Coagulation-fibrinolysis high-balance type freeze-dried plasma as well as preparation method and application thereof

Abstract

The invention relates to the technical field of hemostatic preparations, in particular to blood coagulation-fibrinolysis high-balance type freeze-dried plasma as well as a preparation method and application of the blood coagulation-fibrinolysis high-balance type freeze-dried plasma. According to the blood coagulation-fibrinolysis high-balance type freeze-dried plasma provided by the invention, blood coagulation factors, anticoagulation factors, fibrinolysis factors, fibrinolysis inhibition factors and von willebrand factors with proper concentrations are compounded in the freeze-dried plasma according to different blood coagulation-fibrinolysis system change rules after different types of tissues and organs are damaged; the hemostatic resuscitation agent can be used for hemostatic resuscitation of general severe trauma, severe trauma mainly including liver, severe trauma mainly including severe craniocerebral trauma, severe trauma combined with seawater soaking and other injuries, the hemostatic resuscitation effect is good, thrombus of uninjured parts cannot be caused, and the lactic acid rising amplitude after trauma can be reduced.

Description

Technical Field

[0001] The invention relates to the technical field of hemostatic preparations, and in particular to a coagulation-fibrinolysis high-balance freeze-dried plasma and a preparation method and application thereof. Background Art

[0002] Freeze-dried plasma (FDP) is widely used in pre-hospital and wartime treatment in many countries, showing good resuscitation effects. However, the FDP currently used has a significant decrease in multiple coagulation factors during the freeze-drying process, making it unable to meet the requirements of pre-hospital hemostatic resuscitation. During the freeze-drying and storage process, some coagulation factors of FDP will lose their activity, resulting in a significant decrease in von Willebrand factor (vWF) and coagulation factors II, V, VII, VIII, X, etc. When traumatic coagulopathy occurs in patients with severe trauma, the reduction level of coagulation factors and other factors significantly exceeds the level of related factors in the lost blood. The changes in the coagulation-fibrinolysis system after different types of tissue and organ damage are different. These factors make it difficult for conventional FDP to achieve the goal and effect of hemostatic resuscitation in patients with severe trauma.

[0003] Current studies have shown that FDP has shown a better resuscitation effect in trauma patients. A meta-analysis by Feuerstein et al. [Feuerstein SJ, et al. Freeze-dried plasma in major haemorrhage: a systematic review. Vox Sang. 2020; 115(4): 263-74.] showed that there was no significant difference in mortality between FDP and fresh frozen plasma 30 days after injury. In 2022, a meta-analysis by Mok et al. [MokG, et al. Freeze-dried plasma formajor trauma-Systematic review and meta-analysis. J Trauma Acute Care Surg. 2021; 90(3): 589-602.] showed that there was no significant difference in mortality between trauma patients receiving FDP compared with fresh frozen plasma; and animal experimental results showed that there was no difference in coagulation factor levels and anti-inflammatory properties between FDP and fresh frozen plasma. However, the clinical observation indicator of the above literature is only mortality, and the effects on coagulation function, lactic acid, organ function and other indicators of trauma patients are not observed, and it is limited to comparing the difference in effects between FDP and fresh frozen plasma.

[0004] Sheffield meta-analyzed the effect of FDP in trauma patients in 2024, and included three high-level randomized controlled studies. Among them, the clinical study with the abbreviation code RePHILL included 501 trauma patients in the UK. The observation indicators were mortality and lactate clearance, and the control group used normal saline for resuscitation. The results showed that there was no statistical difference in mortality and lactate clearance between the two groups. The study with the abbreviation code PREHO-PLYO included 150 patients, who were randomly assigned to the hospital to receive up to 4 units of FDP or up to 1000 ml of normal saline. The results showed that there was no significant difference in the international normalized ratio between the two groups. A third study evaluated the feasibility of transfusing freeze-dried plasma and red blood cells in the prehospital setting of the Australian School of Aeronautical Medicine using a randomized controlled design and found that the freeze-dried plasma group may have a lower mortality rate within 24 hours of admission and at discharge [Sheffield WP, ​​et al. Prehospital Freeze-Dried Plasma in Trauma: A Critical Review. Transfus Med Rev. 2024; 38(1): 150807.].

[0005] In summary, although there is no significant difference in mortality between FDP and fresh frozen plasma, FDP does not improve the coagulation status and shock correction effect of the injured compared with normal saline. More importantly, the above studies did not stratify trauma patients according to the severity of injury. The coagulation-anticoagulation-fibrinolysis-antifibrinolysis system disorder in severe trauma patients is more serious and complex than that in milder patients, and the use of traditional FDP resuscitation may not achieve the goal of hemostatic resuscitation strategy. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a coagulation-fibrinolysis high-balance freeze-dried plasma and its preparation method and application. The coagulation-fibrinolysis high-balance freeze-dried plasma provided by the present invention can be used for hemostatic resuscitation of general severe trauma, severe trauma mainly involving the liver, severe trauma mainly involving severe craniocerebral trauma, severe trauma combined with seawater immersion and other types of injuries, and has a good hemostatic resuscitation effect.

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

[0008] The invention provides a coagulation-fibrinolysis high-balance freeze-dried plasma, comprising the following components in parts by mass: freeze-dried plasma, 32-600 parts of coagulation factor II, 33.5-1200 parts of fibrinogen, 0.7-16 parts of coagulation factor V, 0.9-16 parts of coagulation factor X, 3.1-50 parts of antithrombin III, 0-24 parts of plasminogen, plasminogen activator inhibitor-1 and 0.112-2.1 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II to the plasminogen activator inhibitor-1 is 32-600 mg: 104-4000 AU; the freeze-dried plasma prepared from 200 ml of human plasma or 20 ml of rabbit plasma is recorded as 1 unit, and the ratio of the freeze-dried plasma to the coagulation factor II is 1 unit: 32-600 mg.

[0009] Preferably, the coagulation-fibrinolysis high balance freeze-dried plasma comprises one or more of independently packaged preparations 1 to 8:

[0010] The preparation 1 comprises the following components in parts by mass: freeze-dried plasma, 300 parts of coagulation factor II, 300 parts of fibrinogen, 8 parts of coagulation factor V, 8 parts of coagulation factor X, 25 parts of antithrombin III, 12 parts of plasminogen, plasminogen activator inhibitor-1 and 1.05 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and the plasminogen activator inhibitor-1 is 300 mg:1000 AU; the ratio of the freeze-dried plasma to the coagulation factor II is 1 unit:300 mg;

[0011] The preparation 2 comprises the following components in parts by mass: freeze-dried plasma, 600 parts of coagulation factor II, 600 parts of fibrinogen, 16 parts of coagulation factor V, 16 parts of coagulation factor X, 50 parts of antithrombin III, 24 parts of plasminogen, plasminogen activator inhibitor-1 and 2.1 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and the plasminogen activator inhibitor-1 is 600 mg: 2000 AU; the ratio of the freeze-dried plasma to the coagulation factor II is 1 unit: 600 mg;

[0012] The preparation 3 comprises the following components in parts by mass: freeze-dried plasma, 300 parts of coagulation factor II, 1200 parts of fibrinogen, 8 parts of coagulation factor V, 8 parts of coagulation factor X, 100 parts of antithrombin III, 48 parts of plasminogen, plasminogen activator inhibitor-1 and 4.2 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and the plasminogen activator inhibitor-1 is 300 mg:4000 AU; the ratio of the freeze-dried plasma to the coagulation factor II is 1 unit:300 mg;

[0013] The preparation 4 comprises the following components in parts by mass: freeze-dried plasma, 300 parts of coagulation factor II, 300 parts of fibrinogen, 8 parts of coagulation factor V, 8 parts of coagulation factor X, 25 parts of antithrombin III, 0 parts of plasminogen, plasminogen activator inhibitor-1 and 4.2 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and the plasminogen activator inhibitor-1 is 300 mg:4000 AU; the ratio of the freeze-dried plasma to the coagulation factor II is 1 unit:300 mg;

