these results suggest that endocytosis of matrix-associated FBG by alveolar epithelial cells may be involved in the processes of alveolar tissue repair and matrix remodeling.

Binding of tissue-plasminogen activator to fibrin: effect of ultrasound.

Ultrasound reversibly alters the structure of polymerized fibrin, an effect that could influence tissue-plasminogen activator (t-PA) binding. We have, therefore, characterized the effects of ultrasound on binding of t-PA to fibrin using a novel system in which radiolabeled, active-site blocked, single chain tissue-plasminogen activator flowed through a fibrin gel at constant rate, and specific binding was determined by monitoring incorporation of radiolabel. Results using polymerized fibrin were compared with those using a surface of fibrin immobilized on Sepharose beads in a similar system. Interaction of t-PA with surface-immobilized fibrin involved two classes of binding sites (Kd = 31 nmol/L and 244 nmol/L) and a maximum binding ratio of 3.8 mol t-PA/mol fibrin. Ultrasound increased Kd for the high affinity site to 46 nmol/L (P < .0001), but it had no significant effects on the Kd 244 nmol/L site nor on Bmax. Tissue-plasminogen activator binding to noncrosslinked fibrin involved two sites with Kds of 267 nmol/L and 952 nmol/L, while a single Kd 405 nmol/L site was identified for crosslinked fibrin. Ultrasound had no significant effect on the binding affinity for noncrosslinked fibrin, but Bmax was increased in the presence of ultrasound, from 31 mumol/L to 43 mumol/L (P < .0001). Ultrasound decreased the Kd for crosslinked fibrin to 343 nmol/L (P = .026) and also increased Bmax from 22 mumol/L to 25 mumol/L (P = .015). Ultrasound also affected the kinetics of t-PA binding to fibrin, significantly accelerating the rate of dissociation by 77% +/- 5% for noncrosslinked fibrin and by 69% +/- 3% for crosslinked fibrin (P < .001 for each). These results indicate that ultrasound exposure accelerates t-PA binding, alters binding affinity, and increases maximum binding to polymerized fibrin, effects that may result from ultrasound-induced changes in fibrin structure.

Binding of basic fibroblast growth factor to fibrinogen and fibrin.

Fibrin is formed at sites of tissue injury and provides the temporary matrix needed to support the initial endothelial cell responses needed for vessel repair. Basic fibroblast growth factor (bFGF) also acts at sites of injury and stimulates similar vascular cell responses. We have, therefore, investigated whether there are specific interactions between bFGF and fibrinogen and fibrin that could play a role in coordinating these actions. Binding studies were performed using bFGF immobilized on Sepharose beads and soluble 125I-labeled fibrinogen and also using Sepharose-immobilized fibrinogen and soluble 125I-bFGF. Both systems demonstrated specific and saturable binding. Scatchard analysis indicated two classes of binding sites for each with Kd values of 1.3 and 260 nM using immobilized bFGF; and Kd values of 0.9 and 70 nM using immobilized fibrinogen. After conversion of Sepharose-immobilized fibrinogen to fibrin by treatment with thrombin, bFGF also demonstrated specific and saturable binding with two classes of binding sites having Kd values of 0.13 and 83 nM. Fibrin binding was also investigated by clotting a solution of bFGF and fibrinogen, and two classes of binding sites were demonstrated using this system with Kd values of 0.8 and 261 nM. The maximum molar binding ratios of bFGF to fibrinogen were between 2.0 and 4.0 with the four binding systems. We conclude that bFGF binds specifically and saturably to fibrinogen and fibrin with high affinity, and this may have implications regarding the localization of its effect at sites of tissue injury.

Heparin-binding domain of fibrin mediates its binding to endothelial cells.

Spreading of human umbilical vein endothelial cells (ECs) on fibrin requires thrombin cleavage of fibrinopeptide B (FPB) and subsequent exposure of the new beta 15-42 N-terminus. To further understand the interactions between ECs and fibrin beta 15-42 sequences, binding of fibrin(ogen) to EC monolayers was measured with polyclonal anti-fibrinogen (FBG) in parallel with monoclonal anti-FBG (18C6, beta 1-21; J88B, gamma 63-78) and anti-fibrin (T2G1, beta 15-21) antibodies in an indirect enzyme-linked immunosorbent assay. To accomplish this, large, soluble fragments of fibrin were prepared by cyanogen bromide (CNBr) cleavage (fibrin-CNBr); CNBr-cleaved FBG (FBG-CNBr) served as the control ligand. N-terminal fibrin-CNBr bound to EC monolayers and cells in suspension in a dose-dependent and saturable manner. By contrast, FBG-CNBr bound only 50% as well to EC monolayers, with no significant binding of intact FBG, C-terminal FBG plasmic fragment D, or N-terminal plasmic fragment E, which lacks beta 1-53. ECs bound the peptide beta 15-42-bovine serum albumin (BSA) conjugate but neither a scrambled beta 15-42 peptide conjugate nor conjugates of beta 24-42, beta 18-27, or beta 18-31. Binding of fibrin-CNBr was inhibited 54% by the beta 15-42-BSA conjugate and 17% by the B beta 1-42-BSA conjugate but not by free beta 15-42 peptide or RGDS-cell binding peptide. Binding of fibrin-CNBr was inhibited > 95% by heparin in a concentration-dependent manner; the same concentrations of heparin inhibited binding of beta 15-42-BSA by > 75% but not the dose-dependent binding of fibronection to ECs. These data suggest that in their native conformation, FBG B beta 15-42 sequences are unavailable for binding to ECs and that thrombin-induced exposure of beta 15-42 is required for binding by a heparin-dependent, RGD-independent mechanism at the new N-terminus of fibrin.

