Preparation method of freeze-dried platelets and application of freeze-dried platelets in preparation of traumatic hemostatic products

Abstract

The invention relates to the technical field of freeze-dried platelets, in particular to a preparation method of freeze-dried platelets and application of the freeze-dried platelets in preparation of traumatic hemostatic products. According to the preparation method provided by the invention, proper freeze-drying conditions are adopted, the freeze-drying process is faster than the conventional freeze-drying process, the activation ratio of LPs can be increased on the basis of keeping the activity of the LPs, the activation rate is about 70%, the migration ability is higher than that of the conventional LPs, the highest peak value (MA) of blood clots in in-vitro and in-vivo experiments is higher than that of conventional freeze-dried platelets, and the hemostatic effect is good. Besides, after the blood platelet infusion stock solution provided by the invention is infused into a wound simulated animal body, compared with conventional freeze-dried blood platelets, the blood coagulation promoting activity is enhanced, more blood platelets are distributed to a damaged part, blood coagulation is better promoted, post-traumatic blood coagulation dysfunction is improved, the number of blood platelets in blood circulation is small, and then the probability of ectopic thrombus is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of freeze-dried platelets, and in particular to a preparation method of freeze-dried platelets and application of freeze-dried platelets in the preparation of traumatic hemostatic products. Background Art

[0002] Platelets are an important component of blood, and one of their main functions is to participate in the formation of thrombi and the coagulation process. Platelet transfusion is required in the treatment of patients with massive blood loss and low platelet disease. Platelets are easy to activate, unstable, and have a short lifespan, which are not conducive to low-temperature storage. Therefore, it is necessary to find a suitable storage method to extend their service life.

[0003] Freeze drying is a method of preserving cells and proteins by removing solvents such as water from a substance and then freezing it. Lyophilized platelets (LPs) are stable at room temperature, small in size, light in weight, easy to store for a long time, and convenient to transport. They are quick to use in clinical applications, easy to rehydrate, and convenient for pathogen disinfection.

[0004] LPs were initially used mainly for patients with low platelet counts due to various reasons. In the treatment of patients with low platelet counts, the longer the freeze-dried platelets circulate in the body after transfusion, the better the effect, allowing for longer dosing intervals. However, the situation is not exactly the same in traumatic hemorrhage. When using platelets to help control massive hemorrhage, ideal freeze-dried platelets should have the same hemostatic function as fresh platelets, and after transfusion, they should be able to quickly distribute to the local area of ​​injury and then play their role in promoting blood clot formation. At the same time, they should not remain in the body for too long to avoid causing thrombosis in other parts of the body.

[0005] Some scholars believe that platelet activation inhibitors should be used before freeze-drying platelets to avoid platelet activation, so as to maximize the retention of platelet function and increase the retention time of platelets in the body after transfusion. At present, commonly used inhibitors include amiloride, adenosine, sodium nitroprusside and prostaglandin El. These platelet function inhibitors all act on the platelet cyclic nucleotide system, stimulate the production of cAMP and cGMP, and inhibit platelet activation. Among them, prostaglandin El is widely used and has the effect of inhibiting platelet aggregation, thromboxane A production, atherosclerotic lipid plaque formation and immune complexes. In addition, the existing storage conditions are mostly considered to treat diseases such as thrombocytopenia. For example, room temperature of 20-24°C is conducive to increasing circulation time, but negatively affects the coagulation effect. In patients with severe bleeding, the focus is on the hemostatic effect, and the platelet circulation time is a relatively minor indicator, because once the platelets participate in coagulation, they are quickly cleared from the body. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a method for preparing freeze-dried platelets and its application in preparing traumatic hemostatic products. The freeze-dried platelets prepared by the preparation method of the present invention have a water content of 2% to 3.5%, an activation rate of about 70%, a stronger migration ability than conventional LPs, and the maximum amplitude (MA) of blood clots detected by thromboelastography in in vitro and in vivo experiments are higher than those of conventional freeze-dried platelets, and the hemostatic effect is good.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing freeze-dried platelets, comprising the following steps:

[0009] Cooling the fresh platelets to -38°C to -40°C for 60 minutes to obtain the first platelets;

[0010] After the first platelet is evacuated to 4.8-5 Pa, it is maintained at -40°C for 70 minutes to obtain a second platelet;

[0011] The second platelet is first heated to -28°C to -30°C and maintained for 60 minutes to obtain a third platelet;

[0012] The third platelet is subjected to a second heating to -18°C to -20°C and maintained for 60 minutes to obtain a fourth platelet;

[0013] The fourth platelet is subjected to a third heating to -10°C and maintained for 60 minutes to obtain a fifth platelet;

[0014] The fifth platelet is subjected to a fourth heating to 0° C. and maintained for 60 minutes to obtain a sixth platelet;

[0015] The sixth platelet is subjected to a fifth heating to 10° C. and maintained for 60 minutes to obtain a seventh platelet;

[0016] The seventh platelet is heated to 20° C. for the sixth time and maintained until dry to obtain the freeze-dried platelet.

