WHOLE BLOOD THROMBOELASTOGRAPHY IN HEALTHY ADULT CAMELIDS (VICUGNA PACOS AND CAMELUS DROMEDARIUS).

Thromboelastography (TEG) provides a global assessment of hemostasis and fibrinolysis and has broad applications to identify and monitor coagulation dysfunction in veterinary patients. Although alpacas are susceptible to a wide variety of coagulopathies, the assessment of TEG has not been reported in clinically healthy alpacas to date. The purpose of this study was to evaluate the analytical performance of recombinant human tissue factor (rhTF)- and kaolin-activated TEG and to establish reference intervals for TEG parameters (reaction [R] and clotting [K] times, angle [α], maximum amplitude [MA], and shear elastic modulus [G]) in healthy, adult alpacas. Kaolin and rhTF-activated TEG were performed using citrated whole blood samples from 20 clinically healthy, nonpregnant, adult Huacaya alpacas each after 30 min of sample storage at room temperature. Six individuals of a related species, dromedary camels, were also sampled for comparative purposes. All data were presented descriptively, assessed for normality, and compared using either independent-sample t tests or Mann-Whitney U tests, with P ≤ 0.05 considered significant. Reference intervals were calculated using a robust method and Box-Cox-transformed data. Mean TEG values (reference intervals) were determined for rhTF-activated TEG as follows: R 6.99 min (3.41-12.71), K 3.43 min (1.61-6.42), α 48.51° (27.21-67.38), MA 52.05 mm (21.53-65.92), and G 5.71 kdyn/cm2 (1.87-9.60), while mean values (reference intervals) for kaolin-activated TEG included R 7.72 min (4.48-11.43), K 4.24 min (2.03-9.20), α 45.06° (23.66-64.20), MA 52.18 mm (33.49-66.63), and G 5.78 kdyn/cm2 (NR-9.66). None of the measured TEG values differed significantly between activators, suggesting that activator choice may have a limited effect on TEG parameters in healthy alpacas. TEG results in alpacas were comparable to those of dromedary camels. These results will thus provide a useful starting point in the evaluation of hemostasis in adult camelids.

Rotational thromboelastometry can predict the probability of bleeding events in a translational rat model of haemophilia A following gene-based FVIIa prophylaxis.

INTRODUCTION: Monitoring of clinical effectiveness of bypassing agents in haemophilia patients is hampered by the lack of validated laboratory assays. Thromboelastography (TEG) and rotational thromboelastometry (ROTEM) have been evaluated for predicting clinical effectiveness of bypassing agents, however, with limited success. AIM: Application of a longitudinal model-based approach may allow for a quantitative characterization of the link between ROTEM parameters and the probability of bleeding events. METHODS: We analyse longitudinal data from haemophilia A rats receiving gene-based FVIIa prophylaxis in terms of total circulatory levels of FVII/FVIIa, clotting time (CT) measured using ROTEM and the probability of bleeding events. RESULTS: Using population pharmacokinetic-pharmacodynamic (PKPD) modelling, a PK-CT-repeated time-to-event (RTTE) model was developed composed of three submodels (a) a FVII/FVIIa PK model, (b) a PK-CT model describing the relationship between predicted FVIIa expression and CT and (c) a RTTE model describing the probability of bleeding events as a function of CT. The developed PK-CT-RTTE model accurately described the vector dose-dependent plasma concentration-time profile of total FVII/FVIIa and the exposure-response relationship between AAV-derived FVIIa expression and CT. Importantly, the developed model accurately described the occurrence of bleeding events over time in a quantitative manner, revealing a linear relationship between predicted change from baseline CT and the probability of bleeding events. CONCLUSION: Using PK-CT-RTTE modelling, we demonstrated that ROTEM parameters can accurately predict the probability of bleeding events in a translational animal model of haemophilia A.

Rapid inflammation and early degeneration of bone and cartilage revealed in a time-course study of induced haemarthrosis in haemophilic rats.

