JP2001513069A - Platelet stabilization against cold activation - Google Patents

Abstract

(57) Abstract The low-temperature, spontaneous activation of platelets commonly used for blood storage is reduced or eliminated by treating the platelets with a thermal hysteresis protein. Preferred thermal hysteresis proteins are antifreeze proteins and antifreeze glycoproteins from polar fish, and chromatographic fractions 2-6 of the antifreeze glycoprotein have been found to be particularly effective.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the field of blood and blood components with particular emphasis on platelet function and utility. In particular, the present invention addresses the issue of platelet activation during storage and the need to control both unwanted activation and bacterial infection. GOVERNMENT RIGHTS This invention was supported, at least in part, by the United States Government under grant number IBN 93-08581 awarded by the National Science Foundation and grant number N00014-94-1 awarded by the Maritime Research Office. Was done. The United States Government has certain rights in the invention. BACKGROUND OF THE INVENTION Platelets are a fraction of human blood that contribute significantly to hemostasis. Platelets are usually oval to spherical with a diameter of 2-4 μm and contain about 60% protein, 15% lipids, and 8.5% carbohydrates. The chemical composition of platelets contains serotonin, epinephrine, and norepinephrine, each of which helps promote vascular architecture at the site of injury. Platelets also contain thromboplastin, a cephalin-type phosphatide, and platelet factors, including adenosine diphosphate, both of which are important for blood coagulation. Maintaining functional platelets is important when storing whole blood for storage in blood banks and when storing concentrated platelet fractions. However, storage of platelets presents certain problems. The remaining fraction of the blood can be stored for long periods by cryopreservation, but platelets tend to activate at low temperatures and are therefore unusable. To avoid activation, platelets are isolated and stored separately at 20 ° C. as concentrated fractions. However, this creates other risks: bacterial infections and metabolic and enzymatic reactions generally known as "platelet storage damage". As a result, the storage of platelets is generally limited to 5 days. SUMMARY OF THE INVENTION By treating platelets with proteins known as cryoprotective proteins and cryoprotective glycoproteins, platelets are treated at temperatures low enough to control bacterial infection and platelet storage damage without the risk of premature activation. We found that it can be stored for a long time. Processing is easily accomplished in a variety of ways, including suspending the platelets in a solution of the protein. Platelets can be maintained in this state until use. Thus, the need to periodically refresh the stored platelet supply is reduced, and the difficulty in meeting various demands is reduced. Tests performed to demonstrate the invention have shown that the protective effect achieved by cryoprotective glycoproteins depends on the amount of cryoprotective glycoprotein used. It has also been found that high molecular weight antifreeze glycoproteins tend to have a greater protective effect at a given concentration than relatively low molecular weight ones. These and other features and advantages of the invention will be apparent from the description below. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a plot from Fourier transform infrared spectroscopy data showing the phase transition from gel to liquid crystal of human platelets. FIG. 2 shows the same data as FIG. 1 and a plot of platelet activation percentage as a function of temperature. FIG. 3 is a plot of percent activation as a function of temperature for both platelets treated with the present invention and platelets not treated with the present invention. FIG. 4a is a plot of percent activation as a function of temperature for both treated and non-treated platelets of the invention, including different treatment levels. FIG. 4b is a plot of percent activation of platelets treated according to the present invention as a function of treatment agent concentration at two temperatures. FIG. 5 is a plot from Fourier transform infrared spectroscopy data showing the same data as in FIG. 1 and data obtained with platelets treated according to the present invention. FIG. 6 is a plot of fluorescence activated cell sorting data showing percent secretion of labeled protein from treated and untreated platelets. FIG. 7 is another plot of fluorescence activated cell sorting data showing percent secretion of labeled protein as a function of treatment agent concentration. DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS The existence of naturally occurring macromolecular species known as "freeze-protecting proteins", "thermal hysteresis proteins", "freezing-protecting glycoproteins", and "freezing-protecting polypeptides" is known. , Widely reported in the literature. For example, the discovery of an antifreeze glycoprotein was first reported by DeVries, AL, et al., "Freezing Resistance in Some Antarctic Fishes," Science 163: 1073-1075 (March 7, 1969). . Diverse et al. Report that various species of fish that live in water at an average temperature of -1.87 ° C over the course of the year have sodium chloride and other salts at levels insufficient to lower the freezing point by conventional freezing. It was observed that it was alive despite having no low molecular weight substances in the blood. Say that the survival of these species is attributed to the presence of a particular glycated protein with a molecular weight of about 2,500 to about 34,000, now termed the cryoprotective glycoprotein or “AFGP”. I was able to show that. Further investigation revealed that many species of northern temperate and arctic fish have antifreeze compounds in their blood. Some of these compounds are glycoproteins, while others do not contain the sugar moiety of cryoprotective polypeptides or proteins ("AFP") having a molecular weight of about 3,300 to about 12,000. In addition, the term "thermal hysteresis protein" is used because these compounds lower the freezing point but do not affect the melting point. Antifreeze proteins and glycoproteins have been isolated from various types of sources, and the structures of these sources and the various proteins obtained from these sources have been widely reported in the literature. Sources include both fish and non-fish, which are listed in Tables I and II below. Table I Table II The most widely studied and preferred proteins for use in the practice of the present invention are those isolated from fish. As shown in Table I, these proteins include both glycated proteins (AFGPs) and non-glycated proteins (AFPs), the latter of which fall into three general classes: type I, type II and type III as indicated. Belong. AFGPs usually comprise a series of repeating tripeptide units, alanyl-threonyl-alanyl, and the disaccharide β-D-galactosyl- (1 → 3) -α-N-acetyl attached to the hydroxyl group of a threonine residue. Consists of -D-galactosamine, but there are variants. For example, relatively low molecular weight AFGPs contain proline and arginine residues instead of some alanine and threonine residues, respectively. Chromatographic studies of AFGPs from representative fish revealed eight major molecular weight fractions as shown in Table III. Table III AFP is much different from AFGP. As shown in Table I, the residue content of the three types of AFP is different from each other. AFP type I is rich in alanine residues (approximately 65%), most of which consists 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. AFP type II is said to contain many cysteine (actually half cysteine) residues and is homologous to type C lectin. Form AFP II from Deer stag contains 7.6% cysteine, 14.4% alanine, a total of 19% aspartic and glutamic acid, and 8% threonine. The molecular weight ranges from about 14,000 to about 16,000. AFP type III has no cysteine residues and has few alanine residues. There is no significant dominance of particular amino acids, 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 percentages shown in this paragraph are on a molar basis. Antifreeze proteins from insects are mainly of type AFP II, and the typical composition of the amino acid residues is that of Coristoneurafmiferana (Coleoptera moth larva) and Tenebrio molitor (Coleoptera) It is. These are listed in Table IV. Table IV also includes the amino acid composition of the deer stag for comparison. Table IV Antifreeze proteins and glycoproteins can be extracted from fish or insect serum or other body fluids by conventional means. Protein isolation and purification can be readily achieved by chromatographic means and 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 can also be produced synthetically by conventional chemical synthesis methods or methods involving recombinant DNA. The DNA coding sequences of the genes that form these proteins have been elucidated and widely reported. For example, Devrees et al. Biol. Chem. 246: 305 (1971); Lin, Y., et al., Biochem. Biophys. Res. Commun. 46:87 (1972); Yang et al. (Yang, DSC, et al.) Nature 333: 232 (1988); Lynn, Proc. Natl. Acad. Sci. USA. 78: 2825 (1981); Davies, PL, et al., J. Am. Biol. Chem. 79: 335 (1982); Gourlie, B., et al., J. Am. Biol. Chem. 259: 14960 (1984); Scott, GK, et al., Can. J. Fish. Aquat. Sci. 43: 1028 (1986); Scott et al. Mol. Evol. 27:29 (1988). Successful microinjection of the AFP gene into species other than the native species has also been reported. See, for example, Zhu, Z., et al., Angew. Ichthyol. 