Platelet adhesiveness and aggregation in combined factor V and factor VIII deficiency and in combined factor VII and factor VIII deficiency.

Platelet aggregation and adhesiveness were studied in 3 patients with combined factor V and factor VIII deficiency and in 3 patients with combined factor VII and factor VIII deficiency. The first three patients belonged to three different kindreds whereas the second group belonged to the same kindred. Serotonin C14 uptake and release was also found to be normal in these patients. These studies indicate that platelet function is normal in combined defects of factor VIII. These findings were in agreement with the presence of a normal bleeding time and a normal factor VIII antigen level in all these patients.     

Role of factor VIII C2 domain in factor VIII binding to factor Xa.

Factor VIII (FVIII) is activated by proteolytic cleavages with thrombin and factor Xa (FXa) in the intrinsic blood coagulation pathway. The anti-C2 monoclonal antibody ESH8, which recognizes residues 2248-2285 and does not inhibit FVIII binding to von Willebrand factor or phospholipid, inhibited FVIII activation by FXa in a clotting assay. Furthermore, analysis by SDS-polyacrylamide gel electrophoresis showed that ESH8 inhibited FXa cleavage in the presence or absence of phospholipid. The light chain (LCh) fragments (both 80 and 72 kDa) and the recombinant C2 domain dose-dependently bound to immobilized anhydro-FXa, a catalytically inactive derivative of FXa in which dehydroalanine replaces the active-site serine. The affinity (K(d)) values for the 80- and 72-kDa LCh fragments and the C2 domain were 55, 51, and 560 nM, respectively. The heavy chain of FVIII did not bind to anhydro-FXa. Similarly, competitive assays using overlapping synthetic peptides corresponding to ESH8 epitopes (residues 2248-2285) demonstrated that a peptide designated EP-2 (residues 2253-2270; TSMYVKEFLISSSQDGHQ) inhibited the binding of the C2 domain or the 72-kDa LCh to anhydro-FXa by more than 95 and 84%, respectively. Our results provide the first evidence for a direct role of the C2 domain in the association between FVIII and FXa.     

The tissue factor region that interacts with factor Xa in the activation of factor VII

Tissue factor is the cell membrane-anchored cofactor for factor VIIa and triggers the coagulation reactions. The initial step is the conversion of factor VII to factor VIIa which, in vitro, is efficiently catalyzed by low concentrations of factor Xa. To identify the tissue factor region that interacts with the activator factor Xa during this process, we evaluated a panel of soluble tissue factor (1-219) mutants for their ability to support factor Xa-mediated activation of factor VII. The tissue factor residues identified as most important for this interaction (Tyr157, Lys159, Ser163, Gly164, Lys165, Lys166, and Tyr185) were identical to those found to be important for the interaction of substrate factor X with the tissue factor.factor VIIa complex. The residues form a continuous surface-exposed patch with an area of about 500 A(2), which appears to be located outside the tissue factor-factor VII contact zone. In agreement, the two monoclonal antibodies 5G6 and D3H44-F(ab')(2), whose epitopes overlap with this identified region, inhibited the rates of factor VII activation by 86% and 95%, respectively. These antibodies also strongly inhibited the conversion of (125)I-labeled factor VII when cell membrane-expressed, full-length tissue factor (1-263) was employed. Together the results suggest the usage of a common surface region of tissue factor in its dual role-as a cofactor for factor Xa-mediated factor VII activation and as a cofactor for factor VIIa-mediated factor X activation. The finding that factor Xa and factor X may engage in similar, if not identical, molecular interactions with tissue factor further indicates that factor Xa and factor X are similarly oriented toward their respective interaction partners in the ternary catalytic complexes.     

