Increased Thrombin Generation and Fibrin Formation In Premature Aging BMAL1 Deficient Mice

Abstract Introduction The occurrence of venous thromboembolism is strongly age-dependent and has a substantial morbidity and mortality. While the incidence is<1 per 1000 per year up to an age of 50, it thereafter increases rapidly end exponentially. The cause of this increased risk is still unclear, however immobility, decreased muscular tone, aging of the veins and the presence of other or more risk factors and diseases than young individuals all contribute to this increased risk. Whether age-induced circulatory changes and/or increased levels of blood coagulation levels are responsible for the increased thrombogenicity remains unclear. We recently developed a method in which these problems were overcome enabling the measurement of thrombin generation (TG) in a small aliquot of blood. The advantage of the use of this whole blood assay is that the small volume enables us to apply this assay to mice and the use of whole blood enables us to include the effect of blood cells on TG. Objectives By modifying the classic plasma Calibrated Automated Thrombogram (CAT) assay, we were able of measuring TG in whole blood in mice. The objective was to validate this assay in mice blood and to examine the rate and extent TG in a mouse model of premature aging. Methods TG was assayed in 20- to 28-week-old brain and muscle ARNT-like protein-1 (Bmal1)-deficient (knockout, KO) mice and wild-type (WT) littermates. The Bmal1-KO mice have an impaired circadian behavior and demonstrate loss of rhythmicity in the expression of their target genes. They are known to have a reduced lifespan and display symptoms of premature aging. Mice blood samples were taken from the orbital sinus using a capillary anticoagulated with citrate (3.8%). Coagulation and therefore TG was initiated with a solution containing calcium, tissue factor (TF) and a thrombin specific substrate. Varying the TF concentration revealed an optimal of 0.5 pM. At the end of the TG assay, the samples were fixated, prepared for and analysed by scanning electron microscopy (SEM). Results The intra-assay variations (CV%) in mice blood of the endogenous thrombin potential (ETP), peak height, lagtime, time-to-peak and velocity index were 10% or less (n=24). The whole blood TG test showed that Bmal1-KO mice have a significantly (p<0.001) higher ETP (437±7 nM.min; mean±SD, n=5) when compared with wild type mice (ETP=220±45 nM.min; mean±SD, n=5). The peak heights also differed significantly (p=0.027). However, differences in lagtime, TTP and velocity index were not significantly different (p-values were respectively 0.45, 0.15 and 0.74). Applying SEM we found that Bmal1 deficient mice display a more dense fibrin network with smaller pores compared to WT-mice. Conclusions The whole blood TG assay in mice blood revealed to be reproducible and we demonstrated that aging Bmal1-KO mice have a hypercoagulable state when compared with WT mice indicating that a higher thrombotic risk in elderly is partially due to a more procoagulation state. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Whole blood thrombin generation in Bmal1-deficient mice

Thrombosis and Haemostasis ◽

10.1160/th13-11-0910 ◽

2014 ◽

Vol 112 (08) ◽

pp. 271-275 ◽

Cited By ~ 4

Author(s):

Marisa Ninivaggi ◽

Hilde Kelchtermans ◽

Marijke J. Kuijpers ◽

Bianca Hemmeryckx ◽

Johan W. M. Heemskerk ◽

...

Keyword(s):

Whole Blood ◽

Thrombin Generation ◽

Peak Height ◽

Premature Aging ◽

Specific Substrate ◽

Mouse Blood ◽

Time To Peak ◽

Cat Assay ◽

Small Aliquot ◽

Deficient Mice

SummaryThe Calibrated Automated Thrombogram (CAT) assay that measures thrombin generation (TG) in platelet-poor and -rich plasma, is increasingly being recognised as a more sensitive tool to determine the overall function of the haemostatic system. We developed a method enabling the measurement of TG in a small aliquot of blood. The objective was to validate this assay in mouse blood and to examine the rate and extent of TG in a mouse model of premature aging. TG was assayed in blood from 20– to 28-week-old brain and muscle ARNT-like protein-1 (Bmal1)-deficient (knockout, KO) mice and wild-type (WT) littermates. Bmal1-KO mice are known to display symptoms of premature aging. TG was initiated by adding calcium, tissue factor and a thrombin specific substrate. After TG, the samples were prepared for scanning electron microscopy (SEM). The intra-assay variations (%) in mouse blood of the endogenous thrombin potential (ETP), peak height, lag time, time-to-peak and velocity index were 10% or less (n=24). We found that Bmal1-KO mice have a significantly (p<0.001) higher ETP (437 ± 7 nM.min; mean ± SD, n=7) when compared with WT mice (ETP=220 ± 45 nM.min; mean ± SD, n=5). The peak heights also differed significantly (p=0.027). By applying SEM we found that Bmal1 deficient mice display a denser fibrin network with smaller pores compared to WT mice. In conclusion, the whole blood TG assay in mice revealed to be reproducible. As a proof-of-principle we have shown that the whole blood TG assay is capable of detecting a prothrombotic phenotype in Bmal1-KO mice.