[0014] The freeze-dried plasma in the preparations 1 to 4 is human freeze-dried plasma;

[0015] The preparation 5 comprises the following components in parts by mass: freeze-dried plasma, 32 parts of coagulation factor II, 33.5 parts of fibrinogen, 0.7 parts of coagulation factor V, 0.9 parts of coagulation factor X, 3.1 parts of antithrombin III, 1.4 parts of plasminogen, plasminogen activator inhibitor-1 and 0.112 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and plasminogen activator inhibitor-1 is 32 mg:104 AU; the ratio of the freeze-dried plasma to coagulation factor II is 1 unit:32 mg;

[0016] The preparation 6 comprises the following components in parts by mass: freeze-dried plasma, 64 parts of coagulation factor II, 67 parts of fibrinogen, 1.4 parts of coagulation factor V, 1.8 parts of coagulation factor X, 6.2 parts of antithrombin III, 2.8 parts of plasminogen, plasminogen activator inhibitor-1 and 0.208 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and plasminogen activator inhibitor-1 is 64 mg:2084 AU; the ratio of the freeze-dried plasma to coagulation factor II is 1 unit:64 mg;

[0017] The preparation 7 comprises the following components in parts by mass: freeze-dried plasma, 32 parts of coagulation factor II, 134 parts of fibrinogen, 0.7 parts of coagulation factor V, 0.9 parts of coagulation factor X, 12.4 parts of antithrombin III, 5.6 parts of plasminogen, plasminogen activator inhibitor-1 and 0.448 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and plasminogen activator inhibitor-1 is 32 mg:416 AU; the ratio of the freeze-dried plasma to coagulation factor II is 1 unit:32 mg;

[0018] The preparation 8 comprises the following components in parts by mass: freeze-dried plasma, 32 parts of coagulation factor II, 33.5 parts of fibrinogen, 0.7 parts of coagulation factor V, 0.9 parts of coagulation factor X, 3.1 parts of antithrombin III, 0 parts of plasminogen, plasminogen activator inhibitor-1 and 0.448 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and plasminogen activator inhibitor-1 is 32 mg:416 AU; the ratio of the freeze-dried plasma to coagulation factor II is 1 unit:32 mg;

[0019] The freeze-dried plasma in the preparations 5 to 8 is freeze-dried plasma from rabbits.

[0020] Preferably, the raw material for preparing the freeze-dried plasma in the preparation 1 is fresh human plasma or human platelet-rich plasma; the raw material for preparing the freeze-dried plasma in the preparation 5 is fresh rabbit plasma or rabbit platelet-rich plasma.

[0021] Preferably, the raw material for preparing the freeze-dried plasma in Preparations 2 to 4 is fresh human plasma; the raw material for preparing the freeze-dried plasma in Preparations 6 to 8 is fresh rabbit plasma.

[0022] The present invention provides a method for preparing the coagulation-fibrinolysis high-balance freeze-dried plasma described in the above technical solution, comprising: mixing the components in proportion to obtain the coagulation-fibrinolysis high-balance freeze-dried plasma.

[0023] Preferably, the method for preparing freeze-dried plasma in the coagulation-fibrinolysis high balance freeze-dried plasma comprises the following steps:

[0024] Plasma, ascorbic acid, and glycine are mixed to obtain primary plasma;

[0025] The primary plasma was pre-frozen at -40°C for 2 hours, then evacuated to below 30 Pa, and sublimated and dried at -40°C for 10 hours;

[0026] After sublimation drying, the freeze-dried plasma was dried at -35°C for 6 hours to keep the water content of the freeze-dried plasma within 3%, thereby obtaining the freeze-dried plasma.

[0027] Preferably, in the primary plasma, the concentration of ascorbic acid is 2-4 mmol / L, and the concentration of glycine is 60 mmol / L.

[0028] The present invention provides the use of the coagulation-fibrinolysis high-balance freeze-dried plasma described in the above technical solution or the coagulation-fibrinolysis high-balance freeze-dried plasma prepared by the preparation method described in the above technical solution in the preparation of hemostatic products.

[0029] Preferably, the hemostatic product is a hemostatic resuscitation product.

[0030] Preferably, the hemostatic resuscitation product comprises one or more hemostatic resuscitation products for general severe trauma, severe trauma mainly involving the liver, severe trauma mainly involving severe craniocerebral trauma, and severe trauma combined with seawater immersion.

[0031] Beneficial effects:

[0032] The coagulation-fibrinolysis high-balance freeze-dried plasma provided by the present invention is compounded with coagulation factors, anticoagulant factors, fibrinolytic factors, fibrinolysis inhibitors and von Willebrand factor at appropriate concentrations according to the different changing rules of the coagulation-fibrinolysis system after different types of tissue and organ injuries. The freeze-dried plasma can be used for hemostatic resuscitation of general severe trauma, severe trauma mainly involving the liver, severe trauma mainly involving severe craniocerebral trauma, severe trauma combined with seawater immersion and other types of injuries. The hemostatic resuscitation effect is good, thrombosis will not be caused in uninjured parts, and the amplitude of lactic acid increase after trauma can be reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] 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.

[0034] Figure 1 is the R value of each group in the in vitro experiment in Test Example 1;

[0035] Figure 2 is the α angle value of each group in the in vitro experiment in Test Example 1;

[0036] Figure 3 The MA values ​​of each group in the in vitro experiment in Test Example 1;

[0037] Figure 4 is the R value of each group in the in vivo experiment in Test Example 1;

[0038] Figure 5 is the α angle of each group in the in vivo experiment in Test Example 1;

[0039] Figure 6 is the MA value of each group in the in vivo experiment in Test Example 1;

[0040] Figure 7 The blood loss of each group in the in vivo experiment in Test Example 1;

[0041] Figure 8 The changes in lactate concentration in each group of the in vivo experiment in Test Example 1;

[0042] Fig. 9 This is the lung tissue section of the FDP animal with high coagulation-fibrinolysis balance in the ISS18 group + 2.0 times in Test Example 1 (×200);

[0043] Fig.10This is the liver tissue section of the ISS18 group + 2.0 times coagulation-fibrinolysis high balance FDP animal in Test Example 1 (×200);

[0044] Fig.11 This is the kidney tissue section of the ISS18 group + 2.0 times coagulation-fibrinolysis high balance FDP animal in Test Example 1 (×200);

[0045] Fig.12 is the R value of each group in the in vitro experiment in Test Example 2;

[0046] Fig.13 is the α angle value of each group in the in vitro experiment in Test Example 2;

[0047] Fig.14 The MA values ​​of each group in the in vitro experiment in Test Example 2;

[0048] Fig.15 is the R value of each group in the in vivo experiment in Test Example 2;

[0049] Fig.16 is the α angle of each group in the in vivo experiment in Test Example 2;

[0050] Fig.17 is the MA value of each group in the in vivo experiment in Test Example 2;

[0051] Fig.18 The blood loss of each group in the in vivo experiment in Test Example 2;

[0052] Fig.19 The changes in lactate concentration in each group of the in vivo experiment in Test Example 2;

[0053] Fig. 20 This is a lung tissue section of a FDP animal with high balance of coagulation and fibrinolysis and rich in fibrinogen in Test Example 2 (×200);

[0054] Fig.21 This is a liver tissue section of a FDP animal with high coagulation-fibrinolysis balance and rich in fibrinogen in Test Example 2 (×200);

[0055] Fig. 22 This is a kidney tissue section of a FDP animal with high coagulation-fibrinolysis balance and rich in fibrinogen in Test Example 2 (×200);

[0056] Fig.23 is the R value of each group in the in vitro experiment in Test Example 3;

[0057] Fig.24 is the α angle value of each group in the in vitro experiment in Test Example 3;

[0058] Fig.25 The MA values ​​of each group in the in vitro experiment in Test Example 3;

[0059] Fig.26 is the R value of each group in the in vivo experiment in Test Example 3;

[0060] Fig. 27 is the α angle of each group in the in vivo experiment in Test Example 3;

