Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Imbalance in the network of soluble mediators may play a pivotal role in the pathogenesis of Kaposi's sarcoma (KS). In this study, we demonstrated that KS cells grown in vitro produced and in part released platelet activating factor (PAF), a powerful lipid mediator of inflammation and cell-to-cell communication. IL-1, TNF, and thrombin enhanced the synthesis of PAF. PAF receptor mRNA and specific, high affinity binding site for PAF were present in KS cells. Nanomolar concentration of PAF stimulated the chemotaxis and chemokinesis of KS cells, endothelial cells, and vascular smooth muscle cells. The migration response to PAF was inhibited by WEB 2170, a hetrazepinoic PAF receptor antagonist. Because neoangiogenesis is essential for the growth and progression of KS and since PAF can activate vascular endothelial cells, we examined the potential role of PAF as an instrumental mediator of angiogenesis associated with KS. Conditioned medium (CM) from KS cells (KS-CM) or KS cells themselves induced angiogenesis and macrophage recruitment in a murine model in which Matrigel was injected subcutaneously. These effects were inhibited by treating mice with WEB 2170. Synthetic PAF or natural PAF extracted from plasma of patients with classical KS also induced angiogenesis, which in turn was inhibited by WEB 2170. The action of PAF was amplified by expression of other angiogenic factors and chemokines: these included basic and acidic fibroblast growth factor, placental growth factor, vascular endothelial growth factor and its specific receptor flk-1, hepatocyte growth factor, KC, and macrophage inflammatory protein-2. Treatment with WEB 2170 abolished the expression of the transcripts of these molecules within Matrigel containing KS-CM. These results indicate that PAF may cooperate with other angiogenic molecules and chemokines in inducing vascular development in KS.
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PMID:Platelet activating factor produced in vitro by Kaposi's sarcoma cells induces and sustains in vivo angiogenesis. 754 96

Angiogenesis in situ occurs within the interstitial extracellular matrix. The complexity of currently used three-dimensional in vitro angiogenesis systems makes it difficult to quantify cellular growth and neovessel formation. To overcome this problem we were interested to develop an angiogenesis system which allows rapid and reliable quantification of three-dimensional neovessel formation in vitro. Endothelial cells were seeded on gelatine-coated microcarriers (MCs). Cell-coated MCs were suspended in a solution of fibrinogen which was then induced to polymerize by addition of thrombin. By this way, MCs were entrapped in a three-dimensional fibrin matrix. Within a few hours, endothelial cells began to leave their supporting microcarriers and to migrate into the fibrin gel. Without addition of stimulators of angiogenesis, endothelial cells showed incoherent migration into the matrix. In contrast, in response to fibronectin, basic fibroblast growth factor (bFGF), or vascular endothelial growth factor (VEGF), respectively, endothelial cells assembled to form multicellular capillary-like structures occasionally exceeding 1000 microns in length. Each MC gave rise to a limited number of capillaries. A single culture dish contained hundreds of MCs, ensuring that a sufficient number of random samples was present for a reliable statistical evaluation. The angiogenic response could be easily quantified by determination of the average number of capillary-like formations per MC (cap/MC). The capillary count for macrovascular endothelial cells from the bovine pulmonary artery was 0.14 cap/MC when no angiogenic stimulators were contained within the fibrin gel. Addition of 200 micrograms/ml fibronectin increased capillary formation to 0.63 cap/MC (P < 0.0001) at Day 6. Already after 3 days, addition of bFGF (30 ng/ml) yielded a capillary count of 1.05 and addition of VEGF (100 ng/ml) resulted in 0.91 cap/MC. In contrast, addition of hyaluronic acid stimulated migration of dispersed endothelial cells into the fibrin matrix without leading to significant capillary formation (0.09 cap/MC). Hydrocortisone alone or in combination with heparin led to a significant inhibition of bFGF-stimulated angiogenesis. We thus have developed a convenient angiogenesis in vitro system which allows reliable quantification of capillary formation in a three-dimensional environment. Based on this assay we conclude that apart from proliferation and migration of endothelial cells, angiogenesis additionally requires the assembly of cells to form multicellular capillaries. This process is strongly induced by the extracellular matrix protein fibronectin. Hyaluronic acid, on the other hand, promotes migration but not capillary formation (assembly).
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PMID:A novel, microcarrier-based in vitro assay for rapid and reliable quantification of three-dimensional cell migration and angiogenesis. 858 47

