American Diagnostica Research Articles

Tissue Factor Bearing Microparticles in the Myeloproliferative Neoplasms

Abstract 5174 Background:Thrombosis is a well recognized complication in the myeloproliferative neoplasms (MPN), essential thrombocytosis (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). The mechanism for thrombosis is not well-established, nor are there relevant biomarkers to predict risk and/or recurrence. Circulating cellular microparticles (MP) containing procoagulant tissue factor (TF) have been shown to correlate with thrombotic risk in many forms of cancer and cardiovascular diseases. To investigate the role of MP in the MPN, we studied 16 patients (ET=5; PV=6; PMF=2; post-ETMF=1, and MPN NOS=2) and compared results to 15 healthy subjects. Methods:Citrated blood samples were collected from the 16 MPN patients and 15 controls. Platelet poor plasma (PPP) was obtained by centrifuging at 1,500 G for 20 minutes. 50 μL of PPP was added to 200 μL PBS (without Mg/Ca) and centrifuged at 20,000 G for 10 minutes. The sediment containing MP was resuspended in 100μL of buffer for labeling with TF, CD41a (platelets), CD14 (monocytes), CD66b (neutrophils), and CD33 (myeloid lineage). Following incubation, PBS was added to the suspension to a volume of 1ml for flow cytometric analysis (LSR Fortessa, FlowJo software). Electronic triggering was done on side-scatter, and acquisition regions were defined based on sizing beads (0.3 to 1.0 micron) along with annexin A5 positivity. Using MP sediment, TF activity was measured using chromogenic assays (Actichrome TF ELISA, American Diagnostica,) and thrombin generation (TGT) was assayed (Technothrombin TGA, diaPharma), with results expressed as lag phase, velocity-index, peak thrombin, and area under the curve (AUC). The Wilcoxon-Rank Sum test was used to compare group differences (MPN vs. control) in median values. Results:Among the MPN patients, 7(44%) were male, and the median age was 60 years. 11 (69%) were JAK2 V617F positive, and 3 (19%) had a prior history of thrombosis (2 hepatic vein thromboses, 1 myocardial infarction). At the time of collection, 14 (93%) were on aspirin, 1 (6%) was on Coumadin, and 5 (31%) were on Hydroxyurea. The median total MP number was increased in MPN patients vs. controls (243580 vs. 83120; p=0.0057). The median percentage of TF-bearing MP’s was also significantly greater in MPN patients compared to controls (35.5% vs. 12%; p=0.0003). When comparing MPN patients to controls, these TF-bearing MP were derived from CD14 (31.5% vs. 2%; p<0.001) and CD41a (24% vs. 7%; p=0.0157), respectively, reflecting monocyte and platelet origins of the MPs. The TF-bearing MPs in MPN patients (N=10) were functionally active compared to controls (N=10) (median TF activity: 1.7 pM vs. 0.03 pM; p=0.0022). Thrombin generation assays were performed in 13 MPN patients and 9 controls, and were comparable: mean lag phase (14.4 vs. 10.15 minutes; p=0.26); mean velocity index (21.13 vs. 14.78; p=0.31); mean peak thrombin generation (111.66 nM vs. 120.41 nM; p=0.75); and mean AUC (3218.77 vs. 3807.41 p=0.37). Conclusion:Compared to controls, samples of JAK2 V617F-positive and negative MPN patients revealed a higher median total number of microparticles. Further, the proportion of TF-bearing MPs was higher in MPN patients, and of monocyte and platelet origin, suggesting their possible role in thrombotic complications. Though the MPs in MPN patients appear functional, based on higher TF activity, functional assays with thrombin generation testing failed to reveal a difference between MPN patients and controls. A lack of difference in TGT may suggest the presence of one or more inhibitors present in the MPN; the nature of this inhibitor is under investigation. Future studies, with a larger sample size and prospective follow-up are indicated to determine the role of MPs in predicting incident or recurrent thrombosis in the MPN. In addition, with a larger sample size, differences by MPN disease class and JAK2 V617F status will be uncovered. Disclosures:No relevant conflicts of interest to declare.

Fluorescent particles in the antibody solution result in false TF- and CD14-positive microparticles in flow cytometric analysis

