Erythrocyte Participation in Thrombin Generation

Abstract Abstract 374 Introduction: The potential contributions of erythrocytes (RBCs) to coagulation biochemistry have been controversial. We evaluated the potential for RBCs to participate in thrombin generation using minimally altered whole blood. Methods: Platelet poor plasma (PPP), platelet rich plasma (PRP) adjusted to a physiologic concentration with PPP, washed RBCs (PLTs <0.5%) and washed PLTs were prepared from contact pathway inhibited (corn trypsin inhibitor, CTI) whole blood in the absence of other anticoagulants. RBCs and PLTs were evaluated for phosphatidylserine (PS) exposure by FACS using FITC-labeled bovine lactadherin and their ability to support prothrombinase (1.4μM prothrombin, 20nM factor Va, 200pM factor Xa (FXa), 3.4μM antithrombin (AT) at physiologic concentrations of RBCs and PLTs). CTI whole blood and prepared sub fractions (PRP, PPP, PRP+RBC, PPP+RBC) were subjected to a 5pM tissue factor (TF) stimulus and samples analyzed by Western blotting and α-thrombin (αIIa) antithrombin (αTAT) ELISA (Rand et al. Blood, 1996). Results: CTI whole blood (N=3 donors mean±SD) clotted in 4±1.5 min whereas the matching PRP clotted in 8±2 min. In αTAT ELISA analyses, PRP showed a corresponding increase in lag phase and a 50% decrease in the maximum rate (26±9nM/min vs 58±7nM/min) and extent (263±62nM vs 476±86nM) of αTAT formation compared to whole blood. When PRP was reconstituted with physiologic levels of washed RBCs, the rate and extent of αTAT formation as well as the lag phase were restored to that observed in CTI whole blood. Addition of buffy coat to PRP, to test white blood cell contributions, had no effects on any of these parameters. Western blotting showed a significant decrease in prothrombin consumption in the PRP experiments compared to whole blood and PRP with washed RBCs. When RBCs were added to PPP (N=1), there was no significant prolongation of the lag phase and the rate of αTAT formation (21nM/min) was half of that seen in the corresponding whole blood (41nM/min). However, the maximum αTAT generated in RBCs+PPP (267nM) vs whole blood (343nM) differed by only 22%. Collectively, these data indicate a significant role for RBCs in the propagation of thrombin generation in CTI whole blood. FACS analysis of PS exposure on RBCs showed the following: untreated RBCs showed minimal binding to lactadherin (1.3%) compared to the positive control (99.7%; RBCs treated with 10mM N-ethylmaleimide (NEM) and 4μM ionophore A23187); pretreatment of the RBC population with 10nM αIIa or FXa showed a 5-fold increase in lactadherin binding over the untreated control indicating the presence of surface conditions capable of supporting prothrombinase. This proportion (5%) of PS-expressing RBCs represents a similar proportion of PLTs to RBCs (4%) in whole blood. Prothrombin activation (N=2)(1.4μM II, 20nM FVa, 200pM FXa, 3.4μM AT) on untreated RBCs exhibited a 3.5±0.5 minute lag phase followed by αTAT production which reached a rate of 18nM/min and maximum level of 180nM. In experiments performed in the absence of AT, pretreatment of RBCs with either 10nM αIIa or FXa shortened the lag phase by 1±0.25min. PAR-1 (TFLLRN) activated PLTs or activated PLTs+RBCs showed no lag phase, and no difference in the rate of αTAT production (25nM/min) or maximum level (200nM) of αTAT generated. Conclusions: In contact pathway-inhibited whole blood initiated with TF, RBCs appear essential for normal thrombin generation. Our approach rapidly fractionates CTI blood in the absence of other anticoagulants and yields populations of minimally altered RBCs that when subjected to a Tf stimulus produce thrombin more rapidly than PRP. Physiologic levels of washed RBCs pretreated with αIIa or FXa support a level of prothrombin activation similar to that observed with washed activated PLTs. These findings suggest that RBCs participate in thrombin generation and produce a PS-equivalent membrane when treated with αIIa or FXa. Disclosures: Mann: Haematologic Technologies: Chairman of the Board, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; corn trypsin inhibitor: Patents & Royalties; NIH, DOD, Baxter: Research Funding; Merck, Daiichi Sankyo, Baxter, GTI: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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High Molecular Weight Kininogen but Not Factor XII Deficiency Attenuates Acetaminophen-Induced Liver Injury in Mice

Blood ◽

10.1182/blood-2019-127206 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3621-3621

Author(s):

Michael Wayne Henderson ◽

Erica Sparkenbaugh ◽

Denis Noubouossie ◽

Reiner Mailer ◽

Thomas Renne ◽

...

