The long-recognized cold-induced activation and damage of human platelets has recently been shown to be associated with platelets passing through a thermotropic phase transition. This proposal has the long-term objective of developing mechanisms of stabilizing platelets during and after this damaging event, resulting in platelets being more tolerant of cold storage with greatly increased storage life time. Antifreeze glycoproteins (AFGPs) from the blood of certain polar fish are capable of preventing membrane leakage during passage through the phase transition. Recent work shows that AFGPs appear to stabilize platelets during chilling and storage below the phase transition temperature. The proposal seeks to better define the mechanisms involved in the cold storage lesion in platelets including the role of intracellular and extracellular calcium. The anticipated studies will also provide evidence as to whether AFGPs might inhibit temperature change associated events including inhibition of membrane leakage and lateral phase separation and/or phospholipase A2 (PLA2) activity. An array of techniques will be employed to accomplish these objectives. Among these is Fourier-transform infrared spectroscopy (FTIR). This will be used to monitor membrane phase transitions and lateral phase separation. Fluorescent phase spectroscopy will be employed for measuring internal calcium concentration, lateral phase separation, and PLA2 activity. Analytical thin layer chromatography (TLC) and gas chromatography will also be used to measure PLA2 activity and membrane composition. FACScanning will measure the expression of glycoproteins known to be on the platelet surface including GPs Ib and IIb/IIIa, and P-selectin. These will be measured in conjunction with aggregometry to quantify the level of activation and agonist responsiveness in human platelets during and after cold storage.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL057810-02
Application #
2702387
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1997-05-01
Project End
2002-04-30
Budget Start
1998-05-01
Budget End
1999-04-30
Support Year
2
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of California Davis
Department
Anatomy/Cell Biology
Type
Schools of Veterinary Medicine
DUNS #
094878337
City
Davis
State
CA
Country
United States
Zip Code
95618
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Crowe, John H (2007) Trehalose as a ""chemical chaperone"": fact and fantasy. Adv Exp Med Biol 594:143-58
Tang, Minke; Wolkers, Willem F; Crowe, John H et al. (2006) Freeze-dried rehydrated human blood platelets regulate intracellular pH. Transfusion 46:1029-37
Oldenhof, Harriette; Wolkers, Willem F; Bowman, John L et al. (2006) Freezing and desiccation tolerance in the moss Physcomitrella patens: an in situ Fourier transform infrared spectroscopic study. Biochim Biophys Acta 1760:1226-34
Krishnan, V V; Lau, Edmond Y; Tsvetkova, Nelly M et al. (2005) Characterization of the restricted rotation of the dimethyl groups in chemically N-terminal 13C-labeled antifreeze glycoproteins: a temperature-dependent study in water to ice through the supercooled state. J Chem Phys 123:044702
He, Xiaoming; Wolkers, Willem F; Crowe, John H et al. (2004) In situ thermal denaturation of proteins in dunning AT-1 prostate cancer cells: implication for hyperthermic cell injury. Ann Biomed Eng 32:1384-98
Leidy, Chad; Gousset, Karine; Ricker, Josette et al. (2004) Lipid phase behavior and stabilization of domains in membranes of platelets. Cell Biochem Biophys 40:123-48
Wolkers, Willem F; Oliver, Ann E; Tablin, Fern et al. (2004) A Fourier-transform infrared spectroscopy study of sugar glasses. Carbohydr Res 339:1077-85
Gousset, Karine; Tsvetkova, Nelly M; Crowe, John H et al. (2004) Important role of raft aggregation in the signaling events of cold-induced platelet activation. Biochim Biophys Acta 1660:7-15
Wolkers, Willem F; Oldenhof, Harriette; Tablin, Fern et al. (2004) Preservation of dried liposomes in the presence of sugar and phosphate. Biochim Biophys Acta 1661:125-34

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