Monocyte Procoagulant Activity Research Articles

MAP-Kinase Dependent Induction of Monocytic Procoagulant Activity by β2-Integrins

β2-Integrin adhesion molecules play crucial roles in monocyte transmigration and adherence to the inflamed extracellular matrix. While integrin engagement contributes to inflammatory cell activation, little is known about the precise signaling pathways that are important to integrin-dependent monocyte activation. We examined the role of tyrosine phosphorylation and extracellular-signal regulated kinase (ERK) activity in β2-integrin signaling in monocytes. Cross-linking of the LFA-1 (CD11a/CD18) and MAC-1 (CD11b/CD18) integrins on the surface of THP-1 monocytic cells induced the accumulation of tyrosine phosphoproteins. As part of this signal both ERK-1 and ERK-2 are tyrosine phosphorylated.In vitrokinase assays documented an increase in ERK-2 activity following both LFA-1 and MAC-1 cross-linking. β2-Integrin cross-linking also led to a marked increase in 4-h procoagulant activity (PCA) in THP-1 cells and purified human monocytes. Inhibition of tyrosine phosphorylation by genistein (10 μg/ml), or selective ERK inhibition with PD98059 (10 μM), was able to block the integrin-dependent induction of PCA in both THP-1 cells and human monocytes. Thus, β2integrin signaling in monocytic cells can flow through the tyrosine phosphorylation and activation of the ERK mitogen activated protein kinases, which is essential for the subsequent expression of tissue factor. These results suggest that the ERK proteins likely function to integrate various adhesion-dependent signals during the process of monocyte transmigration.

Selective anticoagulation with active site-blocked factor IXa suggests separate roles for intrinsic and extrinsic coagulation pathways in cardiopulmonary bypass

Background: Multiple stimuli converge in cardiopulmonary bypass to create a tremendous prothrombotic stimulus. The ideal anticoagulant for cardiopulmonary bypass should selectively target only the intravascular stimuli, thereby eliminating pathologic clotting in the bypass circuit while preserving hemostasis in the thoracic cavity. We propose the inhibition of factor IX as such a targeted anticoagulant strategy. Methods: We prepared an inhibitor of activated factor IX and applied it to a primate model of cardiopulmonary bypass to confirm the anticoagulant efficacy of activated factor IX in this setting and to assess more subtle markers of thrombin generation, macrophage procoagulant activity, and cellular tissue factor expression. Seven baboons that received activated factor IX (460 μg/kg) and 7 that received heparin (300 IU/kg) and protamine underwent cardiopulmonary bypass for 90 minutes and were followed after the operation for 3 hours. Results: Analysis of plasma factor IX activity demonstrated adequate inhibition (<20%) of factor IX throughout cardiopulmonary bypass. Activated factor IX-treated baboons demonstrated similar circuit patency to heparin-treated baboons but had significantly diminished intraoperative blood loss. Preservation of extravascular hemostasis was further demonstrated in activated factor IX-treated animals by (1) significantly increased levels of thrombin-antithrombin III complex and prothrombin activation peptide (F 1+2) without intravascular thrombosis, (2) significantly greater macrophage procoagulant activity in pericardial-derived monocytes, and (3) immunohistochemical evidence of tissue factor expression in pericardial mesothelial cells and macrophages. Conclusions: Anticoagulation with activated factor IX allows for intravascular anticoagulation with maintenance of extravascular hemostasis. These findings suggest activated factor IX as an agent that not only exemplifies a targeted approach to selective anticoagulation in cardiac surgery but also further characterizes the procoagulant milieu during cardiopulmonary bypass. (J Thorac Cardiovasc Surg 1998;116:860-9)

Procoagulant inflammatory responses of monocytes after direct balloon angioplasty in acute myocardial infarction