[0061] Fig.28 is the MA value of each group in the in vivo experiment in Test Example 3;

[0062] Fig.29 The blood loss of each group in the in vivo experiment in Test Example 3;

[0063] Fig.30 The changes in lactate concentration in each group of the in vivo experiment in Test Example 3;

[0064] Fig.31 This is a lung tissue section of a FDP animal with a high balance of coagulation and fibrinolysis rich in fibrinolysis inhibitor in Test Example 3 (×200);

[0065] Fig.32 The liver tissue section of the FDP animal with high coagulation-fibrinolysis balance and rich in fibrinolysis inhibitor in Test Example 3 (×200);

[0066] Fig.33 This is a kidney tissue section of a FDP animal with a high balance of coagulation and fibrinolysis and rich in fibrinolysis inhibitors in Test Example 3 (×200);

[0067] Fig.34 is the R value of each group in the in vitro experiment in Test Example 4;

[0068] Fig.35 is the α angle value of each group in the in vitro experiment in Test Example 4;

[0069] Fig.36 MA values ​​of each group in the in vitro experiment in Test Example 4;

[0070] Fig.37 is the R value of each group in the in vivo experiment in Test Example 4;

[0071] Fig.38 is the α angle of each group in the in vivo experiment in Test Example 4;

[0072] Fig.39 is the MA value of each group in the in vivo experiment in Test Example 4;

[0073] Fig.40 The blood loss of each group in the in vivo experiment in Test Example 4;

[0074] Fig.41 The changes in lactate concentration in each group of the in vivo experiment in Test Example 4;

[0075] Fig.42This is a lung tissue section of a platelet-rich, coagulation-fibrinolysis high-balance FDP animal in Test Example 4 (×200);

[0076] Fig.43 The liver tissue section of the platelet-rich coagulation-fibrinolysis high-balance FDP animal in Test Example 4 (×200);

[0077] Fig.44 The kidney tissue section of the platelet-rich coagulation-fibrinolysis high-balance FDP animal in Test Example 4 (×200);

[0078] Figure 1 to Figure 3 , Figure 12 to Figure 14 , Figure 23 to Figure 25 , Figure 34 to Figure 36 In the table, * means P < 0.05 compared with group A; ★ means P < 0.05 compared with group B; △ means P < 0.05 compared with group C;

[0079] Figures 4 to 6 , Figure 8 In the table, * means P<0.05 compared with the NC group of the same ISS injury severity, ** means P<0.01 compared with the NC group of the same ISS injury severity; △ means P<0.05 compared with the A group of the same ISS injury severity, △△ means P<0.01 compared with the A group of the same ISS injury severity; ★ means P<0.05 compared with the B group of the same ISS injury severity, ★★ means P<0.01 compared with the B group of the same ISS injury severity; ▲ means P<0.05 compared with the same treatment measure in the same injury model;

[0080] Figure 7 In the table, * indicates P < 0.05 compared with group A with the same ISS injury severity; △ indicates P < 0.05 compared with group B with the same ISS injury severity;

[0081] Figure 15 to Figure 17 , Fig.19 , Figure 26 to Figure 28 , Figure 37 to Figure 39 In the table, * means P<0.05 compared with the NC group, ** means P<0.01 compared with the NC group; ★ means P<0.05 compared with the A group, ★★ means P<0.01 compared with the A group; △ means P<0.05 compared with the B group; ▲ means P<0.05 compared with the C group;

[0082] Fig.18 , Fig.29 , Fig.40 In the table, * means P < 0.05 compared with group A, ** means P < 0.01 compared with group A; ★ means P < 0.05 compared with group B; △ means P < 0.05 compared with group C;

[0083] Fig.19 , Fig.30 , Fig.41 In the table, ** means P < 0.01 compared with the NC group; ★ means P < 0.05 compared with the A group; ★★ means P < 0.01 compared with the A group; △ means P < 0.05 compared with the B group; ▲ means P < 0.05 compared with the C group. DETAILED DESCRIPTION

[0084] The invention provides a coagulation-fibrinolysis high-balance freeze-dried plasma, comprising the following components in parts by mass: freeze-dried plasma (FDP), 32-600 parts of coagulation factor II, 33.5-1200 parts of fibrinogen, 0.7-16 parts of coagulation factor V, 0.9-16 parts of coagulation factor X, 3.1-50 parts of antithrombin III, 0-24 parts of plasminogen, plasminogen activator inhibitor-1 and 0.112-2.1 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and the plasminogen activator inhibitor-1 is 32-600 mg: 104-4000 AU; the freeze-dried plasma prepared from 200 ml of human plasma or 20 ml of rabbit plasma is recorded as 1 unit, and the ratio of the freeze-dried plasma to the coagulation factor II is 1 unit: 32-600 mg.

[0085] As an embodiment, the coagulation-fibrinolysis high balance freeze-dried plasma includes one or more of independently packaged preparations 1 to 8:

[0086] The preparation 1 comprises the following components in parts by mass: freeze-dried plasma, 300 parts of coagulation factor II, 300 parts of fibrinogen, 8 parts of coagulation factor V, 8 parts of coagulation factor X, 25 parts of antithrombin III, 12 parts of plasminogen, plasminogen activator inhibitor-1 and 1.05 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and the plasminogen activator inhibitor-1 is 300 mg:1000 AU; the ratio of the freeze-dried plasma to the coagulation factor II is 1 unit:300 mg;

[0087] The preparation 2 comprises the following components in parts by mass: freeze-dried plasma, 600 parts of coagulation factor II, 600 parts of fibrinogen, 16 parts of coagulation factor V, 16 parts of coagulation factor X, 50 parts of antithrombin III, 24 parts of plasminogen, plasminogen activator inhibitor-1 and 2.1 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and the plasminogen activator inhibitor-1 is 600 mg: 2000 AU; the ratio of the freeze-dried plasma to the coagulation factor II is 1 unit: 600 mg;

[0088] The preparation 3 comprises the following components in parts by mass: freeze-dried plasma, 300 parts of coagulation factor II, 1200 parts of fibrinogen, 8 parts of coagulation factor V, 8 parts of coagulation factor X, 100 parts of antithrombin III, 48 parts of plasminogen, plasminogen activator inhibitor-1 and 4.2 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and the plasminogen activator inhibitor-1 is 300 mg:4000 AU; the ratio of the freeze-dried plasma to the coagulation factor II is 1 unit:300 mg;

[0089] The preparation 4 comprises the following components in parts by mass: freeze-dried plasma, 300 parts of coagulation factor II, 300 parts of fibrinogen, 8 parts of coagulation factor V, 8 parts of coagulation factor X, 25 parts of antithrombin III, 0 parts of plasminogen, plasminogen activator inhibitor-1 and 4.2 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and the plasminogen activator inhibitor-1 is 300 mg:4000 AU; the ratio of the freeze-dried plasma to the coagulation factor II is 1 unit:300 mg;

[0090] The freeze-dried plasma in the preparations 1 to 4 is human freeze-dried plasma;

[0091] The preparation 5 comprises the following components in parts by mass: freeze-dried plasma, 32 parts of coagulation factor II, 33.5 parts of fibrinogen, 0.7 parts of coagulation factor V, 0.9 parts of coagulation factor X, 3.1 parts of antithrombin III, 1.4 parts of plasminogen, plasminogen activator inhibitor-1 and 0.112 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and plasminogen activator inhibitor-1 is 32 mg:104 AU; the ratio of the freeze-dried plasma to coagulation factor II is 1 unit:32 mg;

[0092] The preparation 6 comprises the following components in parts by mass: freeze-dried plasma, 64 parts of coagulation factor II, 67 parts of fibrinogen, 1.4 parts of coagulation factor V, 1.8 parts of coagulation factor X, 6.2 parts of antithrombin III, 2.8 parts of plasminogen, plasminogen activator inhibitor-1 and 0.208 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and plasminogen activator inhibitor-1 is 64 mg:2084 AU; the ratio of the freeze-dried plasma to coagulation factor II is 1 unit:64 mg;

