HU211345A9 - Blood platelet membrane micro fragments - Google Patents

Description

The present invention relates generally to the field of medicine, and more particularly to a platelet membrane microparticle composition for use as a transfusion to control bleeding.

BACKGROUND OF THE INVENTION

An adult human body contains between 4.0 and 5.5 liters of blood, which is made up of about 60% fluid (plasma) and 40% body (red blood cells, white blood cells and platelets). Under normal physiological conditions, the main function of platelets is to prevent haemorrhage (bleeding).

Platelets are formed in bone marrow from so-called megakaryocyte precursor cells and have a circulation life of 8 to 10 days. Due to this short life span, platelet deficiency occurs rapidly when bone marrow platelet production capacity is reduced, for example in cancer patients receiving chemotherapy. The decrease in platelet count can occur in many diseases, for example, when in vivo antibodies are formed against platelet surface glycoproteins or other platelet surface antigens. The reduction or death of platelets results in an inadequate amount of circulating platelets (a condition known as thrombocytopenia, or decreased platelet count), which can lead to uncontrolled bleeding. Platelet deficiency can also be the result of a surgical procedure in which blood is circulated out of the body, which contributes to the damage or destruction of circulating platelets. Clinical treatment of thrombocytopenia is typically performed by transfusion of fresh, intact platelets.

It is strongly believed that only metabolically active, intact platelets can act in vivo to stop bleeding. As a result, transfusion therapy depends on the availability of high quality, fresh, viable platelets. Typically, a transfusion platelet preparation is prepared in two ways: (a) from freshly collected blood units from donors, called random-donor platelets, or (b) single-donor apheresis, called single-donor platelets. These transfusion formulations are plasma suspensions of fresh, concentrated, intact platelets.

The major disadvantage of current platelet transfusion practice is the short shelf life of intact platelets, 3-5 days. Unfortunately, many of the platelet units that have been collected by hospitals and blood banks are destined for destruction due to expiration. Due to the short storage life of platelet units, it is difficult to maintain large stocks of platelets. This problem becomes particularly critical in the case of decentralized use, such as in the military, civil defense, and emergency services.

Both clinically and animal models have attempted to use some substitutes instead of intact, live platelets. In 1956 (1), it was reported that after the clinical administration of lyophilized platelets, hemostasis was achieved, suggesting that the morphological integrity of platelets is not essential in that at least some of the in vivo functions of intact platelets are preserved. The major side effect of intravenous administration of the lyophilized platelet agent was that the patient experienced severe pain at the site of the infusion, probably due to the presence of high levels of serotonin in the lyophilized substance. Contrary to previous results, it was reported in 1959 (2) that fresh ultrasound-degraded whole platelet preparations were unable to reduce erythrocytes in the lymph of thrombocytopenic dogs. Recently, McGill et al. (3) reported that transfusion of platelet membrane concentrates reduced the bleeding time of thrombocytopenic rabbits. The concentrate also contained ghost platelets, approximately the size of normal platelets, containing mitochondria and remnants of a surface switch system. The steps of preparing a Mc Gill concentrate include: (1) centrifuging whole rabbit blood to settle fresh platelets; (2) freezing the deposited platelets at -65 ° C; (3) thawing, then freezing and thawing a second time; and (4) rinsing and re-suspending the precipitated platelets in platelet-free plasma.

The temperature of the platelet membrane fractions described above is always maintained at or below 4 ° C. This temperature is constant when working with biological materials, since activity is usually lost in a very short time if the biological material is exposed to a higher temperature. For example, proteins such as enzymes may be inactivated when heated to about 60 ° C. However, activity may be lost at 4 ° C. For example, it has been reported that partially purified platelet factor 3 (PF-3) loses much of its clotting activity during storage for 5 days at 4 ° C (4). (PF-3 appears to be associated with a platelet membrane complex that provides a catalytic surface to promote thrombin formation). Such loss of activity is a major concern if the platelet fraction is to be used as a pharmaceutical.

Of course, when preparing the platelet fraction for human use, sterile conditions should be employed. Like whole blood transfusion, donor platelet units expose the recipient to transmissible diseases including AIDS, hepatitis, and other transfusions.