This study sought to investigate monocyte procoagulant activity and Mac-1 expression after successful percutaneous transluminal coronary angioplasty (PTCA) in acute myocardial infarction (AMI). An increased leukocyte count is an important risk factor for subsequent adverse cardiac events in AMI. Cellular procoagulant responses may contribute to the risk of thrombotic events after AMI. In 20 patients with AMI serial venous blood samples were obtained before, 4, 8 hours, and daily after direct PTCA. Twenty patients with elective PTCA served as a control group. We measured leukocyte procoagulant activity with a 1-stage clotting assay, Mac-1 expression of monocytes by flow cytometry, concentrations of tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and IL-8 using immunoassays. Forty-eight hours after PTCA in patients with AMI, an increase in systemic IL-6 and C-reactive concentrations was found (p = 0.001, p = 0.008) associated with an increase in monocyte Mac-1 expression by 49 ± 18% (p = 0.04) and followed by an increase in monocyte procoagulant activity by 140 ± 63% 72 hours after PTCA (p = 0.01). None of these changes were detectable in the control group. No changes in the concentrations of the cytokines IL-1β, tumor necrosis factor-α, or IL-8 were found. The present study demonstrates an increase in procoagulant activity along with an increase in Mac-1 expression on circulating monocytes after successful PTCA in AMI associated with an increase in systemic IL-6. These cellular procoagulant responses may limit the clinical benefit from timely reperfusion.

Enhancement of monocyte procoagulant activity by adhesion on vascular smooth muscle cells and intercellular adhesion molecule-1-transfected Chinese hamster ovary cells.

Plaque erosion is a frequent finding in sudden death due to coronary thrombosis. The present study sought to investigate whether monocyte adhesion to human aortic vascular smooth muscle cells (VSMCs) induces procoagulant activity (PCA) and whether this could be mediated by intercellular adhesion molecule-1 (ICAM-1). We incubated mononuclear cells (MNCs) with VSMCs and ICAM-1-transfected Chinese hamster ovary (CHO) cells, investigated monocyte tissue factor (TF) mRNA expression by Northern blot analysis and TF protein expression by ELISA, and measured PCA. Incubation of MNCs with VSMCs for 6 hours increased PCA from 0.7+/-0.1 to 166.0+/-37.9 mU/105 cells (P=0.007), which could be inhibited in a dose-dependent manner by the addition of blocking anti-ICAM-1 monoclonal antibodies. Prestimulation of VSMCs with interleukin-1beta enhanced surface ICAM-1 expression significantly but did not induce PCA in VSMCs. Incubation of MNCs with prestimulated VSMCs led to a further increase in PCA to 239.9+/-27.9 mU/10(5) cells (P=0.02 compared with incubation with unstimulated VSMCs). Incubation of MNCs with VSMCs enhanced TF mRNA after 2 hours and significantly increased TF protein content after 6 hours. Incubation of purified monocytes with ICAM-1-transfected CHO cells increased PCA from 1.2+/-0.2 to 81.9+/-3.3 mU/10(5) cells (P<0.001 compared with incubation with untransfected CHO cells) after 6 hours. This effect could be inhibited significantly by the addition of blocking anti-CD18, anti-CD11b, or anti-CD11c monoclonal antibodies. Similar results were obtained for MNCs. Monocyte adhesion to VSMCs induces TF mRNA and protein expression and monocyte PCA, which is regulated by beta2-integrin-mediated monocyte adhesion to ICAM-1 on VSMCs.

Monocyte tissue factor-like activity in post myocardial infarction patients.

It is widely recognized that thrombosis is the major event in the evolution of stable vascular disease to unstable ischaemic syndromes including myocardial infarction and stroke. The purpose of this case-control study was to establish clinical and laboratory data on the possible relationship between specific components of the haemostatic system and coronary heart disease. The procoagulant activity (PCA) of peripheral monocytes and polymorphonuclear neutrophils was assessed in 21 males who had suffered a myocardial infarction (MI) and in age-matched controls. In addition, total factor VII activity, fibrinogen, tissue factor pathway inhibitor (TFPI). D-dimers, tissue plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1), tumour necrosis factor-alpha (TNF-alpha) and full blood counts were measured. Post MI patients had significantly higher monocyte PCA, higher plasma concentrations of TFPI, fibrinogen, t-PA, T/P100 and also higher total white blood cell and neutrophil counts compared to age-matched controls. This elevated procoagulant state in post MI patients could further exacerbate the disease process and increase the risk of subsequent acute ischaemic events.

