the NADPH Oxidase Catalytic Subunit Nox2 Displays Differential Roles in Arterial Vs. Venous Thrombosis

Abstract NADPH oxidase is a major superoxide generating enzyme in vascular tissues. Deficiency of the Nox2 (gp91phox) catalytic subunit of NADPH oxidase is a genetic cause of X-linked chronic granulomatous disease. These patients are prone to infection due to loss of oxidant production by neutrophils, and recent evidence suggests they also have a defect in platelet adhesion to collagen. However, the role of Nox2 in thrombosis is controversial, with one study demonstrating no effect of murine Nox2 deficiency on platelet activation and another study implicating Nox2 in platelet aggregation as well as thrombosis in small vessels. Given that Nox2 may be important for both platelet activation as well as release of neutrophil extracellular traps (NETs, a mediator of venous thrombosis), we hypothesized that genetic deficiency in murine Nox2 leads to decreased susceptibility to experimental thrombosis in both arterial and venous systems. We studied male Nox2 deficient (Cybb-/y) mice and wild type (Cybb +/y) littermates at 10-14 weeks of age. There were no differences in platelet count (921.9±33.6 x103/µl vs. 978±85.2 x103/µl), hematocrit (36.5±0.6% vs. 38.7±0.8% ) or white blood cell count (5.5±0.3 x103/µl vs. 6.1±0.6 x103/µl) between Cybb+/y and Cybb-/y mice. We next, examined platelet activation in response to thrombin (0.05 and 0.2 U/ml) or collagen (80 and 320 ng/ml) using flow cytometry. We observed similar levels of integrin α2bβ3 activation, fibrinogen binding, and intra-platelet levels of H2O2 in platelets from Cybb+/y and Cybb-/y mice after activation with either agonist, which suggests no alteration in platelet inside-out signaling due to loss of Nox2. No significant difference in susceptibility to carotid artery thrombosis in a photochemical injury model was observed between Cybb+/y and Cybb-/- mice (time to stable occlusion 21.97±4.7 vs 27.6±4.2, P>0.4). In contrast, Cybb-/y mice demonstrated significant decreases in the weight and length of venous thrombi in the inferior vena cava after 48 hours of stenosis (P<0.05). Our findings suggest that Nox2 is not a major mediator of platelet activation or arterial thrombosis but contributes to the development of venous thrombi. Future studies are warranted to examine the role of NETs and the prothrombotic effects of Nox2 in association with other cardiovascular risk factors. Disclosures Lentz: Novo Nordisk: Consultancy, Research Funding; Celgene: Equity Ownership; Opko: Membership on an entity's Board of Directors or advisory committees.

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Abstract 36: Alpha 2-antiplasmin Prevents the Resolution of Deep Vein Thrombosis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.36 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Satish Singh ◽

Aiilyan K Houng ◽

Samantha Howard ◽

B Tyler Emerson ◽

Guy L Reed

Keyword(s):

Venous Thrombosis ◽

Vena Cava ◽

Relative Effect ◽

Vein Thrombosis ◽

Significant Difference ◽

Tissue Plasminogen ◽

Deep Vein ◽

Metalloproteinase 9 ◽

One Way Anova

Introduction: Deep venous thrombosis is a major cause of death and disability. Despite anticoagulation treatment, venous thrombi persist for months, causing chronic venous obstruction, inflammatory remodeling and post-thrombotic syndrome. The mechanisms responsible for impaired clearance of venous thrombi are poorly understood. Alpha 2-antiplasmin (a2AP) is the primary physiological inhibitor of plasmin and a key regulator of the fibrinolytic pathway. The role of a2AP in the resolution of venous thrombosis has not been determined. Hypothesis: We tested the hypothesis that a2AP prevents the resolution of experimental deep venous thrombi. Methods and Results: Thrombus resolution and content were examined in a2AP +/+ and a2AP -/- mice using a well-established, fibrinolytic-resistant, stasis model induced by ligation of the inferior vena cava (IVC). Thrombus weight and composition were determined after 7 days. Data was analyzed by one-way ANOVA with Neumann-Keul’s correction. Thrombus weight was reduced in a2AP -/- mice by >90% when compared to a2AP +/+ mice (p<0.001); there was no significant difference between a2AP -/- mice and shams. Histochemical and immunofluorescence staining showed significant reductions in IVC fibrin content, neutrophil recruitment and matrix metalloproteinase-9 expression in a2AP -/- mice (p<0.001) vs. a2AP +/+ mice. The relative effect of plasminogen activation and a2AP on resolution of preformed venous thrombi was examined in wild-type a2AP +/+ mice treated 24 h after IVC ligation with tissue plasminogen activator (TPA) (1.2 or 5 mg/kg) or a monoclonal antibody inactivating a2AP (10 mg/Kg). By comparison to 7 day old venous thrombi in untreated mice, treatment with TPA at 1.2 mg/kg or 5 mg/kg did not decrease thrombus size after 7 days. In contrast, a2AP inactivation significantly reduced thrombus weight vs. untreated and TPA-treated mice (p<0.01 to p<0.001). Conclusions: In experimental venous thrombosis, a2AP was required for the persistence of venous thrombi 7 days after formation. Venous thrombi resisted TPA, but were sensitive to resolution after a2AP inactivation. This suggests that a2AP may be responsible for the persistence of clinical venous thrombosis in humans.