Tissue factor (TF)-positive microparticles (MPs) are highly procoagulant, and linked to thrombosis in sepsis and cancer. MP-associated TF may be assayed by immunological or functional methods. Several reports have demonstrated discrepancies between TF-protein and TF-activity, which have been explained by antibody binding to "encrypted" or degraded forms of inactive TF-protein. Our goal was to evaluate the possible interference of fluorescent antibody aggregates in solutions containing antibodies against TF and CD14 in flow cytometric analysis. Using monocyte-derived microparticles (MPs) released from human monocytes, incubated with or without lipopolysaccharides (LPS) in vitro, we measured MP-associated TF-protein (flow cytometry) and TF-activity (clot formation assay). MPs released from monocytes exposed to LPS (1 ng mL(-1) ) had ∼14 times higher TF-activity than MPs originated from monocytes exposed to only culture medium. However, using untreated anti-TF antibodies (American Diagnostica and BD) in the flow cytometric analysis, MPs released from unstimulated monocytes had a similar number of TF-positive events as MPs secernated from LPS-stimulated monocytes [∼45,000 events mL(-1) (American Diagnostica); ∼15,000 events mL(-1) (BD)]. These TF-positive events did not exert any TF-activity, and centrifugation (17,000g, 30 min, 4°C) of the antibody solutions prior to use effectively removed the interfering fluorescent events. Removal of fluorescent interference, probably in the form of fluorescent antibody aggregates, from the antibody solutions by centrifugation is essential to prevent the occurrence of false positive flow cytometric events. The events can be mistaken as MP-associated TF-protein, and interpreted as a discrepancy between TF-protein and TF-activity.

ADAMTS13, FVIII, von Willebrand factor, ABO blood group assessment in preeclampsia

IntroductionPreeclampsia (PE) is a multifactorial disease characterized by high blood pressure and proteinuria after the 20th week of pregnancy. PE is associated with fibrin deposition in placental microcirculation and intrauterine fetal growth retardation. We evaluated FVIII activity, VWF and ADAMTS13 plasma levels, according to O and “non O” blood groups, in women with severe PE (sPE). MethodsThis case-control study included 140 women; 55 pregnant with sPE, 35 normotensive pregnant and 50 non-pregnant women. VWF and ADAMTS13 antigen levels were assessed by ELISA (American Diagnostica). FVIII activity was measured by automated coagulometric method (Dade Behring) and ABO blood groups phenotyping was performed by indirect technique. ResultsFVIII activity and VWF levels were significantly higher comparing either sPE to normotensive pregnant (P=0.01; P=0.05) and to non-pregnant women (P=0.00 in both cases) or normotensive pregnant and non-pregnant women (P=0.00 in both cases). A significant decrease in ADAMTS13 levels was observed comparing either sPE to normotensive pregnant (P=0.02) and non-pregnant women (P=0.00) or normotensive pregnant and non-pregnant women (P=0.00). FVIII activity and VWF levels were associated to O and “non O” blood groups only in non-pregnant women. ConclusionsThe increase of FVIII activity and VWF levels and the decrease of ADAMTS13 in sPE are not associated to O and “non O” blood groups. These alterations in hemostatic markers in sPE largely surpass those physiologically determined by ABO blood groups influence and may have masked the effect of O and “non O” groups in this disease. A concomitant analysis of VWF levels and ADAMTS13 activity and antigenic levels will be important to clarify the imbalance between these parameters found in sPE in the present study.

Matrix Gla Protein Metabolism in Vascular Smooth Muscle and Role in Uremic Vascular Calcification

Matrix Gla protein (MGP) is an inhibitor of vascular calcification but its mechanism of action and pathogenic role are unclear. This was examined in cultured rat aortas and in a model of vascular calcification in rats with renal failure. Both carboxylated (GlaMGP) and uncarboxylated (GluMGP) forms were present in aorta and disappeared during culture with warfarin. MGP was also released into the medium and removed by ultracentrifugation, and similarly affected by warfarin. In a high-phosphate medium, warfarin increased aortic calcification but only in the absence of pyrophosphate, another endogenous inhibitor of vascular calcification. Although GlaMGP binds and inactivates bone morphogenic protein (BMP)-2, a proposed mediator of vascular calcification through up-regulation of the osteogenic transcription factor runx2, neither warfarin, BMP-2, nor the BMP-2 antagonist noggin altered runx2 mRNA content in aortas, and noggin did not prevent warfarin-induced calcification. Aortic content of MGP mRNA was increased 5-fold in renal failure but did not differ between calcified and noncalcified aortas. Immunoblots showed increased GlaMGP in noncalcified (5-fold) and calcified (20-fold) aortas from rats with renal failure, with similar increases in GluMGP. We conclude that rat aortic smooth muscle produces both GlaMGP and GluMGP in tissue-bound and soluble, presumably vesicular, forms. MGP inhibits calcification independent of BMP-2-driven osteogenesis and only in the absence of pyrophosphate, consistent with direct inhibition of hydroxyapatite formation. Synthesis of MGP is increased in renal failure and deficiency of GlaMGP is not a primary cause of medial calcification in this condition.

