lymphocytes or lymphocyte supernatants from 5-mo-old mice. Plasma from 5-mo-old but not from 1-mo-old mice was able to induce the production of the lymphokine by cells from 1-mo- old animals. This lymphokine was not interleukin 1,2, or gamma interferon. We conclude that induction of monocyte/macrophageprocoagulantactivity parallels disease
The mechanisms underlying the induction of tyrosine phos- phorylation in response to virus and the precise role of the phosphorylated proteins in cell activation were not investi- gated in these studies. The observation that the increase in phosphotyrosine residues was transient and preceded a rise in PCA expression supports the notion that tyrosine phosphory- lation participates early in the signalling cascade leading to gene expression. This pattern is consistent with that observed for other macrophage stimuli such as LPS and zymosan (27, 31). The receptor for MHV-3 is a 110-kDa glycoprotein which is a member of the murine carcinoembryonic antigen family. Its short intracellular domain precludes its ability to function as a receptor tyrosine kinase, thus suggesting the possible in- volvement of associated nonreceptor protein tyrosine kinases. Further, the lack of a tyrosine residue within the proposed cytoplasmic domain suggests that the MHV receptor is also not a substrate for tyrosine phosphorylation. However, splice vari- ants of the receptor with a long cytoplasmic tail containing tyrosine residues have been reported to serve as MHV recep- tors (3, 13). Phosphorylation of one of these carcinoembryonic antigen-related glycoproteins may have participated directly in the signalling pathways, as has been reported for cell-CAM 105 (11). In our studies, a protein with a molecular mass of ;65 kDa was seen to be tyrosine phosphorylated in the virus- treated cells. Several src family tyrosine kinases known to be present in macrophages (e.g., hck, fgr, and src) lie close to this mass and thus may be substrate proteins (5). The present studies also showed rapid phosphorylation of two proteins (41 and 44 kDa) whose molecular masses correspond to those reported for two distinct mitogen-activated protein kinases in macrophages (31). These kinases are felt to play an important role in cell signalling in a variety of cell types (24). Further
hepatocytes from fully susceptible BALB/cJ mice infected at a multiplicity of infection of 0.1, 1.0, and 10.0. In addition, splenic macrophages recovered from infected and untreated BALB/cJ mice demonstrated a marked augmentation in procoagulantactivity (PCA) from a basal 10 +/- 5 mU/10(6) […]
Activation of the immune coagulation system has been implicated in the pathogenesis of liver injury follow- ing infection of inbred mice with murine hepatitis virus strain 3 (MHV-3). Following MHV-3 infection, macro- phages isolated from MHV-3-susceptible and -semisusceptible inbred strains of mice express increased pro- coagulant activity (PCA), whereas macrophages from resistant strains express no increase in PCA over basal levels. The PCA induced by MHV-3 is a prothrombinase, encoded by the gene Fgl-2, which encodes a fibrinogen- like protein (musfiblp). In this study, MHV-3-resistant A/J mice treated with methylprednisolone prior to infec- tion with MHV-3 developed elevated levels of alanine aminotransferase in serum and died within 10 days of infection, with histological findings of fulminant hepatitis. In vitro, macrophages isolated from A/J mice and pretreated with methylprednisolone produced a marked increase in functional PCA following infection with MHV-3. The PCA was shown to be a prothrombinase by its ability to cleave 125
alkyl alcohols, and exhibited more potent antioxidant and antiproliferative activities [6–8]. The fruits of this plants contained flavonoids, polyphenols, glycosides, triterpe- noids, steroids and fatty acid esters, and exhibited antiox- idant activity [9–13].The leaf of apple has been reported to contain high levels of polyphenols, flavonoids, and exhibit great in vitro antioxidant activity and protective effect in reserpine-induced gastric ulcer in mice [14, 15]. However, chemical constituents and pharmacological effects of M. pumila flowers are still uncertain without a clear theoretical evidence.
Infected and control cells (5 × 10 5 ) were washed twice with HEPES buffer (10 mM HEPES, 137 mM sodium chloride, 5 mM calcium chloride, 4 mM potassium chloride, 10 mM glucose, 0.5% bovine serum albumin, pH 7.4), then procoagulantactivity was measured using a standard two-step amidolytic assay based on FXa generation. In brief, recombinant human activated factor VII (FVIIa, final concentration, 1 nM, Hematologic Technologies) and factor X (FX, final concentration, 75 nM, Hematologic Technologies) diluted in HEPES buffer were added to the cells for 15 min at 37°C. Chromogenic substrate (Spectrozyme-FXa, final concentration, 167 μM, American Diagnostica) was then added and the ensuing color change was measured after 10 minutes at an optical density of 405 nm at 37°C with a plate spectrophotometer (Synergy 2, Biotek, Winooski, VT, USA). Optical density results were converted to the amount of FXa generated (nM) based on a standard curve created from serial dilutions of human recombinant FXa (American Diagnostica) that were incubated with the substrate for 10 min. The standard curve was linear between FXa concentrations of 4.69 and 0.29 nM, the latter yielding optical densities more than twice baseline values (infected or control cells with no exogenous FX or chromogenic substrate). For data analysis, values above and below the linearity of the standard curve were standardized to 4.69 and 0 nM FXa, respectively. Human recombinant TF (Innovin, Dade-Behring) with added reagents was included as a positive assay control.
The present study was designed to evaluate the possible effect of liposomes containing FBN on different hemostatic parameters. The activity of liposomes was evaluated using whole blood and mild thrombocytopenic blood (30,000 platelets/ µ L) anticoagulated with low-molecular-weight heparin (LMWH) or citrate (19 mM), depending on the test used. Blood samples were incubated with MLV or MLV-FBN liposomes and the effects on the coagulation system, platelet activation, thrombus formation, and fibrin generation were assessed by different experimental settings.
TF expression is generally suppressed in endothelial cells under normal physiological conditions but is induced by inflammation (Levi and van der Poll, 2005), endotoxin (Colucci et al., 1983), and exposure to air pollution (Karoly et al., 2007; Sun et al., 2008) in vitro and possibly in vivo (Mackman et al., 2007). Activation of TF from injury or pathological conditions is typically balanced by expression of anticoagulant proteins such as TFPI (Crawley and Lane, 2008); however, our findings show lack of parallel upregulation of these anticoagulant proteins resulting in a procoagulant environment that favors thrombin generation. The TF gene in primary endothelial cells contains binding sites for redox- sensitive transcription factors (Herkert and Gorlach, 2002; Moll et al., 1995). Accordingly, we found that in HCAEC, TF mRNA levels following exposure to soluble UF required both H 2 O 2 and superoxide. This finding supports previous studies showing that TF mRNA
expressed in colon cancer stage I. This augment may at least in part due to colon cancer-derived TF + MPs, since tumor-derived MPs are critical sources of TF in cancer and human TF antigen has been demonstrated to be re- leased into the blood from human colon tumor [17, 45, 46]. Increased MP TF activity has been detected in colon cancer patients . However, we found that anti-TF antibody did not significantly prolong the clotting time of MPs, and further coagulation and fibrin inhibition as- says suggest that PS + platelets and MPs seems to be the main source of excessive PCA in colon cancer patients. One possible explanation is that plasma-exposed TF is frequently encrypted with little or no detectable PCA and is decrypted through the availability of clusters of PS . Moreover, our finding is also supported by our previous studies showing that the expression of activated TF overlaps with PS exposure and TF-dependent FXa generation was decreased after blocking PS [13, 47].
Endpoints: During the experiments, animals were euthanized if they had signs of suffering not immediately controlled by anesthetic or analgesic injection. Indeed, following the CLP surgical procedure, we monitored the sepsis severity score described by Shrum et al. . This score takes into account several parameters, including appearance, level of consciousness, activity, response to stimulus, eyes, respiration rates and respiration quality, grading each parameter from 1 to 4. The rat was eutha- nized if a score greater than 21 was reached or if a score > 3 was reached for the rhythm or the respiratory quality. At the end of the experiment, rats were euthanized with intravenous lethal dose of pentobarbital sodium (100 mg/ kg pentobarbital sodium, Ceva Santé Animal, France).
deposition have not been clarified. We find that the procoagulantactivity of human leukocytes is markedly increased after incubation with immunoglobulin and immune complexes. This procoagulantactivity is evident after 4-24 h incubation in the presence of as little as 0.1 mg/ml of autologous, isologous, or heterologous IgG. At least three of the four subclasses of IgG myeloma proteins are effective. Experiments with purified rabbit and rat antibodies demonstrate that enhancement of procoagulantactivity is significantly greater with soluble antigen-antibody complexes than with immunoglobulin alone. In contrast, insoluble complexes are less affective than immunoglobulin alone. Artifacts due to endotoxin contamination of the IgG preparations were excluded on the basis of the differential sensitivities of immunoglobulin and endotoxin to heat and polymyxin B. Evidence is also presented which shows that enhancement of procoagulantactivity involves the production, rather than a simple release, of leukocyte procoagulantactivity in vitro.
dependent on glomerular macrophage infiltration in anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM GN) in rabbits. Expression of PCA on the surface of glomerular macrophages and/or augmentation of intrinsic glomerular cell PCA by macrophage cytokines (such as IL 1) are potential mechanisms by which macrophages may augment glomerular PCA. Macrophages were isolated from glomeruli of rabbits developing anti-GBM GN to measure their PCA expression. These macrophages were characterized by morphological and functional criteria. Glomerular macrophages expressed markedly
or a homodimer of c-Jun, is activated by shear stress, which up- regulates the TRE-containing promoters (30, 34). kB/Rel p50– p65 complex binds to the SSRE of the PDGF-B promoter in response to shear stress (32). It is interesting to note that TRE has no effect in the TF shear inducibility and that kB has only a minor effect; kB may enhance the Sp1 responses to shear stress by providing a higher basal level. Sp1, which is more proximal to the TATA box in the TF gene than any other identified shear stress responsive elements, plays a critical role in shear stress induction. These findings suggest that the ter- tiary structure of the promoter may determine the contribu- tion of individual transcription factors to the promoter activity in response to shear stress. Furthermore, similar cis-elements may result in opposite effects if they are located in distinct genes. Malek et al. have shown that the shear stress downregu- lation of the ET-1 gene is mediated by a fragment between 22.5 kb and 22.9 kb of the ET-1 promoter (24). A GC-rich re- gion is present in this 400 bp region (46), that confers the nega- tive modulation by both shear stress and TPA (24).
Factor VIII in preparations from normal plasma is a large glycoprotein of greater than 2 million molecular weight which elutes in the exclusion volume of 4% agarose gels at an ionic strength of 0.15. Recent studies have demonstrated that the factor VIII in canine and bovine plasma is a macromolecular complex composed of a large inert carrier protein and a noncovalently bound small fragment which contains the procoagulant active site. This complex is dissociable in 0.25 M CaCl 2 , and conditions for its recombination have been reported. The present study reports the dissociation characteristics of normal human factor VIII preparations in 0.25 M CaCl 2 and the ability to achieve quantitative recombination of the dissociated fragments of normal human and bovine factor VIII after the removal of Ca 2+ . The recombination technique was used to characterize further the defect in hemophilia and von Willebrand's disease. Void volume preparations from human hemophilia A - , canine
Factor VIII/von Willebrand factor protein had markedly reduced von Willebrand factor activity in a ristocetin assay. In the second patient the peak of Factor VIII/von Willebrand factor protein, antigen, and procoagulantactivity eluted from a Sepharose 4B column with an estimated molecular weight of approximately half that of normal. This protein had no von Willebrand factor activity. In both patients the reduced Factor VIII/von Willebrand factor protein subunit was indistinguishable from normal on polyacrylamide gel electrophoresis. These studies indicate that in some patients with von Willebrand's disease there is a qualitative defect of the Factor VII/von Willebrand factor protein; the total amount of protein, antigen, and procoagulantactivity are normal while the von Willebrand factor activity is deficient.
Human coagulation factor (F)V is a large plasma glycoprotein that plays an important role in blood coagulation. In plasma, FV circulates as a single-chain procofactor and is activated by thrombin via limited proteolysis [1–3]. Activated FV (FVa) consists of two polypeptide chains (designated as heavy and light chain), which are non-covalently linked via a Ca 2+ ion . FVa serves as the non-enzymatic cofactor of the serine protease Xa, which is responsible for the proteolytic activation of prothrombin in the prothrombinase complex, consisting of FVa, FXa, and negatively charged phospholipids. Down- regulation of FVa is mediated by activated protein C (APC) through proteolytic cleavage of the heavy chain of FVa at Arg306, Arg506 and Arg679 . Procofactor FV contains also APC-cofactor activity in the inactivation of FVIIIa [6,7].
XIa, factor Xa, and leukocyte elastase were 182, 302, and 273 nM, respectively. Moreover, this tri- ple point mutant prolonged the activated partial thromboplastin time and moderately reduced leu- kocyte elastase-induced endothelial injury. Addi- tionally, favorable conformations created by the- se mutations were speculated using the struc- ture of the Kunitz protease inhibitor domain of protease nexin 2 complexed with factor XIa as a reference. We discovered a novel triple point mu- tant of the second domain of bikunin that has potent inhibitory activities against factor XIa, fac- tor Xa, and leukocyte elastase. This variant exhi- bited anticoagulant activity in plasma and sup- pressed endothelial cell injury.
When Factor VIII/von Willebrand factor (FVIII/vWF) protein is rechromatographed on 4% agarose in 0.25 M CaCl 2 , the protein and vWF activity appear in the void volume, but most of the FVIII procoagulantactivity elutes later. Recent evidence suggests that the delayed FVIII procoagulantactivity is a proteolytically modified form of FVIII/vWF protein that filters anomalously from agarose in 0.25 M CaCl 2 . To test whether or not thrombin is the protease involved, the effect of 0.25 M CaCl 2 on FVIII/vWF and its reaction with thrombin was