EN 211 345 A9 Related diseases. There is also the risk that after several transfusions, the recipient / patient may develop antibodies to the platelets they receive (this condition is called alloinunonisation). Such antibodies can cause rapid degradation of transfused platelets. Furthermore, bacterial contamination of stored platelets poses a significant risk of transfusions.

SUMMARY OF THE INVENTION

The present invention relates to medical and diagnostic products obtained by a novel process from platelet membranes. The products include platelet membrane fragment formulations which have a procoagulant effect and which are capable of replacing whole platelets by transfusion inhibition, may be used topically to promote wound healing, or may be used as in vitro or in vivo diagnostic agents.

In one aspect of the invention, the platelet membrane fragment composition is free of active virus. A preferred method of preparing the composition involves heat treating the platelet membrane fragment composition to reduce or eliminate viral contamination and to reduce or eliminate bacterial infection. Unexpectedly, the composition, although heat-treated, retains its procoagulant and antihypertensive properties. In addition, the heat-treated membrane fragment is much more stable than would be expected. The formulation may be stored in solution at 4 ° C for at least 8 weeks without significant reduction in procoagulant activity and retains more than 90% of its activity after at least 6 months. It retains activity (above 90%) even after lyophilization. In addition, protein and phospholipid content is unaffected by either lyophilization or storage for six months.

In another aspect of the invention, the composition is non-immunogenic and, in particular, non-antigenic with respect to HLA class I antigenic determinants. Accordingly, the applicability of the composition to a given recipient is not limited by the genetic characteristics of the donor.

In another aspect of the invention, the platelet membrane fragment composition, which has procoagulant activity and is capable of inhibiting bleeding, is substantially free of GPIIb / IIIa complex. This is surprising given the widespread belief that the GPIIb / IIIa complex is involved and necessary in bleeding.

The platelet membrane fragment composition is preferably substantially free of "ghost" platelets and contains relatively homogeneous microparticles. Preferably, at least 80% of the microparticles are less than 600 nm in diameter and at least 95% are less than 1 micron in diameter. More preferably, the microparticles have an average diameter of about 300 to about 400 nm. The size of such microparticles is about 1/5 to 1/7 the size of a typical ghost platelet.

The preferred formulation is substantially free of serotonin (less than 0.02% is present in the platelet lysate), thus eliminating the respiratory and vascular problems typically encountered in the transfusion of prior art formulations. Further, the formulations do not scavenge for detectable purine nucleoside phosphorylase activity, a cytoplasmic enzyme marker, and are substantially free of factors V, VIII, IX, and X. In one embodiment, the microparticle fraction comprises 3% carbohydrate, 30% phospholipid, 58% protein and 9% cholesterol and has a protein ratio to phospholipid of 1.97 ± 0.10 (mean + SD, n = 7).

Unexpectedly, platelet membrane fragment formulations may be prepared from platelets after use. Typically, hospitals and other institutions store transfusion platelets at room temperature for 3 to 5 days. Thereafter, platelets are considered unfit for use and discarded as "after-use platelets". It has now been discovered that the pharmaceutical and diagnostic compositions of the present invention may be prepared from platelets beyond such useful life. Thus, the present invention has the advantage that the transfused platelet membrane fraction can be wholly or partially obtained from platelets beyond the useful life, thereby utilizing the large amount of platelets that have been destroyed so far that are unusable.

The present invention also relates to the preparation of platelet-derived compositions according to the invention. In one aspect of the invention, a composition comprising a platelet or platelet membrane fragment having prothrombinase activity is treated under conditions favorable to the inactivation of viruses in the composition. Preferably, the composition is heat treated. Such a composition can also be homogenized, preferably by sonication, so that the resulting platelet membrane microparticles are substantially free of ghost platelets. The composition can also be treated for substantially complete removal of the GPIIb / IIIa surface glycoprotein complex.

The present invention relates to a process for the production of platelet-derived products from platelets or membrane fragments thereof that have an after-use time as a starting material. Platelets beyond the shelf life may be derived from a single donor or may be collected from multiple donors. The method may include viral inactivation, treatment to generate microparticles, and treatment that substantially eliminates GPIIa / IHa.

