Introduction: There is no established laboratory method that can predict the most optimal dose of bypassing agents for treatment of haemophiliaA. The objectives of the study was to develop an assay that can a) differentiate between the haemostatic capacity in blood from healthy individuals and severe and moderate haemophilia patients; b) show a dose-response correlation to rFVIIa addition; and c) show dose response differences of rFVIIa addition to plasma samples from non-inhibitor patients of different severity.Materials and Methods: Citrated whole blood from 25 haemophilia A patients was used in four thrombelastography (TEG) assays initiated with: 1) kaolin, 2) Tissue Factor (TF, Innovin 1: 42,500), 3) TF 1: 42,500 + 1.2nM tPA (tissue plasminogen activator) or 4) TF 1: 200,000. rFVIIa was added to give a final concentration in the range of 0.02-4.8 mu g/ml.Results: The TEG assays showed large differences in clot formation demonstrated by prolonged clotting time (R-time), decreased maximum thrombus generation (MTG) between severe and moderate haemophilia A patients and between haemophilia patients and healthy males. The maximal amplitudes (MA) of the clot and resistance against fibrinolysis were only compromised when TF with tPA was added.Conclusion: In vitro addition of rFVIIa improved all TEG profiles significantly in a dose-dependent manner; but only the TEG assay containing kaolin could differentiate between the rFVIIa doses, showing that blood from severe patients need higher doses of rFVIIa to normalize the clot formation pro. le compared to blood from moderate patients. Kaolin seems to be the most useful TEG assay for monitoring rFVIIa treatment. (C) 2010 Elsevier Ltd. All rights reserved.

Background: Colloid plasma expanders are used to maintain blood pressure and ensure tissue perfusion during hypovolemia, e.g., caused by traumatic bleeding. Although colloids stabilize the cardiovascular system, they can also potentially cause coagulopathy. Consequently, bleeding tendency may increase, as well as the associated risk of morbidity and mortality. Thus, there is a need for hemostatic treatment options for these patients. rFVIIa (NovoSeven, Novo Nordisk A/S, Bagsvaerd, Denmark) is a hemostatic agent that effectively controls bleedings in patients with inhibitor-complicated hemophilia. rFVIIa works by enhancing thrombin generation on the activated platelet surface at the site of injury, leading to the formation of a stable fibrin clot. NN1731 is an rFVIIa analog with increased hemostatic potential and is currently under clinical development. Methods: In this study, the effect of rFVIIa and NN1731 on cuticle bleeding in rabbits 50% hemodiluted with hydroxyethyl starch (molecular weight ∼ 200,000) was tested. Cuticle bleeding was induced after a two-stage hemodilution procedure. After 5 minutes, the animals were treated with rFVIIa (2, 5, or 10 mg/kg), NN1731 (1 or 2 mg/kg), or vehicle, followed by 30 minutes of observation. Results: Hemodilution caused a significant increase in bleeding time and blood loss. rFVIIa dose-dependently reduced bleeding time and blood loss, reaching statistical significance at 10 mg/kg. However, 2 mg/kg NN1731 reduced bleeding time and blood loss significantly and to a similar extent as 10 mg/kg rFVIIa. This increased hemostatic potential of NN1731 compared with rFVIIa and was confirmed by findings using thromboelastography on ex vivo hemodiluted whole blood. Conclusion: In summary, rFVIIa and NN1731 significantly and dose-dependently reduced bleeding in extensively hemodiluted rabbits.

The Thrombelastograph (TEG; Haemoscope Corp.) analyzes clot formation in whole blood (WB) and treatment based on this analysis has been shown to reduce transfusion requirements in liver and cardiac surgery when compared to conventional coagulation analysis. Implementing TEG as a routine laboratory-based analysis, however, requires validation of the activators employed and the effect of storage of the WB sample in citrate before analysis.The effect of kaolin, tissue factor (TF) 1:17,000, or TF 1:42,500 on TEG clotting time (R), Angle (velocity of clot formation), and maximum clot strength (amplitude [MA]) were evaluated, together with day-to-day variation, the coefficient of variance (CV%), and the effect of citrate storage time.Clot formation variables were equally affected by TF 1:17,000 and kaolin activation, whereas R was significantly longer when TF 1:42,500 was used. The CV for the different variables varied from 3 to 13 percent with no significant differences between assays. Storage of citrated WB significantly affected the TEG variables in a hypercoagulable direction. Only the R, however, was significantly affected (12%) when samples rested for 0 and 30 minutes were evaluated with kaolin as the activator.The TEG assays evaluated were reproducible and present with an acceptable CV% for routine clinical practice. Kaolin and TF 1:17,000 equally affected the clot formation variables. Storage of WB for up to 30 minutes in citrate did not, except for R, affect clot formation variables when kaolin was used as activator allowing for immediate analysis when the sample arrives in the laboratory.

