Pharmacodynamic Differences in the Two Generic Brands of Enoxaparin

Abstract Abstract 1251 Several generic versions of enoxaparin have recently become available. While these generic versions of enoxaparin exhibit similar molecular profiles and comparable anti-Xa activities; product specific differences in global anticoagulant (APTT, Heptest and thrombin generation inhibition) have been reported. The purpose of this study was to compare a generic version of enoxaparin Sandoz from Argentina (Fibrinox lot 002) and from the US (enoxaparin lot 914786) in various in vitro whole blood and plasma based clotting tests. Despite comparable molecular profile and anti-Xa potency, product specific differences were noted between the products and the US generic enoxaparin showed a cumulatively stronger activity in most of the assays. To further test the pharmacodynamic profile of these products, individual groups of monkeys (n=4–8) were administered with each product at a 1 mg/kg SC and blood samples were collected for up to 28 hours. Clot based assays such as the APTT, Heptest, thrombin time, amidolytic anti-Xa and anti-IIa activities were carried out. In addition, tissue factor pathway inhibitor (TFPI) antigen, thrombin activatable fibrinolysis inhibitor (TAFI) activity and thrombin generation assays were also performed. Variable differences were noted in the clot based and amidolytic assays. Interestingly, the US generic product exhibited a lower release in the TFPI antigen whereas in the thrombin generation assays it produced a stronger inhibition of thrombin in terms of the AUC. TAFI activity profile also showed wide variations. These differences were more prevalent during the 1–4 hour time period. No differences were noted at >6 hours. The hysterisis PK/PD plots revealed marked differences between the two products. These results indicate that the products for the same generic suppliers may exhibit variations according to market places. Moreover, these observations underscore the need for a more stringent pharmacodynamic profile to demonstrate product equivalence. Disclosures: No relevant conflicts of interest to declare.

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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.

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Comparison of Branded Enoxaparin (Lovenox) and a Biosimilar Version of Enoxaparin (Fibrinox)

Blood ◽

10.1182/blood.v116.21.4385.4385 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4385-4385

Author(s):

Walter Jeske ◽

Elizabeth McGeehan ◽

Omer Iqbal ◽

Debra Hoppensteadt ◽

Jeanine M. Walenga ◽

...

Keyword(s):

Molecular Weight ◽

Thrombin Generation ◽

Conflicts Of Interest ◽

Average Molecular Weight ◽

Molecular Profile ◽

Distribution Analysis ◽

Thrombin Time ◽

Plasma Samples ◽

Branded Product ◽

Inhibition Of Thrombin

Abstract Abstract 4385 Several biosimilar versions of branded enoxaparin (Lovenox, Sanofi-Aventis, Paris, France) have recently become available throughout the world. These biosimilar enoxaparin preparations are distributed by multiple suppliers in Asia and in North and South America. Enoxaparin represents a complex mixture of oligosaccharides obtained by alkaline depolymerization of porcine mucosal heparin. It is the most widely used low molecular weight heparin which has been validated for clinical use in multiple indications. While the molecular profile and anti-Xa potencies of some of the biosimilar versions of enoxaparin are comparable, product based differences have been reported amongst some of the biosimilar versions of enoxaparin. The purpose of this study was to compare the biochemical and pharmacologic profile of one biosimilar version of enoxaparin, namely Fibrinox (Sandoz SA, Buenos Aires, Argentina) with the branded product Lovenox. The products were compared in equigravimetric amounts, assuming equivalent potency (100 AXa U/mg). Both products exhibited comparable molecular weight profiles in terms of average molecular weight and oligosachharide distribution. Analysis of the antithrombin binding hexasaccharide fractions of Fibrinox and Lovenox indicated the presence of eight distinct hexasaccharides. The relative proportions these hexasaccharides differed between Fibrinox and Lovenox. The anti-Xa and anti-IIa activities were comparable. In the whole blood clot-based assays such as TEG and ACT, both agents produced similar anticoagulant effects. In the plasma based assays such as the APTT, Heptest and thrombin time, both products showed comparable anticoagulant effects in the normal human pooled plasma samples. However, in plasma samples collected from patients with liver disease who were apparently anticoagulant free, the two products showed differences in their anticoagulant effects in the APTT assay (p<0.05). In the TF mediated thrombin generation assay, Fibrinox produced a stronger inhibition of thrombin generation compared to Lovenox (IC50; Fibrinox, 1.6 μ g/ml, Lovenox 2.2 μ g/ml). No differences were observed between the two products in the agonist induced platelet aggregation assays. However in the 14C serotonin release study, Fibrinox produced a stronger HIT serum mediated 14C release (p<0.05). Differences in the fibrinokinetic profile and the inhibition of thrombin activatable fibrinolytic inhibitor activation were observed with these LMWHs. These studies suggest while both the molecular profile and the pharmacopoeial potency of Fibrinox is similar to the branded product, these drugs can be differentiated in some of the other assays and should be evaluated in terms of additional pharmacologic mechanisims to demonstrate bioequivalence. Disclosures: No relevant conflicts of interest to declare.