1:31 (1985); Schrout et al. (Chourrout, D., et al.), Aquaculture 51: 143 (1986); Duman et al. (Dumman, RA, et al.), Trans. Am. Fish. Soc. 116: 87 (1987); Fletcher, GL, et al., Can. J. Fish Aquat. Sci. 45: 352 (1988); McLean, ND, et al., Bio Technology 5: 257 (1987); Stuart, et al. (Stuart, GW, et al.), Development 103: 403 (1988); McKevoy. (McEvoy, T., et al.), Aquaculture 68:27 (1988); Ozato, K, et al., Cell Differ. 19: 237 (1986). Platelets can be isolated and concentrated according to conventional techniques such as those used to isolate and count platelets. According to one such technique, blood is collected in an anticoagulant and then centrifuged at a speed selected to produce a platelet-rich supernatant. Further concentration can be achieved by collecting the supernatant after further centrifugation. Other methods are known to those skilled in the art. Treatment of platelets with antifreeze proteins and glycoproteins according to the present invention is readily accomplished by incubating platelets as a suspension in an aqueous solution of the treatment. For suspensions where the platelets make up about 0.1 mg / ml to about 1.0 mg / ml of the suspension, the cryoprotective protein or glycoprotein is present in about 0. Preferably, it is present in an amount ranging from 1 mg / ml to about 30 mg / ml, and is preferably about 0.1 mg / ml. An amount ranging from 5 mg / ml to about 20 mg / ml is more preferred, with an amount ranging from about 0.5 mg / ml to about 10 mg / ml being most preferred. Incubations are performed at a temperature low enough to avoid platelet activation by the cryoprotecting compound, noting that the cryoprotecting compound tends to activate platelets at 37 ° C. Thus, platelets formed at a temperature in this range or above are cooled to room temperature before adding the antifreeze compound. After incubation, the platelets can be kept in suspension and cooled to a temperature below 20 ° C. until use, or cooled after further concentration or recovery from the suspension. If necessary, the platelets can be cooled to a temperature below or below the thermotropic phase transition temperature where the platelets transition from the liquid crystal phase to the gel phase. For human platelets, this temperature is about 17 ° C. The preferred temperature range for storage is from about 1C to about 10C, with the most typical storage conditions being from about 1C to about 6C. If platelets are needed for clinical use, it is most preferred to warm up immediately before use, by diluting the chilled sample approximately 10-fold with 37 ° C. buffer. Other means of treating platelets with cryoprotective proteins or glycoproteins to achieve equivalent results will be readily apparent to those skilled in treating platelets. The following examples are provided by way of illustration, not limitation. EXAMPLES Human blood was collected by non-vacuum venipuncture and placed in ACD buffer (55 mM citrate, 110 mM sodium citrate, 170 mM dextrose). Platelet-rich plasma was extracted from the blood diluted with buffer by light centrifugation and the platelets were washed twice with the following solutions: NaCl 100 mM KCl 10 mM EGTA 10 mM (ethylene glycol bis (β-aminoethyl ether ) N, N, N ', with N'- tetraacetic acid) imidazole 10 mM pH = 6.8 Fourier transform infrared spectroscopy, by plotting the extent of the frequency versus temperature symmetric CH _2, a phase transition of the platelet membrane decision did. This plot is shown in FIG. The midpoint of the frequency change occurring at 17 ° C. was taken as the phase transition temperature from the gel of the film to the liquid crystal. This confirms that the destabilization is due to a phase transition, since this temperature exactly corresponds to the temperature that destabilizes human platelets. Initially, platelet activation as a function of temperature was determined using a morphological assay. The washed platelets were incubated at various temperatures for 1 hour. Next, these were fixed overnight with Karnofsky's fixative, and the numbers were examined with a Zeiss optical microscope using a Nomarski optical system with a 100-times objective. At each incubation temperature, a total of 250 platelets were counted. If the platelets were spherical, ie, had two or more pseudopodia, they were considered "activated". If the platelets were disk-shaped and had 0-1 pseudopodia, they were considered "stable". A plot of percent activity versus temperature is shown in FIG. Here, a circle (●) represents the percentage of activation, and an inverted triangle (▽) is a copy of the data of FIG. The activation curves confirm that the percent activity increases as the incubation temperature