[0017] Preferably, the cooling rate is -2℃ / min; the first heating rate is 0.2℃ / min; the second heating rate is 0.2℃ / min; the third heating rate is 0.2℃ / min; the fourth heating rate is 0.2℃ / min; the fifth heating rate is 0.2℃ / min; and the sixth heating rate is 0.2℃ / min.

[0018] Preferably, the moisture content of the freeze-dried platelets is 2 to 3.5 wt.%.

[0019] The present invention provides a platelet transfusion stock solution, comprising a platelet activator, an interstitial cytokine and frozen platelets prepared by the preparation method described in the above technical scheme.

[0020] Preferably, the platelet activator comprises one or more of thrombin, adenosine diphosphate and ristocetin.

[0021] Preferably, the concentration of thrombin in the platelet transfusion stock solution is 1 U / ml, the concentration of adenosine diphosphate is 200-500 μmol / L, and the concentration of ristocetin is 1.5-2 mg / ml.

[0022] Preferably, the concentration of interstitial cytokines in the platelet transfusion stock solution is 150 ng / ml.

[0023] Preferably, the concentration of frozen platelets in the platelet transfusion stock solution is (2-2.5)×10 11 Pieces / L.

[0024] Preferably, the solvent of the platelet transfusion stock solution is physiological saline.

[0025] The present invention provides frozen platelets prepared by the preparation method described in the above technical solution or the use of the platelet transfusion stock solution described in the above technical solution in the preparation of hemostatic products, and the hemostatic products include traumatic hemostatic products.

[0026] Beneficial effects:

[0027] The preparation method provided by the present invention adopts appropriate freeze-drying conditions, and the freeze-drying process is faster than the conventional freeze-drying process. The activation ratio of LPs can be increased on the basis of maintaining the activity of LPs, the activation rate is about 70%, the migration ability is stronger than that of conventional LPs, the MA values ​​of in vitro and in vivo experiments are higher than those of conventional freeze-dried platelets, and the hemostatic effect is good.

[0028] Furthermore, the platelet transfusion stock solution provided by the present invention can store transfused platelets and migrate to the bleeding site through platelet activators and interstitial cytokines, thereby increasing its hemostatic effect and reducing the incidence of thrombosis in unexpected locations. Experimental results show that after the platelet transfusion stock solution provided by the present invention is transfused into trauma-simulated animals, compared with conventional freeze-dried platelets, it promotes enhanced coagulation activity, is more distributed to the injury site, better promotes coagulation, improves post-traumatic coagulation dysfunction, and has a small number of platelets in the blood circulation, thereby reducing the probability of ectopic thrombosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0030] Figure 1 The results of adhesion experiments of different platelets are shown;

[0031] Figure 2 The results of the migration experiment of different platelets; compared with group A, * indicates P < 0.05, ** indicates P < 0.01; compared with group C, ★ indicates P < 0.05; compared with group F, ▲ indicates P < 0.05;

[0032] Figure 3 The highest peak results for blood clots of different platelets;

[0033] Figure 4 The mean arterial pressure results of rabbits in each group at different times;

[0034] Figure 5 Blood gas values ​​of rabbits in each group at different times; A: lactic acid value, B: base excess value;

[0035] Figure 6 The results of TEG parameter changes of rabbits in each group at different times; A: MA value, B: R value, C: α-angle value;

[0036] Figure 7 The blood coagulation values ​​of rabbits in each group at different times; A: APTT value, B: PT value;

[0037] Figure 8 The results of platelet and hemoglobin counts of rabbits in each group at different times; A: platelet count, B: hemoglobin count;

[0038] Fig. 9 The urea nitrogen index changes of rabbits in each group at different times;

[0039] Figure 1 or Figure 3 In the table, ns indicates P > 0.05, * indicates P < 0.05, and *** indicates P < 0.001; Figures 4 to 9 In, n=10, Δ: compared with group A at the same time, P<0.05; ▲: compared with group B at the same time, P<0.05, ◆: compared with the 0h value of this group, P<0.05; ◇: compared with group D at the same time, P<0.05. DETAILED DESCRIPTION

[0040] The present invention provides a method for preparing freeze-dried platelets, comprising the following steps:

[0041] Cooling the fresh platelets to -38°C to -40°C for 60 minutes to obtain the first platelets;