Keyword(s):

Liver Injury ◽

Board Of Directors ◽

Plasma Levels ◽

Thrombin Generation ◽

Mass Spectrometry Analysis ◽

High Molecular Weight Kininogen ◽

Advisory Committees ◽

Spectrometry Analysis ◽

Contact Pathway ◽

Coagulation Pathway

Acetaminophen (APAP) toxicity is a common cause of acute liver failure (ALF). Dysregulation of coagulation is well documented in ALF patients. Recent reports indicate that small subset of ALF patients (~10%) exhibit spontaneous bleeding, while thrombotic complications are more frequent. Animal studies demonstrated that the inhibition of tissue factor-initiated thrombin generation attenuates APAP-induced liver injury in mice via both fibrin- and protease activated receptor 1-dependent mechanisms. Activation of FXII leads to two events: propagation of coagulation by activation of FXI (intrinsic pathway) and activation of plasma prekallikrein to kallikrein, with subsequent cleavage of high molecular weight kininogen (HK) into bradykinin (BK) and cleaved HK fragments (contact pathway). There is a growing interest in therapeutic targeting of FXII or FXI to prevent thrombosis, primarily because this goal may be attainable without incurring a significant bleeding risk. In this study, we evaluated if the intrinsic coagulation pathway contributes to the activation of coagulation and liver injury in a mouse model of APAP-induced ALF. We postulated that targeting FXII would attenuate coagulation and reduce liver injury without affecting hemostasis. In addition, FXII deficiency could provide a further protection by preventing activation of contact pathway and subsequent reduction of inflammatory response. We used 12 weeks old FXII, FXI, prekallkrein and HK deficient male mice and their respective WT controls. Mice were fasted for 16 hours followed by a single intraperitoneal injection of APAP (400 mg/kg) or sterile saline. Livers and plasma samples were collected 6 and 24 hours after injections. Hepatocellular necrosis was determined on liver sections stained with H&E. Plasma levels of alanine transaminase (ALT- marker of liver injury), thrombin-antithrombin (TAT) complexes, plasmin-α2 antiplasmin (PAP) complexes and interleukin-6 were analyzed using commercially available assays. HK cleavage was determined using BK ELISA, Western blot, and mass spectrometry analysis. Consistently with previously published data, plasma levels of ALT, TAT and IL-6, as well as liver injury scores were significantly elevated in APAP-challenged mice compared to saline-injected WT mice at both 6 and 24 hours. Surprisingly, neither FXII, FXI, nor prekallikrein deficiency had statistically significant effects on any of these parameters in APAP-challenged mice at either time point. Interestingly, however, plasma levels of ALT were significantly attenuated in HK-/- mice (n=23-34) compared to HK+/+ (n=19-31) controls at both 6 (1278 ± 184 vs. 1850 ± 235 U/L; p<0.05) and 24 hours (1546 ± 302 vs. 3857 ± 493 U/L; p<0.01) after injection of APAP. The attenuation of liver injury was observed despite the lack of a significant effect on APAP-induced coagulation activation (plasma TAT levels [mean±SEM]: 72.9 ± 8.2 μg/L in HK+/+ vs. 70.8 ± 4.3 μg/L in HK-/-). HK deficiency also reduced plasma levels of IL-6 (75.8%; p<0.001), number of infiltrating neutrophils in liver (59.8%; p<0.001) and liver necrotic area (28.1%; p<0.001) 24 hours after APAP challenge. Importantly, the protective effects of HK deficiency were completely reversed by the injection of HK protein into APAP-treated HK-/- mice. It has been previously shown that plasminogen deficiency protects against APAP-induced liver injury. Indeed, APAP administration activated the fibrinolytic system in WT mice as demonstrated by increased plasma levels of active tissue plasminogen activator (19.5 fold; p<0.01) and PAP complexes (64.4 fold; p<0.01). Using Western blotting analysis, BK ELISA, and mass spectrometry analysis we demonstrated that plasmin efficiently cleaves HK in a buffer system as well as in mouse and human plasma. Importantly, plasmin mediated cleavage of HK resulted in the release of BK and generation of cleaved HK fragments. In summary, our data indicate that the extrinsic but not the intrinsic coagulation pathway drives the coagulation-mediated pathologies associated with APAP-induced liver injury in mice. HK contributes to the liver injury independently of thrombin generation. We propose that in APAP-challenged mice, HK cleavage and downstream hepatotoxicity, are mediated by plasmin rather than FXIIa-dependent generation of kallikrein. Disclosures McCrae: Sanofi Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Pfizer Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Dova Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Rigel Pharmaceutical: Membership on an entity's Board of Directors or advisory committees. Key:Uniqure BV: Research Funding.

