ALBERT

Description: Introduction: We have recently shown that low TFPI is a weak risk factor for deep vein thrombosis (DVT)(Dahm et al Blood2003;101;4387–92). Plasma contains free, full-length TFPI (FL-TFPI) and truncated and lipoprotein associated TFPI. Since free, FL-TFPI has a much stronger ability to prolong clotting time in diluted prothrombin time (dPT) assay than other types of TFPI, it has been suggested that FL-TFPI biologically plays a more important role than other forms of TFPI. Aims: To determine the fraction of free, FL-TFPI in plasma and to determine the role of TFPI anticoagulant activity in the prevention of DVT. Materials and Methods: Normalized TFPI Anticoagulant Activity (n-TFPIac) Ratio was assayed using a dPT assay after incubation of plasma in the absence and the presence of neutralizing anti-TFPI antibodies. Results were expressed as a ratio with dPT in the presence of anti-TFPI as the denominator. The ratio was normalized against a ratio obtained with a reference plasma within each run. TFPI chromogenic substrate (TFPIcs) activity assay was determined by the quantification of residual TF/FVIIa catalytic activity after the incubation of diluted plasma (containing TFPI) with TF, FVIIa, and FXa. TFPI antigen assays: TFPI free antigen (full-length TFPI) and TFPI total antigen (full-length + truncated TFPI) were assayed with commercial kits from Stago, France. Bound TFPI was calculated as the difference between TFPI total antigen and TFPI free antigen. Study population: Individuals included in The Leiden Thrombophila Study (LETS), which is a case-control study of 474 patients with DVT and 474 controls. 363 controls and 362 cases were available for the OR calculations and 473 controls for the estimation of the fraction of free, FL-TFPI. Statistics: The fraction of free, FL-TFPI was calculated by dividing TFPI free antigen by TFPI total antigen. Odds ratios (OR) for DVT were calculated for individuals with TFPI values below the 10th percentile as compared with those above. Results: The fraction of free, FL-TFPI in plasma was 19%, but with large variations due to hormonal state (table 1). Normalized TFPIac ratio below the 10th percentile gave an OR of 1.5 (95% CI 0.97-2.4) for DVT, which was comparable to the ORs obtained with other TFPI assays. Individuals with low TFPI in both activity assays had an OR of 5.9 (95% CI 1.7–20) for DVT (table 2). Conclusion: Approximately 20% of TFPI in plasma was free, FL-TFPI. Low n-TFPIac ratio was a weak risk factor for DVT, but was not a stronger risk factor than low TFPI in the other assays. However, combined low n-TFPIac ratio and low TFPIcs activity seemed to be a strong risk factor for DVT. Table 1. Mean (95% CI) plasma fraction of TFPI free antigen in controls OC users (n=54) OC nonusers (n=99) Postmenopausal women (n=89) Men (n=201) All (n=473) TFPI Free Antigen/TFPI total TFPI antigen 0.125 (0.115–0.135) 0.175 (0.165–0.184) 0.197 (0.186–0.208) 0.206 (0.199–0.209) 0.187 (0.182–0.192) Table 2. OR (95% CI) for DVT for different TFPI parameters below the 10th percentile nTFPIac ratio TFPI free antigen TFPI total antigen TFPIcs activity Bound TFPI n-TFPIac ratio + TFPIcs activity 1.5 (0.97–2.4) 1.3 (0.83–2.1) 1.3 (0.85–2.1) 1.2 (0.75–1.9) 1.4 (0.90–2.2) 5.9 (1.7–20)

Description: Abstract 4203 Background: Venous thrombosis (VT) is the leading cause of maternal death in the western world. Genetic causes of pregnancy related VT are insufficiently understood. Objectives: To investigate if recently discovered associations between single nucleotide polymorphisms (SNPs) and thrombotic disease influenced the risk for pregnancy related VT. To investigate candidate genes for new associations between SNPs and pregnancy related VT. Methods: A hospital based case control study. Cases were women with objectively verified VT during pregnancy or puerperium, controls were women giving birth without having VT. We selected 49 candidate genes involved in coagulation (35 genes), inflammation (4 genes), and hormonal metabolism (10 genes). In these genes, 463 tag-SNPs were selected from the CEU population in Hapmap and analyzed in 313 cases and 353 controls. Odds ratios for VT were calculated with the most common genotype as the reference genotype for each SNP. P-values were adjusted for multiple testing to false discovery rates. Results: Of the SNPs previously shown by others to be associated to thrombotic disease, rs2289252 in the FXI gene (F11), rs3917643 in the tissue factor gene (F3), rs1613662 in the glycoprotein VI gene, rs2001490 in NAT8B, rs4524 in the FV gene (F5), and rs3087505 in the prekallikrein gene were associated to pregnancy related VT. After analyzing all 463 tag-SNPs, 13 SNPs were associated to