OBJECTIVES: Detailed knowledge of the sequential cell and tissue responses following haemarthrosis is important for a deep understanding of the pathological process initiated upon extensive bleeding into the joint causing haemophilic arthropathy (HA). The underlying pathobiology driving haemarthrosis towards HA has been difficult to establish in detail, although animal models have shed light on some processes. Previous studies have focused on a single or a few distant time points and often only characterizing one tissue type of the joint. The objective of this study was, therefore, to carefully map early onset of synovitis and HA following induced haemarthrosis. METHODS: One hundred and thirty haemophilia A rats were subjected to induced haemarthrosis or a sham procedure in full anaesthesia and euthanized from 30 min to 7 days after the procedure. Pathological changes of the joints were visualized using micro-computed tomography, histology and immunohistochemistry. RESULTS: Synovitis developed within 24 h and was dominated by myeloid cell infiltrations. Cartilage and bone pathology were evident as early as 48-96 h after haemarthrosis, and the pathology rapidly progressed with extensive periosteal bone formation and formation of subchondral cysts. CONCLUSION: Fast, extensive and simultaneous cartilage and bone degeneration developed shortly after haemarthrosis, as shown by the detailed mapping of the early pathogenesis of HA. The almost immediate loss of cartilage and the pathological bone turnover suggest a direct influence of blood on these processes and are unlikely to be attributed simply to an indirect effect of inflammation.

Oral Tolerance Induction in Hemophilia B Dogs Fed with Transplastomic Lettuce.

Anti-drug antibodies in hemophilia patients substantially complicate treatment. Their elimination through immune tolerance induction (ITI) protocols poses enormous costs, and ITI is often ineffective for factor IX (FIX) inhibitors. Moreover, there is no prophylactic ITI protocol to prevent anti-drug antibody (ADA) formation. Using general immune suppression is problematic. To address this urgent unmet medical need, we delivered antigen bioencapsulated in plant cells to hemophilia B dogs. Commercial-scale production of CTB-FIX fusion expressed in lettuce chloroplasts was done in a hydroponic facility. CTB-FIX (∼1 mg/g) in lyophilized cells was stable with proper folding, disulfide bonds, and pentamer assembly after 30-month storage at ambient temperature. Robust suppression of immunoglobulin G (IgG)/inhibitor and IgE formation against intravenous FIX was observed in three of four hemophilia B dogs fed with lyophilized lettuce cells expressing CTB-FIX. No side effects were detected after feeding CTB-FIX-lyophilized plant cells for >300 days. Coagulation times were markedly shortened by intravenous FIX in orally tolerized treated dogs, in contrast to control dogs that formed high-titer antibodies to FIX. Commercial-scale production, stability, prolonged storage of lyophilized cells, and efficacy in tolerance induction in a large, non-rodent model of human disease offer a novel concept for oral tolerance and low-cost production and delivery of biopharmaceuticals.

Pulmonary vasculature in dogs assessed by three-dimensional fractal analysis and chemometrics.

Fractal analysis of canine pulmonary vessels could allow quantification of their space-filling properties. Aims of this prospective, analytical, cross-sectional study were to describe methods for reconstructing three dimensional pulmonary arterial vascular trees from computed tomographic pulmonary angiogram, applying fractal analyses of these vascular trees in dogs with and without diseases that are known to predispose to thromboembolism, and testing the hypothesis that diseased dogs would have a different fractal dimension than healthy dogs. A total of 34 dogs were sampled. Based on computed tomographic pulmonary angiograms findings, dogs were divided in three groups: diseased with pulmonary thromboembolism (n = 7), diseased but without pulmonary thromboembolism (n = 21), and healthy (n = 6). An observer who was aware of group status created three-dimensional pulmonary artery vascular trees for each dog using a semiautomated segmentation technique. Vascular three-dimensional reconstructions were then evaluated using fractal analysis. Fractal dimensions were analyzed, by group, using analysis of variance and principal component analysis. Fractal dimensions were significantly different among the three groups taken together (P = 0.001), but not between the diseased dogs alone (P = 0.203). The principal component analysis showed a tendency of separation between healthy control and diseased groups, but not between groups of dogs with and without pulmonary thromboembolism. Findings indicated that computed tomographic pulmonary angiogram images can be used to reconstruct three-dimensional pulmonary arterial vascular trees in dogs and that fractal analysis of these three-dimensional vascular trees is a feasible method for quantifying the spatial relationships of pulmonary arteries. These methods could be applied in further research studies on pulmonary and vascular diseases in dogs.