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Factor XI Activation In Tissue Factor-Initiated Blood Coagulation

Blood ◽

10.1182/blood.v118.21.2239.2239 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2239-2239 ◽

Cited By ~ 1

Author(s):

Thomas Orfeo ◽

Matthew Gissel ◽

Matthew Whelihan ◽

Saulius Butenas ◽

Kenneth G. Mann

Keyword(s):

Computational Model ◽

Board Of Directors ◽

Tissue Factor ◽

Active Site ◽

Thrombin Generation ◽

Zymogen Activation ◽

Advisory Committees ◽

Initiation Phase ◽

Contact Pathway ◽

Feedback Activation

Abstract Abstract 2239 Introduction. In in vitro models of tissue factor (TF)-initiated coagulation, FXI activation has been linked to increased thrombin generation. However the effects of FXI in experimental models of normal hemostasis have often been subtle, prompting ongoing investigations to define contributing cofactors, potential collaborating activators and/or reaction conditions (e.g. low TF concentrations). In this study, predictions from a computational model of TF-initiated thrombin generation that includes thrombin dependent FXI activation are used to direct an investigation of the role of FXI in two empirical models of TF-initiated coagulation. Methods. FXI activation and FXIa interactions were computationally modeled by adding the appropriate sets of equations describing thrombin activation of FXI, FXIa activation of FIX, antithrombin inhibition of FXIa, and high molecular weight kininogen binding to FXI to the existing framework of differential equations. The efficacy of FXIa in promoting thrombin generation (α- thrombin-antithrombin, αTAT) was assessed via titration in contact pathway inhibited whole blood and compared to activation by TF, FIXa, FXa and α-thrombin. TF-initiated reactions and their resupply were performed as described previously (Orfeo T et al. J. Biol. Chem., 2008) in either contact pathway inhibited blood ± an inhibitory anti-FXI antibody) or in synthetic coagulation proteome (SCP) mixtures (± FXI), allowing FXI effects on both the TF dependent phase and the resulting procoagulant pool of catalysts to be evaluated. Results. The computational model (± FXI pathway) was validated by showing congruence between computational thrombin generation profiles initiated with 5 pM TF and corresponding SCP reconstructions, which showed the lag phase shortened by 30 to 60 s and maximum thrombin levels increased when FXI was present. Similarly there was good correspondence between computational and SCP thrombin generation when FXIa (4 pM by active site) was the initiator. A computational analysis provided the following ranking of effectiveness in initiating thrombin generation: FXIa>TF>FIXa>FXa>α-thrombin. When tested in contact pathway inhibited blood, addition of 5 pM FXIa (by active site) resulted in clot times and 20 min αTAT levels similar to those observed with 5 pM TF, while similar outcomes required 25 pM FIXa, 100 pM FXa or between 10–100 nM α-thrombin. The computational modeling made clear two consequences of FXI feedback activation. The first was mechanistic, demonstrating that the amplification of thrombin generation was achieved by better coordinating the initial activations of FIX and FVIII; limited early initiation phase activation of FXI (<0.01% zymogen activation) increases the amount of FIXa available during the activation of FVIII, yielding higher concentrations of intrinsic tenase earlier in the initiation phase. The second consequence was increased FIXa accumulation as the reaction proceeds. This prediction was confirmed in two ways: 1) Western blot analysis of SCP reactions containing FXI showed ∼20% consumption of FIX over 20 min compared to no detectable FIX consumption in reactions without FXI; and 2) Resupply of TF-initiated SCP reactions (± 30 nM FXI) demonstrated that both the FVIII dependence and the long-term stability of the observed re-initiation of thrombin generation depended on the presence of FXI. Resupply studies were then performed in contact pathway inhibited blood (N=4 individuals) by using an inhibitory anti-FXI antibody to negate FXI contributions to both the TF-initiated and resupply dynamics. No effect of blocking FXI function on the time courses of αTAT formation was observed in the TF initiated phase. Two of the four individuals showed attenuated αTAT formation upon resupply when FXI function was blocked from the onset of TF initiation. Conclusions. FXIa is a potent initiator of coagulation, and the computational and SCP analyses predict the potential for feedback activation by thrombin after TF initiation to somewhat enhance the propagation phase of thrombin generation and more dramatically enhance the resupply response. However in contact pathway inhibited blood, FXI contributions to Tf-initiated thrombin generation are not discernible and the effect on the resupply response is variable between individuals suggesting additional mechanisms suppressing FXI activation in blood. Disclosures: Mann: Haematologic Technologies: Chairman of the Board, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; corn trypsin inhibitor: Patents & Royalties; NIH, DOD, Baxter: Research Funding; Merck, Daiichi Sankyo, Baxter, GTI: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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The effect of corn trypsin inhibitor and inhibiting antibodies for FXIa and FXIIa on coagulation of plasma and whole blood: comment

