Mikael Tranholm

Thromboelastographic Evaluation of Hemostatic Function in Dogs with Disseminated Intravascular Coagulation

BACKGROUND There is considerable variation in the coagulation profile of dogs with disseminated intravascular coagulation (DIC), making it difficult to assess overall hemostatic function. OBJECTIVES To characterize the overall hemostatic state in dogs with DIC, by use of tissue factor-activated thromboelastography (TF-TEG), and to determine whether there is an association between hemostasis and outcome. ANIMALS 50 dogs with DIC. METHODS Dogs admitted to the intensive care units, with an underlying disease known to predispose to DIC, were prospectively assessed with TF-TEG. Citrated blood samples were collected daily during hospitalization and an extended coagulation panel and TF-TEG were performed. Diagnosis of DIC was based on expert opinion. RESULTS Hemostatic dysfunction was observed on the TF-TEG profile in 33/50 of the dogs, of which 22/50 were hypercoagulable and 11/50 were hypocoagulable based on the TF-TEG G value alone. There were significant differences in k, alpha, and MA values (P < .0001) among hypo-, normo-, and hypercoagulable dogs. There was a significant difference in case fatality rate between hypo- (64%) and hypercoagulable (32%) dogs (relative risk = 2.38; P= .04). Dogs that died had significantly lower antithrombin activity (P= .03) and higher d-dimer concentration (P= .03) than survivors. CONCLUSIONS The most common overall hemostatic abnormality in dogs diagnosed with DIC was hypercoagulability, and there was significant difference in survival between hyper- and hypocoagulable dogs. The results suggest TF-TEG is valuable in the assessment of hemostatic function in dogs diagnosed with DIC.

Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide

Current management of hemophilia B entails multiple weekly infusions of factor IX (FIX) to prevent bleeding episodes. In an attempt to make a longer acting recombinant FIX (rFIX), we have explored a new releasable protraction concept using the native N-glycans in the activation peptide as sites for attachment of polyethylene glycol (PEG). Release of the activation peptide by physiologic activators converted glycoPEGylated rFIX (N9-GP) to native rFIXa and proceeded with normal kinetics for FXIa, while the K(m) for activation by FVIIa-tissue factor (TF) was increased by 2-fold. Consistent with minimal perturbation of rFIX by the attached PEG, N9-GP retained 73%-100% specific activity in plasma and whole-blood-based assays and showed efficacy comparable with rFIX in stopping acute bleeds in hemophilia B mice. In animal models N9-GP exhibited up to 2-fold increased in vivo recovery and a markedly prolonged half-life in mini-pig (76 hours) and hemophilia B dog (113 hours) compared with rFIX (16 hours). The extended circulation time of N9-GP was reflected in prolonged correction of coagulation parameters in hemophilia B dog and duration of effect in hemophilia B mice. Collectively, these results suggest that N9-GP has the potential to offer efficacious prophylactic and acute treatment of hemophilia B patients at a reduced dosing frequency.

A novel B-domain O-glycoPEGylated FVIII (N8-GP) demonstrates full efficacy and prolonged effect in hemophilic mice models

Frequent infusions of intravenous factor VIII (FVIII) are required to prevent bleeding associated with hemophilia A. To reduce the treatment burden, recombinant FVIII with a longer half-life was developed without changing the protein structure. FVIII-polyethylene glycol (PEG) conjugates were prepared using an enzymatic process coupling PEG (ranging from 10 to 80 kDa) selectively to a unique O-linked glycan in the FVIII B-domain. Binding to von Willebrand factor (VWF) was maintained for all conjugates. Upon cleavage by thrombin, the B-domain and the associated PEG were released, generating activated FVIII (FVIIIa) with the same primary structure and specific activity as native FVIIIa. In both FVIII- and VWF-deficient mice, the half-life was found to increase with the size of PEG. In vivo potency and efficacy of FVIII conjugated with a 40-kDa PEG (N8-GP) and unmodified FVIII were not different. N8-GP had a longer duration of effect in FVIII-deficient mouse models, approximately a twofold prolonged half-life in mice, rabbits, and cynomolgus monkeys; however, the prolongation was less pronounced in rats. Binding capacity of N8-GP on human monocyte-derived dendritic cells was reduced compared with unmodified FVIII, resulting in several-fold reduced cellular uptake. In conclusion, N8-GP has the potential to offer efficacious prevention and treatment of bleeds in hemophilia A at reduced dosing frequency.

IL-1β, IL-6, KC and MCP-1 are elevated in synovial fluid from haemophilic mice with experimentally induced haemarthrosis

Summary.  The hallmark of haemophilia is the joint morbidity resulting from haemarthrosis that accounts for the majority of the bleeds. The exact mechanisms underlying changes are not fully elucidated. Cytokines are speculated to be involved in the progression and in vitro studies have confirmed the presence of elevated levels of cytokines in synovial tissue and cartilage from patients with haemophilic synovitis. In this study, the presence of selected cytokines in synovial fluid from haemophilia A mice with experimentally induced haemarthroses treated with rFVIII, rFVIIa and an rFVIIa analogue were investigated. Ten cytokines previously shown to be involved in arthritic syndromes were evaluated. Interleukin (IL)‐1β, IL‐2, IL‐4, IL‐6, IL‐10, IL‐17, Tumor Necrosis Factor‐alpha (TNF‐ α), keratinocyte‐derived chemokine (KC), Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES) and monocyte chemotactic protein‐1 (MCP‐1) were included. In this article, we demonstrate, for the first time, that bleeding in knee joints of haemophilia A mice resulted in correlated increased levels of the pro‐inflammatory cytokines: IL‐1β, IL‐6, KC and the MCP‐1 in synovial fluid. These results suggest an important role of MCP‐1 in the recruitment of monocytes and furthermore that the inflamed synovium releases IL‐1β, IL‐6 and KC, which in turn might contribute to further progression of the inflammatory process.

Faster onset of effect and greater efficacy of NN1731 compared with rFVIIa, aPCC and FVIII in tail bleeding in hemophilic mice

Summary.  Background: Recombinant factor VIIa (rFVIIa, Novoseven®) is currently used to control bleeding in hemophiliacs with inhibitors. A new rFVIIa variant, NN1731, with increased activity on the surface of activated platelets, has demonstrated a more potent and faster onset of reactivity than rFVIIa in various in vitro models. The present study aimed to investigate whether this translates into greater efficacy and faster promotion of hemostasis in vivo. Method and results: In a severe tail‐bleeding model in hemophilia A mice, NN1731 demonstrated significantly greater efficacy than rFVIIa, plasma‐derived activated prothrombin complex concentrate (pd‐aPCC, FEIBA®) or FVIII (Refacto®). Assessment of the blood loss over time showed that NN1731 significantly and dose‐dependently reduced the blood loss in the first 5‐min observation period, whereas the effect of rFVIIa, FVIII and pd‐aPCC first became evident 5–10 min after injury. Conclusion: 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.

Recombinant factor VIIa reduces bleeding in severely thrombocytopenic rabbits.

Severe thrombocytopenia is a common complication to intensive chemotherapeutic regimens. For bleeding episodes associated with severe thrombocytopenia, the current standard treatment is platelet transfusion. However, due to several transfusion complications such as transfusion-transmitted diseases, platelet refractoriness and immunomodulation, as well as increasing problems with sufficient supply of platelet products, it is imperative to search for alternatives to platelet transfusion. To test the efficacy of recombinant activated human coagulation factor VII (rFVIIa, NovoSeven) in thrombocytopenia, a preclinical study was conducted in thrombocytopenic rabbits. Thrombocytopenia was induced by a combination of gamma-irradiation and the use of platelet antibodies, and the effect of rFVIIa on nail cuticle bleeding was determined. Administration of rFVIIa at 2 mg/kg significantly shortened the prolonged bleeding time in thrombocytopenic animals (rFVIIa vs. control, median 23 min 41 s vs. 60 min, p=0.016) as well as significantly reducing the blood loss (rFVIIa vs. control, median: 8.8 vs. 12.2 nmol hemoglobin/ml, p=0.016). This effect was also reflected by a significant reduction of the prothrombin time, activated partial thromboplastin time, as well as improvement in clotting parameters in an in vitro thromboelastography thrombocytopenia model. Histopathological evaluation of kidney biopsies for the presence of micro thrombi did not reveal evidence of prothrombotic effects of rFVIIa in this model. These data demonstrate the haemostatic efficacy of rFVIIa in a rabbit model of severe thrombocytopenia. Clinical trials will be needed to further explore the potential of NovoSeven as a haemostatic agent in thrombocytopenic patients.

Development of a model based scoring system for diagnosis of canine disseminated intravascular coagulation with independent assessment of sensitivity and specificity