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A Generic Version of Recombinant FVIIa Is Similar to the Branded Product (NovoSeven)

Blood ◽

10.1182/blood.v126.23.4746.4746 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4746-4746

Author(s):

Nasir Sadeghi ◽

Paul O'Malley ◽

Daniel Kahn ◽

Debra Hoppensteadt ◽

Jawed Fareed

Keyword(s):

Protein Content ◽

Thrombin Generation ◽

Conflicts Of Interest ◽

Factor Viia ◽

Generic Version ◽

Factor Vii ◽

Molecular Profile ◽

The Us ◽

Branded Product

Abstract Background: Commercially available recombinant factor VIIa (Novoseven) is widely used in the management of hemophilia patients with inhibitors. Recently several generic versions of recombinant VIIa (rFVIIa) have become available. The generic versions of rFVIIa are claimed to be biosimilar to the barnded Novoseven (Novo Nordisk, Copenhagen, Denmark). The purpose of this study is to compare the US and European Novoseven products with a generic version of rFVIIa namely, Aryoseven (Aryogen, Tehran, Iran). Methods: Four commercially available random lots of Novoseven were obtained from the US and European sources. Four different batches of Aryoseven were obtained from Aryogen. All individual rFVIIa preparations were diluted to obtain working concentrations of 100, 10, 1 and 0.1 ug/ml. Protein content (Lowry's method), molecular profile using surface enhanced laser desorption ionization (SELDI), gel electrophoretic profile (GEP), factor VII related antigen level (FVII:Ag), factor VII correction studies in depleted plasma and thrombin generation (TG) studies were carried out. In addition, VIIa/tissue factor mediated thrombin generation studies were carried out in various prothrombin complex concentrates such as Beriplex and Prothromplex. Results: The protein content and SELDI mass spectrophotometric profile of all 4 rFVIIa preparations were comparable. There was no differences in the Novoseven obtained from the US and European sources. The GEP of the two groups of agents showed a comparable profile with distinct peaks at 50 KDa and 25 KDa. The FVII related antigen levels were also comparable in the Novoseven and Aryoseven preparations. Supplementation of both the Novoseven and Aryoseven preparations at 10 and 100 ug/ml resulted in a comparable correction of the factor VII deficient plasma as measured by PT(INR). Thrombin generation was comparable in the branded and generic product. Conclusions: These results demonstrate that the US and European Aryoseven are comparable. Four batches of Novoseven and 4 individual clinical batches of Aryoseven were found to be comparable. When the US purchased Novoseven preparation was compared with the European Novoseven product, no differences were noted. Thus, the generic Aryoseven is biosimilar to barnded Novoseven and warrant in vivo validation studies. Disclosures No relevant conflicts of interest to declare.

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Abstract P683: Thrombin Generation in Plasma of Stroke Patients With Atrial Fibrillation

Stroke ◽

10.1161/str.52.suppl_1.p683 ◽

2021 ◽

Vol 52 (Suppl_1) ◽

Author(s):

Sarina Falcione ◽

Gina Sykes ◽

Joseph Kamtchum Tatuene ◽

Danielle Munsterman ◽

Twinkle Joy ◽

...