Thromboelastography in Selected Avian Species.

Currently available assay methods and reagents are not optimized for evaluating avian hemostasis; therefore, assessing avian coagulopathies is challenging. Recently, thromboelastography (TEG), which measures the viscoelastic properties of blood, has been used clinically in mammalian species to diagnose and characterize hemostatic disorders. To evaluate TEG in healthy individuals of 6 avian species, we modified existing mammalian TEG protocols to allow analysis of citrated, avian whole-blood samples collected from scarlet ibis (Eudocimus ruber) (n = 13), American flamingos ( Phoenicopterus ruber ) (n = 13), helmeted Guinea fowl ( Numida meleagris ) (n = 12), Amazon parrots (Amazona species) (n = 9), Humboldt penguins ( Spheniscus humboldti ) (n = 6), and domestic chickens (n = 16). Activated partial thromboplastin time, prothrombin time, and fibrinogen were measured as a means of comparison. Regardless of the mode of activation, clot formation in the species studied was markedly delayed compared with mammals. Because of prolonged reaction time (14.7-52.7 minutes) with kaolin and diluted tissue factor, undiluted human tissue factor was used in all avian samples because it provided the shortest reaction time. Species differed significantly in reaction time (P = .007), clotting rate (P < .001), rate of clot formation (α angle; P < .001), and maximum amplitude (P < .001) values, indicating that species-specific reference intervals are necessary. Based on these results, TEG with specific reference intervals could prove useful in evaluating avian hemostatic disorders.

Clinical implications of infection with a novel metastrongyloid species in the red panda (Ailurus fulgens).

In a recent survey, 30% of the European red panda (Ailurus fulgens) population was found to be infected with a newly discovered metastrongyloid nematode. In a following prospective study, four naturally infected captive-bred red pandas infected with this parasite were examined and compared with two uninfected control animals. On clinical examination, no abnormalities were detected with respect to vital parameters and cardiovascular system in all six examined animals. Similarly, few and nonspecific changes were recorded on serum biochemistry. No changes on pulmonary pattern were noted on thoracic radiographs. Vertebral heart scores were between 7.2 to 8.6, and no difference was noted between infected and control animals. Two animals had slightly prolonged clotting time and reaction time on thromboelastography but not likely to be of clinical relevance. In conclusion, infection with the newly identified metastrongyloid nematode in the red pandas seems to have little or no clinical importance.

Thromboelastography in veterinary medicine.

Thromboelastography (TEG) has been used in experimental animal studies since the early 1960s and in a routine clinical setting for the past decade. From the data currently available, it is clear that both the scope and limitations of TEG in animals resemble those observed in humans. TEG has been used to diagnose hypercoagulability in animals with disseminated intravascular coagulation, various types of cancer, and critical illness. Its ability to detect and monitor animals with various types of coagulopathies has been well established, both clinically and in experimental studies. TEG is often used in animals to monitor the effect of different pro- and anticoagulant drugs and often performs better at this task than conventional coagulation assays. TEG is already well established in veterinary medicine, and with the rapid dissemination of the technique currently taking place, we can expect to see a wide variety of interesting animal data published in the near future.

FVIII activity following FVIII protein infusion or FVIII gene transfer predicts the bleeding risk in hemophilia A rats.