Zymogen factor IX potentiates factor IXa-catalyzed factor X activation

Intrinsic factor X activation is accelerated >10(7)-fold by assembly of the entire complex on the activated platelet surface. We have now observed that increasing the concentration of zymogen factor IX to physiologic levels ( approximately 100 nM) potentiates factor IXa-catalyzed activation of factor X on both activated platelets and on negatively charged phospholipid vesicles. In the presence and absence of factor VIIIa, factor IX (100 nM) lowered the K(d,appFIXa) approximately 4-fold on platelets and 2-10-fold on lipid vesicles. Treatment of two factor IX preparations with active-site inhibitors did not affect these observations. Autoradiographs of PAGE-separated reactions containing either (125)I-labeled factor IX or (125)I-labeled factor X showed that the increased factor X activation was not due to factor Xa-mediated feedback activation of factor IX and that there was increased cleavage of factor X heavy chain in the presence of factor IX in comparison with control reactions but only in the presence of both the enzyme and the surface. Since plasma concentrations of prothrombin, factor VII, protein C, or protein S did not by themselves potentiate factor Xa generation and did not interfere with the potentiation of the reaction of factor IX, the effect is specific for factor IX and is not attributable to the Gla domain of all vitamin K-dependent proteins. These observations indicate that under physiologic conditions, plasma levels of the zymogen factor IX specifically increase the affinity of factor IXa for the intrinsic factor X activation complex.     

Definition of a factor Va binding site in factor Xa

We reported previously that residue 347 in activated fX (fXa) contributes to binding of the cofactor, factor Va (fVa) (Rudolph, A. E., Porche-Sorbet, R. and Miletich, J. P. (2000) Biochemistry 39, 2861-2867). Four additional residues that participate in fVa binding have now been identified by mutagenesis. All five resulting fX species, fX(R306A), fX(E310N), fX(R347N), fX(K351A), and fX(K414A), are activated and inhibited normally. However, the rate of inhibition by antithrombin III in the presence of submaximal concentrations of heparin is reduced for all the enzymes. In the absence of fVa, all of the enzymes bind and activate prothrombin similarly except fXa(E310N), which has a reduced apparent affinity ( approximately 3-fold) for prothrombin compared with wild type fXa (fXa(WT)). In the absence of phospholipid, fVa enhances the catalytic activity of fXa(WT) significantly, but the response of the variant enzymes was greatly diminished. On addition of 100 nm PC:PS (3:1) vesicles, fVa enhanced fXa(WT), fXa(R306A), and fXa(E310N) similarly, whereas fXa(R347N), fXa(K351A), and fXa(K414A) demonstrated near-normal catalytic activity but reduced apparent affinity for fVa under these conditions. All enzymes function similarly to fXa(WT) on activated platelets, which provide saturating fVa on an ideal surface. Loss of binding affinity for fVa as a result of the substitutions in residues Arg-347, Lys-351, and Lys-414 was verified by a competition binding assay. Thus, Arg-347, Lys-351, and Lys-414 are likely part of a core fVa binding site, whereas Arg-306 and Glu-310 serve a less critical role.     

Hepatoma-derived growth factor is a neurotrophic factor harbored in the nucleus

Hepatoma-derived growth factor (HDGF) is a heparin-binding proliferating factor originally isolated from conditioned medium of the hepatoma-derived cell line HuH-7. HDGF has greatest homology in an amino acid sequence with high mobility group 1 (HMG1), which has been characterized as a DNA-binding, inflammatory, and potent neurite outgrowth molecule. HDGF is reported to be widely expressed and act as a growth factor in many kinds of cells. However, it has not been investigated in the nervous system. Here, we show by Western blot analysis that HDGF is present in the mouse brain from the embryonic period until adulthood. In situ hybridization and immunohistochemical analyses revealed that HDGF was expressed mainly in neurons, and HDGF protein was localized to the nucleus. HDGF and high mobility group 1 were secreted under physiological conditions and released extracellularly in necrotic conditions. Furthermore, we showed that exogenously supplied HDGF had a neurotrophic effect and was able to partially prevent the cell death of neurons in which endogenous HDGF was suppressed. Therefore, we propose that HDGF is a novel type of neurotrophic factor, on account of its localization in the nucleus and its potential to function in an autocrine manner under both physiological and pathological conditions throughout life.     

