Source: UNIV OF CALIFORNIA (VET-MED) submitted to
A NOVEL METHOD FOR PRESERVING HUMAN BLOOD PLATELETS BY FREEZE-DRYING
Sponsoring Institution
Cooperating Schools of Veterinary Medicine
Project Status
COMPLETE
Funding Source
STATE
Reporting Frequency
Annual
Accession No.
0194736
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Mar 1, 2000
Project End Date
Jul 30, 2005
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIV OF CALIFORNIA (VET-MED)
(N/A)
DAVIS,CA 95616
Performing Department
ANATOMY, PHYSIOLOGY AND CELL BIOLOGY
Non Technical Summary
Human platelets are stored in blood banks at 20-24 degrees C for a maximum of five days, after which they are discarded. Even during this short storage time, they are morphologically and functionally degraded. This work will likely improve means for storing human platelets for clinical applications. The need is particularly acute among cancer patients, trauma patients and those who have been immuno-compromised
Animal Health Component
(N/A)
Research Effort Categories
Basic
50%
Applied
(N/A)
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3053840102010%
3053840103050%
3053840104010%
3053840109010%
3053840110010%
3053840116010%
Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
3840 - Laboratory animals;

Field Of Science
1040 - Molecular biology; 1090 - Immunology; 1030 - Cellular biology; 1160 - Pathology; 1100 - Bacteriology; 1020 - Physiology;
Goals / Objectives
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 project 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. This project seeks to better define the mechanisms involved in the cold storage lesion in platelets including the role of intracellular and extracellular calcium. The 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.
Project Methods
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 freeze drying.

Progress 03/01/00 to 07/30/05

Outputs
The project was designed to develop methods of stabilizing platelets in the dry state. Human platelets were loaded with trehalose ( a non-reducing disaccharide of glucose) and freeze-dried in a mixture of trehalose and one percent human albumin in a physiological platelet buffer. Freeze-dried platelets could be stored for up to 6 months, under nitrogen at room temperature in the dark. Rehydrated cells were able to respond to hypotonic shock, as well as to activate subsequent to the addition of platelet agonists. These responses were within normal limits. In addition, the cells contained a normal number of integrin receptors and bound fibrinogen in a manner virtually identical to that of fresh platelets. Freeze-dried rehydrated platelets did have a diminished ability to generate a rise in intracellular calcium, suggesting that intracellular platelet signaling may have been impaired. Furthermore, freeze-dried rehydrated platelets were able to regulate their intracellular pH in a manner similar to that of fresh platelets. Overall, this technology may provide for improved platelet storage in the dry state.

Impacts
We anticipate that freeze-dried rehydrated human and animal platelets will be available for use in wound healing (bandages containing growth factors) as well as for transfusion. Autologous platelet transfusions will greatly aid recovery from chemotherapy in all species.

Publications

  • Crowe, J.H., Tablin, F., Wolkers, W.F., Gousset, K., Tsvetkova, N.M., and J. Ricker. Stabilization of membranes in human platelets freeze-dried with trehalose. Chemistry and Physics of Lipids 1122:41-52, 2003.
  • Wolkers, W.F., S.A. Looper, A.E. McKiernan, N.M. Tsvetkova, F. Tablin and J.H. Crowe. Membrane and protein properties of freeze-dried mouse platelets. Molecular Membrane Biology 19:201-210, 2003.
  • Wolkers, W.F., S.A. Looper, R.A. Fontanilla, N.M. Tsvetkova, F. Tablin and J.H. Crowe. Temperature dependence of fluid phase endocytosis coincindes with membrane properties of pig platelets. Biochemica et Biophysica Acta 1612:154-163, 2003.
  • Wolkers, W.F., N.J. Walker, Y. Tamari, F. Tablin and J.H. Crowe. Towards a clinical application of freeze-dried platelets. Cell Preservation Technology 1:175-188, 2003.
  • Tang, M., Wolkers, W.F., Crowe, J.H. and F. Tablin. Freeze-dried rehydrated human blood platelets regulate intracellular pH. Transfusion, 2005 in press.


Progress 01/01/04 to 12/31/04

Outputs
Studies from the Center for Biostabilization funded by this DARPA grant are designed to improve stability of red blood cells and platelets in the dry state. To this end we have successfully dried human platelets which have been loaded with trehalose and freeze-dried in a mixture of trehalose and albumin. When rehydrated, these cells are responsive to platelet agonists, thrombin, ADP, and collagen by formation of a clot in the presence of plasma. Further, the cells are able to accommodate osmotic stress in a fashion virtually identical to fresh human platelets. Red blood cell studies are continuing to improve the formulation required for freeze-drying. Currently trehalose loaded human red blood cells are freeze-dried in a mixture of trehalose, albumin and hydroxyethyl starch. While these cells are intact and viable upon rehydration they are very fragile, and efforts are underway to improve the rehydration conditions for further assessment. Rehydrated red blood cells have a low hemolytic index, and relatively low methemoglobin (7-10%). Our studies on both of these cellular systems are continuing and we anticipate further funding from DARPA for this work.

Impacts
We anticipate that freeze-dried rehydrated human and animal platelets will be available for use in wound healing (bandages containing growth factors) as well as for transfusion. Autologous platelet transfusions will greatly aid recovery from chemotherapy in all species.