BACKGROUND: Prophylactic replacement therapy in hemophilia A (HA) patients does not adequately prevent bleeds and arthropathic complications. A more refined understanding of the relationship between coagulation factor VIII (FVIII) levels and bleeding risk during protein prophylaxis, or with gene therapy, is needed to improve patient care. OBJECTIVES: Investigate this relationship in the HA rat, a model exhibiting spontaneous bleeds and development of arthropathy similar to HA patients. METHODS: Human B domain-deleted FVIII was delivered to HA rats via adeno-associated virus (AAV)-mediated gene transfer or multiple intravenous protein injections. RESULTS AND CONCLUSIONS: After 12 weeks of observation, both approaches significantly reduced bleeds per animal and increased the proportion of bleed-free animals compared with controls (43% vs 0%, respectively [AAV]; 75% vs 8%, respectively [injection]). Both approaches resulted in an anti-FVIII inhibitory response in 20% to 37% of treated animals, similar to HA patients. Inhibitory antibodies were refractory to clinical improvement (reduction of bleeds) only in the AAV-based prophylaxis. An integrated model-based analysis of data on FVIII exposure and bleeding events was performed. This predicted the bleeding risk at any given circulating FVIII activity. Specifically, 4.8 or 10 IU/dL FVIII (0.048 and 0.1 IU/mL, respectively) were predicted to reduce bleeding risk by 90% or 95%, respectively, compared with untreated controls. Our data establish the utility of the HA rat model in FVIII prophylaxis studies and describe how FVIII activity affects bleeding risk in this setting. These enable further studies on FVIII prophylaxis focusing on disease complications for an optimized treatment of HA patients.

Tissue factor activated thromboelastography correlates to clinical signs of bleeding in dogs.

The ability of a laboratory assay to correlate to clinical phenotype is crucial for the accurate diagnosis and monitoring of haemostasis and is therefore challenging with currently used routine haemostasis assays. Thromboelastography (TEG) is increasingly used to evaluate haemostasis in humans and may well be of value in the workup of dogs suspected of having a haemostatic disorder. This study was undertaken to evaluate prospectively how tissue factor (TF) activated TEG correlated to clinical signs of bleeding in dogs, compared to a routine coagulation profile. A prospective case-control study was performed over a 2 year period from 2004-2006. Eligible dogs were those where the primary clinician requested a coagulation profile to evaluate haemostasis. The dogs were simultaneously evaluated with a TF-activated TEG assay. Twenty-seven dogs, characterised as hypo-coagulable based on the TEG parameter G (<3.2 Kdyn/cm(2)), were included in the study as cases. Size matched control groups of TEG normo- (G=3.2K-7.2 Kdyn/cm(2)) and hyper-coagulable (G>7.2 Kdyn/cm(2)) dogs were selected retrospectively from the eligible dogs. For all dogs, clinical signs of bleeding were noted at time of analysis. There were statistically significant differences between all TEG values of hypo- and normo- and hyper-coagulable dogs. Thromboelastography correctly identified dogs with clinical signs of bleeding with a positive predictive value (PPV) of 89% and a negative predictive value (NPV) of 98% based on G alone. In comparison, the coagulation profile had a PPV between 50-81% and a NPV between 92-93% for detection of bleeding, depending on the observer. In conclusion, a TF-activated TEG G value<3.2K dyn/cm(2) correctly identified dogs with clinical signs of bleeding with very high PPV and NPV, irrespective of observer. The findings strongly suggest that TF- activated TEG may be of value in the workup of dogs suspected of having a haemostatic disorder.

D-Dimer Concentrations and Thromboelastography in Five Dogs With Ischemic Stroke.

Ischemic stroke is a condition increasingly recognized in dogs; however, the number of publications on dogs with ischemic stroke is still limited and hemostatic parameters are infrequently reported. D-dimer levels have been shown to be elevated in people with acute ischemic stroke compared to a healthy control population and it has been proposed that a normal D-dimer can be used to exclude thromboembolism in dogs. In this case series, we report hemostatic parameters, including D-dimer and thromboelastography (TEG) along with clinical and imaging findings for five dogs diagnosed with ischemic stroke. All dogs had a normal D-dimer concentration on presentation. A hypercoagulable state was identified in two dogs based on the results of the TEG, and was suspected in the remaining three cases based on a shortened TEG clot reaction time. Based on the findings in the present cases, a D-dimer within the normal reference range does not seem an appropriate negative predictor for canine ischemic stroke. The demonstration of a possible hypercoagulable state, as identified by the TEG, is an interesting finding which should be explored further to help reveal predisposing hypercoagulable conditions in dogs with ischemic stroke.

Gene-based FVIIa prophylaxis modulates the spontaneous bleeding phenotype of hemophilia A rats.

