STABILIZATION OF BLOOD PLATELETS AGAINST LOW TEMPERATURE ACTIVATION

Abstract: Spontaneous activation of platelets at the low temperatures normally used for blood storage is reduced or eliminated by treating the platelets with thermal hysteresis proteins. Preferred thermal hysteresis proteins are antifreeze proteins and antifreeze glycoproteins from polar fish species, and chromatographic fractions Nos. 2-6 of antifreeze glycoproteins have been found to be particularly effective.

Description: STABILIZATION OF BLOOD PLATELETS AGAINST LOW TEMPERATURE ACTIVATION This invention lies in the field of blood and blood components, with special emphasis on the function and usefulness of platelets. In particular, this invention addresses the problem of platelet activation during storage and the need to control both undesired activation and bacterial infection. GOVERNMENT RIGHTS This invention was made at least in part with United States Government support under Grant No. IBN 93-08581 awarded by the National Science Foundation, and Grant No. N00014-94-1, awarded by the Office of Naval Research. The United States Government has certain rights in this invention. BACKGROUND OF THE INVENTION Platelets, or thrombocytes, are a fraction of human blood which are important contributors to hemostasis . Platelets are generally oval to spherical in shape, with a diameter of 2-4 μm, and contain about 60% protein, 15% lipid, and 8.5% carbohydrate. Included in the chemical composition of platelets are serotonin, epinephrine, and norepinephrine, each of which aids in promoting the construction of blood vessels at the site of injury. Platelets also contain platelet factors, including platelet thromboplastin, which is a cephalin-type phosphatide, and adenosine diphosphate, both of which are important in blood coagulation. The maintenance of functional platelets is important in preserving whole blood for storage in blood banks, and in preserving concentrated platelet fractions. The storage of platelets raises certain problems, however. While the remaining fractions of blood can be preserved by cold storage for extended periods of time, platelets tend to become activated at low temperatures and thereby useless. To avoid activation, platelets are isolated and stored separately as concentrated fractions at 20°C. This raises other risks, however, namely bacterial infection as well as metabolic and enzymatic reactions known collectively as "platelet storage lesion." As a result, platelet storage is generally limited to five days. SUMMARY OF THE INVENTION It has now been discovered that platelets can be stored for extended periods of time at temperatures sufficiently low to control bacterial infection and platelet storage lesion, without risk of premature activation, by treating the platelets with proteins known as antifreeze proteins and antifreeze glycoproteins. Treatment is readily achieved in a variety of ways, including suspending the platelets in a liquid solution in which the proteins are dissolved. The platelets can be maintained in this condition until ready for use. The need for periodic renewal of a stored platelet supply is thus reduced, as are the difficulties in meeting varying demands. Tests performed to verify the invention show that the protective effect achieved by antifreeze glycoproteins varies with the amount of antifreeze glycoprotein used. It is also shown that antifreeze glycoproteins with a higher molecular weight tend to have a greater protective effect at a given concentration than those of relatively low molecular weight. These and other features and advantages of the invention will become apparent from the description which follows. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a plot taken from Fourier transform infrared spectroscopy data, showing the gel to liquid- crystalline phase transition in human platelets. FIG. 2 shows the same data as FIG. 1 plus a plot of percent activation of platelets as a function of temperature. FIG. 3 is a plot of percent activation of platelets as a function of temperature, both with and without treatment in accordance with the invention. FIG. 4a is a plot of percent activation as a function of temperature, both with and without treatment in accordance with the invention, including different levels of treatment. FIG. 4b is a plot of percent activation of platelets treated in accordance with the invention, as a function of the concentration of the treatment agent, at two temperatures. FIG. 5 is a plot taken from Fourier transform infrared spectroscopy data, showing the same data as FIG. 1 together with data taken with platelets treated in accordance with the invention. FIG. 6 is a plot of fluorescence-activated cell sorting data, showing the percent