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The (Patho)Biology of SRC Kinase in Platelets and Megakaryocytes

Medicina ◽

10.3390/medicina56120633 ◽

2020 ◽

Vol 56 (12) ◽

pp. 633

Author(s):

Lore De Kock ◽

Kathleen Freson

Keyword(s):

Platelet Activation ◽

Knockout Mice ◽

Missense Variant ◽

Surface Receptors ◽

Src Signaling ◽

Normal Platelet ◽

Fibrinogen Binding ◽

Cellular Environment ◽

Proplatelet Formation

Proto-oncogene tyrosine-protein kinase SRC (SRC), as other members of the SRC family kinases (SFK), plays an important role in regulating signal transduction by different cell surface receptors after changes in the cellular environment. Here, we reviewed the role of SRC in platelets and megakaryocytes (MK). In platelets, inactive closed SRC is coupled to the β subunit of integrin αIIbβ3 while upon fibrinogen binding during platelet activation, αIIbβ3-mediated outside-in signaling is initiated by activation of SRC. Active open SRC now further stimulates many downstream effectors via tyrosine phosphorylation of enzymes, adaptors, and especially cytoskeletal components. Functional platelet studies using SRC knockout mice or broad spectrum SFK inhibitors pointed out that SRC mediates their spreading on fibrinogen. On the other hand, an activating pathological SRC missense variant E527K in humans that causes bleeding inhibits collagen-induced platelet activation while stimulating platelet spreading. The role of SRC in megakaryopoiesis is much less studied. SRC knockout mice have a normal platelet count though studies with SFK inhibitors point out that SRC could interfere with MK polyploidization and proplatelet formation but these inhibitors are not specific. Patients with the SRC E527K variant have thrombocytopenia due to hyperactive SRC that inhibits proplatelet formation after increased spreading of MK on fibrinogen and enhanced formation of podosomes. Studies in humans have contributed significantly to our understanding of SRC signaling in platelets and MK.

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Bortezomib-Thalidomide-Dexamethasone Compared with Thalidomide-Dexamethasone as Induction and Consolidation Therapy Before and After Double Autologous Transplantation In Newly Diagnosed Multiple Myeloma: Results From a Randomized Phase 3 Study

Blood ◽

10.1182/blood.v116.21.42.42 ◽

2010 ◽

Vol 116 (21) ◽

pp. 42-42 ◽

Cited By ~ 4

Author(s):

Michele Cavo ◽

Giulia Perrone ◽

Silvia Buttignol ◽

Elisabetta Calabrese ◽

Monica Galli ◽

...

Keyword(s):