Secretory Leukocyte Protease Inhibitor (SLPI) Expression and Tumor Invasion in Oral Squamous Cell Carcinoma

Differential expression of secretory leukocyte protease inhibitor (SLPI) impacts on tumor progression. SLPI directly inhibits elastase and other serine proteases, and regulates matrix metalloproteinases, plasminogen activation, and plasmin downstream targets to influence invasion. We examined tissues from human oral squamous cell carcinoma (OSCC) for SLPI expression in parallel with proteases associated with tumor progression and evaluated their relationships using tumor cell lines. Significantly decreased SLPI was detected in OSCC compared to normal oral epithelium. Furthermore, an inverse correlation between SLPI and histological parameters associated with tumor progression, including stage of invasion, pattern of invasion, invasive cell grade, and composite histological tumor score was evident. Conversely, elevated plasmin and elastase were positively correlated with histological parameters of tumor invasion. In addition to its known inhibition of elastase, we identify SLPI as a novel inhibitor of plasminogen activation through its interaction with annexin A2 with concomitant reduced plasmin generation by macrophages and OSCC cell lines. In an in vitro assay measuring invasive activity, SLPI blocked protease-dependent tumor cell migration. Our data suggest that SLPI may possess antitumorigenic activity by virtue of its ability to interfere with multiple requisite proteolytic steps underlying tumor cell invasion and may provide insight into potential stratification of oral cancer according to risk of occult metastasis, guiding treatment strategies.

Soluble Tumor Necrosis Factor-like Weak Inducer of Apoptosis Plasma Levels as a Novel Biomarker of Endothelial Function in Prevalent Orthotopic Heart Transplant Recipients

Background Nonrenal solid organ transplantation is now an established method of therapy with significantly improved outcome over the last years; however, an increasingly prevalent complication in this population is chronic kidney disease. Endothelial dysfunction is highly prevalent in both cardiovascular disease and chronic kidney disease. Tumor necrosis factor (TNF)–like weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily of cytokines. Materials and Methods The aim of the study was to assess TWEAK concentration in 134 prevalent heart transplant recipients in relation to kidney function. Complete blood count, urea, serum lipids, fasting glucose, creatinine, NT-proBNP were studied. Soluble TWEAK was assayed using commercially available kits from Bender MedSystems (VIenna, Austria). High-sensitivity C-reactive protein was assayed using commercially available kits from American Diagnostica (Greenwich, Conn, USA). Results Heart transplant recipients had significantly higher serum creatinine, urea, cholesterol, triglycerides, fasting glucose, white blood cell count, lower TWEAK levels and lower estimated glomerular filtration rate (eGFR) than the control group. Serum TWEAK levels fell together with New York Heart Association (NYHA) class and rise in GFR. Serum TWEAK was related to kidney function, NYHA class, NT-proBNP, and triglycerides. Kidney function and NYHA class turn out to be predictors of TWEAK in heart transplant recipients. Conclusion TWEAK level is dependent on kidney and heart function. It might also represent a surrogate marker of endothelial dysfunction and atherosclerosis.

Increased thrombin generation measured in the presence of thrombomodulin in women with early pregnancy loss

Compared with 537 parous controls with no history of pregnancy loss, a lower thrombomodulin-related inhibition of the endogenous thrombin potential was measured in 264 cases with previous unexplained pregnancy loss, especially when losses occurred between 9 and 12 weeks of gestation. Adjusting age, protein S, factor VIII, factor V Leiden, and prothrombin G20210A did not change the results.

Inhibition of Endothelial Cell Tube Formation by ADAMTS13 Occurs Via Interaction with VEGF

AbstractAbstract 4307The von Willebrand factor-cleaving protease, also known as ADAMTS13, is synthesized, in part, by endothelial cells (EC). We previously reported that proliferating EC secreted ∼3-fold more ADAMTS13 (antigen and activity) than confluent EC, and that this synthesis was transcriptionally regulated (SJ Kling et al, Pathophysiol Haemost Thromb 2008; 36: 233.) Thrombospondin domains, a defining feature of the ADAMTS protease family, in other ADAMTS family members mediate inhibition of angiogenesis. In particular, ADAMTS1 inhibits angiogenesis by sequestering vascular endothelial growth factor (VEGF) (A Luque et al, J Biol Chem, 2003; 278: 23656). Since ADAMTS13 also has thrombospondin domains, we hypothesized that ADAMTS13 might also mediate anti-angiogenic activity. Human umbilical vein EC angiogenesis was quantified by Angioquant analysis of fluorescence microscopy tube images in a Matrigel assay. Treatment of EC with inhibitors of angiogenesis (anti-VEGF, vasostatin) did not alter EC production of ADAMTS13. However, exogenous recombinant ADAMTS13 inhibited angiogenesis in a dose-dependent manner (Figure 1), in media containing VEGF. In VEGF-free media, ADAMTS13 had no effect on EC tube formation. Preincubation of ADAMTS13 with an antibody to the ADAMTS13 thrombospondin domains 5–7 partially reversed the inhibition of tube formation, implicating these domains in the anti-angiogenic interaction (Figure 2). We also found that VEGF co-immunoprecipitates with ADAMTS13, providing strong evidence that these two proteins interact. When EC lysates were crosslinked with DTSSP prior to immunoprecipitation, anti-ADAMTS13 immunoprecipitated as much VEGF as did anti-ADAMTS1, while an antibody to ADAM17, a similar protein that lacks thrombospondin domains, failed to co-immunoprecipitate VEGF (Figure 3). These data indicate that as with other ADAMTS family members, ADAMTS13 inhibits tubule formation - a parameter of angiogenesis – through its interaction with VEGF, an effect likely mediated by its thrombospondin domains. Inhibition of angiogenesis adds to the expanding roles of ADAMTS13 in down-regulation of thrombosis and inflammation. [Display omitted] [Display omitted] [Display omitted] Disclosures:Fryer:American Diagnostica Inc: Employment. Greenfield:American Diagnostica Inc: Employment.