The most preferred process for the preparation of the products of the invention involves re-freezing-thawing and washing the platelets to obtain primarily ghost platelets and a lysate. The ghost platelets are then separated from the lysate and resuspended in solution. The resulting suspension of ghost platelets is heated to at least 60 ° C and maintained at this temperature for at least 2 hours for viral contamination.

EN 211 345 A9 divorce. Heat treatment also involves precipitation. However, the precipitate is not removed at this point of the process since the precipitate contains a significant portion of the desired activity. Instead, the suspension containing the precipitate is first homogenized, preferably by ultrasound, and then the precipitate is separated from the suspension. The suspension can then be stored or used for transfusion.

The platelet membrane fragment composition can be used in a therapeutically effective amount to treat animals and humans to prevent bleeding. When the composition of the invention is used as a pharmaceutical composition for transfusion, the sterile composition may be suspended in any physiologically compatible solution, such as saline or plasma. The preparation may be used alone or in combination with other active substances, including whole platelets. It is an ideal supplement for artificial blood. The composition can also be applied topically to stop bleeding and treat wounds. For such use, the compositions may be suspended in, or impregnated with, a pharmaceutically acceptable carrier such as a gel or ointment, such as gauze. The compositions may also be used as carriers for delivery of the active ingredient, or may be used in a labeled condition for diagnostic purposes, such as in the imaging agent for locating a blood clot.

BRIEF DESCRIPTION OF THE FIGURES Figure 1.

Fig. 4A is a standard curve of thrombin clotting time;

Figure 2 shows the effect of heat treatment on the specific activity of PF-3;

Figure 3 shows the platelet count and bleeding time of rabbits with normal and reduced platelet counts;

Figure 4 shows the effect of transfused platelet membrane microparticles on the bleeding time of animals with doxorubicin hydrochloride-induced thrombocytopenia;

5.

Fig. 2A shows the effect of transfused platelet membrane microparticles on animals with antibody-induced thrombocytopenia; and Figure 6 shows the dose-dependent effect of transfused platelet membrane microparticles on the bleeding time of busulfan-induced severe thrombocytopenia.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION The product of the present invention is prepared from whole blood platelets or whole blood platelet membrane derivatives. Whole platelets may be freshly harvested platelets or platelets after a period of use.

Platelets after the shelf life are those platelets that have been stored between 4 ° C and room temperature for at least 3 days. Platelets beyond the shelf-life are discarded according to accepted practice as they are no longer usable as transfusion material. Platelet derivatives refer to non-intact platelets, such as ghost platelets or platelet membrane fragment containing platelet membrane microparticles.

The products of the invention are administered to the subject being treated in an effective amount. The term "being" refers to any living organism in which the ability to stop bleeding is mediated, at least in part, by platelets, such as humans, dogs, cats, horses, and the like. By "effective amount" is meant an amount capable of meeting the therapeutic or diagnostic purpose for which the product is being administered. By an effective amount of transfusion in general, is meant an amount that restores bleeding function to substantially the same level as would be achieved by transfusion of whole platelets. By effective amount in patients with thrombocytopenia, is meant an amount capable of reducing the patient's bleeding time, preferably to a non-dangerous level, more preferably to a level that is normal to the individual. The effective amount will be determined on an individual basis, based at least in part on the size of the subject being treated, the severity of the symptoms, and the results to be achieved. Thus, the effective dose may be determined by one of ordinary skill in the art by routine assays.

The products of the invention can be treated to be free of active virus. Active virus-free means that the product has been subjected to satisfactory conditions to inactivate or kill any viruses present, such as HBV, NANBHV, CMV, and HIV. These conditions include treatment with antibodies, treatment with ethanol, ultraviolet light, organic solvent detergent, and phenanthroline chelating agents [see Viral Inactivation in Plasma Products, J.-J. Morgenthaler, Current Studies in Hemotology and Blood Transfusion No. 56, Karger, NY 1989, incorporated herein by reference]. Preferred methods for inactivating viruses include heat treatment of a duration and degree that inactivates or destroys the virus.