decreases. The plot also shows that the temperature at which activation rises most rapidly is the same as the temperature at which the phase transition of the platelet membrane occurs. To investigate the effect of cryoprotective proteins and glycoproteins on platelet activation, AFGP-binding fractions 2-6 and 5-7 from Trematomus Vernakey and AFP type I from Pseudopleuronectus americans were used. And did a test. Platelets suspended in a solution of the antifreeze compound in 5 × 10 ⁷ platelets / ml suspension were frozen at a temperature of 0 ° C. to 40 ° C. for 1 hour at a concentration of 1-4 mg compound / ml suspension. A similar experiment was performed without the antifreeze compound, while incubating in the presence of the antifreeze compound. The results of the bound fractions AFGP2-6 are shown in FIG. Here, an inverted triangle (▽) indicates a test performed using the antifreeze compound, and a circle (●) indicates a test performed without any antifreeze compound. This plot shows that at an incubation temperature of 5 ° C, AFGPs 2-6 reduce the activation level by 43%. A comparison between the different antifreeze compounds at 5 ° C. is shown in Table I below. TABLE I Effect of cryoprotectant compounds on percent platelet activation after 1 hour incubation at 5 ° C Figures 4a and 4b show the change in the protective effect with increasing concentrations of the antifreeze compound. In FIG. 4a, the bound fractions 5-7 were used at various concentrations during a 1 hour incubation at a temperature between 5 ° C. and 37 ° C. The black circles (●) in the plot represent control tests performed without the antifreeze compound, the white squares (□) represent tests performed at a concentration of 0.2 mg / ml, and the black squares (■) represent 0.5 mg / ml. The white circle () represents the test performed at 1 mg / ml, the white triangle (△) represents the test performed at 2 mg / ml, and the black triangle (▼) represents the test performed at 4 mg / ml. The data in FIG. 4a shows that as the concentration increases, the activity of platelets decreases at temperatures below 20 ° C. FIG. 4b shows similar data plotted as concentration response curves for 5 ° C. and 15 ° C. incubations. These curves show that at both temperatures, the percentage of activity decreases with increasing concentration of the antifreeze compound, which means an increase in the percentage of platelet protection. To determine whether the cryoprotectant compound actually prevents platelet activation under normal activation conditions, platelets are incubated at 5 ° C. for 1 hour, then re-warmed to 37 ° C. and at this temperature The response of platelets to thrombin was examined. Platelets incubated in the presence of 1 mg / ml of AFGP5-7 showed 96% activation, and platelets incubated at the same temperature without AFGP showed 97% activation with little difference. In conclusion, AFPG did not interfere with the physiological activation cascade as measured by morphological evaluation. In order to establish that the antifreeze compound does not gain its effect by lowering the phase transition temperature of the membrane, the phase transition temperature in both the presence (AFGP 5-72 mg / ml) and in the absence of AFGP, Determined by Fourier transform infrared spectroscopy. Similar to the data obtained in FIG. 1, the frequency versus temperature of the symmetric CH ₂ spread is shown in FIG. There is no significant difference between the two curves, confirming that AFGP did not change the phase transition temperature. The above experiments show the serious effect of AFGP on morphological changes upon cooling. To examine subtle changes in storing platelets below the lipid phase transition, labels for secretion with activation were used. The label selected was α-granular protein, gp53. Normally, this protein is in the membrane-bound vesicles of the cytoplasm of platelets, but is secreted to the cell surface upon activation. In a series of experiments, the presence of proteins on the cell surface was detected by a fluorescently labeled antibody against gp53, commercially available from Immunotech, Inc. (Westbrook, ME, USA). The interaction of the fluorescently labeled antibody with secreted gp53 was detected by sorting fluorescently activated cells. FIG. 6 shows the percent secreted label as a function of days of 5 ° C. incubation performed after platelet formation. In this figure, platelets treated with the AFGP-bound fraction from Trematmus vernaquee represented by white squares (□) are compared to control platelets not treated with the cryoprotectant compound represented by solid circles (●). The data show that about 15% of the platelets in both sets secreted gp53 immediately after cooling, which is slightly higher than that seen before cooling. After storage at 5 ° C. for 7 