Biomedical subjects

Charles W Francis

Publications and source records attributed to Charles W Francis.

At least 19 recordsLinked to original sources

PubMed2006

The platelet as a therapeutic target for treating vascular diseases and the role of eicosanoid and synthetic PPARgamma ligands.

The platelet was traditionally thought only to serve as the instigator of thrombus formation, but now is emerging as a pivotal player in cardiovascular disease and diabetes by inciting and maintaining inflammation. Upon activation, platelets synthesize eicosanoids such as thromboxane A2 (TXA2) and PGE2 and release pro-inflammatory mediators including CD40 ligand (CD40L). These mediators activate not only platelets, but also stimulate vascular endothelial cells and leukocytes. These autocrine and paracrine activation processes make platelets an important target for attenuating inflammation. The growing interest and recent discoveries in platelet biology has lead to the search for therapeutic platelet targets. Recently, platelets, although anucleate, were discovered to possess the transcription factor PPARgamma. Treatment with eicosanoid and synthetic PPARgamma ligands blunts platelet release of the bioactive mediators, soluble (s) CD40L and TXA2, in thrombin-activated platelets. PPARgamma ligand treatment may prove useful for dampening unwanted platelet activation and chronic inflammatory diseases such as cardiovascular disease.

Blood Platelets↗

PubMed2006

Thromboembolism in hospitalized neutropenic cancer patients.

PURPOSE: Cancer is associated with thrombosis, but the frequency of thromboembolism in hospitalized cancer patients receiving current chemotherapy regimens is not known. We investigated venous and arterial thromboembolism and associated outcomes in hospitalized cancer patients actively receiving therapy, as identified by neutropenia. METHODS: We conducted a retrospective cohort study using the discharge database of the University HealthSystem Consortium. This included 66,106 adult neutropenic cancer patients with 88,074 hospitalizations between 1995 and 2002 at 115 medical centers in the United States. RESULTS: Thromboembolism was reported in 5,272 patients (8%), with 5.4% patients developing venous thromboembolism and 1.5% developing arterial thromboembolism during the first hospitalization. Patients with lymphoma and leukemia accounted for one third of venous and nearly one half of arterial events. Clinical variables most frequently associated with thromboembolism were age > or = 65 years; primary site of cancer, including lung, GI, gynecologic, and brain; and comorbidities, including infection, pulmonary and renal disease, and obesity. In-hospital mortality was significantly greater in patients with venous (odds ratio [OR] = 2.01; 95% CI, 1.83 to 2.22) or arterial thromboembolism (OR = 5.04; 95% CI, 4.38 to 5.79). From 1995 to 2002, there was a 36% increase in venous events and a 124% increase in arterial events (P < .0001 for trend). CONCLUSION: Thromboembolism is frequent in hospitalized neutropenic cancer patients, including in perceived low-risk subgroups such as patients with hematologic malignancies and nonmetastatic disease, and seems to be increasing. Thromboembolism is associated with increased in-hospital mortality. Increased efforts at thromboprophylaxis are warranted.

Aged↗

PubMed2006

Venous thromboembolism in patients with diffuse large B-cell lymphoma.

We conducted a retrospective record review to determine the frequency of venous thromboembolism (VTE) in patients with diffuse large B-cell lymphoma (DLBCL). All records from 1990 to 2001 of patients with the diagnosis of DLBCL at a tertiary care hospital were reviewed. Those with transformation from low-grade lymphoma, central nervous system lymphoma, HIV-related lymphoma or with incomplete records were excluded. All episodes of symptomatic VTE confirmed by imaging studies that were either present at diagnosis or occurred during initial treatment were identified. VTE occurred in 27 of 211 patients (12.8%). Stage I disease was associated with a low risk, whereas a high international prognostic index score increased risk. Of patients with VTE, thrombosis was present at diagnosis in 37% and occurred during the first chemotherapy cycle in 22% and during the first three cycles in 82%. The median survival of patients with VTE was 1.04 years [95% confidence interval (CI) = 0.75 - 1.33] compared to 5.2 years (95% CI 1.8 - 8.6) for those without VTE (P = 0.038). We conclude that VTE is a frequent complication of DLBCL that occurs particularly at diagnosis and during initial therapy, and it is associated with a worse prognosis.

Adult↗

PubMed2005

Risk factors for chemotherapy-associated venous thromboembolism in a prospective observational study.