The products of the present invention may also be subjected to treatments that reduce or eliminate their immunogenicity when used as transfusions. Preferably, the products of the present invention are prepared from harvested platelets, which, when administered by transfusion to most individuals, would normally elicit an antibody response. Currently, alloimmunization is a major problem in the treatment of thrombocytopenic patients who require repeated platelet transfusions. Platelets have HLA-A and HLA-B antigens that are either hematopoietic

They are part of the A9 necrotic membranes or are absorbed from the plasma. The development of antibodies against HLAA or HLA-B (the primary cause of platelet alloimmunization) results in a rapid degradation of transfused platelets, thus reducing the effect of platelet transfusion on the suppression of bleeding. Contamination of platelet concentrates with white blood cells (a source of HLA antigens) may also contribute to the development of alloimmunization.

Preferred products of the present invention are non-immunogens, non-immunoimmunogens, and non-immunogens for HLA determinants (particularly class I HLA determinants). By "non-immunogenic" is meant that the transfused entity does not accumulate, after some transfusions, an immunological response sufficient to counteract the therapeutic effect of the product administered by transfusion. By "non-alloimmunogen" is meant that the transfected entity does not exhibit a detectable immunological response to alloantigens after some transfusions of material from an alloimmunogen source. By detectability, it is meant that serum antibody to alloantigens cannot be detected by standard procedures, such as a DOT assay, or that any immunoassay against any alloantigen is not sufficient to counteract the therapeutic effect of the product administered by transfusion.

In the following, the preferred process for preparing the preferred product of the invention is described. Freshly collected, stored (1-5 days at 20-25'C) or after shelf life (stored at 4'C for 5 days) platelet concentrates (50-60 ml / concentrate) are collected in 600 ml blood bags (Fenwall transfer pack unit, 4R2023, Fenwall Laboratories, Deerfield, Illinois) using sterile plasma transfer equipment (Fenwall 4C2243, Fenwall Laboratories, supra). Each bag contains a total of 500-600 ml of platelet machine concentrate (hereafter referred to as 600 ml unit). The 600 ml unit was centrifuged at 1000 rpm for 11 minutes at 22 ° C to remove red blood cell and white blood cell impurities (PR7000, International Equipment Company, Needham Heights, Massachusetts). The platelet-containing supernatant is then transferred to a new 600 ml blood bag, centrifuged at 3000 rpm for 25 minutes at 22 ° C to separate the platelets from plasma. Plasma-poor plasma is removed, and the platelet-containing precipitate is gently resuspended in 20 ml of 0.9% sodium chloride solution (physiological saline), diluted to 100 ml with additional saline, and collected in 300 ml blood bags (three 100 ml samples per bag). three original units of 600 ml). The resuspended platelets were resuspended by centrifugation at 3000 rpm for 20 minutes at 22 ° C. The supernatant was removed and the platelet-containing precipitate was washed twice with physiological saline by repeated resuspension and centrifugation.

Finally, the washed platelets are resuspended in physiological saline (each 600 ml unit in 25 ml), then re-frozen (-80 ° C for at least 6 hours) and thawed (25 ° C for at least 1 hour). thawing operations are repeated three times. The frozen-thawed suspension was diluted with physiological saline (every 600 mL unit to 100 mL) and centrifuged at 3000 rpm for 30 min to collect ghost platelets. This ghost platelet precipitate was resuspended in physiological saline (100 mL of saline for every 600 mL) and washed twice by resuspension and centrifugation.

Other methods may be used to destroy platelet membranes and isolate the platelet ghost fraction. For example, platelets can be equilibrated with glycerol and then hypotonically destroyed by rapid dilution of extracellular glycerol. Such an operation creates an osmotic pressure gradient across the outer membrane of the platelet, leading to the destruction of the cell membrane. In addition, many other chemical agents (e.g., sodium chloride) can be used in a similar manner to induce osmotic shock and to disrupt platelets. In a preferred embodiment of the invention, repeated freezing and thawing are used.

