Procoagulant Activity Of Cells Research Articles

IONIZING RADIATION INCREASES CONCENTRATION OF PLASMA VON WILLEBRAND FACTOR IN CEBUS APELLA PARAGUAYANUS MONKEYS

Radiation damage to the vasculature results in a decrease in the anticoagulant activity and an increase in the procoagulant activity of endothelial cells [ 1 ELDOR A. VLODAVSKY I. HYAM E. ATZMON R. FUKS Z. The effect of radiation on prostacyclin (PGI2) production by cultured endothelial cells. Prostaglandins. 1983; 25: 263-279 Crossref PubMed Scopus (43) Google Scholar , 2 SPORN I.A. RUBIN P. MARDER V.J. WAGNER D.D. Irradiation induces release of von Willebrand protein from endothelial cells in culture. Blood. 1984; 64: 567-570 Crossref PubMed Google Scholar , 3 RAYMOND J. YOON S.C. TS'AO C.H. Stimulation of radiation impaired plasminogen activator release by phorbol ester in aortic endothelial cells. Proc Soc Exp Biol Med. 1990; 195: 213-217 Crossref PubMed Scopus (8) Google Scholar ]. The effects of ionizing radiation on endothelial cell functions have been studied “in vitro” and include: decreased capacity to secrete plasminogen activator [ 4 TS'AO C. WARD W.F. Acute radiation effects on the content and release of plasminogen activator in cultured aortic endothelial cells. Radiat Res. 1985; 101: 394-401 Crossref PubMed Scopus (33) Google Scholar ] and prostacyclin production [ 1 ELDOR A. VLODAVSKY I. HYAM E. ATZMON R. FUKS Z. The effect of radiation on prostacyclin (PGI2) production by cultured endothelial cells. Prostaglandins. 1983; 25: 263-279 Crossref PubMed Scopus (43) Google Scholar ], loss of thrombomodulin activity [ 5 ZHOU Q. ZHOA Y. LI P. BAI X. RUAN C. Thrombomodulin as a marker of radiation-induced endothelial cell injury. Radiat Res. 1992; 131: 285-289 Crossref PubMed Scopus (59) Google Scholar ] and increased von Willebrand factor (vWf) release [ 6 VERHEIJ M. DEWIT L.G.H. BOOMGAARD M.N. BRINKMAN H.J.M. VAN MOURIK J.A. Ionizing radiation enhances platelet adhesion to the extracellular matrix of human endothelial cells by an increase in the release of von Willebrand Factor. Radiat Res. 1994; 137: 202-207 Crossref PubMed Scopus (100) Google Scholar ]. Radiation has been also shown to induce increased release of vWf from cultured human umbilical vein endothelial cells into the medium 48 hr after a single dose of 20 Gy [ 2 SPORN I.A. RUBIN P. MARDER V.J. WAGNER D.D. Irradiation induces release of von Willebrand protein from endothelial cells in culture. Blood. 1984; 64: 567-570 Crossref PubMed Google Scholar ]. “In vivo” effects were studied in four Cebus Apella Paraguayanus monkeys exposed to a dose of 4 Gy. We evaluated changes in t-PA, fibrinogen and vWf before and after radiation.

Dilazep, an Antiplatelet Agent, Inhibits Tissue Factor Expression in Endothelial Cells and Monocytes

AbstractDilazep, an antiplatelet agent, is generally used as an antithrombotic drug in clinical practice. Dilazep is also known to exert cytoprotective and antioxidant effects on endothelial cells. However, its effect on the endothelial or monocyte procoagulant activity is unknown. In the current study, the effect of dilazep on the expression of tissue factor (TF ) in human umbilical vein endothelial cells (HUVECs) after the stimulation with tumor necrosis factor-α (TNF ), thrombin, or phorbol 12-myristate 13-acetate (PMA) was evaluated. We also evaluated the effect of dilazep on TNF (1,000 U/mL)-induced TF expression on monocytes. Dilazep inhibited TF activity induced on HUVECs by each stimulant, TNF (1000 U/mL), thrombin (25 nmol/L), or PMA (5 nmol/L) in a dose-dependent fashion (1 to 100 μg/mL). TF activity decreased to approximately 10% after treating with 100 μg/mL of dilazep. Dilazep also blocked the expression of TF antigen induced by each stimulant on the surface of HUVECs as determined by flow cytometric analysis. In addition, in HUVECs, it significantly decreased the expression of TF mRNA and the total TF antigen induced by thrombin or PMA, but not those induced by TNF, suggesting that dilazep blocks the TF expression induced by PMA or thrombin at a transcriptional level and that induced by TNF at a posttranscriptional level. Western blot analysis showed that dilazep reduces the accumulation of native TF but increases that in lower molecular weight TF derivatives. The adenosine receptor antagonist, 8-(p-sulfophenyl) theophylline, partially counteracted the anticoagulant activity of dilazep on HUVECs, thereby suggesting that the inhibitory effect of dilazep on TF expression in HUVECs depends, at least in part, on its adenosine potentiating activity. Dilazep also inhibited TNF-induced TF expression on monocytes in a dose-dependent fashion (0.1 to 100 μg/mL). In brief, the current study showed for the first time that dilazep, a commonly used antiplatelet drug, strongly inhibits the TF expression in HUVECs and monocytes. Dilazep may have a potent therapeutic value in patients with hypercoagulable state for its inhibitory property on the procoagulant activity of endothelial cells and monocytes.

Dilazep, an Antiplatelet Agent, Inhibits Tissue Factor Expression in Endothelial Cells and Monocytes

Dilazep, an antiplatelet agent, is generally used as an antithrombotic drug in clinical practice. Dilazep is also known to exert cytoprotective and antioxidant effects on endothelial cells. However, its effect on the endothelial or monocyte procoagulant activity is unknown. In the current study, the effect of dilazep on the expression of tissue factor (TF ) in human umbilical vein endothelial cells (HUVECs) after the stimulation with tumor necrosis factor-α (TNF ), thrombin, or phorbol 12-myristate 13-acetate (PMA) was evaluated. We also evaluated the effect of dilazep on TNF (1,000 U/mL)-induced TF expression on monocytes. Dilazep inhibited TF activity induced on HUVECs by each stimulant, TNF (1000 U/mL), thrombin (25 nmol/L), or PMA (5 nmol/L) in a dose-dependent fashion (1 to 100 μg/mL). TF activity decreased to approximately 10% after treating with 100 μg/mL of dilazep. Dilazep also blocked the expression of TF antigen induced by each stimulant on the surface of HUVECs as determined by flow cytometric analysis. In addition, in HUVECs, it significantly decreased the expression of TF mRNA and the total TF antigen induced by thrombin or PMA, but not those induced by TNF, suggesting that dilazep blocks the TF expression induced by PMA or thrombin at a transcriptional level and that induced by TNF at a posttranscriptional level. Western blot analysis showed that dilazep reduces the accumulation of native TF but increases that in lower molecular weight TF derivatives. The adenosine receptor antagonist, 8-(p-sulfophenyl) theophylline, partially counteracted the anticoagulant activity of dilazep on HUVECs, thereby suggesting that the inhibitory effect of dilazep on TF expression in HUVECs depends, at least in part, on its adenosine potentiating activity. Dilazep also inhibited TNF-induced TF expression on monocytes in a dose-dependent fashion (0.1 to 100 μg/mL). In brief, the current study showed for the first time that dilazep, a commonly used antiplatelet drug, strongly inhibits the TF expression in HUVECs and monocytes. Dilazep may have a potent therapeutic value in patients with hypercoagulable state for its inhibitory property on the procoagulant activity of endothelial cells and monocytes.

Effects of iron on bacterial endotoxin

Bacterial endotoxin, a major modulator of morbidity and mortality during bacterial sepsis, has affinity for cations. Binding of certain cations to LPS can cause alterations of LPS size and ultrastructure, and can affect LPS biological potency. Although the addition of iron to LPS has been shown previously to result in decreased LPSmediated lethality in mice, it is not certain whether this represents a direct effect of iron on LPS biological activity. To examine this possibility, we measured binding of ferrous or ferric iron to Escherichia coli LPS in vitro. 1.5-2 moles of iron (regardless of oxidation state) was shown to bind per mole LPS. Binding of iron to LPS produced a dose-dependent decrease in two measures of LPS biological activity, activation of Limulus amebocyte lysate (which decreased to 10% of control when LPS was saturated with Fe) and stimulation of endothelial cell procoagulant activity (which decreased to 1-10% of control when LPS was saturated with Fe).

Induction of human endothelial tissue factor expression byNeisseria meningitidis: the influence of bacterial killing and adherence to the endothelium

Tissue factor (TF), a small membrane bound high affinity receptor for factor VII, has an important procoagulant role in the haemostatic dysfunction associated with severe sepsis. Using anin vitromodel of human endothelial TF expression, defined strains ofNeisseria meningitidiswere found to upregulate endothelial cell procoagulant activity (PCA) in a dose dependent manner. This TF response was detected with as little as 104cfu/ml and reached similar levels to those seen with high concentrations of purified endotoxin (>1 ng/ml). Treatment ofN. meningitidiswith either adult donor immune serum, penicillin or gentamicin failed to enhance this PCA. Limulus amoebocyte lysate assay of lipopolysaccharide in bacterial culture filtrates together with polymyxin B inhibition experiments suggest that endotoxin is largely responsible for endothelial TF induction byN. meningitidis. Incubation of endothelial cells withN. meningitidisB1940 and B1940siaD- (an eight-fold more adherent unencapsulated isogenic strain), revealed a significantly greater TF response to B1940siaD- (P<0.01). In conclusion, bacterial adhesion to the vessel wall and therefore local levels of endotoxin may be important determinants of the endothelial procoagulant response toN. meningitidisand the consequent coagulopathy commonly associated with the disease.

Shear stress induction of the tissue factor gene.

Using flow channel, we report that the application of a laminar shear stress induced a transient increase of tissue factor (TF) procoagulant activity in human umbilical vein endothelial cells (HUVEC), which was accompanied by a rapid and transient induction of the TF mRNA in the HUVEC. Functional analysis of the 2.2 kb TF 5' promoter indicated that a GC-rich region containing three copies each of the EGR-1 and Sp1 sites was required for induction. Mutation of the Sp1 sites, but not the EGR-1 sites, attenuated the response of TF promoter to shear stress. Thus, Sp1 is a newly defined shear stress responsive element. Electrophoretic mobility shift assays showed there was no increase in binding of nuclear extracts from sheared cells to an Sp1 consensus site. In contrast, immunoblotting of these nuclear extracts with antibody against transcription factor Sp1 demonstrated that shear stress increased the phosphorylation of Sp1. We also showed that shear stress, like the phosphatase inhibitor okadaic acid, increased the transcriptional activity of Sp1. These findings suggest that the shear stress induction of TF gene expression is mediated through an increased Sp1 transcriptional activity with a concomitant hyperphosphorylation of Sp1.

Effects of pentosan polysulfate on peripheral blood leukocyte populations and mononuclear cell procoagulant activity in patients with osteoarthritis

Pentosan polysulfate (PPS) is a new antiarthritis drug whose mechanism of action is still under investigation. The objective of this study was to determine the effects of PPS on peripheral blood mononuclear cell procoagulant activity (MPA) and individual blood leukocyte populations in 11 patients with osteoarthritis (OA) before treatment, over 24 hours, and 4 weeks after four weekly intramuscular injections (3 mg/kg) of the drug. In all patients, MPA was reduced statistically significantly, relative to baseline values, for up to 24 hours after a single PPS injection. However, in those patients with moderate OA (Kellgren and Lawrence, grade 2), this reduction in MPA was significantly sustained for up to 4 weeks after the last injection of PPS. Following the initial PPS injection the levels of circulating leukocytes increased in the blood of all OA patients, reaching a maximum after 8 hours. The major cellular contribution to this elevation was from lymphocytes, because monocyte levels initially decreased and neutrophils remained unchanged. Four weeks after the last of the four PPS injections, lymphocyte populations were still significantly increased, whereas neutrophils and monocyte levels were decreased in the majority of patients compared with their baseline values. Although platelet levels remained unchanged during the initial 24 hours after PPS injection, a small but statistically significant increase was observed 4 weeks after the last injection. These data demonstrate the ability of PPS to selectively recruit lymphocytes into the circulation and modulate the expression of MPA. We suggest that these activities may contribute to the mechanism of action of the drug.

Ionizing Irradiation Increases Transcription of the von Willebrand Factor Gene in Endothelial Cells

Ionizing irradiation damage to the vasculature results in an increase in procoagulant activity of endothelial cells, including elevated von Willebrand factor (vWf) secretion. We investigated the mechanism of irradiation induction of vWf release and demonstrated that vWf mRNA levels were increased when either human or bovine endothelial cells were exposed to 20 Gy irradiation. This response to irradiation was independent to de novo protein synthesis, but required new transcription. Nuclear run-on experiments indicated that increased vWf transcriptional activity was partly responsible for the higher levels of the mRNA accumulation. Transfection analyses with plasmids in which a human growth hormone structural gene was under the control of the endothelial-cell-specific vWf promoter demonstrated that irradiation increased promoter activity. Deletion analyses demonstrated that sequences necessary for irradiation induction of the promoter activity were located within the 112-bp sequences (-90 to +22) that constitute the non-endothelial-cell-specific core promoter region of the vWf gene. Results of gel mobility assays and deletion analyses demonstrated that a site in the vWf promoter other than the putative NF-kB binding site is involved in the mechanism of irradiation induction of the vWf.

Modulation of human endothelial cell procoagulant activity in tumour models in vitro

Modulation of human endothelial cell procoagulant activity in tumour models in vitro

Modulation of human endothelial cell procoagulant activity in tumour models in vitro.

Several tumour-derived factors have recently been identified which induce tissue factor (TF) expression in endothelial cells in vitro. However, there is only limited evidence that endothelial cells lining tumour blood vessels express elevated procoagulant activity (PCA) in vivo. We have investigated the effects of human breast and small cell lung cancer cell lines on the PCA of human micro- and macrovessel endothelial cell monolayers using a one-stage clotting assay, as well as detection of TF mRNA by RT-PCR. Only conditioned medium from the MDA-MB-231 breast adenocarcinoma cell line produced a consistent although transient increase in endothelial cell surface PCA, which was maximal by 6-9 hr. TF mRNA was detectable in the endothelial cells after 1 hr incubation with MDA-MB-231-conditioned medium and subsequently fell below detectable levels. Following 24 hr stimulation, nearly half the endothelial cell PCA was due to the presence of TF-containing membrane vesicles shed by the MDA-MB-231 cells. Consistent with these findings, the MDA-MB-231 cell line expressed high levels of cell surface-associated TF activity. Co-culture of MDA-MB-231 and endothelial cells for up to 5 days increased (approx. 118-fold) PCA associated with endothelial cell monolayers, due mainly to sequestration of shed tumour cell vesicles. Our results suggest that induction of TF de novo is not a common feature in the supporting endothelium of these tumour types.

COMPARISON OF THE CAPACITY OF rhTNF-α AND ESCHERICHIA COLI TO INDUCE PROCOAGULANT ACTIVITY BY BABOON MONONUCLEAR CELLS IN VIVO AND IN VITRO

The procoagulant activity of mononuclear cells (MNCs) may play an important role in the disseminated intravascular coagulation seen in septic shock. This study compares the capacity of Escherichia coli (E. coli) and recombinant human TNF-alpha (rhTNF-alpha) to induce procoagulant activity by baboon MNCs. In vivo studies showed that MNC procoagulant activity was significantly increased at T + 120 min after LD100 E. coli infusion into baboons. Most of this procoagulant activity was attributable to tissue factor. In contrast, a bolus infusion of rhTNF-alpha (150 micrograms/kg) and a monoclonal antibody to activated protein C (2 mg/kg) did not induce any increase of MNC procoagulant activity at T + 120 min even though the plasma TNF-alpha level was 10 times higher than that seen following infusion of E. coli. In vitro studies showed that E. coli at concentrations comparable to that observed in the vivo study and LPS at a concentration of 2.5 ng/mL induced more intense tissue factor expression by both human and baboon monocytes than rhTNF-alpha in the concentrations ranging from 10 to 1,000 ng/mL. These results suggest that TNF-alpha alone is not sufficient to induced noticeable MNC procoagulant activity, at least, in the early stage of this septic shock model.

All-Trans-Retinoic Acid Counteracts Endothelial Cell Procoagulant Activity Induced by a Human Promyelocytic Leukemia-Derived Cell Line (NB4)

Therapy with all-trans-retinoic acid (ATRA) can rapidly improve the coagulopathy of acute promyelocytic leukemia (APL). This study was designed to evaluate whether the APL cell line NB4 induces the procoagulant activity (PCA) of human endothelial cells (ECs) in vitro, and whether this property is modified after ATRA-induced NB4 maturation. EC monolayers were incubated for 4 hours at 37 degrees C with the conditioned media (CM) of NB4 treated with 1 mumol/L ATRA (ATRA-NB4-CM) or the vehicle (control-NB4-CM). EC lysates were tested for PCA. ATRA-NB4-CM induced significantly more PCA:tissue factor (TF) than control-NB4-CM (P < .01). To identify the cause of TF induction, interleukin (IL)-1 beta antigen levels were measured in CM samples. ATRA-NB4-CM contained significantly more IL-1 beta than control-NB4-CM. EC PCA was significantly inhibited by an anti-IL-1 beta antibody. The addition to the media of 10 mumol/L ATRA counteracted the EC TF expression induced by NB4-CM. These data indicate that ATRA increases the promyelocyte-induced EC TF, partly through increased IL-1 beta production. However, ATRA can protect the endothelium from the procoagulant stimulus of leukemic cells.

Procoagulant activity of mononuclear cells is increased in myeloproliferative and myelodysplastic diseases.

Procoagulant activity (PCA) of peripheral mononuclear cells (PMC) was evaluated in patients with primary thrombocythemia (PT, group A), polycythemia vera (PV), idiopathic myelofibrosis (IM) and myelodysplastic syndromes (group B), and in 15 healthy subjects as control group. PCA of PMC was assayed under basal conditions and after agonist-induced stimulation: bacterial lipopolysaccharide, glycosylated granulocyte-macrophage colony-stimulating factor, recombinant alpha-interferon. PCA was similar in the control group and group A when no stimulation was used, while PCA was found significantly higher in group B patients in the same conditions. In group A patients and in the control group, but not in group B patients, a lower PCA expression was found when PMC were simultaneously coincubated with LPS and alpha-interferon with respect to LPS incubation alone.

Protein kinase c regulates cytokine-induced tissue factor transcription and procoagulant activity in human endothelial cells

Interleukin-lα (IL-lα) and tumor necrosis factor-α (TNF-α) induce tissue factor in endothelium, which results in activation of the coagulation cascade. Despite extensive investigation, in which various stimuli that induce tissue factor have been defined, the intracellular processes that control tissue factor expression are not well understood. It has been proposed that protein kinase C regulates tissue factor expression primarily because phorbol myristate acetate, the protein kinase C activator, induces tissue factor expression. In this study we examined whether IL-1α- or TNF-α-stimulated tissue factor production is regulated through a protein kinase C-dependent mechanism. Northern blot analysis showed that cytokine-induced tissue factor mRNA was significantly reduced in human umbilical vein endothelial cells treated with calphostin C, a specific protein kinase C inhibitor. Tissue factor functional activity was decreased in the presence of calphostin C as well. Calphostin C also inhibited phorbol myristate acetate-induced tissue factor expression. In contrast, calphostin C did not alter cytokine induction of E-selectin or prostacyclin release. Because calcium stimulates protein kinase C binding to the membrane and its resulting catalytic activity, human umbilical vein endothelial cells were exposed to IL-1α or TNF-α in the presence of calcium ionophore A23187. A23187 had little effect alone but significantly augmented cytokine stimulation of tissue factor mRNA. Okadaic acid, a phosphatase inhibitor, increased cytokine-induced tissue factor mRNA compared with cytokine alone, which suggests that a phosphorylation event is important in tissue factor expression. These results indicate that protein kinase C is involved in cytokine activation of endothelial cell tissue factor expression.

Hemoglobin Enhances the Binding of Bacterial Endotoxin to Human Endothelial Cells

Human endothelial cells, when incubated with bacterial endotoxin (lipopolysaccharide, LPS), modify their surface in association with prominent production of procoagulant tissue factor (TF) activity. This deleterious biological effect of LPS has been shown previously to be enhanced approximately 10-fold by the presence of hemoglobin (Hb), a recently recognized LPS binding protein that causes disaggregation of LPS and increases the biological activity of LPS in a number of in vitro assays. The present study was performed to test the hypothesis that Hb enhances the LPS-induced procoagulant activity of human umbilical vein endothelial cells (HUVEC) by increasing LPS binding to the cells. The binding of 3H-LPS to HUVEC was determined in the absence or presence of Hb or two other known LPS-binding proteins, human serum albumin (HSA) and IgG. LPS binding was substantially increased in the presence of Hb, in a Hb concentration-dependent manner, but was not increased by HSA or IgG. Hb enhancement of LPS binding was observed in serum-free medium, indicating that there was no additional requirement for any of the serum factors known to participate in the interaction of LPS with cells (e.g., lipopolysaccharide (LPS)-binding protein (LBP) and soluble CD 14 (sCD14)). Hb enhancement of LPS binding also was observed in the more physiologic condition of 100% plasma. LPS-induced TF activity was stimulated by Hb, but not by HSA or IgG. In serum-free medium, TF activity was not stimulated under any of the conditions tested. Ultrafiltration of LPS was dramatically increased after incubation with Hb but not with HSA or IgG, suggesting that LPS disaggregation by Hb was responsible for the enhanced binding of LPS to HUVEC and the subsequent stimulation of TF activity.

Effects of tissue factor induced by oxygen free radicals on coronary flow during reperfusion.

Tissue factor is a transmembrane protein that activates the extrinsic coagulation pathway by binding factor VII. Endothelial cells, being in contact with circulating blood, do not normally express tissue factor. Here we provide evidence that oxygen free radicals induce tissue factor messenger RNA transcription and expression of tissue factor procoagulant activity in endothelial cells in culture. Isolated, perfused rabbit hearts exposed to exogenous oxygen free radicals also showed a marked increase in tissue factor activity within the coronary circulation. Furthermore, in ex vivo and in vivo hearts subjected to ischemia and reperfusion, a condition associated with a production of oxygen free radicals in large amounts, a marked increase in tissue factor activity occurred. This phenomenon could be abolished by oxygen radical scavengers. This increase in tissue factor activity during postischemic reperfusion was accompanied by a significant decrease in coronary flow, suggesting that increase in tissue factor activity with the consequent activation of the coagulation cascade might impair coronary flow during reperfusion and possibly contribute to the occurrence of reperfusion injury.

Increased Procoagulant Activity of Red Blood Cells from Patients with Homozygous Sickle Cell Disease and β-Thalassemia

It has recently been proved that, in vitro, red blood cells (RBCs) from patients with homozygous beta-thalassemia behave as procoagulant cells. The procoagulant activity of beta-thalassemia RBCs might be the result of an increased exposure of procoagulant phospholipids (i.e. phosphatidylserine) in the outer leaflet of the membrane. In order to test this hypothesis, we compared the catalytic properties of RBCs of patients with beta-thalassemia and homozygous sickle cell disease (SS-RBCs) with that of controls. The catalytic parameters (Km, kcat) of prothrombin activation by factor Xa were determined both in the absence and in the presence of RBCs. The turn-over number (kcat) of the reaction was not modified by normal, SS- or beta-thalassemia RBCs. The Km was lower in the presence of normal RBCs (mean value: 9.1 microM) than in the absence of cells (26 microM). The Km measured in the presence of either SS-RBCs (mean value: 1.6 microM) or beta-thalassemia RBCs (mean value: 1.5 microM) was significantly lower compared to normal RBCs (p < 0.001). No significant difference was observed between SS-RBCs and beta-thalassemia RBCs. Annexin V, a protein with high affinity and specificity for anionic phospholipids, inhibited the procoagulant activity of both SS-RBCs and beta-thalassemia RBCs, in a dose-dependent manner. More than 95% inhibition was achieved at nanomolar concentrations of annexin V. These results indicate that the procoagulant activity of both beta-thalassemia RBCs and SS-RBCs may be fully ascribed to an abnormal exposure of phosphatidylserine at the outer surface of the red cells.

Inhibition of factor Xa-mediated procoagulant activity of human lung fibroblasts and pleural mesothelial cells.

Extravascular fibrin deposition characterizes diverse forms of lung and pleural injury. Fibrin formation in these compartments is locally potentiated by the assembly and expression of the prothrombinase procoagulant complex (factors Xa, Va and II) at the surface of human lung fibroblasts and pleural mesothelial cells. We sought to identify structural domains on factor Xa that mediate expression of prothrombinase activity by these cells. In order to accomplish this objective, we used panels of monoclonal antibodies (MoAbs) to factor X to block prothrombinase assembly and function on the surface of cultured human lung fibroblasts and pleural mesothelial cells. Of 30 factor X MoAbs that recognized native factors X and Xa, 10 completely inhibited factor Xa function (prothrombin activation), and five others neutralized Xa function without affecting cell-binding, presumably by blocking the prothrombin binding site. Western blots showed that these inhibitory MoAbs reacted with the Xa heavy-chain. One MoAb that recognized the factor Xa light-chain blocked prothrombin activation at the factor Va binding site. Our results indicate that prothrombinase activity at the surface of lung parachymal or pleural cells can be blocked by MoAbs that interact with either the heavy- or light-chain of factors X. Antibodies that neutralize cell surface-expressed prothrombin activation offer a potential means to arrest pericellular fibrin formation in the lung and pleural space.

A simplified and low-cost one-stage chromogenic assay for tissue factor dependent procoagulant activity of endothelial cells

We developed a sensitive tissue factor (TF) chromogenic assay on a limited number of endothelial cells (EC), performed in microtiter plates, and which uses normal pooled human plasma instead of purified concentrates as a source of coagulation factors. Primary cultures of human umbilical vein EC (HUVEC), both unstimulated and stimulated by lipopolysaccharide (LPS), were incubated with 50 μl of diluted normal human plasma (NHP) and 50 μl of Factor Xa-specific chromogenic substrate (CBS 31–39, Stago, France). Hirudin was added at 4 U/ml to the plasma/CBS 31–39 mixture to inhibit thrombin generation. Optical densities were read at 405 nm and corresponding amounts of generated factor Xa were expressed in mU Xa/well using a standard curve established with purified human Factor Xa. The following parameters were then defined: the number of EC to plate (10 4 EC/well of a 96-well plate), the plasma-test dilution (1:20), the concentration of CBS 31–39 (0.50 mM) and the incubation time of reagents with EC (2 hours). The procoagulant activity (PCA) measured was only dependent on TF since it was no longer detectable either when FVII-deficient plasma was tested instead of normal human plasma or when PCA assays were performed in the presence of a blocking anti-human TF monoclonal antibody. This method allowed detection of a TF-dependent PCA on as few as 1000 EC per well. In addition, TF expression equal to 50% of maximal values was measured with LPS concentrations as low as 1 ng/ml, supporting the high sensitivity of the assay.

Blood and tissue fibrinolytic profiles in patients with colorectal carcinoma

In patients with colorectal cancer, profound alterations of the plasminogen activator system have been described at the tumor level, but conflicting results have been obtained for fibrinolytic parameters in plasma. Components of the fibrinolytic system, including tissue-type and urokinase-type plasminogen activators and their inhibitors type 1 and 2, were measured in tissue and/or plasma from 41 patients with colorectal cancer and in 40 controls. Procoagulant activity of freshly isolated mononuclear cells (basal activity) and the procoagulant activity and fibrinolytic proteins produced by the cells after incubation for 18 h without exogenous stimulation were also evaluated. Malignant tissue extracts had significantly higher levels of urokinase-type plasminogen activator and plasminogen activator inhibitor-1, but lower levels of tissue-type plasminogen activator than normal mucosa. Plasminogen activator inhibitor-1 alone was higher in advanced (Dukes' stages C + D) than limited (B) tumors. Plasminogen activator inhibitor-2 was not different in malignant tissue and normal mucosa. Plasma levels of plasminogen activator inhibitor-1 antigen were significantly increased in cancer patients compared with controls, but there were no differences in tissue-type and urokinase-type plasminogen activator, in plasminogen activator inhibitor-2, and D-dimer levels. Intra-patient analysis revealed no significant correlation between tumor and plasma levels of plasminogen activators or type 1 inhibitor. Tissue-type plasminogen activator, but not the urokinase type or inhibitor type 1, was higher in venous than in arterial blood collected at the tumor site during surgery. Basal procoagulant activity of mononuclear cells and the procoagulant activity and inhibitor type-2 produced by the cells after short-term culture were comparable in patients and controls. These findings indicate that, at least in our patients with colorectal cancer, the profound changes occurring at tumor level are barely detectable in the blood. Thus, the clinical relevance of plasma fibrinolytic parameters, especially urokinase-type plasminogen activator antigen, as tumor markers in colorectal cancer remains to be established.