CN1318035C - Human platelet substitute and its preparation method - Google Patents
Human platelet substitute and its preparation method Download PDFInfo
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- CN1318035C CN1318035C CNB031571964A CN03157196A CN1318035C CN 1318035 C CN1318035 C CN 1318035C CN B031571964 A CNB031571964 A CN B031571964A CN 03157196 A CN03157196 A CN 03157196A CN 1318035 C CN1318035 C CN 1318035C
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Abstract
The present invention discloses a human platelet substitute and a preparing method thereof. The human platelet substitute of the present invention is a platelet glycoprotein liposome composed of hog platelet membrane glycoprotein and lipids. The method for preparing the human platelet substitute comprises the steps: step 1, platelet solution is extracted from hog blood; step 2, glycoprotein in the platelet solution obtained in the step 1 is purified to obtain hog platelet membrane glycoprotein; step 3, hog platelet membrane glycoprotein obtained in the step 2 is connected with lipids to obtain the hog platelet glycoprotein liposome, namely the human platelet substitute. Experiments indicate that the human platelet substitute of the present invention has the functions of promoting blood coagulation, etc. and the advantages of high safety and reliability.
Description
Technical field
The present invention relates to a kind of human blood platelets succedaneum and preparation method thereof in the field of medicaments.
Background technology
Platelet is absolutely necessary in multiple physiology, pathological processes such as thrombosis and hemostasis, and platelet membrane glycoprotein (Glycoprotein GP) plays an important role in this course.
Platelet membrane albumen not only has some physiology receptors, but also plays the effect of transmission information between platelet.The platelet membrane abnormal protein also can cause some diseases in addition; as (Ruan Changgeng, blood platelet disorder progress of research, Chinese Journal of Hematologies such as Bernard Soulier syndrome, platelet type angiohemophilia, thrombocytasthenia, platelet the 3rd factor deficiency diseases; 22:767,1983).Have multiple glycoprotein on the platelet membrane surface, this type of glycoprotein mainly comprises GPIb, GPIIb, GPIIIa, GPIV, GPIX (Li Jiazeng, He Shilin, the sharp chief editor of letter, thrombotic disease is learned, Science Press 1998) etc., wherein main sugar is sialic acid, galactose, N-acetylgalactosamine and N-acetylglucosamine in the glycoprotein ibalpha, and wheat germ agglutinin (WGA) is the protein of a kind of coagulation cell in the Fructus Hordei Germinatus, can be single-minded in conjunction with N-acetylglucosamine (GLcNAc) and sialic acid (NANA).The proteic biochemical analysis of platelet membrane is the powerful measure of carrying out the platelet cell biological study, but the actual many problems analytically such as slightly solubility as platelet membrane, memebrane protein purification difficult that still exist.
The platelet membrane protein SDS-PAGE of various animals with Coomassie brilliant blue (Coomassie blue, CB) and periodic acid-Schiff (PAS) dye and can obtain 2-3 bar zone of protein usually.But forming, the platelet glycoprotein of different genera has than big-difference, wherein (comprise Ia with GP I, Ib, Ic) difference maximum, felid comprises lion, leopard does not all have GP I, also difference is bigger to have its molecular weight of kind of GP I, pig, Cavia porcellus also has some accessory glycoprotein district bands (Suzhou Medical College thrombosis and hemostasis seminar: the research of platelet glycoprotein (I), the hematoblastic polyacrylamide gel electrophoresis of different genera (II), the reactivity of not agnate platelet and monoclonal antibody AN51 and J15, the doctor of Soviet Union scientific research data, 35 phase 28-29,1982).
Platelet adhesion relates generally to three factor: GPIb in blood vessel endothelium undertissue, vWF (von willebandfactor) and subendothelial tissue.