Multiple Myeloma ◽

Multivariate Analysis ◽

Board Of Directors ◽

Induction Therapy ◽

Autologous Transplantation ◽

Consolidation Therapy ◽

Newly Diagnosed ◽

Advisory Committees ◽

Significant Difference

Abstract Abstract 42 We prospectively compared thalidomide-dexamethasone (TD) with bortezomib-thalidomide-dexamethasone (VTD) as induction therapy before, and consolidation after, double autologous stem-cell transplantation (ASCT) in patients with newly diagnosed multiple myeloma (MM). Three 21-d cycles of either VTD (V, 1.3 mg/m2 twice-weekly; T, 200 mg/d through d 1 to 63; D, 320 mg/cycle) or TD were given as induction therapy. Consolidation therapy comprised two 35-d cycles of VTD (V, 1.3 mg/m2 once-weekly; T, 100 mg/d through d 1 to 70; D, 320 mg/cycle) or TD. 474 patients randomized to the VTD (n=236) or TD (n=238) arm were analyzed on an intention-to-treat basis for response rate, PFS and OS. Centrally reassessed CR/nCR rate was significantly higher in the VTD compared with the TD arm after all treatment phases, including induction therapy (30% vs 10%, p<0.0001), double autotransplantation (54% vs 42%, p=0.008) and consolidation therapy (60% vs 44%, p=0.001). Best confirmed overall CR/nCR rate was 71% in the VTD arm compared with 52% in the TD arm (p<0.0001); the corresponding values for VGPR or better were 89% vs 72%, respectively (p<0.0001). To evaluate the role of consolidation therapy we performed a per-protocol analysis of 323 patients, 161 treated with VTD and 162 with TD. Overall, upgraded responses with VTD and TD as consolidation therapy were observed in 55% vs 37% of patients, respectively (p=0.01; OR:1.15-3.77). Furthermore, the probability to improve responses from less than CR before consolidation to CR after consolidation was 28% with VTD vs 15% with TD (p=0.02; OR:1.07-4.57) (p=0.003 using the Mc Nemar's test). Post-consolidation molecular detection of minimal residual disease was the objective of a substudy; detailed results are reported in a separate abstract. Briefly, both qualitative and quantitative analyses confirmed the statistically significant superiority of VTD over TD in effecting higher rates of molecular remissions and reducing the burden of residual myeloma cells after ASCT. Any grade 3–4 non-hematologic adverse events were 10% with VTD (peripheral neuropathy: 1.3%, skin rash: 0.6%) vs 12% with TD. With a median follow-up of 31 months, median PFS was 42 months in the TD arm and was not yet reached in the VTD arm (44-month projected rate: 61%) (HR: 0.62 [CI: 0.45–0.87], p=0.006). Superior PFS in the VTD vs TD arm was retained across patient subgroups with poor prognosis, including those with t(4;14) and/or del(17p). Randomization to VTD overcome the adverse influence of t(4;14) on PFS (40-month projected rates: 69% vs 67% according to the presence or absence of this abnormality, respectively; p=0.6). By the opposite, in the TD arm corresponding median PFS values were 24.5 vs 41.5 months, respectively (p=0.01). The small numbers of patients with del(17p) in both arms of the study precluded a statistical comparison with del(17p)-negative group. In a multivariate analysis, variables favorably influencing PFS were beta2-m lower than 3.5 mg/L (HR:0.47; p=0.000), absence of t(4;14) and/or del(17p) (HR:0.52; p=0.000), randomization to VTD arm (HR:0.57; p=0.002), attainment of at least VGPR (HR:0.50; p=0.009) and CR (HR:0.8; p=0.01). No statistically significant difference between the overall treatment protocols was seen in terms of OS, although curves seemed to initially diverge after 40 months (44-month projected rates: 84% vs 74% for VTD and TD arms, respectively). A multivariate analysis showed the independent role of absence of t(4;14) and/or del(17p) (HR:0.42; p=0.003), ISS stage1-2 (HR:0.49; p=0.02) and randomization to VTD (HR:0.53; p=0.04) in prolonging OS. When time-dependent CR entered the model, absence of t(4;14) and/or del(17p) and less advanced ISS stage retained their positive prognostic value; attainment of CR (strictly related to VTD randomization) was an additional favorable variable. In conclusion, in comparison with the TD arm of the study, 1) VTD induction emerges as a new standard of care for maximizing the degree and speedy of tumor reduction in preparation for ASCT; 2) VTD consolidation effected significantly higher rates of upgraded responses, including CR, and of molecular remissions; 3) double ASCT incorporating VTD as induction and consolidation therapy resulted in significantly longer PFS, a benefit confirmed in a multivariate regression analysis and maintained in the subgroup of patients with adverse cytogenetic abnormalities. Disclosures: Cavo: Janssen-Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Off Label Use: Use of bortezomib and thalidomide as induction therapy before, and consolidation after, autologous transplantation in newly diagnosed multiple myeloma. Baccarani:NOVARTIS: Honoraria; BRISTOL MYERS SQUIBB: Honoraria.

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Chronically Elevated Interleukin-6 Disturbs the Coagulation Cascade in Mice

Blood ◽

10.1182/blood-2021-149628 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2065-2065

Author(s):

Tanja Knopp ◽

Jeremy Lagrange ◽

Rebecca Jung ◽

Johannes Wild ◽

Heidi Rossmann ◽

...

Keyword(s):