A template for a scoring system for disseminated intravascular coagulation (DIC) in humans has been proposed by the International Society on Thrombosis and Haemostasis (ISTH). The objective of this study was to develop and validate a similar objective scoring system based on generally available coagulation tests for the diagnosis of DIC in dogs. To develop the scoring system, 100 dogs consecutively admitted to an intensive care unit (ICU) with diseases predisposing for DIC were enrolled prospectively (group A). The validation involved 50 dogs consecutively diagnosed with diseases predisposing for DIC, admitted to a different ICU (group B). Citrated blood samples were collected daily during hospitalisation and diagnosis of DIC was based on the expert evaluation of an extended coagulation panel. A multiple logistic regression model was developed in group A for DIC diagnosis. The integrity and diagnostic accuracy of the model was subsequently evaluated in a separate prospective study at a different ICU (group B) and was carried out according to The Standards for Reporting of Diagnostic Accuracy (STARD) criteria. Thirty-seven dogs were excluded from group A and four from group B due to missing data. Based on expert opinion, 23/63 dogs (37%) had DIC. The final multiple logistic regression model was based on activated partial thromboplastin time, prothrombin time, D-Dimer and fibrinogen. The model had a diagnostic sensitivity and specificity of 90.9% and 90.0%, respectively. The diagnostic accuracy of the model was sustained by prospective evaluation in group B (sensitivity 83.3%, specificity 77.3%). Based on commonly used, plasma-based coagulation assays, it was possible to design an objective diagnostic scoring system for canine DIC with a high sensitivity and specificity.

rFVIIa and a new enhanced rFVIIa-analogue, NN1731, reduce bleeding in clopidogrel-treated and in thrombocytopenic rats

Summary.  Background: The pharmacological effect of rFVIIa occurs at the surface of activated platelets by enhancing thrombin generation at the site of vascular damage. It is therefore important to study the effects of rFVIIa in platelet‐related bleeding situations. We examined the effect of rFVIIa and an rFVIIa‐analogue, NN1731, on clopidogrel‐induced and thrombocytopenic bleeding in rats. Methods and results: Clopidogrel [10 mg kg−1; per oral (p.o.)] severely inhibited platelet aggregation and increased blood loss after tail‐transection four hours after administration. Treatment with rFVIIa (5, 10, 20 mg kg−1) or NN1731 (1, 5, 10 mg  kg−1), administered five minutes after induction of bleeding, reduced blood loss significantly and dose‐dependently. NN1731 had an increased hemostatic potential compared with rFVIIa, reducing blood loss to the control level, whereas this was not even achieved with the highest dose of rFVIIa. Antibody‐induced thrombocytopenia reduced platelet numbers by more than 90% and increased the blood loss after tail‐transection. Treatment with 10 and 20 mg kg−1 rFVIIa significantly reduced blood loss, whereas 10 mg kg−1 NN1731 reduced the bleeding to control levels. Conclusions: The hemostatic effect of rFVIIa and NN1731 was demonstrated in thrombocytopenic and clopidogrel‐treated rats, showing efficacy in situations with decreased platelet number or functionality. Our findings are consistent with the hypothesis that rFVIIa/NN1731 contribute to hemostasis by thrombin generation even in situations with platelet disorders. Furthermore, NN1731 demonstrated a higher hemostatic potential than rFVIIa.

A sensitive venous bleeding model in haemophilia A mice: effects of two recombinant FVIII products (N8 and Advate(®)).

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.

Pharmacokinetics and pharmacodynamics of a new recombinant FVIII (N8) in haemophilia A mice

Summary.  N8 is a new recombinant factor VIII (rFVIII) compound produced and formulated without human‐ or animal‐derived protein. The aims of the present studies were to evaluate the pharmacokinetics and pharmacodynamics properties of N8 and to compare with a commercially available rFVIII product (Advate®) in haemophilia A mice. The pharmacokinetics were evaluated after single i.v. administration of 80, 120 and 280 IU kg−1 of N8 and Advate® and measurements of FVIII blood concentrations as a function of time. The efficacy and dose response curves of N8 and Advate® (1–200 IU kg−1) were evaluated in a tail bleeding model. Furthermore, the effects in a newly developed haemophilia knee joint haemarthrosis model were investigated. No significant differences were found in the pharmacokinetic parameters between N8 and Advate®. The clearances were 11 ± 1 vs. 10 ± 2 mL h−1 kg−1 (P = 0.14) and the half‐lives 7.2 ± 0.9 vs. 7.7 ± 1.4 h (P = 0.31) after administration of N8 and Advate® respectively. Dose‐independent pharmacokinetics was shown, and comparable efficacy and potency were shown between N8 and Advate® in the tail bleeding model. Both compounds normalized the bleeding at the dose of 200 IU kg−1, and for blood loss ED50 values of 27 IU kg−1 (N8) and 28 IU/kg (Advate®) were found (P = 0.97). In the haemarthrosis model, treatment with N8 and Advate® at 200 IU kg−1 reduced the mean increase in the joint diameter significantly from 1.23 ± 0.19 to 0.32 ± 0.08 mm (P < 0.01) and 0.25 ± 0.08 mm (P < 0.001) respectively. Pharmacokinetics and pharmacodynamics of N8 and Advate® were comparable after i.v. administration to haemophilia A mice.