EP0871706A1 - Stabilisation des plaquettes sanguines vis-a-vis de l'activation a basse temperature - Google Patents

Stabilisation des plaquettes sanguines vis-a-vis de l'activation a basse temperature

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Publication number
EP0871706A1
EP0871706A1 EP95944344A EP95944344A EP0871706A1 EP 0871706 A1 EP0871706 A1 EP 0871706A1 EP 95944344 A EP95944344 A EP 95944344A EP 95944344 A EP95944344 A EP 95944344A EP 0871706 A1 EP0871706 A1 EP 0871706A1
Authority
EP
European Patent Office
Prior art keywords
platelets
antifreeze
thermal hysteresis
proteins
purified
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95944344A
Other languages
German (de)
English (en)
Other versions
EP0871706A4 (fr
Inventor
Fern Tablin
John H. Crowe
Ann E. Oliver
Lisa M. Hays
Lois M. Crowe
Robert E. Feeney
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of California
Original Assignee
University of California
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of California filed Critical University of California
Publication of EP0871706A1 publication Critical patent/EP0871706A1/fr
Publication of EP0871706A4 publication Critical patent/EP0871706A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/19—Platelets; Megacaryocytes

Definitions

  • This invention lies in the field of blood and blood components, with special emphasis on the function and usefulness of platelets.
  • this invention addresses the problem of platelet activation during storage and the need to conmrol both undesired activation and bacterial infection.
  • Platelets 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 ⁇ m, 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.
  • 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.
  • 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.
  • 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.
  • antifreeze proteins 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.
  • glycosylated proteins having molecular weights ranging from about 2,500 to about 34,000, which are now referred to as antifreeze glycoproteins or "AFGPs.”
  • AFGPs antifreeze glycoproteins
  • AFPs antifreeze polypeptides or 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.
  • 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.
  • AFGPs glycosylated proteins
  • AFPs non-glycosylated proteins
  • the AFGPs generally consist of a series of repeats of the tripeptide unit alanyl-threonyl-alanyl, with the disaccharide ⁇ -D-galactosyl- (1 ⁇ 3)- ⁇ -N-acetyl- D- ⁇ alactosamine attached to the hydroxyl group of the threonine residue, although variations exist.
  • 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.
  • 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 Tenebrio moli tor (beetle). These are listed in Table IV, which also includes the amino acid composition of the sea raven for comparison.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ omarski optics with a 100 ⁇ 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 ( ⁇ ) 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.
  • FIGS. 4a and 4b illustrate the variation of protective effect with increasing concentration of the antifreeze compounds.
  • 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 ( ⁇ ) represent the test performed at 0.2 mg/mL concentration, the filled squares
  • FIG. 4a shows 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.
  • 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.
  • 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 two curves show no significant difference, confirming that the AFGPs did not alter the phase transition temperature.
  • 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.
  • platelets treated with AFGP combined fractions 5-7 from Trematomus bernachii represented by open squares ( ⁇ ) 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%.
  • 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.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
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  • Wood Science & Technology (AREA)
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  • Cell Biology (AREA)
  • Biotechnology (AREA)
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Abstract

L'activation spontanée des plaquettes sanguines aux températures de stockage normalement peu élevées peut s'éliminer par le traitement des plaquettes à l'aide de protéines à hystérèse thermique. Les protéines de ce type préférées sont des protéines et des glycoprotéines antigel provenant d'espèces de poissons polaires. Les fractions chromatographiques N° 2 à 6 des glycoprotéines antigel s'avèrent particulièrement efficaces.
EP95944344A 1995-01-05 1995-12-18 Stabilisation des plaquettes sanguines vis-a-vis de l'activation a basse temperature Withdrawn EP0871706A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US36894195A 1995-01-05 1995-01-05
US368941 1995-01-05
PCT/US1995/016519 WO1996021001A1 (fr) 1995-01-05 1995-12-18 Stabilisation des plaquettes sanguines vis-a-vis de l'activation a basse temperature

Publications (2)

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EP0871706A1 true EP0871706A1 (fr) 1998-10-21
EP0871706A4 EP0871706A4 (fr) 2001-05-02

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EP95944344A Withdrawn EP0871706A4 (fr) 1995-01-05 1995-12-18 Stabilisation des plaquettes sanguines vis-a-vis de l'activation a basse temperature

Country Status (6)

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EP (1) EP0871706A4 (fr)
JP (1) JP2001513069A (fr)
CN (1) CN1220696A (fr)
AU (1) AU696094B2 (fr)
CA (1) CA2207892A1 (fr)
WO (1) WO1996021001A1 (fr)

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DE102009059274B4 (de) * 2009-12-22 2012-07-19 Max-Joseph Kraus Verfahren zur Messung der Dynamik der Änderungen von Blutplättchen
CN113712023B (zh) * 2021-04-02 2023-07-04 苏州大学 一种血小板的保存方法

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US5358931A (en) * 1990-01-17 1994-10-25 The Regents Of The University Of California Interaction of thermal hysteresis proteins with cells and cell membranes and associated applications

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No further relevant documents disclosed *
See also references of WO9621001A1 *

Also Published As

Publication number Publication date
AU696094B2 (en) 1998-09-03
CN1220696A (zh) 1999-06-23
AU4641596A (en) 1996-07-24
CA2207892A1 (fr) 1996-07-11
WO1996021001A1 (fr) 1996-07-11
EP0871706A4 (fr) 2001-05-02
JP2001513069A (ja) 2001-08-28

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