Characterization of the Procoagulant Activity of Two Different Microparticle Species.

AbstractAbstract 1143 Background:Microparticles (MP) exhibit procoagulant activity by exposure of tissue factor (TF) and consecutive factor (F) × activation via binding to activated FVII (FVIIa). We have shown that TF-negative MP derived from endothelial cells (EMP) after exposure to cisplatin induce thrombin generation in vitro in a TF-independent manner (Lechner et al., J. Thromb. Haemost. 2007). The procoagulant properties of TF-negative MP are not well characterized. We therefore aimed to investigate the mechanisms of coagulation activation by TF-negative EMP (obtained from human pulmonary microvascular endothelial cells - HMVEC-L) in comparison to TF-abundant MP derived from a cancer cell line (A431-MP). Methods:MP were obtained by ultracentrifugation (100,000 × g for 1 h) of cell culture supernatant. The procoagulant activity was measured in normal plasma and in plasmas deficient in coagulation factors VII, VIII, IX, X, XI, and XII, respectively, by an in vitro thrombin generation assay (Technothrombin TGA, Technoclone, Austria), by activated partial thromboplastin (aPTT), prothrombin time (PT) and a chromogenic FIX activation assay. Antibodies to TF and FVII (both American Diagnostica, USA) were used. Plasmas used in the thrombin generation assay were MP-depleted by ultracentrifugation before use. Results:EMP and A431-MP induced in vitro thrombin generation in normal plasma. In vitro thrombin generation induced by EMP was abolished in plasmas deficient in FVIII or FIX, and was markedly reduced in FXI-deficient plasma. In FVII-deficient plasma, normal thrombin generation after addition of EMP was seen. In contrast, A431-MP-triggered thrombin generation was abolished in FVII-deficient plasma, but was not influenced by depletion of FVIII, FIX or FXI. In FX-deficient plasma, thrombin generation could not be triggered by addition of either EMP or A431-MP. Thrombin generation was normal after addition of EMP or A431-MP to FXII-deficient plasma (Figure). In a modified aPTT system using kaolin as surface activator, EMP and A431-MP induced clot formation in normal plasma. In a modified PT system only A431-MP but not EMP induced clot formation. We then investigated whether MP can directly activate FIX. In a plasma-free environment FIX activation by A431-MP was much more pronounced than by EMP (14-fold vs. 2.4 fold increase compared to a control experiment with cell culture medium only). FIX activation by A431-MP was blocked by the addition of antibodies to TF and FVII, while no such effect was seen after addition to EMP. Conclusions:Our findings show that both TF-positive and TF-negative MP exhibit procoagulant activity. If TF is expressed (A431-MP), coagulation activation is triggered via FVII/FVIIa, which is abolished in FVII-deficient plasma or by the addition of antibodies to TF or FVII. TF-negative EMP induce thrombin generation via the intrinsic coagulation pathway by activating FXI and FIX, as shown by the generation of activated FIX in a FIX activation assay. EMP are also able to induce clot formation in an aPTT based system. We surmise that the procoagulatory effect of EMP may be due to their high phospholipid content or to a specific phospholipid surface composition. The results of our FIX activation assay also confirm a direct FIX activation by TF/FVIIa, which can be abolished by addition of antibodies to TF or FVII. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

93 Cytokines Associate with Tissue Factor But Not with Thrombin Formation in Preterm Infants

Background: Tissue factor (TF), the in vivo initiator of coagulation cascade, is a central link between inflammatory and coagulation pathways.Objective: To evaluate associations between cytokine concentrations, TF and thrombin formation in preterm infants.Methods: We measured proinflammatory cytokines (IL-6, IL-8, TNF-alpha), anti-inflammatory cytokine IL-10, free TF and, as indicator of thrombin generation, prothrombin fragment 1+2 (F1+2) in 56 VLBW infants (GA 23+5-31+5 wks, BW 600-1500g). Cord blood and blood samples on day 1, 3, and 5-7 were collected. From plasma cytokines were analyzed with Luminex technique, free TF and F1+2 were analyzed with Elisa tests (American Diagnostica, Dade Behring).Results: IL-6 correlated with free TF in cord samples and on days 1 and 3 (R=0.53, p=0.013, n=21; R=0.380, p=0.019, n=38; R=0.373, p=0.042, n=30). IL-8 correlated with free TF on days 1 and 3 (R=0.597, p< 0.001, n=38; R=0.462, p=0.009, n=31). TNF-alpha and IL-10 both correlated with free TF in cord samples (R=0.536, p=0.012, n=31, and R=0.610, p=0.003, n=22). No correlations were found between cytokines and F1+2.Conclusions: Both pro- and anti-inflammatory cytokines correlated with high TF concentration. A difference seems to exist between in utero (cord) and postnatal TF regulation: in cord plasma TNF-alpha as well as IL-10 showed strong associations with TF but lost this role to IL-8 postnatally. Free TF failed to contribute to the rate of thrombin generation. This shift of TF from coagulation to inflammation may have effects on pathogenesis of lung injury, as well as on extrapulmonary morbidity in preterm infants.