days, about 50% of the control platelets showed activation, but the treated platelets remained at the initial value of 15%. To achieve this stabilization, the cooled platelets had to be rapidly re-warmed. This was done by diluting the chilled platelets at 37 ° C. in a relatively large volume (10 times the stored volume) of buffer. This rapidly warmed the platelets via lipid phase transition and diluted the AFGP. The protective effect of AFGP depends on the concentration of AFGP. The protective effect of the same AFGPs 5-7 used above, stored at 5 ° C. for 4 days and then rapidly warmed to 37 ° C., is shown in FIG. At a concentration of 1.5 mg / ml AFGP, secretion was reduced to about 5%. The effect of AFPG on gp53 secretion varied significantly with each donor, but in each case studied, the qualitative results were found to be identical. The foregoing is provided primarily for purposes of illustration. Further altering or substituting the selection, proportions, processing methods, and other parameters of the proteins of the invention described herein in various ways without departing from the spirit and scope of the invention. Will be readily apparent to those skilled in the art.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, M C, NL, PT, SE), OA (BF, BJ, CF, CG , CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, SZ, U G), AL, AM, AT, AU, BB, BG, BR, B Y, CA, CH, CN, CZ, DE, DK, EE, ES , FI, GB, GE, HU, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LS, LT, LU, L V, MD, MG, MK, MN, MW, MX, NO, NZ , PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TT, UA, UG, UZ, VN (72) Inventor Oliver, Ann E.             United States 95616 California             State Davis Russell Park 413-             4 (72) Inventor Hayes, Lisa M.             United States 95616 California             Davis Sea Street 117 (72) Inventor Klaue, Royce M.             United States 95616 California             State Davis Mulberry Lane 764 (72) Inventor Feeney, Robert E.             United States 95616 California             Elvis Wood Drive, Davis, Oregon             721

Claims (1)

[Claims]   1. Blood platelets that reduce the occurrence of spontaneous activation at temperatures below 20 ° C The method of claim 1, wherein the platelets are activated with a reduced amount of one or more thermal hysteresis stampers. A method for incorporating a thermohysteresis protein into a biological material by contacting the same with a substrate. .   2. More than one heat hysteresis protein is isolated and purified from polar fish 2. The protein having a molecular structure of a heat hysteresis protein. The method described.   3. Polar fishes include Antarctic Nototenioids, North Sea cod, Flatfish, and Swordfish The method of claim 1, wherein the member is a member selected from the group consisting of Agonaceae.   4. One or more thermohysteresis proteins are selected from the group consisting of (a) Pagotenia Volkgrev Ink, Trematmus Bork Grevinki, Trematmus Bernakie and Di Isolated from a member selected from the group consisting of (B) pseudopreuronectus americanus and Isolated and purified from a member selected from the group consisting of rimanda ferguinaire Antifreeze polypeptide type I; (c) isolated from hemitripterus americanus Purified antifreeze polypeptide type II; and (d) macrozoarces americanus , A member selected from the group consisting of Rigophyla de Alborni and Ricodesporalis Selected from the group consisting of antifreeze polypeptide type III isolated and purified from a bar 2. The method of claim 1, wherein said member is a member.   5. One or more thermal hysteresis proteins are (a) Isolated from a member selected from the group consisting of Soni and Trematmus Bernakie (B) Pseudopreuronectus America Cryoprotective polypeptide type I isolated and purified from C. heus; (c) hemitripterus An antifreeze polypeptide type II isolated and purified from Americanus; Is antifreeze polypeptide type III isolated and purified from Rozoarces americanus? The method of claim 1, wherein the member is a member selected from the group consisting of:   6. The method of claim 1, wherein the one or more heat-hysteresis proteins is an antifreeze glycoprotein. The method of claim 1.   7. One or more thermal hysteresis proteins are in frozen protein fraction 2 The method of claim 1, wherein the number is 6.   8. Incubating the platelets with an aqueous solution of the thermal hysteresis protein, 2. The method of claim 1, comprising forming an aqueous suspension of platelets.   9. The thermal hysteresis protein comprises about 0.1 mg / ml to about 30 mg of the suspension. 9. The method of claim 8, wherein the method comprises: 10. The thermal hysteresis protein comprises about 0.5 mg / ml to about 20 mg of the suspension. 9. The method of claim 8, wherein the method comprises: