Pathogenesis Of Cancer-associated Thrombosis Research Articles

The pathogenesis of cancer-associated thrombosis.

Patients with cancer have a higher risk of venous thromboembolism (VTE), including deep vein thrombosis (DVT) and pulmonary embolism (PE), compared to the general population. Cancer-associated thrombosis (CAT) is a thrombotic event that occurs as a complication of cancer or cancer therapy. Major factors determining VTE risk in cancer patients include not only treatment history and patient characteristics, but also cancer type and site. Cancer types can be broadly divided into three groups based on VTE risk: high risk (pancreatic, ovarian, brain, stomach, gynecologic, and hematologic), intermediate risk (colon and lung), and low risk (breast and prostate). This implies that the mechanism of VTE differs between cancer types and that specific VTE pathways may exist for different cancer types. This review summarizes the specific pathways that contribute to VTE in cancer patients, with a particular focus on leukocytosis, neutrophil extracellular traps (NETs), tissue factor (TF), thrombocytosis, podoplanin (PDPN), plasminogen activator inhibitor-1 (PAI-1), the intrinsic coagulation pathway, and von Willebrand factor (VWF).

The effectiveness of atorvastatin for the prevention of deep vein thrombosis in cancer patients undergoing chemotherapy

BackgroundDeep vein thrombosis (DVT) is a common complication in cancer. Although thromboprophylaxis in cancer patients is recommended by the guidelines, clinicians’ use of thromboprophylaxis remains limited due to cost, bleeding complications, and reluctance to give injectable anticoagulants. Inflammation plays essential roles in the pathogenesis of cancer-associated thrombosis. Owing to its ability to decrease proinflammatory cytokines, statins have anti-inflammatory properties. Thus, statins can be possibly utilized as thromboprophylaxis therapy in cancer patients undergoing chemotherapy.ObjectiveTo compare the effectiveness of atorvastatin and rivaroxaban for DVT prevention in high-risk thrombosis patients with cancer undergoing chemotherapy.MethodsDouble-blind, randomized controlled trial involving cancer patients with high-risk of thrombosis undergoing chemotherapy. We randomly assigned patients without deep-vein thrombosis at screening to receive atorvastatin 20 mg or rivaroxaban 10 mg daily for up to 90 days. Doppler ultrasonography was performed 90 days following chemotherapy to diagnose DVT. Average cost-effectiveness analysis was performed to analyze the cost of atorvastatin compared to rivaroxaban.ResultsOf the eighty six patients who underwent randomization, primary efficacy end point was observed in 1 of 42 patients (2.3%) in the atorvastatin group and in 1 of 44 (2.2%) in the rivaroxaban group (Odds Ratio [OR], 0.953; 95% confidence interval [CI], 0.240 to 3.971; p = 1.000). There was a significant difference in the incidence of major bleeding, 2 of 42 patients (4.8%) in the atorvastatin group and 12 of 44 (27.3%) in the rivaroxaban group (OR, 0.257; 95% CI, 0.07 to 0.94; p = 0.007). The average cost-effectiveness ratio of using atorvastatin was lower than that of rivaroxaban.ConclusionAtorvastatin did not differ significantly from rivaroxaban in reducing the incidence of DVT, lower bleeding risk, and cost-effectiveness for thromboprophylaxis in high-risk thrombosis patients with cancer undergoing chemotherapy. The presence of limited statistical power and wide confidence intervals in this study needs further study to strengthen the efficacy of atorvastatin as DVT prophylaxis in cancer patients.Trial registrationISRCTN71891829, Registration Date: 17/12/2020.

CML-271: Neutrophil Extracellular Traps Are Increased in Chronic Myeloid Leukemia and Are Differentially Affected by Tyrosine Kinase Inhibitors

Context: Cardiovascular (CVS) complications are increasingly reported with the use of certain tyrosine kinase inhibitors (TKIs) to treat chronic myeloid leukemia (CML). Neutrophil extracellular traps (NETs) have previously been implicated in the pathogenesis of cancer-associated thrombosis. Objective: To assess NET formation in neutrophils from patients with CML, to characterize the role of reactive oxygen species (ROS) and peptidyl arginine deiminase 4 (PAD4) in NET formation in CML, and to assess the effects of TKIs on NET formation in CML. Design: Blood samples were obtained from patients with newly diagnosed CML and from age/gender-matched controls. NETs were assessed after stimulation with phorbol 12-myristate 13-acetate (PMA) or ionomycin (IO), with or without pre-treatment with imatinib, ponatinib, nilotinib, and dasatinib at clinically relevant concentrations. NET formation was assessed by morphology, immunofluorescence (IF), and the expression of proteins known to be associated with NET formation (NET-associated elastase, citrullinated histone H3 [H3cit], etc.). ER-HoxB8 conditionally immortalized murine myeloid progenitor cell line was differentiated into functional neutrophils and transduced with a BCR-ABL1 oncogene (or empty vector control). Results: Neutrophils isolated from treatment-naive patients with CML showed a significant increase in NET formation compared to matched controls at baseline and after stimulation with IO as assessed by IF and after stimulation with PMA as assessed by NET-associated elastase expression. Expression of H3cit, PAD4, and ROS was significantly higher in CML samples compared to controls. Pre-treatment of neutrophils with TKIs was associated with a differential effect on NET formation. Exposure to ponatinib significantly augmented NET-associated elastase and ROS levels as compared to control and other TKIs in neutrophils derived from patients with CML. BCR-ABL1 retrovirally transduced HoxB8-neutrophils demonstrated increased H3cit and myeloperoxidase expression, consistent with excess NET formation. This was inhibited by Cl-amidine, a PAD4 inhibitor, but not by the NADPH inhibitor diphenyleneiodonium (DPI). Ponatinib and nilotinib pre-exposure significantly increased H3cit expression in HoxB8-BCR-ABL1 cells after stimulation with IO. Conclusions: CML is associated with increased NET formation, which is further augmented by ponatinib and nilotinib, suggesting a possible role for NETs in promoting vascular toxicity in CML. In the ER-HoxB8-BCR-ABL1 model, NET formation is PAD4- but not ROS-dependent.

Alteration of Endothelial Cell Hemostatic Equilibrium By Cancer Cell-Derived Microparticles, a New Insight in Pathogenesis of Cancer Associated Thrombosis

Background: Cancer is associated with hypercoagulable state and frequently complicated by venous thromboembolism (VTE). Microparticles (MPs) are implicated in the pathogenesis of cancer associated thrombosis. However, not much is known about their interaction with the endothelium.Aim: To study the effect of cancer cell derived MPs on the hemostatic balance of endothelial cells.Methods: Microparticles released in conditioned medium from pancreas adenocarcinoma cells (BXPC3) and human breast cancer cells (MCF7) were isolated by differential centrifugation from cell cultures at 90% confluence. The pellets were washed twice in phosphate-buffered saline (BSA) and re-suspended in 1 ml Endothelial cell Basal Medium (EBM-2). Human umbilical vein endothelial cells (HUVEC) were cultured in 96-well plates in presence or absence of MPs derived from BXPC3 or MCF7 cells for 72h (C0). Then they were washed twice in BSA. and re-suspended in serum RPMI-1640 medium and re-cultivated over three cycles (C1-C3) following the same procedure of washing and culture.Thrombin generation (TG) of normal platelet poor plasma (PPP) added in wells carrying HUVEC were assessed by the Calibrated Automated Thrombogram (Rousseau et al Thromb Res. 2015;136:1273-9). Furthermore, TF activity (TFa) was assessed with a clotting based assay (from Diagnostica Stago, France). Cells and MPs were analyzed by flow cytometry for TF and annexin V expression using specific antibodies.Results: Some traces of TFa were found in HUVEC cells (0.20±0.03pM) and HUVEC MPs (1.2±1.0pM) being comparable to normal PPP (0.19±0.02pM). Abundant amounts of TFa were found in BXPC3 cells (2.6±0.05pM) and BXPC3 MPs (920±50pM). MCF7 cells expressed low amounts of TFa (1.21±0.05pM) whereas TFa levels in MCF7 MPs were significantly higher (12.1±2.2pM). Flow cytometry analysis for TF expression showed similar results for the three types of cells and the corresponding MPs. No procoagulant phospholipids were found on the surface of the three cell lines. In contrast, procoagulant phospholipids were expressed by BXPC3 and MCF7 MPs but not by HUVEC derived MPs. HUVEC exposed to cancer cell derived MPs enhanced thrombin generation. This procoagulant property of HUVEC was acquired after exposure to MPs and remained significant after three cycles of cells culture. MPs derived from BXPC3 had more pronounced effect on HUVEC as compared to MPs derived from MCF7 (Table 1).Conclusion:Cancer cell-derived microparticles possess significantly higher procoagulant activity as compared to the cells of origin. They are characterized by high expression of TF and procoagulant phospholipids. Cancer cell-derived MPs induced shift of HUVEC hemostatic balance. Quiescent HUVEC were transformed to potent procoagulant cells and enhanced thrombin generation. The procoagulant properties acquired by HUVEC are transferred to the following cells generations. To the best of our knowledge, the present study shows for the first time that the inherent procoagulant properties of MPs could alter vascular functions. The impact of MPs on the procoagulant phenotype of the HUVEC varies according to the type of cancer cell line. Overall, the findings of the present study provide novel information regarding the specific contribution of tumor-derived MPs to the pathogenesis of cancer-associated thrombosis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