Redox control of β2-glycoprotein I-von Willebrand factor interaction by thioredoxin-1.

Background:β2-Glycoprotein I (β2GPI) is an abundant plasma protein that is closely linked to blood clotting, as it interacts with various protein and cellular components of the coagulation system. However, the role of β2GPI in thrombus formation is unknown. We have recently shown that β2GPI is susceptible to reduction by the thiol oxidoreductases thioredoxin-1 and protein disulfide isomerase, and that reduction of β2GPI can take place on the platelet surface. Methods:β2GPI, reduced by thioredoxin-1, was labeled with the selective sulfhydryl probe Na-(3-maleimidylpropionyl)biocytin and subjected to mass spectrometry to identify the specific cysteines involved in the thiol exchange reaction. Binding assays were used to examine the affinity of reduced β2GPI for von Willebrand factor (VWF) and the effect of reduced β2GPI on glycoprotein (GP)Ibα binding to VWF. Platelet adhesion to ristocetin-activated VWF was studied in the presence of reduced β2GPI. Results: We demonstrate that the Cys288–Cys326 disulfide in domain V of β2GPI is the predominant disulfide reduced by thioredoxin-1. Reduced β2GPI in vitro displays increased binding to VWF that is dependent on disulfide bond formation. β2GPI reduced by thioredoxin-1, in comparison with non-reduced β2GPI, leads to increased binding of GPIbα to VWF and increased platelet adhesion to activated VWF. Conclusions: Given the importance of thiol oxidoreductases in thrombus formation, we provide preliminary evidence that the thiol-dependent interaction of β2GPI with VWF may contribute to the redox regulation of platelet adhesion.

PO-85 Effects of an anthocyanin (delphinidin-3-glucoside) from wild blueberries on the proangiogenic and prothrombotic properties of endothelial cells

CFPAC1 (Table). The clotting times (CT) mirrored TF expression levels with CFPAC1 giving the fastest time and MIAPaCA-2 the slowest. Blocking of cell-expressed TF with a saturating concentration of a polyclonal antibody (American diagnostica) increased CT in all cases. The highest cell invasion was also observed with CFPAC1, followed by AsPC1 and MIAPaCa-2. Blocking of TF decreased cell invasion to approximately 50% in both CFPAC1 and AsPC1, but showed minimal effect on the already low levels observed with MIAPaCa-2. Conclusions: Clotting time and cell invasion appear to be strongly correlated, with both processes being mediated, at least in part, via TF.

Preface

The 22nd International Congress on Heterocyclic Chemistry (ICHC-22) was held 2-7 August 2009 in St. John's, Newfoundland and Labrador, Canada. St. John's, the capital of Canada's youngest Province, Newfoundland and Labrador, is also Canada’s oldest and North America’s most easterly city. The Conference was chaired by Prof. Mohsen Daneshtalab (School of Pharmacy, Memorial University of Newfoundland) and was organized by the School of Pharmacy and the Chemistry Department at Memorial University of Newfoundland.Approximately 260 participants from over 30 different countries attended. The scientific program consisted of 10 plenary lectures, 19 invited lectures, 52 short communications, and 115 posters. Prof. Samuel Danishefsky (Sloan-Kettering Institute for Cancer Research, Columbia University) was honored with the 2009 ISHC Senior Award in Heterocyclic Chemistry, and Prof. John Wood (Colorado State University) was the 2009 Katritzky Junior Award winner. A special symposium entitled "Focus on heterocycles in organic synthesis today and tomorrow" was held during the Congress as a tribute to Prof. Victor Snieckus (Queen's University, Kingston) for his research accomplishments and long-time contribution to the International Society of Heterocyclic Chemistry (ISHC).The five Congress themes were:- New Methods in Heterocyclic Chemistry- Biologically Active Heterocycles (Pharmaceuticals/Agrochemicals)- Heterocyclic Natural Products and their Analogues- Applications of Heterocycles in Organic Synthesis- Heterocycles in Materials ScienceBesides the collection of 9 papers that are based on the plenary and invited lectures included in this issue of Pure and Applied Chemistry, the ICHC-22 Book of Abstracts is available online and can be downloaded for free from http://www.ichc2009.ca/abstract_book.pdf in pdf format.ICHC-23 will be held in Glasgow, Scotland, 31 July to 5 August 2011 with the following five main themes of heterocyclic chemistry: synthetic methodology, natural products and complex molecule synthesis, materials, medicinal chemistry, and nanochemistry. The conference will be chaired by Prof. Colin Suckling (University of Strathclyde).The organizers are grateful to all who contributed to a successful scientific program, especially to the speakers and to our public and private sponsors: City of St. John's, Memorial University of Newfoundland, IUPAC, Thieme, Wiley-Blackwell, Elsevier, Taiho Pharmaceutical Co., ChemRoutres Corporation, and American Diagnostica, Inc.Paris E. GeorghiouConference Editor

Risk Assessment in Node-Negative Breast Cancer by the Invasion Factors (Urokinase-Type Plasminogen Activator) and Its Inhibitor PAI-1. Correlation to the Tumor Characteristics in the Prospective NNBC 3-Europe Trial. On Behalf of the NNBC-3 Trial Group.

Abstract Background:ASCO Tumor Marker Guidelines 2007 recommended clinical routine use of the invasion markers uPA and PAI-1 for risk assessment in N0 breast cancer patients (Harris et al. JCO 2007; 25:5287). We conducted the prospective NNBC 3-Europe trial addressing the direct comparison between risk-assessment by uPA/PAI-1 and clinico-pathological factors, and optimization of adjuvant chemotherapy for high-risk N0 pts: FEC*3-Doc*3 vs. standard FE100C*6.Study Design:Risk assessment was based on grade, and in G2 tumors either by uPA/PAI-1 status (UP) or by a St. Gallen adapted algorithm (CP). Type of assessment was decided by each centre prior to study participation. High-risk patients received adjuvant chemotherapy according to randomisation. Adjuvant endocrine therapy is given according to guidelines. Use of fresh tissue sampling was necessary for determination of uPA/PAI-1 by ELISA. All laboratories took part on the central quality assurance program. HER2 overexpression was confirmed centrally.Results:From Dec 2002 to Jan 2009, 153 centres recruited 4,150 pts (4,145 evaluable). In 2,497 pts., risk was assessed by UP, and in 1,648 by CP. By UP, 38.7% were assigned to the low-risk group, whilst 61.3% of the patients still had to receive adjuvant chemotherapy; by CP-assessment (n=1,648) the risk groups comprised 31.3%, and 68.7 %.Tab.1. Tumor characteristics.risk groupUP lowUP highCP lowCP hightotaln9671,5305161,1324,145age (median)54 (31-70)53 (21-70)57 (34-70)53 (21-70)54 (21-70)pT2178 (18.4%)544 (35.6%)24 (4.7%)603 (53.3%)1,349 (33%)Steroid hormone receptor status pos.951 (98.3%)1,116 (72.9%)513 (99.4%)739 (65.3%)3,331 (80.4%)HER2 overexpression45 (4.7%)284 (18.6%)6 (1.2%)247 (21.8%)582 (14.0%)triple negative (TNBC)15 (1.6%)319 (20.8%)2 (0.4%)295 (26.1%)631 (15.2%)Grading 1417 (43.1%)-164 (32.0%)15 (1.3%)596 (14.4%)Grading 2550 (56.9%)750 (48.9%)345 (67.4%)464 (40.7%)2,115 (51.0%)Grading 3-780 (50.9%)7 (1.4%)639 (56.1%)1,424 (34.4%)uPA value [ng/mg] median1.33.8--2.4PAI-1 value [ng/mg]median9.121.5--15.4 More pT2 tumors were listed as low risk by the UP method. Typical high risk groups (e.g. TNBC, HER2) were identified by UP. A substantial number of HER2 overexpressing tumors (4.7%) in UP were assigned to the low risk group, suggesting an independent effect of UP and HER2. In the QA programm, mean coefficients of variations were 12% for uPA and PAI-1 determination. In total 1,335 pts. were randomised to receive FEC-Doc, and 1.327 to receive FEC. The chemotherapy was well tolerated. However, we observed four therapy related deaths.Conclusions:Determination of uPA and PAI-1 in frozen tissue is feasible and reliable. Using UP for additional prognostic information may prevent chemotherapy from many patients. The effect of the taxane containing regimen will be evaluated with longer follow-up.Cooperation: EORTC Patho-Biology Group, AGO Breast Group, GBG, Unrestricted grants by Sanofi-Aventis, Pfizer, American Diagnostica, Martin-Luther-University Halle-Wittenberg. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4040.

Gla-Rich Protein Is a Novel Vitamin K-Dependent Protein Present in Serum That Accumulates at Sites of Pathological Calcifications

Mineralization of soft tissues is an abnormal process that occurs in any body tissue and can greatly increase morbidity and mortality. Vitamin K-dependent (VKD) proteins play a crucial role in these processes; matrix Gla protein is considered one of the most relevant physiological inhibitors of soft tissue calcification know to date. Several studies have suggested that other, still unknown, VKD proteins might also be involved in soft tissue calcification pathologies. We have recently identified in sturgeon a new VKD protein, Gla-rich protein (GRP), which contains the highest ratio between number of Gla residues and size of the mature protein so far identified. Although mainly expressed in cartilaginous tissues of sturgeon, in rat GRP is present in both cartilage and bone. We now show that GRP is a circulating protein that is also expressed and accumulated in soft tissues of rats and humans, including the skin and vascular system in which, when affected by pathological calcifications, GRP accumulates at high levels at sites of mineral deposition, indicating an association with calcification processes. The high number of Gla residues and consequent mineral binding affinity properties strongly suggest that GRP may directly influence mineral formation, thereby playing a role in processes involving connective tissue mineralization.