Board Of Directors ◽

Interleukin 6 ◽

Thrombus Formation ◽

Vena Cava ◽

Coagulation Cascade ◽

Bleeding Complications ◽

Thrombin Inhibitor ◽

Advisory Committees ◽

Hemostatic System

Abstract Introduction: Pro-inflammatory cytokines play an essential role as activators of the hemostatic system and in the regulation of physiological antithrombotic mechanisms. Interleukin-6 (IL-6) influences platelet production and platelet activation. It was associated with accelerated clotting and intravascular coagulation in tissue factor (TF)-driven murine thrombosis models. However, the precise role of myeloid cell-derived IL-6 on thrombosis formation and the hemostatic system is still unknown. Methods and Results: To better understand the role of IL-6 in thrombosis and the hemostatic system, we developed a new mouse strain with Cre-recombinase driven constitutive IL-6 expression specifically in myeloid cells (LysM-IL-6 OE, Control mice: IL-6 OE). LysM-IL-6 OE mice had a prolonged tail bleeding time and lacked venous thrombus formation induced by inferior vena cava (IVC) stenosis. There were no differences in D-Dimer levels in LysM-IL-6 OE mice neither on baseline level nor after IVC ligation. However, we found unstoppable post-operative bleedings in LysM-IL-6 OE. They showed a prolonged aPTT, a significantly increased INR and a prolonged thrombin converting time. The Factor V and IX expression were reduced, but von Willebrand factor, antithrombin and fibrinogen expression were up-regulated and could not explain the missing thrombus formation. We found significantly elevated erythrocyte sedimentation in line with erythrocytes aggregates, which seemed to be mediated by IL-6 and α2M. Most importantly, hepatic levels of thrombin inhibitor α2 macroglobulin (α2M) mRNA and protein were increased in LysM-IL-6 OE/+ mice compared to control mice. In parallel, Platelet erythrocyte interaction seemed to be essential in the development of the bleeding phenotype. Conclusions: These findings show the role of chronically elevated IL-6 in driving the accumulation of A2m on the surface of erythrocytes, thereby mediating a phenotype of increased bleeding complications. This work was supported by the DFG KA4035/1-1 and by the German Ministry for Education and Research (BMBF 01EO1503) Disclosures Lämmle: Takeda: Membership on an entity's Board of Directors or advisory committees; Ablynx: Membership on an entity's Board of Directors or advisory committees, Other: Travel Support, Speakers Bureau; Baxter: Other: Travel Support, Speakers Bureau; Alexion: Other: Travel Support, Speakers Bureau; Siemens: Other: Travel Support, Speakers Bureau; Bayer: Other: Travel Support, Speakers Bureau; Roche: Other: Travel Support, Speakers Bureau; Sanofi: Other: Travel Support, Speakers Bureau. Ruf: ARCA bioscience: Consultancy, Patents & Royalties; ICONIC Therapeutics: Consultancy; MeruVasimmune: Current holder of individual stocks in a privately-held company.

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Protective Effects of Kininogen-1 Gene Deficiency in Mouse Models of Venous Thrombosis

Blood ◽

10.1182/blood-2021-153316 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 289-289

Author(s):

Aatira Vijay ◽

Mohamad B Kassab ◽

Young Jun Shim ◽

Shadi Swaidani ◽

Adam Mauskapf ◽

...

Keyword(s):

