AbstractHemophilia is treated by IV replacement therapy with Factor VIII (FVIII) or Factor IX (FIX), either on demand to resolve bleeding, or as prophylaxis. Improved treatment may be provided by drugs designed for subcutaneous and less frequent administration with a reduced risk of inhibitor formation. Tissue factor pathway inhibitor (TFPI) down-regulates the initiation of coagulation by inhibition of Factor VIIa (FVIIa)/tissue factor/Factor Xa (FVIIa/TF/FXa). Blockage of TFPI inhibition may facilitate thrombin generation in a hemophilic setting. A high-affinity (KD = 25pM) mAb, mAb 2021, against TFPI was investigated. Binding of mAb 2021 to TFPI effectively prevented inhibition of FVIIa/TF/FXa and improved clot formation in hemophilia blood and plasma. The binding epitope on the Kunitz-type protease inhibitor domain 2 of TFPI was mapped by crystallography, and showed an extensive overlap with the FXa contact region highlighting a structural basis for its mechanism of action. In a rabbit hemophilia model, an intravenous or subcutaneous dose significantly reduced cuticle bleeding. mAb 2021 showed an effect comparable with that of rFVIIa. Cuticle bleeding in the model was reduced for at least 7 days by a single intravenous dose of mAb 2021. This study suggests that neutralization of TFPI by mAb 2021 may constitute a novel treatment option in hemophilia.

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Summary.  Haemostatic effect of compounds for treating haemophilia can be evaluated in various bleeding models in haemophilic mice. However, the doses of factor VIII (FVIII) for normalizing bleeding used in some of these models are reported to be relatively high. The aim of this study was to establish a sensitive venous bleeding model in FVIII knock out (F8‐KO) mice, with the ability to detect effect on bleeding at low plasma FVIII concentrations. We studied the effect of two recombinant FVIII products, N8 and Advate®, after injury to the saphenous vein. We found that F8‐KO mice treated with increasing doses of either N8 or Advate® showed a dose‐dependent increase in the number of clot formations and a reduction in both average and maximum bleeding time, as well as in average blood loss. For both compounds, significant effect was found at doses as low as 5 IU kg−1 when compared with vehicle‐treated F8‐KO mice. Normalization of maximum bleeding time was found at doses equal to or above 10 IU kg−1 N8 or Advate®, corresponding to plasma concentrations of approximately 10% of the level in wild type mice. The present study adds a new model to the armamentarium of bleeding models used for evaluation of pro‐coagulant compounds for treatment of haemophilia. Interestingly, the vena saphena model proved to be sensitive towards FVIII in plasma levels that approach the levels preventing bleeding in haemophilia patients, and may, thus, in particular be valuable for testing of new long‐acting variants of e.g. FVIII that are intended for prophylaxis.

This study shows that NN1731 has a greater efficacy and faster resolution of bleeding in a severe bleeding model in hemophilia A mice compared with any of the other agents tested.

Abstract:The surgical stress response is the neurophysiologic reflex response to surgery, which involves activation of the hypothalamic-pituitary-adrenal axis and is regulated by the hypothalamic paraventricular nucleus. The effect of preoperative use of local anaesthetics on activation of neurones in the paraventricular nucleus during surgery was studied by quantification of the neuronal expression of the c-fos-gene after a standardized plantar incision in rats. Furthermore, cfos expression in the spinal dorsal horn was used as a measure of spinal nociception. Six halothane-anaesthetized animals underwent surgery following infiltration with lidocaine and bupivacaine, six animals were operated without local anaesthetics, and six control animals were subjected to the anaesthetic procedures. After two hours, the animals were perfused with 4% formaldehyde and the spinal cords and brains were collected and processed by immunohistochemistry for stereological quantification of the number of neurones with Fos-like immunoreactivity. Furthermore, brain and spinal cord were sampled from nine control animals right after induction of halothane anaesthesia. Surgery without local anaesthetics caused a significant increased number of neurones with Fos-like immunoreactivity in the spinal cord (4258∫1710; mean∫S.D.; PϽ0.01) compared to the anaesthesia control group (1204∫436). Local anaesthetics reduced this number to 2029∫919 (PϽ0.05), which was not significantly different from the anaesthesia control group. After surgery, the number of neurones with Fos-like immunoreactivity in paraventricular nucleus increased from 2948∫1365 in the anaesthetized control group to 5550∫3875 and 5191∫1558 in the surgery and local anaesthetics plus surgery group, respectively, although significance was only reached for the group receiving local anaesthetics (PϽ0.05). In conclusion, preoperative local anaesthetic infiltration did not reduce the surgery-induced c-fos expression in paraventricular nucleus after paw surgery in rats, although spinal nociception was reduced.Post-operative complications, leading to increased morbidity and mortality, are common problems after surgery (Holte & Kehlet 2002). Some of these complications can be ascribed to the surgical stress response (Kehlet 1988), which is defined as the neurophysiologic reflex response to surgery involving activation of the hypothalamic-pituitary-adrenal axis (Wilmore et al. 1976;Herman & Cullinan 1997), implying extensive hormonal and metabolic changes (Kehlet 1984;Chrousos 1998;Desborough 2000). Thus, activated neurones in the hypothalamic paraventricular nucleus synthesize and release corticotrophin-releasing factor, which via secretion of adrenocorticotrophic hormone (ACTH) from the pituitary gland initiates release of cortisol (corticosterone in rats) from the adrenal cortex. Activation of neurones in the paraventricular nucleus is associated with expression of the immediate-early gene c-fos, which consequently has been used as a marker of stress . Similarly, c-fos expression in neur...