Microalbuminuria in IDDM is associated with increased expression of monocyte procoagulant activity.

Microalbuminuria, the early phase of diabetic nephropathy, is associated with an increased risk of atherothrombosis. Monocytes play an important part in the pathogenesis of atherosclerosis and in the activation of haemostasis. However, procoagulant activity is poorly understood in Type I (insulin-dependent) diabetes mellitus, particularly in the presence of microalbuminuria. This study aimed to evaluate spontaneous and endotoxin-induced monocyte procoagulant activity in insulin-dependent diabetic patients with normoalbuminuria or microalbuminuria. Seventeen patients with microalbuminuria, 28 with normoalbuminuria and 26 healthy control subjects matched for age, sex, body mass index and smoking habit were studied. Mononuclear cells from peripheral venous blood were incubated with or without bacterial lypopolysaccharide. Spontaneous procoagulant activity and procoagulant activity after 3 h and 6 h of incubation were calculated. Spontaneous procoagulant activity values were similar in the three groups. After 3 h and 6 h incubation with bacterial lypopolysaccharide, procoagulant activity values were slightly, but not statistically significantly, higher in the normoalbuminuric diabetic group than in control group, and significantly higher in microalbuminuric diabetic group than in control group (p < 0.01). The increased endotoxin-induced monocyte procoagulant activity helps to explain the link between microalbuminuria and the increased risk of atherothrombosis in patients with Type I diabetes.

Direct activation of factor X by monocytes occurs during cardiopulmonary bypass.

We hypothesized that monocyte procoagulant activity, which includes up-regulation of tissue factor and direct activation of factor X by CD11b. is an activator of coagulation during cardiopulmonary bypass (CPB), because recent studies have cast doubt on the presumption that the surfaces of CPB activate the intrinsic pathway. Sequential samples were taken from 17 patients undergoing cardiac surgery. During CPB a significant increase in thrombin-antithrombin complexes occurred (P < 0.0005). Factor XIIa levels increased (P < 0.005) but remained within the normal range. Total monocyte procoagulant activity was measured and a functional assay was developed to detect direct activation of factor X alone. There was a significant increase in total procoagulant activity on circulating monocytes from the start of CPB (P < 0.005) to which direct factor X activation was a major contributor (P < 0.005). Direct activation of factor X was inhibited by CD11b blocking peptides. Using flow cytometry, up-regulation of monocyte CD11b (P < 0.0005). but not up-regulation of tissue factor, was found on circulating monocytes. Monocytes adherent on the oxygenator fibres showed increased CD11b expression (P < 0.0001), but no tissue factor when assessed by fluorescent image analysis. In conclusion, direct activation of factor X through monocyte CD11b occurs during CPB and appears to contribute to thrombin generation during CPB.

Dual Regulation of Ligand Binding by CD11b I Domain: INHIBITION OF INTERCELLULAR ADHESION AND MONOCYTE PROCOAGULANT ACTIVITY BY A FACTOR X-DERIVED PEPTIDE