A sizable proportion of hemophilia inhibitor patients fails immune tolerance induction and requires bypass agents for long-term bleed management. Recombinant human-activated coagulation Factor VII (rhFVIIa) is an on-demand bypass hemostatic agent for bleeds in hemophilia inhibitor patients. Prophylactic use of rhFVIIa may enable sustained hemostatic management of inhibitor patients, but the critical relationship of rhFVIIa circulating levels and clinical outcome in that setting remains unclear. To address this in vivo, we used the rat hemophilia A (HA) model that exhibits spontaneous bleeds and allows longitudinal studies with sufficient statistical power. We simulated activated Factor VII (FVIIa) prophylaxis by adeno-associated virus (AAV) gene transfer of a rat FVIIa transgene. Compared with naive HA animals, rat FVIIa continuous expression affected the overall observed bleeds, which were resolved with on-demand administration of recombinant rat FVIIa. Specifically, although 91% of naive animals exhibited bleeds, this was reduced to 83% and 33% in animals expressing less than 708 ng/mL (<14 nM) and at least 708 ng/mL (≥14 nM) rat FVIIa, respectively. No bleeds occurred in animals expressing higher than 1250 ng/mL (>25 nM). Rat FVIIa expression of at least 708 ng/mL was also sufficient to normalize the blood loss after a tail vein injury. Continuous, AAV-mediated rat FVIIa transgene expression had no apparent adverse effects in the hemostatic system of HA rats. This work establishes for the first time a dose dependency and threshold of circulating FVIIa antigen levels for reduction or complete elimination of bleeds in a setting of FVIIa-based HA prophylaxis.

In vitro heparinization of canine whole blood with low molecular weight heparin (dalteparin) significantly and dose-dependently prolongs heparinase-modified tissue factor-activated thromboelastography parameters and prothrombinase-induced clotting time.

BACKGROUND: Low-molecular-weight heparin (LMWH) is being used increasingly in veterinary medicine for both treatment and prophylaxis of thromboembolic disease, but no predictable patient-side method exists to monitor its effect. OBJECTIVES: The aim of this study was to evaluate thromboelastography (TEG) and prothombinase-induced clotting time (PiCT) assays for detecting hemostatic alterations following in vitro heparinization of canine whole blood with dalteparin (Fragmin). METHODS: Citrated whole-blood samples were collected from 7 clinically healthy dogs. Dalteparin was added at concentrations of 0, 0.156, 0.625, 1.25, and 2.5 U/mL of whole blood. TEG was performed using heparinase cups with tissue factor (TF, 1:50,000) and kaolin as activators. Reaction time (R), clotting time (K), angle (alpha), and maximum amplitude (MA) were recorded. PiCT and anti-FXa activity were measured in plasma. RESULTS: With TF, increasing concentrations of dalteparin significantly prolonged R and K and significantly decreased alpha and MA. K, alpha, and MA ratios were significantly different from baseline at all dalteparin concentrations and R was significantly different from baseline at concentrations of 0.625, 1.25, and 2.5 U/mL. With kaolin, only R was significantly different from baseline at dalteparin concentrations of 0.625 and 2.5 U/mL. PiCT detected dalteparin concentrations < or = 0.625 U/mL, with a good linear correlation (r(2)=.96, P<.0001). CONCLUSION: These results suggest that TF-activated TEG and PiCT assays should be further evaluated as promising new methods for evaluating the effect of LMWH, using doses in the recommended clinical range and prospective clinical studies.

The influence of inflammation and hematocrit on clot strength in canine thromboelastographic hypercoagulability.