The haemostatic effect of recombinant activated factor VII (rFVIIa;NovoSeven) in thrombocytopenic patients has been a matter of controversy. Haemostasis by rFVIIa occurs via FVIIa-mediated thrombin generation in a platelet-dependent manner and may therefore be suboptimal in patients without functional platelets. Under such conditions, a clot-stabilizing agent, such as factor XIII (FXIII), may supplement the effect ofrFVIIa and improve haemostasis. Recombinant factor XIII (rFXIII-A2) is produced as an A2 homodimer of the FXIII A subunit and is equivalent to cellular FXIII normally found in platelets. The combined effects of rFVIIa andrFXIII-A2 were evaluated in clot lysis assays using factor XIII-deficient plasma and by whole blood thrombelastography (TEG) analysis from normal donors and thrombocytopenic stem cell transplantation patients. Clotting time was shortened by rFVIIa (0.6-10 microg/ml). rFVIIa only modestly improved anti-fibrinolysis,whereas rFXIII-A2 (0-20 microg/ml) enhanced anti-fibrinolysis without effect on clotting time. TEG analysis showed rFVIIa shortened the clotting time, and enhanced clot development, maximal mechanical strength and resistance to fibrinolysis, whereas, rFXIII-A2 enhanced clot development,maximal mechanical strength and markedly enhanced resistance to fibrinolysis. These data illustrate that rFVIIa and rFXIII-A2 contribute to clot formation and stability by different mechanisms suggesting enhanced haemostatic efficacy by combining these agents.

Predicting the clinical effect of bypassing agents such as recombinant activated factor VII in haemophilia patients with inhibitors is hampered by the limited availability of reliable laboratory monitoring tools. This multicentre, open-label trial aimed to explore the dose–response relationship between recombinant activated factor VII concentration and thromboelastography parameters in blood samples from patients with haemophilia A or B with inhibitors in a nonbleeding state. Citrated whole blood samples from 16 patients (≥16 years) with haemophilia A or B were spiked ex vivo with recombinant activated factor VII (1.2, 1.6, 2.0, 2.6, 3.0, 3.5 μg/ml), corresponding approximately to doses of 90–270 μg/kg. Samples were analysed by Thromboelastograph or Rotation Thromboelastography (three United States and three European centres, respectively) within 30 min (final lipidated recombinant tissue factor 1: 17 000; final CaCl2 15 mM). Thromboelastograph/Rotation Thromboelastography parameters showed large intersubject variation in the baseline profiles. There was a clear effect when recombinant activated factor VII was added; however, a clear concentration–response relationship was only detected for one patient. This is likely due to the fact that the curves were not sufficiently abnormal that led to reduced assay sensitivity. Our preliminary results suggest that thromboelastography may potentially be a clinically useful tool for monitoring changing concentrations of recombinant activated factor VII in haemophilia patients, but only when the baseline curve is significantly abnormal. Thus, test conditions may need to be optimized before Thromboelastograph/Rotation Thromboelastography can be utilized for all inhibitor patients.

