Hans Prydz

Coagulation factors VIIa and Xa induce cell signaling leading to up-regulation of the egr-1 gene.

Intracellular signaling induced by the coagulation factors (F) VIIa and Xa is poorly understood. We report here studies on these processes in a human keratinocyte line (HaCaT), which is a constitutive producer of tissue factor (TF) and responds to both FVIIa and FXa with elevation of cytosolic Ca2+, phosphorylation of extracellular signal-regulated kinase (Erk) 1/2, p38MAPK, and c-Jun N-terminal kinase, and up-regulation of transcription of the early growth response gene-1 (egr-1). Using egr-1 as end point, we observed with both agonists that phosphatidylinositol-specific phospholipase C and the mitogen-activated protein kinase/Erk kinase/Erk pathway were mediators of the responses. The responses to FVIIa were TF-dependent and up-regulation of egr-1 mRNA did not require presence of the TF cytoplasmic domain. Antibodies to EPR-1 and factor V had no effect on the response to FXa. We have provided evidence that TF is not the sole component of the FVIIa receptor. The requirement for proteolytic activity of both FVIIa and FXa suggests that protease-activated receptors may be involved. We now report evidence suggesting that protease-activated receptor 2 or a close homologue may be a necessary but not sufficient component of this particular signal transduction pathway. The up-regulation of egr-1 describes one way by which the initiation of blood coagulation may influence gene transcription. The ability of these coagulation proteases to induce intracellular signals at concentrations at or below the plasma concentrations of their zymogen precursors suggests that these processes may occur also in vivo.

Coagulation Factors VII and X Induce Ca2+ Oscillations in Madin-Darby Canine Kidney Cells Only When Proteolytically Active

We have recently reported that the activated serine protease and blood coagulation Factor VII (FVIIa) can induce Ca2+ oscillations in Madin-Darby canine kidney cells. We now demonstrate a similar response by Madin-Darby canine kidney cells to the active coagulation Factor X (FXa), which is also a serine protease and a substrate of the tissue factor (TF)·FVIIa complex in the initiation of the coagulation cascade. The phosphatidyl inositol-specific phospholipase C inhibitor U73122 inhibited the signals elicited by both FVIIa and FXa. Lack of sensibility to the tyrosine kinase inhibitors herbimycin A, genistein, and the tyrphostin AG18 and discordance between TF expression and FVIIa responsiveness argued against TF acting as a cytokine-like receptor, with tyrosine kinase-mediated activation by FVIIa. As demonstrated using the protease inhibitor benzamidine and by specific active site inhibition with 1,5-dansyl-Glu-Gly-Arg chloromethyl ketone, both FVIIa and FXa lost their ability to elicit a calcium response when devoid of their proteolytic activity. Consistent with this, the native (zymogen) form of Factor X did not induce Ca2+ transients. Homologous but not heterologous inhibition of FVIIa- and FXa-evoked Ca2+ signals by 1,5-dansyl-Glu-Gly-Arg chloromethyl ketone-inactivated FVIIa and FXa suggested that each factor had its own specific cell surface anchoring receptor. The two coagulation factors did not show homologous desensitization as seen for thrombin stimulation. Studies with hirudin excluded involvement of the established activation pathway through thrombin itself. Lack of desensitization of the response to FVIIa or FXa by thrombin ruled out any involvement of proteinase activated receptor-1 (PAR-1), the thrombin receptor. We speculate that FXa and FVIIa may work via a receptor (possibly common) analogous to PAR-1 or its functional homologue PAR-2. Although TF is essential for the FVIIa-induced signaling event, its role in the phosphatidyl inositol-specific phospholipase C-mediated Ca2+ signal may be in anchoring FVIIa to the cell surface rather than in transmembrane signal mediation.

Cellular cooperation in endothelial cell thromboplastin synthesis

Summary. Endothelial cells from human umbilical veins produce a procoagulant identified as thromboplastin (tissue factor, factor III) when stimulated with the phorbol ester 12‐0‐tetradecanoyl‐phorbol‐13‐acetate (TPA), phytohaemagglutinin (PHA) or endotoxin. Inducible thromboplastin synthesis (i.e. synthesis of the protein component of thromboplastin, apoprotein III) was totally inhibited by cycloheximide and actinomycin D, indicating that de novo protein and RNA syntheses are necessary. Serum enhanced the induced apoprotein synthesis.

Alternative transcripts of the candidate tumor suppressor gene, WWOX, are expressed at high levels in human breast tumors

The presence of putative tumor-suppressor genes on chromosome 16q23.2-24.1 has been suggested by LOH analysis in several cancer types. This region overlaps with the fragile site FRA16D and the region of homozygous deletions found in several cancer types. The candidate gene WWOX/FOR has been mapped within this region. The mouse homologue of the WWOX protein has been defined as an apoptogenic protein and an essential partner of p53 in cell death, supporting WWOX as a tumor suppressor gene candidate. We performed an expression study of the WWOX/FOR gene in a series of human breast tumors and breast cancer cell lines, and detected reduced expression of the WWOX/FOR transcript in a series of breast cancer cells. Furthermore, identification of two distinct alternative WWOX transcripts expressed at high levels in human tumors suggests an involvement of the WWOX gene in breast cancer progression.