Granulocyte-macrophage colony-stimulating factor as an autocrine survival-growth factor in human gliomas

We studied the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptors (GM-CSF.R) in 20 human brain gliomas with different tumor gradings and demonstrated constitutive high levels of both mRNA gene expression and protein production exclusively in the highest-grade tumors (WHO, III-IV grade). Five astrocytic cell lines were isolated in vitro from glioma cells, which had selectively adhered to plates pre-coated with rhGM-CSF. These cells were tumorigenic when xenografted to athymic mice, and produced GM-CSF constitutively in culture. Two lines, particularly lines AS1 and PG1, each from a patient with glioblastoma multiforme, constitutively over-expressed both GM-CSF and GM-CSF.R genes and secreted into their culture media biologically active GM-CSF. Different clones of the AS1 line, isolated after subsequent passages in vitro and then transplanted to athymic mice, demonstrated higher tumorigenic capacity with increasing passages in vivo. Cell proliferation was stimulated by rhGM-CSF in late-stage malignant clones, whereas apoptosis occurred at high frequency in the presence of blocking anti-GM-CSF antibodies. In contrast, rhGM-CSF did not induce any apparent effect in early-stage clones expressing neither GM-CSF nor GM-CSF.R. The addition of rhGM-CSF or rhIL-1β, to cultures induced the overproduction of both GM-CSF and its receptors and increased gene activation for several functional proteins (e.g. NGF, VEGF, VEGF.R1, G-CSF, MHC-II), indicating that these cells may undergo dynamic changes in response to environmental stimuli. These findings thus revealed: (1) that the co-expression of both autocrine GM-CSF and GM-CSF.R correlates with the advanced tumor stage; (2) that an important contribution of GM-CSF in malignant glioma cells is the prevention of apoptosis. These results imply that GM-CSF has an effective role in the evolution and pathogenesis of gliomas.     

Catabolism of factor VIIa bound to tissue factor in fibroblasts in the presence and absence of tissue factor pathway inhibitor

Vascular injury leads to the exposure of blood to fibroblasts and smooth muscle cells within the vessel wall. These cells constitutively express tissue factor (TF), the cellular receptor for plasma clotting factor VIIa (FVIIa). Formation of TF.FVIIa complexes on cell surfaces triggers the blood coagulation cascade. In the present study, we have investigated the fate of TF.FVIIa complexes formed on the cell surface of fibroblasts in the presence and absence of plasma inhibitor, tissue factor pathway inhibitor (TFPI). FVIIa bound to TF on the cell surface was internalized and degraded without depleting the cell surface TF antigen and activity. TFPI significantly enhanced the TF-specific internalization and degradation of FVIIa. TFPI-enhanced internalization and degradation of FVIIa requires the C-terminal domain of TFPI and factor Xa. TFPI. Xa-mediated internalization of FVIIa was associated with the depletion of TF from the cell surface. A majority of the internalized FVIIa was degraded, but a small portion of the internalized FVIIa recycles back to the cell surface as an intact protein. In addition to TF, other cell surface components, such as low density lipoprotein receptor-related protein (LRP) and heparan sulfates, are essential for TFPI.Xa-induced internalization of FVIIa. Acidification of cytosol, which selectively inhibits the endocytotic pathway via coated pits, inhibited TFPI.Xa-mediated internalization but not the basal internalization of FVIIa. Overall, our data support the concept that FVIIa bound to cell surface TF was endocytosed by two different pathways. FVIIa complexed with TF in the absence of the inhibitor was internalized via a LRP-independent and probably noncoated pit pathway, whereas FVIIa complexed with TF along with the inhibitor was internalized via LRP-dependent coated pit pathway.     

Endogenous platelet-activating factor and anti-platelet-activating factor in patients with renovascular hypertension

Renovascular hypertension is relieved by percutaneous transluminal renal angioplasty. In four patients with renovascular hypertension, platelet-activating factor (PAF) was found to be released into the ipsilateral renal venous blood after percutaneous transluminal renal angioplasty, but was not found in the contralateral renal venous blood following this procedure. Anti-platelet-activating factor with a lipid-like property was also found, and its polarity was slightly lower than that of PAF judging by its behavior on thin layer chromatography. Anti-platelet-activating factor completely blocked the aggregation of rabbit platelets induced by PAF, ADP or arachidonic acid. These results indicate that PAF and anti-platelet-activating factor are released into renal venous blood following percutaneous transluminal renal angioplasty in patients with renovascular hypertension.     

Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis

Fibroblast growth factors (FGFs) are a family of heparin-binding growth factors. FGFs exert their pro-angiogenic activity by interacting with various endothelial cell surface receptors, including tyrosine kinase receptors, heparan-sulfate proteoglycans, and integrins. Their activity is modulated by a variety of free and extracellular matrix-associated molecules. Also, the cross-talk among FGFs, vascular endothelial growth factors (VEGFs), and inflammatory cytokines/chemokines may play a role in the modulation of blood vessel growth in different pathological conditions, including cancer. Indeed, several experimental evidences point to a role for FGFs in tumor growth and angiogenesis. This review will focus on the relevance of the FGF/FGF receptor system in adult angiogenesis and its contribution to tumor vascularization.     

Species-specific aggregation factor in sponges. Sialyltransferase associated with aggregation factor.

The sialyltransferase (= glycoprotein-sialic acid transferase) was studied in the sponge Geodia cydonium, a mesozoan organism. The experiments were performed both in intact cellular and in isolated enzyme systems. It is shown, that desialylated cells show a lower aggregation potency than the controls. During aggregation enzymic sialylation of desialylated sponge cells occurs in the presence of an aggregation factor, which is associated with a high molecular weight particle. The sialylation process is temperature-dependent and can be inhibited by N-ethylmaleimide. Sialylation occurs predominantly at a distinct cell surface component, the aggregation receptor. The sialyltransferase was isolated and purified by the following steps: Sepharose 4B, CM-cellulose, Nonidet treatment, and Sephadex G-100. By this procedure the enzyme was purified 680-fold with a 31% yield. The sialyltransferase is originally associated with the high molecular weight particle also carrying the aggregation factor. In the last step the aggregation factor was separated from the sialyltransferase. The enzyme catalyzes the transfer of sialic acid from CMP-sialic acid to the desialylated aggregation receptor. The molecular weight of the sialyltransferase has been determined to be 52,000. Kinetic studies revealed no lag phase and a dependence on enzyme concentration. The purified transferase has a pH optimum of 7.75 and requires 200 mM NaCl for activity. No requirement for Mg2+ or Ca2+ could be observed. The reaction is inhibited by 10 micronM N-ethylmaleimide.     

Tissue factor is not involved in the mitogenic activity of factor VIIa

The present study was undertaken to evaluate in vitro the importance of tissue factor in the mitogenic effect of factor VIIa for embryonic fibroblasts. For that purpose, embryonic fibroblasts were isolated from either wild-type or transgenic mice showing a single inactivation of the tissue factor gene or expressing a truncated form (lacking the cytosolic domain) of this protein. Factor VIIa stimulated in a dose-dependent manner the growth of the 3 types of fibroblasts, thus showing that TF is not involved in the mitogenic activity of factor VIIa. The mitogenic activity of factor VIIa disappeared in serum immunopurified in factor X and was almost totally inhibited by DX9065, a selective factor Xa inhibitor, showing that this effect of factor VIIa occurred via factor Xa generated during the incubation period. Hirudin did not show any significant effect on factor VIIa-induced fibroblast proliferation, thus showing that the effect observed for factor VIIa was selectively mediated by factor Xa and was not due to thrombin formation. Our results therefore represent the first evidence for the possible importance of factor Xa in the mitogenic effect of factor VIIa and show the negligible role of tissue factor in this process.     