Under the normal condition platelet can not with the blood vessel endothelium tissue bond, after the vascular injury, vWF at first is incorporated into interior subcutaneous collagen fiber, so that configuration takes place and changes in vWF, this moment, platelet GPIb promptly combined with vWF, the performance adhesive attraction, discharge ADP simultaneously, improve calcium ion concentration in the kytoplasm, the GPIIb-IIIa that the activation calcium ion relies on, expose fibrinogen deceptor, Fibrinogen can be simultaneously and at least two GPIIb-IIIa form albumen bridge chain between the platelet in conjunction with making, cause hematoblastic (the period-luminosity discipline of sticking and assemble, the huge platelet of the beautiful summary of horse syndromic molecular variant foreign medical science physiological and pathological science and clinical fascicle 2000:20 (3): 248-251), form white thrombus (Schade et al.Cytoplasmic Truncation of glycoprotein Iba weakens itsinteraction with von willeband factor and impairs celladhesion.Biochemistry, 2003; 42 (7): 2244-2251).Platelet membrane glycoprotein IIb and IIIa (GPIIb-IIIa), plasma fibrinogen and extracellular calcium ion play an important role in the platelet aggregation process; Platelet membrane glycoprotein Iba is the receptor of vWF, thrombin, palatelet-selectin, Mac-1, the XII factor and high molecular weight kininogen, be main adhesion receptor (the Suzhou Medical College thrombosis and hemostasis seminar: the research of platelet glycoprotein (I) that participates in adhesive attraction, the hematoblastic polyacrylamide gel electrophoresis of different genera (II), the reactivity of not agnate platelet and monoclonal antibody AN51 and J15, the doctor of Soviet Union scientific research data, 35 phase 28-29,1982).
Acceptor interaction on various derivants and the platelet membrane makes platelet activation, and adenosine diphosphate sodium salt (ADP) induced platelet must have Fibrinogen to exist when assembling, and GPIIb-IIIa is a fibrinogen deceptor.The receptor of vWF on platelet membrane is GP I b (Yang Shen, et al.Requirement of leucine-rich repeatsof glycoprotein (GP) Ib α for shear-dependent and static binding of vonWilleband factor to the platelet membrane GP Ib-IX-V complex.Blood.2000:95:903-910), need ristomycin (Ristocetin) just to can be incorporated on the platelet receptor at external vWF as cofactor.
Platelet counts reduces or dysfunction of platelet can cause multiple disease, severe loss of blood that causes thus even entail dangers to life, the most effective Therapeutic Method of this type of patient be exactly the input blood donor provide the hematoblastic blood of a large amount of normal functions arranged.Yet this Therapeutic Method can bring a series of blood supply and safety problem.The easy inactivation of platelet needs separate from fresh blood in 8 hours, and isolated platelet life span has only several days, and expired platelet just can only be discarded.The platelet that this type of patient once imports needs ten blood donors to provide, and therefore needs a large amount of blood donors.The platelet of input also might have hepatitis virus, HIV (human immunodeficiency virus) (Human Immunodeficiency Virus) etc., and present detection means also is difficult to control fully these viruses.Therefore, because platelet supply and safety problem are badly in need of the platelet succedaneum that a kind of safety is easy to get now.
Pig and people are very approaching on physiology and structure, so the donor that tissue of pig and internal organs are transplanted as the human organ is the focus of research always.
The innovation and creation content
The purpose of this invention is to provide a kind of human blood platelets succedaneum and preparation method thereof.
Human blood platelets succedaneum provided by the present invention, the Sanguis sus domestica platelet glycoprotein liposome of forming by Sanguis sus domestica platelet membrane glycoprotein and lipid.
Wherein, the ratio of weight and number of Sanguis sus domestica platelet membrane glycoprotein and lipid is 1: 100-1: 5, be preferably 1: 10.
Described lipid can be lecithin, cholesterol, phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, Phosphatidylserine, phosphatidyl glycerol and phosphatidic acid etc., is preferably lecithin and cholesterol, and its ratio of weight and number is 4: 1-1: 1.
Described Sanguis sus domestica platelet membrane glycoprotein obtains by following steps:
(1) from Sanguis sus domestica, extracts platelet solution;
(2) glycoprotein in the platelet solution that obtains in the purification step (1) obtains Sanguis sus domestica platelet membrane glycoprotein.
In the above-mentioned steps (1), adopt 1%TritonX-100 dissolving platelet membrane albumen; Adopt wheat germ agglutinin affinitive layer purification Sanguis sus domestica platelet glycoprotein in the step (2), wherein adopt 0.3M N-acetylglucosamine to carry out eluting.