Journal of Thrombosis and Haemostasis ◽

10.1111/jth.12812 ◽

2015 ◽

Vol 13 (3) ◽

pp. 487-488 ◽

Cited By ~ 8

Author(s):

S. Butenas ◽

K. G. Mann

Keyword(s):

Trypsin Inhibitor ◽

Whole Blood ◽

Corn Trypsin Inhibitor

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Inhibition of the Proteasome in Bone Marrow-Derived CD138+ Tumor Cells Following Carfilzomib Administration in Relapsed or Refractory Myeloma Patients.

Blood ◽

10.1182/blood.v114.22.1845.1845 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1845-1845

Author(s):

Suzanne Trudel ◽

Susan Lee ◽

Christopher J. Kirk ◽

Nashat Gabrail ◽

Sagar Lonial ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Tumor Cells ◽

Whole Blood ◽

Research Funding ◽

Proteasome Inhibition ◽

Proteasome Activity ◽

Advisory Committees ◽

Post Dose ◽

Blood Samples

Abstract Abstract 1845 Poster Board I-871 Background: Proteasome inhibition is an effective strategy for the treatment of multiple myeloma. In patients, proteasome inhibition has primarily been measured in peripheral blood samples (whole blood or mononuclear cells). However, it is unknown whether myeloma cells in the bone marrow (BM) are equally sensitive to proteasome inhibitors such as bortezomib (BTZ) and carfilzomib (CFZ). Aim: To measure proteasome inhibition in purified tumor cells from BM samples taken from patients enrolled in two ongoing Phase 2 trials of single agent CFZ in relapsed or refractory myeloma: PX-171-003 (003) and PX-171-004 (004). Methods: CFZ was administered as an IV bolus of 20 mg/m2 on Days 1, 2, 8, 9, 15 and 16 of a 28-day cycle on both trials. Bone marrow samples, from an optional sub-study of both trials, were taken during screening and Day 2 (post-treatment) and sorted into CD138+ and CD138− cells. Proteasome activity was measured by an enzymatic assay using a fluorogenic substrate (LLVY-AMC) for the chymotrypsin-like (CT-L) activity and an active site ELISA (ProCISE) to quantitate levels of the CT-L subunits of the constitutive proteasome (Beta5) and immunoproteasome (LMP7) and the immunoproteasome subunit MECL1. Results: Whole blood samples from patients treated with CFZ showed inhibition of CT-L activity of ∼80+, similar to values obtained in Phase 1 studies. A total of 10 CD138+ screening samples, 6 from 004 and 4 from 003, and 9 post-dose samples, 5 from 004 and 4 from 003, were analyzed for proteasome levels and activity. In addition, 15 CD138−screening samples, 7 from 004 and 8 from 003, and 9 post-dose samples, 5 from 004 and 4 from 003, were analyzed. When compared to the average base-line activity, CFZ treatment resulted in 88% CT-L inhibition in CD-138+tumor cells from 004 patients (P = 0.0212 by unpaired t-test) and 59% CT-L inhibition in CD-138+ tumor cells from 003 patients (P = 0.25). Baseline CT-L activity in CD138+ tumor cells was 3-fold higher in 004 than 003, which includes a more heavily pre-treated patient population with greater prior exposure to BTZ. Higher specific enzymatic activity was due to increased levels of both constitutive and immunoproteasomes in tumor cells, where immunoproteasomes account for >75% of total cellular proteasomes. No differences between trials were seen in baseline CT-L activity from non-tumor (CD138−) cells. Inhibition in CD138− cells was 84% (P = 0.0380 and 42% (P = 0.38) in 004 and 003, respectively. Using ProCISE, we measured inhibition of LMP7 (66%), beta5 (48%) and MECL1 (64%) in CD138+ tumor cells from 004 patients. Three patients from 004 and one from 003 had both a screening and post-dose tumor cell samples available for analysis. Inhibition of CT-L activity was >80% in two of the 3 patients on 004; the third patient showed no proteasome inhibition by ProCISE and was unavailable for analysis by CT-L. CT-L activity in the CD138+ tumor cells in the 003 patient was not inhibited, however, inhibition was seen in non-tumor cells. Conclusions: CFZ inhibits the proteasome activity of myeloma cells in the bone marrow of relapsed and refractory myeloma patients. The levels of inhibition were similar to those measured in whole blood samples, supporting the use of the blood-based assay as a surrogate marker for proteasome inhibition in tumor cells. CFZ treatment resulted in inhibition of both CT-L subunits as well as additional subunits of the immunoproteasome in tumor cells. Reduced baseline activity in the more heavily pretreated 003 patients may reflect reduced tumor-dependency on the proteasome and may be related to prior treatment with BTZ in these patients. More samples are needed in order to make correlations between levels of proteasome inhibition in bone marrow tumor cells and prior therapies or response. These observations support further evaluation of proteasome activity and the effects of this promising new agent in primary tumors cells from myeloma patients. Disclosures: Trudel: Celgene: Honoraria, Speakers Bureau; Ortho Biotech: Honoraria. Lee:Proteolix, Inc.: Employment. Kirk:Proteolix, Inc.: Employment. Lonial:Celgene: Consultancy; Millennium: Consultancy, Research Funding; BMS: Consultancy; Novartis: Consultancy; Gloucester: Research Funding. Wang:Proteolix, Inc.: Research Funding. Kukreti:Celgene: Honoraria. Stewart:Genzyme, Celgene, Millenium, Proteolix: Honoraria; Takeda, Millenium: Research Funding; Takeda-Millenium, Celgene, Novartis, Amgen: Consultancy. Jagannath:Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. McDonagh:Proteolix: Research Funding. Zonder:Celgene: Speakers Bureau; Pfizer: Consultancy; Seattle Genetics, Inc.: Research Funding; Amgen: Consultancy; Millennium: Research Funding. Bennett:Proteolix: Employment.

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Is Serum Hepcidin a Useful Diagnostic Test for Iron Deficiency?

Blood ◽

10.1182/blood.v116.21.2056.2056 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2056-2056

Author(s):

Sant-Rayn S Pasricha ◽

Zoe McQuilten ◽

Mark Westerman ◽

Anthony Keller ◽

Elizabeta Nemeth ◽

...

Keyword(s):

Iron Deficiency ◽

Board Of Directors ◽

Diagnostic Test ◽

Sensitivity And Specificity ◽

Whole Blood ◽

Blood Donation ◽

Iron Status ◽

Life Sciences ◽

Normal Subjects ◽

Advisory Committees

Abstract Abstract 2056 Introduction: Iron deficiency remains the commonest blood disorder worldwide. Hepcidin is a key regulator of iron homeostasis. In iron depletion, decreased hepcidin facilitates increased iron absorption and recycling. Hepcidin is detectable in whole blood, serum & urine, and although assays have been developed, the utility and clinically appropriate cutoffs for diagnosis of iron deficiency remain to be established. Blood donors are at particular risk of iron deficiency, yet early diagnosis remains challenging in this setting; thus donors are an ideal population in which to evaluate a new diagnostic test of iron deficiency. We evaluated hepcidin as a diagnostic test of iron deficiency in female blood donors. Methods: Subjects: Premenopausal, non-anemic females accepted for whole blood donation by the Australian Red Cross Blood Service, not taking iron supplements and with no history of hemochromatosis. Iron status assessment: Serum ferritin (chemiluminescence), soluble transferrin receptor (sTfR) (immunoturbidometry) and serum hepcidin (competitive ELISA). Analysis: Diagnostic utility of hepcidin, compared with ‘gold standards’ ferritin, sTfR and sTfR/log(ferritin) index, was evaluated by Area under Receiver Operating Characteristic curves (AUCROC). Potential hepcidin cutoffs were identified, and their sensitivities and specificities evaluated. Results: We recruited 261 donors: 22.6% had ferritin<15ng/mL, 10.3% had sTfR>4.4mg/mL, and 20.3% had sTfR/log(ferritin) index>3.2. The 95% range of hepcidin values was <5.4-175.0ng/mL (overall); 9.3–203.0ng/mL (if ferritin≥15ng/mL); and 8.1–198.5ng/mL (if sTfR/log(ferritin)index≤3.2). By linear regression, log(hepcidin) was associated with log(ferritin) (coefficient +1.08, P<0.001); log(sTfR) (coefficient -2.02, P<-0.001) and log(sTfR/ferritin index) (coefficient -1.58, P<0.001). The AUCROC for hepcidin, compared with sTfR/log(ferritin) index>3.2 was 0.89, compared with ferritin<15ng/mL was 0.87 and compared with sTfR>4.4mg/mL was 0.81. An undetectable hepcidin (<5.4ng/mL) had sensitivity and specificity of 32.2% and 99.9% respectively for identification of sTfR/log(ferritin) index>3.2; hepcidin<8.1ng/mL had sensitivity and specificity of 41.5% and 97.5% respectively, and hepcidin<20ng/mL had sensitivity and specificity 74.6% and 83.2% respectively. Conclusions: Hepcidin shows promise as a diagnostic test for iron deficiency. Further work is needed to select suitable cutoffs for this assay, however a cutoff of <8.1ng/mL seems to accurately identify normal subjects, whilst <20ng/mL offers a balance between appropriate identification of cases and normal subjects. Hepcidin may become a valuable clinical index of iron status. Rapid diagnosis of iron deficiency with point of care whole blood or urine hepcidin assays may be achievable and useful in various settings, including blood donation. Prevention of donor iron deficiency is a high priority for the Australian Red Cross Blood Service and is being addressed through a comprehensive strategy. Disclosures: Westerman: Intrinsic Life Sciences: Employment, Membership on an entity's Board of Directors or advisory committees. Nemeth:Intrinsic Life Sciences: Employment, Membership on an entity's Board of Directors or advisory committees. Ganz:Intrinsic Life Sciences: Employment, Membership on an entity's Board of Directors or advisory committees.

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Evidence Of Relative Iron Deficiency in Apheresis Platelet Donors Correlates With Donation Frequency

Blood ◽

10.1182/blood.v122.21.1155.1155 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1155-1155

Author(s):

Frances Condon ◽

Huihui Li ◽

Debra Kessler ◽

Vijay Nandi ◽

Tomas Ganz ◽

...

Keyword(s):

Platelet Count ◽

Iron Deficiency ◽

Board Of Directors ◽

Serum Ferritin ◽

Whole Blood ◽

Advisory Committees ◽

Donor Pool ◽

Donation Frequency ◽

Serum Hepcidin ◽

Equity Ownership

Abstract A minimal hemoglobin (Hb) of 12.5 g/dL is required to protect blood donors from iron-deficiency and anemia and ensure collection of an adequate red cell product. The effects of whole blood or red blood cell (RBC) donation on donor Hb concentration and iron stores have been extensively studied. These changes have not been well characterized in platelet donors. Because platelet donation can occur as frequently as every 72 hours up to 24 times per year, tubes taken for donor testing (approximately 50 mL) at each donation may result in the loss of blood volume equivalent to 2-3 units of whole blood (500 mL each) in frequent donors. We hypothesized that iron deficiency and its associated thrombocytosis is underappreciated in platelet donors. To test this hypothesis, we proposed to 1) analyze the degree of iron deficiency / depletion in platelet donors, 2) assess the correlation between pre-donation platelet count with iron stores, and 3) evaluate the effect of platelet donation frequency on erythropoiesis- and iron-related parameters in white males age 40-65 years, typically representative of the platelet donor pool. Eligible donors were selected from a donor pool who had not donated whole blood / RBCs in the previous 12 months prior to study enrollment. Prospective participants with a history of iron-related pathology (e.g. iron deficiency, hereditary hemochromatosis, anemia, bleeding, or abnormal colonoscopy findings) were excluded from the study. Eligible donors who had not donated any blood products in the prior 12 months were enrolled as controls. Analysis of circulating RBC parameters, serum iron, serum ferritin, serum transferrin concentration and saturation, serum hepcidin, and soluble serum TfR1 were performed and correlations analyzed. The “TfR1 Ferritin Index” (i.e. log(sTfR1/ferritin)) was also evaluated, representing iron restricted erythropoiesis in the absence of frank iron deficiency. Statistical significance was measured using a student t-test; data is presented as mean ± s.e.m. and p<0.05 was considered statistically significant. Fifty eligible platelet donors and eight controls were enrolled in the study. Average age of platelet donors was 56±1 years, no different from that of controls (54±2 years; p=0.6). More donors (22/50 (44%)) were taking multivitamins compared to controls (2/8 (25%)). Only a small number of platelet donors (2/50 (4%)) had previously been deferred for low Hb. Although within the normal range, platelet donors were found to have a lower serum ferritin (54±6 vs.169±60 ng/mL; p<0.0001), transferrin saturation (30±1 vs. 40±7%; p=0.04), and serum hepcidin (35±3 vs. 57±11 mg/mL; p=0.006) relative to controls. In addition, TfR1 ferritin index was suggestive of relatively iron restricted erythropoiesis in platelet donors relative to controls (p=0.005). These results support our premise that platelet donors are relatively more iron deficient. Furthermore, among donors, lower MCV (89±1 vs. 93±1 fL; p=0.03) and CHr (31±0.4 vs. 32±0.4 pg; p=0.03) were observed in those who had donated platelets more than ten times relative to those who had donated once or twice. Lower hepcidin concentration (24±3 vs. 48±6 mg/mL, p=0.005), serum ferritin (38±7 vs. 77±16 ng/mL; p=0.04), and hepcidin/log ferritin (16.0±1.6 vs. 27.0±3.4, p=0.01) were observed in those who had donated platelets more than ten donations relative to those who had donated once or twice. Correlations between hepcidin and donation frequency (r=-0.396), serum ferritin and donation frequency (r=-0.323), and confirmatory hepcidin and log ferritin (r=0.454) were noted in platelet donors. Lastly, pre-donation platelet count correlated with sTfR1 (r=0.418), suggesting that thrombocytopoiesis is stimulated in the setting of relative iron restricted erythropoiesis. Taken together, this pilot study for the first time demonstrates evidence of iron restricted erythropoiesis in frequent apheresis platelet donors. An analysis of iron- and erythropoiesis-related parameters in a broader population of frequent platelet donors (i.e. male and female, white and non-white donors) may demonstrate a potential utility of iron replacement. Disclosures: Ganz: Intrinsic LifeSciences: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Nemeth:Intrinsic LifeSciences: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Westerman:Intrinsic LifeSciences: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