total pregnancy related VT with a false discovery rate ≤ 0.20. These SNPs belonged to F5, SELP (coding for P-selectin), F3, F8 (coding for factor VIII), and F11. When we studied antenatal and postnatal VT separately, 17 SNPs were associated with antenatal VT, while only 1 SNP was associated with postnatal VT. Thirteen SNPs in F5, SELP, or the gene for E-selectin were associated with thrombosis. After excluding carriers of factor V Leiden, 4 of these SNPs remained associated. Two SNPs in the gene for epidermal growth factor receptor (EGFR) were associated to antenatal thrombosis. Conclusion: Genetic variation seems more important for antenatal than for postnatal VT. We confirmed the importance of some SNPs for the risk for pregnancy related VT, in particular rs2289252 in F11. The EGFR-gene appears to be a new genetic locus associated with pregnancy related VT. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract LBA-1 Background: Following acute deep vein thrombosis (DVT) of the lower limb, approximately 1 in 4 patients treated with anticoagulation (AC) and elastic compression stockings (ECS) in accordance with current guidelines, are still at risk for developing a chronically reduced functional outcome, i.e., the post-thrombotic syndrome (PTS). Additional treatment with catheter-directed thrombolysis (CDT) enhances clot removal and is suggested to favor venous competence and patency, thereby reducing the risk for PTS. This interventional therapy is expensive, associated with life-threatening bleeding, and converts an outpatient disease to an inpatient disease. However, it has become standard care in some centers despite a complete lack of evidence from randomized, controlled trials (RCT). The CaVenT study, representing the first RCT in this area, aimed to evaluate whether additional CDT with alteplase improved the functional outcome by reducing PTS development following acute iliofemoral DVT. Methods: The CaVenT study was an open, multicenter RCT that recruited patients from 20 hospitals in the Norwegian south-eastern health region. Patients of age 18–75 years with a first-time objectively verified acute iliofemoral DVT above mid-thigh level and symptoms for up to 21 days were eligible for recruitment. Study patients were randomly assigned with a 1:1 ratio to standard (control) treatment with AC and ECS grade II (30 mmHg) or to CDT with alteplase in addition to standard treatment. The present report concerns the primary clinical end-point; the frequency of PTS after 24 months follow-up. The study was designed to detect a reduction in PTS from 25% to 10% with a 5% significance level and with 80% power. Follow-up visits were conducted at 6 months ± 2 weeks and 24 months ± 4 weeks and included evaluation of PTS by the Villalta scale as recommended by the International Society on Thrombosis and Haemostasis. Iliofemoral patency was assessed with ultrasonography and air-plethysmography. A two-sided uncorrected Chi-square test was used for comparing dichotomous variables in the two treatment groups. Results: 209 patients with acute iliofemoral DVT were randomized during 2006–2009; 101 patients were allocated the CDT arm and 108 the control arm. At the completion of 24 months follow-up, data on clinical status were available and included in the intention to treat analyzes for 90 patients in the CDT arm and 99 control patients. Mean age was 51.5 years (SD 15.8), 36% were female, and mean duration of symptoms was 6.6 days (SD 4.6). 80/90 patients receiving CDT had successful lysis. At 24 months follow-up 37 (41.1%, 95% CI 31.5–51.4%) allocated additional CDT presented with PTS compared to 55 (55.6%, 95% CI 45.7–65.0%) in the control group (p=0.047), including one control with severe PTS. The difference in PTS corresponds to an absolute risk reduction of 14.4% (95% CI 0.2–27.9), and the number needed to treat was 7 (95% CI 4–502). No patients presented with venous ulcer. In total 20 bleeding complications were reported; 3 were classified as major and 5 as clinically relevant. The majority of bleedings were related to the puncture site. The major bleedings included 1 abdominal wall hematoma requiring blood transfusion, 1 compartment syndrome of the calf requiring surgery, and 1 inguinal puncture site hematoma. No bleeding led to a permanently reduced outcome, and there were no deaths, pulmonary embolism or cerebral hemorrhage related to CDT. Patients who had regained venous patency after 6 months, developed PTS in 38/103 (36.9%, 95% CI 28.2–46.5%) as compared to 49/80 (61.3%, 95% CI 50.3–71.2%) of patients with insufficient recanalization (p

