CN116794333A - Activator formula for comprehensively activating platelet-based aggregation function and preparation method thereof - Google Patents
Activator formula for comprehensively activating platelet-based aggregation function and preparation method thereof Download PDFInfo
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- 238000004220 aggregation Methods 0.000 title description 14
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Abstract
The invention provides an activator formula for comprehensively activating the basic platelet aggregation function and a preparation method thereof, wherein the activator formula can be matched with the Feiteju series products of the company to be used as the activator of a baseline channel of the platelet aggregation function, can also be used for generating the maximum platelet aggregation level in other platelet function detection reagents, and can calculate the platelet inhibition rate under a specific platelet activator approach.
Description
Technical Field
The invention belongs to the technical field of medical materials, and relates to an activator formula for comprehensively activating basic platelet aggregation function and a preparation method thereof.
Background
Platelets play a critical role in maintaining normal hemostasis. When exposed to a damaged blood vessel, platelets will adhere to the exposed subendothelial matrix, and subsequently, various factors (e.g., thrombin, ADP, collagen, etc.) released or produced at the site of the injury activate the platelets. Upon platelet activation, the glycoprotein GPIIb/IIIa receptor on the platelet undergoes a conformational change such that it binds to multivalent fibrinogen, causing adjacent platelets to recruit to the site of injury and aggregate to form a tampon or thrombus.
In vitro platelet aggregation is activated in dependence of activation of platelets by an activator. Among them, common activators for in vitro activation include thrombin receptor activators (thrombin receptor activating peptide 1 and thrombin receptor activating peptide 4), epinephrine, collagen, arachidonic Acid (AA), adenosine Diphosphate (ADP), ristocetin, and the like.
Most platelet aggregation detection methods in the current market adopt a single activator to perform activation detection, so that the platelet aggregation function under the activation action way of the single activator is detected. For example: the activator ADP activates platelets through a P2Y12 receptor pathway, so that the activator ADP can be used for evaluating the drug effect of patients taking P2Y12 receptor inhibitor drugs (such as clopidogrel and ticagrelor). For example: the activator AA activates platelets through the cyclooxygenase-1 pathway, so that the medicine effect of a patient taking a cyclooxygenase-1 inhibitor medicine (such as aspirin) can be evaluated. In addition to the results reported by activation alone, some assays report a corresponding platelet inhibition rate. The report of inhibition provides the most intuitive form of report for doctors and patients to directly understand the proportion of the level of aggregation in the platelet basal aggregation level at the corresponding activation pathway of platelets.
If it is desired to evaluate the basic platelet aggregation function of a patient, it is necessary to collect the level of platelet aggregation before the patient takes a drug affecting the platelet aggregation function, or to activate and collect the level of platelet aggregation using an activator capable of activating the maximum platelet aggregation function.
At present, only the Feiteju@P2Y12 receptor platelet aggregation detection reagent card (nephelometry) and the VerifyNow@P2Y12 receptor related platelet aggregation function detection kit (nephelometry) of China are available on the market (no continuous registration in China at present), and the inhibition rate is reported by setting a basic aggregation function acquisition channel of platelets.
The invention relates to a more general and more reliable activator formula capable of activating platelets to show the basic aggregation level of the platelets and a preparation method thereof.
Disclosure of Invention
The invention aims to provide an activator formula for comprehensively activating the basic agglutination function of platelets and a preparation method thereof.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in one aspect, the present invention provides an activator formulation for the overall activation of platelet-based agglutination function, wherein the major activator components comprise thrombin receptor activators (PAR 1 and PAR 4), collagen, epinephrine, ADP, AA, ristocetin, and the like, and wherein one, several, and all of the activators are combined in a composite form to form different activator combinations. The amount of activator and combination may be such that the maximum aggregation function of platelets is assessed over different reaction times.
On the other hand, the activator formula is in the form of liquid or freeze-dried products, wherein the freeze-dried products are freeze-dried cakes, balls, powder and the like. The activator may be used in liquid form or lyophilized form. The liquid form solution can be ultrapure water, physiological saline, buffer solution and other basic liquids. Additives such as preservative, protein protectant, surfactant, and adhesion promoter can be added. The lyophilized form solution may be a base solution such as ultrapure water, physiological saline, buffer solution, etc. Additives such as preservative, protein protectant, surfactant, adhesion promoter, and filler can be added. The freeze-dried product is prepared by combining the components such as an activating agent, a liquid matrix, auxiliary materials and the like.