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Defective TAFI Activation in Hemophilia Worsens Joint Bleeding

Blood ◽

10.1182/blood.v128.22.3752.3752 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3752-3752

Author(s):

Tine L Wyseure ◽

Esther J Cooke ◽

Paul J Declerck ◽

Joost CM Meijers ◽

Annette von Drygalski ◽

...

Keyword(s):

Board Of Directors ◽

Thrombin Generation ◽

Hemophilia A ◽

Bleeding Tendency ◽

Protective Effects ◽

Acquired Hemophilia ◽

Advisory Committees ◽

Fviii Inhibitor ◽

Vascular Beds ◽

The Difference

Abstract Background: Joint bleeds are common in hemophilia A or B and cause hemophilic arthropathy. It is clinically well recognized that patients with acquired hemophilia generally do not suffer from joint bleeding, but the molecular mechanisms responsible for the difference in joint bleeding tendency between acquired and congenital hemophilia are ill defined. FVIII deficiency causes defective thrombin generation, impaired coagulation, and increased fibrinolysis. The latter is caused by impaired activation of thrombin activatable fibrinolysis inhibitor (TAFI). Our previous plasma-based analyses showed that clotting and thrombin generation were readily inhibited by an anti-FVIII antibody, whereas a 10-fold higher antibody concentration was required to inhibit thrombin-mediated TAFI activation. We hypothesize that residual TAFI activation occurring in acquired hemophilia, but not in congenital hemophilia, protects against joint bleeding. Here, we determine whether TAFI activation prevents joint bleeding in a mouse model of acquired hemophilia. Methods and results: A transient (anti-FVIII) acquired hemophilia A model was set up to compare joint bleeding in wild type (WT) vs. TAFI-/- mice. Joint bleeding was induced by a subpatellar needle puncture in the right knee. This model caused considerable joint bleeding in FVIII-/- mice as evidenced by the decreased hematocrit (Hct) 2 days post injury (D2 Hct) (D2 Hct= 29 ± 11 % (n= 9) vs. baseline Hct (46 ± 2 %); p< 0.0001). A single injection of the FVIII inhibiting antibody (GMA-8015; 0.25 mg/kg) in WT mice caused acquired hemophilia for up to 72 hours as evident from increased tail bleeding similar to that observed in FVIII-/- mice. Consistent with clinical findings, only minimal joint bleeding was observed in inhibitor-treated WT mice (D2 Hct= 44 ± 4 % (n= 15) for BALB/c and 40 ± 4 % (n= 17) for C57Bl/6J). Significant joint bleeding (D2 Hct= 36 ± 9% (n= 12) for C57Bl/6J; p< 0.05) could be induced by a higher dose of inhibitor (1 mg/kg), however bleeding remained considerably less severe than that observed in FVIII-/-mice. In vitro, the FVIII inhibitor readily inhibited thrombin generation but was relatively ineffective in inhibiting TAFI activation. Therefore, we tested our hypothesis that continued TAFI activation prevented severe joint bleeding in the inhibitor-treated WT mice. Indeed, administration of the FVIII inhibitor (0.25 mg/kg) in TAFI-/-mice resulted in excessive joint bleeding (D2 Hct= 25 ± 8 %; n= 14; p< 0.0001). Similarly, joint bleeding in WT mice was increased significantly when the FVIII inhibitor was co-administered with an inhibitory antibody against TAFI (D2 Hct= 34 ± 7 %; n= 13; p< 0.01). In contrast, TAFI deficiency did not increase tail bleeding with or without FVIII inhibitor, as determined by acute blood loss, 24-hour mortality, and Hct of the survivors at 24 hours post tail resection. These data clearly demonstrate that different vascular beds empower different mechanisms to curb bleeding and suggest that the protective effects of TAFI are specifically relevant for the vascular beds of the synovial joint. Activated TAFI (TAFIa) conveys multiple functions, including