A novel form of factor VII in plasma from men at risk for cardiovascular disease

Summary. In the Northwick Park Heart Study high activity of the coagulation factor VII was associated with cardiovascular deaths. We confirm here that men at high risk for such disease have increased levels of factor VII activity and demonstrate for the first time that this is due to a phospholipid–factor VII complex in their plasma. This complex increases the specific activity of its factor VII significantly. It is not inhibited by a neutralizing antiserum to thromboplastin and thus does not represent a factor VII‐thromboplastin complex. Gel filtration data suggest an apparent Mr of 66–72 000 for the complex. Factor VII in this phospholipid complex is more sensitive than native factor VII to the serine protease inhibitor diisopropyl‐fluorophosphate, consistent with a more accessible active site conformation (and a higher specific activity) for factor VII in the complex. The level of phospholipid–factor VII complex showed a significant positive correlation with plasma triglycerides and correlation of borderline significance with cholesterol.

Studies on tissue thromboplastin: I. Solubilization with sodium deoxycholate

Abstract Treatment of tissue thromboplastin from human brain with deoxycholate released the thromboplastic activity from the microsomal cell fraction, giving a 5-fold purification. Gel filtration of such preparations on columns of Sephadex G-200 was possible. This resulted in a further 3- to 4-fold purification, giving preparations with a specific activity of 150–190 units per mg substance. The size of the structures carrying the thromboplastic activity was greatly reduced in the presence of deoxycholate. Reaggregation took place when the deoxycholate was removed.

Species Specificity of Tissue Thromboplastin

The species specificity of thromboplastin (tissue factor) is an important fact to take into consideration when clotting assays or experiments are planned. We have (re)investigated all possible combinations of thromboplastin and plasma from several of the most commonly used species of experimental animals.

Factor VIIa Induces Tissue Factor-dependent Up-regulation of Interleukin-8 in a Human Keratinocyte Line

Tissue factor (TF), a transmembrane receptor for the serine protease coagulation factor VII(a) (FVIIa), is the main initiator of the coagulation cascade. Through incompletely elucidated mechanisms, TF serves additional functions in tumor-associated angiogenesis and metastasis. We have studied interleukin-8 (IL-8) as a possible link between TF-FVIIa complex formation and subsequent processes. Recombinant human FVIIa induced the up-regulation of both IL-8 mRNA and protein in a FVIIa dose- and time-dependent fashion. A neutralizing antibody to TF reduced this induction by 93 ± 5%. Active site-inhibited FVIIa had no stimulatory effect and completely blocked that of FVIIa. This confirms that the increased IL-8 production was dependent on the formation of TF-FVIIa complexes and the proteolytic activity of FVIIa. The IL-8 promoter contains DNA binding sites for nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). In response to FVIIa, the DNA binding activity of both NF-κB and AP-1 was enhanced in an electrophoretic mobility shift assay. In addition, theIL-8 promoter was transcriptionally activated both in a luciferase reporter system and a nuclear run-off assay. Moreover, IL-8 mRNA stability was significantly enhanced by FVIIa-induced activation of the mitogen-activated protein kinases ERK1/2 and p38. Taken together, TF-FVIIa signaling induced increased transcription as well as mRNA stabilization leading to the significant up-regulation of IL-8 protein synthesis.

The protein component of human brain thromboplastin

Abstract The protein component of human brain tissue thromboplastin (factor III) has been purified by deoxycholate (DOC) extraction, ultracentrifugation, gel filtration and finally repeated preparative polyacrylamide gel electrophoresis (PGE) in the presence of sodium dodecylsulphate (SDS). The final preparations gave one band in analytical PGE. Reduced and alkylated protein appeared as a band of molecular weight about 53 000 in SDS-PGE. The protein had a low solubility in aqueous solutions in the absence of detergents. When recombined with an optimal amount of the phospholipid fraction of tissue thromboplastin (fraction B) the procoagulant thromboplastin activity was regained. Neither alone nor after recombination with phospholipid did the protein catalyze the hydrolysis of aminoacyl-β-naphthylamides or casein.

In vitro stimulation of tissue thromboplastin (factor III) activity in human monocytes by immune complexes and lectins

Abstract Human monocytes isolated from peripheral blood can be stimulated in vitro to produce a procoagulant which is very similar or identical to tissue thromboplastin. Immune complexes formed by transferrin/antitransferrin and albumin/antialbumin at equivalence induce a marked increase in this thromboplastic activity. Lectins (phytohaemagglutinin, wheat germ agglutinin and concanavalin A) have a similar effect. The specific thromboplastin activity of homogenates of stimulated monocytes reaches levels equivalent to or higher than those of ordinary crude brain thromboplastin preparations. The induction of this activity in monocytes by immune complexes and other endogenous stimulants may be of clinical significance.