PF800 SULFATED NON‐ANTICOAGULANT LOW MOLECULAR WEIGHT HEPARIN (S‐NACH) IN COMBATING CANCER ASSOCIATED THROMBOSIS WITHOUT EFFECTS ON HEMOSTASIS

Background:Cancer‐associated thrombosis (CAT) accounts for about 20% of all cases of Venous Thromboembolism (VTE). Cancer patients are 4 – 7 folds more likely to have VTE compared to patients without cancer. The pathogenesis of CAT is a complex process as cancer itself is a heterogeneous disease. Tissue factor (TF) was proven to be highly expressed on the angiogenic endothelial wall. Subsequent theories proposed that the cancer cells activate the coagulation cascade, as the oncogenes cause tumor transformation, which in turn causes the cancer tissues to be able to express different pro‐coagulant proteins. The VTE risk factors can be divided into cancer‐related, patient‐related and therapy‐related factors. Chemotherapy, surgery, hormonal therapy, anti‐angiogenic agents, immunomodulatory drugs, or transfusions, central venous catheters, and even hospitalization are reported to increase the VTE risk. To combat thrombosis complications, heparins and/or oral anticoagulants are used in therapy for cancer patients but these drugs have limited use due to an increased risk of bleeding complications in cancer patient.Aims:To investigate the effects of sulfated non‐anticoagulant low molecular weight (S‐NACH)on cancer‐associated thrombosis‐mediated by human pancreatic cancer cells. S‐NACH lacks anti‐thrombin binding and devoid of systemic anticoagulant effects on factor IIa and Xa in comparison to enoxaparinMethods:Pancreatic cancer cell line (SUIT2) and human endothelial cells (HUVEC) were obtained from ATCC and maintained in RPMI medium/10% FBS and EBM2 medium/ 20%FBS, respectively. For the Thrombelastography (TEG) Assay, we used SUIT2 cells to test the efficiency of blood coagulation in the presence of S‐NACH, and Enoxaparin at 1–10 μg. TEG measures clotting time, R, in minutes and clot strength, MA, in mm. Furthermore, we used human endothelial Cells (HUVEC), to test the release of TFPI‐1 and 2 in the presence of S‐NACH and Enoxaparin at different concentrations (1–10 μg). Additionally, we examined the in vivo effects of S‐NACH on release kinetics of plasma TFPI‐1 and 2.Results:Human whole blood clot kinetics using TEG assay shows significantly decreased clotting time (R‐value) and increased clot strength in blood from cancer patients or blood from healthy donors when adding pancreatic SUIT2 cells. The TEG assay demonstrated lack of effects of S‐NACH or TFPI alone on clot kinetics in human but significant changes with enoxaparin at 10 μg. In contrast, S‐NACH (1, 3 and 10 μg) in the presence of 0.01–0.1 ng TFPI‐1 or 2 resulted in a dose‐dependent suppression of clot strength and prolongation of clotting time. The introduction of TFPI had no effect on the MA values for the positive control, Enoxaparin (Figure 1).In the TFPI release assay, the concentration of the TFPI released at 1 hours and 3 hours increased as the concentration of S‐NACH and enoxaparin increased from 1 μg to 10 μg, with greater release with S‐NACH versus enoxaparin. In addition, we demonstrated in a previous study that, of S‐NACH did not affect the activity of factor IIa and Xa in comparison to Enoxaparin.Summary/Conclusion:Our data indicated that S‐NACH prevents cancer‐associated thrombosis without any effects on hemostasis. S‐NACH have no effect on homeostasis and work locally on in vivo on releasing vascular TFPI‐1 and 2. S‐NACH enhances the affinity of TFP‐1 and TFPI‐2 in suppressing and blocking cancer‐associated thrombosis.image