We have identified several mechanisms by which the angiogenic cytokine vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) likely regulates endothelial cells (EC) migration. VPF/VEGF induced dermal microvascular EC expression of mRNAs encoding the alphav and beta3 integrin subunits resulting in increased levels of the alphavbeta3 heterodimer at the cell surface, and VPF/VEGF also induced mRNA encoding osteopontin (OPN), an alphavbeta3 ligand. OPN promoted EC migration in vitro; and VPF/VEGF induction of alphavbeta3 was accompanied by increased EC migration toward OPN. Because thrombin cleavage of OPN results in substantial enhancement of OPN's adhesive properties, and because VPF/VEGF promotes increased microvascular permeability leading to activation of the extrinsic coagulation pathway, we also investigated whether VPF/VEGF facilitates thrombin cleavage of OPN in vivo. Consistent with this hypothesis, co-injection of VPF/VEGF together with OPN resulted in rapid cleavage of OPN by endogenous thrombin. Furthermore, in comparison with native OPN, thrombin-cleaved OPN stimulated a greater rate of EC migration in vitro, which was additive to the increased migration associated with induction of alpha v beta 3. Thus, these data demonstrate cooperative mechanisms for VPF/VEGF regulation of EC migration involving the alphavbeta3 integrin, the alphavbeta3 ligand OPN, and thrombin cleavage of OPN. These findings also illustrate an operational link between VPF/VEGF induction of EC gene expression and VPF/VEGF enhancement of microvascular permeability, suggesting that these distinct biological activities may act accordingly to stimulate EC migration during angiogenesis.
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PMID:Stimulation of endothelial cell migration by vascular permeability factor/vascular endothelial growth factor through cooperative mechanisms involving the alphavbeta3 integrin, osteopontin, and thrombin. 868 54

In the living organism, capillary growth frequently occurs in a fibrin-rich extracellular matrix. The structure and the mechanical properties of fibrin clots are influenced by various macromolecules (i.e., hyaluronic acid and thrombospondin) and also by pH, ionic strength, and thrombin concentrations of the milieu in which they polymerize. The configuration (three-dimensional architecture) and the rigidity of fibrin clots correlate with their opacity measured by spectrophotometric absorbance readings at 350 nm. By using bovine pulmonary artery endothelial cells and bovine fibrinogen, we show here that transparent fibrin clots (A(350) < 1.0), polymerized at > or = pH 7.5 or in the presence of increased thrombin or sodium chloride concentrations, strongly stimulated capillary morphogenesis in vitro. In contrast, opaque fibrin gels (A(350) > 1.5), polymerized at pH 7.2 or in the presence of dextran, stimulated only the migration of endothelial cells but not capillary morphogenesis. We demonstrate that the angiomorphogenic effects of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) are strongly dependent on the structure of the fibrin clots. Our findings suggest that bFGF/VEGF primarily stimulate the proliferation of endothelial cells, whereas the three-dimensional architecture of the fibrin matrix is decisive for capillary morphogenesis.
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PMID:The configuration of fibrin clots determines capillary morphogenesis and endothelial cell migration. 899 33

Thrombomodulin (TM) is a cell-surface receptor that plays a critical role in endothelial cell anticoagulant activity through its cofactor role in the thrombin-catalyzed activation of human protein C. In this study, we examined the effect of vascular endothelial growth factor (VEGF), a potent angiogenic factor, on surface anticoagulant activity and thrombomodulin expression. We show that thrombin-dependent activation of human protein C, measured on the endothelial cell surface, increased from 50 to 80% following exposure of cells to VEGF for 24 h. The effect was concentration dependent with the half-maximal stimulatory effect at approximately 100 pM. This increase in thrombin-dependent aPC generation correlated with a proportional and concentration-dependent increase in the level of cell-surface TM antigen. Both the total cellular TM antigen and the total cellular TM mRNA levels increased approximately 2.5-fold in VEGF-treated cells suggesting that most if not all of the regulation was at the message level. We further show that VEGF blocked IL-1 beta-induced suppression of both TM surface antigen and mRNA and was similarly capable of antagonizing the down-regulation of TM by TGF-beta and from cell activation by LPS. Our data suggest that VEGF regulation of TM may contribute to mechanisms that would maintain local hemostasis during angiogenesis and revascularization and could play a role in minimizing loss of vessel anticoagulant function during inflammatory processes.
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PMID:Thrombomodulin-dependent anticoagulant activity is regulated by vascular endothelial growth factor. 945 83