The role of coagulation factor X as a ligand for CD11b/CD18 (Mac-1, alphaMbeta2) in leukocyte adhesion was investigated. A factor X peptide, (G)L238YQAKRFKV246(G), blocked ligand binding to CD11b/CD18 and prevented monocyte procoagulant activity. This peptide also inhibited monocytic THP-1 cell adhesion to tumor necrosis factor alpha-stimulated endothelium and blocked neutrophil migration through tumor necrosis factor alpha-activated endothelial cell monolayers. In contrast, other factor X-derived peptides were ineffective. Radiolabeled peptide (G)LYQAKRFKV(G) bound specifically and saturably to isolated recombinant CD11b I domain. Functionally, the factor X sequence (G)LYQAKRFKV(G) dose-dependently inhibited THP-1 cell attachment to intercellular adhesion molecule 1 (ICAM-1) transfectants (IC50 = approximately 50 microg/ml), indistinguishably from anti-CD18 monoclonal antibodies 60.3 and IB4. In contrast, peptide (G)LYQAKRFKV(G) failed to reduce binding of 125I-fibrinogen to immobilized CD11b I domain, which was abolished by the fibrinogen-derived peptide KYG190WTVFQKRLDGSV202. By Lineweaver-Burke analysis, peptide (G)LYQAKRFKV(G) inhibited factor X binding to CD11b/CD18 in a noncompetitive fashion, and intact factor X did not reduce monocyte-endothelial cell interaction. These data suggest that the factor X sequence (G)LYQAKRFKV(G) defines an ICAM-1-binding site on CD11b I domain physically distinct from and nonoverlapping with the fibrinogen interacting region(s). Engagement of this site induces a conformational change in the holoreceptor, which disrupts a distant factor X-binding site required for monocyte procoagulant activity. These observations demonstrate a dual regulatory role of CD11b I domain in ligand binding and provide a molecular basis for the recently reported anti-inflammatory properties of factor X homologous sequences in vivo.

Granulocyte-macrophage colony-stimulating factor induces activation and restores respiratory burst activity in monocytes from septic patients.

Monocyte activation in response to recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) was examined in vitro in septic shock patients. These monocytes exhibited a greater respiratory burst activity than monocytes from healthy subjects; the response to secondary stimulation with bacterial stimuli was attenuated. GM-CSF restored the ability of monocytes to respond appropriately to secondary stimulation. Expression of certain integrin adhesion molecules, L-selectin, and Fcgamma receptors was increased on monocytes of septic shock patients; expression of CD11c was reduced. GM-CSF up-regulated integrin expression and decreased L-selectin, FcgammaRII, and FcgammaRIII expression. Septic patients exhibited greater biologic activity of monocyte tissue factor than did healthy subjects. Priming monocytes with GM-CSF accelerated tissue factor activation following stimulation with lipopolysaccharide and bacterial culture supernatant. Certain parameters of monocyte function may be restored by exposure to GM-CSF. This benefit may be offset by an increase in monocyte procoagulant activity.

Monocyte Procoagulant Activity Induced by Adherence to an Artificial Surface Is Reduced by End-point Immobilized Heparin-coating of the Surface

Heparin-coating improves the biocompatibility of blood contacting artificial surfaces. This led us to investigate the impact of heparin-coating (Carmeda AB, Stockholm) of polymetylmetacrylate on the expression of monocyte tissue factor procoagulant activity (TF-PCA) by surface adhesion. Also, the anticoagulant effect of heparin-coating in the presence or absence of adherent procoagulant monocytes was assessed. This is of particular interest, since activation of extrinsic coagulation by adherent monocyte TF-PCA may play a significant role in thrombin generation during extracorporeal circulation. Monocytes exposed to heparin-coated or non-coated polymetylmetacrylate expressed TF-PCA. The heparin coat did not affect the rate of monocyte adhesion. However, heparin-coating reduced the induction of TF-PCA of non-adherent and adherent monocytes by 17 and 33% (p <0.001 and p <0.0003), respectively. Heparin-coating in the absence of monocytes, totally inhibited the clotting of recalcified plasma (p <0.003). In contrast, in the presence of adherent monocytes expressing TF-PCA, surface-bound heparin did not inhibit clotting. However, inclusion of heparin in a plasma concentration of 8.9 IU/ml totally inhibited the activation of coagulation. It is apparent that heparin-coating of an artificial surface is an efficient means to inhibit coagulation of recalcified plasma, but much less so when procoagulant monocytes are adherent to the coated surface. The present findings are of clinical relevance, since monocytes will adhere to blood contacting surfaces of extracorporeal circuits or to implanted vascular prostheses and subsequently express TF-PCA, and this may promote thromboembolism.

Effect of human recombinant interleukin-6 and interleukin-8 on monocyte procoagulant activity.