BACKGROUND: The incidence of venous thromboembolism (VTE) is increased in cancer, but little information is available about risk factors in cancer patients on chemotherapy. METHODS: We analyzed data from a prospective, multicenter observational study to determine the frequency and risk factors for VTE in ambulatory cancer patients initiating a new chemotherapy regimen. The association of VTE with clinical variables was characterized using univariate and multivariate analysis. RESULTS: Among 3003 patients treated with at least one cycle of chemotherapy, VTE occurred in 58 (1.93%) over a median follow-up of 2.4 months (0.8%/mo). The incidence varied significantly by site of cancer (P = 0.01) with highest rates in upper gastrointestinal (2.3%/mo) and lung cancer (1.2%/mo), and lymphoma (1.1%/mo). An elevated prechemotherapy platelet count was significantly associated with an increased rate of VTE (P for trend = 0.005). The incidence of VTE was 3.98% (1.66%/mo) for patients with a prechemotherapy platelet count > or = 350,000, compared with 1.25% (0.52%/mo) for patients with platelet counts of < 200,000 (P for trend=0.0003). In multivariate analysis, a prechemotherapy platelet count of > or = 350,000/mm(3) (adjusted OR 2.81, 95% CI 1.63-4.93, P = 0.0002), site of cancer, hemoglobin < 10 g/dL or use of erythropoietin, and use of white cell growth factors in high-risk sites of cancer were significantly associated with VTE. CONCLUSIONS: Symptomatic VTE is a frequent complication of chemotherapy. The prechemotherapy platelet count is a unique risk factor and can help identify high-risk patients for future trials of thromboprophylaxis.

Age Distribution↗

PubMed2005

FGF-2 binding to fibrin(ogen) is required for augmented angiogenesis.

We have shown previously that fibrin(ogen) binds fibroblast growth factor 2 (FGF-2) and potentiates stimulation of endothelial-cell (EC) proliferation. We have now used 2 FGF-2 mutants differing only in the 5 residues constituting the binding site to characterize the importance of this interaction in angiogenesis. The nonbinding (2212) and binding (221*2) mutants stimulated EC proliferation by 2.2 +/- 0.4-fold and 2.9 +/- 0.3-fold over control, respectively, and both were similar to wild-type (wt) FGF-2 (2.5 +/- 0.3-fold). Proliferation was augmented by fibrinogen to 5.3 +/- 1.2-fold and 4.8 +/- 0.8-fold with wtFGF-2 and 221*2, whereas no augmentation occurred with 2212 and fibrinogen. Using a placental explant model in a fibrin matrix, wtFGF-2 resulted in 2.6 +/- 0.9-fold more growth over control, and 221*2 increased growth 3.3 plus or minus 0.9-fold. Vessel outgrowth with 2212 was minimal and comparable to control. Similarly, fibrinogen potentiated wtFGF-2 or 221*2-mediated angiogenesis in the chicken chorioallantoic membrane model. In a mouse Matrigel implant model, fibrinogen significantly increased angiogenesis with either wtFGF-2 or 221*2, whereas there was no augmentation with 2212. These results demonstrate that binding of FGF-2 to fibrin(ogen) mediated by the 5-residue FGF-2-fibrin(ogen) interactive site is required for augmented angiogenesis.

Animals↗

PubMed2005

Endothelial cell activation by IL-1beta in the presence of fibrinogen requires alphavbeta3.

OBJECTIVE: The purpose of this study was to investigate the receptor requirements for enhanced IL-1beta-induced secretion of nitric oxide (NO) by endothelial cells (ECs) in the presence of fibrinogen. METHODS AND RESULTS: ECs were exposed to IL-1beta with or without fibrinogen and NO was measured as nitrite. NO production by EC exposed to fibrinogen (0.3+/-0.1 micromol/L) was comparable concentration to control (0.2+/-0.1 micromol/L), but IL-1beta significantly increased NO production (0.8+/-0.1 micromol/L), and the combination of both fibrinogen and IL-1beta resulted in a further increase to 2.2+/-0.2 micromol/L (P<0.002). 7E3 or LM609, antibodies to alphavbeta3, inhibited NO production stimulated by fibrinogen-bound IL-1beta to 0.2+/-0.1 micromol/L (P<0.001) or 0.2+/-0.03 micromol/L (P<0.0001), respectively. These levels were comparable to control and significantly less than with IL-1beta (P<0.002). EC or fibroblasts exposed to both fibrinogen and IL-1beta, but not vitronectin and IL-1beta, demonstrated positive Western blotting for alphavbeta3 after immunopurification with anti- IL-1R, indicating specific association between alphavbeta3 and IL-1R. Dual immunofluorescence also revealed colocalization of alphavbeta3 and IL-1R only when the cells were exposed to both fibrinogen and IL-1beta. CONCLUSIONS: The enhanced NO production by ECs in the presence of fibrinogen-bound IL-1beta requires the coordinated effects of colocalized alphavbeta3 and IL-1R.