secretion of a marker protein from treated and untreated platelets. FIG. 7 is another plot of fluorescence-activated cell sorting data, showing percent secretion of the marker protein as a function of the concentration of the treatment agent . DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS The existence of naturally-occurring macromolecular species known as "antifreeze proteins, " "thermal hysteresis proteins," "antifreeze glycoproteins," and "antifreeze polypeptides" is well known and widely reported in the literature. The discovery of antifreeze glycoproteins, for example, was first reported by DeVries, A.L., et al. , in "Freezing Resistance in Some Antarctic Fishes," Science 163:1073-1075 (7 March 1969). DeVries, et al . observed that various species of fish surviving in water at temperatures averaging -1.87°C over the course of a year did so despite having insufficient levels of sodium chloride and other low molecular weight substances in their blood to depress the freezing point by conventional freezing point depression. DeVries, et al . were able to attribute the survival of these species to the presence of certain glycosylated proteins having molecular weights ranging from about 2,500 to about 34,000, which are now referred to as antifreeze glycoproteins or "AFGPs. " Further investigations revealed that many species of north temperate and Arctic fishes carry antifreeze compounds in their blood. Some of these compounds are glycoproteins while others contain no sugar moieties and are referred to as antifreeze polypeptides or proteins ("AFPs") , with molecular weights ranging from about 3,300 to about 12,000. Furthermore, while the compounds lower the freezing point, the melting point remains unaffected, hence the term "thermal hysteresis proteins." Antifreeze proteins and glycoproteins have been isolated from a wide variety of sources, and these sources and the structures of the various proteins obtained from them have been reported extensively in the literature. The sources include both fish species and non-fish species, and are listed in Tables I and II below. TABLE I THERMAL HYSTERESIS PROTEINS OF FISH SPECIES Protein Type, Composition Trivial Name of and Size Source Fish Species Fish species Antifreeze glycoproteins Antarctic notothenioids: (AFGPs): Pagothema borchgrevmki contain alaniπe, Trematomus borchgrevmk i Antarctic cod threonine and Gal-GalNAc Tremato us berπachn disaccharide: Dissostichus mawsom H.U.: 2,600-33,700 Northern ocean gadoids: Gadus agac Greenland cod Gadus morhua Atlantic cod Microgadus tomcod Atlantic tomcod Boreogadus saida Arctic polar cod El igenus graci l is Saffron cod Antifreeze Polypeptides Righteye flounders: (AFPs), Type I: Pseudop leuronectus Winter flounder alaπine-rich; amencan s M. .: 3,300-6,000 L imanda ferrugmea Yellowtail flounder Cottids: Myoxycepha lus scorpius Shorthorn sculpin Myoxycepha 1 us aenaeus Grubby sculpin Myoxycepha lus scorp i odes Arctin sculpin Antifreeze Polypeptides Cottid: (AFPs), Type II: Herm tnpterus amencanus Sea raven cysteine-rich; Osmerus mordex Smelt homologous to C-type Clupea hareπgus harengus Herring lectins; M.W.: 14,000-16,000 Antifreeze Polypeptides Eel pouts: (AFPs), Type III: Macrozoarces amencanus Ocean pout no cysteines, and not rich R igop π la dearborm Antarctic eel pout in alanines; Ly 'codes polans Arctic eel pout M.W.: 5,000-6,700 TABLE II NON-FISH SOURCES OF THERMAL HYSTERESIS PROTEINS Insects Other Than Beetles: Order Species Collembola 7 spp. Plecoptera Arcynopteryx compacta Orthoptera Parcoblata pennsylvanica 10 Hemiptera Oncopel tus fasciatus Mecoptera Boreus westwoodi Lepidoptera Choristoneura fumiferana B. Coleoptera (Beetles) 15 Family Species Tenebrionidae Tenebrio moli tor Meracantha co tracta Uloma impressa Platydema sp. Elateridae A pedus lineatus Ampedus sp. Lepidσtus discoideus Melanotus sp. Cucujidae Cucujuε clavipes 20 Pyrochridae Dendroides canadensis Lampyridae Photinus sp. Coccinellidae Coccinella novemnotata Scolytidae Ips acuminatus Cerambycidae Rhagium inquisi tor 25 Non-Insect Arthropods: Animal Species Spiders Philodromus sp . Clubiona sp . Bolyphantes index Centipede Li thobius forfica tus Mite Alaskozetes antarcticus D. Other Invertebrates: Mussel Mytilus edulis The proteins which have been the most extensively studied, and which are the preferred proteins for use in the practice of the present invention, are those isolated from fish species. As indicated in Table I, these proteins include both glycosylated proteins (AFGPs) and non-glycosylated proteins (AFPs) , and the latter fall within three general categories, designated Type I, Type II, and Type III. The AFGPs generally consist of a series of repeats of the tripeptide unit alanyl-threonyl-alanyl, with the disaccharide S-D-galactosyl - (1→3) -o.