Interleukin (IL)-6 and IL-8 are important regulators of inflammatory responses in myocardial infarction. Induction of monocyte procoagulant activity (PCA) by these cytokines could present a mechanism that links inflammatory responses to thrombotic events. We therefore investigated the effect of IL-6 and IL-8 on monocyte tissue factor (TF) expression. Recombinant human IL-6 and IL-8 caused a time- and dose-dependent increase in PCA (recalcification time) of monocytic U937 cells and of mononuclear leukocytes. Using blocking anti-TF monoclonal antibodies and factor VII-deficient control plasma, this PCA was shown to be TF dependent. Compared with unstimulated cells, mononuclear cell PCA increased by 4.5-fold to 17 +/- 2 mU/5x10(5) cells after exposure to 100 ng/L IL-6 for 4 hours and by 6.6-fold to 27 +/- 4 mU/5x10(5) cells after exposure to IL-8 under the same conditions. Northern blot analysis showed an increase in TF mRNA after stimulation with IL-6 or IL-8 for 2 hours, and after 4 hours an increase in cellular TF protein content was found by immunoassay. Flow cytometry demonstrated that IL-6 and IL-8 induced an increase in TF surface expression on monocytes. Thus, IL-6 and IL-8 induce monocyte PCA by increasing mRNA, protein content, and surface expression of TF.

Simvastatin reduces monocyte-tissue-factor expression type IIa hypercholesterolaemia

Inhibitors of 3-hydroxy-methyl-glutaryl coenzyme A reductase have been shown to reduce cardiovascular events in patients with raised and average serum cholesterol. It is unclear whether this effect is attributable solely to reduction of serum cholesterol or to other mechanisms. We analysed whether simvastatin directly affects expression of monocyte tissue factor (TF). After 4 weeks of diet alone (fat intake <30% of total calories, cholesterol <300 mg daily, polyunsaturated/ saturated fatty acids ratio=1·0) (run-in phase), 24 patients (12 men and 12 women; mean age 52 [SD 8] range 35–70 years) with polygenic hypercholesterolaemia were randomised to simvastatin (20 mg daily) or to continue diet for 8 weeks. Before and after treatment, expression of TF antigen and activity as well as the circulating concentrations of prothrombin fragment F1+2, a marker of thrombin generation, were measured. Nine parts of blood were mixed in a pre-cooled vacutainer (Becton Dickinson Vacutainer System, France) with 1 part of 3·8% sodium citrate and centrifuged for 20 min at 2000 g at –4oC. Plasma samples for measurement of F1+2 (Enzygnost F1+2, Behringwerke ag, Marburg) were stored at –40oC. To measure monocyte TF, peripheral blood mononuclear cells were isolated from heparinised venous blood (2 10 cells/mL) and incubated for 6 h with 0·4 ng/mL lipopolysacchariae (Escherichia coli [OB11:B4, Sigma, St Louis, MI, USA). In the lysate of the cells, TF antigen was measured by an ELISA test (Imubind Tissue Factor ELISA kit, American Diagnostica Inc, Greenwich, CT) and TF activity was determined by measuring monocyte procoagulant activity with one stage clotting assay. Hypercholesterolaemic patients showed significantly higher concentrations of F1+2 (mean [SD] 2·2 [0·4] vs 0·6 [0·3] nmol/L: p<0·0001), TF antigen (median [range] 63 [41–89] vs 16 [10–39] pg/2 10 monocytes; p<0·0001) and activity (median [range] 31 [20–44] vs 9 [4–20] U/2 10 monocytes; p<0·0001) than 24 controls matched for age and sex (unpaired t test and Mann-Whitney U test). Patients randomised to diet or diet plus simvastatin had similar clinical (not shown) and laboratory variables (table). In patients continuing the follow-up by diet alone, no significant changes of the variables were observed. In patients taking simvastatin there was a significant decrease of cholesterol, LDL cholesterol, F1+2, and monocyte TF expression. Comparing the diet and simvastatin groups at the end of the treatment period, simvastatin group showed significantly lower concentrations of F1+2, TF antigen, and activity. This study shows that simvastatin reduces the expression of monocyte TF, an effect which may explain the reduction of cardiovascular events elicited by simvastatin.

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.