[0042] After the first platelet is evacuated to 4.8-5 Pa, it is maintained at -40°C for 70 minutes to obtain a second platelet;

[0043] The second platelet is first heated to -28°C to -30°C and maintained for 60 minutes to obtain a third platelet;

[0044] The third platelet is subjected to a second heating to -18°C to -20°C and maintained for 60 minutes to obtain a fourth platelet;

[0045] The fourth platelet is subjected to a third heating to -10°C and maintained for 60 minutes to obtain a fifth platelet;

[0046] The fifth platelet is subjected to a fourth heating to 0° C. and maintained for 60 minutes to obtain a sixth platelet;

[0047] The sixth platelet is subjected to a fifth heating to 10° C. and maintained for 60 minutes to obtain a seventh platelet;

[0048] The seventh platelet is heated to 20° C. for the sixth time and maintained until dry to obtain the freeze-dried platelet.

[0049] The present invention cools fresh platelets to -38°C to -40°C and maintains the temperature for 60 minutes to obtain first platelets. As an embodiment, the fresh platelets are cooled to -40°C and maintained for 60 minutes. As an embodiment, the cooling rate is -2°C / min.

[0050] After obtaining the first platelet, the present invention evacuates the first platelet to 4.8-5 Pa and maintains the platelet at -40°C for 70 minutes to obtain the second platelet. As an embodiment, the first platelet is evacuated to 5 Pa.

[0051] After obtaining the second platelet, the present invention first heats the second platelet to -28°C to -30°C and maintains it for 60 minutes to obtain the third platelet. As an embodiment, the second platelet is first heated to -30°C and maintained for 60 minutes. As an embodiment, the first heating rate is 0.2°C / min.

[0052] After obtaining the third platelet, the present invention performs a second temperature increase of the third platelet to -18°C to -20°C and maintains for 60 minutes to obtain a fourth platelet. As an embodiment, the third platelet is subjected to a second temperature increase of -20°C and maintained for 60 minutes. As an embodiment, the second temperature increase rate is 0.2°C / min.

[0053] After obtaining the fourth platelet, the present invention performs a third heating of the fourth platelet to -10°C and maintains the temperature for 60 minutes to obtain the fifth platelet. As an embodiment, the third heating rate is 0.2°C / min.

[0054] After obtaining the fifth platelet, the present invention performs a fourth heating of the fifth platelet to 0°C and maintains the temperature for 60 minutes to obtain the sixth platelet. As an embodiment, the fourth heating rate is 0.2°C / min.

[0055] After obtaining the sixth platelet, the present invention performs a fifth heating of the sixth platelet to 10° C. and maintains the temperature for 60 minutes to obtain the seventh platelet. As an embodiment, the fifth heating rate is 0.2° C. / min.

[0056] After obtaining the seventh platelet, the present invention heats the seventh platelet to 20°C for the sixth time and maintains it until it is dry to obtain the freeze-dried platelet. As an embodiment, the speed of the sixth temperature increase is 0.2°C / min. As an embodiment, the water content of the freeze-dried platelet is 2 to 3.5wt.%.

[0057] The preparation method provided by the present invention adopts appropriate freeze-drying conditions, and the freeze-drying process is faster than the conventional freeze-drying process. The activation ratio of LPs can be increased while maintaining the activity of LPs, with an activation rate of about 70%, and the migration ability is stronger than that of conventional LPs.

[0058] The present invention provides a platelet transfusion stock solution, comprising a platelet activator, an interstitial cytokine, and frozen platelets prepared by the preparation method described in the above technical solution. One problem that needs to be avoided when using freeze-dried platelets to treat bleeding is to prevent the formation of thrombi in non-injured parts of important organs such as the brain and lungs. The platelet transfusion stock solution provided by the present invention can store transfused platelets and migrate to the bleeding site through platelet activators and interstitial cytokines, thereby increasing its hemostatic effect and reducing the incidence of thrombi in unexpected sites.

[0059] As an embodiment, the platelet activator includes one or more of thrombin, adenosine diphosphate and ristocetin. As another embodiment, the platelet activator is thrombin.

[0060] As an embodiment, the concentration of thrombin in the platelet transfusion stock solution is 1 U / ml, the concentration of adenosine diphosphate is 200-500 μmol / L, and the concentration of ristocetin is 1.5-2 mg / ml.

[0061] As an embodiment, the concentration of stromal-derived cytokines in the platelet transfusion stock solution is 150 ng / ml.

[0062] As an embodiment, the concentration of frozen platelets in the platelet transfusion stock solution is (2-2.5)×10 11 Pieces / L.

[0063] As an embodiment, the solvent of the platelet transfusion stock solution is physiological saline.