Publications

  • No publications reported this period


Progress 01/01/03 to 12/31/03

Outputs
We have continued to scale up our freeze-drying process for human platelets and now freeze-dry up to a unit of platelet concentrate. Freeze-dried rehydrated human platelets demonstrate a rise in intracellular calcium in response to agonists and also show a clear binding of fibrinogen in response to agonist. The fibrinogen binding data are virtually indistinguishable from fresh controls, while the calcium binding data show a diminished response of freeze-dried platelets when compared with fresh control platelets. Freeze-dried rehydrated human platelets are able to maintain their intracellular pH in a manner identical to fresh platelets. Furthermore, the Na+/H+ membrane antiporter is entirely functional as these cells respond to increasing sodium by raising their intracellular pH. We are now proceeding to circulation studies to be conducted in Yucatan mini-pigs.

Impacts
We anticipate that freeze-dried rehydrated human and animal platelets will be available for use in wound healing (bandages containing growth factors) as well as for transfusion. Autologous platelet transfusions will greatly aid recovery from chemotherapy in all species.

Publications

  • Wolkers, W.F., N.J. Walker, Y. Tamari, F. Tablin and J.H. Crowe. 2003 Towards a clinical application of freeze-dried platelets. Cell Preservation Technology 1:175-188.
  • Wolkers, W.F., S.A. Looper, R.A. Fontanilla, N.M. Tsvetkova, F. Tablin and J.H. Crowe. 2003 Temperature dependence of fluid phase endocytosis coincindes with membrane properties of pig platelets. Biochemica et Biophysica Acta 1612:154-163.
  • Wolkers, W.F., S.A. Looper, A.E. McKiernan, N.M. Tsvetkova, F. Tablin and J.H. Crowe. 2003 Membrane and protein properties of freeze-dried mouse platelets. Molecular Membrane Biology 19:201-210.
  • Crowe, J.H., F. Tablin, W.F. Wolkers, K. Gousset, N.M. Tsvetkova and J. Ricker. 2003 Stabilization of membranes in human platelets freeze-dried with trehalose. Chemistry and Physics of Lipids 1122:41-52.
  • Tablin, F., W.F. Wolkers, N.J. Walker, A.E. Oliver, N.M. Tsvetkovak, K. Gousset, L.M. Crowe and J.H. Crowe.2002 Membrane reorganization during chilling: implications for long-term stabilization of platelets. Cryobiology 43:114-123.


Progress 01/01/02 to 12/31/02

Outputs
We have made considerable progress in the stabilization of human platelets by freeze-drying. Our most recent studies, conducted in vitro for human platelets, demonstrate that these cells are able to maintain a response to thrombin (a physiological agonist) as demonstrated by aggregometry (99% of the cells are incorporated into the aggregate) as well as by a rise in intracellular calcium. Studies have also demonstrated that freeze-dried human platelets maintain their intracellular ATP levels subsequent to rehydration. We have also continue our animal model studies - in both mice and pigs. We have successfully freeze-dried mouse platelets, which respond in vitro, in a virtuallly identical manner to human platelets. In addition, freeze-dried rehydrated mouse platelets labeled with the fluorescent dye CMFDA, do circulate in vivo, with half-lives of up to 20 hours. In vitro studies using pig platelets, have shown that these cells also respond to agonist and we are currently engaging in circulation studies in this animal model as well.

Impacts
We anticipate that we will have freeze-dried human platelets available for human clinical trials within the next 20 months.

Publications

  • Tsvetkova, N.M., N.J. Walker, J.H. Crowe, C.L. Field, Y. Shi and F. Tablin. Lipid phase separation correlates with activation in platelets during chilling. 2000 Molecular Membrane Biology 17:209-218.
  • Wolkers, W.F., N.J. Walker, F. Tablin and J.H. Crowe. 2001 Human platelets loaded with trehalose survive freeze-drying. Cryobiology 42:79-87.
  • Wolkers, W.F., F.Tablin and J.H. Crowe. From anhydrobiosis to freeze-drying of eukaryotic cells. 2002 Comparative Biochemistry and Physiology Part A Molecular & Integrative Physiology 131A(3):535-543.
  • Gousset, K., W.F. Wolkers, N.M. Tsvetkova, A.E. Oliver, C.L. field, N.J. Walker, J.H. Crowe and F. Tablin. 2002 Evidence for a physiological role for membrane rafts in human platelets. Journal of Cellular Physiology 190:117-128.
  • Crowe, J.H., L.M. Crowe, A.E. Oliver, N. Tsvetkova, W. Wolkers and F. Tablin. 2002 The trehalose myth revisited: introduction to a symposium on stabilization of cells in the dry state. Cryobiology 43:89-105.
  • Tablin, F., W.F. Wolkers, N.J. Walker, A.E. Oliver, N.M. Tsvetkovak, K. Gousset, L.M. Crowe and J.H. Crowe. 2002 Membrane reorganization during chilling: implications for long-term stabilization of platelets. Cryobiology 43:114-123.
  • Crowe, J.H. A.E. Oliver and F. Tablin. 2002 Is there a single biochemical adaptation to anhydrobiosis? Integrative and Comparative Biology 42:497-503.
  • Crowe, J.H., F. Tablin, W.F.Wolkers, K. Gousset, N.M. Tsvetkova, and J. Ricker. 2003 Stabilization of membranes in human platelets freeze-dried with trehalose. Chemistry and Physics of Lipids 1122:41-52.
  • Tsvetkova N.M., Horbath, I., Torok, Z., Wolkers, W.F., Balogi, Z., Shigapova, N., Crowe, L.M., Tablin, F., Vierling, E., Crowe, J.H., and L. Vigh. 2002 Small heat shock proteins regulate membrane lipid polymorphism. Proceedings of the National Academy of Sciences USA 99:13504-13509.