Pharmacodynamic Differences of An Anti-Xa Enriched Low Molecular Weight Heparin, AVE 5026 in Comparison to Enoxaparin and Unfractionated Heparin.

Abstract 4171AVE5026 (Sanofi-Aventis, Paris, France) represents an anti-Factor (F) Xa enriched ultra low molecular weight heparin (ULMWH) (Mw=2.4 Kda; anti-FXa activity ∼160 U/mg). In comparison to Enoxaparin it has a lower anti-FIIa activity (∼2 U/mg). The oligosaccharide composition of AVE5026 also differs from Enoxaparin and other LMWHs. Besides the molecular and compositional differences, the biologic half-life of AVE5026 (18-20 hours) is significantly longer than Enoxaparin (4-6 hours). In order to compare the other pharmacodynamic differences between AVE5026, Enoxaparin and unfractionated heparin (UFH), a primate model (macaca mulatto) was used since its tissue factor pathway inhibitor (TFPI) profile is comparable to the human response. Individual groups of primates (n=6) were administered with 1 mg/kg SC of either AVE5026, Enoxaparin or UFH. Heptest and APTT measurements were determined on whole blood (WB) and plasma was analyzed for APTT, Heptest, thrombin time (TT), anti-FXa and anti-FIIa effects at varying periods up to 28 hours. TFPI antigen was measured using the assay from Stago (Parsipanny, NJ). Functional TFPI measurements were determined using the kit from American Diagnostica (Stamford, CT). In contrast to UFH, in the WB assays, neither the AVE5026 nor the Enoxaparin produced a strong effect on the APTT and TT, however both demonstrated a strong effect on the heptest assay. AVE5026 produced a much stronger effect with a longer half-life (T½=11 hrs) in comparison to Enoxaparin (T½=6 hrs). In the plasma based systems only UFH produced a measurable effect on the APTT and TT. However, in the heptest and anti-FXa assays, both AVE5026 and Enoxaparin produced a stronger effect, which was much longer with AVE5026 (2-3 fold increase). The plasma time course of TFPI antigen release was longer with AVE5026 in comparison to Enoxaparin and UFH. The ratios of immunologic to functional TFPI levels were also higher in the primates administered with AVE 5026. In the thrombin generation test, AVE5026 produced a sustained effect which lasted longer than Enoxaparin (T½ =16.8 hrs vs. 9.2 hrs.). These results show that AVE5026 produces stronger anti-FXa effects in primates which are associated with a higher circulating level of TFPI and more pronounced suppression of thrombin generation compared to Enoxaparin and UFH. Disclosures:Hoppensteadt:Sanofi-Aventis: Research Funding.

Vascular Endothelial Growth Factor (VEGF) Receptor-2 Tyrosine 1175 Signaling Controls VEGF-induced von Willebrand Factor Release from Endothelial Cells via Phospholipase C-γ1- and Protein Kinase A-dependent Pathways

There is increasing evidence that vascular endothelial growth factor (VEGF) contributes to inflammation independent of its angiogenic functions. Targeting some of the components in endothelial Weibel-Palade bodies (WPBs) effectively inhibits VEGF-induced inflammation, but little is known about how VEGF regulates WPB exocytosis. In this study, we showed that VEGF receptor-2 (VEGFR2), but not VEGFR1, is responsible for VEGF-induced release of von Willebrand factor (vWF), a major marker of WPBs. This is in good contrast to VEGF-stimulated interleukin-6 release from endothelium, which is selectively mediated through VEGFR1. We further demonstrated that VEGFR2-initiated phospholipase C-gamma1 (PLCgamma1)/calcium signaling is important but insufficient for full vWF release, suggesting the possible participation of another effector pathway. We found that cAMP/protein kinase A (PKA) signaling is required for full vWF release. Importantly, a single mutation of Tyr(1175) in the C terminus of VEGFR2, a tyrosine residue crucial for embryonic vasculogenesis, abolished vWF release, concomitant with defective activations of both PLCgamma1 and PKA. These data suggest that Tyr(1175) mediates both PLCgamma1-dependent and PKA-dependent signaling pathways. Taken together, our results not only reveal a novel Tyr(1175)-mediated signaling pathway but also highlight a potentially new therapeutic target for the management of vascular inflammation.