-N-acetyl - D-galactosamine attached to the hydroxyl group of the threonine residue, although variations exist. For example, AFGPs of relatively low molecular weight contain proline and arginine residues in place of some of the alanine and threonine residues, respectively. Chromatographic studies of the AFGPs from representative fish species have revealed eight major molecular weight fractions, as indicated in Table III. TABLE III Molecular Weight Fractions of AFGPs From Pagothenia borchgrevinki The AFPs differ from one another to a larger degree than do the AFGPs. As indicated in Table I, the three types of AFPs differ from each other in their residue content. Type I AFPs are rich in alanine residues (about 65%) , with most of the remainder consisting of polar residues such as aspartic acid, glutamic acid, lysine, serine and threonine. The molecular weight ranges from about 3,300 to about 6,000. Type II AFPs are considered to be rich in cysteine (actually half-cysteine) residues, and are homologous to C-type lectins . Type II AFPs from the sea raven contain 7.6% cysteine, 14.4% alanine, 19% total of aspartic and glutamic acids, and 8% threonine. The molecular weight ranges from about 14,000 to about 16,000. Type III AFPs are devoid of cysteine residues and not rich in alanine residues. No conspicuous dominance of any particular amino acid is evident, and the amino acid content is evenly divided between polar and non-polar residues. The molecular weight ranges from about 5,000 to about 6,700. All percents referred to in this paragraph are on a mole basis. Antifreeze proteins from insects are primarily AFPs of Type II, and typical compositions in terms of amino acid residues are those of the Choristoneura fumiferana (spruce budworm) and Tenejbrio moli tor (beetle) . These are listed in Table IV, which also includes the amino acid composition of the sea raven for comparison. TABLE IV Comparative Amino Acid Compositions of Type II AFPs Antifreeze proteins and glycoproteins can be extracted from the sera or other bodily fluids of fish or insects by conventional means. Isolation and purification of the proteins is readily achievable by chromatographic means, as well as by absorption, precipitation, and evaporation. Other methods, many of which are described in the literature, will be readily apparent to those skilled in the art. Thermal hysteresis proteins may also be produced synthetically, either by conventional chemical synthesis methods or by methods involving recombinant DNA. The DNA coding sequences of the genes which form these proteins have been elucidated and are extensively reported. See, for example, DeVries, A.L., et al . , J. Biol. Chem. et al . , Biochem. Biophys. Res. Yang, D.S.C., et al . , Nature Proc. Natl. Acad. Sci. U.S.A. P.L., et al . , J. Biol. Chem. B., et al., J. Biol. Chem. 259:14960 (1984) ; Scott, G.K., et al . , Can. J. Fish. Aquat. Sci. 43:1028 (1986) ; Scott, G.K., et al . , J. Mol . Evol . 27:29 (1988) . Successful microinjection of the AFP gene into species other than its native species has also been reported. See, for example, Zhu, Z., et al . , Angew. Ichthyol . 1:31 (1985) ; Chourrout, D., et al . , Aquaculture 51:143 (1986) ; Dumman, R.A. , et al . , Trans. Am. Fish. Soc. 116:87 (1987) ; Fletcher, G.L., et al . , Can. J. Fish Aquat. Sci. 45:352 (1988) ; MacLean, N.D., et al . , Bio Technology 5:257 (1987) ; Stuart, G.W., et al . , Development 103:403 (1988) ; McEvoy, T., et al . , Aquaculture 68:27 (1988) ; Ozato, K, et al . , Cell Differ. 19:237 (1986) . Platelets may be isolated and conentrated according to conventional techniques such as those used to isolate platelets for platelet counting. According to one such technique, blood is collected in an anticoagulant, then centrifuged at a speed which is selected to produce a supernatant which is platelet-rich. Further concentration can be achieved by recovery of the supernatant followed by further centrifuging. Other methods are known to those skilled in the art . Treatment of the platelets with the antifreeze proteins and glycoproteins in accordance with this invention is readily accomplished by incubation of the platelets as a suspension in an aqueous solution of the treatment agent. For suspensions in which the platelets constitute from about 0.1 mg/mL to about 1.0 mg/mL of the suspension, the antifreeze proteins or glycoproteins will be present in an amount preferably ranging from about 0.1 mg/mL to about 30 mg/mL of the suspension, more preferably from about 0.5 mg/mL to about 20 mg/mL, and most preferably from about 0.5 mg/mL to about 10 mg/mL. The incubation is performed at a temperature low enough to avoid activation of the platelets by the antifreeze