Described Sanguis sus domestica platelet glycoprotein liposome is that described Sanguis sus domestica platelet glycoprotein is connected as follows with described lipid and obtains: 4-1g lecithin and 1g cholesterol are dissolved in the 1-100mL chloroform, 0.01-1g Sanguis sus domestica platelet glycoprotein is dissolved in adds behind the PBS in described lecithin, the cholesterol chloroformic solution, after supersound process, remove organic solvent, placed 20-40 minute, and finished connection.
A kind of method for preparing the human blood platelets succedaneum may further comprise the steps:
(1) from Sanguis sus domestica, extracts platelet solution;
(2) glycoprotein in the platelet solution that obtains in the purification step (1) obtains Sanguis sus domestica platelet membrane glycoprotein;
(3) the Sanguis sus domestica platelet membrane glycoprotein that obtains in the step (2) is connected with lipid, obtaining Sanguis sus domestica platelet glycoprotein liposome is the human blood platelets succedaneum.
In the step (1), adopt 1%TritonX-100 dissolving platelet membrane albumen.Adopt wheat germ agglutinin affinitive layer purification Sanguis sus domestica platelet glycoprotein in the step (2), wherein adopt 0.3M N-acetylglucosamine to carry out eluting.In the step (3), described lipid can be lecithin, cholesterol, phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, Phosphatidylserine, phosphatidyl glycerol and phosphatidic acid etc., is preferably lecithin and cholesterol, and its ratio of weight and number is 4: 1-1: 1; The ratio of weight and number of described Sanguis sus domestica platelet membrane glycoprotein and lipid is 1: 100-1: 5.The liposome of Sanguis sus domestica platelet glycoprotein described in the step (3) is that described Sanguis sus domestica platelet glycoprotein is connected as follows with described lipid and obtains: 4-1g lecithin and 1g cholesterol are dissolved in chloroform, 0.01-1g Sanguis sus domestica platelet glycoprotein is dissolved in adds behind the PBS in described lecithin, the cholesterol chloroformic solution, after supersound process, remove organic solvent, placed 20-40 minute, and finished connection.
The present invention is dissolved platelet membrane albumen with non-ionic surface active agent TritonX-100, and has been obtained than pure blood platelet glycoprotein by wheat germ agglutinin affinity chromatography platelet purification glycoprotein.Because platelet membrane albumen and membrane phospholipid are combined closely, proteic hydrophobic part inserts in the phospholipid bilayer, must make it dissolving or impose external force and could make platelet solution by isolating platelet with detergent, the used non-ionic surface active agent TritonX-100 of the present invention can make platelet membrane albumen well dissolve.
The present invention shows the composition of Sanguis sus domestica platelet membrane glycoprotein and molecular weight and physiognomy seemingly; And can with the combination of mouse-anti people GP I b monoclonal antibody; And the aggregation of inhibition adenosine diphosphate sodium salt (ADP) and the inductive human blood platelets of ristomycin (Ristocetin); Show that Sanguis sus domestica platelet membrane glycoprotein has similar 26S Proteasome Structure and Function to the human blood platelets membrane glycoprotein.
Experiment shows that human blood platelets succedaneum of the present invention has the effect that can promote aspects such as blood coagulation, is a kind of safe and reliable human blood platelets succedaneum.
Description of drawings
Fig. 1 is the Sanguis sus domestica platelet glycoprotein dot blotting hybridization figure through the wheat germ agglutinin affinitive layer purification
Fig. 2 is the Sanguis sus domestica platelet glycoprotein SDS-PAGE electrophoretogram of purification
Fig. 3 is that iodic acid-Schiff (PAS) method is identified Sanguis sus domestica platelet glycoprotein figure as a result
Fig. 4 is an ADP positive control curve of platelet aggregation
Fig. 5 is the inhibition curve of the preceding sample of purification to the inductive platelet aggregation of ADP
Fig. 6 be behind the purification sample to the inhibition curve of the inductive platelet aggregation of ADP
Fig. 7 is a Ristocetin positive control curve of platelet aggregation
Fig. 8 is the inhibition curve of the preceding sample of purification to the inductive platelet aggregation of Ristocetin
Fig. 9 be behind the purification sample to the inhibition curve of the inductive platelet aggregation of Ristocetin
The gathering curve of Figure 10 glycoprotein liposome solutions
The gathering curve that adds the PHN89 monoclonal antibody in Figure 11 glycoprotein liposome solutions
The gathering curve that adds vWF antibody in Figure 12 glycoprotein liposome solutions
The specific embodiment
Material
EDTA-2Na anticoagulant (54mmol/mlEDTA-2Na, 120mmol/1NaCl), PMSF (Phenylmethanesulfonyl fluoride U.S. Sigma), TritionX-100 (U.S. Sigma), SDS (sodium lauryl sulphate), acrylamide (Fluka), Bradford determination of protein concentration test kit (the green skies), mouse-anti people GPIb monoclonal antibody PHN89 (" Chinese experimental clinical immunology magazine " 1989 the 1st the 3rd phases of volume), the adenosine diphosphate sodium salt, ristomycin (Ristocetin U.S. Sigma), Sephorse 4B wheat germ agglutinin (Amersham), other reagent are homemade analytical pure.
The preparation of embodiment 1, human blood platelets succedaneum
1, the preparation of platelet solution
Collect fresh Sanguis sus domestica, with the 10%EDTA-2Na anticoagulant.22 ℃ of centrifugal 10min of 100g isolate platelet rich plasma (PRP), and PRP is with 22 ℃ of centrifugal 10min precipitation platelet of 1500g, platelet be will precipitate and bufferA (PH6.5,4.8mM glucose, 3mM KCl will be suspended from, 100mM NaCl, 10mM EDTA, 30mM sodium citrate), washing is again with bufferB (PH6.5,30mM glucose, 120mM NaCl, 10mM EDTA, 5mM sodium citrate) wash 2 times, bufferC (PH7.4,134mM NaCl, 10mM EDTA, 10mM Tris-HCl) washes 2 times, after this add and the isopyknic bufferD of platelet (PH7.4,124mM NaCl, 20mM EDTA, 2mM PMSF, 2%TritonX-100,10mM Tris-HCl, 2mM N-ethyl-maleimide), stirring at room 30min, centrifugal 2 hours of 4 ℃ of 2500g, further 4 ℃ 100 of supernatant, super centrifugal 1 hour of 000g.The bufferE (PH7.4,154mM NaCl, the 1mM EDTA that 20ml are contained 6% sucrose, 0.06%TritonX-100,10mM Tris-HCl) is heated to 35 ℃ in the adding 50ml centrifuge tube, carefully adds the supercentrifugal platelet supernatant fluid of 20ml, 30 ℃ of centrifugal 10min of 1000g.Collect supernatant and add 1%TritonX-100, the bufferE that this supernatant reuse contains 6% sucrose is centrifugal by the same method, collects supernatant and is platelet solution.
2, the glycoprotein in the affinitive layer purification platelet solution
Wheat germ agglutinin Sephorse4B adorns post, with binding buffer (PH7.4,20mM Tris-HCl, 0.1MNaCl) go up sample behind the balance pillar, with foreign protein in the binding buffer flushing affinity column and not in conjunction with last glycoprotein, again with the glycoprotein of elution buffer (0.3M N-acetylglucosamine) elution of bound to the agglutinin, collect instrument with semiautomatic computer and collect elution samples, every pipe 1ml, and be one anti-with mouse-anti people GPIb monoclonal antibody PHN89, do dot blotting, the result shows the fully GPIb of elution of bound to the agglutinin of 0.3 M N-acetylglucosamine as shown in Figure 1.Four points in the lower right corner are the sample before the purification among the figure.The collection liquid that amalgamation result is positive concentrates, the dialysis desalination, and filtration sterilization is preserved or the lyophilizing preservation for-80 ℃.