[0064] The present invention preferably also provides a method for preparing the platelet transfusion stock solution described in the above technical solution, comprising the following steps:

[0065] Dissolving the platelet activator and the interstitial-derived cytokine in proportion to obtain a stimulator;

[0066] The stimulant and the frozen platelets prepared by the preparation method described in the above technical solution are mixed for 10 to 30 minutes to obtain the platelet transfusion stock solution.

[0067] As an embodiment, the volume mass ratio of the stimulator and the frozen platelets is 1 ml:1 g.

[0068] As an embodiment, the platelet transfusion stock solution is first diluted with physiological saline before infusion; based on the mass of frozen platelets, the mass volume ratio of the platelet transfusion stock solution and physiological saline is 1g:100ml.

[0069] The present invention provides the use of frozen platelets prepared by the preparation method described in the above technical solution or the platelet transfusion stock solution described in the above technical solution in the preparation of hemostatic products, and the hemostatic products include traumatic hemostatic products. As an embodiment, the traumatic hemostatic product can be a traumatic massive bleeding hemostatic product, and the applicable animal model is a soft tissue injury combined with hemorrhagic shock and seawater immersion injury model. As an embodiment, the dosage form of the product can be an injection.

[0070] After the platelet transfusion stock solution provided by the present invention is infused into trauma simulated animals, compared with conventional freeze-dried platelets, it promotes enhanced coagulation activity, is more distributed to the injury site, better promotes coagulation, improves post-traumatic coagulation dysfunction, and has a small number of platelets in the blood circulation, thereby reducing the probability of ectopic thrombosis.

[0071] The present invention also preferably provides a hemostasis method, comprising: preparing frozen platelets prepared by the preparation method described in the above technical solution or the platelet infusion stock solution described in the above technical solution, and infusing them in combination with lactated Ringer's solution. The freeze-dried platelets provided by the present invention combined with lactated Ringer's solution can significantly increase the mean arterial pressure of rabbits and reduce their acidosis. The freeze-dried platelets can continuously improve the coagulation function after pre-activation.

[0072] To further illustrate the present invention, a method for preparing freeze-dried platelets provided by the present invention and its application in preparing traumatic hemostatic products are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0073] Example 1

[0074] 1. Collecting platelets: After the rabbit is anesthetized, the femoral artery is cannulated to collect the whole blood of the rabbit. The whole blood of the rabbit is mixed with 40g / L sodium citrate solution at a volume ratio of 9:1, centrifuged at 3500rpm for 20min and then centrifuged for 10min. After the plasma is clear, the plasma and the buffy coat layer are separated. After the buffy coat is collected, centrifuged again and the buffy coat is removed. 10ml of lyophilized pretreatment solution is added to each 50ml centrifuge tube, incubated in an oscillating box at 37℃ for 1h, and then centrifuged at 3000rpm for 5min, the precipitate is removed, 10ml of lyophilized buffer is added, and the precipitate is placed in a small bowl and spread flat, and the thickness of the liquid surface does not exceed 1cm; the lyophilized pretreatment solution is composed of the following concentrations: 40mmol / L trehalose, 100mmol / L NaCl, 10mmol / L KCl, 10mmol / L EGTA, 10mmol / L imidazole (pH=6.8), 3% (v / v) DMSO; the lyophilization buffer consists of the following components in concentration: 9.5mmol / L HEPEs, 142.5mmol / L NaCl, 4.8mmol / L KCI, 1.0mmol / L MgCl2, 30mmol / L trehalose, 1% (v / v) bovine serum albumin, pH=6.8.

[0075] 2. Preparation of freeze-dried platelets: Freeze the platelets collected in step 1 from 22°C to -40°C at a freezing rate of -2°C / min and maintain for 60 min; evacuate to 5 Pa and maintain at -40°C for 70 min, heat to -30°C at a heating rate of 0.2°C / min and maintain for 60 min, heat to -20°C at a heating rate of 0.2°C / min and maintain for 60 min, heat to -10°C at a heating rate of 0.2°C / min and maintain for 60 min, heat to 0°C at a heating rate of 0.2°C / min and maintain for 60 min, heat to 10°C at a heating rate of 0.2°C / min and maintain for 60 min, heat to 20°C at a heating rate of 0.2°C / min and maintain until dry (water content 3wt.%), to obtain freeze-dried platelets (LPs).

[0076] Example 2

[0077] A frozen platelet stimulator consists of the following components: 1U / ml thrombin and 150ng / mL interstitial derived cytokine (SDF-1), and the solvent is normal saline.

[0078] Comparative Example 1

[0079] 1. Collect platelets: the method is the same as step 1 in Example 1.