Keyword(s):

Atrial Fibrillation ◽

Ischemic Stroke ◽

Thrombin Generation ◽

Thrombus Formation ◽

Lag Time ◽

Thrombin Time ◽

Stroke Patients ◽

Time To Peak ◽

Generation Capacity

Background and Purpose: Thrombus formation is central to pathophysiology of stroke in patients with atrial fibrillation. Whether factors in plasma contribute to thrombus generation in patients with atrial fibrillation remains unclear. In this study we sought to determine whether plasma contributes to thrombin generation in patients with atrial fibrillation. Methods: There were 78 acute ischemic strokes with atrial fibrillation and 37 non-stroke controls. Plasma thrombin generation was measured by thrombin generation assay, resulting lag time, peak thrombin, time to peak and area under the curve was assessed. Thrombin generation capacity was compared in stroke patients with atrial fibrillation to non-stroke controls. The relationship to anticoagulation was assessed. In vitro, the effect of anticoagulation on plasma thrombin generation was determined. Results: Thrombin generation capacity was increased (shorter lag time and time to peak) in ischemic stroke patients with atrial fibrillation compared to non-stroke atrial-fibrillation controls (p<0.05 and p<0.01, respectively). Anticoagulation decreased plasma induced thrombin generation. Ischemic stroke patients with atrial fibrillation treated with anticoagulation (DOAC or warfarin) had lower plasma induced thrombin generation compared to atrial-fibrillation patients not on anticoagulation (p<0.05). Thrombin generation by plasma could be further reduced by DOAC in an in-vitro assay. Conclusions: Stroke patients with atrial fibrillation have a higher plasma induced thrombin generation compared to atrial fibrillation controls. Factors in plasma such as leukocyte derived tissue factor likely contribute to thrombus formation in patients with atrial fibrillation. As such, components in plasma may represent new targets to reduce thrombus formation and stroke risk in patients with atrial fibrillation.

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Effect of Heparin on TAFI-Dependent Inhibition of Fibrinolysis: Relative Importance of TAFIa Generated by Clot-Bound and Fluid Phase Thrombin

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613199 ◽

2002 ◽

Vol 88 (08) ◽

pp. 282-287 ◽

Cited By ~ 14

Author(s):

Anna Pentimone ◽

Bianca Binetti ◽

Marialisa Cramarossa ◽

Donatella Piro ◽

Nicola Semeraro ◽

...

Keyword(s):

Thrombin Generation ◽

Blood Clot ◽

Fluid Phase ◽

Clot Lysis ◽

Relative Importance ◽

Thrombin Activatable Fibrinolysis Inhibitor ◽

Dependent Inhibition ◽

Fibrinolysis Inhibitor ◽

Dependent Mechanism

SummaryHeparin has been proposed to enhance thrombolysis by inhibiting thrombin-dependent generation of activated TAFI (thrombin activatable fibrinolysis inhibitor), a carboxypeptidase that inhibits fibrinolysis. We evaluated the effect of heparin in an in vitro thrombolysis model consisting of a radiolabelled blood clot submerged in defibrinated plasma. Fibrinolysis was induced by adding t-PA (250 ng/ml) and calcium to the plasma bath. Control experiments indicated that thrombin generation induced by recalcification caused significant TAFI activation and inhibited clot lysis. Heparin (up to 1 U/ml), added to the plasma bath, failed to enhance clot lysis. Thrombin generation in the fluid phase was totally inhibited by heparin at concentrations > 0.5 U/ml. In contrast, thrombin generation on the clot surface was not inhibited by heparin (1 U/ml). TAFIa generation did occur in heparin-containing samples (1 U/ml) and amounted to about 10% of TAFIa formed in control samples. This low amount of TAFIa did exert antifibrinolytic activity as indicated by the observation that the addition of a specific TAFIa inhibitor (PTI) along with heparin enhanced clot lysis. Hirudin (10 µg/ml), at variance with heparin, inhibited clot-bound thrombin and enhanced clot lysis. These data show that heparin is unable to stimulate fibrinolysis through a TAFI-dependent mechanism, most likely because of its inefficiency in inhibiting thrombin generation on the clot surface. Moreover, they suggest that clot-bound thrombin plays a major role in TAFI-mediated inhibition of fibrinolysis through “localized” TAFIa generation.

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Reversion of the Experimental Hemodilutional Coagulopathy Induced by Crystalloids and Colloids Using Different Coagulation Factor Concentrates

Blood ◽

10.1182/blood.v118.21.4349.4349 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4349-4349

Author(s):

Carolina Caballo ◽

Ana M Galan ◽

Maribel Diaz-Ricart ◽

Irene Lopez-Vilchez ◽

Miguel Lozano ◽

...