3, polyacrylamide gel electrophoresis (SDS-PAGE)
Glycoprotein behind the purification in the step 2 is concentrated glue 5%, and 10% separation gel carries out SDS-PAGE and reaches the bottom to tracer dye under the 50V voltage, Coomassie brilliant blue (Coomassie blue, CB) dyeing.The result shows visible 2 obvious zone of protein behind the glycoprotein purification in the sample platelet solution as shown in Figure 2, and wherein 200KDa district band is actin-binding protein, and it is GPIb α that about 145KDa district is with.Among Fig. 2,1 expression protein standard, 2 expression platelet solutions, glycoprotein behind the 3 expression purification.
The known protein district about 200KDa of band is an actin-binding protein, and about 145KDa is GPIb α, and about 90-120KDa is GPIb fragment, IIb, IIIa, and about 45KDa is actin and GPIb α fragment.
4, Sanguis sus domestica platelet glycoprotein solution concentration behind the mensuration purification
Bradford Coomassie brilliant blue G250 development process is a standard with BSA, Sanguis sus domestica platelet glycoprotein solution behind the 595nm mensuration purification.Bradford test kit description by green skies company is specifically measured.The result shows glycoprotein concentration average out to 0.2mg/ml behind the purification.
5, Sanguis sus domestica platelet glycoprotein is qualitative
Adopt periodic acid-Schiff (PAS) method (Ruan Changgeng chief editor platelet-basis and clinical Shanghai science tech publishing house 1987).After the SDS-PAGE electrophoresis finishes, place 12.5% trichloroacetic acid fixing in glue, the 3% acetic acid oxidation of 1% periodic acid, room temperature Schiff reagent dyeing.It is reported (Suzhou Medical College thrombosis with the hemostasis seminar: the research of platelet glycoprotein (I), the hematoblastic polyacrylamide gel electrophoresis of different genera (II), the reactivity of not agnate platelet and monoclonal antibody AN51 and J15, the doctor of Soviet Union scientific research data, 35 phase 28-29,1982) platelet solution of various animals utilization SDS-PAGE and PAS dyeing can obtain 2-3 bar glycoprotein district band.The result shows the visible 2 glycoprotein district bands of platelet solution glycoprotein as shown in Figure 3, and about 145KDa is GPIb α, and about 120KDa is GPIb fragment, IIb, IIIa.
6, the Sanguis sus domestica platelet solution before and after the purification is to the inhibition test of ADP and the inductive platelet aggregation of Ristocetin
Gathering whole blood from healthy people is with 3.14% sodium citrate to mix at 1: 10 by volume, and 100g collected and is rich in hematoblastic supernatant (PRP) in centrifugal 10 minutes.Regulating PC with the serum (PPP) of no platelet is 300 * 10
6/ ml.Sample protein matter (concentration is 1.2mg/ml) earlier with PRP incubated at room 5 minutes, after derivant is added PRP.On platelet aggregation instrument, measure 10 minutes coagulation rates, analyze inhibitory action platelet aggregation.The derivant that adds is ADP 30 μ mol, Ristocetin 1.2mol/ml.
The result is shown in Fig. 4-9, abscissa express time among Fig. 4-9 (minute), vertical coordinate is represented percentage rate (%), Fig. 4-6 shows that ADP positive control platelet aggregation rate reaches 74%, sample platelet aggregation rate before and after the purification is respectively 42% and 3%, Fig. 7-9 shows that Ristocetin positive control platelet aggregation rate is up to 100%, sample platelet aggregation rate before and after the purification is respectively 25% and 3%, illustrating before and after the platelet solution purification has inhibitory action to ADP and the inductive platelet aggregation of Ristocetin, shows that the prepared glycoprotein of this method has biologic activity.
7, the preparation of glycoprotein liposome
Adopt reverse phase evaporation.
4g lecithin and 1g cholesterol are dissolved in the 10mL chloroform, the Sanguis sus domestica platelet glycoprotein of 0.05g purification is dissolved in PBS to add in above-mentioned lecithin, the cholesterol chloroformic solution, supersound process is 5 minutes in the formula of bath Ultrasound Instrument, organic solvent is removed in decompression, obtain a kind of stiff jelly, adding the 10mLPBS buffer continues reduction vaporization again and removes micro-organic solvent, placed 30 minutes, the liposome suspension of gained is passed through sephadex column (SephadexG-50), separate and remove the glycoprotein that does not wrap into, obtain the glycoprotein liposome.