Antibodies, Monoclonal↗

PubMed2005

Atherosclerotic risk genotypes and recurrent coronary events after myocardial infarction.

The association of a group of prespecified atherosclerotic risk genotypes with recurrent coronary events (coronary-related death, nonfatal myocardial infarction, or unstable angina) was investigated in a cohort of 1,008 patients after infarction during an average follow-up of 28 months. We used a carrier-ship approach with time-dependent survivorship analysis to evaluate the average risk of each carried genotype. Contrary to expectation, the hazard ratio for recurrent coronary events per carried versus noncarried genotype was 0.89 (95% confidence interval 0.80 to 0.99, p = 0.03) after adjustment for relevant genetic, clinical, and environmental covariates. This hazard ratio, derived from the 7 prespecified genotypes, indicated an average 11% reduction in the risk of recurrent coronary events per carried versus noncarried genotype. At 1 year after hospital discharge, the cumulative probability of recurrent coronary events was 26% in those who carried < or =1, 20% for those with 2 to 4, and 13% for those with > or =5 of these genotypes (p = 0.02). This unexpected risk reversal is a likely consequence of changes in the mix of risk factors in pre- and postinfarction populations. In conclusion, this under appreciated, population-based, risk-reversal phenomenon may explain the inconsistent associations of genetic risk factors with outcome events in previous reports involving coronary populations with different risk attributes.

Aged↗

PubMed2005

Ximelagatran vs low-molecular-weight heparin and warfarin for the treatment of deep vein thrombosis: a randomized trial.

CONTEXT: Ximelagatran, an oral direct thrombin inhibitor with a rapid onset of action and predictable antithrombotic effect, has the potential to be a simple therapeutic alternative to current standard treatment of acute venous thromboembolism. OBJECTIVE: To compare the efficacy and safety of ximelagatran with standard enoxaparin/warfarin treatment for the prevention of recurrent venous thromboembolism. DESIGN, SETTING, AND PATIENTS: Randomized, double-blind, noninferiority trial (Thrombin Inhibitor in Venous Thromboembolism [THRIVE] Treatment Study) of 2489 patients with acute deep vein thrombosis, of whom approximately one third had concomitant pulmonary embolism. The study was conducted at 279 centers in 28 countries from September 2000 through December 2002. INTERVENTIONS: Patients were randomized to receive 6 months of treatment with either oral ximelagatran, 36 mg twice daily, or subcutaneous enoxaparin, 1 mg/kg twice daily, for 5 to 20 days followed by warfarin adjusted to maintain an international normalized ratio of 2.0 to 3.0. MAIN OUTCOME MEASURES: Recurrent venous thromboembolism, bleeding, and mortality. RESULTS: Venous thromboembolism recurred in 26 of the 1240 patients assigned to receive ximelagatran (estimated cumulative risk, 2.1%) and in 24 of the 1249 patients assigned to receive enoxaparin/warfarin (2.0%). The absolute difference between ximelagatran and enoxaparin/warfarin was 0.2% (95% confidence interval [CI], -1.0% to 1.3%). This met the prespecified criterion for noninferiority. Corresponding values for major bleeding were 1.3% and 2.2% (difference, -1.0%; 95% CI, -2.1% to 0.1%), and for mortality were 2.3% and 3.4% (difference, -1.1%; 95% CI, -2.4% to 0.2%). Alanine aminotransferase levels increased to more than 3 times the upper limit of normal in 119 patients (9.6%) and 25 patients (2.0%) receiving ximelagatran and enoxaparin/warfarin, respectively. Increased enzyme levels were mainly asymptomatic. Retrospective analysis of locally reported adverse events showed a higher rate of serious coronary events with ximelagatran (10/1240 patients) compared with enoxaparin/warfarin (1/1249 patients). CONCLUSIONS: Oral ximelagatran administered in a fixed dose without coagulation monitoring, was as effective as enoxaparin/warfarin for treatment of deep vein thrombosis with or without pulmonary embolism and showed similar, low rates of bleeding. Increased levels of liver enzymes in 9.6% of ximelagatran-treated patients require regular monitoring; the mechanism requires further evaluation. Prospective assessment of coronary events in future studies is warranted.