Keyword(s):

Thrombin Generation ◽

Conflicts Of Interest ◽

Coagulation Factor ◽

Human Albumin ◽

Massive Bleeding ◽

Trauma Patients ◽

Beneficial Effects ◽

Ringer’S Lactate ◽

The Impact

Abstract Abstract 4349 BACKGROUND: Massive bleeding and subsequent coagulopathy are responsible for 35% of deaths in trauma patients. Hemodilution during resuscitation may worsen the coagulopathy and perpetuate bleeding. STUDY DESIGN AND METHODS: Blood samples from healthy donors were diluted (30–60%) using crystalloids (saline, Ringer’s lactate, Plasmalyte™) or colloids (6%hydroxyethylstarch (HES130/0.4), 5% human albumin, and gelatin). The impact of hemodilution on platelet adhesion, thrombin generation (TG), and clot viscoelastic properties by thromboelastometry (TEM) was analyzed. Effects of fibrinogen (Fbn), prothrombin complex concentrates (PCCs), rFVIIa, or cryoprecipates (cryo) on hemodilution were also assessed. RESULTS: Hemodilution caused a significant decrease in platelet interaction that was not improved by the addition of any of the plasma derivatives. A decrease in TG and important alterations of TEM were also observed. HES130/0.4 was the expander with the most deleterious action. TG was significantly enhanced by PCCs and their combination with Fbn whereas rFVIIa only slightly accelerated it. Fbn restored the alterations of TEM caused by hemodilution including those more deeply altered by HES 130/0.4. The combination of Fbn with PCC or rFVIIa did not have an additional effect in TEM. Cryo significantly improved the alterations caused by hemodilution on TG and TEM parameters. Effects of cryo on TG disappeared after ultracentrifugation, suggesting that contaminating microvesicular material could account for this effect. CONCLUSION: Hemostatic alterations caused by hemodilution are multifactorial and affect both blood cells and coagulation. In our in vitro approach, HES 130/0.4 seemed to exert a more deleterious effect on hemostasis. None of the concentrates improved platelet-mediated hemostasis, although they always showed variable beneficial effects on coagulation parameters. Our data indicate that PCC, rFVIIa and cryo enhance or accelerate thrombin generation. Fbn concentrates could be useful to preserve blood clotting abilities during fluid resuscitation of critically ill patients without exposing them to enhanced thrombin generation. Grants: PET(2008_0231), FIS(CP04-00112, PS09/00664), SAF2009-10365, RD06/0009 Disclosures: No relevant conflicts of interest to declare.

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Trace FVIII Augments Rfviia Induced Thrombin Generation and Improves Hemostasis in Hemophilia A Patients with Inhibitors: A clinical Cohort Study

Blood ◽

10.1182/blood.v120.21.40.40 ◽

2012 ◽

Vol 120 (21) ◽

pp. 40-40

Author(s):

Tami Livnat ◽

Uri Martinowitz ◽

Shirley Azar-Avivi ◽

Ariela Zivelin ◽

Gili Kenet

Keyword(s):

Thrombin Generation ◽

Hemophilia A ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Therapy Response ◽