Transmission of Procoagulant Properties to Daughter Generations of Endothelial Cells upon Exposure to Cancer Cell-Derived Microparticles. a New Implication of Cancer Derived Microparticles in the Pathogenesis of Cancer Associated Thrombosis

Introduction & Objectives: Endothelium activation by cancer cell derived MPs (CaCe-dMP) is essential in pathogenesis of cancer associated thrombosis (CAT). Recent studies demonstrated that exposed endothelial cells to CaCe-dMP acquire a new procoagulant phenotype characterized by endocytosis of tissue factor (TF) and enhancement of thrombin generation.The aim of the present study was to investigate whereas the procoagulant properties acquired upon exposure of endothelial cells to CaCe-dMP are transferred to daughter generations of the endothelial cells.Materials & Methods: Pancreas adenocarcinoma cells (BXPC3) and human breast cancer cells (MCF7) were cultured in 75 cm2 culture flasks at 37˚C in a 100% humidified atmosphere with 5% CO2 and were used for experiments at a confluence of 70%. Microparticles released in conditioned medium from BXPC3 and MCF7 cells were isolated by differential centrifugation. The pellets were washed twice and re-suspended in 1 ml endothelium cell basal medium (EBM-2). Human umbilical vein endothelial cells (HUVEC) were cultured for 72 h in the presence or absence of CaCe-dMP (mother generation : C0). Subsequently, they were washed and re-cultivated over three cycles to get daughter cell generations (Daughter Generations : C1, C2, C3). Tissue factor activity (TFa) was assessed with a chromogenic assay in the absence or presence of cells in normal PPP. Procoagulant phospholipid-dependent clotting time (Proag-PPL): was measured with STA®Procoag-PPL assay. Thrombin generation was studied as described elsewhere using Calibrated Automated Thrombogram®. All assays were from Diagnostica Stago (Asnières, France).Results: The HUVEC and their microparticles manifested faint amounts of TFa (0.18±0.03 pM and 1.2±1.0 pM respectively). At steady state, HUVEC did not induce any detectable thrombin generation. Abundant TFa was showed by BXPC3-dMP (92±50 pM). The levels of TFa were significantly lower at MCF7-dMP (12.1±2.2 pM; p<0.05). Following exposure to BXPC3-dMP, the HUVEC abundantly displayed TFa as compared to non-exposed cells (25±3 pM versus 0.18±0.03 respectively; p<0.001) and showed significantly shorter Procoag-PPL® as compared to non-exposed cells (56±2 sec versus 102±10 sec. respectively ; p<0.001). HUVEC exposed to MCF7-dMP showed significantly increased TFa as compared to non-exposed cells (0.75±0.2 pM versus 0.18±0.03 respectively; p<0.001) and reduced the Procoag-PPL® clotting time as compared to non-exposed cells (65±3 sec versus 102±10 sec respectively; p<0.001). HUVEC exposed to CaCe-dMP significantly increased thrombin generation as compared to non-exposed HUVEC. Data of thrombin generation experiments are summarized in Table 1.Daughter HUVEC from mother cells exposed to CaCe-dMP exhibited significantly higher TFa as compared to daughter generations from HUVEC non-exposed to CaCe-dMPs. The high TFa was conserved in three generations of cells derived from those exposed to CaCe-dMPs (Figure 1). Daughter generations of HUVEC exposed to CaCe-dMP induced significantly higher thrombin generation as compared to that induced by daughter generations of non exposed HUVEC (Table 1). The TFa of daughter cells stemming from HUVEC exposed to BXPC3 was significantly higher as compared to that of daughter cells stemming from HUVEC exposed to MCF7 (Figure 1). The Procoag-PPL® did not significantly change in daughter HUVEC exposed to microparticles derived either from BXPC3 or MCF7.Conclusion: Upon exposure to CaCe-dMP, endothelial cells acquire a procoagulant phenotype marked by abundant TFa and enhanced thrombin generation potential. The procoagulant properties are transferred to the daughter generations of cells. These results support the concept that CaCe-dMPs lead to a procoagulant shift of endothelial cells which, upon exposure, present high TF activity and enhance thrombin generation. The procoagulant shift of endothelial cells can be transferred to next generations of endothelial cells and could be an additional mechanism - together with cancer-induced blood hypercoagulability - in the pathogenesis of cancer associated thrombosis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pathogenesis of cancer‐associated thrombosis