Preferably, the activator concentrations are selected and combined according to the table below:
| activator name | Activator concentration |
| Thrombin receptor I (PAR 1) activators | 1～10μM |
| Thrombin receptor IV (PAR 4) activators | 1～100μM |
| Collagen | 1～50μg/mL |
| Epinephrine system | 0.5～10μM |
| ADP | 0.5～20μM |
| AA | 0.5～2mM |
| Ruistomilast | 0.5～1.5mg/mL |
Preferably, the auxiliary materials of the freeze-dried product comprise additive components such as preservative, protein protectant, surfactant, adhesion promoter, filler and the like. The ingredients and concentrations can be referred to, but are not limited to, the following table:
| name of the name | Content of |
| Microcrystalline cellulose | 0～5％ |
| Sucrose | 0～10％ |
| Lactose and lactose | 0～5％ |
| PEG8000 | 0～5％ |
| PEG20000 | 0～5％ |
| Tween 80 | 0～0.5％ |
| BSA | 0～3％ |
| Sodium azide | 0～0.5％ |
Preferably, the dispersion matrix of the activator formulation is one or a mixture of buffers such as physiological saline, PBS, TRIS, boric acid, carbonic acid, MES, HEPES, etc. Wherein, one or more additives such as preservative, protein protectant, surfactant, adhesion promoter, etc. can be added.
Preferably, the present formulation may be added to concentrated platelet, platelet-containing plasma, and whole blood waiting samples for use, or the samples may be introduced into an activator-containing device for use.
Preferably, the formula is matched with relevant products such as a platelet function detection product Feiteling@series product, a Sifenos@thromboelastography series product and the like of the company for use.
Drawings
FIG. 1 is a schematic diagram of a Feitegel @ reagent card. The activator can be preset in the detection channels (1, 2, 3 and 4), and the activator and the sample are mixed to play the role of activation after the sample enters.
FIG. 2 is a graph showing the effect of activation time for different activator formulations. Different activator formulations, due to the different activation rates, can achieve the maximum basal aggregation rate levels at different test durations.
FIG. 3 is a graph showing the effect of activating aggregation at different activator formulations. Different activator formulations, due to the different degrees of activation, can achieve different basal aggregation rate levels for the same test duration.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
Example 1
The formula consists of a thrombin receptor activator PAR1 activation peptide and a PAR4 activation peptide as main components. Wherein: PAR1 stock concentration was 1mM; PAR4 stock concentration was 1mM. The dispersion matrix was 0.01M PBS (ph=7.4, 0.5% bsa, 0.5% pvp, 0.5% tween 20 and 0.1% sodium azide as protectant and stabilizer). In the activation experiment, 100 mu L of activator is added into 1.9mL of fresh sodium citrate anticoagulated whole blood, and the detection is carried out by using the Feitegel detection platform of the company. The detection result shows that the maximum aggregation rate can be reached within 1.5-2 minutes.
Example 2
The formula consists of a thrombin receptor activator PAR1 activation peptide, a PAR4 activation peptide, epinephrine and collagen as main components. Wherein: PAR1 activating peptide mother liquor concentration is 1mM; PAR4 activation peptide stock concentration was 1mM; epinephrine mother liquor concentration was 0.1mM; the concentration of the mother liquor of collagen is 0.5mg/mL. The dispersion matrix was 0.05M tris (ph=7.5, 5% sucrose, 0.5% sodium caseinate, 1% peg20000, 0.5% triton and 0.2% sodium azide as protectant and stabilizer). In the activation experiment, after four activator mother solutions are mixed, 1 mu L of the mixed activator mother solution is added into 1.0mL of fresh sodium citrate anticoagulated whole blood, and the mixture is detected by using a Feitegel detection platform of the company. The detection result shows that the maximum aggregation rate can be reached within 10-20 minutes.