OBJECTIVE: To investigate parameters causing canine thromboelastographic hypercoagulability and to investigate whether thromboelastography (TEG) with Cytochalasin D (Cyt D) added is related to parameters of platelet activity. DESIGN: Prospective observational study on hemostatic and inflammatory parameters. Data were collected between November 2012 and July 2013. SETTING: University teaching hospital. ANIMALS: Twenty-eight dogs suffering from diseases predisposing to thrombosis and 19 clinically healthy dogs. Diseased dogs were enrolled if they fulfilled inclusion criteria regarding age, size, informed client consent, and obtained a diagnosis of a disease that has been associated with thrombosis or hypercoagulability. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Parameters of coagulation and anticoagulation, fibrinolysis, and antifibrinolysis, platelet activity, inflammation, platelet count, and hematocrit were measured using CBC, TEG, platelet aggregation on multiplate, platelet activity on flow cytometry, and hemostatic and inflammatory markers on plasma and serum analyses. ANOVA and multilinear regression analyses indicated that especially hematocrit and the inflammatory parameters C-reactive protein and interleukin-8 showed best association with overall clot strength in diseased dogs with hypercoagulable TEG tracings. Ratios presumed to reflect platelet contribution to the TEG tracing obtained in TEG analyses with Cyt D were related especially with hematocrit and P-selectin expression of platelets measured after γ-Thrombin activation on flow cytometry. CONCLUSION: Overall clot strength in TEG analyses of the hypercoagulable dogs included in the present study appears to be primarily associated with inflammation as well as hematocrit. Furthermore, the ratio between standard TEG analyses and TEG analyses with Cyt D may reflect some degree of platelet activity.

Evaluation of the TEG platelet mapping assay in blood donors.

BACKGROUND: Monitoring of antiplatelet therapy in patients at cardiovascular risk is difficult because existing platelet function tests are too sophisticated for clinical routine. The whole blood TEG Platelet Mapping assay measures clot strength as maximal amplitude (MA) and enables for quantification of platelet function, including the contribution of the adenosine diphosphate (ADP) and thromboxane A2 (TxA2) receptors to clot formation. METHODS: In 43 healthy blood donors, the analytical (CVa) and inter-individual variability (CVg) of the TEG Platelet Mapping assay were determined together with platelet receptor inhibition in response to arachidonic acid (AA) and ADP. RESULTS: The CVa of the assay for maximal platelet contribution to clot strength (MAThrombin) was 3.5%, for the fibrin contribution to clot strength (MAFibrin) 5.2%, for MAAA 4.5% and for MAADP it was 6.6%. The MAThrombin CVg was 2.8%, MAFibrin 4.7%, MAAA 6.6% and for MAADP it was 26.2%. Females had a higher MAThrombin compared to males (62.8 vs. 58.4 mm, p = 0.005). The platelet TxA2 receptor inhibition was 1.2% (range 0-10%) and lower than for the ADP receptor (18.6% (0-58%); p < 0.0001). CONCLUSION: The high variability in ADP receptor inhibition may explain both the differences in response to ADP receptor inhibitor therapy and why major bleeding sometimes develops during surgery in patients not treated with ADP receptor inhibitors. An analytical variation of ~5 % for the TEG(R) enables, however, for routine monitoring of the variability in ADP receptor inhibition and of antiplatelet therapy.

Study on biological variation of haemostatic parameters in clinically healthy dogs.

Thromboelastography (TEG) may be a valuable supplement to the coagulation assays activated partial thromboplastin time (aPTT), prothrombin time (PT), thrombin time (TT), fibrinogen, antithrombin (AT) and D-Dimer currently used in most clinical pathology laboratories. Allowable imprecision and bias reference limits for analytical tests can be calculated based on measurements of biological variation. No studies to date have examined the effect of biological variation on these haemostasis parameters in the same group of dogs. Plasma samples were collected after a set protocol once weekly for five consecutive weeks from eight healthy dogs (four males and four females) and stored at -80 degrees C until analysis. Randomized duplicate coagulation tests and TEG analyses were performed on all plasma samples within one run. The data were analyzed for outliers and subsequently subjected to nested analysis of variance to obtain the coefficient of analytical, intra-individual and inter-individual variation. From these objective analytical performance standards for imprecision, critical difference, total error and the index of individuality were calculated to assess the utility of conventional population-based reference ranges. All the clotting times (aPTT, PT and TT), fibrinogen, AT and D-Dimer showed a degree of individuality, which may make the use of population-based reference ranges alone an insensitive interpretation criterion, whereas a population-based reference interval seems to be sensitive for interpreting all TEG parameters. Analytical performance standards for imprecision were only met for one of the coagulation assays, whereas all TEG parameters except the alpha angle, alpha achieved this analytical goal.