Program in Thrombosis and Hemostasis, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA This is an open access Abstract Book distributed under the Attribution-NonCommercial-NoDerivs (CCBY-NC-ND), which allows third parties to download the abstracts and share them with others as long as they credit the author and the abstract book, but they cannot change the content in any way or use them commercially. http://creativecommons.org/licenses/by-nc-nd/4.0

Citrullinated histone H3, a biomarker of neutrophil extracellular trap formation, predicts the risk of venous thromboembolism in cancer patients

Background Neutrophil extracellular traps (NETs) are decondensed chromatin fibers that might play a role in the prothrombotic state of cancer patients. Objectives To investigate whether the levels of citrullinated histone H3 (H3Cit), a biomarker for NET formation, cell-free DNA (cfDNA) and nucleosomes predict venous thromboembolism (VTE) in cancer patients. Patients/Methods Nine-hundred and forty-six patients with newly diagnosed cancer or progression after remission were enrolled in this prospective observational cohort study. H3Cit, cfDNA and nucleosome levels were determined at study inclusion, and patients were followed for 2 years. VTE occurred in 89 patients; the cumulative 3-month, 6-month, 12-month and 24-month incidence rates of VTE were 3.7%, 6.0%, 8.1%, and 10.0%, respectively. Results Patients with elevated H3Cit levels (> 75th percentile of its distribution, n = 236) experienced a higher cumulative incidence of VTE (2-year risk of 14.5%) than patients with levels below this cut-off (2-year risk of 8.5%, n = 710). In a competing-risk regression analysis, a 100 ng mL-1 increase in H3Cit level was associated with a 13% relative increase in VTE risk (subdistribution hazard ratio [SHR] 1.13, 95% confidence interval [CI] 1.04-1.22). This association remained after adjustment for high VTE risk and very high VTE risk tumor sites, D-dimer level, and soluble P-selectin level (SHR 1.13, 95% CI 1.04-1.22). The association of elevated nucleosome and cfDNA levels with VTE risk was time-dependent, with associations with a higher risk of VTE only during the first 3-6 months. Conclusion These data suggest that biomarkers of NET formation are associated with the occurrence of VTE in cancer patients, indicating a role of NETs in the pathogenesis of cancer-associated thrombosis.

Mechanisms and risk factors of thrombosis in cancer.