anti-fibrinolytic effects and numerous anti-inflammatory activities. Interestingly, tranexamic acid (TXA), a Lys analogue and potent anti-fibrinolytic agent, added at 50 mg/ml to the drinking water, did not reduce joint bleeding in FVIII-/- mice or TAFI-/- mice with the FVIII inhibitor, whereas TXA did correct tail bleeding in these mice. This suggests that the protective effects of TAFI on joint bleeding were independent of its anti-fibrinolytic effects and may result from its anti-inflammatory activities. This is supported by histological analysis at day 7 showing increased stromal proliferation and inflammatory cell recruitment in the joints of TAFI-/-mice. Conclusions:TAFI activation is impaired in congenital hemophilia but not in acquired hemophilia. Abrogation of TAFIa activity, either genetically or pharmaceutically, increased joint bleeding in mice with acquired hemophilia, indicating that TAFI may be responsible for the difference in joint bleeding tendency between acquired and congenital hemophilia. Protective effects of TAFI were vascular bed specific and independent of its anti-fibrinolytic effects, suggesting that one or more of TAFIa's other substrates promote hemophilic joint bleeding. Disclosures von Drygalski: Novo Nordisk: Consultancy, Honoraria, Speakers Bureau; CSL-Behring: Consultancy, Honoraria, Speakers Bureau; Hematherix LLC: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Speakers Bureau; Biogen: Consultancy, Honoraria, Speakers Bureau; Bayer: Consultancy, Honoraria, Speakers Bureau; Baxalta/Shire: Consultancy, Honoraria, Speakers Bureau. Mosnier:The Scripps Research Institute: Patents & Royalties; Hematherix LLC: Membership on an entity's Board of Directors or advisory committees; Bayer: Honoraria, Speakers Bureau; Baxalta: Honoraria, Speakers Bureau.

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Prothrombin activation in blood coagulation: the erythrocyte contribution to thrombin generation

Blood ◽

10.1182/blood-2012-05-427856 ◽

2012 ◽

Vol 120 (18) ◽

pp. 3837-3845 ◽

Cited By ~ 83

Author(s):

Matthew F. Whelihan ◽

Vicentios Zachary ◽

Thomas Orfeo ◽

Kenneth G. Mann

Keyword(s):

Blood Cells ◽

Thrombin Generation ◽

Marked Decrease ◽

Platelet Rich Plasma ◽

Buffy Coat ◽

Glycophorin A ◽

Initiation Phase ◽

Platelet Concentration ◽

Corn Trypsin Inhibitor ◽

Prothrombin Activation

Abstract Prothrombin activation can proceed through the intermediates meizothrombin or prethrombin-2. To assess the contributions that these 2 intermediates make to prothrombin activation in tissue factor (Tf)–activated blood, immunoassays were developed that measure the meizothrombin antithrombin (mTAT) and α-thrombin antithrombin (αTAT) complexes. We determined that Tf-activated blood produced both αTAT and mTAT. The presence of mTAT suggested that nonplatelet surfaces were contributing to approximately 35% of prothrombin activation. Corn trypsin inhibitor–treated blood was fractionated to yield red blood cells (RBCs), platelet-rich plasma (PRP), platelet-poor plasma (PPP), and buffy coat. Compared with blood, PRP reconstituted with PPP to a physiologic platelet concentration showed a 2-fold prolongation in the initiation phase and a marked decrease in the rate and extent of αTAT formation. Only the addition of RBCs to PRP was capable of normalizing αTAT generation. FACS on glycophorin A–positive cells showed that approximately 0.6% of the RBC population expresses phosphatidylserine and binds prothrombinase (FITC Xa·factor Va). These data indicate that RBCs participate in thrombin generation in Tf-activated blood, producing a membrane that supports prothrombin activation through the meizothrombin pathway.

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