The Urokinase Receptor Promotes Cancer Metastasis Independently of Urokinase-Type Plasminogen Activator in Mice

The urokinase receptor (uPAR) promotes metastasis of human malignancies; however, its mechanism of action remains incompletely understood. Established models focus on the ability of uPAR to bind urokinase-type plasminogen activator (uPA) and promote protease activation in the tumor cell microenvironment; however, uPAR also regulates cell signaling and migration by both uPA-dependent and -independent mechanisms in vitro. The significance of uPAR as a cell-signaling receptor in vivo remains unclear. In this study, we expressed either human or mouse uPAR in human embryonic kidney (HEK-293) cells. We selected HEK-293 cells because, unlike most cancer cells, they do not express uPA or uPAR endogenously. Both mouse and human uPAR increased cell adhesion and migration on vitronectin. Rac1 was activated and responsible for the increase in cell migration. HEK-293 cells that did not express uPAR formed palpable tumors in severe combined immunodeficient mice; however, metastases were exceedingly rare. The xenografts contained abundant mouse uPA, produced by infiltrating mouse cells, but no human uPA. Mouse uPA bound only to mouse uPAR and not human uPAR and, thus, could not interact with human uPAR-expressing HEK-293 cells in xenografts. Nevertheless, both mouse and human uPAR significantly increased HEK-293 cell metastasis into the lungs. The activity of human uPAR suggests that uPAR may promote cancer metastasis independent of uPA. Candidate mechanisms include its effects on adhesion, migration, and Rac1 activation.

ASCO-recommended prognostic factors uPA/PAI-1 in node-negative (N0) breast cancer patients (pts) compared to clinicopathological risk assessment within the NNBC 3-Europe trial

544 Background: ASCO Tumor Marker Guidelines 2007 recommended clinical routine use of the invasion markers uPA (urokinase-type plasminogen activator) and its inhibitor PAI-1 for risk assessment in N0 breast cancer pts (Harris et al, J Clin Oncol. 2007;25:5287). Ten-year follow-up data of the confirmatory prospective clinical uPA/PAI-1 trial (CHEMO-N0) will be presented at the ASCO 2009 Meeting (Harbeck et al.). We conducted a further prospective trial (NNBC 3-Europe) addressing the direct comparison between risk-assessment by uPA/PAI-1 (UP) and clinico-pathological factors (C), and optimization of adjuvant chemotherapy for high-risk N0 pts: FEC*3-Doc*3 vs. standard FEC*6. Methods: From December 2002 to January 2009, 4,040 pts were recruited by 151 centres. For uPA and PAI-1 ELISA, tumors samples were snap frozen und processed. Risk assessment was based on grade and in G2 tumors, either by UP or by a St. Gallen adapted C algorithm. High-risk pts were randomized for adjuvant chemotherapy. All laboratories took part in central quality assurance (QA) for uPA/PAI-1 determination. Results: Risk assessment was performed for 1,590 pts based on C and for 2,445 pts based on UP. End of accrual was planned when 2,572 high risk pts were randomised for chemotherapy and thus ends Jan 31, 2009. Current status shows 1,291 high-risk pts in the FEC-Doc and 1,294 in the FEC arm. In the low-risk arm, 1,450 pts have endocrine therapy, only. By both risk stratifications, 42% of the G2 tumors were assigned as low risk, but in total, more pts were classified as low-risk by UP (39%) than by C (31%). Median uPA level were at 2.40 ng/mg protein (below cutoff), median PAI-1 at 15.9 (above cutoff). In the QA programm, mean coefficients of variations are 12% for uPA and PAI-1 determination. Conclusions: The prospective biomarker trial using uPA/PAI-1 has just ended accrual with more than 4,000 patients. Routine uPA/PAI-1 determination is reliable and feasible even in core needle specimens. Using tumor grade and uPA/PAI-1, adjuvant chemotherapy may be avoided in about 39% of N0 breast cancer pts. Cooperation: EORTC Patho-Biology Group, AGO Breast Group, GBG, Unrestricted grants by Sanofi-Aventis, Pfizer, American Diagnostica, Martin-Luther-Universitaet Halle-Wittenberg. [Table: see text]

Plasmin-mediated Proteolysis Is Required for Hepatocyte Growth Factor Activation during Liver Repair

The physiological relevance of the activation of hepatocyte growth factor (Hgf) by the plasminogen (Plg) system of proteases and its contribution to tissue repair are largely undefined. Here, we investigated whether the defective liver repair in mice lacking Plg is due to impaired activation of Hgf. Loss of Plg in vivo suppressed Hgf activation and signaling through its Met tyrosine kinase receptor. Without Plg, hepatocytes were unresponsive to Hgf-induced proliferation and migration, with a more pronounced impairment in hepatocyte movement within the hepatic environment. Most notably, circumventing the defect in proteolytic activation of Hgf by the downstream expression of an activated Met receptor corrected the functional deficits and improved liver repair in Plg-deficient mice. These findings support a fibrinolysis-unrelated role for Plg in modulating cell proliferation and migration by activation of Hgf.