Response To Therapy ◽

Individual Therapy ◽

Severe Bleeding ◽

Inhibitor Level

Abstract Abstract 40 Treatment of Hemophilia A patients with inhibitors is challenging, as correlation between inhibitor level and hemostatic response to therapy may be limited. Thrombin generation (TG) assays may be used to monitor hemostasis and/or predict patients' response to various bypass agents. Since combination of excess FVIII and bypassing agents may potentiate improved TG in inhibitor plasma tested in-vitro, we aimed to define the therapeutic feasibility of co-administration of rFVIIa and FVIII in hemophilia A patients with inhibitors. Patients and Methods: Following consent, blood was sampled from 15 hemophilia patients (age: 0.5–46y) with inhibitor (0.5–668 BU). Platelet poor plasma (PPP) was prepared, spiked and incubated with excess FVIII. Ex-vivo kinetics of FVIII neutralization over time was evaluated by sequential measurements of residual FVIII activity. We then used recalcification induced-TG (performed in PPP supplemented with 4μM phospholipids), to measure the ex-vivo response to increasing concentrations of FVIII (0–200%) and rFVIIa (0–6.8μg/ml), alone or in combination. Based upon these ex-vivo studies, an individually tailored therapeutic regimen of concomitant bolus doses of rFVIIa and FVIII was applied to nine hemophilia patients with inhibitors. Results: FVIII ex- vivo measurements post incubation detected either rapid or slow neutralization- not correlating with inhibitor level. Flat baseline TG curves were recorded for all inhibitor patients, with variable responses to FVIII and/or rFVIIa. Combined spiking with FVIII and rFVIIa dramatically increased rFVIIa induced ETP (762.7 ±305.7 as compared to 339.3±179.9 nM/min with rFVIIa only) and peak height (48.7±23.6 vs 23.7±16.6) in all patients' plasma samples. Based upon individual ex vivo assays, concomitant bolus doses of rFVIIa (120–200 mcg/kg) and FVIII (50–100 U/Kg), were applied to 9 patients, for a total of 333 episodes during study period (February 2010-Septemeber 2012). Patients during immune tolerance received rFVIIa prophylaxis with combined rFVIIa/FVIII dosing applied 3 times weekly. For most mild- moderate joint bleeds hemostasis was defined as satisfactory following a single combined dose. Severe bleeding episodes or target joint bleeds responded to 2–8 (median:3) combined doses, applied every 12 hours. During study period the median number of spontaneous joint bleeds decreased from 4 to 1 per month. Neither thrombosis nor any other complications evolved. Conclusions: Prediction of individual therapy response may be achieved by pre-analytical studies, assessing FVIII neutralization kinetics as well as ex-vivo TG responses to combined bypass/FVIII therapy. Such studies enabled treatment of inhibitor patients according to individually tailored regimens. We confirmed for the first time that the in- vitro advantage of combining FVIII and rFVIIa, indeed accounts for improved hemostasis and may safely be applied to inhibitor patients. Disclosures: No relevant conflicts of interest to declare.

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Hemoglobinuria and Mechanical Hemolysis Associated with Red Blood Cell Transfusion in Pediatric Patients

Blood ◽

10.1182/blood.v120.21.3429.3429 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3429-3429 ◽

Cited By ~ 1

Author(s):

Janet McNaughton ◽

Jonathan Hughes ◽

Jennifer C. Andrews ◽

Tracy I. George ◽

Cassandra Bergero ◽

...

Keyword(s):

Blood Cell ◽

Red Blood Cell ◽

Conflicts Of Interest ◽

Infusion Pump ◽

Red Blood Cell Transfusion ◽

Treatment Center ◽

Outpatient Unit ◽

Time Period ◽

Additive Solution

Abstract Abstract 3429 Introduction: Mechanical hemolysis is a non-immune mediated destruction of red blood cells that can be associated with blood transfusion. Various etiologies include the use of blood warmers, small bore needles or high infusion rates. We report the investigation of hemoglobinuria cases observed post-packed red blood cell (PRBC) transfusion with subsequent changes in management. Case report: Over a time period of 2 months, 6 events of hemoglobinuria and hemolysis were reported in 5 patients in the pediatric hematology/oncology treatment center. Each child presented with abnormally tea-colored urine following PRBC transfusion. Transfusion reaction workup included a direct antiglobulin test (DAT), urinalysis (UA), serum haptoglobin (H), lactate dehydrogenase (LDH), and total bilirubin (T-B). All patients had a negative or unchanged DAT, negative RBC antibody screen, decreased H, increased T-B, and increased LDH (4 out of 6), suggestive of intravascular hemolysis (Table). In spite of hemolysis, hemoglobin (Hgb)/hematocrit(Hct) increased normally. Multiple factors were investigated to determine a cause: collection and processing of blood units; handling of blood at the transfusion service; and blood administration by nursing staff. The only significant change identified was the implementation of a new infusion pump (pump A) replacing a model that was phased out. A hospital-wide retrospective review of urinalysis (UA) was performed over a one month time period, along with prospective UA surveillance in the outpatient unit. Results: In the 6 cases, a total of 10 irradiated PRBC units (7 with citrate phosphate dextrose buffer (CPDA1, HCT 65–80%) and 3 with additive solution (AS-5, HCT 55–65%)) were transfused. Although Pump A had been validated by the manufacturer with non-irradiated additive solution units, an in vitro study with irradiated CPDA1 RBC units was performed to compare pump A with an alternative pump B. Samples were taken after infusion using the pumps only (no needle or catheter) and dripped directly into the test tube for measurement of free hemoglobin (FHb). Irradiated CPDA1 units infused at a low rate (50ml/hr) showed an increase in FHb level at 1998mg/dL with pump A versus 496 mg/dL with pump B. Non-irradiated AS-5 units tested as controls resulted in a FHb level at 246mg/dL with pump A versus 117mg/dL with pump B. CPDA1 units were subsequently replaced with AS-5 units for transfusion. Finally, pump A was replaced by pump B in the outpatient hematology oncology unit. In vitro studies are still ongoing to determine if irradiation of the PRBC may also play a role in the mechanical hemolysis observed with pump A. One month retrospective UA review found no association between patients with hemoglobinuria and RBC transfusion. The UA prospective surveillance performed in the outpatient unit revealed that 7 of 26 patients had trace hemoglobinuria. Each one had a confounding factor (chronic hemolysis) for hemoglobinuria. Conclusion: Preliminary investigation determined that the infusion of highly concentrated irradiated RBC (CDPA1) using a specific commercial pump was associated with mechanical hemolysis. The change to less concentrated RBC units through an alternative pump has been to date, an effective corrective action. Disclosures: No relevant conflicts of interest to declare.