Venous Thrombosis ◽

Thrombus Formation ◽

Vena Cava ◽

Continuous Light ◽

Light Irradiation ◽

Fluorescence Angiography ◽

Contact Activation ◽

Tail Vein ◽

Platelet Accumulation

Abstract Background- High molecular weight Kininogen (HK) is a nonenzymatic co-factor of the contact activation system. HK binds prekallikrein (PK) and FXI to surfaces in proximity to FXII, amplifying PK activation by FXIIa and the reciprocal activation of FXII by activated PK (PKa), as well as FXI activation of FXIIa. PKa cleavage of HK also liberates bradykinin-a proinflammatory and vasoactive nanopeptide. The aim of this study was to define the pro-thrombotic role of kininogen in venous thrombosis (VT) and to use in vivo serial analysis of thrombus development to understand the recruitment and retention of platelets in the growing thrombus in the absence and presence of kininogen. Methods- The development of VT in mice deficient in kininogen (mKng1-/-) was compared to that in their wild-type littermates. A femoral-saphenous stasis VT model was prepared by ligating both saphenous and femoral veins. Next VT formation, growth, and dissolution (n=3 for each group) was monitored using intravital microscopy (IVM) via a multichannel epifluorescence microscope (Nikon Eclipse 90i). To induce stasis VT, FITC-dextran (10 mg/kg, ex/em 488/520 nm) was injected retro-orbitally, and then continuous light irradiation (20x objective, 475nm/35nm) of the saphenous vein was applied for 5 minutes. FITC-dextran fluorescence angiography monitored thrombus formation and dissolution. Immediately after VT formation, platelet accumulation at the thrombus site was monitored in the Cy5 channel (630/38 nm) via injection of a GPIbβ antibody conjugated with Dylight-649 (150nmol/kg), over time. All images were identically windowed in each channel, and thrombus area was measured using NIH ImageJ software. To corroborate IVM studies, we also evaluated a complete stasis model of inferior vena cava (IVC) ligation (n=7-8 per group). Thrombi were harvested after 48 hours and thrombus weight and length were measured to estimate thrombus mass. FXI circulates in blood as a homodimer along with HK. We determined the effect of kininogen deficiency on FXI activity. FXI activity assay used a combination of inhibitors, serially, to monitor the cleavage of substrate specific to activated FXI and release of chromogen, as a function of FXI activity. Finally, to determine the effects of Kng1 deficiency on bleeding, tail vein bleeding times were also determined (n=8 per group). Results- In femoral-saphenous stasis VT, thrombus developed in both groups immediately following FITC-channel light irradiation. However, thrombus size was smaller in Kng1-/- as compared to WT (Figure 1). Results from serial IVM of VT indicated faster thrombus dissolution in the Kng1-/- group. Lower platelet signals, as shown at 2 and 6 hours in the Kng1-/- mice may be consistent with this hypothesis. Thrombus area analysis suggested decreased thrombus formation in the Kng1-/- animals, and temporal analysis indicated faster dissolution by 6 hours (Figure 2). IVC ligation results corroborated the findings of femoral-saphenous DVT model, demonstrating that thrombus weight was significantly lower in Kng1-/- mice as compared to WT (p<0.001, Figure 3). FXI activity was also decreased in the Kng1-/- group (p<0.10). Tail vein bleeding times, however, showed no increased bleeding in Kng1-/- mice. Conclusion- These initial results suggest a pro-thrombotic role of kininogen and a protective role of kininogen deficiency in two murine venous thrombosis models, without incurring a bleeding penalty. Thrombus dissolution was faster and platelet accumulation was inhibited in Kng1-/- mice. These findings suggest that targeting kininogen may provide a new approach to prevent and treat venous thrombosis. Figure 1 Figure 1. Disclosures McCrae: Dova, Novartis, Rigel, and Sanofi Genzyme: Consultancy; Sanofi, Novartis, Alexion, and Johnson & Johnson: Consultancy, Honoraria. Jaffer: Mercator, Inc.: Other: Sponsred research.

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HIF-1α Is Not Essential For The Establishment Of MLL-Leukaemic Stem Cells

Blood ◽

10.1182/blood.v122.21.3767.3767 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3767-3767

Author(s):

Deniz Gezer ◽

Amelie V Guitart ◽

Milica Vukovic ◽

Chithra Subramani ◽

Karen Dunn ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Myeloid Leukaemia ◽

Progenitor Cells ◽

Board Of Directors ◽

Advisory Committees ◽

Hypoxic Conditions ◽

Significant Difference

Abstract Haematopoietic stem cells (HSCs) reside in hypoxic niches in the bone marrow (BM) and sustain long-life haematopoiesis. HSCs are largely quiescent, self-renew, undergo apoptosis and generate progenitor cells, which differentiate to multiple blood lineages. The strict regulation of the balance between these fate decisions is essential for haematopoiesis and their dysregulation in HSCs and progenitor cells can result in leukaemic transformation. HSCs and leukemic stem cells (LSCs) are suggested to share the same niche and are in need to adapt to hypoxic conditions. Hypoxia-inducible-factor-1α (HIF-1α) is a key mediator of cellular responses to hypoxia and is important for the maintenance of HSC functions under stressful conditions. Furthermore, in chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) HIF-1α is essential for LSC maintenance and ablation or knockdown of HIF-1α leads to exhaustion of established LSCs. The aim of this study was to investigate the requirement for HIF-1α in the generation of pre-LSCs and the establishment of LSCs. To investigate the role of HIF-1α in the generation of pre-LSCs we retrovirally transduced haematopoietic stem and progenitor cells (HSPCs) from either WT or HIF1-αfl/fl Vav-iCre with MLL-ENL retroviruses. Next we performed serial re-plating assays under normoxic and hypoxic conditions to generate pre-LSCs. Surprisingly, WT and HIF-1α deficient HSPCs generated comparable numbers of colonies in normoxia and hypoxia (Fig. 1a). In addition no significant difference was found in the immunophenotypic profile of colonies (Figure 1b). Furthermore, microscopic examination indicated that colonies of all genotypes were dense consistent with their transformed shape (Fig. 1c). WT and HIF-1α-deficient pre-LSCs cultured under normoxia and hypoxia had similar cloning efficiency, which is known to directly correlate with the numbers of LSCs in vivo (Fig. 2). These results indicate that HIF-1α is dispensable for the generation of pre-LSCs. To test the role of HIF-1α in establishment of LSCs from pre-LSCs we transplanted pre-LSCs into lethally irradiated mice together with support BM and monitored the mice for disease development. No significant difference was found in disease latency (Fig. 3a) or frequency of LSCs in peripheral blood, bone marrow or spleens (Fig. 3b) indicating that pre-LSCs lacking HIF-1α can efficiently generate LSCs that cause aggressive AML. In conclusion, we provide genetic evidence that HIF-1α is dispensable for the generation of pre-LSCs and the establishment of LSCs from pre-LSCs. These surprising findings, together with published results indicating that HIF-1α is essential for maintenance of LSCs, imply that HIF-1α has different roles at different stages of leukaemic transformation. Further studies are required to explain the distinct roles of HIF-1α in different stages of leukaemogenesis. Disclosures: Ratcliffe: RedOx: Founder Other. Holyoake:Novartis: Membership on an entity’s Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity’s Board of Directors or advisory committees; Ariad: Membership on an entity’s Board of Directors or advisory committees.

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INTEREST OF THE RATIO OF INCREASE OF (β-THROMBOGLOBULIN (Δ+/βTG) AND OF FIBRINOPEPTIDE A (Δ+ FPA) FOR DIAGNOSIS AND TREATMENT OF THR0MB0-EMB0LIC DISEASES

10.1055/s-0038-1643050 ◽

1987 ◽

Author(s):

E Lavenne-Pardonge ◽

C Col-De Beys ◽

M Moriau

Keyword(s):

Venous Thrombosis ◽

Platelet Activation ◽

Hip Prosthesis ◽

Diagnosis And Treatment ◽

Minor Role ◽

Leading Role ◽

Two Parameters ◽

A Minor ◽

Valvular Replacement

The interest of release of TG and FPA for the diagnosis and treatment of prethrombotic and thrombotic disorders is well knownThe ratio of increase of these two parameters (Δ+βTG/+ FPA) seems to bring some additional informations.This ratio, normally about 1, increases in isolated or preponderant platelet activation and decreases when platelet activation plays a minor role than the plasmatic factors. A more logical choice of therapeutics and a better control of its effectiveness are so possible.This study includes 91 cases of established thrombosis (20 arterial and 71 venous) and 272 cases of prethrombotic disorders (58 Raynaud syndromes, 54 cases of venous insufficiency, 60 of hip prosthesis, 40 of coronary by-pass, 60 of valvular replacement). The ratio + TG/ + FPA was calculated before, during and after efficacious or inefficacious treatment. In the cases of established thrombosis, our results confirm the leading role of platelets in the development of arterial thrombosis. The cases of venous thrombosis may be divided in two groups : simple venous thrombosis when the plasmatic factors play a leading role and complicated or recurrent venous thrombosis where the platelets play an equivalent or even a greater role.In the cases of prethrombotic states, the role of the platelets which is important on the arterial side is generally far from negligeable on the venous side. In cases of valvular replacement or of coronary by-pass the modification of the ratio lead us very frequently to modify our prophylactic therapeutics

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Increased Soluble GPVI Levels in Cirrhosis: Evidence for Collagen Induced Platelet Activation In Vivo

Blood ◽

10.1182/blood.v126.23.1114.1114 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1114-1114

Author(s):

Karl Egan ◽

Eimear Dunne ◽

Audrey Dillon ◽

Barry Kevane ◽

Zita Galvin ◽

...

Keyword(s):