Adult↗

PubMed2005

Oral direct thrombin inhibitor ximelagatran compared with warfarin for the prevention of venous thromboembolism after total knee arthroplasty.

BACKGROUND: Warfarin, which requires coagulation monitoring, is associated with relatively high rates of thromboembolism despite providing adequate prophylaxis. This study compared an oral direct thrombin inhibitor, ximelagatran, with warfarin in order to evaluate the safety and efficacy of the medication for the prevention of venous thromboembolism in patients undergoing total knee arthroplasty. METHODS: Following surgery, patients were randomly assigned to fixed-dose oral ximelagatran (36 mg twice daily) or warfarin (target international normalized ratio, 2.5), both administered for seven to twelve days in a double-blind, double-dummy design. Warfarin was initiated on the evening of the day of surgery, and ximelagatran, on the morning after surgery. The primary efficacy end point was the incidence of asymptomatic deep-vein thrombosis determined by bilateral venography, objectively confirmed symptomatic deep-vein thrombosis or pulmonary embolism, and death from all causes during treatment. RESULTS: Adequate venograms or confirmed symptomatic events (efficacy population) were obtained for 1949 patients. Venous thromboembolism and death from all causes occurred in 22.5% (221) of 982 ximelagatran-treated patients and in 31.9% (308) of 967 warfarin-treated patients (p < 0.001). Proximal deep-vein thrombosis and pulmonary embolism were observed in 3.1% (thirty) and 0.2%, respectively, of the patients in the ximelagatran group and in 3.4% (thirty-three) and 0.4%, respectively, of the patients in the warfarin group. The six deaths from all causes included 0.3% (four) of the ximelagatran-treated patients and 0.2% (two) of the warfarin-treated patients. Major bleeding was noted in 1% (twelve) of the ximelagatran-treated patients and in 0.4% (five) of the warfarin-treated patients (p = 0.09). CONCLUSIONS: Oral ximelagatran (36 mg twice daily), administered without coagulation monitoring or dose adjustment and started the day after total knee arthroplasty, demonstrates superior efficacy compared with warfarin prophylaxis, with no wound complications and no significant difference with respect to bleeding events, although the rate of major bleeding events was greater with ximelagatran than with warfarin. LEVEL OF EVIDENCE: Therapeutic Level I.

Aged↗

PubMed2004

Ultrasound energy improves myocardial perfusion in the presence of coronary occlusion.

OBJECTIVES: We evaluated whether ultrasound improves myocardial tissue perfusion in 14 animals with coronary artery occlusion. BACKGROUND: A recent study demonstrated that low-frequency ultrasound improves tissue perfusion in the rabbit ischemic limb, but there are no data on ultrasound enhancement of myocardial perfusion. METHODS: Fourteen animals (9 dogs, 5 pigs) underwent thoracotomy and occlusion of a diagonal branch of the left anterior descending coronary artery. Myocardial tissue perfusion units (TPUs) and pH were measured before coronary occlusion, after occlusion, and after direct exposure of the ischemic myocardium in the presence of fixed occlusion to low-frequency ultrasound (27 kHz). RESULTS: The TPU decreased from 100.9 +/- 13 at baseline to 71.1 +/- 13 (p < 0.01) after 60 min occlusion but rose by 19.7% to 85.1 +/- 8 (p < 0.01) after ultrasound exposure for 60 min. After 60-min coronary occlusion, myocardial pH fell from 7.43 +/- 14 to 7.05 +/- 0.15 (p < 0.01) but then improved to normal (7.46 +/- 0.32) after ultrasound for 60 min. Administration of L-Nomega-nitro-arginine methyl esther (L-NAME), an inhibitor of nitric oxide synthase, before ultrasound exposure, blocked improvement in myocardial tissue perfusion and pH by ultrasound. Quantitative histomorphology showed a significant increase in the capillary area of myocardium exposed to ultrasound versus non-exposed myocardium (16.2 +/- 7.9 vs. 8.2 +/- 2.1, p < 0.02). CONCLUSIONS: Low-frequency, low-intensity ultrasound improves myocardial tissue perfusion and pH in the presence of a fixed coronary artery occlusion.