[0080] 2. Preparation of freeze-dried platelets: Freeze the platelets collected in step 1 from room temperature to -45°C at a cooling rate of 20°C / h and pre-freeze for 4 hours; after evacuating to 5Pa, heat to -40°C and maintain for 1 hour, heat to -30°C and maintain for 1 hour, heat to -20°C and maintain for 2 hours, heat to -10°C and maintain for 2 hours, heat to 0°C and maintain for 3 hours, heat to 10°C and maintain for 3 hours, heat to 20°C and maintain for 4 hours, heat to 25°C and maintain until dry to obtain freeze-dried platelets; the heating rate after evacuating to 5Pa is 1°C / min.

[0081] Test Example 1 In vitro experiment

[0082] 1. Grouping and Stimulation of LPs

[0083] The in vitro experiment was divided into three groups: Group A was fresh platelets, Group B was LPs prepared in Comparative Example 1 (denoted as Scheme 1), and Group C was pre-stimulated LPs, specifically: 1 g of LPs prepared in Example 1 (denoted as Scheme 2) and 1 ml of the frozen platelet stimulant in Example 2 were mixed for 30 minutes to obtain the pre-stimulated LPs.

[0084] 2. Resuspension and Counting of Freeze-dried Platelets

[0085] Platelet-poor plasma (PPP) was used to dissolve and resuspend LPs or pre-stimulated LPs, and platelet count was performed using a hemocytometer according to the ratio of 0.05 g of freeze-dried powder to 1 ml of PPP.

[0086] 3. Platelet Adhesion Assay

[0087] Add 1mg / ml collagen to the EP tube, and then add it to the 96-well plate and incubate at 37℃ for 1h. Rinse the excess collagen with PBS buffer, add 100μl platelets (fresh platelets or resuspended freeze-dried platelets) to each well and incubate at 37℃ for 2h. After incubation, rinse with PBS buffer to remove the suspension, add 200μl 1% tritonX-100 to each well, and lyse at 37℃ for 30min. Take 120μl of the supernatant, add 60μl of the detection working solution in the Biyuntian LDH Cytotoxicity Detection Kit, mix well, incubate at room temperature in the dark for 30min, and then use an enzyme marker to measure the absorbance value of each sample. Prepare a series of known concentrations of platelet-containing plasma (PRP), measure the absorbance value after lysis, and calculate the number of adherent platelets in the sample after making a standard curve with the known platelet number and the absorbance value.

[0088] 4. Platelet Migration Assay

[0089] Take the resuspended freeze-dried platelet solution or fresh platelets and dilute it to (1-2) × 10 11 Within the concentration range of 100ng / L, the platelet concentration of each group was controlled to be similar as much as possible. The platelets after different dilutions were divided into the following treatment groups: Group A (fresh platelets + SDF-1 stimulation), Group B (fresh platelets + no SDF-1 stimulation), Group C (pre-stimulated LPs + SDF-1 stimulation), Group D (pre-stimulated LPs + no SDF-1 stimulation), Group E (LPs prepared in Comparative Example 1 + SDF-1 stimulation), Group F (LPs prepared in Comparative Example 1 + no SDF-1 stimulation), the group with SDF-1 stimulation was to add SDF-1 to the diluted platelets to a final concentration of 150ng / ml (simulating the up-regulation of local SDF-1 expression after in vivo trauma), and the group without SDF-1 stimulation was not added with SDF-1. The treatment methods of different groups are as follows:

[0090] Prepare a transwell cell migration chamber with a pore size of 8 μm and a 6-well plate, add 500 μl of physiological saline, incubate for 30 minutes for hydration, remove the liquid, and check for leakage. Take 1.5 ml of PPP and add it to the lower chamber of the migration chamber. Take 1 ml of diluted platelets from each treatment group and put them into the transwell chamber respectively; after preparation, put the migration chamber together with the cell culture plate into a 37°C incubator for 2 hours, then detect the number of platelets in the lower chamber and calculate the migration rate of each group.

[0091] 5. Thromboelastography of platelet suspension

[0092] After resuspending the two batches of platelets in group B and group C, PRP and PPP samples were added for thromboelastography (TEG) testing. 1 ml of each sample was added to the activated coagulation reagent tube for reaction for 3 minutes, and then 20 μl of 0.1M calcium chloride was added to the thromboelastography sample cup, and then 340 μl of the sample was taken from the activated reagent tube and added to the test cup to measure the thromboelastography parameters such as coagulation reaction time (R value), maximum peak value of blood clot (MA) and blood clot formation rate (α-angle).

[0093] The experimental results are as follows:

[0094] 1. Platelet adhesion test results Figure 1 And as shown in Table 1.

[0095] Table 1 Adhesion number of fresh platelets and freeze-dried platelets (n=6)

[0096] Group Group A Group B Group C <![CDATA[Number of adherent platelets (10 6 )]]> 18.56±3.45 0.88±0.27 2.46±0.66

[0097] The results showed that compared with fresh platelets (group A), the adhesion ability of LPs freeze-dried by both methods was significantly decreased (P<0.001, Figure 1There was no significant difference in adhesion ability between group B and group C (P>0.05, Figure 1 ).

[0098] 2. Platelet migration test results are shown in Figure 2 . The results showed that after SDF-1 stimulation, the migration ability of fresh platelets, freeze-dried platelets in comparative example 1 and freeze-dried platelets in example 1 was significantly increased. Compared with, the migration ability of the two groups of freeze-dried platelets after stimulation was significantly lower than that of fresh platelets after stimulation. Compared with conventional freeze-dried platelets (comparative example 1), the migration ability of freeze-dried platelets prepared in example 1 of the present invention after stimulation was significantly enhanced.

[0099] 3. The results of the thromboelastogram MA value of the platelet suspension are shown in Figure 3 and Table 2.

[0100] Table 2 MA values ​​of freeze-dried platelets, fresh platelets, and plasma-deficient platelets in two groups (n=6)

[0101] project Platelet-free plasma Platelet-rich plasma Example 1 Comparative Example 1 MA / mm 23.65±6.96 82.08±3.28 67.02±7.47 46.65±9.20

[0102] There was no significant difference in the α-angle and R values ​​of platelets in each group, indicating that the freeze-drying process and pre-stimulation had no significant effect on the coagulation rate and the initiation speed of coagulation. The MA values ​​reflecting platelet function showed differences (Table 1 and Figure 2 ): The MA values ​​of groups B and C were significantly lower than those of group A, while that of group B was significantly lower than that of group C. It can be seen that pre-stimulation can improve the coagulation activity of LPs, increase the MA value, and thus improve the quality of blood clots and reduce bleeding.

[0103] Test Example 1 In vivo experiment

[0104] 1. Experimental Animal Grouping Design

[0105] The experimental animals were 50 New Zealand white rabbits, provided by the Animal Experiment Center of the Army Medical University, with a weight of (2.25±0.16) kg, an age of 3 to 4 months, and no gender restrictions. The 50 rabbits were randomly divided into five groups, A, B, C, D, and E, with 10 rabbits in each group. The animal model used the rabbit soft tissue injury combined with hemorrhagic shock and seawater immersion injury model established in the laboratory in the early stage, which is briefly described as follows: 3% sodium pentobarbital was used to anesthetize the rabbits at a dose of 1 ml / kg body weight, and then the left lower limb soft tissue injury was given, and gauze was used to stop bleeding, and the blood loss of the lower limb soft tissue injury was measured. The right lower limb was cannulated with a femoral artery, and then blood was drawn according to 26% of the total blood volume of the rabbit (the blood loss of the lower limb soft tissue injury was included), and then the rabbits were vertically immersed in 3% simulated seawater, and the seawater submerged the xiphoid process, and the rabbits were fished out after 15 minutes. The preparation method of the 3% simulated seawater is as follows: artificial sea salt is added to tap water in proportion to prepare artificial simulated seawater with a salinity of 3%. Crushed ice is added within 1 hour before the start of the experiment and during the experiment to adjust the water temperature to maintain at 22°C to 23°C.

[0106] The established animal models were treated as follows according to the groups (kg in the following different groups refers to the weight of rabbits):

[0107] Group A: no resuscitation (i.e. no treatment);

[0108] Group B: rabbits were infused with 20 ml / kg of Ringer's solution;

[0109] Group C: 20 ml / kg of Ringer's solution and 3.78 × 10 8 Fresh platelets / kg were transfused into rabbits;

[0110] Group D: 20 ml / kg Ringer's solution and 3.78 × 10 8 LPs / kg were infused into rabbits; the LPs were prepared in Example 1, and physiological saline was used as a rehydrating agent for rehydration during infusion, LPs: physiological saline = 1 g: 100 ml;

[0111] Group E: 20 ml / kg Ringer's solution and 3.78 × 10 8 Pre-stimulated LPs / kg were infused into rabbits; the preparation method of the pre-stimulated LPs was as follows: 1g of LPs prepared in Example 1 was mixed with 1ml of the frozen platelet stimulator described in Example 2 for 10 minutes to obtain the pre-stimulated LPs; during the infusion, normal saline was used as a rehydrating agent for dilution, and the mass of LPs before pre-stimulation was calculated as follows: pre-stimulated LPs: normal saline = 1g: 100ml.

[0112] The changes of injury condition in different groups were observed 4 hours after infusion for 20 minutes. The animals used in this experiment were approved by the Ethics Committee of Army Medical University, and the experimental process complied with animal ethics requirements.