[0093] The preparation 7 comprises the following components in parts by mass: freeze-dried plasma, 32 parts of coagulation factor II, 134 parts of fibrinogen, 0.7 parts of coagulation factor V, 0.9 parts of coagulation factor X, 12.4 parts of antithrombin III, 5.6 parts of plasminogen, plasminogen activator inhibitor-1 and 0.448 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and plasminogen activator inhibitor-1 is 32 mg:416 AU; the ratio of the freeze-dried plasma to coagulation factor II is 1 unit:32 mg;

[0094] The preparation 8 comprises the following components in parts by mass: freeze-dried plasma, 32 parts of coagulation factor II, 33.5 parts of fibrinogen, 0.7 parts of coagulation factor V, 0.9 parts of coagulation factor X, 3.1 parts of antithrombin III, 0 parts of plasminogen, plasminogen activator inhibitor-1 and 0.448 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and plasminogen activator inhibitor-1 is 32 mg:416 AU; the ratio of the freeze-dried plasma to coagulation factor II is 1 unit:32 mg;

[0095] The freeze-dried plasma in the preparations 5 to 8 is freeze-dried plasma from rabbits.

[0096] As an embodiment, the raw material for preparing the freeze-dried plasma in the preparation 1 is fresh human plasma or human platelet-rich plasma; the raw material for preparing the freeze-dried plasma in the preparation 5 is fresh rabbit plasma or rabbit platelet-rich plasma. As another embodiment, when the raw material for preparing the freeze-dried plasma in the preparation 1 is fresh human plasma, the preparation 1 is recorded as preparation 1-1; when the raw material for preparing the freeze-dried plasma in the preparation 1 is human platelet-rich plasma, the preparation 1 is recorded as preparation 1-2; when the raw material for preparing the freeze-dried plasma in the preparation 5 is fresh rabbit plasma, the preparation 5 is recorded as preparation 5-1; when the raw material for preparing the freeze-dried plasma in the preparation 5 is rabbit platelet-rich plasma, the preparation 5 is recorded as preparation 5-2.

[0097] As an embodiment, the raw material for preparing the freeze-dried plasma in Preparations 2 to 4 is fresh human plasma; the raw material for preparing the freeze-dried plasma in Preparations 6 to 8 is fresh rabbit plasma.

[0098] During the resuscitation process, increasing the content of coagulation factors in FDP helps to achieve the goal of hemostatic resuscitation. However, coagulation-anticoagulation-fibrinolysis-antifibrinolysis is in a dynamic balance, and increasing coagulation may increase the possibility of ectopic thrombus formation. The disinfection, freeze-drying and storage processes of FDP will affect the activity of coagulation-related factors. Compared with normal plasma, vWF and coagulation factors II, V, VII, and VIII in FDP decrease most significantly. The present invention compounds coagulation factors, anticoagulation factors, fibrinolytic factors, fibrinolysis inhibitors and von Willebrand factor with appropriate concentrations in freeze-dried plasma, which can be used for hemostatic resuscitation of general severe trauma, severe trauma mainly caused by liver, severe trauma mainly caused by severe craniocerebral trauma, severe trauma combined with seawater immersion and other types of injuries, and has a good hemostatic resuscitation effect, and will not cause thrombosis in uninjured parts, and can reduce the amplitude of post-traumatic lactic acid increase.

[0099] The present invention provides a method for preparing the coagulation-fibrinolysis high-balance freeze-dried plasma described in the above technical solution, comprising: mixing the components in proportion to obtain the coagulation-fibrinolysis high-balance freeze-dried plasma.

[0100] As an embodiment, the method for preparing freeze-dried plasma in the coagulation-fibrinolysis high balance freeze-dried plasma comprises the following steps:

[0101] Plasma, ascorbic acid, and glycine are mixed to obtain primary plasma;

[0102] The primary plasma was pre-frozen at -40°C for 2 hours, then evacuated to below 30 Pa, and sublimated and dried at -40°C for 10 hours;

[0103] After sublimation drying, the freeze-dried plasma was dried at -35°C for 6 hours to keep the water content of the freeze-dried plasma within 3%, thereby obtaining the freeze-dried plasma.

[0104] As an embodiment, in the primary plasma, the concentration of ascorbic acid is 2-4 mmol / L, and the concentration of glycine is 60 mmol / L.

[0105] The present invention provides the use of the coagulation-fibrinolysis high-balance freeze-dried plasma described in the above technical solution or the coagulation-fibrinolysis high-balance freeze-dried plasma prepared by the preparation method described in the above technical solution in the preparation of hemostatic products.

[0106] As an embodiment, the hemostatic product is a hemostatic resuscitation product.

[0107] As an embodiment, the hemostatic resuscitation product includes one or more hemostatic resuscitation products for general severe trauma, severe trauma mainly involving the liver, severe trauma mainly involving severe craniocerebral trauma, and severe trauma combined with seawater immersion.

[0108] As an embodiment, the preparations 1-1, 2, 5-1 and 6 described in the above technical solution can be used for general severe trauma (see the animal model of Test Example 1), the preparations 3 and 7 described in the above technical solution can be used for severe trauma mainly involving the liver (see the animal model of Test Example 2), the preparations 4 and 8 described in the above technical solution can be used for severe trauma mainly involving severe craniocerebral trauma (see the animal model of Test Example 3), and the preparations 1-2 and 5-2 described in the above technical solution can be used for severe trauma combined with seawater immersion (see the animal model of Test Example 4).

[0109] According to the different changing rules of the coagulation-fibrinolysis system after different types of tissue and organ injuries, the present invention compounds coagulation factors, anticoagulant factors, fibrinolytic factors, fibrinolysis inhibitors and von Willebrand factor at appropriate concentrations in freeze-dried plasma to obtain different coagulation-fibrinolysis high-balance freeze-dried plasma preparations, which can be used for hemostatic resuscitation of general severe trauma, severe trauma mainly involving the liver, severe trauma mainly involving severe craniocerebral trauma, severe trauma combined with seawater immersion and other types of injuries, have good hemostatic resuscitation effects, will not cause thrombosis in uninjured parts, and can reduce the amplitude of lactic acid increase after trauma.

[0110] To further illustrate the present invention, a coagulation-fibrinolysis high-balance freeze-dried plasma provided by the present invention and its preparation method and application 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.

[0111] Example 1

[0112] 1. Prepare basic freeze-dried plasma (FDP) as follows:

[0113] Add ascorbic acid with a final concentration of 2-4 mmol / L (to maintain pH at 7.4-7.6) and glycine with a final concentration of 60 mmol / L (to maintain protein stability) to fresh plasma, and then freeze-dry: pre-freeze at -40°C for 2 hours, then pre-vacuum below 30 Pa, and sublimate at -40°C for 10 hours; after sublimation, decompose and dry at -35°C for 6 hours to keep the moisture content of freeze-dried plasma within 3%. Collect and package freeze-dried plasma 60 Sterilize with Coγ ray.

[0114] For the convenience of preparation, the FDP obtained from 200 ml of human plasma is recorded as 1 unit. The FDP obtained from every 20 ml of fresh rabbit plasma is compared with the body weight of a human, and both are recorded as basal FDP.

[0115] 2. The concentrations of various factors of the coagulation-fibrinolysis system in fresh plasma were determined by ELISA. The results are shown in Table 1.

[0116] Table 1 Concentrations of factors in the normal coagulation-fibrinolysis system

[0117] factor people Rabbit Coagulation Factor II 3mg / ml 3.2mg / ml Fibrinogen 3.3g / L 3.35g / L Coagulation Factor V 0.08mg / ml 0.07mg / ml Coagulation Factor X 0.08mg / ml 0.09mg / ml Antithrombin III 0.25mg / ml 0.31mg / ml Plasminogen 0.12mg / ml 0.14mg / ml Plasminogen Activator Inhibitor-1 (PAI-1) 10.0AU / ml 10.4AU / ml Von Willebrand Factor (vWF) 10.5μg / ml 11.2μg / ml

[0118] 3. Changes in the coagulation-fibrinolysis system after different types of injury

[0119] The multifunctional trauma-inducing machine developed in the early stage of the laboratory was used, and the built-in infrared aiming and other settings were used to accurately prepare the following five rabbit models: lower limb fracture + lung injury (injury severity score 18), lower limb fracture + lung injury + intestinal injury + hemorrhagic shock (injury severity score 34), severe liver injury (injury severity score 16), severe craniocerebral penetrating injury (injury severity score 16), and severe soft tissue injury + seawater immersion injury (injury severity score 16).The multifunctional trauma-injury machine is disclosed in the literature [Ye Z, et al. Characterization of Combined Blast-and Fragment-Induced Pelvic Injuries and Hemostatic Resuscitation in Rabbits. J Surg Res. 2023; 285: 158-167.], and the method of use is described in the literature [Du WQ, Zhong X, Jiang RQ, Zong ZW, Jia YJ, Ye Z, Zhou XL. Animal model-based simulation training for three emergent and urgent operations of penetrating thoracic injuries. Chin J Traumatol. 2022 Aug 2; S1008-1275 (22) 00084-0.], and the preparation methods of different rabbit models are described in the literature [Du WQ, Jiang RQ, Zong ZW, Zhang L, Ye Z, Zhong X, Jia YJ. Establishment of a combat damage control surgery training platform for explosive combined thoraco-abdominal injuries. Chin J Traumatol. 2022 Jul; 25(4): 193-200.] and [Yang Haoyang, Du Wenqiong, Ye Zhao, et al. Analysis of changes in coagulation-fibrinolysis function of rabbit soft tissue injury combined with hemorrhagic modification and seawater immersion. Journal of the Third Military Medical University 2023; 24]. For the injury severity scoring standard, please refer to the literature [Du WQ, Jiang RQ, Zong ZW, Zhang L, Ye Z, Zhong X, Jia YJ. Establishment of a combat damage control surgery training platform for explosive combined thoraco-abdominal injuries. Chin J Traumatol. 2022 Jul; 25(4): 193-200.].

[0120] Blood was collected 1 hour after injury to measure routine coagulation phase and thromboelastogram, and plasma was collected for ELISA to detect the content of coagulation factors, anticoagulation factors, fibrinolytic factors and antifibrinolytic factors. The results are as follows: The changes in the coagulation-fibrinolytic system were different in the five types of injuries.

[0121] One hour after injury, the levels of core coagulation factors and anticoagulation factors in patients with lower limb fracture + lung injury were approximately 0.52 to 0.55 times the normal level, while the levels of core coagulation factors and anticoagulation factors in patients with lower limb fracture + lung injury + intestinal injury + hemorrhagic shock further decreased to approximately 0.33 to 0.35 times the normal level. This result indicates that as the severity of injury increases, the changes in the coagulation-fibrinolysis system become more severe, and a higher multiple of balanced FDP is required for resuscitation.

[0122] In the plasma of animals with severe liver damage, the levels of core coagulation factors and anticoagulation factors were similar to those of animals with lower limb fractures + lung damage, but the fibrinogen level dropped to 0.45 g / L, which is about 1 / 7 of the normal value.

[0123] In the plasma of animals with severe craniocerebral penetrating injuries, the levels of core coagulation factors and anticoagulant factors were similar to those of animals with lower limb fractures + lung injuries, but the plasmin level was elevated to about 1.6 times the normal level.

[0124] In the plasma of animals with severe soft tissue injury and seawater immersion, the levels of core coagulation factors and anticoagulant factors were similar to those of animals with lower limb fracture and lung injury, but the platelet level decreased more than that of animals with lower limb fracture and lung injury, with a decrease of about 45×10 9 Pieces / L.

[0125] Example 2

[0126] A universal coagulation-fibrinolysis high-balance FDP, including a complementary universal coagulation-fibrinolysis high-balance FDP or a 2.0-fold coagulation-fibrinolysis high-balance FDP, is prepared by adding coagulation-fibrinolysis system factors to the basic FDP described in Example 1 according to the proportions in Table 2 under strict aseptic conditions, and then mixing and encapsulating.

[0127] Table 2 General coagulation-fibrinolysis high balance FDP

[0128]

[0129]

[0130] Note: The addition amount of various factors in Table 2 is based on 1 unit of basic FDP. The same applies to Table 3.

[0131] Example 3

[0132] It can be seen from the results of Example 1 that when the liver is damaged, the various coagulation factors synthesized by the liver will be severely reduced, especially fibrinogen; in severe brain tissue damage, compared with conventional damage, the concentration of plasma plasmin and the like increases, and other changes are the same as conventional damage. In view of these changing characteristics, the present invention provides a coagulation-fibrinolysis high-balance FDP rich in fibrinogen and a coagulation-fibrinolysis high-balance FDP rich in fibrinolysis inhibitors. The preparation method is similar to that of Example 1. The amount of coagulation-fibrinolysis system factors added to 1 unit of basic FDP is shown in Table 3.

[0133] Table 3 Highly balanced coagulation-fibrinolysis FDPs rich in fibrinogen and highly balanced coagulation-fibrinolysis FDPs rich in fibrinolysis inhibitors

[0134]

[0135] Example 4

[0136] As can be seen from Example 1, when seawater immersion is combined with tissue damage, the decrease in platelet count is more obvious than when there is no seawater immersion. In view of the above-mentioned change characteristics, the present invention provides a platelet-rich coagulation-fibrinolysis high-balance FDP, the preparation method of which is similar to the complementary universal coagulation-fibrinolysis high-balance FDP in Example 2, the only difference being that the basic FDP is replaced with a platelet-rich FDP.

[0137] The preparation method of the platelet-rich FDP is similar to that of the basic FDP in Example 1, except that fresh plasma is replaced with platelet-rich plasma (PRP). The platelet-rich FDP obtained from 200 ml of PRP in humans is recorded as 1 unit, and the platelet-rich FDP obtained from 20 ml of PRP in rabbits is recorded as 1 unit of FDP.

[0138] The preparation method of PRP is as follows: whole blood is collected from a human or rabbit, and sodium citrate anticoagulant (4%) is mixed with the whole blood at a volume ratio of 1:9, and the plasma is obtained after centrifugation at 450g for 10 minutes, which is platelet-rich plasma.

[0139] There is no significant difference in appearance between the basic FDP and the four different types of FDP prepared in Examples 2 to 4. The water content is between 3.5% and 4.2%, and there is no significant difference between the five FDPs.

[0140] Test Example 1 Observation experiment on the effect of universal coagulation-fibrinolysis high-balance FDP

[0141] 1. Experimental Methods

[0142] 1. Separation of platelets, red blood cells and plasma

[0143] After the rabbit is anesthetized, the femoral artery is cannulated and the whole blood of the rabbit is collected and mixed with 4% sodium citrate in a volume ratio of 9:1 in a 50ml centrifuge tube, and centrifuged at 3500rpm for 20min and then for 10min. At this time, the whole blood is separated into three layers, namely plasma, platelets (including some white blood cells), and red blood cells from top to bottom, and the plasma and platelets are collected separately for later use.

[0144] 2. Ex Vivo Thromboelastography

[0145] The in vitro thromboelastogram (TEG) indices of normal plasma (Group A), the basic FDP in Example 1 (Group B), the complementary general coagulation-fibrinolysis high balance FDP in Example 2 (Group C) and the 2.0-fold coagulation-fibrinolysis high balance FDP in Example 2 (Group D) were compared.

