Center for Blood Research

Vancouver, Canada

Center for Blood Research

Vancouver, Canada
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Panwar P.,University of British Columbia | Panwar P.,Center for Blood Research | Panwar P.,University of Southern Denmark | Soe K.,University of Southern Denmark | And 5 more authors.
British Journal of Pharmacology | Year: 2016

Background and Purpose Cathepsin K (CatK) is a major drug target for the treatment of osteoporosis. Potent active site-directed inhibitors have been developed and showed variable success in clinical trials. These inhibitors block the entire activity of CatK and thus may interfere with other pathways. The present study investigates the antiresorptive effect of an exosite inhibitor that selectively inhibits only the therapeutically relevant collagenase activity of CatK. Experimental Approach Human osteoclasts and fibroblasts were used to analyse the effect of the exosite inhibitor, ortho-dihydrotanshinone (DHT1), and the active site inhibitor, odanacatib (ODN), on bone resorption and TGF-ß1 degradation. Cell cultures, Western blot, light and scanning electron microscopy as well as energy dispersive X-ray spectroscopy, molecular modelling and enzymatic assays were used to evaluate the inhibitors. Key Results DHT1 selectively inhibited the collagenase activity of CatK, without affecting the viability of osteoclasts. Both inhibitors abolished the formation of resorption trenches, with DHT1 having a slightly higher IC50 value than ODN. Maximal reductions of other resorption parameters by DHT1 and ODN were comparable, respectively 41% and 33% for total resorption surface, 46% and 48% for resorption depths, and 83% and 61% for C-terminal telopetide fragment (CTX) release. DHT1 did not affect the turnover of fibrosis-associated TGF-ß1 in fibroblasts, whereas 500 nM ODN was inhibitory. Conclusions and Implications Our study shows that an exosite inhibitor of CatK can specifically block bone resorption without interfering with other pathways. © 2015 The British Pharmacological Society.


Williamson L.M.,National Health Service Blood and Transplant | Devine D.V.,Center for Blood Research
The Lancet | Year: 2013

Although blood suppliers are seeing short-term reductions in blood demand as a result of initiatives in patient blood management, modelling suggests that during the next 5-10 years, blood availability in developed countries will need to increase again to meet the demands of ageing populations. Increasing of the blood supply raises many challenges; new approaches to recruitment and retainment of future generations of blood donors will be needed, and care will be necessary to avoid taking too much blood from these donors. Integrated approaches in blood stock management between transfusion services and hospitals will be important to minimise wastage - eg, by use of supply chain solutions from industry. Cross-disciplinary systems for patient blood management need to be developed to lessen the need for transfusion - eg, by early identification and reversal of anaemia with haematinics or by reversal of the underlying cause. Personalised medicine could be applied to match donors to patients, not only with extended blood typing, but also by using genetically determined storage characteristics of blood components. Growing of red cells or platelets in large quantities from stem cells is a possibility in the future, but challenges of cost, scaling up, and reproducibility remain to be solved.


Madsen E.H.,Aarhus University Hospital | Saw J.,Vancouver General Hospital | Kristensen S.R.,Aarhus University Hospital | Schmidt E.B.,Aarhus University Hospital | And 3 more authors.
Clinical Chemistry | Year: 2010

BACKGROUND: A reduced response to aspirin and clopidogrel predicts ischemic events, but reliable tests are needed to identify low responders. We compared 3 platelet-function tests during long-term dual treatment with aspirin and clopidogrel. METHODS: Patients who underwent a percutaneous coronary intervention and were receiving a combination of 325 mg/day aspirin and 75 mg/day clopidogrel were followed for 1 year. Blood was sampled 5 times during this period for 3 tests: light transmission aggregometry (LTA) assay, with 5.0 μmol/L ADP or 1.0 mmol/L arachidonic acid (AA) used as an agonist; VerifyNow™ assay, with the P2Y12 or aspirin cartridge (Accumetrics); and thrombelastography (TEG), stimulated by 2.0 μmol/L ADP or 1.0 mmol/L AA. RESULTS: Twenty-six of 33 patients completed all scheduled visits. A low response to clopidogrel was found in a few patients at variable frequencies and at different visits, depending on the method and criteria used. We found a moderate correlation between the LTA (ADP) and VerifyNow (P2Y 12 cartridge) results, but the TEG (ADP) results correlated poorly with the LTA and VerifyNow results. A low response to aspirin was found with the VerifyNow (aspirin cartridge) and TEG (AA) methods on 6 and 2 occasions, respectively, but not with the LTA (AA) method, except for 1 occasion caused by probable noncompliance. CONCLUSIONS: Detecting a low response to clopidogrel depends largely on the method used. Which method best predicts ischemic events remains uncertain. A low response to aspirin is rare with AA-dependent methods used at the chosen cutoffs. In some patients, the response to clopidogrel or aspirin may be classified differently at different times, even with the same method. © 2010 American Association for Clinical Chemistry.