compounds, noting that the compounds tend to activate the platelets at 37°C. Platelets formed at temperatures in this range or higher are therefore cooled to room temperature before the antifreeze compounds are added. After incubation, the platelets can be maintained in the suspension and cooled to a temperature below 20°C until ready for use, or cooled after being further concentrated or recovered from the suspension. If desired, the platelets can be cooled to a temperature at or below the thermotropic phase transition temperature at which platelets undergo a transition from the liquid crystalline phase to the gel phase. For human platelets, this temperature is approximately 17°C. A preferred temperature range for storage purposes is about 1°C to about 10°C, with most typical storage conditions being about 1°C to about 6°C. When platelets are required for clinical use, they are most preferably warmed rapidly immediately prior to use by diluting the chilled sample about ten-fold with buffer at 37°C. Other means of treating the platelets with antifreeze proteins or glycoproteins to achieve an equivalent result will be readily apparent to those skilled in the handling of platelets. The following examples are offered by way of illustration rather than limitation. EXAMPLES Human blood was collected into acid citrate dextrose buffer (55 mM citric acid, 110 mM sodium citrate, 170 mM dextrose) by venous puncture without vacuum. Platelet- rich plasma was extracted from the buffer-diluted blood by gentle centrifugation, and the platelets were washed twice with the following solution: 100 mM NaCl 10 mM KC1 10 mM EGTA (ethylene glycol bis (/3-aminoethyl ether) N, N, N' , N' - tetraacetic acid) 10 mM imidazole pH = 6.8 The phase transition of the platelet membranes was determined by Fourier transform infrared spectroscopy, plotting the frequency of the symmetric CH 2 stretch against temperature. The plot is shown in FIG. 1. The midpoint of the frequency shift, which occurs at 17°C, was taken as the gel to liquid crystalline phase transition temperature of the membrane. Since this temperature corresponds exactly to the temperature at which human platelets are destabilized, this confirms that the destabilization is due to passage through the phase transition. Platelet activation as a function of temperature was initially determined using a morphological assay. The washed platelets were incubated at various temperatures for one hour. They were then fixed overnight with Karnovsky's fixative and counted with a Zeiss light microscope, using Νomarski optics with a lOOx objective. A total of 250 platelets were counted for each incubation temperature. If the platelets were globular, or had two or more pseudopodia, they were considered "activated". If the platelets were discoid and had zero to one pseudopodium, there were considered "resting" . A plot of the percent activated vs . temperature is given in FIG. 2, where the circles (•) represent the percent activated and the inverted triangles (V) are a repeat of the data in FIG. 1. The activation curve confirms that percent activation increases as the incubation temperature decreases. The plot also shows that the temperature at which activation increases most steeply is the same temperature at which the phase transition of the platelet membrane occurs . To test the effect of antifreeze proteins and glycoproteins on platelet activation, tests were performed using AFGP combined fractions 2-6 and 5-7 from rre_T.a o.Tius J ernachii and AFP Type I from Pseudopleuronectus americanus . Platelets suspended in solutions of the antifreeze compounds at -5 x 10 7 platelets/mL of suspension were incubated for one hour at temperatures ranging from 0°C to 40°C, in the presence of antifreeze compounds at concentrations of 1 to 4 mg of the compounds per mL of suspension, while parallel experiments wer performed without the antifreeze compounds. The results for the combined fractions AFGP 2-6 are shown in FIG. 3, where the inverted triangles (V) represent the tests performed with the antifreeze compounds and the circles (•) the tests performed without any antifreeze compounds. The plot shows that at an incubation temperature of 5°C, AFGP 2-6 decreases the level of activation by 43%. A comparison among different antifreeze compounds at 5°C is shown in Table I below. TABLE I Effect of Antifreeze Compounds on Percent Platelet Activation After Incubation at 5°C for l Hour FIGS. 4a and 4b illustrate the variation of protective effect with increasing concentration of the antifreeze compounds. In FIG. 4a, the combined fractions AFGP 5-7 were used at various concentrations in one-hour incubations at temperatures ranging from 5°C to 37°C. The filled circles (•) in the plot represent the control tests performed in the absence of the antifreeze compounds, the open squares (D) represent the test performed at 0.2 mg/mL concentration, the filled squares ( ■ ) the tests performed at 0.5 mg/mL concentration, the open circles (O) the tests performed at 1 mg/mL, the open triangles (Δ) the tests performed at 2 mg/mL, and the filled triangles (▼) the tests performed at 4 mg/mL. The data in FIG. 4a show that at increasing concentration of AFGP, there is a decrease in the percent activation of platelets at temperatures below 20°C. FIG. 4b shows similar data plotted as dose response curves for incubations at 5°C and 15°C. These curves show that at both temperatures, increasing the concentration of the antifreeze compounds results in a decrease in the percent activation, which means an increase in the percent protection of the platelets. To determine whether the antifreeze compounds actually interfere with platelet activation under normal activation conditions, platelets were incubated for one hour at 5°C and then rewarmed to 37°C, at which temperature their activation response to thrombin was tested. Platelets which had been incubated in the presence of 1 mg/mL AFGP 5-7 exhibited 96% activation, while platelets which had the same temperature exposure but in the absence of the AFGPs exhibited 97% activation, an insignificant difference. The conclusion is that the AFPGs did not interfere with physiological activation cascades as measured by morphological assessment. To establish that the antifreeze compounds do not achieve their effect by lowering the phase transition temperature of the membrane, the phase transition temperatures both in the presence of AFGPs (2 mg/mL AFGP 5-7) and in their absence were determined by Fourier transform infrared spectroscopy. The frequency of the symmetric CH 2 stretch vs . temperature, in the same manner as the data obtained for FIG. 1, is shown in FIG. 5. The two curves show no significant difference, confirming that the AFGPs did not alter the phase transition temperature. The foregoing experiments illustrate acute effects of AFGPs on morphological changes during chilling. In order to investigate more subtle changes during storage of the platelets below the lipid phase transition, a marker for secretion, which accompanies activation, was used. The marker chosen was an α.-granule protein, gp53. This protein is normally in membrane-bound vesicles in the cytoplasm of platelets, but is secreted to the cell surface during activation. In a series of experiments, the presence of the protein on the cell surface was detected by a fluorescent antibody to gp53, which is commercially available from Immunotech, Inc. (Westbrook, Maine, USA) . Interaction of the fluorescent antibody with secreted gp53 was detected by fluorescent activated cell sorting. FIG. 6 shows the percent of marker secreted as a function of the number of days of incubation at 5°C following the formation of the platelets. In this figure, platelets treated with AFGP combined fractions 5-7 from Tre.mato_τιus bernachii , represented by open squares (D) are compared with control platelets which were not treated with antifreeze compounds, represented by filled circles (•) . The data indicates that about 15% of the platelets of both sets showed secretion of gp53 immediately after chilling, a value that is only slightly higher than that seen in the platelets before chilling. After seven days at 5°C, nearly 50% of the control platelets showed activation showed activation, while the treated platelets remained at the initial value of 15%. To achieve this stabilization, re-warming of the chilled platelets had to be performed rapidly. This was done by diluting the chilled platelets into a relatively large volume (ten times the storage volume) of buffer at 37°C. This produced rapid warming of the platelets through the lipid phase transition, and also diluted the AFGPs. The protective effect of the AFGPs varies with the AFGP concentration, and this is shown in FIG. 7 for the same AFGP 5-7 fraction used above, after storage at 5°C for four days, followed by rapid warming to 37°C. At an AFGP concentration of 1.5 mg/mL, secretion was reduced to about 5%. The effect of the AFPGs on gp53 secretion shows considerable variability from one individual donor to the next, but in every case studied, the qualitative results were shown to be the same. The foregoing is offered primarily for purposes of illustration. It will be readily apparent to those skilled in the art that the choice of proteins, proportions, methods of treatment, and other parameters of the invention described herein may be further modified or substituted in various ways without departing from the spirit and scope of the invention.

