Vascular endothelial growth factor production by fibroblasts in response to factor VIIa binding to tissue factor involves thrombin and factor Xa.

Tissue factor (TF) assembled with activated factor VII (FVIIa) initiates the coagulation cascade. We recently showed that TF was essential for FVIIa-induced vascular endothelial growth factor (VEGF) production by human fibroblasts. We investigated whether this production resulted from TF activation by its binding to FVIIa or from the production of clotting factors activated downstream. Incubation of fibroblasts with a plasma-derived FVIIa concentrate induced the generation of activated factor X (FXa) and thrombin and the secretion of VEGF, which was inhibited by hirudin and FXa inhibitors. By contrast, the addition of recombinant FVIIa to fibroblasts did not induce VEGF secretion unless factor X was present. Moreover, thrombin and FXa induced VEGF secretion and VEGF mRNA accumulation, which were blocked by hirudin and FXa inhibitors, respectively. The effect of thrombin was mediated by its specific receptor, protease-activated receptor-1; in contrast, the effect of FXa did not appear to involve effector cell protease receptor-1, because it was not affected by an anti-effector cell protease receptor-1 antibody. An increase in intracellular calcium with the calcium ionophore A23187 or intracellular calcium chelation by BAPTA-AM had no effect on either basal or FXa-induced VEGF secretion, suggesting that the calcium signaling pathway was not sufficient to induce VEGF secretion. Finally, FVIIa, by itself, had no effect on mitogen-activated protein (MAP) kinase activation, contrary to thrombin and FXa, which activate the p44/p42 MAP kinase pathway, as shown by the blocking effect of PD 98059 and by Western blotting of activated MAP kinases. These findings indicate that FVIIa protease induction of VEGF expression is mediated by thrombin and FXa generated in response to FVIIa binding to TF-expressing fibroblasts; they also exclude a direct signaling involving MAP kinase activation via the intracellular domain of TF when expressed by these cells.

Tissue factor-dependent vascular endothelial growth factor production by human fibroblasts in response to activated factor VII.

The transmembrane protein tissue factor (TF) is the cell surface receptor for coagulation factor VII (FVII) and activated factor VII (FVIIa). Recently, TF has been identified as a regulator of angiogenesis, tumor growth, and metastasis. This study was designed to link the binding of FVII(a) to its receptor, TF, with the subsequent triggering of angiogenesis through vascular endothelial growth factor (VEGF) production by human lung fibroblasts. We report that incubation of fibroblasts, which express constitutive surface TF, with FVII(a) induces VEGF synthesis. FVII(a)-induced VEGF secretion, assessed by a specific enzyme-linked immunosorbent assay, was time- and concentration-dependent. VEGF secretion was maximal after 24 hours of incubation of the cells with 100 nmol/L FVII(a) and represented a threefold induction of the basal VEGF level. Reverse transcriptase-polymerase chain reaction analysis of VEGF detected three mRNA species of 180, 312, and 384 bp corresponding, respectively, to VEGF121, VEGF165, and VEGF189. A 2.5- to 3.5-fold increase was observed for the 180- and 312-bp transcripts at 12 and 24 hours, respectively. FVII(a)-dependent VEGF production was inhibited by a pool of antibodies against TF, pointing to the involvement of this receptor. On specific active-site inhibition with dansyl-glutamyl-glycinyl-arginyl chloromethyl ketone, FVIIa lost 70% of its capacity to elicit VEGF production. Consistent with this, the native form (zymogen) of FVII only had a 1.8-fold stimulating effect. Protein tyrosine kinase and protein kinase C are involved in signal transduction leading to VEGF production, as shown by the inhibitory effects of genistein and GF 109203X. The results of this study indicate that TF is essential for VIIa-induced VEGF production by human fibroblasts and that its role is mainly linked to the proteolytic activity of the TF-VIIa complex.

Properties of a new fibrin glue stable in liquid state.

A pasteurized preparation of fibrin glue composed of two separate stable, liquid components: highly purified human thrombin and fibrinogen concentrate is described. The components are mixed extemporaneously during application. Thrombin was prepared using a prothrombin complex concentrate as starting material which was activated by calcification and then heated in solution during 10 hours at 60 degrees C in the presence of stabilizers. The isolation of thrombin was carried out using a column of benzamidine-Sepharose 6B. The eluate contained thrombin with a high degree of purity (more than 95% assessed by SDS-PAGE) with a specific activity > 2,500 IU/mg protein. The purified liquid thrombin preparation remained stable for at least 6 months. The fibrinogen concentrate was prepared from cryoprecipitate after removal of factor VIII and then virally inactivated by pasteurization in the presence of glucose and sorbitol. After purification the concentrate containing a high level of fibrinogen was formulated with urea 0.5 M or arginine 5% before conditioning. Both components of the fibrin glue kept its biological properties for more than 6 months at +4 degrees C.

Aprotinin is a competitive inhibitor of the factor VIIa-tissue factor complex.

A highly purified preparation of human plasma factor VIIa was submitted to chromogenic assays with S-2288 factors IXa, Xa, activated protein C and thrombin being absent. Factor VIIa alone or in the presence of calcium, kept its activity even in the presence of high concentrations of aprotinin, inhibition appeared only in the presence of a factor VIIa-tissue factor complex. A two-stage amidolytic assay using activation of purified factor X and hydrolysis of S-2765 chromogenic substrate by the generated Xa was used to show a competitive inhibition with a Ki value of 30 microM. Aprotinin had no effect on factor Xa amidolytic activity per se. The factor VIIa-tissue factor complex could be adsorbed to immobilized aprotinin and removed by a chaotropic ion like KSCN 3 M. The assays with the DFP inactivated VIIa-tissue factor complex proved that the interaction involved the active site of factor VIIa. The inhibition of the VIIa-tissue factor complex was demonstrated in a clotting assay using aprotinin enriched normal or factor VIII deficient plasma.

Inhibition of activated protein C by aprotinin and the use of the insolubilized inhibitor for its purification.

The study of the interaction between activated protein C (APC) and non-plasmatic inhibitors allowed us to demonstrate that aprotinin is a potent competitive inhibitor of APC with a Ki of 1.35 mumol/L. It was possible to adsorb immunopurified protein C (PC) activated by venom activator to insolubilized aprotinin and to recover the active enzyme after elution by HCl 0.1 N or by a chaotropic ion, for example KSCN 3 mol/L. The interaction involved the active-site of the enzyme since PC and DIP-APC did not bind to the matrix. Thus, APC could be purified, after activation, in a one-stage procedure out of a mixture of protein such as a prothrombin complex concentrate.

A human factor VIIa concentrate and its effects in the hemophilic A dog.

Properties of a new concentrate of FVII/FVIIa, obtained by adsorption onto an inorganic adsorbent followed by a chromatographic step onto Q-Sepharose using a by-product of routine fractionation as starting material, are described. This fraction contains only small amounts of the other components of the prothrombin complex but it is enriched in Proteins C and S. This preparation is essentially free of FVIIICag. It has been submitted to animal experiments including those carried out on normal and hemophilic A dogs. A normalization of the buccal bleeding time was seen after injection of a minimal dose of 4 uFVIIa/kg. No adverse reaction was observed at any dose employed. This concentrate might thus be indicated for the treatment of Haemophilia A patients with inhibitors. Its viral inactivation has been achieved.

Functional Kunitz inhibitor binding domain in plasmin-derived light chain as shown by affinity chromatography.

The light chain of plasmin, prepared by selective reduction of the interchain disulfide bridges, can be separated from the heavy chain by affinity adsorption onto Kunitz inhibitor/Sepharose. This adsorption involves the active center of plasmin because it does not occur if the light chain is derived from a plasmin previously blocked by the active site titrant p-nitrophenyl-p'-guanidinobenzoate. It can be deduced that the conformation of the inhibitor binding domain of plasmin is preserved in the free light chain.