[0113] 2. Detection indicators and detection methods

[0114] Mean arterial pressure (MAP) was monitored at four time points: before injury, 1 h, 2 h, and 4 h after injury. Blood was drawn for testing of five coagulation tests, routine blood tests, blood gas tests, 11 emergency tests, and thromboelastograms. The number of red blood cells and platelets was measured using the Mindray BC-5180CRP fully automatic blood cell analyzer (Mindray, Shenzhen, China). 1.5 ml of venous blood was collected at each time phase, and after centrifugation, the prothrombin time (PT) and activated partial clotting time (APTT) were measured using the Raydu coagulation analyzer (Raydu, Shenzhen, China). The pH, lactate concentration (Lac), base excess (BE), and blood urea nitrogen (BUN) at the four time phases were measured using the i-STAT handheld blood gas analyzer (Flextronics Manufacturing, Singapore) and its matching CHEM8+ and CG4+ test cards. TEG analysis was performed using a thromboelastograph (CFMS LEPU-8800, Lepu Medical Technology Co., Ltd., Beijing, China). For each test, 1 ml of anticoagulated blood was drawn into the thromboelastograph activation coagulation actual bottle to react for 3 to 5 minutes. Subsequently, a common cup was installed and 20 μl of calcium chloride was added thereto. After 3 to 5 minutes, 340 μl of blood in the activation reagent bottle was drawn into the common cup and loaded for testing. The coagulation reaction time (R value), the highest peak value of blood clot (MA) and the blood clot formation rate (α-angle) were recorded.

[0115] The experimental results are as follows:

[0116] 1. The results of MAP changes in each group of rabbits are shown in Figure 4 .

[0117] The results showed that the baseline MAP values ​​of the groups before injury were not statistically significant. The MAP of groups C, D, and E was higher than that of group A at 1 hour after injury, and there was no significant difference in MAP between groups A and B. At 2 hours after injury, the MAP of groups C, D, and E was higher than that of group A, and the MAP of groups C and D was higher than that of group B. At 4 hours after injury, the MAP of groups B, C, D, and E was higher than that of group A, and the MAP of groups C, D, and E was higher than that of group B.

[0118] 2. The results of blood gas analysis of each group of rabbits after treatment are shown in Figure 5 .

[0119] The results showed that the lactate baseline values ​​of the rabbits in each group before injury were not statistically significant; at 1 hour after injury, the lactate value of group C was lower than that of groups A and B; at 2 hours after injury, the lactate levels of groups C, D, and E were lower than those of group A, and the lactate levels of groups C and E were lower than those of group B; at 4 hours after injury, the lactate levels of groups C, D, and E were all lower than those of groups A and B ( Figure 5 Base excess (BE) is another auxiliary indicator of acidosis. The results show that at 1h and 2h after injury, the negative BE values ​​of groups C, D, and E were lower than those of groups A and B. At 4h after injury, the negative BE values ​​of groups C, D, and E were still lower than those of group A, and the negative BE value of group E was lower than that of group B ( Figure 5 (middle B).

[0120] 3. The trend of TEG changes in each group of rabbits after treatment is shown in Figure 6 .

[0121] The results showed that MA reflected platelet function. Among the five groups, the MA values ​​of group A and group D decreased significantly at 2h and 4h after injury compared with the initial time. There was no significant difference between the MA values ​​of the other three groups, B, C, and E. At 2h, the MA value of group C was significantly higher than that of group A. At 4h, the MA values ​​of groups B, C, and E were higher than those of group A ( Figure 6 The R value represents the coagulation reaction time, and it changes dynamically over time in each group. In group A, the R value tends to be shortened, but it is not statistically significant. At 1 hour after injury, the R values ​​of groups C and E tend to be prolonged compared with the initial values ​​of each group. At 2 hours and 4 hours after injury, the R value of group D also tends to be prolonged compared with the initial value ( Figure 6 The α-angle value represents the blood clot production rate. It tends to decrease over time in groups A and B. At 4 hours, the α-angle value of group B is significantly lower than that of group C at 0 hours. There is no significant difference in the α-angle values ​​of the other groups at different times. Figure 6 Middle C).

[0122] 4. The results of the changes in coagulation function of rabbits in each group after treatment are shown in Figure 7 .

[0123] Activated partial coagulation time (APTT) reflects the endogenous coagulation status. In groups A and D, APTT at 2h and 4h after injury was prolonged compared with the initial value. APTT in other groups showed a tendency to prolong after injury, but there was no significant statistical significance ( Figure 7 Prothrombin time (PT) reflects the exogenous coagulation status in vivo. In group A, PT was prolonged at 1h and 2h after injury, while no significant PT prolongation was observed in the other groups ( Figure 7 (middle B).