The close relationship between cancer and thrombosis is known since more than a century. Venous thromboembolism (VTE) may be the first manifestation of an occult malignancy in an otherwise healthy individual. Cancer patients commonly present with abnormalities of laboratory coagulation tests, indicating an ongoing subclinical hypercoagulable condition. The results of laboratory tests demonstrate that a process of fibrin formation and removal parallels the development of malignancy, which is of particular interest since fibrin and other clotting products are important for both thrombogenesis and tumor progression. Besides general clinical risk factors (i.e. age, previous VTE, immobility, etc.), other factors typical of cancer can increase the thrombotic risk in these patients, including the type of cancer, advanced disease stage, and cancer therapies. In addition, biological factors, including tumor cell-specific prothrombotic properties and the host cell inflammatory response to the tumor, play a central role in the pathogenesis of cancer-associated thrombosis. Cancer cells produce and release procoagulant and fibrinolytic proteins, as well as inflammatory cytokines. In addition, they are capable of directly adhering to host cells (i.e. endothelial cells, monocytes, platelets, and neutrophils), thereby stimulating additional prothrombotic properties of the host effector cells. Tumor-shed procoagulant microparticles also contribute to the patient hypercoagulable state. Finally, the changes of stromal cells of the tumor 'niche' induced by tissue factor (TF) highlight new interactions between hemostasis and cancer. Of interest, most of these mechanisms, besides activating the hemostatic system, also promote tumor growth and metastasis, and are regulated by oncogenic events. Indeed, molecular studies demonstrate that oncogenes responsible for the cellular neoplastic transformation drive the programs of hemostatic protein expression and microparticle liberation by cancer tissues. Human and animal experimental models demonstrate that activation of cancer-associated prothrombotic mechanisms parallels the development of overt thrombotic syndromes in vivo.

Development of Venous Thromboembolic Events in Cancer Patients with Elevated Levels of Tissue Factor-Bearing Microparticles

Cancer cells shed procoagulant vesicles containing tissue factor, and these tissue factor-bearing microparticles (TFMP) may play a role in thrombus formation in vivo. Using impedance-based flow cytometry to quantify microparticles and a high affinity monoclonal antibody specific for tissue factor, we previously demonstrated the presence of tissue factor-bearing microparticles in platelet-poor plasma in cancer patients. In this case control study, tissue factor-bearing microparticles represented a 4-fold risk factor for venous thromboembolic events (VTE) in cancer patients with acute VTE compared to age, stage, sex, diagnosis-matched controls with cancer but without acute VTE. To further assess the relationship between tissue factor-bearing microparticles and VTE, we performed a retrospective analysis of deep venous thrombosis or pulmonary emboli diagnosed in cancer patients initially enrolled without evidence of VTE. All radiographic reports for the cancer-no VTE group in the 2 years following enrollment were analyzed by a reviewer blinded to microparticle status. Only documented evidence of a new proximal extremity deep vein thrombosis or pulmonary embolism was included in the analysis. The TFMP and no-TFMP groups did not differ significantly for age, sex, active cancer treatment, smoking status, diabetes, or the presence of metastatic disease at time of enrollment. Sixteen of the 60 patients in this group had measurable tissue factor-bearing microparticles, 4 (4/16; 25%) of which subsequently developed radiographic evidence of VTE within 12 months of enrollment. No thrombotic events were recorded among the 44 patients without detectable tissue factor-bearing microparticles within the initial 12 months; however, one patient developed a pulmonary embolism 17 months following enrollment. Identifying death without VTE as a competing risk, the one-year estimate of the rate of VTE in cancer patients with detectable tissue factor-bearing microparticles was 34.8%; among the same group without detectable tissue factor-bearing microparticles, the 1-year rate was 0% (Log Rank p-value=0.002). The presence of tissue factor-bearing microparticles in cancer patients initially thrombosis-free predicted a 7-fold increased risk of thrombosis over cancer patients who were negative for tissue factor-bearing microparticles (OR 7.00, 95% CI 0.85–82.74, P=0.02). These tissue factor-bearing microparticles appear to be derived from the underlying malignancy since samples analyzed from patients with pancreatic cancer demonstrated co-expression of both tissue factor and MUC-1, a transmembrane glycoprotein overexpressed in epithelial malignancies. These data further support the role of tissue factor-bearing microparticles in the pathogenesis of cancer-associated thrombosis and as a biomarker for the prediction of cancer patients at risk of thrombosis. A prospective clinical study, currently being initiated, is required to evaluate this biomarker for the prediction of VTE risk in cancer patients and the utility of thromboprophylaxis in patients with elevated numbers of tissue factor-bearing microparticles.