Animals↗

PubMed2004

Stimulation of endothelial cell proliferation by FGF-2 in the presence of fibrinogen requires alphavbeta3.

We have shown previously that fibrin(ogen) binding potentiates the capacity of fibroblast growth factor 2 (FGF-2) to stimulate endothelial cell (EC) proliferation. We have now investigated the receptor requirement for EC proliferation by fibrinogen-bound FGF-2. ECs were cultured with 25 ng/mL FGF-2 with or without 10 microg/mL fibrinogen, and proliferation was measured as (3)H-thymidine incorporation. Proliferation was increased 2.4 +/- 0.5-fold over medium alone with FGF-2 and increased significantly more to 4.0 +/- 0.7-fold with fibrinogen and FGF-2 (P < .005). Addition of 7E3 or LM609, antibodies to alpha(v)beta(3), inhibited EC proliferation with fibrinogen-bound FGF-2 by 80% +/- 8% (P < .001) or 67% +/- 14% (P < .002), respectively, to levels significantly less than that observed with FGF-2 alone (P < .001). Neither LM609 nor 7E3 exhibited any inhibition of activity with FGF-2 alone. Peptide GRGDS caused dose-dependent inhibition of proliferation by fibrinogen-bound FGF-2 of 31% +/- 8%, 45% +/- 9%, and 68% +/- 11% at 0.25, 0.5, and 1 mM, respectively. Coimmunoprecipitation and immunofluorescence studies demonstrated a direct specific association between alpha(v)beta(3) and FGF receptor 1 (FGFR1) in ECs and fibroblasts when exposed to both FGF-2 and fibrinogen but not with vitronectin. We conclude that fibrinogen binding of FGF-2 enhances EC proliferation through the coordinated effects of colocalized alpha(v)beta(3) and FGFR1.

Cell Proliferation↗

PubMed2004

NF-kappaB activation suppresses host cell apoptosis during Rickettsia rickettsii infection via regulatory effects on intracellular localization or levels of apoptogenic and anti-apoptotic proteins.

Rickettsia rickettsii, a gram-negative and obligate intracellular bacterium, is the causative agent of Rocky Mountain spotted fever. In human infections, the primary target of R. rickettsii infection is vascular endothelium. Our laboratory has shown that activation of nuclear transcription factor-kappa B (NF-kappaB) during R. rickettsii infection of cultured human endothelial cells protects against apoptosis by preventing the activation of apical caspases-8 and -9, and the effector caspase-3. To understand upstream signaling mechanisms, we have determined the effect of NF-kappaB blockade on the status of different Bcl-2 (B-cell lymphoma 2) proteins in this study. Quantitative analysis following TUNEL and Hoechst staining confirmed that infection of endothelial cells with R. rickettsii for 6 h in the presence of a specific NF-kappaB inhibitor, MG132, resulted in induction of apoptosis. Infection-induced apoptosis of EC was associated with decreased level of Bid and accumulation of Bad, while cytosolic level of Bax remained relatively unchanged. Further, the cellular levels of apoptosis antagonist Bcl-2 were found to be down-regulated and apoptogenic mitochondrial proteins Smac and cytochrome c were released into cytoplasm. These results implicate an important regulatory role for NF-kappaB in controlling the intracellular levels and/or localization of pro- as well as anti-apoptotic proteins of Bcl-2 family, the intricate balance of which is a critical determinant of downstream signaling mechanisms governing cell fate during intracellular infection.