The glycoprotein liposome solutions is adjusted to 2 * 10
12Individual/ml, add 50 μ g/ml vWF, derivant Ristocetin (1.2mol/ml) is added after 5 minutes in incubated at room.On platelet aggregation instrument, measure 10 minutes coagulation rates, analyze its aggregation rate.
Same anti--vWF or the PHN89 monoclonal antibody that adds 50 μ g/ml adds derivant Ristocetin (1.2mol/ml) after 5 minutes in incubated at room.On platelet aggregation instrument, measure 10 minutes coagulation rates, analyze its aggregation rate.
Result such as Figure 10, Figure 11 and shown in Figure 12, the maximum agglutination rate that shows the glycoprotein liposome is 80%, and the maximum agglutination rate that the glycoprotein liposome adds the PHN89 monoclonal antibody is 19%, and the maximum agglutination rate that the glycoprotein liposome adds vWF antibody is 35%.Illustrate that the maximum agglutination rate that is added with anti--vWF or PHN89 monoclonal antibody is starkly lower than the aggregation rate of glycoprotein liposome, show that the glycoprotein liposome can play hematoblastic aggregation under anti--vWF and Ristocetin induce.
Claims (6)
1, a kind of human blood platelets succedaneum is 1 by ratio of weight and number: 100-1: the Sanguis sus domestica platelet glycoprotein liposome that 5 Sanguis sus domestica platelet membrane glycoprotein and lipid are formed; Wherein, described Sanguis sus domestica platelet membrane glycoprotein obtains by following steps:
(1) with the platelet membrane albumen in the 1%TritonX-100 dissolving Sanguis sus domestica, obtains platelet solution;
(2) employing wheat germ agglutinin affinitive layer purification obtains the Sanguis sus domestica platelet glycoprotein in the platelet solution, and the eluent of described affinity chromatography is a 0.3M N-acetylglucosamine.
2, human blood platelets succedaneum according to claim 1 is characterized in that: the ratio of weight and number of described Sanguis sus domestica platelet membrane glycoprotein and lipid is 1: 10.
3, human blood platelets succedaneum according to claim 1 and 2 is characterized in that: described lipid is lecithin and cholesterol, and the ratio of weight and number of lecithin and cholesterol is 4: 1-1: 1.
4, human blood platelets succedaneum according to claim 3, it is characterized in that: described Sanguis sus domestica platelet glycoprotein liposome is that described Sanguis sus domestica platelet glycoprotein is connected as follows with described lipid and obtains: 4-1g lecithin and 1g cholesterol are dissolved in chloroform, 0.01-1g Sanguis sus domestica platelet glycoprotein is dissolved in adds behind the PBS in described lecithin, the cholesterol chloroformic solution, after supersound process, remove organic solvent, placed 20-40 minute, and finished connection.
5, a kind of method for preparing the human blood platelets succedaneum may further comprise the steps:
(1) with the platelet membrane albumen in the 1%TritonX-100 dissolving Sanguis sus domestica, obtains platelet solution;
(2) employing wheat germ agglutinin affinitive layer purification obtains the Sanguis sus domestica platelet glycoprotein in the platelet solution, and the eluent of described affinity chromatography is a 0.3M N-acetylglucosamine;
(3) the Sanguis sus domestica platelet membrane glycoprotein that obtains in the step (2) is connected with lipid, obtaining Sanguis sus domestica platelet glycoprotein liposome is the human blood platelets succedaneum.
6, method according to claim 5 is characterized in that: in the described step (3), lipid is lecithin and cholesterol, and the weight ratio of lecithin and cholesterol is 4: 1-1: 1; Sanguis sus domestica platelet glycoprotein is connected according to following method with described lipid: 4-1g lecithin and 1g cholesterol are dissolved in chloroform, 0.01-1g Sanguis sus domestica platelet glycoprotein is dissolved in adds behind the PBS in described lecithin, the cholesterol chloroformic solution, after supersound process, remove organic solvent, placed 20-40 minute, and finished connection.
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