The thrombin generation capability of the Chacma baboon (Papio ursinus): implications for haemostatic disease models.

Baboon models are often used to investigate haemostatic diseases, such as acquired thrombotic thrombocytopenic purpura or bacterial sepsis-induced disseminated intravascular coagulation, and their potential treatment with novel drugs. Thrombin generation is vital for these models, and an important potential therapeutic target. We investigated the thrombin generation profile of the Chacma baboon (Papio ursinus - a common pre-clinical model) including the effects of sex and ABO blood group. Thrombin generation curves, lag times, peak heights, times-to-peak, velocity indexes and Endogenous Thrombin Potentials (ETPs) of 40 adult Chacma baboons were assessed and compared with normal human plasma, using a low concentration of tissue factor (1 pM) and phospholipids. Reference intervals were calculated, and results compared between O and non-O ABO blood groups, and between males and females. Lag times of all baboons fell within the human reference interval. Most animals (n = 32; 80%) had times-to-peak above, and velocity indexes and peak heights markedly below (n = 27; 68%) the human range. However, 97.5% of baboons had an ETP above the human reference interval, indicating greater overall thrombin generation. ABO blood group had no effect, but males (n = 14; 35%) had less potent thrombin generation than females (n = 26; 65%), with significantly longer lag times (p = 0.0475), lower peak thrombin concentrations (p = 0.0203), and lower ETPs (p = 0.0238). Chacma baboons have greater overall endogenous thrombin generation potentials than humans, which is even more prominent in females. This should be considered when designing future baboon model experiments involving the haemostatic system, or when evaluating novel therapies in these animals.

The effects of streptokinase in a Chacma baboon (Papio ursinus) model of acquired thrombotic thrombocytopenic purpura.

TTP is a life-threatening disorder with limited pharmaceutical treatment options. Recently, the potential of streptokinase in the treatment of acquired TTP was demonstrated in humans in vitro, and in vivo in a mouse model. We aimed to determine the in vitro and in vivo effects of streptokinase in an established Papio ursinus model of acquired TTP. In vitro: VWF activities & multimer patterns and thromboelastograms were assessed with increasing concentrations of streptokinase. In vivo: After induction of TTP, escalating streptokinase doses (ranging from 50,000 to 900,000 IU) were administered, and the effects of streptokinase assessed on peripheral blood counts, fibrinolysis, VWF activities & multimer patterns and thromboelastograms. In an extension of the study, high-dose streptokinase (1,500,000-3,000,000 IU) was administered to another baboon. After spiking, fibrinolysis with loss of large VWF multimers was observed at [2200 IU/mL]-roughly equivalent to 1,500,000 IU. However, administration of escalating intravenous streptokinase doses had no in vivo effect on the TTP phenotype, and in vivo increases in plasmin activity were mild when compared with baseline, even at high doses. Minimal effect on VWF multimer patterns was observed but only at doses ≥ 1500,000 IU. Streptokinase is not effective in resolving TTP in a Papio ursinus model of TTP, possibly due to limited activation of the baboon fibrinolytic system. Modifications to this model, the use of alternative higher animal models, or alternative thrombolytics, should be considered to establish proof-of-concept.

Comparison of ADAMTS13 and Von Willebrand factor levels and activities, and plasminogen levels, in plasma products currently available for the treatment of thrombotic thrombocytopenic purpura in South Africa.

OBJECTIVE: Thrombotic thrombocytopenic purpura (TTP) results from a deficiency in the Von Willebrand factor (VWF) cleaving protease, ADAMTS13. Treatment involves plasma exchange (PEX) therapy with either fresh frozen plasma (FFP), cryosupernatant (CSP) or solvent/detergent-treated plasma (SDP), available in South Africa as Bioplasma FDP. The aim of the study was to generate in vitro data on these products, and to explore possible differences between the products that may offer treatment advantages. METHODS: Twenty samples per product (FFP, CSP and Bioplasma FDP) were analysed for levels and activities of ADAMTS13 and VWF. Plasminogen levels, a proposed physiological back-up system for ADAMTS13, were also determined. FFP and CSP samples were subanalysed according to ABO blood group. Samples were analysed by means of commercially available ELISA assays. RESULTS: All samples had normal/high ADAMTS13 activity (Median activity for SDP = 94.0%, CSP = 80.5%, FFP = 122.0%) and plasminogen levels. The VWF content was mostly normal for Bioplasma FDP, typically deficient for CSP and mostly deficient for FFP, which was an unexpected finding. Depending on the parameter, Bioplasma FDP was the most standardised, with coefficients of variation (CV) from 14.1% to 27.3%, while FFP showed great inter-individual variation (CV 24.6% to 208.6%). Statistically significant differences were found across products (P ≤ 0.0095), and ABO blood groups (P = 0.0001). CONCLUSION: All three products can be used for the treatment of TTP. The choice of product depends on the need for additional viral safety, costs, product availability and the perceived impact of within-product variations.

The Use of Phage Display and Yeast Based Expression System for the Development of a Von Willebrand Factor Propeptide Assay: Development of a Von Willebrand Factor Propeptide Assay.

BACKGROUND: The diagnosis of von Willebrand disease is complex due to the heterogeneity of the disease. About eighty percent of von Willebrand disease patients are diagnosed with a quantitative defect of von Willebrand factor (VWF) where fifty percent is due to an increased clearance of von Willebrand factor. These patients do not respond well to the treatment of choice, Desmopressin (DDAVP) due to decreased efficacy. The ratio between the VWF propeptide and the mature VWF antigen is used to diagnose these patients. Commercial VWF propeptide assays are too expensive for use in developing countries. In this study, we developed a cost-effective ELISA assay. METHODS: We first displayed VWF propeptide on yeast. Antibody fragments were selected against the displayed VWF propeptide by using phage display technology. The antibodies were used to develop a cost-effective VWF propeptide assay and compared to a commercial VWF propeptide assay. RESULTS: Two of these antibody fragments bound specific to the VWF propeptide and not to the yeast used for the expression of the propeptides. These purified antibody fragments were able to detect VWF propeptide in normal plasma. CONCLUSION: Our assay performed well when compared to a commercial kit. It also showed a higher binding affinity for VWF propeptide in plasma at especially lower plasma concentrations.

Sites of elimination and pharmacokinetics of recombinant [131I]lepirudin in baboons.

Lepirudin has a short half-life, and only 50-60% of the intravenously administered dose is excreted by the kidneys. The fate of the remainder is unknown. We designed a study to determine the fate of this lepirudin. In each of six baboons, [131I]lepirudin was given intravenously as a bolus or infused over 30 min, 24 h apart. The in vivo redistribution of [131I]lepirudin was determined and quantified by scintillation camera imaging. In all studies, the half-life of [131I]lepirudin, as determined from the disappearance of radioactivity, was 21 +/- 3 min. The half-life determined from the disappearance of lepirudin, measured by the Ecarin Clotting Time (ECT) method, was similar at 23 +/- 8 min. Results obtained with the labeled lepirudin are therefore comparable with those obtained using the plasma concentration of lepirudin. When lepirudin was administered as a bolus, the half-life was 18 +/- 4 min, and lepirudin was cleared from the plasma at a rate of 42 +/- 12 mL/min and by the kidneys at 23 +/- 2 mL/min. Following infusion over 30 min, the half-life and total and renal clearances were not significantly different. In both studies, between 50 and 60% of the administered lepirudin was excreted by the kidney. Studies on sacrificed baboons showed that appreciable amounts of lepirudin were present in the bile, indicating the liver as a contributor to the elimination of lepirudin.