[0124] 5. Changes in platelet and hemoglobin counts in each group of rabbits after treatment Figure 8 .

[0125] The platelet counts of each group decreased significantly after injury, and at 2 h after injury, the platelet count of group C was higher than that of group D. There was no significant difference in the platelet counts of each group at other time points ( Figure 8 The hemoglobin (Hb) counts in each group also showed a downward trend after injury. 2 hours after injury, the Hb counts in group B were lower than those in group D ( Figure 8 (middle B).

[0126] 6. Changes of blood urea nitrogen (BUN) in each group of rabbits after treatment Fig. 9 .

[0127] At 4 hours after injury, the BUN values ​​of rabbits in each group increased to varying degrees. At the final 4 hours, the BUN values ​​of groups C, D, and E were all lower than those of group A ( Fig. 9 ).

[0128] In summary, the mean arterial pressure of group A, which was not resuscitated after injury, decreased to (53.12±8.38) mmHg at 1 h after injury, lactate increased, maximum amplitude (MA) decreased, and activated partial coagulation time (APTT) prolonged (P<0.05). In contrast, the mean arterial pressure of groups C, D, and E was higher than that of group A at 1 h, 2 h, and 4 h after resuscitation (P<0.05). At the same time, the lactate levels of groups C, D, and E were lower than those of groups A and B at 4 h after injury (P<0.05). The MA values ​​of groups A and D at 4 h were significantly decreased compared with their initial values ​​(P<0.05). The APTT and R values ​​of group D at 4 h were significantly prolonged compared with those at 0 h (P<0.05). It can be seen that platelets combined with lactated Ringer's solution can significantly increase the mean arterial pressure of rabbits and reduce their acidosis. Freeze-dried platelets can continuously improve coagulation function after pre-activation.

[0129] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing freeze-dried platelets, characterized in that: The following steps are involved: Cooling the fresh platelets to -38°C to -40°C for 60 minutes to obtain the first platelets; After the first platelet is evacuated to 4.8-5 Pa, it is maintained at -40°C for 70 minutes to obtain a second platelet; The second platelet is first heated to -28°C to -30°C and maintained for 60 minutes to obtain a third platelet; The third platelet is subjected to a second heating to -18°C to -20°C and maintained for 60 minutes to obtain a fourth platelet; The fourth platelet is subjected to a third heating to -10°C and maintained for 60 minutes to obtain a fifth platelet; The fifth platelet is subjected to a fourth heating to 0° C. and maintained for 60 minutes to obtain a sixth platelet; The sixth platelet is subjected to a fifth heating to 10° C. and maintained for 60 minutes to obtain a seventh platelet; The seventh platelet is heated to 20° C. for the sixth time and maintained until dry to obtain the freeze-dried platelet.

2. The preparation method according to claim 1, characterized in that: The cooling rate is -2℃ / min; the first heating rate is 0.2℃ / min; the second heating rate is 0.2℃ / min; the third heating rate is 0.2℃ / min; the fourth heating rate is 0.2℃ / min; the fifth heating rate is 0.2℃ / min; and the sixth heating rate is 0.2℃ / min.

3. The preparation method according to claim 1, characterized in that: The water content of the freeze-dried platelets is 2-3.5 wt.%.

4. A platelet transfusion stock solution, characterized in that: The invention comprises a platelet activator, an interstitial cytokine and frozen platelets prepared by the preparation method according to any one of claims 1 to 3.

5. The platelet transfusion stock solution according to claim 4, characterized in that: The platelet activator comprises one or more of thrombin, adenosine diphosphate and ristocetin.

6. The platelet transfusion stock solution according to claim 5, characterized in that: The concentration of thrombin in the platelet transfusion stock solution is 1 U / ml, the concentration of adenosine diphosphate is 200-500 μmol / L, and the concentration of ristocetin is 1.5-2 mg / ml.

7. The platelet transfusion stock solution according to claim 4, characterized in that: The concentration of the stromal-derived cytokines in the platelet transfusion stock solution is 150 ng / ml.

8. The platelet transfusion stock solution according to claim 4, characterized in that: The concentration of frozen platelets in the platelet transfusion stock solution is (2-2.5)×10 11 Pieces / L.

9. The platelet transfusion stock solution according to any one of claims 4 to 8, characterized in that: The solvent of the platelet transfusion stock solution is normal saline.

10. Use of frozen platelets prepared by the preparation method according to any one of claims 1 to 3 or the platelet transfusion stock solution according to any one of claims 4 to 9 in the preparation of hemostatic products, wherein the hemostatic products include traumatic hemostatic products.

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