ALBERT

Description: Open-heart surgery performed with cardiopulmonary extracorporeal bypass (CPB) of anticoagulated blood often is complicated by excessive postoperative hemorrhage due to an acquired platelet function defect and thrombocytopenia inherent to the procedure. One strategy for reducing excessive blood loss is to protect the platelets pharmacologically from activation and damage during the surgical procedure and the CPB, but most inhibitors of platelet activation used in this way would exacerbate bleeding because of the duration of their effect. INS50589 is a competitive and selective P2Y12 receptor antagonist in development by Inspire Pharmaceuticals Inc. Previous studies in normal dogs indicate that 20μM ADP induced ex vivo whole blood platelet aggregation is completely inhibited by the administration of INS50589 and that the effect is quickly reversed upon discontinuation of the administration. This drug is now being tested for efficacy and safety in a canine model of CPB established at East Carolina University. Subjects in the range of 20–30 kg are prepared by sternotomy for extracorporeal recirculation of blood after systemic heparinization. CPB occurs for ninety minutes, followed by weaning from the pump and maintenance for a four hour postoperative period. INS50589 was administered by continuous infusion at a rate of 17 mg/kg/min at initiation of sternotomy and throughout CPB until weaned. Special testing in all subjects includes ex vivo whole blood aggregometry with two doses of ADP, thromboelastography with the TEG® ADP Mapping kit, bleeding times using a 23 gauge puncture of the exposed jugular vein, and measurements of blood loss from the surgical fields. In the placebo treated group, there was a significant loss of platelet response to ADP during CPB that remained in the post-op period at approx 50% of initial values; in contrast, the drug-treated subjects show post-op recovery of ADP response to within 90% of initial values (p

Description: It has much more advantages to preserve human red blood cells (RBC) in freezing and drying form than conventional blood storage, even though it is still under developing. In the present study, we successfully used 0.045% glutaraldehyde(GA) to fix RBCs under appropriate conditions prior to lyophilization and regained the bulk of RBCs membrane filterability. The phlebotomized whole blood was anti-coagulated with CPDA-1 and the plasma was removed by centrifugation. White blood cells were removed using Leucocytes filter (Pall Corporation). 1 x 10 9 cells/ml were separately incubated with 0.03%, 0.045% and 0.05% GA at 37 °C for 10 min; RBCs at concentrations 0.5 x 10 9, 1 x 10 9 and 2 x 10 9 cell/ml were incubated with 0.045% GA at 37 °C for 10 min; Bovine serum albumin (BSA), trehalose, hydroxyethyl starch and dextran were used as protective additive in freezing and drying RBCs, respectively; ethanolamine, glycine, lysine, BSA, glutamic acid and homocysteic acid were used to recover membrane deformability due to GA fixation. Recovery RBCs yield after reconstituion was determined by a cell counter. Fee hemoglobin , glutathione (GSH), 2,3-diphosphoglycerate (2,3-DPG), ATP, and Glucose-6-phosphate dehydrogenase (G-6-PDH) in RBCs were determined before and after lyophilization. Membrane flexibility was assayed by osmotic fragility test. RBCs filterability was determined using positive filter apparatus through 5 micrometer pore size nitrocellulose membrane. Our results suggested that RBCs at 1 x 10 9 cells/ml fixed by 0.045% GA for10 min at pH 7.8 were enough to resist freezing and drying damages and the bulk of RBCs’s membrane filterability were remained. The effective fixation of GA is dependent on GA’s concentration, RBCs concentration, incubation period and pH etc. BSA is the most potential additive in preserving RBCs. More interestingly, the following reagents can recover the reduced RBCs membrane filterability by GA fixation. The potency of recovery is in the order of : 10 mM ethanolamine 〉 5 mM lysine 〉 0.5%BSA 〉 10 mM glutamatic acid 〉 5 mM homcysteic acid 〉 2% glycine. After reconstitution, 85 ± 2.3% RBCs yield is achieved, 71 ± 4.1% of these RBCs can freely pass through 5 micrometer pore size filter membrane. Biochemical function indexes as GSH, 2,3-DPG, ATP, and G-6-PDH are saved by 20 ± 1.2%. Lyophilized human red blood cells yields from reconstitution by different treatment. 1x 109 cells/ml fixed at 0.03% glutaraldehyde 44.7 ± 4.1 0.045%glutaraldehyde 84.7 ± 5.8 0.05%glutaraldehyde 88.7 ± 7.6 0.045% glutaraldehyde fix: 0.5x 109 cells/ml 91 ± 4.3 1x 109 cells/ml 84.7 ± 5.8 2 x 109 cells/ml 35.2 ± 2.3 Effects of various treatment on lyophilized RBCs after reconstitution. Group Filterability(%) 1x9 cells/ml fixed by: N = 5. 0.03%glutaraldehyde 88.3 ± 1.2 0.045%glutaraldehyde 73.3 ± 1.8 0.05%glutaraldehyde 44.7 ± 2.8 0.045%glutaraldehyde fixation at: 0.5x109 cells/ml 0 1x109 cells/ml 73.3 ± 1.8 2x109 cells/ml 83.9 ± 4.6

Description: The experiments presented here were undertaken to determine if factor VIIa (rFVIIa, the Novo Nordisk product NovoSeven™) will directly bind to rehydrated, lyophilized (RL) platelets (Stasix™ platelets, Entegrion, Inc. trade) for the formation of a catalytic surface with an enhanced ability to generate thrombin. The relationship of rFVIIa to the RL platelet surface was examined by measuring equilibrium and non-equilibrium binding of the coagulation factor to the cells, by studying the subcellular localization of the coagulation factor on RL platelets, and by following the effects of the surface modification on the kinetics of thrombin generation. The association of rFVIIa with RL platelets occurred with an on rate of 3.6x103 sec−1moles−1. Saturation occurred in minutes and was calcium dependent. Disassociation (in plasma or citrated saline) was slow, with over half of the coagulation factor remaining bound after two hours (with slow and fast rate constants of 5.0x10−5 and 4.1x10−4 sec−5 respectively). These results define a binding site with an apparent equilibrium constants of 110 nM. Equilibrium binding of rFVIIa to RL platelets was analyzed with flow cytometry and Western analysis. The rFVIIa was bound to RL platelets in a dose-dependent manner when incubated at concentrations of 0.3 to 10.0 uM rFVIIa and 3x104 to 106 RL platelets/ul in citrated saline. When high concentrations of rFVIIa were bound to RL platelets densities of over one million molecules of rFVIIa per RL platelet was obtained. Fluorescent microscopy analysis revealed that the rFVIIa was localized to the surface membrane and that some rFVIIa localized internally to the outer surface of the surface connected open canalicular system and/or sites of internal trafficking. Flow cytometric analysis with annexin V demonstrated that considerable quantities of phosphatidylserine were present on the external surface of the RL platelet membrane for potential facilitation of rFVIIa binding. The effect of RL platelet surface modification by rFVIIa on thrombin generation was investigated by following the hydrolysis of the thrombin-specific fluorogenic substate D-phe-pro-arg-ANSNHin plasma. rFVIIa and RL platelets accelerated thrombin generation in this system with rFVIIa being approximately twice as effective (per molecule of the recombinant protein) when added to the assay system pre-bound to RL platelets as compared to being initially free in the plasma. Similar results were obtained when free and RL platelet bound rFVIIa were tested in factor IX-deficient plasma. These experiments show that rFVIIa retains activity when super-saturated on the RL platelet membrane. The results of the studies presented here suggest that RL platelets can be used to concentrate rFVIIa at sites of vascular injury.