Example 3
The formula consists of a thrombin receptor activator PAR1 activation peptide, a PAR4 activation peptide, ADP, AA and epinephrine as main components. Wherein: PAR1 activating peptide mother liquor concentration is 1mM; PAR4 activation peptide stock concentration was 1mM; AA stock solution concentration was 5mM; ADP mother liquor concentration was 0.5mM; the concentration of epinephrine mother liquor was 1mM. The dispersion matrix is normal saline. The auxiliary materials are shown in the following table:
| name of the name | Content of |
| Microcrystalline cellulose | 1.50％ |
| Sucrose | 5.00％ |
| Lactose and lactose | 1.00％ |
| PEG8000 | 2％ |
| PEG20000 | 1％ |
| Tween 80 | 0.50％ |
| BSA | 1％ |
| Sodium azide | 0.10％ |
100 mu L of each activator mother solution is added into 5mL of uniformly mixed auxiliary materials, vortex mixing is carried out, and then a liquid dispenser is used for dripping the activator composite solution into liquid nitrogen at a constant speed according to the volume of 10 mu L of each drop. After the droplets were frozen, the frozen spheres were transferred to a 1.5mL centrifuge tube at low temperature and lyophilized. Precooling for 30 minutes, freeze-drying for 48 hours, and then heating to 24 ℃ at a heating rate of 2 ℃ per minute, and finishing freeze-drying. The freeze-dried pellet assumes a spherical or nearly spherical freeze-dried pellet after lyophilization is completed.
In the activation experiment, 3 freeze-dried balls are taken and added into 0.1mL of fresh sodium citrate anticoagulated whole blood, and the detection is carried out by using the Feitegel detection platform of the company. The results of the test show that the maximum aggregation rate can be reached in 5 minutes. 1 particle of freeze-dried balls are taken and added into 0.1mL of fresh sodium citrate anticoagulated whole blood, and the detection is carried out by using the Feitegel detection platform of the company. The detection result shows that 45% of the maximum aggregation rate can be achieved in 5 minutes.
The applicant states that the present invention is illustrated by the above examples as to the preparation process and method of use of the present invention, but the present invention is not limited to the above process steps, i.e. it is not meant that the present invention must be carried out in dependence on the above process steps. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected raw materials, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the present invention and the scope of disclosure.
Claims (10)
1. The main activator components in the activator formula comprise thrombin receptor (PAR 1 and PAR 4) activators, collagen, epinephrine, ADP, AA and ristocetin, and the activator comprises any one or more of the activator components and different activator combination forms including a compound form of the common collocation of all the activators.
2. The method according to claim 1, wherein: the activator formulation is formed from any of the activators.
3. The method according to claim 1, wherein: the activation formulation comprises a binary complex activator form of a thrombin receptor activator in combination with any of several other activators, or any 2 of the activators described in claim 1.
4. The method according to claim 1, wherein: the activation formulation comprises a ternary complex activator form of a thrombin receptor activator in combination with collagen and any other of the compounds of claim 1 or any 3 of the activators of claim 1.
5. The method according to claim 1, wherein: the activation formulation comprises a quaternary complex activator form comprising a thrombin receptor activator, collagen, epinephrine, and any other combination of any of the compounds of claim 1 or any 4 of the activators of claim 1.
6. The method according to claim 1, wherein: the activating formulation comprises a five-membered complex activator form comprising thrombin receptor activator, collagen, epinephrine, ADP and any of the other combinations of claim 1 or any 5 of the activators of claim 1.
7. The method according to claim 1, wherein: the activation formulation comprises a six-member complex activator form comprising thrombin receptor activator, collagen, epinephrine, ADP, AA, and any other combination of any of the compounds recited in claim 1 or any 6 of the activators recited in claim 1.
8. The method according to claims 1-7, characterized in that: the apparent form of any activator combination is liquid or freeze-dried; wherein the freeze-dried product is in the forms of freeze-dried cake, freeze-dried ball, freeze-dried powder and the like; wherein the base liquid is ultrapure water, normal saline, buffer solution and other base liquids; wherein the additive comprises the following components: additives such as preservative, protein protectant, surfactant, adhesion promoter, and filler.
9. The method of claim 1-8, wherein the activator formulation is used with platelet function test products of the company and is also used for other products related to the market and related new products.
10. The activator formulation according to claims 1-9, which is suitable for concentrating platelets, platelet-containing plasma and whole blood samples.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN117368454A (en) * | 2023-12-08 | 2024-01-09 | 普迈德（北京）科技有限公司 | Thromboelastography rapid activated blood coagulation reagent card, preparation method and application |
| CN117368454B (en) * | 2023-12-08 | 2024-04-23 | 普迈德（北京）科技有限公司 | Thromboelastography rapid activated blood coagulation reagent card, preparation method and application |
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