Tissue factor-bearing microparticles derived from tumor cells: impact on coagulation activation.

Tissue factor (TF)-bearing microparticles (MP) from different origins are thought to be involved in the pathogenesis of cancer-associated thrombosis. However, the role of circulating tumor cell-derived TF is not well understood. TF antigen and activity were measured in MP generated in vitro from human TF-expressing cancer cells by ELISA and clotting or thrombin generation assays, respectively. TF antigen and activity were also measured in vivo in cell-free plasmas from mice previously injected with in vitro-generated MP or in cell-free plasmas from nude mice bearing orthotopically injected human cancer cells. Tumor cell-derived MP (TMP) exhibited strong TF-dependent procoagulant activity (PCA) in vitro and in vivo. Injection of TMP into mice was associated with acute thrombocytopenia and signs of shock, which were prevented by prior heparinization. Human TF antigen and activity could be detected in mouse cell-free plasmas up to 30 min after TMP injections. Human TF was detected in the spleen of injected mice and its clearance from circulation was delayed in splenectomized mice, suggesting the involvement of the spleen in the rapid clearance of circulating MP in vivo. Detectable levels of TF-dependent PCA and thrombin-antithrombin complex were found in cell-free plasmas from mice growing pancreatic human tumors, suggesting that circulating tumor-derived TF causes coagulation activation in vivo. MP derived from certain cancer cells exhibit TF-dependent PCA both in vitro and in vivo. These results provide new information about the specific contribution of tumor-derived MP to the hypercoagulable state observed in cancer.

Increased blood levels of inflammatory cytokines CRP, TNF-α, CD40L, MCP-1 and Nitric Oxide in malignancy-associated thrombosis and their modulation by a low molecular weight heparin, Enoxaparin

6077 Background: Endogenous generation of inflammatory cytokines and induction of NO synthase (NOS, EC 1.1.4. 13.49) resulting in enhanced formation of NO free radicals contribute to the pathogenesis of vascular and thrombotic events in these patients. Patients recruited in an open label, multidose active comparator parallel design study were randomized to receive a low molecular weight heparin (LMWH) namely enoxaparin at a dose of 1–1.5 mg/kg SC for one week. Methods: Blood samples were collected prior to and 3–7 days after initiation of LMWH. These samples were analyzed for NO measurement utilizing a chemical method (Sievers 280i, Ionics Corp. Boulder, CO) and ELISA methods for MCP-1, CD40L, TNF-α (R&D Systems, Minneapolis, MN) and hsCRP (American Diagnostica, Stamford, CT). In addition to the control group (n=50), a group of medical patients with thrombosis without cancer were included (n=50). Results: The relative increase of these inflammatory markers in the cancer patients with thrombosis was higher in comparison to the two other groups. In particular, the CD40L levels were found to be 10-fold higher in comparison to normals. Patients with thrombosis without cancer exhibited only a 4-fold increase in comparison to normal. The MCP-1 levels showed a 5-fold increase in the cancer with thrombosis group, whereas cancer patients without thrombosis exhibited a 40% increase. HsCRP was much higher in the cancer patients with thrombosis when compared with cancer patients without thrombosis. The TNF-α levels were increased in both groups. Interestingly, the relative increase in the NO level was significantly higher in cancer patients with thrombosis 63.1± 50.1 μ M for ONCENOX and 39.8± 16.2 μ M for CORTES study. Cancer patients with thrombosis treated with LMWH for 3–7 days exhibited a marked downregulation of MCP-1, CD40L, hsCRP and NO. In the group of patients with thrombosis without cancer a decreased trend was noted. Conclusions: This observation clearly suggests that the pathogenesis of cancer-associated thrombosis is strongly influenced by inflammatory cytokines. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Aventis Aventis; Mitsubishi; Pfizer