[0146] Methods: Take 5 ml of each of the four types of plasma, add platelets and red blood cells in the corresponding proportion of whole blood, mix well, then take 1 ml of the sample into the TEG analysis cup, add it to the activated coagulation reagent tube to react for 3 minutes, and then add 20 μl of 0.1M calcium chloride to the thromboelastogram sample cup, then take out 340 μl of the sample from the activated reagent tube and add it to the test cup to measure MA and other thromboelastogram parameters. The three indicators of R value, α angle (reflecting the rate of blood clot formation), and maximum amplitude value (MA, reflecting the strength of blood clot) were analyzed in particular. The analysis method can be found in the literature [Ye Z, et al. Characterization of Combined Blast-andFragment-Induced Pelvic Injuries and Hemostatic Resuscitation in Rabbits. J Surg Res. 2023; 285: 158-167.].

[0147] 3. Animal Model Preparation and Intervention Measures

[0148] Two animal models of lower limb fracture + lung injury (recorded as ISS18) and lower limb fracture + lung injury + intestinal injury + hemorrhagic shock (recorded as ISS 36) were established by the method of Example 1. 30 minutes after injury, the animals were resuscitated by infusion of no FDP (Group A), infusion of basic FDP (Group B), infusion of complementary general coagulation-fibrinolysis high balance FDP (Group C) and infusion of 2.0 times coagulation-fibrinolysis high balance FDP (Group D). There were 8 rabbits in each group. At the same time, 8 normal rabbits were taken as the normal control group, without injury and resuscitation, and only blood was taken for the following tests, which was recorded as the normal control group (NC group). The group without FDP infusion was infused with lactated Ringer's solution; the infusion volume of each group was 10ml / kg body weight.

[0149] 4. Biochemical detection indicators and detection methods

[0150] Blood pressure was monitored before and 4 h after injury, and blood was drawn for routine blood tests, blood gas analysis, thromboelastography, and other indicators. The hematocrit was recorded using the Mindray BC-5180CRP fully automatic blood cell analyzer (Mindray, Shenzhen, China). The lactate concentration was detected using the i-STAT handheld blood gas analyzer (Flextronics Manufacturing, Singapore) and its matching CHEM8+ and CG4+ test cards.

[0151] TEG detection method: Take 1 ml of whole blood from each group of animals at different time points, perform the same operation as above, and observe the same indicators as above.

[0152] 5. Thrombosis detection

[0153] The animals were killed by an overdose of potassium chloride injection 4 hours after injury, and then the kidneys, lungs on the uninjured side and other organs were quickly removed. First, a gross dissection was performed to observe the presence of thrombosis with the naked eye, and then the tissues were fixed, embedded and sectioned with routine 4% paraformaldehyde, and then HE staining was used to observe the presence of ectopic thrombosis.

[0154] 2. Experimental Results

[0155] 1. In vitro TEG results are shown in Figure 1 to Figure 3 .

[0156] The results showed that compared with normal plasma, the R value of the basic FDP group increased significantly, the α angle was smaller than that of normal plasma, and the MA value was significantly lower than that of normal plasma. In the complementary general coagulation-fibrinolysis high balance FDP group and the 2.0-fold coagulation-fibrinolysis high balance FDP group, the R value was significantly shorter than that of normal plasma and basic FDP, the α angle was significantly larger than that of the other two groups, and the MA value was significantly higher than that of the other two groups. The change trend of the 2.0-fold coagulation-fibrinolysis high balance FDP was more obvious than that of the complementary general coagulation-fibrinolysis high balance FDP.

[0157] 2. The results of in vivo coagulation index test are shown in Figures 4 to 6 .

[0158] The results showed that the R values ​​of the animals in the ISS18 group and the ISS34 group after injury were significantly higher than those of the normal animals, and the R value of the animals in the ISS34 group was significantly higher than that of the animals in the ISS18 group ( Figure 4 ). The α angle and MA value also show the same trend ( Figure 5 and Figure 6 ). This result suggests that as the severity of injury increases, the coagulation disorder becomes more severe, which is manifested by a more obvious prolongation of the R value and a more obvious decrease in the α angle and MA value.

[0159] In the ISS18 group, the complementary universal coagulation-fibrinolysis high balance FDP could be restored to normal values ​​better than the basic FDP, and the 2.0-fold coagulation-fibrinolysis high balance FDPR value was significantly lower than the normal value ( Figure 4 ). This result suggests that for animals with an injury severity of about 18, the use of a complementary universal coagulation-fibrinolysis high-balance FDP is sufficient to correct the R value to a normal value, while the use of a 2.0-fold coagulation-fibrinolysis high-balance FDP shortens the R value too much, which is an overcorrection. This phenomenon also shows a similar trend in the changes in MA value and α angle ( Figure 5 and Figure 6 ).

[0160] In the ISS 34 group, although the complementary universal coagulation-fibrinolysis high balance FDP restored the R value better than the basic FDP, it was still significantly different from the normal value, while the 2.0-fold coagulation-fibrinolysis high balance FDP restored the R value to a level close to the normal level, with no significant difference from the normal value ( Figure 4 ). This result suggests that for animals with injury severity of about 34, the use of 2.0 times the coagulation-fibrinolysis high balance FDP can correct the R value to a close to normal level. This phenomenon also shows a similar trend in the changes of MA value and α angle ( Figure 5 and Figure 6 ).

[0161] 3. The results of blood loss are shown in Figure 7 .

[0162] The results of blood loss calculated by hematocrit showed that in animals with lower limb fractures and lung injuries, the complementary universal coagulation-fibrinolysis high balance FDP could significantly reduce blood loss compared with the basic FDP and the 2.0-fold coagulation-fibrinolysis high balance FDP, and there was no significant difference between the two ( Figure 7 ). This result suggests that it is not necessary to use a 2.0-fold high-balance FDP for coagulation and fibrinolysis in animals with moderate injury severity, such as lower limb fractures and lung injuries.

[0163] In animals with lower limb fracture + lung injury + intestinal injury + hemorrhagic shock, although the complementary general coagulation-fibrinolysis high balance FDP was better than the basic FDP in reducing blood loss, its blood loss was significantly higher than 2.0 times the blood loss of the coagulation-fibrinolysis high balance FDP ( Figure 7 ). This result suggests that in animals with severe injuries such as lower limb fractures + lung injuries + intestinal injuries + hemorrhagic shock, a high-multiple balanced FDP is required.

[0164] 4. Lactic acid changes are shown in Figure 8 .

[0165] The results showed that the lactate concentration in each injury group increased significantly. After the use of conventional FDP, the lactate concentration decreased to a certain extent, but there was no significant difference with the control group without plasma infusion. After the use of balanced FDP, the lactate concentration decreased further, which was significantly different from the group without plasma infusion.

[0166] 5. Thrombosis test results are shown in Figures 9 to 11 .

[0167] The results showed that HE staining showed that no thrombosis was found in the kidneys, lungs, livers and other organs of the animals in each group.

[0168] Test Example 2 Observation experiment on the effect of high-balanced coagulation-fibrinolysis FDP rich in fibrinogen

[0169] 1. Experimental Methods

[0170] 1. Separate platelets, red blood cells and plasma using the same method as in Test Example 1.

[0171] 2. Ex vivo thromboelastogram, similar to that in Test Example 1, except that the 2.0-fold coagulation-fibrinolysis high-balance FDP in Group D was replaced with the fibrinogen-rich coagulation-fibrinolysis high-balance FDP in Example 3.

[0172] 3. Animal Model Preparation and Intervention Measures

[0173] The animal model of severe liver injury was established by the method of Example 1. 30 minutes after injury, the animals were resuscitated by the infusion group without FDP, the infusion group with basic FDP, the replenishing type universal coagulation-fibrinolysis high balance FDP and the coagulation-fibrinolysis high balance FDP rich in fibrinogen, respectively, and were named A to D groups. There were 8 rabbits in each group. At the same time, 8 normal rabbits were taken as the normal control group, without injury and resuscitation, and only blood was taken for the following tests, which was recorded as the normal control group (NC group). The group without FDP infusion was infused with lactated Ringer's solution; the infusion volume of each group was 10 ml / kg body weight.

[0174] 4. Biochemical detection indicators and detection methods are the same as those in Test Example 1.

[0175] 5. Thrombosis detection: the method is the same as test example 1.