Introduction: Acidosis is associated with high mortality in trauma patients. Therefore there is a major interest in generating acidosis models in vitro and in vivo to determine the effect of acidosis on coagulation and to develop treatments. The aim of this study was to examine the effect of acidosis induction in human whole blood using HCl versus Hepes and to analyze the subsequent effect of rFVIIa (NovoSeven®). Materials and Methods: Native human whole blood was obtained from healthy volunteers (n=6) and pH was adjusted to 6.8 using 1 M HCl or 1 M Hepes (pH 6.8). Coagulation was triggered with kaolin or tissue factor (TF, Innovin, final dilution 1:42500) and measured by thrombelastography (TEG, Haemoscope®). Furthermore, the effect of rFVIIa (25nM ∼ 90 mcg/kg) was measured. The TEG parameters R (sec), angle (deg) and maximum amplitude (MA, mm) were recorded and presented as mean±SD. A shorter R and greater angle and MA values are indicative of a more robust clot formation. Statistical analysis was performed by a two-way ANOVA-model. Platelet function was analyzed by platelet aggregation using Multiplate (Dynabyte Medical). Exposure of P-selectin, negatively charged phospholipids (annexin A5 binding) and induction of the active conformation of the fibrinogen receptor GPIIb/IIIa (PAC-1 binding) on platelets after TRAP-stimulation of whole blood was analyzed using a FACS Canto flow cytometer (BD). Results: TEG, platelet aggregation and flow cytometry indicated that lowering the pH to 6.8 by HCl affected the blood significantly different than when pH was lowered by addition of Hepes. HCl-treated blood triggered with either kaolin or TF showed a significantly decreased R value (378±45 or 661±130 vs 539±98 or 888±353 in untreated controls), significantly decreased MA (52±6 or 51±9 vs 66±8 or 62±13) and decreased angle (50±7 or 36±10 vs control 57±10 or 44±19, not significant). Hepes-treated blood triggered with kaolin showed no difference in R (458±52), angle (64±4) and MA (58±9) compared to untreated controls, whereas blood triggered with TF showed significantly shortened R-value (461±91) and enhanced angle (63±5) compared to untreated controls. Hepes treatment had no effect on MA (64±12). rFVIIa significantly shortened R irrespective of the acidosis inducer or clot trigger(HCl/kaolin 283±34, HCl/TF 307±52; Hepes/kaolin 363±32, Hepes/TF 313±46). Although the other TEG parameters were also improved, the effect was only significant when blood was treated with HCl and clotting initiated with TF (angle 48±11, MA 56±10). HCl-induced acidosis abolished platelet aggregation, whereas Hepes-induced acidosis did not alter platelet aggregation compared to normal blood. Flow cytometry showed that platelets from HCl-treated blood were pre-activated as evidence by expression of P-selectin on 70% of the platelets, annexin A5 binding to 14% of the platelets and PAC-1 binding to 62% of the platelets before stimulation. TRAP-stimulation increased P-selectin expression, and PAC1 and Annexin A5 binding to platelets in HCl-treated blood. In contrast, Hepes-treatment did not pre-activate the platelets and the increase in P-selectin expression, and annexin A5 and PAC-1 binding after TRAP-stimulation was as seen for control blood. Conclusion: The method used to lower pH in human blood strongly influences the functionality of the platelets and coagulation factors independent of the final pH. It is therefore important in experimental in vitro and in vivo models to be aware of these dramatically different effects in order to draw correct conclusions.

Introduction: The objective of this pilot study was to evaluate and compare the pharmacokinetic and pharmacodynamic (PK/PD) profile of rFVIIa and NN1731 in two hemophilia A dogs. In addition, it was the aim to evaluate the use of TEG for monitoring rFVIIa/NN1731 activity after in vivo administration and to compare with ex vivo spiking data from a previous study. NN1731 is a new rFVIIa analoge with enhanced activity (Allen et al. Arterioscler. Thromb. Vasc. Biol. 2007; 27:683–689). In hemophilia patients as well as hemophilia dogs the clot formation is impaired and reflected in coagulation assays such as thromboelastography (TEG) and APTT. The choice of hemophilia dogs is based upon the knowledge that the pharmacokinetics of human coagulations factors (FVIII, FIX and rFVIIa) as well as the effective dose is similar to that in humans. In normal dogs, it is not possible to evaluate the effect of these procoagulant proteins in coagulation assays as no impaired clotting is observed.

Benzo[b]thiophene-2-carboxamides and benzo[b]furan-2-carboxamides have been found to be antagonists on the human histamine-3-receptor, showing a Ki value of as low as 4 nM.