Liver Disease ◽

Board Of Directors ◽

Platelet Activation ◽

Research Funding ◽

Decompensated Cirrhosis ◽

Dependent Manner ◽

Healthy Controls ◽

Advisory Committees ◽

Significant Difference

Abstract Background Cirrhosis is a consequence of prolonged inflammation arising from chronic liver disease of different aetiologies. It is characterised by tissue fibrosis, the deposition of collagen-rich extracellular matrix tissue within the liver. Glycoprotein VI (GPVI) is platelet-specific collagen receptor that is shed from the platelet surface in a metalloproteinase-dependent manner in response to GPVI ligation. The shed extracellular region of GPVI can be detected in plasma and used as a measure of GPVI-dependent platelet activation in vivo. Several lines of evidence suggest that GPVI-dependent platelet activation occurs in cirrhosis. Platelets have been shown to accumulate at sites of collagen-rich fibrotic tissue. Circulating levels of collagen are increased in cirrhosis. Collagen-induced platelet aggregation responses are reduced in vitro with cirrhosis. Based on these results, we hypothesised that soluble GPVI (sGPVI) levels are increased in patients with cirrhosis. As such, the aim of this study was to quantify sGPVI levels in patients with cirrhosis and compare to healthy controls. Methods Compensated cirrhotic patients were recruited at the Mater Misericordiae University Hospital, Dublin, Ireland. The diagnosis of cirrhosis was based on clinical examination, blood tests, and radiological examination (nodular surface, larger right lobe, coarse echotexture). Exclusion criteria were decompensated cirrhosis, recent thrombotic events, and antiplatelet and/or anticoagulant therapies. Healthy controls were recruited at the Royal College of Surgeons in Ireland and Beaumont Hospital, Dublin, Ireland. Blood samples were collected into vacutainers containing 3.2 % sodium citrate as anticoagulant. sGPVI levels in platelet poor plasma were measured using an in house custom ELISA. Results 57 patients with mixed aetiology cirrhosis and 55 healthy controls were recruited. In the patient group, 42% of patients had alcoholic liver disease (ALD), 30% had hepatitis C (HCV), 7% had non alcoholic fatty liver disease (NAFLD), 5% had Hepatitis B (HBV), 5% had autoimmune hepatitis (AIH), 5% had cryptogenic liver disease, 4% had hereditary haemochromatosis (HH), and 2% had primary biliary cholangitis (PBC). sGPVI levels were significantly increased in patients with cirrhosis (5.8 ± 0.6 ng/ml, n = 57) compared to healthy controls (3.2 ± 0.4 ng/ml, n = 55, p < 0.0001). There was no significant difference between sGPVI levels in AIH (4 ± 1 ng/ml, n = 3), ALD (5.6 ± 1 ng/ml, n = 24), cryptogenic (12 ± 5 ng/ml, n = 3), HBV (3.1 ± 1 ng/ml, n = 3), HCV (5 ± 0.6 ng/ml), or NAFLD (5.3 ± 1.1 ng/ml, n = 4). sGPVI levels did not correlate with platelet count (r = 0.12, p = 0.3) or parameters of liver cell function (albumin, bilirubin, prothrombin time, and liver stiffness measurements). Conclusion sGPVI levels are significantly increased in patients with mixed aetiology cirrhosis. This indicates collagen induced platelet activation is occurring in vivo and suggests the presence of an underlying coagulopathy in patients with cirrhosis. Disclosures Ní Áinle: Actelion Pharma: Research Funding; Leo Pharma: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees.

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Abstract 141: Anti-thrombin Perfluorocarbon Nanoparticles Decrease Clot Burden in a Murine Model of Venous Thrombosis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.141 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Chandu Vemuri ◽

Batool Arif ◽

Susannah A Grathwohl ◽

John S Allen ◽

Peter K Henke ◽

...

Keyword(s):

Venous Thrombosis ◽

Murine Model ◽

Safety Margin ◽

Vena Cava ◽

Clot Lysis ◽

Control Group ◽

Treatment Regimens ◽

Significant Difference ◽

Initial Work ◽

Clot Burden

Objective: Venous thromboembolism (VTE) afflicts nearly an million Americans with significant mortality and long-term morbidity. Current medical treatment regimens pose significant bleeding risks and recurrence risks. The purpose of this work is to determine if anti-thrombin perfluorocarbon nanoparticles (NP-PPACK) can attenuate clot progression after vascular injury in a murine model of venous thrombosis. Methods: Male, C57 black-6 mice underwent inferior vena cava (IVC) ligation through an institutionally approved protocol. Following ligation, groups of ten mice were randomized to receive intravenous, weight-based (1 ml/kg) tail vein injections of saline, plain nanoparticles, NP-PPACK or heparin (80 units/kg). After 6 hours the animals were sacrificed, IVC with clot excised and weight and length recorded. Clot integrity (N=4) analysis was then performed by incubating clots with 750 units of streptokinase for 90 minutes at 37 degrees Celsius, removing liquid clot and recording the percent change in clot weight. Results: There was a significant difference in clot burden between NP-PPACK and the control group (0.59 mg/mm ± 0.063 vs. 1.26mg/mm ± 0.85, p=.0001). Immunofluorescent histology performed on a subgroup of animals verified nanoparticle tracking to venous thrombus. Additionally, using exogenous clot lysis as a surrogate for clot integrity, NP-PPACK treated animals exhibited a trend towards enhanced lysis over saline treatments (change in clot weight over 90 minutes: 57.8 ±14.4 vs. 21.5 ± 7.49, NP PPACK vs saline p=.067). Conclusions: This initial work demonstrates that NP-PPACK significantly decreases clot burden by local targeting and reduces clot strength in this model of VTE. We have shown previously that the system is locally active for hours against thrombosis yet produces no sustained systemic anticoagulant effect beyond 60 minutes, indicative of its significant safety margin for clinical application.

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Role of Microenvironment-Associated Chemokines and Cytokines for Binet Stage A CLL Patients Included in a Prospective Trial (CLL1 trial) of the German CLL Study Group (GCLLSG): sIl2Ralpha Is An Independent Predictor of Progression-Free Survival (PFS),

Blood ◽

10.1182/blood.v118.21.3869.3869 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3869-3869

Author(s):

Till M Seiler ◽

Roland Aydin ◽

Tobias Herold ◽

Raymonde Busch ◽

Markus Schwarz ◽

...