Calcium modulates plasmin cleavage of the fibrinogen D fragment gamma chain N-terminus: mapping of monoclonal antibody J88B to a plasmin sensitive domain of the gamma chain.

Plasmin sensitive sites are found on the A alpha, B beta and gamma chains of fibrinogen at regions joining the two C-terminal D fragments with the central E fragment. We have developed a monoclonal antibody (MoAb) reactive with this plasmin sensitive region on the human fibrinogen gamma chain and mapped its epitope. MoAb J88B reacts with gamma chains of both native as well as with reduced and denatured fibrinogen and fibrin, the CNBr fragment of the fibrinogen central domain, plasmin cleaved fragments D, gamma-gamma dimers, but not with plasmic fragments E. These data indicate that J88B maps to the plasmin sensitive domain localized to gamma 63-78. MoAb J88B failed to react with synthetic peptide gamma 70-78, which suggests that the epitope includes the newly exposed N-terminal residues gamma 63-70 of the early plasmic fragment D1A. As calcium has a marked influence on plasmin cleavage of C-terminal sites on the gamma chain, the effects of calcium on modulating plasmin cleavage of D1A to D1 were assessed in the absence or presence of J88B. The results indicated that calcium delays and J88B (+/- calcium) protects the gamma chain from plasmin cleavage at the N-terminus of D1A, suggesting that this enzymatically labile site is calcium-sensitive. Thus, MoAb J88B should prove useful in studies examining the structure of plasmin cleaved fibrinogen and fibrin.

Thrombin cleavage enhances exposure of a heparin binding domain in the N-terminus of the fibrin beta chain.

Thrombin (IIa)-cleavage of fibrinogen (FBG) to form polymerized fibrin promotes endothelial cell spreading, proliferation, and von Willebrand factor release, requiring the exposure of the beta 15-42 domain. Studies reported here indicate that IIa-cleavage of fibrinopeptide B enhances exposure of a heparin binding domain at the beta 15-42 neo-N-terminus of fibrin. Crossed immunoelectrophoresis showed heparin-induced mobility shifts indicative of complexing with FBG and with N-terminal CNBr fragments of FBG (NDSK) and of fibrin (IIa-NDSK), but not evidence of heparin complexing with FBG lacking B beta 1-42 or with FBG fragments D and E was seen. Elution from heparin-agarose with a linear gradient of NaCl showed that bound portions of both intact FBG and D fragments eluted below physiologic salt concentrations, whereas E3 fragments lacking B beta 1-53 did not bind. NDSK bound with higher affinity than did intact FBG, whereas binding of IIa-NDSK was maximal in this system. Binding of fibrin(ogen) to heparin agarose was saturable as well as inhibitable in a dose-dependent manner with both FBG and heparin. Scatchard analysis indicated a single class of binding site, with dissociation constants (kd) of 0.3 mumol/L for IIa-NDSK, 0.8 mumol/L for NDSK, and 18 mumol/L for FBG. Immobilized fibrin had twofold more heparin binding sites than did immobilized FBG and required a 5.5-fold higher concentration of heparin to inhibit by 50% the binding of labeled heparin. Together, the results indicate that IIa-cleavage results in enhanced exposure of two heparin binding domains (HBDs) with approximately threefold higher affinity in fibrin than in FBG. Synthetic peptide beta 15-42 showed highest binding to heparin-agarose followed by B beta 1-42, whereas peptides beta 18-31, beta 18-27, and beta 24-42 did not bind. Thus, the primary structure of beta 15-42 is required for specificity of heparin binding. Basic residues within the beta 15-32 region segregate primarily to one side of an alpha-helix in a helical wheel diagram, as is typical for authentic HBDs. Desulfated heparin and heparan sulfate bound more fibrin(ogen) than did other proteoglycans; however, heparin bound sixfold more Ila-NDSK than NDSK. These results confirm that fibrin binds to heparin with higher affinity than does FBG and that fibrin binding is not solely dependent on charge interactions of beta 15-42 with the negatively charged glycosaminoglycan.

Immunochemical characterisation of a murine monoclonal anti-idiotypic antibody.

Y7, a murine monoclonal IgG1 kappa antibody against a human monoclonal IgM lambda DJ molecule, was affinity purified on an IgM lambda immunoaffinity column. As detected by enzyme-linked immunosorbent assay (ELISA) the isolated Y7 monoclonal antibody was shown to be not cross-reactive with human IgG, human secretory IgA, mu chain, lambda + kappa chains and another human monoclonal IgM lambda BR. Binding to the polyclonal human IgM standard in the same assay was about 30 percent. The epitope specificity of affinity purified and biotinylated Y7 MoAb was localized only in the nonreduced pepsin Fab fragments of IgM lambda DJ immunogen. As the immunogen was determined to be a specific antibody to phosphorylcholine, the specificity of Y7 MoAb was further ascertained in its capacity to induce 95% inhibition of immunogen binding for phosphorylcholine.