Global measurement of coagulation in plasma from normal and haemophilia dogs using a novel modified thrombin generation test - Demonstrated in vitro and ex vivo.

INTRODUCTION: Canine models of severe haemophilia resemble their human equivalents both regarding clinical bleeding phenotype and response to treatment. Therefore pre-clinical studies in haemophilia dogs have allowed researchers to make valuable translational predictions regarding the potency and efficacy of new anti-haemophilia drugs (AHDs) in humans. To refine in vivo experiments and reduce number of animals, such translational studies are ideally preceded by in vitro prediction of compound efficacy using a plasma based global coagulation method. One such widely used method is the thrombin generation test (TGT). Unfortunately, commercially available TGTs are incapable of distinguishing between normal and haemophilia canine plasma, and therefore in vitro prediction using TGT has so far not been possible in canine plasma material. AIM: Establish a modified TGT capable of: 1) distinguishing between normal and haemophilia canine plasma, 2) monitoring correlation between canine plasma levels of coagulation factor VIII (FVIII) and IX (FIX) and thrombin generation, 3) assessing for agreement between compound activity and thrombin generation in ex vivo samples. METHODS: A modified TGT assay was established where coagulation was triggered using a commercially available activated partial thromboplastin time reagent. RESULTS: With the modified TGT a significant difference was observed in thrombin generation between normal and haemophilia canine plasma. A dose dependent thrombin generation was observed when assessing haemophilia A and B plasma spiked with dilution series of FVIII and FIX, respectively. Correlation between FVIII activity and thrombin generation was observed when analyzing samples from haemophilia A dogs dosed with canine FVIII. Limit of detection was 0.1% (v/v) FVIII or FIX. CONCLUSION: A novel modified TGT suitable for monitoring and prediction of replacement therapy efficacy in plasma from haemophilia A and B dogs was established.

Acute Haemarthrosis in the Haemophilia A Rat Generates a Local and Systemic Proinflammatory Response.

Background Replacement therapy with coagulation factor VIII (FVIII) concurrent with bleeds (on-demand) in haemophilia A (HA) patients has been hypothesized to increase the risk for antidrug antibodies (inhibitors). A danger signal environment, characterized by tissue damage and inflammation at the site of a bleed, is thought to contribute to the anti-FVIII response. The nature of this inflammatory reaction is, however, not fully known, and new insights will be valuable for both managing inhibitors and understanding arthropathy development. Objective To characterize the inflammatory response, locally and systemically, during the first 24 hours following a joint bleed in the HA rat. Methods HA rats received a needle-induced knee joint bleed (n = 83) or a sham procedure (n = 41). Blood samples were collected at selected time points from 0 to 24 hours post injury/sham. Synovial fluid, intra-articular knee tissue and popliteal lymph nodes were collected at 24 hours. Cytokine/chemokine concentrations and gene expression were measured. Results Gene expression analysis revealed a rapid inflammatory response in the injured knees, accompanied by significantly increased levels of specific gene products in the synovial fluid; IL-1ß, TNFα, KC/GRO, IL-6, Eotaxin, MCP-1, MCP-3, MIP-1α, MIP-2, RANTES, A2M and AGP. Plasma analysis demonstrated significantly increased systemic levels of KC/GRO and IL-6 in injured rats already after 5 to 6 hours. Conclusion A rapid proinflammatory response, locally and systemically, characteristic of innate immunity, was demonstrated. Results reveal a more comprehensive inflammatory picture than previously shown, with resemblance to human haemophilic arthropathy, and with unique correlation between gene expression level, synovial concentration and plasma concentration in individual rats.

PET Imaging of Tissue Factor in Pancreatic Cancer Using 64Cu-Labeled Active Site-Inhibited Factor VII.