Keyword(s):

T Cells ◽

Progression Free Survival ◽

Serum Levels ◽

Healthy Controls ◽

Advisory Committees ◽

Cytokines And Chemokines ◽

Small Subgroup ◽

Median Serum ◽

Significant Difference

Abstract Abstract 3869 Background: In CLL, proliferation of the leukemic cell clone occurs in the bone marrow and lymphatic tissues rather than peripheral blood. In this microenvironment, CLL cells interact with accessory cells, such as T cells and CD68+ nurselike cells (NLCs) and display signs of B cell receptor (BCR) activation, suggesting that CLL proliferation is T cell- and BCR-driven. In addition, cytokines and chemokines secreted by leukemic cells, stromal cells and T cells are essential in forming the disease-specific microenvironment. However, the exact biological role of cytokines and chemokines needs to be defined, especially in the light of distinct prognostic and biological subgroups of patients (pts). Methods: In order to address this issue, we measured serum levels of chemokines and cytokines in a prospective cohort of 157 previously untreated Binet stage A pts., a small subgroup of the risk-stratified CLL1 trial of the GCLLSG. Median follow-up time of that subgroup was 50.3 months. Median time to progression was 61.3 months. Median time from diagnosis to study entry was 1 year. Serum samples had been centrally collected at study entry and stored at −80°C. Sera were analyzed on a luminex-based multiplex platform, allowing simultaneous screening of multiple serum parameters. In a pilot phase, sera of 21 pts were pre-analytically screened for a total of 62 different chemokines. Median serum levels of 27 different chemokines and cytokines were found to differ from healthy controls in a significant manner. Those 27 chemokines and cytokines were subsequently analyzed in 157 pts. For all parameters univariate and multivariate analyses were performed for progression-free survival. Results: Serum levels of CCL3 and CCL4, chemokines known to be secreted by CLL cells upon BCR engagement, were elevated compared to healthy controls. High CCL3 levels correlated strongly with high CCL4 levels (p<0.001) and tended to be associated with unmutated IgHV status (p=0.06), supporting the role of BCR triggering in biologically selected CLL pts. Concerning CCL3 and CCL4, no significant difference in PFS was detected. Serum levels of CCL2 and CCL17, both binding chemokine receptor CCR4, were concordantly elevated compared to healthy controls (p<0.001), suggesting a possible role of chemoattraction of CCR4+ T cells towards these chemokines in CLL. CXCL12, CCL21, and CXCL10, chemokines associated with chemotaxis of CLL cells, were concordantly elevated compared to healthy controls. Pts with serum levels of CCL21 beyond the median also had higher levels of CXCL10 (p<0.001) and CXCL12 (p=0.02). CLL pts have been found to have abnormal neovascularization in the bone marrow and lymph nodes. Elevated VEGF levels were strongly associated with elevated EGF level (p<0.001). High VEGF levels correlated with high white blood cell counts (p=0.008) and showed a trend towards association with del(11q) (p=0.07) and lymphadenopathy (p=0.165). Concerning VEGF, no significant difference in PFS was detected. In contrast, pts with high levels of sIl2R alpha showed a significant shorter PFS. When confirmed by conventional ELISA, median PFS in pts within the highest quartile of sIl2Ralpha levels were 22 months compared to 72 months in the lower three quartiles (p<0.001). When we analyzed sIl2R alpha together with genetic abnormalities like del(11q), trisomy 12, del(13q), del(17p), the risk stratification model used in CLL1 (high risk versus low risk for disease progression), the hierarchical model as published by Döhner et al., and IgHV status in a multivariate Cox regression, we found that sIl2R alpha (OR 2.5, 95% CI 1.4–4.8, p=0.004) and IgHV mutational status (OR 4.0, 95% CI 2.2–7.3, p<0.001) were both independent prognostic variables. Conclusion: The assessment of sera in a small subgroup of the CLL1 study cohort of the GCLLSG revealed that the levels of several chemokines and cytokines were elevated when compared to healthy controls. Median serum levels of different chemokines correlated with distinct biological characteristics of CLL pts, like genetic abnormalities or clinical parameters. In addition, sIl2R alpha could be identified as an independent prognostic variable for PFS in early CLL. Disclosures: Eichhorst: Hoffmann La Roche: Honoraria, Research Funding, Travel Grants; Mundipharma: Research Funding, Travel Grants; Gilead: Consultancy. Stilgenbauer:Hoffmann La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel Grants. Hallek:Hoffmann La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Bergmann:Celgene: Honoraria.

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