Abciximab inhibits platelet aggregation by binding to GPIIb/IIIa. Thus, Abciximab is effective in prevention and treatment of thromboembolic events. Although major bleeding complications are rare, Abciximab has the potential to increase bleeding which often is managed by temporary discontinuation of administration of drug. However, an effective and instant haemostatic intervention may be needed in some cases of acute serious bleeding. The aim of this study was to investigate 1) the effect of in vitro addition of Abciximab to whole blood (WB) from 8 healthy donors, 2) to determine the haemostatic potential of ex vivo addition of 10, 25 (25nM≈90ug/kg), and 100nM rFVIIa (NovoSeven®, Novo Nordisk) to WB treated with 4μg/ml of Abciximab (ReoPro®, Lilly) and 3) to compare the response of rFVIIa in TEG assays containing tissue factor (TF, Innovin 1:42500) or Kaolin (standard initiator provided by Haemsoscope) with or without tPA (0.75nM). TEG Platelet Mapping was performed on all Abciximab-treated blood samples before conducting TEG demonstrating 70–100% inhibition of ADP-induced platelet aggregation. The following TEG parameters were used: Reaction time (R-time), Maximum Thrombin generation (MTG), Maximum Amplitude (MA) and Area under the TEG fibrinolysis curve calculated from MA (AUC lysis). The statistical analysis was performed by a two-way ANOVA model. A significant effect of adding rFVIIa (10 nM) to Abciximab-treated blood was observed for all clot formation parameters and with both TF and Kaolin as initiator. rFVIIa demonstrated a significant dose-dependent improvement of the TEG parameters in the assay containing kaolin, in kaolin+tPA (except MA) and in TF (except for MTG). rFVIIa showed significant dose-dependent protection against clot lysis (AUC lysis) in the assay containing kaolin+tPA, whereas no significant effect on clot lysis was observed in the assay containing TF. In conclusion, addition of rFVIIa to WB treated with Abciximab significantly improves most of the TEG parameters. Comparison of Abciximab+rFVIIa TEG parameters with TEG parameters obtained in normal WB (n=59) showed normalization of the R-value, however, none of the other TEG parameters reached normal values although a significant effect was observed after addition of rFVIIa. The TEG assay using kaolin as initiator showed significant dose-dependent effect of rFVIIa on all TEG parameters. In contrast, in the TEG assay initiated with TF the dose-dependent response of rFVIIa was only significant for some of the parameters. This may be due to a strong initiating effect of TF in the assay thus lowering the sensibility to rFVIIa. In contrast, in the assay initiated with Kaolin only TF-independent effects of rFVIIa are measured. The design of a TEG assay therefore seems to be important for evaluating the in vitro effect of rFVIIa. The clinical relevance of these findings is currently unknown and needs to be evaluated further.

We recently reported the birth of a tammar wallaby produced by intrauterine AI [Paris DBBP et al., Australian Mammal Society Conference, July 2002]. The use of AI has great potential for the conservation of rare and endangered marsupials, but a non-surgical method of insemination is needed if such a technique is to be widely adopted. In this study we compared the anatomy of the reproductive tract of two monovular marsupials: the tammar wallaby (Macropus eugenii, family Macropodidae) and southern hairy-nosed wombat (Lasiorhinus latifrons, family Vombatidae), in relation to the ability to deposit semen non-surgically by catheter. These species are ideal models for endangered marsupials such as the brush-tailed rock wallaby (Petrogale penicillata ), long-nosed potoroo (Potorous tridactylus) and critically endangered northern hairy-nosed wombat (Lasiorhi-nus krefftii). As in all marsupials studied, tammars and wombats have two completely separate uteri each opening into the anterior vaginal culs desac (AVC) through two distinct cervices. The vaginal canals, unique and highly variable in marsupials, consist of two lateral vaginae (LV) and a third median vagina (MV) connecting the AVC to the urogenital sinus and opening. Adult female reproductive tracts were examined from 13 tammars and 5 wombats. A silicon balloon HSG catheter (5 French 30 cm; Cook, Australia) was introduced at the urogenital opening and navigated through the MV to the AVC, proximal to the cervices. The balloon was inflated to seal off the posterior end of the MV. Two milliliter (tammar) or 1 ml (wombat) of 0.1% Coomassie brilliant blue (Sigma, Australia) was injected into the AVC to stain and simulate the site of insemination. The balloon was defeated, catheter retracted and the extent of dye distribution in the tracts noted. In the tammar, as in all macropodids, the MV is a single open canal, allowing both cervices to draw semen. However, a septum divides the MV in wombats and interestingly, the MV was partially fused at the caudal end. In the tammar, Coomassie blue penetrated the MV, the AVC and cervices. These lie a distance of 7:6 +/-0:3cm from the urogenital opening (see table). In the wombat, the MV was fused caudally and so was difficult to penetrate. The cervices lay 13:3 +/- 0:3 cm from the urogenital opening. Coomassie blue was localized to only one side of the MVand AVC, confirming that the medium septum effectively separates the cervices. These results have important implications for AI using non-surgical catheters for semen deposition. In the tammar wallaby and potentially other macropodids, cervical insemination via the MV is feasible, requiring no ultrasoni cguidan ce. However in the southern hairy-nosed wombat and other vombatids, cervical insemination is unlikely to be successful due to the fused MV. Presence of the septum would require the side of ovulation and thus catheter delivery to be determined by ultrasound.