The washed ghost platelet precipitate is resuspended in physiological saline (40-50 ml per 600 ml unit) and kept in a water bath at 60 ° C for 20 hours. Alternatively, the ghost platelet suspension may be heated at 100 ° C for 5 minutes. These conditions are sufficient to inactivate any viral contamination. Significant precipitation occurs during heat treatment. The heat-treated ghost platelet suspension is then sonicated (Ultrasonic Processor Model W-385, Heat Systems, Inc., Farmingdale, New York) using a 1.27 cm destructive horn (Model 800B, Heat Systems). The ultrasound system is first flushed with nitrogen and then injected into the ghost platelet suspension. The suspension is then sonicated with a 20 kHz pulse for 5 minutes 43 seconds (in 2-second cycles, 1.4 seconds on, 0.6 seconds off) at power level 4 to give a double amplitude of 48 micrometers. Subsequently, the sonicated preparation is centrifuged at 3000 rpm for 30 minutes at 22 ° C to separate the precipitated material from the formed platelet membrane microparticles, which remain in the supernatant. The supernatant is then removed and stored in sealed containers at 4 'C, -20' C or -80 'C under nitrogen or can be lyophilized under a nitrogen atmosphere. Unless otherwise noted, the following procedures employ microparticles stored at 4'C.

The platelet microparticle fraction prepared in the above manner is free of active virus. Virtually free of ghost platelets and microparticles5

More than 80% of the A9 is less than 600 nm in diameter and more than 95% is less than 1000 nm in diameter. The average diameter of microparticles made from platelets that have been slightly over (up to 2 weeks) during the shelf life is 7 to 400 nm for 7 different formulations. The average diameter of the 7 formulations is 341 nm.

The platelet microparticle fraction is also substantially free of serotonin, GPIIb / IIIa (a surface glycoprotein), purine nucleoside phosphorylase, coagulation factors V, VIII, IX and X and thrombospondin (an alpha granule component). On the other hand, GPIb (another surface glycoprotein) is present in the formulation, and β-glucuronidase (a lysosome marker) is also detectable (about 25% of the lysate). The lack of GPIIb / IIIa was surprising, since GPIIb / IIIa was thought to be required to relieve bleeding.

The platelet microparticulate fraction preparation was analyzed in two separate assays for certain components, and our results are reported in Table I below.

Table 1

As a percentage of total (% by weight)

test Carbohydrate phospholipid pid Protein Koleszte- rin N = 4 3,310,14 30,010,9 57,810,9 9 (estimated) N = 8 2,610,3 30,112,5 53,211,7 9,910,9

The procoagulant activity of the preferred platelet microparticulate fraction from platelets with a short lifetime was determined by the Russel viper poison time method (5), which is used to measure PF-3 activity. The Russel assay is a thrombin formation assay whose end point is determined by fibrinogen clotting. Specific activity is determined relative to a standard thrombin curve and is expressed in terms of thrombin / mg platelet protein formed or thrombin / mg platelet phospholipid produced. The specific activity of PF-3 per mg of protein (E / mg) in the first set of assays was found to be 148.1 ± 13.4 (n = 7). In the second set of tests, this value was 175 ± 8 (n = 8). The specific activity of PF-3 on phospholipid 1 mg (E / mg) was 291.3 ± 40.0 in the first set of experiments and 310 ± 23 in the second set of experiments. These specific activity values are far higher than those obtained for fresh, intact whole platelets. This is believed to be due to the release of procoagulant membrane fragments not normally available in intact platelets.

Specific activity is preserved even after lyophilization, although a preservative (0.4 g sucrose / 100 ml, more than 90% of the activity is preserved) is required to maintain a specific activity greater than about 60%.

The composition of the phospholipid portion of the preparation was determined in two different assays, the results of which are shown in Table II. See Table 1.

//. Spreadsheet

As% of total phospholipid (wt%)

examination see E.g PS PE PC SP N = 4 5,811,0 10,110,9 22,511,5 45,814,0 15,410,7 N = 8 6,810,5 12,210,7 23,812,1 40,612,3 16,611,5

Eg: phosphatidylinositol,

PE: phosphatidyl ethanolamine

SP: Sphingomiel

PS: Phosphatidylserine

PC: phosphatidylcholine

The procoagulant activity of platelet microparticle fractions and their ability to attenuate bleeding in thrombocytopenic animals were tested. The procoagulant activity of microparticle fractions made from platelets with little after-use time was found to be comparable with that of fresh platelets. The procoagulant activity of the fresh platelets and of the platelets with little after-use time can be measured by the activity of whole platelets. Transfusion of such a fraction reduced bleeding time in all recipient animals.