Imran ul-haq M.,Center for Blood Research | Lai B.F.,Center for Blood Research | Kizhakkedathu J.N.,Center for Blood Research
Macromolecular bioscience | Year: 2014

Multifunctional polymers with defined structure and biocompatibility are critical to the development of drug delivery systems and bioconjugates. In this article, the synthesis, in vitro blood compatibility, cell viability, in vivo circulation, biodistribution, and clearance of hybrid copolymers based on linear and branched polyglycerol are reported. Hybrid polyglycerols (M(n) ≈ 100 kDa) are synthesized with different compositions (15-80 mol% linear polyglycerol). Relatively small hydrodynamic size and radius of gyration of the hybrid polyglycerols suggest that they are highly compact functional nanostructures. The hybrid polyglycerols show excellent blood compatibility as determined by measuring their effects on blood coagulation, red blood cell aggregation, hemolysis, platelet, and complement activation. The cell viability in presence of hybrid polyglycerols is excellent up to 10 mg mL(-1) concentration and is similar to both dextran and polyvinyl alcohol. Furthermore, tritium labeled hybrid polyglycerol shows long blood circulation (t(1/2β)= 34 h) with minimal organ accumulation in mice. Multifunctionality, compact nature, biocompatibility, and the long blood circulation make these polymers attractive for the development of bioconjugates and drug delivery systems. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Lai B.F.L.,Center for Blood Research | Zou Y.,Center for Blood Research | Brooks D.E.,Center for Blood Research | Brooks D.E.,University of British Columbia | Kizhakkedathu J.N.,Center for Blood Research
Biomaterials | Year: 2010

Poly-N-[(2,2-dimethyl-1,3-dioxolane)methyl]acrylamide (PDMDOMA) is a neutral synthetic water-soluble polymer. In this report, we evaluated the influence of PDMDOMA on blood hemostasis by studying the fibrin polymerization process, the three-dimensional clot structure, and the mechanical properties and fibrinolysis. PDMDOMA altered the normal fibrin polymerization by changing the rate of protofibril aggregation and resulting in a 5-fold increase in the overall turbidity. Fibrin clots formed in presence of PDMDOMA exhibited thinner fibers with less branching which resulted in a more porous and heterogeneous clot structure in scanning electron micrographs. The overall strength and rigidity of the whole blood clot also decreased up to 10-fold. When a combination of plasminogen and tissue-plasminogen activators were included in clotting reactions, fibrin clots formed in the presence of PDMDOMA exhibited highly shortened clot lysis times and was supported by the enhanced clot lysis measured by thromboelastography in whole blood. Further evidence of the altered clot structure and clot cross-linking was obtained from the significant decrease in d-dimer levels measured from degraded plasma clot. Thus, PDMDOMA may play an important role as an antithrombotic agent useful in prophylactic treatments for thrombosis by modulating fibrin clot structure to enhance fibrinolysis. © 2010 Elsevier Ltd.


Liu Z.,Center for Blood Research | Janzen J.,Center for Blood Research | Brooks D.E.,Center for Blood Research | Brooks D.E.,University of British Columbia
Biomaterials | Year: 2010

Hydrophobically derivatized hyperbranched polyglycerol (HPG)-polyethylene glycol (PEG) polymers bearing stearoyl chains (HPG-C18-PEG) were originally developed as human serum albumin substitutes and further as a unimolecular drug delivery system. In view of these in vivo applications and the potential for membrane interaction by these materials due to their amphiphilic structure, determining the adsorption of the polymers to human red blood cells (RBCs) is an important issue. This paper reports on the in vitro adsorption to RBCs of tritium-radiolabeled HPG-C18-PEG polymers. The morphological changes of RBCs associated with the adsorption were also examined by light and scanning electron microscopy (SEM). Laser scanning confocal microscopy (LSCM) suggests that the binding site of the polymers on RBCs is the cell membrane. Adsorption experiments show that, in the medium of either saline or plasma, the binding amount of the polymers to RBCs increases with increased polymer concentration in a manner which implies simple Langmurian behavior. The binding amount in saline is of the order of 105 molecules/cell at an equilibrium concentration of 1 mg/mL of HPG-C18-PEG polymer. The RBC morphology depends on the adsorbed amount; the cells become crenated in high concentrations (5 and 10 mg/mL) of the polymer solutions in the absence of plasma proteins. Interestingly, a large amount of polymers remain bound to RBCs even after washes with plasma (of the order of 104 molecules/cell). Thus, the bound polymers might have an extended circulating time by "hitchhiking" on RBCs in the bloodstream. These results provide significant information and insight for related studies of the interaction of amphiphilic molecules with cell membranes and for in vivo applications of biopolymers as drug delivery systems. © 2010 Elsevier Ltd. All rights reserved.


Gyongyossy-Issa M.I.C.,Center for Blood Research
Transfusion and Apheresis Science | Year: 2011

The metabolic conversion of glucose to energy and reducing power by platelets is examined. Although platelets concurrently metabolize glucose aerobically and anaerobically, the balance between the cytosolic and mitochondrial pathways is affected not only by physiological activation but also by conditions prevailing during in vitro storage. The development of platelet additive solutions and pathogen reduction technologies point to increased glucose metabolism and consequent high levels of lactate production as the effect of platelet damage, rather than the cause. Consequently a different perspective of the data suggests that reduction rather than support of platelet metabolism in vitro would result in a better quality of stored platelets. © 2011 Elsevier Ltd.