Fibrinolysis, inhibitors of blood coagulation, and monocyte derived coagulant activity in acute malaria.

Different parameters of fibrinolytic systems like t-PA, PAI, D-dimer, and inhibitors of blood coagulation, i.e., protein C (PC), protein S(PS), and antithrombin III (AT-III), have been studied in cases of acute malaria due to Plasmodium falciparum and plasmodium vivax infection, and these patients were followed up. It was observed that the plasma PAI-1 was very high in cases of P. falciparum malaria infection as compared to normal controls and P. vivax infection. The changes in complicated cases of P. falciparum were remarkable as compared to uncomplicated ones. The PC, PS, and AT-III levels were also low in P. falciparum, particularly so in complicated cases, and were normal in P. vivax infection. The factor VIII R:Ag levels were invariably high in acute malaria. On follow-up of some of these cases the values came back to normal after the antiparasite treatment. The monocyte procoagulant activity was found to be significantly higher in P. falciparum infection as compared to that of P. vivax infection. All these findings therefore contribute towards the production of a hypercoagulable state in P. falciparum infection and partly explain the complications of P. falciparum infection like cerebral malaria.

CD40 engagement induces monocyte procoagulant activity through an interleukin-10 resistant pathway.

Interactions between membrane-bound molecules were previously shown to be involved in the induction of tissue factor-dependent monocyte procoagulant activity (PCA) by activated T cells. To investigate the potential role of the CD40/CD40 ligand (CD40L) pathway in this process, we first determined the effects of blocking anti-CD40 or anti-CD40L monoclonal antibodies (mAb) on the development of monocyte PCA during mixed lymphocyte reaction (MLR) between allogeneic peripheral blood mononuclear cells (PBMC). The strong inhibitory effect exerted by both mAb (mean percentages of inhibition: 88 and 91% for anti-CD40 and anti-CD40L mAb, respectively) indicates that CD40/CD40L interactions are required for the induction of PCA in MLR. These data led us to measure monocyte PCA after incubation of PBMC or purified monocytes with a stimulating anti-CD40 mAb (BL-C4) or with 3T6 fibroblasts transfected with the gene encoding CD40L. In both systems, we found that CD40 engagement strongly induced monocyte PCA which was related to tissue factor expression as shown by flow cytometric analysis. Finally, we observed that recombinant interleukin (IL)-10, which inhibits lipopolysaccharide-induced PCA, did not significantly influence CD40-dependent PCA. We conclude that CD40 engagement on monocytes induces tissue factor-dependent PCA through an IL-10-resistant pathway. These findings have implications for the control of coagulation events triggered by interactions between T cells and monocytes.

Increased induced monocyte tissue factor expression by plasma from patients with severe ovarian hyperstimulation syndrome

To investigate whether monocyte expression of tissue factor is increased by plasma from patients with severe ovarian hyperstimulation syndrome (OHSS). Prospective longitudinal study. Assisted Reproduction Unit of the Hospital Clínic i Provincial in Barcelona, a tertiary care setting. Nine IVF patients with severe OHSS. Pretreatment with leuprolide acetate was followed by gonadotropin treatment for ovarian follicular stimulation. After administration of hCG, a standard IVF-ET procedure was performed. Measurement during the syndrome and 4 to 5 weeks after recovery of induced monocyte tissue factor expression. In each of the nine patients, plasma obtained during the syndrome induced a significantly higher proportion of monocytes expressing tissue factor and a significantly higher intensity of tissue factor expression on monocytes than plasma obtained after recovery and control plasma. Procoagulant activity of blood monocytes, which is mediated principally by tissue factor expression, is increased in patients with severe OHSS. This fact may be important in thrombotic events associated with the syndrome.