Download Full-text

An Engineered Factor Fva Prevents Bleeding Induced By Anticoagulant Wild Type Activated Protein C

Blood ◽

10.1182/blood.v122.21.203.203 ◽

2013 ◽

Vol 122 (21) ◽

pp. 203-203

Author(s):

Annette Von Drygalski ◽

Andrew J. Gale ◽

Laurent Burnier ◽

Thomas J. Cramer ◽

John H. Griffin ◽

...

Keyword(s):

Blood Loss ◽

Whole Blood ◽

Thrombin Generation ◽

Conflicts Of Interest ◽

Unmet Need ◽

Wild Type ◽

Risk Of Bleeding ◽

Liver Laceration ◽

Increased Risk ◽

Tail Clip

Abstract Increased risk of bleeding is observed in patients receiving therapy with a variety of anticoagulant drugs, and there is an unmet need for prohemostatic agents that reduce bleeding risk. In some clinical trials, wild type (wt) APC therapy was associated with an increased risk of serious bleeding. In most animal models of inflammatory injury and disease where APC was beneficial, APC’s cytoprotective effects were responsible for the protective effects of APC therapy whereas its anticoagulant effects were neither required nor contributing. Thus, wt APC’s anticoagulant activities and associated risk of bleeding may be a limiting factor for potential novel wt APC therapies. The availability of a wt APC-anticoagulant specific antidote or reversal agent that does not affect wt APC’s cytoprotective activities would be highly desirable. We hypothesized that superFVa, an engineered FVa-variant that potently normalizes hemostasis in hemophilia, fits the criteria for a prohemostatic biologic that could reduce wt APC-anticoagulant-induced bleeding, and here we test this hypothesis. SuperFVa has enhanced specific activity compared to wt FVa due to an engineered disulfide bond (Cys609-Cys1691) between the A2 and A3 domains (FV(A2-SS-A3)) and, its biological activity is augmented by mutations of the APC cleavage sites (Arg506/306/679Gln). As a result of these modifications, superFVa was found to be resistant to wt APC and normalized hemostatic properties and prevented bleeding in a hemophilic mouse model much more efficiently than wt FVa. In aPTT clotting assays both superFVa and wt FVa dose-dependently normalized clotting times when wt-APC was used to prolong clotting (aPTT > 100 sec). However, superFVa normalized the aPTT at 100-fold lower concentrations compared to wt FVa. In thrombin generation assays using either human or murine plasma, superFVa fully restored thrombin generation at concentrations where wt FVa did not show effects. In an ex vivo whole blood aPTT assay, intravenous (iv) injection of murine recombinant wt APC (0.5 mg/kg) in Balb/c mice doubled clotting times from 30 sec to ∼ 60 sec (n=40). Addition of superFVa (1 nM) to whole blood significantly normalized aPTT clotting times whereas wt FVa failed to show a significant effect. In a tail clip-bleeding model in Balb/c mice, injection (iv) of human recombinant or plasma-derived wt APC induced significant bleeding at 1.25 mg/kg and mean blood loss increased from 3.4 µL/g with saline to 27 µL/g with wt APC treatment. SuperFVa injected (iv 3.5 mg/kg) 2 min prior to wt APC administration reduced bleeding significantly to 9.2 µL/g (n∼10 per group). In another bleeding model, liver laceration was used because it provides important information concerning microvessel-mediated bleeding after acute organ injury. After adaptation and technical modifications of the model used in rats and rabbits for mouse anatomy and validation in hemophilia versus control mice, this model provided a reliable assessment of bleeding with a ∼ 4-fold reduced inter-individual range of blood loss compared to the tail clip-bleeding model. Injection (iv) of human wt APC increased blood loss from 29 µL/g to 49 µL/g, and the excessive bleeding was associated with a ∼40% mortality rate. SuperFVa reduced wt APC-induced bleeding after 20 min significantly to 29 µL/g and abolished bleeding-induced mortality. Remarkably, bleeding patterns in the tail clip and liver laceration models were different when blood loss was determined separately for the 1st and 2nd 10 min after injury. In the tail clip-model wt-APC-induced bleeding during both 1st and 2nd 10 min after tail clip and superFVa decreased blood loss during both phases. In the liver laceration model, most blood loss occurred immediately after injury and bleeding during the 2nd 10 min was less pronounced. In this model, however, superFVa corrected blood loss entirely during the 1st 10 min phase and fully prevented bleeding during the 2nd 10 min phase. Our results provide proof of principle that superFVa is effective in the prevention and reversal of wt APC-induced bleeding. Thus, in addition to improving hemostasis in hemophilia, superFVa protects against bleeding in 2 different mouse models where bleeding was induced by wt APC. Hence, superFVa deserves future consideration for development as a prohemostatic agent in situations where bleeding is a serious risk. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Abstract 1129: Obesity-related Elevations in Soluble P-selectin are Derived From Endothelium and Dependent on Expression of Leukocyte P-selectin Glycoprotein Ligand-1