Imran Ul-Haq M.,Center for Blood Research | Hamilton J.L.,Center for Blood Research | Lai B.F.L.,Center for Blood Research | Shenoi R.A.,Center for Blood Research | And 5 more authors.
ACS Nano | Year: 2013

Patients requiring chronic red blood cell (RBC) transfusions for inherited or acquired anemias are at risk of developing transfusional iron overload, which may impact negatively on organ function and survival. Current iron chelators are suboptimal due to the inconvenient mode of administration and/or side effects. Herein, we report a strategy to engineer low molecular weight iron chelators with long circulation lifetime for the removal of excess iron in vivo using a multifunctional dendritic nanopolymer scaffold. Desferoxamine (DFO) was conjugated to hyperbranched polyglycerol (HPG) and the plasma half-life (t 1/2) in mice is defined by the structural features of the scaffold. There was a 484 fold increase in t1/2 between the DFO (5 min) versus the HPG-DFO (44 h). In an iron overloaded mouse model, efficient iron excretion by HPG-DFO in the urine and feces was demonstrated (p = 0.0002 and 0.003, respectively) as was a reduction in liver, heart, kidney, and pancreas iron content, and plasma ferritin level (p = 0.003, 0.001, 0.001, 0.001, and 0.003, respectively) compared to DFO. Conjugates showed no apparent toxicity in several analyses including body weight, serum lactate dehydrogenase level, necropsy analysis, and by histopathological examination of organs. These findings were supported by in vitro biocompatibility analyses, including blood coagulation, platelet activation, complement activation, red blood cell aggregation, hemolysis, and cell viability. This nanopolymer-based chelating system would potentially benefit patients suffering from transfusional iron overload. © 2013 American Chemical Society.


Han V.,Center for Blood Research | Serrano K.,Center for Blood Research | Devine D.V.,Center for Blood Research
Vox Sanguinis | Year: 2010

Background and Objectives There is no standardized method of measuring the parameters for haemolysis determination of red cell concentrate (RCC). Three haemoglobin quantification methods (automated analyser, Harboe and Drabkin's) and two methods of haematocrit measurement (automated analyser and microcapillary centrifugation) were evaluated for use with RCC. Materials and Methods Twenty stored RCC were assayed for total haemoglobin, supernatant haemoglobin and haematocrit. Results Drabkin's and Harboe methods were linear (r2≥ 0·995) over 0·015-220 g/l haemoglobin. Overestimation by Drabkin's increased from 0% at 220 g/l to 137% at 0·015 g/l haemoglobin. Harboe values generally stayed within 6% of expected while haematology analyser values had a maximum 11% underestimation above 10 g/l. Analyser total haemoglobin was significantly lower (202 ± 22 g/l) than Drabkin's (224 ± 24 g/l) and Harboe (222 ± 22 g/l) values. Haematocrit was greater via the analyser (65·7 ± 5·7%) than with microcapillary centrifugation (59·3 ± 5·7%). Conclusions Harboe and Drabkin's methods are suitable for measuring total haemoglobin and supernatant haemoglobin in RCC. The analyser gave higher haematocrit values (11% on average) than did microcapillary centrifugation. © 2009 International Society of Blood Transfusion.


Kyluik-Price D.L.,Canadian Blood Services | Kyluik-Price D.L.,Center for Blood Research | Kyluik-Price D.L.,University of British Columbia | Scott M.D.,Canadian Blood Services | And 2 more authors.
Biomaterials | Year: 2016

Tissue rejection occurs subsequent to the recognition of foreign antigens via receptor-ligand contacts between APC (antigen presenting cells) and T cells, resulting in initialization of signaling cascades and T cell proliferation. Bioengineering of donor cells by the covalent attachment of methoxypolyethylene glycol (mPEG) to membrane proteins (PEGylation) provides a novel means to attenuate these interactions consequent to mPEG-induced charge and steric camouflage. While previous studies demonstrated that polymer-mediated immunocamouflage decreased immune recognition both in vitro and in vivo, these studies monitored late events in immune recognition and activation such as T cell proliferation. Consequently little information has been provided concerning the early cellular events governing this response. Therefore, the effect of PEGylation was assessed by examining initial cell-cell interactions, changes to activation pathways, and apoptosis to understand the role that each may play in the decreased proliferative response observed in modified cells during the course of a mixed lymphocyte reaction (MLR). The mPEG-modified T cells resulted in significant immunocamouflage of lymphocyte surface proteins and decreased interactions with APC. Furthermore, mPEG-MLR exhibited decreased NFκB pathway activation, while exhibiting no significant differences in degree of cell death compared to the control MLR. These results suggest that PEGylation may prevent the direct recognition of foreign alloantigens by decreasing the stability and duration of initial cell-cell interactions. © 2015 The Authors.

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