The effect of snow treatment and ultrasound treatment on procoagulant activity was also investigated. The microparticulate fraction from platelets with a short lifetime was found to be relatively stable to snow treatment. It has further been found that the procoagulant activity of this platelet microparticle fraction is significantly reduced when heat treatment is preceded by homogenization. This and other properties of the microparticle fraction of the present invention are described in more detail in the following examples.

The product prepared according to the procedure described above, as well as its various intermediates, were tested for the presence of GPIIb / IIIa by immunological DOT. GPIIb / IIIa was present in intact platelet preparations, in the platelet lysate, in the supernatant of the platelet lysate, and in the ghost fraction of the platelet membrane. No detectable GPIIb / IIIat was detected in the heat treated platelet ghost membrane fraction or in the final product.

Immunological DOT assays were also performed on the final product and intermediates for the presence of HLA antigens (class I). HLA antigens were present in washed, intact platelet preparations, in the platelet lysate, in the platelet lysate supernatant, and in the platelet membrane ghost fraction. HLA antigens were not detected in the ghost fraction of the heat-treated platelet membrane nor in the final product.

In addition, the final product and various intermediates were tested for immunobiological DOT in the presence of GPIb. All samples tested, including the snow-treated sample, showed the presence of GPIb.

The foregoing description is a preferred embodiment of the invention. However, it is not limited to this in the broad sense of the invention. It is an object of the present invention to provide, on the one hand, heat-treated platelets or platelet membrane fractions to eliminate viral infections. Although also6

A9 is that such heat treatment generally inactivates the viral infection and such treatment has never been used with platelet preparations. Therefore, the present invention provides for the first time a transfusion-compatible virus-inoculated platelet fraction.

The invention also relates to a transfusion composition made from platelets after a period of use. The invention first provides a high mass of platelets beyond the shelf life, which is usually discarded to make them suitable for transfusion. Furthermore, since the likelihood of viral infection of platelets beyond the shelf life is increased during storage, the heat treatment step of the present invention also contributes to the utilization of the platelets beyond the shelf life to assure their virus-free status.

In accordance with the invention, the heat inactivation and homogenization steps are combined to produce a platelet microparticle fraction substantially free of ghost platelets. The composition contains homogeneous microparticles and is substantially free of unwanted cytoplasmic material. In a preferred embodiment of the invention, homogenization (ultrasonic homogenization) follows the heat inactivation step, thereby providing, on the one hand, homogeneous size of the microparticles and, on the other hand, preventing a significant amount of activity from attaching to the precipitate formed during the heat inactivation step.

As described above, the microparticle fraction can be prepared stepwise. As a first step, the platelet membrane is partially degraded to produce a ghost platelet fraction and a lysate containing cytoplasmic substances. Once the ghost platelet fraction is separated from the cytoplasmic material, the ghost platelets are homogenized to obtain a substantially ghost platelet fraction containing microparticles of substantially uniform size. Note, however, that the first step, partial destruction, can be omitted. Thus, platelets are sonicated and the resulting microparticles can be isolated from the lysate.

It is believed that the platelet membrane microparticle fraction of the present invention is substantially non-immunogenic relative to whole platelets and therefore can be prepared from platelets collected from various matching blood donors. Thorough washing with complete removal of white blood cells may contribute to obtaining a non-immunogenic preparation.

Preferred products of the present invention have procoagulant activity, are virus-free, non-alloimmunogens, and are so stable that they can be commercially prepared, lyophilized, and stored in suitable containers.

Example 7

APF-3 (platelet factor 3) procoagulant activity was measured by the Russel viperin poison time assay, which is a thrombin formation assay. The end point of the assay was visually determined by the clotting of fibrinogen present in the collected human plasma sample.

A 0.025 M stock solution of calcium chloride in imidazole buffer pH 7.3 was maintained at 37 ° C. The collected human plasma and platelet membrane microparticle fraction were stored as a 25 µg / ml solution at room temperature. Russel viper venom (RW; 10 pg / ml in saline; Wellcome Diagnostics, Dartford, England) was kept on ice.