Thrombin-catalyzed, cross-linked fibrin (XLF) formation is a characteristic histopathological finding in many human and experimental tumors and is thought to be of importance in the local host defense response. Although the pathogenesis of tumor-associated fibrin deposition is not entirely clear, several tumor procoagulants have been described as likely primary stimuli for the generation of thrombin (and XLF) in the tumor microenvironment (TME). In a previous study of a variety of human tumors we have shown that tissue factor (TF) is the major procoagulant. However, the relative contribution to fibrin deposition in the TME of tumor cell TF and host cell TF (eg, macrophage-derived) was not established. In addition, recent evidence has implicated TF in the regulation of the synthesis of the pro-angiogenic factor vascular endothelial growth factor (VEGF) by tumor cells. In the current study we used in situ techniques to determine the cellular localization of XLF, TF, VEGF, and an alternative tumor procoagulant, so-called cancer procoagulant (CP), a cysteine protease that activates clotting factor X. In lung cancer we have found XLF localized predominantly to the surface of tumor-associated macrophages, as well as to some endothelial cells and perivascular fibroblasts in the stromal area of the tumors co-distributed with TF at the interface of the tumor and host cells. Cancer pro-coagulant was localized to tumor cells in several cases but not in conjunction with the deposition of XLF. TF and VEGF were co-localized in both lung cancer and breast cancer cells by in situ hybridization and immunohistochemical staining. Furthermore, a strong relationship was found between the synthesis of TF and VEGF levels in human breast cancer cell lines (r2 = 0.84; P < 0.0001). Taken together, these data are consistent with a highly complex interaction between tumor cells, macrophages, and endothelial cells in the TME leading to fibrin formation and tumor angiogenesis.
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PMID:Activation of coagulation and angiogenesis in cancer: immunohistochemical localization in situ of clotting proteins and vascular endothelial growth factor in human cancer. 946 66

Production of vascular endothelial growth factor (VEGF) by cancer cells at invasive and metastatic sites is an important aspect of tumor angiogenesis. Although known primarily as a mitogen and a vascular permeability factor (VPF) for endothelial cells, VEGF/VPF has been proposed to induce the expression of procoagulant factors in endothelial cells. In this study, we have explored the ramifications of VEGF induction of tissue factor (TF) in human umbilical vein endothelial cells (HUVECs) and subsequent activation of progelatinase A. Within 3 hr of incubation with VEGF/VPF, endothelial cells accelerate TF generation as measured using chromogenic substrate assays for coagulation factors Xa and thrombin. Incubation of VEGF/VPF-pre-treated cells with prothrombin and factors X, Va, and VIIa at 37 degrees C and subsequent generation of thrombin resulted in activation of secreted endothelial progelatinase A as demonstrated by gelatin zymography. Anti-thrombin III or antibodies to TF inhibited thrombin generation and progelatinase A activation. VEGF/VPF also directly increased HUVEC secretion of interstitial collagenase, tissue inhibitor of metalloproteinases (TIMP-1) and, to a lesser extent, gelatinase A. The effect of thrombin on endothelial proliferation in serum-free media was examined. Thrombin was a growth factor for HUVECs at a lower dose than that required for progelatinase A activation. Whereas TIMP-2 abrogated thrombin-induced progelatinase A activation, it had no significant effect on thrombin-induced endothelial cell growth. We propose that an early step in tumor angiogenesis involves VEGF-induced thrombin generation and increased MMP production with subsequent activation of endothelial progelatinase A and degradation of the underlying basement membrane.
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PMID:Vascular endothelial growth factor induces tissue factor and matrix metalloproteinase production in endothelial cells: conversion of prothrombin to thrombin results in progelatinase A activation and cell proliferation. 949 49

1. Dysregulated vascular endothelial growth factor (VEGF) expression has been reported in several pathological states based upon evidence of elevated serum VEGF levels. Using two immunoassays for VEGF, this study determines normal plasma and serum VEGF ranges, determines which are more likely to reflect circulating VEGF levels and investigates a potential contribution of VEGF from platelets to VEGF levels detected in serum. 2. The presence of soluble VEGF receptor, sflt-1, at a molar excess of 7:1 significantly reduced measured VEGF levels in both assays. Serum VEGF levels were higher than plasma levels in children [(mean +/- S.E.M.) 306.1 +/- 39.4 versus 107.4 +/- 24.9 pg/ml, P < 0.0001] and adults (249.4 +/- 46.4 versus 76.1 +/- 10.7 pg/ml, P < 0.0001). Serum VEGF increased with clotting time (P = 0.0005 t0 compared with 2 h samples); plasma VEGF levels were not affected by time between sampling and centrifugation. 3. Calcium-induced clotting of platelet-rich but not platelet-poor plasma induced VEGF release with a proportional response between platelet count and VEGF level and isolated platelets released significant quantities of VEGF upon incubation with thrombin. Reverse transcriptase-PCR studies confirmed that platelets express VEGF121 and VEGF165 mRNA. 4. These data suggest that plasma is the preferred medium to measure VEGF levels; a significant and highly variable platelet-mediated secretion of VEGF during the clotting process invalidates the use of serum as an indicator of circulating VEGF levels in disease states.
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PMID:Vascular endothelial growth factor (VEGF) is released from platelets during blood clotting: implications for measurement of circulating VEGF levels in clinical disease. 964 Mar 45