[0176] 2. Experimental Results

[0177] 1. In vitro TEG results are shown in Figure 12 to Figure 14 .

[0178] The results showed that compared with normal plasma, the R value of the basic FDP group increased significantly, the α angle was smaller than that of normal plasma, and the MA value was significantly lower than that of normal plasma. The R value of the complementary general coagulation-fibrinolysis high balance FDP group and the fibrinogen-rich coagulation-fibrinolysis high balance FDP group was significantly shorter than that of normal plasma and basic FDP, the α angle was significantly larger than that of the other two groups, and the MA value was significantly higher than that of the other two groups. The MA value of the fibrinogen-rich coagulation-fibrinolysis high balance FDP group was significantly higher than that of the complementary general coagulation-fibrinolysis high balance FDP group, while there was no significant difference in the R value and α angle between the two groups.

[0179] 2. The results of in vivo coagulation index test are shown in Figure 15 to Figure 17 .

[0180] The results of in vivo experiments showed that in the animal model of severe liver damage, the basic FDP can restore various coagulation and fibrinolysis indicators well, but there is a large gap with the normal values. The complementary general coagulation-fibrinolysis high-balance FDP, except that the MA value did not return to the normal level, the coagulation indicators were basically close to the normal values, with no significant difference. The coagulation-fibrinolysis high-balance FDP rich in fibrinogen made various indicators including MA basically return to normal, which was basically close to the normal values, with no significant difference.

[0181] 3. Blood loss results are shown in Fig.18 .

[0182] The results of blood loss calculated by hematocrit showed that the blood loss of the fibrinogen-rich coagulation-fibrinolysis high balance FDP was the least, less than the complementary general coagulation-fibrinolysis high balance FDP and the basic FDP, which was related to its effective improvement of MA value. The blood loss of the complementary general coagulation-fibrinolysis high balance FDP was less than that of the basic FDP.

[0183] 4. Lactic acid concentration results are shown in Fig.19 .

[0184] In this model, lactate levels increased significantly after injury, significantly higher than normal lactate levels. The use of a complementary universal coagulation-fibrinolysis high-balance FDP partially reduced lactate levels, significantly lower than those in the basal FDP and non-FDP groups, while the use of a fibrinogen-rich coagulation-fibrinolysis high-balance FDP further reduced lactate levels, lower than those in the basal FDP and complementary universal coagulation-fibrinolysis high-balance FDP groups.

[0185] 5. Thrombosis test results are shown in Figures 20-22 .

[0186] HE staining showed that no thrombosis was found in the kidneys, lungs, livers and other organs of the animals in each group.

[0187] Test Example 3 Observation experiment on the effect of FDP rich in fibrinolysis inhibitors and high-balance coagulation-fibrinolysis

[0188] 1. Experimental Methods

[0189] 1. Separate platelets, red blood cells and plasma using the same method as in Test Example 1.

[0190] 2. Ex vivo thromboelastogram, similar to Test Example 1, except that the 2.0-fold coagulation-fibrinolysis high-balance FDP in Group D was replaced with the coagulation-fibrinolysis high-balance FDP rich in fibrinolysis inhibitor in Example 3.

[0191] 3. Animal Model Preparation and Intervention Measures

[0192] The animal model of severe craniocerebral injury was established by the method of Example 1. 30 minutes after the injury, the animals were resuscitated by the infusion group without FDP, the infusion group with basic FDP, the supplementary general coagulation-fibrinolysis high balance FDP, and the coagulation-fibrinolysis high balance FDP rich in fibrinolysis inhibitors, respectively, and were named A to D groups. There were 8 rabbits in each group. At the same time, 8 normal rabbits were taken as the normal control group, without injury and resuscitation, and only blood was taken for the following tests, recorded as the normal control group (NC group). The group without FDP infusion was infused with lactated Ringer's solution; the infusion volume of each group was 10 ml / kg body weight.

[0193] 4. Biochemical detection indicators and detection methods are the same as those in Test Example 1.

[0194] 5. Thrombosis detection: the method is the same as test example 1.

[0195] 2. Experimental Results

[0196] 1. In vitro TEG results are shown in Figure 23 to Figure 25 .

[0197] The results showed that compared with normal plasma, the R value of the basic FDP group increased significantly, the α angle was smaller than that of normal plasma, and the MA value was significantly lower than that of normal plasma. The R value of the complementary general coagulation-fibrinolysis high balance FDP group and the coagulation-fibrinolysis high balance FDP rich in fibrinolysis inhibitors was significantly shorter than that of the basic FDP group, the α angle was significantly larger than that of the basic FDP group, and the MA value was significantly higher than that of the basic FDP group. Compared with the coagulation-fibrinolysis high balance FDP rich in fibrinolysis inhibitors, there were no significant differences in the R value, α angle, and MA value between the two groups.

[0198] 2. The results of in vivo coagulation index test are shown in Figure 26 to Figure 28 .

[0199] In group A, which did not use FDP for resuscitation, the R value, α angle, and MA after injury were significantly different from normal values, while basic FDP could restore various coagulation and fibrinolysis indicators to a certain extent, but there was a large gap with normal values, and the R value, α angle, and MA values ​​were significantly different from normal values. The R value, α angle, and MA values ​​of group B animals resuscitated with basic FDP were better than those of group A resuscitated with FDP, but there was no statistical difference.

[0200] The indicators of the complementary general coagulation-fibrinolysis high balance FDP group and the coagulation-fibrinolysis high balance FDP group rich in fibrinolysis inhibitors were close to normal values, and the R value, α angle, and MA value were not significantly different from the normal values, and were significantly better than those of group A without FDP resuscitation. Compared with the animals in group B resuscitated with basic FDP, the R value, α angle, and MA value of the complementary general coagulation-fibrinolysis high balance FDP group and the coagulation-fibrinolysis high balance FDP group rich in fibrinolysis inhibitors were significantly better than those of group B.

[0201] The indicators of the complementary general coagulation-fibrinolysis high balance FDP group and the coagulation-fibrinolysis high balance FDP group rich in fibrinolysis inhibitors were similar, with no significant differences.

[0202] 3. The results of blood loss are shown in Fig.29 .

[0203] The results of blood loss calculated by hematocrit showed that the blood loss of the FDP with high coagulation-fibrinolysis balance, which was rich in fibrinolysis inhibitors, was the least, significantly less than that of the FDP resuscitation group without FDP. This may be because it is rich in fibrinolysis inhibitors, which inhibit thrombus dissolution and thus reduce the amount of bleeding.

[0204] The blood loss in the complementary universal coagulation-fibrinolysis high balance FDP group was significantly lower than that in the non-FDP resuscitation group. The blood loss in the complementary universal coagulation-fibrinolysis high balance FDP group was less than that in the basic FDP group, but there was no significant difference.

[0205] 4. Results of lactate concentration are shown in Fig.30 .

[0206] In this model, lactate levels increased significantly after injury, significantly higher than normal lactate concentrations. The use of basic FDP can slightly reduce lactate concentrations, but there is no significant difference from the non-FDP resuscitation group, and it is still significantly higher than normal values.

[0207] After using the supplementary FDP, the lactate concentration was significantly lower than that in the non-FDP resuscitation group. The lactate concentration in the group using the coagulation-fibrinolysis high-balance FDP rich in fibrinolysis inhibitors was further reduced and was significantly lower than that in groups A, B, and C, which was related to the fact that the amount of bleeding was the least in each group.

[0208] 5. Thrombosis test results are shown in Figure 31 to Figure 33 .

[0209] HE staining showed that no thrombosis was found in the kidneys, lungs, livers and other organs of the animals in each group.

[0210] Test Example 4 Effect Observation Experiment of Platelet-Rich Coagulation-Fibrinolysis High Balance FDP

[0211] 1. Experimental Methods

[0212] 1. Separate platelets, red blood cells and plasma using the same method as in Test Example 1.