The assay was started by mixing 0.1 mL of collected plasma with 0.1 mL of platelet membrane microparticle solution (12 x 75 mm) in a borosilicate glass tube and incubating at 37 ° C for 5-10 min. Then 0.1 ml of RW solution was added and the mixture was incubated at 37 ° C for 30 seconds and then 0.1 ml of CaCl ₂ solution was added. The time interval between the addition of CaCl ₂ -solution and fibrin clot was measured. The thrombin unit produced by the assay system was calculated from the standard thrombin clotting time curve of Figure 1 using purified bovine thrombin (Sigma 850-1; Sigma Chemical Co., St. Louis, MO) and collected human plasma.

The effect of heat treatment and sonication on procoagulant activity (PF-3) was measured by Russel's viper venom time. As shown in Figure 2, heat treatment followed by ultrasound treatment yielded a platelet membrane microparticle fraction that retained a significant portion of its procoagulant activity. However, when ultrasound treatment precedes heat treatment (not shown), the procoagulant activity of the platelet membrane microparticle fraction is dramatically altered, with a 50% decrease in procoagulant activity.

Example 2

The ability of platelet microparticles to attenuate antibody-induced bleeding in thrombocytopenic animals was investigated. In rabbits, thrombocytopenia was induced by anti-rabbit platelet antiserum. First, platelet count and bleeding time (platelet count 548,000 ± 97,000 / μΙ; bleeding time 153 ± 47 s; mean ± SD) were determined in 10 normal rabbits (3.53 ± 0.41 kg body weight). Anti-rabbit platelet antiserum from commercial sources was administered intravenously to 0.2 rabbits in an amount of 0.2-0.4 ml / kg body weight. At 2 hours after injection of antiserum, different degrees of thrombocytopenia were observed in all animals. Based on the data, groups were formed according to the degree of "Reverse Thrombocytopenia": Group 1: Platelet count <80,000 / μ1; Group 2: platelet count between 100,000 and 200,000 / μ1 and group 3: platelet count greater than 200,000 / μΙ (as shown in Figure 3).

All 10 rabbits treated with antisera also exhibit prolonged bleeding time (measured 2 hours after antiserum injection). Significant elongation occurred only in the first group of animals (severe thrombocytopenia, platelet count <75 ΟΟΟ / μΙ), and a moderate prolongation was observed in group 2 and 3 animals (see Figure 3).

HU 211 345 A9

Group 1 animals with severe thrombocytopenia (2 mg protein / kg body weight) were administered transfusions of platelet microparticle fraction of platelets slightly below the shelf-life. As shown in Figure 4, 2 hours after antiserum injection, platelet counts were 41,000, 73,000, and 56,000 / μl in animals 1, 2, and 3, respectively. During the same period, bleeding time was significantly prolonged in all three rabbits, especially rabbit 2, where the incisional wound was heavily bleed even after 20 minutes, and local bleeding was required to stop bleeding. After administration of platelet microparticles, the bleeding time of animals 1 and 3 was significantly reduced, whereas in rabbit 2, bleeding stopped spontaneously after 20 minutes without applying local pressure. Thus, it was observed in all animals tested that the transfusion time of the platelet microparticles of the invention is reduced.

Example 3

The effect of platelet microparticles on reducing the bleeding of animals with doxorubicin hydrochloride-induced thrombocytopenia was investigated. Nine rabbits were dosed intravenously with doxorubicin hydrochloride (Adriamycin; 2 mg / kg body weight) to induce thrombocytopenia. The basal platelet count before injection of doxorubicin is 488,000 + 94,000 / μ1. The baseline bleeding time was 128 ± 21 s. One week after doxorubicin injection, the platelet count decreased to 130,000 ± 31 ΟΟΟ / μΙ (p <0.01), while the bleeding time increased 2.4-fold compared to thrombocytopenic control (311 ± 89 s, n = 9, p <0.01).

Rabbits suffering from doxorubicin-induced thrombocytopenia (2 mg membrane protein / kg body weight) were administered transfusions of platelet microparticulate fraction or saline preparation with a slightly over-life platelet. In all animals to which procoagulant membranes were administered, a haemostatic effect was observed. A significant reduction in bleeding time at 15 minutes, 2 hours, and 4 hours after transfusion was observed relative to thrombocytopenic control (p <0.01) (Figure 5). In contrast, no significant change was observed in thrombocytopenic rabbits administered saline infusion.