Description: Background: Several strategies for managing patients with suspected PE have been validated. However, most of these strategies are complicated, involving multiple rounds of tests, and are thus, time consuming, costly and difficult to apply in clinical practice. Aiming to introduce a simple, fast and cost-effective strategy, we adopted a new diagnostic approach combining clinical probability assessment, D-Dimer and multi-slice spiral CT (MSSCT) scanning. Aims: The aim of this study was to assess the safety and efficacy of this management strategy by a prospective outcome study with 3-month follow-up. Methods: 495 consecutive patients referred to the Emergency Department at Østfold Hospital, Fredrikstad, Norway, for suspected PE, between Feb 2002 and Dec 2003, were considered for inclusion. 63 (12.7%) patients were excluded and the final cohort consisted of 432 patients. Patients were managed by serial non-invasive testing starting with D-Dimer test. Normal plasma D-Dimer (Liatest, latex agglutination assay, Stago-France, cut-off

Description: Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. Cell trafficking and homing of CLL cells play a critical role in organ infiltration and contribute to the clinical course of CLL. Interaction between CLL cells and endothelial cells affects gene expression in CLL cells and further regulates cell trafficking. Endothelial cells are the main source of tissue factor (TF) pathway inhibitor (TFPI), which is the primary inhibitor of TF. However, the role of TFPI in leukemic cell trafficking has not been explored before. CXCL12 and its receptor CXCR4 play an important role in CLL cell trafficking and homing. To assess the effect of TFPI on CXCL12-mediated migration of leukemic cells, we investigated transendothelial migration (TEM) of B cells from CLL patients by using CXCL12 as a chemoattractant. We found that full-length recombinant TFPI (rTFPI) increased CXCL12-mediated migration of CLL cells dose-dependently by using MACS flow cytometry to count the migrated cells. Accordingly, Western blot showed the binding of rTFPI to the CLL cells after 24 h treatment. However, the chemokine receptor CXCR4 expression in CLL cells was not increased or even slightly decreased by rTFPI treatment, suggesting that TFPI enhances leukemic cell migration probably through interacting with other factor(s) than overexpressing CXCR4 in CLL cells. Surface expression and intracellular trafficking of TFPI is regulated by glycosylphosphatidylinositol (GPI)-anchored co-receptor(s). Glypican-3, a member of proteoglycan family, binds to the plasma membrane by a GPI anchor and its heparan sulfate (HS)-type glycosaminoglycan chains are considered to mediate the interaction with other cell membrane proteins. To determine whether TFPI interacts with glypican-3, immunofluorescence staining and co-immunoprecipitation were performed in K562 and HL60 cells. Immunofluorescence analysis revealed colocalization of TFPI and glypican-3 on the surface of K562 and HL60 cells. Co-immunoprecipitation confirmed the binding between TFPI and glypican-3 in K562 cells. Moreover, co-localized TFPI and glypican-3 were found in bone marrow biopsies of leukemia patients. We further employed an antibody against HS chains of glypican-3, which inhibits the activity of glypican-3. Western blot showed that rTFPI treatment failed to increase TFPI protein in CLL cells when glypican-3 inhibitor HS20 was applied. In addition, TEM assay showed that HS20 treatment abolished the effect of TFPI on cell migration, which suggests that inhibition of glypican-3 blocks the binding of TFPI to CLL cells and then abolishes the effect of TFPI on cell migration. It indicates that TFPI affects CLL cell migration through binding to glypican-3. Using a factor Xa generation assay, we found that HS20 suppressed TF activity on the surface of HL60 cells, suggesting that the inhibition of glypican-3 impaired the procoagulant activity of leukemic cells. In addition, Western blot and RT-PCR showed that HS20 downregulated TF mRNA and protein expression in CLL cells. Finally, we explored the role of Wnt/β-catenin signaling, which has been shown to regulate tumor progression and metastasis. We found a dose-dependent increase of activated β-catenin in the CLL cells after rTFPI treatment. Inhibition of Wnt/β-catenin signaling by a potent inhibitor IWP4 strongly impaired the migration of CLL cells induced by rTFPI. In this study, we show that TFPI enhances CLL cell migration through binding to glypican-3 and activating Wnt/β-catenin signaling pathway. Our findings suggest a novel molecular mechanism regulating the transmigration of leukemic cells. It may open a new window for therapeutic targeting in the treatment of organ infiltration of CLL patients. Disclosures No relevant conflicts of interest to declare.