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SummaryAfter a search on Medline, it appears that intraperitoneal injection of sodium pentobarbitone is often used for anaesthesia and euthanasia of rodents. In the present pilot study in rats, spinal nociception after intraperitoneal injection of sodium pentobarbitone, with and without lidocaine, was examined by estimation of the number of c-fos-expressing neurones in the spinal dorsal horn. One group of rats received an intraperitoneal injection of 0.4 mL/kg sodium pentobarbitone (100 mg/mL; n ¼ 4). Another group of rats received a similar intraperitoneal injection of sodium pentobarbitone formulated with lidocaine 10 mg/mL (n ¼ 4); a control group received a similar intraperitoneal injection of 0.9% saline (n ¼ 4). After 3 h, the animals were re-anaesthetized and perfused with 4% formaldehyde, and the spinal cord was collected and processed by immunohistochemistry for stereological quantification of the number of neurones with c-fos-like immunoreactivity (FLI). Intraperitoneal injection of the sodium pentobarbitone formulation caused a significantly increased number of neurones with FLI in the spinal cord (39307247; mean7SEM; Po0.001) compared with the saline control group (7657131). The lidocaine added to the sodium pentobarbitone formulation significantly reduced the number to 27167393 (Po0.05). In conclusion, intraperitoneal injection of sodium pentobarbitone caused a significant increase in nociception which was lowered by adding lidocaine to the formulation, although it was still significantly higher than the control level. Further studies are needed with the aim of optimizing the lidocaine concentration and also to examine the effect of the combination of lidocaine with a long-acting local anaesthetic agent, e.g. bupivacaine.

To cite this article: Lauritzen B, Hedner U, Johansen PB, Tranholm M, Ezban M. Recombinant human factor VIIa and a factor VIIa-analogue reduces heparin and low molecular weight heparin (LMWH)-induced bleeding in rats. J Thromb Haemost 2008; 6: 804-11.Summary. Background: Heparin and low molecular weight heparin (LMWH) are widely used for prevention and treatment of thromboemobolic events, but may occasionally cause uncontrollable bleeding. Heparin can readily be antagonized with protamine, but this is less effective against LMWH. Objectives: To test the effects of rFVIIa or an analogue of rFVIIa, NN1731, on heparin-and LMWH-induced bleeding in rats. Methods: Initially the doses of heparin and tinzaparin (a LMWH) were determined by dose-titration. Following pretreatment with heparin or tinzaparin in rats, tail-transection was performed, and the effect of rFVIIa and NN1731 on the bleeding was observed. Results: rFVIIa (5, 10 and 20 mg kg tinzaparin, 10 mg kg )1 NN1731 reduced and normalized the bleeding, while the effect of 20 mg kg )1 rFVIIa failed to reach statistical significance. These data are consistent with the hypothesis that rFVIIa/NN1731 are capable of generating sufficient thrombin locally on the surface of activated platelets to induce hemostasis in the presence of heparin/LMWH. Conclusions: This study suggests that rFVIIa and NN1731 may have the potential to control bleedings caused by heparin or LMWH.