Claims: WHAT IS CLAIMED IS: 1. A method for the treatment of blood platelets to reduce the incidence of spontaneous activation at temperatures below 20°C, said method comprising contacting said platelets with an activation-reducing amount of one or more thermal hysteresis proteins to incorporate said thermal hysteresis proteins into said biological materials. 2. A method in accordance with claim 1 in which said one or more thermal hysteresis proteins are proteins having the molecular structure of thermal hysteresis proteins isolated and purified from a polar fish species. 3. A method in accordance with claim 1 in which said polar fish species is a member selected from the group consisting of Antarctic notothenioids, northern ocean gadoids, righteye flounders, cottids and eel pouts. 4. A method in accordance with claim 1 in which said one or more thermal hysteresis proteins are members selected from the group consisting of: (a) antifreeze glycoproteins isolated and purified from a member selected from the group consisting of Pagothenia borchgrevinki, Trematomus borchgrevinki , Trematomus bernachii, and Dissostichus mawsoni; (b) Type I antifreeze polypeptides isolated and purified from a member selected from the group consisting of Pseudopleuronectus americanus and Limanda ferruginea; (c) Type II antifreeze polypeptides isolated and purified from Hemitripterus americanus; and (d) Type III antifreeze polypeptides isolated and purified from a member selected from the group consisting of Macrozoarces americanus, Rhigophila dearborni and Lycodes polaris. 5. A method in accordance with claim 1 in which said one or more thermal hysteresis proteins are members selected from the group consisting of: (a) antifreeze glycoproteins isolated and purified from a member selected from the group consisting of Dissostichus mawsoni and Trematomus bernachii; (b) Type I antifreeze polypeptides isolated and purified from Pseudopleuronectus americanus; (c) Type II antifreeze polypeptides isolated and purified from Hemitripterus americanus; and (d) Type III antifreeze polypeptides isolated and purified from Macrozoarces americanus. 6. A method in accordance with claim 1 in which said one or more thermal hysteresis proteins are antifreeze glycoproteins. 7. A method in accordance with claim 1 in which said one or more thermal hysteresis proteins are antifreeze glycoproteins fractions 2 through 6. 8. A method in accordance with claim 1 comprising incubating said platelets with an aqueous solution of said thermal hysteresis proteins to form an aqueous suspension of said platelets. 9. A method in accordance with claim 8 in which said thermal hysteresis proteins comprise from about 0.1 mg/mL to about 30 mg/mL of said suspension. 10. A method in accordance with claim 8 in which said thermal hysteresis proteins comprise from about 0.5 mg/mL to about 20 mg/mL of said suspension.

Inventors: CROWE JOHN H, CROWE LOIS M, FEENEY ROBERT E, HAYS LISA M, OLIVER ANN E, TABLIN FERN, CROWE, JOHN, H., CROWE, LOIS, M., FEENEY, ROBERT, E., HAYS, LISA, M., OLIVER, ANN, E., TABLIN, FERN
Applicants: UNIV CALIFORNIA, THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Application year: 1995
Main classification: C12N5
IPC codes: A01N1, A61K35, A61M1, C07K14, C12N5