Circulation ◽

10.1161/circ.116.suppl_16.ii_227-b ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Kevin J Wickenheiser ◽

Peter F Bodary ◽

Kristina Bahrou ◽

Daniel T Eitzman

Keyword(s):

Bone Marrow ◽

Body Weight ◽

Leptin Receptor ◽

The Body ◽

Wild Type ◽

Post Transplant ◽

Time Period ◽

Increased Risk ◽

Soluble P ◽

Deficient Mice

Background : Obesity is associated with proinflammatory changes and an increased risk for vascular disease complications. The tissue source and mechanism by which soluble P-selectin (sPsel) is generated in obesity are unclear. Methods and Results : Soluble p-selectin (sPsel) levels were measured in the circulation from lean wild type and obese leptin receptor deficient mice (LepR−/−) at 4 and 10 weeks of age. In wild-type mice body weight increases from 13+/−2 to 20+/−3 grams over this time period while the body weight increases from 15+/−2 to 38+/−5 grams in LepR−/− mice. At 4 weeks of age sPsel levels were 103+/−8mg/mL in wild-type mice vs. 138+/−9 ng/mL in LepR−/− mice, p=0.048. By 10 wks of age sPsel increased to 112 +/− 2 in wild-type mice and 182 +/− 9 in LepR−/− mice, p=0.00005. In order to determine if the obesity-induced rise in sPsel is regulated by leukocyte Psgl-1, bone marrow transplantation was performed from Psgl+/+ or Psgl−/− donors into irradiated LepR−/−recipients. At 4 weeks post-transplant, sPsel levels were 166 +/−6 ng/mL in LepR−/− mice receiving Psgl+/+ marrow and 45 +/− 4 ng/mL in LepR−/− mice receiving Psgl−/− marrow, p=0.0000004. In order to determine if the sPsel in LepR−/− mice originated from the endothelium versus platelets, we transplanted Psel−/− bone marrow into irradiated LepR−/−mice. At 4 weeks post transplant, sPsel levels were 153 +/−3 ng/mL in LepR−/− mice receiving Psel−/− bone marrow and were not significantly different from LepR−/− mice receiving Psel+/+ bone marrow (166 +/−6 ng/mL, p=0.06). By 10 weeks post transplant, mice gained even more weight and levels were 377+/−51 ng/mL in LepR−/− mice receiving Psel+/+ bone marrow and 370+/−73 ng/mL in LepR−/− mice receiving Psel−/− bone marrow, p=0.87. Conclusions : These data suggest that the increase in sPsel observed in obesity is primarily derived from the endothelium and that this process is regulated by leukocyte Psgl-1.

Download Full-text

Mef2C Is a Lineage-Restricted Target Gene of Scl/Tal1 and Regulates Megakaryopoiesis and B-Cell Homeostasis

Blood ◽

10.1182/blood.v112.11.278.278 ◽

2008 ◽

Vol 112 (11) ◽

pp. 278-278

Author(s):

Katrin E Rhodes ◽

Christos Gekas ◽

Laurraine Gereige ◽

Hildur Helgadottir ◽

Roberto Ferrari ◽

...

Keyword(s):

Transcription Factor ◽

B Cell ◽

Muscle Development ◽

Target Genes ◽

Fetal Liver ◽

Premature Aging ◽

Bhlh Transcription Factor ◽

Functional Requirements ◽

B Lymphoid ◽

Deficient Mice

Abstract The bHLH transcription factor stem cell leukemia/T-cell acute leukemia gene (Scl/Tal1) is a master regulator for hematopoiesis, essential for hematopoietic specification and proper differentiation of the erythroid and megakaryocyte lineages. However, the critical downstream targets of Scl remain undefined. To identify Scl target genes in hematopoietic cells, we performed gene expression analysis on HOX11-immortalized Sclfl/fl fetal liver cell lines. Analysis of the top 50 downregulated genes revealed several genes related to hematopoiesis including erythroid and megakaryocyte development, vasculogenesis, as well as genes/unknown ESTs that have not been previously linked to blood development. One of the top downregulated genes was transcription factor myocyte enhancer factor 2C (Mef2C). Mef2C−/− embryos die at E9.5, the same time as Scl−/− embryos, and exhibit severe defects in cardiac and muscle development. Analysis of Mef2C−/− embryos showed that, Mef2C, in contrast to Scl, is not required for specification into primitive or definitive hematopoietic lineages. To bypass the embryonic lethality, we utilized a conditionally targeted Mef2Cfl/fl strain and crossed it with a hematopoietic cell-specific VavCre strain that deactivates Mef2C shortly after the emergence of HSCs. Interestingly, adult VavCre+Mef2Cfl/fl mice exhibited severe platelet defects highly reminiscent to those observed in Scl deficient mice. The platelet counts were reduced, while platelet size was increased and the platelet shape and granularity was altered. Furthermore, megakaryopoiesis was severely impaired in vitro. ChIP-on-chip analysis revealed that Mef2C is directly regulated by Scl in megakaryocytic cells, but not in erythroid cells. In addition, an Scl independent requirement for Mef2C in B-lymphoid homeostasis was observed in Mef2C-deficient mice, characterized as severe age-dependent reductions of specific B-cell progenitor populations reminiscent of premature aging. In summary, this work identifies Mef2C as an integral member of hematopoietic transcription factors with distinct upstream regulatory mechanisms and functional requirements in megakaryocyte and B-lymphoid lineages.

Download Full-text

Generic and Branded Versions of Argatroban Exhibit Differential Anticoagulant Effects In Whole Blood, Plasma Based Assays and Thrombin Generation Assays

Blood ◽

10.1182/blood.v116.21.5129.5129 ◽

2010 ◽

Vol 116 (21) ◽

pp. 5129-5129

Author(s):

Jawed Fareed ◽

Debra Hoppensteadt ◽

Omer Iqbal ◽

Jeanine M. Walenga ◽

Bruce E Lewis

Keyword(s):

Protein Interactions ◽

Whole Blood ◽

Thrombin Generation ◽

Conflicts Of Interest ◽

Anticoagulant Effect ◽

Thrombin Time ◽

Clotting Time ◽

Generic Products

Abstract Abstract 5129 Several generic versions of argatroban) (Mitsubishi; Tokyo, Japan) have been introduced in Japan (Argaron, Gartban, Slovastan). In addition, other generic versions of argatroban are being considered by the European and North American regulatory bodies. While the generic versions of argatroban exhibit similar antithrombin potency (Ki values), because of the differential compositional variations their anticoagulant effects in whole blood systems may differ due to their cellular and plasmatic protein interactions. Branded and generic versions of argatroban may exhibit differential anticoagulant actions in the whole blood and plasma based assays due to their differential interactions with blood cells, platelets and plasma proteins. Three generic versions of argatroban that are commercially available in Japan namely Argaron, Gartban and Slovastan and a powdered version of generic argatroban (Lundbeck) were compared with the branded argatroban. Native whole blood thrombelastographic (TEG) analysis was carried out at 0.1 ug/mL, the Activated Clotting Time (ACT) assay was carried out in a concentration range of 0–10 ug/mL, and such coagulation tests as the PT/INR, aPTT, Heptest, and calcium thrombin time were performed. Plasma retrieved from the supplemented whole blood was also assayed. Ratios of the clotting time test values from whole blood and plasma were calculated. Retrieved plasma samples were also assayed in the thrombin generation assays (TGA). All of the different versions of argatroban produced a concentration dependent anticoagulant effect in the native whole blood TEG and ACT. In the TEG, while argatroban and Slovastan showed a similar effect, Gartban, Argaron and a powdered generic showed weaker effects. Argatroban was also different in the ACT assay. At a concentration of 5 ug/ml the ACTs were, Arg 340+15.2 secs, S 297+10.5 secs, G 292.0+19.1 secs and A 285.2+21.7 secs. In the citrated whole blood systems, all agents produced a concentration dependent anticoagulant effect; however, the generic versions produced a stronger anticoagulant effect in comparison to branded argatroban (p<0.001). In the PT assay at 5 ug/mL, argatroban showed 32 ± 3 sec vs 40–50 sec for the generic products. Similarly in the aPTT, Heptest and thrombin time tests argatroban was weaker than the generic products. Differences among generic versions were also evident. Similar results were obtained in the retrieved plasma, however the ratio of whole blood over plasma varied from product to product. The IC50 of the generic and branded argatrobans in the TGA were also different. These results show that while in the thrombin inhibition assays generic and branded argatroban may show similar effects, these agents exhibit assay dependent differences in the whole blood and plasma based assays. Such differences may be more evident in the in vivo studirs where endothelial cells and other interactions may contribute to product individuality. Therefore, based on the in vitro antiprotease assays, generic argatrobans may not be considered equivalent and require a multi-parametric study. Currently available generic argatrobans may not be equivalent in the in vivo anticoagulant effects. Therefore, clinical validation of the clinical equivalence for these drugs is warranted. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Angiogenesis and Tumor Growth Are Increased in Kininogen Deficient Mice