Platelet aggregation is a cardinal feature of both vascular repair and vascular disease. During aggregation platelets release a variety of vasoactive substances; some of these promote angiogenesis, endothelial permeability, and endothelial growth, actions shared by vascular endothelial growth factor (VEGF). This study was undertaken to investigate the hypothesis that VEGF is released by aggregating platelets. We found that VEGF was secreted during the in vitro aggregation of platelet-rich plasma induced by thrombin, collagen, epinephrine, and ADP (range 23-518 pg VEGF/ml). Furthermore, serum VEGF levels were elevated compared with plasma (230 +/- 63 vs. 38 +/- 8 pg VEGF/ml), indicative of VEGF release during whole blood coagulation. Lysates of apheresed, leukocyte-poor platelet units contained significant amounts of VEGF (2.4 +/- 0.8 pg VEGF/mg protein). VEGF message and protein were also present in a megakaryocytic cell line (Dami cell). These results suggest constitutive roles for platelet VEGF in the repair of intimal vessel injury and in the altered permeability and intimal proliferation seen at sites of platelet aggregation and thrombosis.
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PMID:In vitro release of vascular endothelial growth factor during platelet aggregation. 972 13

Tumors do not grow without inducing a new vessel formation. The postulation of Dr. Folkman in 1971-that tumor growth is angiogenesis-dependent-has been widely accepted, more than two decades later. The question now becomes, "Is it possible to treat cancer by attacking its blood supply?" Many pharmaceutical companies directed their research to antiangiogenic therapy in the past years. Despite increasing knowledge of tumor-induced angiogenesis, the mechanism as to how antiangiogenic agents inhibit new vessel formation remains unknown. Even the mechanisms of two of the most potent preclinical antiangiogenic drugs, angiostatin and endostatin, are still unknown. Many factors are involved in new vessel formation and experimental models are not sophisticated enough to take into account all factors that play a role in spontaneously occurring tumors. Translational research from the clinic to the laboratory is warranted for the discovery of new potent antiangiogenic agents. Our translational angiogenesis research started two years ago, when we hypothesized that circulating concentrations of vascular endothelial growth factor (VEGF), an important angiogenic factor, if initially elevated, would decrease during therapy in cancer patients. Until then, several investigators tried to correlate serum concentrations of VEGF with the prognosis of cancer patients. Fascinatingly, we found a specific pattern of VEGF concentrations that correlated exactly with the platelet counts of these patients during therapy. No relationship with tumor burden was detected, indicating that circulating levels of VEGF are not influenced by tumor cells, but are mainly dependent on platelet contents. In addition, it was shown by others that thrombin activation of platelets causes VEGF release.What then is the role of circulating VEGF carried by platelets? VEGF has been shown to induce permeability, has mitogenic and chemotactic activity on endothelial cells, and also has procoagulatory activity. Platelets play a critical role in wound healing and, if they are activated, they release upon activation, in addition to VEGF, other growth factors that are involved in angiogenesis (e.g., platelet-derived endothelial cell growth factor, thrombospondin, and platelet factor 4). On the other hand, in the clinic it was found that platelet counts have prognostic significance for cancer patients and that coagulation abnormalities are regularly found in cancer patients. In preclinical studies the tumor-platelet interactions have been studied extensively and a relationship between metastasis formation and platelet-tumor interaction has been reported. We are currently investigating whether a specific tumor endothelium-platelet interaction can contribute to tumor-induced angiogenesis.Although these translational studies have no direct impact on clinical cancer therapy, oncologists should be aware of a potential role for platelets in cancer growth. For example, bone marrow-supportive agents, currently used in high-dose chemotherapy, contribute to platelet production and thereby may influence response to therapy. At this time we investigate in our hospital the pretreatment platelet counts in cancer patients, and we are studying how bone marrow-supportive agents during chemotherapy affect these counts in relation to the response to therapy. We would be pleased to learn of your observations.
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PMID:Tumor Growth: A Putative Role for Platelets? 1038 96


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