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FXIIa Differentially Regulates Thrombin Generation during Steady State and Vaso-Occlusive Crisis in Sickle Cell Mice

Blood ◽

10.1182/blood.v128.22.162.162 ◽

2016 ◽

Vol 128 (22) ◽

pp. 162-162 ◽

Cited By ~ 4

Author(s):

Erica M Sparkenbaugh ◽

Camille Faes ◽

Denis Noubouossie ◽

Daniel K. Kirchhofer ◽

András Gruber ◽

...

Keyword(s):

Steady State ◽

Mouse Model ◽

Vascular Permeability ◽

Sickle Cell ◽

Plasma Levels ◽

Thrombin Generation ◽

Conflicts Of Interest ◽

Control Group ◽

Liver And Kidney

Abstract Sickle cell disease (SCD) is associated with chronic activation of coagulation. Previously, we demonstrated that inhibition of tissue factor (TF) attenuates thrombin generation (measured by plasma levels of thrombin-antithrombin complexes [TAT]) in a mouse model of SCD during steady state. Furthermore, we showed that neither inhibition of FXIIa-dependent activation of FXI (using 14E11 antibody) nor FXI deficiency reduces thrombin generation (TG) in sickle mice. In contrast, genetic deficiency of FXII or kininogen (HK) reduced plasma TAT levels. These data suggest that during steady state, FXIIa contributes to TG in sickle mice via activation of the kallikrein/HK pathway, but not FXI. In the present study, we further investigated the mechanisms of HK-induced TG at steady state, and increased TG observed during vaso-occlusive crisis (VOC). All experiments were performed using 4-5 month old Townes SS (sickle) and AA (control) mice. Kallikrein cleaves HK into HK fragments (HKFs) and bradykinin (BK). First, we investigated whether a BK-mediated increase in vascular permeability contributes to TG by exposing perivascular TF. This hypothesis was disproved by data demonstrating no difference in vascular permeability (measured by the extravasation of Evans blue in the heart, lung, liver and kidney) between AA (n=8) and SS (n=10) mice. HKFs were shown to induce leukocyte TF expression in vitro via binding to CD11b/CD18 (Mac-1). Therefore, we investigated whether Mac-1 inhibition affects TG in SS mice. AA and SS mice were treated with an inhibitory anti Mac-1 (M1/70) or IgG control antibody on days 0, 3 and 6 (i.p. 1 mg/kg) and TG was analyzed 1 day after the last injection. In the control group, SS mice demonstrated higher plasma TAT levels compared to AA mice (8.1±1.6 vs 4.2±0.6 ng/mL, n=10-11, p<0.05), but inhibition of Mac-1 significantly reduced plasma TAT levels in SS mice (4.6±0.7 ng/mL, n=11, p<0.05). These data suggest that HK might contribute to TG during steady state via Mac-1-dependent induction of monocyte TF. The steady state of SCD is interspersed with acute periods of VOC. Clinical data demonstrate that compared to the steady state, plasma levels of cell free DNA (cfDNA), activation of the contact system, and TG are further enhanced during VOC. To determine the mechanism of increased TG during VOC, we used the previously characterized mouse model of TNFα -induced VOC. Townes AA and SS mice were injected with recombinant TNFα (2 µg/g body weight) or the same volume of PBS, and plasma was collected 5 hours later. TNFα not only dramatically increased plasma levels of cfDNA in SS mice (14.78 ± 1.64 vs 679 ± 300 ng/mL; p<0.01), but also further increased plasma TAT levels compared to those observed in PBS-treated SS mice (2.9 fold, p<0.001, n=8). Importantly, there was a significant positive correlation between cfDNA and TAT in SS mice (r2 =0.65, p<0.001). Since cfDNA can activate FXII, we determined whether FXIIa-dependent activation of FXI contributes to TG during VOC. AA and SS mice received 14E11 or IgG control (4 mg/kg) 30 minutes before TNFα (2 μg/g) or PBS injection, and plasma TAT was assessed 5 hours later. Strikingly, 14E11 attenuated the increased TAT level in TNFα-treated SS mice, to the level observed in SS mice injected with PBS and IgG (IgG/SS/PBS: 9 ng/mL ± 1.8 vs. IgG/SS/TNF: 18.9 ± 3.6, p<0.001; 14E11/SS/TNF: 9.86 ± 0.72, p<0.05 vs. IgG/SS/TNF). We also determined if TF activity is required for the increased TG observed during VOC. Interestingly, inhibition of TF with an inhibitory 1H1 antibody (25 or 75 mg/kg injected i.p. 1 or 18 hours prior to TNFα, respectively) had no effect on the increased TG observed in TNFα treated SS mice. In aggregate, our data suggest that during the steady state of SCD, FXII-dependent TG is not FXI-dependent, but instead is mediated by a pathway involving HK, Mac-1 integrin and leukocyte TF. Furthermore, we propose that during VOC the massive release of cfDNA results in FXIIa-dependent FXI activation and enhances TG independently of TF. This study provides mechanistic insight into the initiators of TG in SCD. Moreover, it implicates FXIIa as a potential therapeutic target to reduce the prothrombotic state in SCD, during both steady state and VOC. Disclosures No relevant conflicts of interest to declare.