Blood ◽

10.1182/blood.v118.21.371.371 ◽

2011 ◽

Vol 118 (21) ◽

pp. 371-371

Author(s):

Wan-Ming Zhang ◽

Linda Hsi ◽

Keith R. McCrae

Keyword(s):

Growth Factor ◽

Tumor Growth ◽

Conflicts Of Interest ◽

Vascular Endothelial Cell ◽

Vascular Endothelial ◽

Wild Type ◽

Contact Phase ◽

Tumor Implantation ◽

Antiangiogenic Activity ◽

Deficient Mice

Abstract Abstract 371 High molecular weight kininogen (HK) is an abundant plasma protein and member of the contact phase of coagulation that is involved in a number of important biological processes. We have previously reported that kallikrein-cleaved HK (HKa) induces rapid apoptosis of proliferating endothelial cells and inhibits angiogenesis. We have recently produced a kininogen deficient mouse by deleting the murine gene for kininogen that is expressed in liver, mKng1. These mice lack detectable kininogen, despite persistent expression of mRNA from the mKng2 gene, primarily expressed in kidney. To further determine the importance of kininogen in the regulation of angiogenesis, we assessed angiogenesis in mKng1−/− mice. These mice demonstrated increased angiogenesis in subcutaneously-implanted Matrigel plugs containing vascular endothelial cell growth factor (VEGF) or basic fibroblast growth factor (bFGF), compared to wild-type littermates. We also assessed the growth of B16F10 melanoma cells and Lewis lung carcinomas planted subcutaneously in mKng1−/− mice. In both cases, in particular with the B16F10 implants, tumor growth occurred substantially faster in the setting of kininogen deficiency (mean tumor volume of mKng1−/− mice 4820 ± 728 mm3 vs 1990 ± 412 mm3 in wild type mice; P = 0.003). Differences in growth rates were apparent approximately 8 days after tumor implantation, and were associated with increases in tumor microvascular density. Several reports suggest that the urokinase receptor (uPAR), which binds HKa, mediates its antiangiogenic activity. However, using Matrigel plug assays we observed that HKa inhibited angiogenesis equally well in wild type and uPAR−/− mice. These studies demonstrate that kininogen serves as an endogenous regulator of angiogenesis. Current studies are focused on better defining the mechanisms of this activity. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Inhibition of Platelet-Prothrombianse by a Covalent Antithrombin-Heparin Complex.

Blood ◽

10.1182/blood.v120.21.2212.2212 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2212-2212

Author(s):

Ivan Stevic ◽

Howard H.W. Chan ◽

Ankush Chander ◽

Leslie R. Berry ◽

Anthony K.C. Chan

Keyword(s):