Apoptosis↗

PubMed2004

Human bone marrow megakaryocytes and platelets express PPARgamma, and PPARgamma agonists blunt platelet release of CD40 ligand and thromboxanes.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor important in lipid metabolism, diabetes, and inflammation. We evaluated whether human platelets and megakaryocytes express PPARgamma and whether PPARgamma agonists influence platelet release of bioactive mediators. Although PPARgamma is mainly considered a nuclear receptor, we show that enucleate platelets highly express PPARgamma protein as shown by Western blotting, flow cytometry, and immunocytochemistry. Meg-01 megakaryocyte cells and human bone marrow megakaryocytes also express PPARgamma. Platelet and Meg-01 PPARgamma bound the PPARgamma DNA consensus sequence, and this was enhanced by PPARgamma agonists. Platelets are essential not only for clotting, but have an emerging role in inflammation in part due to their release or production of the proinflammatory and proatherogenic mediators CD40 ligand (CD40L) and thromboxanes (TXs). Platelet incubation with a natural PPARgamma agonist, 15d-PGJ(2), or with a potent synthetic PPARgamma ligand, rosiglitazone, prevented thrombin-induced CD40L surface expression and release of CD40L and thromboxane B(2) (TXB(2)). 15d-PGJ(2) also inhibited platelet aggregation and adenosine triphosphate (ATP) release. Our results show that human platelets express PPARgamma and that PPARgamma agonists such as the thiazolidinedione class of antidiabetic drugs have a new target cell, the platelet. This may represent a novel mechanism for treatment of inflammation, thrombosis, and vascular disease in high-risk patients.

Adenosine Triphosphate↗

PubMed2004

Interleukin-1beta but not IL-1alpha binds to fibrinogen and fibrin and has enhanced activity in the bound form.

Fibrin is formed at sites of injury or inflammation and provides the temporary matrix to support vascular cell responses that are also mediated by cytokines including interleukin-1 (IL-1). We have shown previously that fibroblast growth factor 2 (FGF-2) binds with high affinity to fibrin(ogen). Because IL-1 has a structure similar to FGF-2, we have investigated the possible binding of IL-1 to fibrin(ogen). Experiments using IL-1 immobilized on Sepharose beads and soluble iodine 125 ((125)I)-labeled fibrinogen demonstrated no specific interaction of IL-1alpha with fibrinogen, but IL-1beta showed saturable and specific binding. Scatchard analysis indicated a single binding site with an apparent K(d) = 1.5 nM and a maximum molar binding ratio of IL-1beta to fibrinogen of 1.8:1. Binding of (125)I-IL-1beta to Sepharose-immobilized fibrinogen also demonstrated a single binding site with an apparent K(d) of 3.5 nM. IL-1beta also bound specifically to fibrin monomer and polymerized fibrin with apparent K(d)s of 3.4 nM and 2.3 nM, respectively. IL-1beta displaced FGF-2 for binding to fibrin, indicating an interaction with the same or a closely related site. Compared with free form, fibrinogen-bound IL-1beta stimulated increased activation of endothelial cell nuclear factor kappaB (NF-kappaB), monocyte chemoattractant protein-1 (MCP-1) secretion, and nitric oxide (NO) synthesis. We conclude that IL-1beta binds with high affinity to fibrin(ogen) and demonstrates increased activity in the bound form.

Binding, Competitive↗

PubMed2004

Ximelagatran: a new oral anticoagulant.

Vitamin K antagonists are effective oral anticoagulants, but they have limitations related to a narrow therapeutic range, food and drug interactions, slow onset of action and the need for routine coagulation monitoring. Ximelagatran is a promising new oral anticoagulant under investigation in advanced clinical trials. It is a prodrug that is converted after oral administration to melagatran, a direct thrombin inhibitor, with a peak effect after 2 hours and a half-life of approximately 3 hours with primarily renal excretion. Administration results in prolongation of coagulation tests, but routine monitoring is not required because of reliable absorption and predictable effects. A large clinical trials program has demonstrated effectiveness in prophylaxis of deep vein thrombosis (DVT) following major orthopedic surgery, treatment of symptomatic DVT, prevention of embolism in patients with atrial fibrillation, and prophylaxis of recurrent events after acute myocardial infarction. Bleeding complications have been similar to those with standard therapy, with no unexpected adverse effects except for elevation of serum transaminase levels in over 6% of patients beginning after 1 month of therapy. Ximelagatran may be an alternative oral anticoagulant for patients currently taking vitamin K antagonists.

Anticoagulants↗

PubMed2004

Fibrinogen regulates the expression of inflammatory chemokines through NF-kappaB activation of endothelial cells.