UNLABELLED: Tissue factor (TF) is the main initiator of the extrinsic coagulation cascade. However, TF also plays an important role in cancer. TF expression has been reported in 53%-89% of all pancreatic adenocarcinomas, and the expression level of TF has in clinical studies correlated with advanced stage, increased microvessel density, metastasis, and poor overall survival. Imaging of TF expression is of clinical relevance as a prognostic biomarker and as a companion diagnostic for TF-directed therapies currently under clinical development. Factor VII (FVII) is the natural ligand to TF. The purpose of this study was to investigate the possibility of using active site-inhibited FVII (FVIIai) labeled with (64)Cu for PET imaging of TF expression. METHODS: FVIIai was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and labeled with (64)Cu ((64)Cu-NOTA-FVIIai). Longitudinal in vivo PET imaging was performed at 1, 4, 15, and 36 h after injection of (64)Cu-NOTA-FVIIai in mice with pancreatic adenocarcinomas (BxPC-3). The specificity of TF imaging with (64)Cu-NOTA-FVIIai was investigated in subcutaneous pancreatic tumor models with different levels of TF expression and in a competition experiment. In addition, imaging of orthotopic pancreatic tumors was performed using (64)Cu-NOTA-FVIIai and PET/MRI. In vivo imaging data were supported by ex vivo biodistribution, flow cytometry, and immunohistochemistry. RESULTS: Longitudinal PET imaging with (64)Cu-NOTA-FVIIai showed a tumor uptake of 2.3 ± 0.2, 3.7 ± 0.3, 3.4 ± 0.3, and 2.4 ± 0.3 percentage injected dose per gram at 1, 4, 15, and 36 h after injection, respectively. An increase in tumor-to-normal-tissue contrast was observed over the imaging time course. Competition with unlabeled FVIIai significantly (P < 0.001) reduced the tumor uptake. The tumor uptake observed in models with different TF expression levels was significantly different from each other (P < 0.001) and was in agreement with the TF level evaluated by TF immunohistochemistry staining. Orthotopic tumors were clearly visible on the PET/MR images, and the uptake of (64)Cu-NOTA-FVIIai was colocalized with viable tumor tissue. CONCLUSION: (64)Cu-NOTA-FVIIai is well suited for PET imaging of tumor TF expression, and imaging is capable of distinguishing the TF expression level of various pancreatic tumor models.

Quantitative analysis of inflammatory and immune responses in dogs with gastritis and their relationship to Helicobacter spp. infection.

The present study sought to quantitatively examine mucosal inflammatory and immune responses in dogs with gastritis and the relationship of these responses to infection with Helicobacter. Gastric biopsies from 30 dogs were evaluated for B- and T-lymphocytes, neutrophils, eosinophils, macrophages, and mast cells. Mucosal atrophy, fibrosis, cellularity, and severity of gastritis were graded qualitatively. Messenger-RNA (mRNA) for actin, interleukin-1beta (IL-1beta), IL-4, IL-8, and IL-10, transforming growth factor beta (TGF-beta), and interferon gamma (IFN-gamma) was quantified by polymerase chain reaction (PCR). The presence of Helicobacter spp. was determined by urease activity, histology, PCR, and enzyme-linked immunosorbent assay. mRNA for IL-1beta, IL-8, IL-10, TGF-beta, and IFN-gamma was detected in most dogs. IL-4 mRNA was detected in only 1 dog. Correlations were observed for IL-1beta versus IL-8 and IL-10; IL-8 versus IL-10, IFN-gamma, and TGF-beta; and IL-10 versus IFN-y. Mucosal pathology was related to cytokine mRNA expression (neutrophils to IL-8 and IFN-gamma, macrophages and lymphocytes to IFN-gamma, and fibrosis to IL-1beta). Gastritis was categorized as lymphoplasmacytic in all dogs, and its histologic severity correlated with atrophy, infiltration with lymphocytes and macrophages, and expression of IL-10 and IFN-gamma. Of the dogs examined, 76.7% were infected with Helicobacter spp. Infection was associated with increased expression of TGF-beta and fibrosis. Circulating anti-Helicobacter immunoglobulin G titers were higher in uninfected than infected dogs. We conclude that lymphoplasmacytic gastritis in dogs is characterized by concurrent activation of proinflammatory and immunomodulatory cytokines, with increased mRNA expression related to mucosal pathology. No significant associations between Helicobacter infection and proinflammatory cytokine expression, severity of gastritis, or differences in the pathogenicity of different Helicobacter spp. were found.