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Comparative Studies on the Anticoagulant Profile of Branded Enoxaparin and a New Biosimilar Version

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/1076029620960820 ◽

2020 ◽

Vol 26 ◽

pp. 107602962096082

Author(s):

Dalia Qneibi ◽

Eduardo Ramacciotti ◽

Ariane Scarlatelli Macedo ◽

Roberto Augusto Caffaro ◽

Leandro Barile Agati ◽

...

Keyword(s):

Molecular Weight ◽

High Performance ◽

Thrombin Generation ◽

Area Under The Curve ◽

Factor Xa ◽

In Vivo Studies ◽

In Vitro Tests ◽

Thrombin Time

Low molecular weight heparins (LMWH) represent depolymerized heparin prepared by various methods that exhibit differential, biochemical and pharmacological profiles. Enoxaparin is prepared by benzylation followed by alkaline depolymerization of porcine heparin. Upon the expiration of its patent, several biosimilar versions of enoxaparin have become available. Heparinox (Sodic enoxaparine; Cristália Produtos Químicos Farmacêuticos LTDA, Sao Paulo, Brazil) is a new biosimilar form of enoxaparin. We assessed the molecular weight and the biochemical profile of Heparinox and compared its properties to the original branded enoxaparin (Lovenox; Sanofi, Paris, France). Clotting profiles compared included activated clotting time, activated partial thromboplastin time (aPTT), and thrombin time (TT). Anti-protease assays included anti-factor Xa and anti-factor IIa activities. Thrombin generation was measured using a calibrated automated thrombogram and thrombokinetic profile included peak thrombin, lag time and area under the curve. USP potency was determined using commercially available assay kits. Molecular weight profiling was determined using high performance liquid chromatography. We determined that Heparinox and Lovenox were comparable in their molecular weight profile. Th anticoagulant profile of the branded and biosimilar version were also similar in the clot based aPTT and TT. Similarly, the anti-Xa and anti-IIa activities were comparable in the products. No differences were noted in the thrombin generation inhibitory profile of the branded and biosimilar versions of enoxaparin. Our studies suggest that Heparinox is bioequivalent to the original branded enoxaparin based upon in vitro tests however will require further in vivo studies in animal models and humans to determine their clinical bioequivalence.

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