Example 4

The bleeding time reduction effect of the platelet microparticles of the invention was further demonstrated in a dose-dependent effect of transfusion of the platelet microparticles of the invention to severe thrombocytopenic rabbits. In rabbits, thrombocytopenia was induced by two subcutaneous injections of busulfan (total 35 mg / kg body weight) (platelet count below 25,000 / μl). In severely thrombocytopenic rabbits, platelet microparticles were administered in three different concentrations at doses of 0.5 mg / kg, 1.0 mg / kg and 2.0 mg / kg body weight, respectively, and as a control solution saline in the form of a transfusion. Baseline platelet count was measured on the day of transfusion, with an order of magnitude of 15,000 / μΙ for each animal.

Figure 6 shows that the antihypertensive effect of platelet microparticles is directly dependent on the dose administered by transfusion. In severely thrombocytopenic rabbits, the bleeding time of saline was not reduced. On the other hand, prolonged bleeding time in severely thrombocytopenic rabbits following transfusion of platelet microparticles decreased in a dose-dependent manner.

The above transfusion studies in thrombocytopenic rabbits confirm the antihypertensive activity of the platelet membrane fragment of the invention.

The embodiment of the invention described above and illustrated in the drawings can, of course, be accomplished by a number of modifications and changes within the skill of the art. Therefore, it is intended that the material in the foregoing description and shown in the drawings be construed as illustrative and not limiting.

references

1) Klein, E. "Farber, S., Djersasi, I., Toch, R., Freeman, G." Amold, P., "Materials for the Preparation and Clinical Administration of Lyophilized Pediatric to Children with Acute Leukemia and Aplastic Anemia", J. Pediatrics, 49, 517 (1956).

2) Hjort, P. Perman, V., and Cronkite, E., Fresh, Disintegrated Platelets and Radiation Thrombocytopenia: Correction of Prothrombin Consumption without Correction of Bleeding, 1959.

3) McGill, M. Fugman, D., Vittorio, N., and Darrow, C., "Piatelet Membrane Vesicles Reduced Microvascular Bleeding Times in Thrombocytopenic Rabbits",

J. Foot. Clin. Med., 109, 127-133 (1987).

4) Wu, V-Y., Mc Coy, L. E., Platelet Factor 3: Quantitation and Characterization, Thromb. Rev., 11, 581-593 (1977).

5) Spaet, T.H., Cintron, J., Studies on Platelet Factor3 Availability, Brit. J. Hamematol, 11, 269 (1965).

Claims (14)

PATENT CLAIMS CLAIMS 1. A platelet membrane product derived from the platelets of a platelet of a mammalian species having platelets, comprising isolated platelet membrane fragments having procoagulant activity that are inactive against the HLA antigens of the species from which the platelets are derived. Platelet membrane product according to claim 1, characterized in that the isolated fragments are non-alloimmunogenic. Platelet membrane product according to claim 1, characterized in that the isolated fragment is non-immunogenic. HU 211 345 A9 Platelet membrane product according to any one of claims 1, 2 or 3, characterized in that the isolated fragments are free of GP γ / nia activity. The membrane product of any one of claims 1, 2 or 3, characterized in that it is substantially free of ghost platelets. The membrane product according to any one of claims 1, 2 or 3, wherein the isolated fragments are isolated microparticles having a diameter of less than 1 micron. Platelet membrane product according to claim 6, characterized in that it is free of microparticles with a diameter greater than 1 micron 8. The platelet membrane of claim 4 wherein the ghost is platelet-free. The platelet membrane according to claim 4, wherein the isolated fragments are isolated microparticles having a diameter of less than 1 micron. Platelet membrane product according to claim 9, characterized in that it is greater than 1 microns Free from microparticles with a diameter of 5. 11. A platelet membrane product having procoagulant activity and comprising an isolated membrane fragment free of GPIIb / IIIa complex. The platelet membrane product according to claim 11, wherein the ghost is free of platelets. 13. The platelet membrane of claim 11, wherein the isolated fragments are isolated microparticles having a diameter of less than 1 micron. Platelet membrane product according to claim 13, characterized in that it is free of microparticles with a diameter greater than 1 micron.