N-3 PUFA supplementation, monocyte PCA expression and interleukin-6 production

n-3 polyunsaturated fatty acids (PUFA) can affect several monocyte functions and the biochemistry of blood cells, thus possibly influencing the initiation of thrombosis, inflammatory disease and atherosclerosis. In this study, we have investigated the effect of dietary supplementation with n-3 PUFA ethyl esters on procoagulant activity (PCA) and interleukin-6 (IL-6) production by human mononuclear cells. Nine healthy volunteers received 4 g/d of n-3 PUFA ethyl esters (4 × 1 g capsules with at least 85% eicosapentaenoic + docosahexaenoic acid ethyl esters) for 18 weeks. Before and at the end of the treatment, mononuclear cells were obtained from peripheral citrated blood by Ficoll-Hypaque density gradient centrifugation. Cellular suspensions (10 7 cells/ml) were incubated at 37°C for 4 h in the absence and presence of lipopolysaccharide (10 μg/ml); PCA was determined by one-stage clotting assay and IL-6 concentrations were assayed in supernatants by specific ELISA. After 18-week treatment, both unstimulated and stimulated monocyte PCA were significantly reduced by 66% and 63%, respectively ( P < 0.01). Similarly, a significant inhibitory effect by n-3 PUFA treatment on basal and LPS-stimulated IL-6 monocyte production was observed (50% and 46%, respectively, P < 0.05). These data indicate that 18-week n-3 PUFA supplementation may influence monocyte activities, which play a specific role in atherosclerosis and its thrombotic complications.

Antiphospholipid antibodies (aPL) increase the potential monocyte procoagulant activity in patients with systemic lupus erythematosus.

Lupus anticoagulant (LA) and anticardiolipin antibodies (aCL) are frequently detected in sera from patients affected by systemic lupus erythematosus (SLE). However, the role of antiphospholipid antibodies (aPL) in thrombus formation has not been defined as yet. Twenty-two patients affected by SLE, all fulfilling the 1982 ARA revised criteria, and twenty healthy subjects were investigated for the presence of LA, aCL and other aPLs. Monocyte procoagulant activity-PCA (Tissue Factor production) was evaluated by one stage plasma recalcification time. In all patients the plasma levels of F1 + 2 and of plasminogen activator inhibitor (PAI) were also determined. Monocyte PCA was significantly higher in SLE patients with LA and/or aCL in comparison to SLE patients without LA and/or aCL (p < 0.01) and to controls (p < 0.05). However, no connection was observed between PCA expression by mononuclear cells and LA or aCL levels. No differences in F1 + 2 and PAI plasma levels were found between SLE patients with or without aPL and controls. In our SLE patients LA and/or aCL positivity appears strictly related to an increased monocyte activation that could play an important role in the occurrence of thrombotic events.

Monocyte Procoagulant Activity Induced by In Vivo Administration of the OKT3 Monoclonal Antibody

The first injection of OKT3 in kidney transplant recipients activates the common pathway of coagulation. This may result in early thrombosis of graft vessels. To this day, the cells involved in this phenomenon have not been identified. The aim of this study was to investigate whether circulating monocytes participated in this OKT3-induced coagulopathy. The procoagulant activity (PCA) of circulating monocytes rose from (mean +/- SEM) 0.15 +/- 0.02 mU/mL to 0.40 +/- 0.05 mU/mL at 3 hours (P = .002) and 0.56 +/- 0.21 at 5 hours (P = .045) after the initial OKT3 injection. These monocytes displayed increased tissue factor expression at the same moments (mean flourescence intensity: 14 +/- 2 before OKT3 injection versus 54 +/- 14 at 3 hours, P = .008 and 34 +/- 7 at 5 hours, P = .01). Tissue factor mRNA was detected in blood by reverse transcriptase-polymerase chain reaction as early as 2 hours after OKT3 administration. The circulating monocytes also displayed a steady increase in membrane expression upregulation of ICAM-1, CD29, CD11b, and CD11c. In vitro experiments showed that OKT3 as well as 2 mitogenic, humanized anti-CD3 antibodies potently induced monocytic PCA whereas the 4 nonmitogenic anti-CD3 antibodies tested were over 1,000-fold less potent than OKT3. We conclude that (1) OKT3 induces in vivo tissue factor gene upregulation and membrane expression resulting in increased PCA of circulating monocytes; and (2) nonmitogenic anti-CD3 antibodies seem devoid of significant procoagulant properties.