Description: LBA-1 Background: Following acute deep vein thrombosis (DVT) of the lower limb, approximately 1 in 4 patients treated with anticoagulation (AC) and elastic compression stockings (ECS) in accordance with current guidelines, are still at risk for developing a chronically reduced functional outcome, i.e., the post-thrombotic syndrome (PTS). Additional treatment with catheter-directed thrombolysis (CDT) enhances clot removal and is suggested to favor venous competence and patency, thereby reducing the risk for PTS. This interventional therapy is expensive, associated with life-threatening bleeding, and converts an outpatient disease to an inpatient disease. However, it has become standard care in some centers despite a complete lack of evidence from randomized, controlled trials (RCT). The CaVenT study, representing the first RCT in this area, aimed to evaluate whether additional CDT with alteplase improved the functional outcome by reducing PTS development following acute iliofemoral DVT. Methods: The CaVenT study was an open, multicenter RCT that recruited patients from 20 hospitals in the Norwegian south-eastern health region. Patients of age 18–75 years with a first-time objectively verified acute iliofemoral DVT above mid-thigh level and symptoms for up to 21 days were eligible for recruitment. Study patients were randomly assigned with a 1:1 ratio to standard (control) treatment with AC and ECS grade II (30 mmHg) or to CDT with alteplase in addition to standard treatment. The present report concerns the primary clinical end-point; the frequency of PTS after 24 months follow-up. The study was designed to detect a reduction in PTS from 25% to 10% with a 5% significance level and with 80% power. Follow-up visits were conducted at 6 months ± 2 weeks and 24 months ± 4 weeks and included evaluation of PTS by the Villalta scale as recommended by the International Society on Thrombosis and Haemostasis. Iliofemoral patency was assessed with ultrasonography and air-plethysmography. A two-sided uncorrected Chi-square test was used for comparing dichotomous variables in the two treatment groups. Results: 209 patients with acute iliofemoral DVT were randomized during 2006–2009; 101 patients were allocated the CDT arm and 108 the control arm. At the completion of 24 months follow-up, data on clinical status were available and included in the intention to treat analyzes for 90 patients in the CDT arm and 99 control patients. Mean age was 51.5 years (SD 15.8), 36% were female, and mean duration of symptoms was 6.6 days (SD 4.6). 80/90 patients receiving CDT had successful lysis. At 24 months follow-up 37 (41.1%, 95% CI 31.5–51.4%) allocated additional CDT presented with PTS compared to 55 (55.6%, 95% CI 45.7–65.0%) in the control group (p=0.047), including one control with severe PTS. The difference in PTS corresponds to an absolute risk reduction of 14.4% (95% CI 0.2–27.9), and the number needed to treat was 7 (95% CI 4–502). No patients presented with venous ulcer. In total 20 bleeding complications were reported; 3 were classified as major and 5 as clinically relevant. The majority of bleedings were related to the puncture site. The major bleedings included 1 abdominal wall hematoma requiring blood transfusion, 1 compartment syndrome of the calf requiring surgery, and 1 inguinal puncture site hematoma. No bleeding led to a permanently reduced outcome, and there were no deaths, pulmonary embolism or cerebral hemorrhage related to CDT. Patients who had regained venous patency after 6 months, developed PTS in 38/103 (36.9%, 95% CI 28.2–46.5%) as compared to 49/80 (61.3%, 95% CI 50.3–71.2%) of patients with insufficient recanalization (p

Description: Background: Angiogenesis is a potential target for therapy, both in solid tumors and in hematologic malignancies. However, the regulation of angiogenesis is complex, different cancers are inherently heterogeneous, and these drugs target different angiogenic regulators. Therefore, a detailed characterization of angiogenesis in individual cancers is needed. Materials and methods: The study cohort consisted of 93 consecutive patients with hematologic neoplasia; i.e. acute myeloid leukemia (n=20), chronic lymphatic leukemia (n=14), multiple myeloma (n=11), and non-Hodgkin’s lymphoma (NHL, n=48). We determined the microvessel density (MVD) in bone marrow biopsy specimens by immunohistochemical staining for CD34. In addition, we measured the plasma concentrations of eight putative angiogenesis regulators using Luminex multiplex assay, and the expression in peripheral blood mononuclear cells (PBMNC) of 40 angiogenesisrelated mRNAs using quantitative RT-PCR. Results: Untreated patients had increased bone marrow MVD (mean 39 microvessels/mm2) compared to healthy subjects (mean 20 microvessels/mm2; p