The objective of this study was to characterize the role of fibrinogen in stimulating expression of inflammatory chemokines in endothelial cells through NF-kappaB activation. Human umbilical vein endothelial cells (HUVEC) were exposed to fibrinogen up to 3,000 microg/ml, and NF-kappaB activation was assessed using electrophoretic mobility shift assay (EMSA). Fibrinogen exposure resulted in a concentration dependent increase in NF-kappaB activation that reached a maximum at 1,000 microg/ml after 4 hours and was sustained up to 24 hours. The effect was inhibited by antibodies to alpha(v)beta(3) and alpha(5)beta(1) and by the GRGDS peptide, indicating integrin involvement. Preincubation with Mn(2+) lowered the fibrinogen concentration-dependence, consistent with integrin activation. Supershift assays demonstrated involvement of the p50, p65 and c-Rel components of NF-kappaB. Fibrinogen exposure also resulted in up-regulation of expression of monocyte chemoattractant protein-1 (MCP-1) and of interleukin-8 as shown by RNase protection assays and by real-time RT-PCR. Increased secretion of MCP-1 was confirmed by ELISA. Parthenolide, an IkappaB kinase inhibitor, prevented up-regulation of MCP-1 by fibrinogen, linking this response to NF-kappaB activation. From our findings, we conclude that fibrinogen regulates NF-kappaB activation and expression of inflammatory chemokines in endothelial cells and may be involved in mediating inflammatory processes.

Chemokine CCL2↗

PubMed2003

Identification of a binding site on human FGF-2 for fibrinogen.

Endothelial cell adhesive interactions are mediated by both fibrinogen and fibrin, and growth is stimulated by fibroblast growth factor 2 (FGF-2). We have shown previously that FGF-2 binds specifically and with high affinity to fibrinogen and fibrin and that fibrinogen potentiates the proliferative capacity of FGF-2 and also protects it from proteolytic degradation. To further characterize this interaction we have performed FGF-2 mutagenesis to identify the interactive site. Because FGF-1 has a similar structure to FGF-2 but does not bind to fibrinogen, we used a strategy of cassette and site-directed mutagenesis, exchanging residues from FGF-1 and FGF-2 and correlating structural changes with fibrinogen binding. Two cassette interchange mutants, 2212 and 2211, contained either the third cassette or both the third and fourth cassettes from FGF-1, and neither exhibited any affinity for fibrinogen. Exchange of 5 residues (Phe95, Ser100, Asn102, Arg107, and Arg109) from FGF-2 into the corresponding sites in the third cassette of FGF-1 imparted high-affinity binding with apparent dissociation constants (Kd) of 5.3 nM and 8.6 nM, respectively, compared with 1.3 nM for wild-type FGF-2. We conclude that these 5 residues define a high-affinity binding site in FGF-2 for fibrinogen.

Amino Acid Sequence↗

PubMed2003

Comparison of ximelagatran with warfarin for the prevention of venous thromboembolism after total knee replacement.

BACKGROUND: In a previous study of the prevention of venous thromboembolism after total knee replacement, the efficacy of ximelagatran, an oral direct thrombin inhibitor that does not require monitoring of coagulation or dose adjustment, was found to be similar to that of warfarin at a dose of 24 mg of ximelagatran twice daily. The purpose of the present study was to determine whether a higher dose of ximelagatran is superior to warfarin. METHODS: This randomized, double-blind trial compared a regimen of 7 to 12 days of oral ximelagatran, at a dose of 24 or 36 mg twice daily, starting the morning after surgery, with warfarin therapy started the evening of the day of surgery. The composite end point of venous thromboembolism and death from all causes and the incidence of bleeding were the primary outcome measures. RESULTS: Among the 1851 patients in the efficacy analysis, oral ximelagatran at a dose of 36 mg twice daily was superior to warfarin with respect to the primary composite end point of venous thromboembolism and death from all causes (20.3 percent vs. 27.6 percent; P=0.003). There were no significant differences between these two groups with respect to major bleeding (incidence, 0.8 percent and 0.7 percent, respectively), perioperative indicators of bleeding, wound characteristics, or the composite secondary end point of proximal deep-vein thrombosis, pulmonary embolism, and death (2.7 percent vs. 4.1 percent; P=0.17). CONCLUSIONS: The efficacy of oral ximelagatran, administered starting the morning after total knee replacement, was superior to that of warfarin for prevention of venous thromboembolism. Rates of hemorrhagic complications with the two drugs were similar.

Administration, Oral↗