Thrombin Generation ◽

Conflicts Of Interest ◽

Anticoagulant Activity ◽

Calcium Ionophore ◽

Thrombin Inhibitor ◽

Ionophore A23187 ◽

Specific Substrate ◽

Prothrombinase Complex ◽

Activated Platelets

Abstract Abstract 2212 Introduction: Factor Xa is protected within the prothrombinase complex from inhibition by heparin. We have developed a covalent antithrombin-heparin complex (ATH) with enhanced anticoagulant activity. Previously, we have shown that ATH is able to inhibit coagulation enzymes much more efficiently than regular antithrombin+heparin (AT+UFH). For example, ATH inhibited TF/VIIa ∼30-fold faster compared to AT+UFH. Furthermore, we have also demonstrated that ATH is capable of inhibiting Xa within a prothrombinase complex assembled on synthetic phospholipid vesicles better than AT+UFH. However, ATH's effect on prothrombinase when the complex is formed on a more native system such as platelets has never been explored. Thus, the objective of the present study is to determine the ability of ATH vs AT+UFH to inhibit Xa within the prothrombinase complex when the enzyme complex is assembled on the platelet system. Methods: Discontinuous second order rate constant assays were performed to obtain k2-values for inhibition of free or prothrombinase-bound Xa by AT+UFH or ATH. Freshly prepared resting platelets were subjected to inhibition analysis by first incubating them with Xa, Va, Ca2+ and pefabloc®-TH (thrombin inhibitor) in different wells of a 96-well plate for 3 min at 37 °C. Prothrombin was then simultaneously added to all wells to initiate thrombin generation, followed by addition of AT+UFH or ATH inhibitors to each well at specific time intervals. Reactions were neutralized by simultaneous addition of polybrene, Na2EDTA and Xa-specific substrate S-2222™ in buffer. The remaining Xa enzyme activity was obtained and final k2-values calculated. For experiments requiring activated platelets, freshly isolated platelets were activated with 5 μM calcium ionophore A23187 + 4 mM CaCl2 for 15 min at room temperature. The activated platelets were then tested in inhibition assays as described above. To investigate the roles of individual components of the prothrombinase complex on the anticoagulant effects of AT+UFH and ATH, additional experiments were performed where components of the complex (prothrombin, activated platelets or Va) were omitted prior to reaction with inhibitors. Thrombin generation was used to assess functionality of the activated platelet-prothrombinase system in the presence of inhibitors using a thrombin-specific substrate S-2238™. Results: The k2-values (×108M−1min−1) for inhibition of free Xa or resting platelet-prothrombinase were similar for both inhibitors, although the overall inhibition rates achieved by the ATH were 2-fold faster than AT+UFH (p<0.001). Since activated platelets are required for enhanced prothrombinase function, we then compared inhibition of free vs activated platelet-prothrombinase by the two inhibitors (platelet activation was confirmed with flow cytometry using an anti CD-41 antibody). No differences were observed in the k2-values between free Xa (3.96±0.23) and activated platelet prothrombinase (3.83±0.39) for ATH reactions. However, the k2-values for inhibition of free Xa by AT+UFH was 2.37±0.32, and assembly of Xa within the activated platelet-prothrombinase resulted in a reduction in the k2-values to 0.99±0.22 (p<0.001), thus confirming a moderate 60% protection of Xa by the prothrombinase components. However, omitting the components (prothrombin, activated platelets or Va) from the complex resulted in higher k2-values (1.76±0.37, 2.29±0.26 and 2.52±0.32, respectively p<0.01) for AT+UFH, and as expected, no net effect was observed for ATH. Thrombin generation was inhibited significantly by both AT+UFH and ATH compared to the control (p<0.001), but further analysis of thrombin potential yielded greater inhibition by ATH compared to AT+UFH (p<0.05). Conclusion: In this study, we report inhibition of the prothrombinase complex on the surface of resting and activated platelets. Consistent with previous investigations, a moderate protection of Xa was observed when the activated platelet-prothrombinase was inhibited by AT+UFH. ATH on the other hand, targets and inhibits prothrombinase complexed-Xa as fast as free Xa, and at inhibition rates that were significantly faster than AT+UFH. Thus, overall the covalent conjugate enhances anticoagulation of surface-bound enzymes and offers advantages over conventional heparin for the treatment of cell-based coagulation in vivo. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Increased Tumorigenesis In Ribosomal Proteins L5 and S24 Heterozygous Mice

Blood ◽

10.1182/blood.v122.21.1227.1227 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1227-1227 ◽

Cited By ~ 1

Author(s):

Shideh Kazerounian ◽

Pedro Ciarlini ◽

Roxanne Ghazvinian ◽

Meritxell Alberich-Jorda ◽

Daniel Yuan ◽

...

Keyword(s):

Ribosomal Protein ◽

Ribosomal Proteins ◽

Conflicts Of Interest ◽

Normal Skin ◽

Gene Mutations ◽

Normal Function ◽

Spindle Cell Sarcoma ◽

Wild Type ◽

Increased Risk ◽

Risk Of Cancer

Abstract Diamond-Blackfan anemia (DBA) is a congenital red blood cell aplasia inherited in an autosomal dominant pattern caused by mutations in ribosomal protein (RP) genes and in an X-linked recessive pattern by GATA1 mutations. Heterozygous mutations and large deletions in 11 RP genes, RPS19, RPS24, RPS17, RPL5, RPL11, RPL35A, RPS7, RPS10, RPS26, RPL26, and RPL15, are present in ∼65% of DBA patients. DBA is associated with congenital abnormalities in ∼50% of patients and with increased risk of malignancy. To investigate the molecular pathogenesis of RPL5 and RPS24 gene mutations, we generated two murine lines of heterozygous mice, Rpl5 and Rps24, by knocking-out exons 1-8 in the Rpl5 gene and exons 2-3 in the Rps24 gene in C57BL/6 mice. Knock-out of both alleles of Rpl5 and Rps24 genes are embryonic lethal. In contrast, heterozygous mice exhibited normal hematological phenotype, as well as normal Rpl5 and Rps24 RNA and protein levels in their tissues, suggesting that the presence of one allele was sufficient to support the normal function of ribosomal proteins L5 and S24 in mice. To evaluate the risk of cancer development in Rpl5 +/- and Rps24 +/- mice, we monitored these mice and wild type mice until late age. Out of 21 Rpl5 +/- mice (between the ages of 14 and 26 months), two mice developed tumors at 22 months of age and two mice were euthanized due to severe dermatitis at the same age. Similarly, we have been monitoring 23 Rps24 +/- mice between 15 and 26 months of age. One of these mice developed a tumor at 17 months of age, five mice were euthanized due to severe dermatitis between the ages of 17 and 19 months, and two mice were euthanized due to injuries at ages 15 and 29 months. We also monitored 20 control wild-type mice ranging from 13 to 26 months of age. To this date, no tumors have been detected in wild-type mice, although nine of these mice developed severe dermatitis and were euthanized. Histological and immunohistochemical studies were performed to determine the nature of tumors in Rpl5 +/- and Rps24 +/- mice. Comparison of tumor tissues with normal skin from wild-type or Rpl5 +/- and Rps24 +/- with no detected tumors showed that all tissues had normal epidermis and underlying dermis, but connective tissues from tumor sections consisted of a densely cellular neoplasms composed of predominantly atypical spindle shaped cells arranged in intersecting fascicles. The tumor cells had strong cytoplasmic reactivity for vimentin and negative staining for S100, CD45, and pan-keratin, consistent with a high-grade spindle cell sarcoma. Recent studies conducted by the DBA Registry of North America revealed that out of 608 DBA patients, 18 with median age of 41 years developed various types of cancer including sarcomas, colon cancer, and acute myeloid leukemia. The relative risk of cancer in DBA was increased 5.4 fold compared to general population (Vlachos, et al., 2012). Our studies also suggest the correlation between ribosomal protein gene mutations and cancer. However, further studies are required to better understand the underlying molecular mechanism. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Thrombin Generation Is Directly Correlated To Proteinuria Severity In An Experimental Model Of Nephrotic Syndrome

Blood ◽

10.1182/blood.v122.21.3615.3615 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3615-3615

Author(s):

Amanda P. Waller ◽

William E. Smoyer ◽

Melinda A. Chanley ◽

Marvin T. Nieman ◽

Bryce A. Kerlin

Keyword(s):

Nephrotic Syndrome ◽

Thrombin Generation ◽

Conflicts Of Interest ◽

Linear Regression Analysis ◽

Vena Cava ◽

Surrogate Marker ◽

Urinary Protein ◽

Thrombotic Risk ◽

Α Angle ◽

Dose Dependent

Abstract Introduction Thromboembolism is a common complication of nephrotic syndrome (NS). The massive urinary protein loss (proteinuria) of NS results in an acquired, complex hypercoagulopathy. Recent epidemiologic studies have demonstrated that severity of proteinuria in patients with NS is independently predictive for thrombotic risk. Nephrotic-range proteinuria is known to result in acquired deficiencies of antithrombin and free protein S as well as accumulation of procoagulants such as factors V and VIII and von Willebrand factor. However, published cohort studies reveal that the severity of these derangements are quite variable, thus the net effect of proteinuria on thrombotic potential remains unknown. Therefore, we hypothesized that proteinuria severity directly correlates with extent of hypercoagulopathy. Methods PAN (puromycin aminonucleoside)-induced rat NS is known to induce glomerular injury with peak proteinuria at day ∼9 following intravenous injection. Severity of proteinuria and global hemostasis were compared in five groups of male Wistar rats (body weight ∼150 g) receiving a single i.v. injection of PAN at 0 (saline), 25, 50, 100, or 150 mg/kg (n=4/group). Morning spot urines collected on days 0 (before PAN injection) & 9 were analyzed for urinary [protein]:[creatinine] ratio (uPr:Cr). After urine collection on day 9 the rats were anesthetized with isoflurane and blood was collected from the inferior vena cava into 0.32% NaCitrate/1.45 µM Corn Trypsin Inhibitor [final concentrations]. Rotational thromboelastometry (ROTEM) was performed on whole blood within 20 min of collection, using the INTEM (activated intrinsic pathway) assay with and without urokinase (35 ng). Platelet poor plasma (PPP) was prepared from the remaining blood sample. Thrombin generation assays (Technothrombin TGA) were performed on PPP diluted 1:1 with buffer. Results As expected, PAN-treated rats displayed escalating dose-dependent increases in proteinuria at 9 days post-injection (Fig A). The highest dose groups exhibited differential derangements in ROTEM parameters, such that clot formation time (CFT) was decreased and α-angle was increased in rats receiving 100 & 150 mg/kg PAN vs. the sham group (P<0.05; Table). Maximum clot firmness (MCF), amplitude at 10 min (A10) & 20 min (A20), and lysis index at 60 min (LI60) were also significantly higher with the 100 & 150 mg/kg doses, compared to the 0, 25, & 50 groups (P<0.05). Linear regression analysis of uPr:Cr and all measured ROTEM parameters demonstrated that proteinuria was negatively correlated with CFT (R2=0.612, P<0.001), and positively correlated with MCF, A10, A20, & α-angle (respective R2=0.662, R2=0.496, R2=0.536, & R2=0.674; P<0.001 for each parameter). The amount of urokinase-induced fibrinolysis at 60 min was inversely related to proteinuria (LI60; R2=0.674, P<0.001). TGA parameters also exhibited a dose-dependent effect (Table & Fig B). Peak thrombin and endogenous thrombin potential (ETP) were higher in the 100 & 150 mg/kg dose groups vs. 0, 25 & 50 mg/kg groups (P<0.05). There was a positive correlation between uPr:Cr and parameters of thrombin generation (peak thrombin R2=0.751; ETP R2=0.854; P<0.001; Fig C & D). Conclusion These experiments demonstrate that proteinuria severity in the PAN-induced rat NS model is directly proportional to hypercoagulability as assessed by ROTEM and TGA. This suggests that proteinuria may have biologic relevance as an easily measured surrogate marker for hypercoagulopathy severity in NS. Analysis of inducible thrombus formation in PAN-induced rat NS is currently underway to determine the physiologic relevance of these findings. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Hemophilia a Clots Generated with Recombinant Factor VIIa Differed in Structure and Composition from Those Formed with Factor VIII

Blood ◽

10.1182/blood.v128.22.3798.3798 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3798-3798

Author(s):

Lilley Leong ◽

Irina N. Chernysh ◽

Yifan Xu ◽

Cornell Mallari ◽

Billy Wong ◽

...

Keyword(s):

Factor Viii ◽

Whole Blood ◽

Neutralizing Antibodies ◽

Research Funding ◽

Thrombin Generation ◽

Hemophilia A ◽

Clot Formation ◽

Factor Vii ◽

Wild Type ◽

Independent Mechanism

Abstract Patients with severe factor VIII (FVIII) deficiency (hemophilia A [HemA]) develop neutralizing antibodies (inhibitors) against FVIII in up to ~30% of cases. For HemA patients with inhibitors, activated recombinant factor VII (rFVIIa) is a treatment option. High levels of rFVIIa are required for treating HemA patients with inhibitors to induce direct activation of factor X on the surface of activated platelets via a tissue factor (TF)-independent mechanism (Hoffman M, Monroe DM. Thromb Res. 2010;125(suppl 1):S16-S18). To assess how rFVIIa-mediated clot formation in HemA patients with inhibitors may differ from unaffected individuals, we compared the effect of rFVIIa on HemA versus control (or HemA supplemented with 100% FVIII) clot formation in human and/or mouse systems. By TF-induced thrombin generation assay, increasing rFVIIa from 5 nM to 100 nM did not appreciably alter the kinetics or extent of thrombin generation compared with the same human HemA plasma containing 100% FVIII. Confocal microscopy of human HemA plasma clots generated with 75 nM rFVIIa and TF showed few branching fibrin fibers and an open fibrin meshwork. In contrast, TF-induced coagulation of the same HemA plasma containing 100% FVIII formed fibrin clots with numerous branches, interconnecting to form a dense meshwork. To confirm that these findings reflect rFVIIa-mediated clot formation in vivo, we assessed the intrinsic coagulation of mouse HemA whole blood collected without anticoagulant and spiked with rFVIIa. Intrinsic coagulation with rFVIIa was assessed by T2 magnetic resonance (T2MR), a technique capable of monitoring the separation of whole blood into serum, loose-clot, and tight-clot compartments during coagulation (Skewis et al. Clin Chem. 2014;60:1174-1182; Cines et al. Blood. 2014;123:1596-1603). By T2MR, rFVIIa induced the separation of HemA whole blood into the serum and clot compartments, indicating that the reduced fibrin generation with rFVIIa did not interfere with whole blood coagulation. Furthermore, saphenous vein puncture of HemA mice treated with rFVIIa showed a dose-dependent decrease in clot times. Scanning electron microscopy of the clots extracted from these HemA mice indicated markedly different composition than clots extracted from wild-type mice. In wild-type clots, fibrin and polyhedral erythrocytes formed a large proportion of the total structures. In contrast, clots from rFVIIa-treated HemA mice consisted primarily of platelets and erythrocytes with forms intermediate between discoid and polyhedral but, surprisingly, low fibrin content. Taken together, these data suggest that rFVIIa-mediated clot formation may require greater activated platelet involvement, which would be consistent with the TF-independent mechanism of action proposed for rFVIIa in HemA. Finally, the compositional difference between clots from wild-type versus HemA mice dosed with rFVIIa suggest that evaluating HemA therapies for their ability to form more physiologic clots could be an approach to improve treatment options for patients with HemA. Disclosures Leong: Bayer: Employment. Xu:Bayer: Employment. Mallari:Bayer: Employment. Wong:Bayer: Employment. Sim:Bayer: Employment. Cuker:Stago: Consultancy; Genzyme: Consultancy; Amgen: Consultancy; Biogen-Idec: Consultancy, Research Funding; T2 Biosystems: Research Funding. Marturano:T2 Biosystems: Employment. Lowery:T2 Biosystems: Employment. Kauser:Bayer: Employment. Weisel:Bayer: Research Funding.

Download Full-text