Hypoxia-inducible factor 1-alpha and vascular endothelial growth factor in cartilage tumors

Neoangiogenesis has been demonstrated in chondrosarcoma. Anti-angiogenic therapies are being tested in clinical trials for chondrosarcomas. Studies of the underlying mechanisms have been performed almost exclusively in cell lines. We immunostained 20 samples of chondrosarcoma and 20 samples of enchondromas with antibodies against hypoxia-inducible factor 1-alpha (HIF-1-alpha) and vascular endothelial growth factor (VEGF). The immunohistochemical staining of HIF-1-alpha and VEGF were highly correlated. Enchondromas were HIF-1-alpha and VEGF negative, whereas all chondrosarcoma exhibited HIF-1-alpha and VEGF immunostaining. HIF-1-alpha/VEGF double positive cases were almost exclusively chondrosarcomas with a high tumor grade. We suggest that HIF-1-alpha is a marker of malignancy in chondrosarcomas that correlates with tumor neo-angiogenesis. Our findings also suggest that a HIF-1-alpha/VEGF angiogenic pathway may exist in chondrosarcoma in vivo as in other malignant tumors. The inclusion of novel inhibitors to HIF-1-alpha and other factors may optimize anti-angiogenic interventions in chondrosarcoma.     

Tenascin-C induction by the diffusible factor epidermal growth factor in stromal-epithelial interactions

Tenascin-C, a six-armed extracellular matrix glycoprotein, is expressed in a temporally and spatially restricted pattern during carcinogenesis and invasion or metastasis of carcinoma cells in association with stromal-epithelial interactions. The human epidermoid carcinoma-derived cell lines, A431 and HEp-2, which do not express tenascin-C by themselves in vitro, do express tenascin-C after transplantation into nude mice, and transforming growth factor β1 (TGF-β1) induces them to express tenascin-C in vitro. Epidermal growth factor (EGF) induced tenascin-C in these cells more effectively (about 3.5-fold greater) than did TGF-β1. Hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF) had little effect on the induction of tenascin-C. EGF also induced other extracellular matrix components, fibronectin and laminin. Tenascin-C was also induced when the carcinoma cells were co-cultured with embryonic fibroblasts from mice which were homozygous for a null mutation in the tenascin-C gene, or when the conditioned medium from these cells was added. The induction of tenascin-C in the co-culture was reduced by treating the cells with antibodies against EGF or its receptor. The addition of EGF caused both cell types to disrupt their cytoskeleton and focal contacts as evidenced by the loss of stress fibers and vinculin plaques. EGF did neither induce tenascin-C nor affect the morphology in tenascin-C-nonproducing A549 carcinoma cells, which did not produce tenascin-C after transplantation. Thus, EGF induces tenascin-C in tenascin-C-nonproducing human carcinoma cells through EGF receptors. Furthermore, in stromalepithelial interactions, the diffusible factor EGF participates in the induction of human tenascin-C in these cells through EGF receptors. © 1995 Wiley-Liss Inc.     

Epidermal growth factor in blood

The presence of receptors for epidermal growth factor (EGF) in a wide variety of human tissues and also some tumours indicates an as yet undefined role for EGF and it is therefore necessary to know precise concentrations in blood and other fluids. We have investigated the occurrence of EGF in the circulation and found that in platelet rich plasma, EGF levels were 51 +/- 5 pmol/l (mean +/- S.E.M., n = 6) while in platelet poor plasma levels were 2.9 +/- 0.9 pmol/1. In contrast, serum EGF was 37 +/- 7 pmol/l if separated at 30 min and rose to 117 +/- 5 pmol/l if separated at 270 min. Gel chromatography showed that all residual EGF immunoreactivity in platelet poor plasma resided in the high molecular weight form thought to be non biologically active. In serum, delay in separation resulted in an increase in the proportion of EGF immunoreactivity co-eluting with EGF standard. These results suggest that EGF in the circulation is associated with platelets and that the process of blood coagulation leads to release of free EGF.