[0213] 2. Ex vivo thromboelastogram, similar to that in Test Example 1, except that the 2.0-fold coagulation-fibrinolysis high-balance FDP in Group D was replaced with the platelet-rich coagulation-fibrinolysis high-balance FDP in Example 4.

[0214] 3. Animal Model Preparation and Intervention Measures

[0215] The animal model of severe soft tissue injury was established by the method of Example 1, and the animals were immediately immersed in simulated seawater for 10 minutes after injury. 30 minutes after successful injury, the animals were resuscitated by the non-FDP infusion group, the basic FDP infusion group, the complementary general coagulation-fibrinolysis high balance FDP, and the platelet-rich coagulation-fibrinolysis high balance FDP, respectively, and were named A to D groups. There were 8 rabbits in each group. At the same time, 8 normal rabbits were taken as the normal control group, without injury and resuscitation, and only blood was taken for the following tests, recorded as the normal control group (NC group). The non-FDP infusion group was infused with lactated Ringer's solution; the infusion volume of each group was 10ml / kg body weight.

[0216] 4. Biochemical detection indicators and detection methods are the same as those in Test Example 1.

[0217] 5. Thrombosis detection: the method is the same as test example 1.

[0218] 2. Experimental Results

[0219] 1. In vitro TEG results are shown in Figure 34 to Figure 36 .

[0220] The results showed that compared with normal plasma, the R value of the basic FDP group increased significantly, the α angle was smaller than that of normal plasma, and the MA value was significantly lower than that of normal plasma. In the complementary general coagulation-fibrinolysis high balance FDP group and the platelet-rich coagulation-fibrinolysis high balance FDP group, the R value was significantly shorter than that of the basic FDP group, and the α angle was significantly larger than that of the basic FDP group.

[0221] Compared with the platelet-rich coagulation-fibrinolysis high balance FDP group, there were no significant differences in R value and α angle between the two groups, but the MA value of the platelet-rich coagulation-fibrinolysis high balance FDP group was significantly higher than that of the complementing general coagulation-fibrinolysis high balance FDP group, which is related to the high platelet content in the platelet-rich coagulation-fibrinolysis high balance FDP.

[0222] 2. The results of in vivo coagulation index test are shown in Figure 37 to Figure 39 .

[0223] In group A, which did not use FDP for resuscitation, the R value, α angle, and MA after injury were significantly different from normal values, while basic FDP could restore various coagulation and fibrinolysis indicators to a certain extent, but there was a large gap with normal values, and the R value, α angle, and MA values ​​were significantly different from normal values. The R value, α angle, and MA values ​​of group B animals resuscitated with basic FDP were better than those of group A without FDP resuscitation.

[0224] There were no significant differences in R value and α angle between the platelet-rich coagulation-fibrinolysis high balance FDP group (group D) and the complementary general coagulation-fibrinolysis high balance FDP group (group C), but the MA value of the former was significantly higher than that of the latter.

[0225] 3. Blood loss results are shown in Fig.40 .

[0226] The results of blood loss calculated by hematocrit showed that the platelet-rich coagulation-fibrinolysis high balance type FDP had the least blood loss, which was less than the complementary type universal coagulation-fibrinolysis high balance FDP and the basic type FDP. This is related to its effective improvement of MA value and thus the quality of thrombosis. The blood loss of the complementary type universal coagulation-fibrinolysis high balance FDP group was less than that of the basic type FDP.

[0227] 4. Lactic acid concentration results are shown in Fig.41 .

[0228] In this model, lactate levels increased significantly after injury, significantly higher than normal lactate concentrations. The use of basic FDP can slightly reduce lactate concentrations, but there is no significant difference from the non-FDP resuscitation group, and it is still significantly higher than normal values.

[0229] After using the complementary universal coagulation-fibrinolysis high-balance FDP, the lactate concentration was significantly lower than that in the non-FDP resuscitation group. The lactate concentration in the platelet-rich coagulation-fibrinolysis high-balance FDP group was further reduced and significantly lower than that in groups A, B, and C, which was related to the fact that the amount of bleeding was the least in each group.

[0230] 5. Thrombosis test results are shown in Figure 42 to Figure 44 .

[0231] HE staining showed that no thrombosis was found in the kidneys, lungs, livers and other organs of the animals in each group.

[0232] In summary, the coagulation-fibrinolysis high-balance freeze-dried plasma provided by the present invention can be used for hemostatic resuscitation of general severe trauma, severe trauma mainly involving the liver, severe trauma mainly involving severe craniocerebral trauma, severe trauma combined with seawater immersion and other types of injuries, and has a good hemostatic resuscitation effect.

[0233] 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 coagulation-fibrinolysis high-balance freeze-dried plasma, characterized in that: The invention comprises the following components in parts by weight: freeze-dried plasma, 32 to 600 parts of coagulation factor II, 33.5 to 1200 parts of fibrinogen, 0.7 to 16 parts of coagulation factor V, 0.9 to 16 parts of coagulation factor X, 3.1 to 50 parts of antithrombin III, 0 to 24 parts of plasminogen, plasminogen activator inhibitor-1 and 0.112 to 2.1 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II to the plasminogen activator inhibitor-1 is 32 to 600 mg: 104 to 4000 AU; the freeze-dried plasma prepared from 200 ml of human plasma or 20 ml of rabbit plasma is recorded as 1 unit, and the ratio of the freeze-dried plasma to the coagulation factor II is 1 unit: 32 to 600 mg.

2. The coagulation-fibrinolysis high-balance freeze-dried plasma according to claim 1, characterized in that: The coagulation-fibrinolysis high-balance freeze-dried plasma includes one or more of independently packaged preparations 1 to 8: The preparation 1 comprises the following components in parts by mass: freeze-dried plasma, 300 parts of coagulation factor II, 300 parts of fibrinogen, 8 parts of coagulation factor V, 8 parts of coagulation factor X, 25 parts of antithrombin III, 12 parts of plasminogen, plasminogen activator inhibitor-1 and 1.05 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and the plasminogen activator inhibitor-1 is 300 mg:1000 AU; the ratio of the freeze-dried plasma to the coagulation factor II is 1 unit:300 mg; The preparation 2 comprises the following components in parts by mass: freeze-dried plasma, 600 parts of coagulation factor II, 600 parts of fibrinogen, 16 parts of coagulation factor V, 16 parts of coagulation factor X, 50 parts of antithrombin III, 24 parts of plasminogen, plasminogen activator inhibitor-1 and 2.1 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and plasminogen activator inhibitor-1 is 600 mg: 2000 AU; The ratio of the freeze-dried plasma to coagulation factor II is 1 unit: 600 mg; The preparation 3 comprises the following components in parts by mass: freeze-dried plasma, 300 parts of coagulation factor II, 1200 parts of fibrinogen, 8 parts of coagulation factor V, 8 parts of coagulation factor X, 100 parts of antithrombin III, 48 parts of plasminogen, plasminogen activator inhibitor-1 and 4.2 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and the plasminogen activator inhibitor-1 is 300 mg:4000 AU; the ratio of the freeze-dried plasma to the coagulation factor II is 1 unit:300 mg; The preparation 4 comprises the following components in parts by mass: freeze-dried plasma, 300 parts of coagulation factor II, 300 parts of fibrinogen, 8 parts of coagulation factor V, 8 parts of coagulation factor X, 25 parts of antithrombin III, 0 parts of plasminogen, plasminogen activator inhibitor-1 and 4.2 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and the plasminogen activator inhibitor-1 is 300 mg:4000 AU; the ratio of the freeze-dried plasma to the coagulation factor II is 1 unit:300 mg; The freeze-dried plasma in the preparations 1 to 4 is human freeze-dried plasma; The preparation 5 comprises the following components in parts by mass: freeze-dried plasma, 32 parts of coagulation factor II, 33.5 parts of fibrinogen, 0.7 parts of coagulation factor V, 0.9 parts of coagulation factor X, 3.1 parts of antithrombin III, 1.4 parts of plasminogen, plasminogen activator inhibitor-1 and 0.112 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and plasminogen activator inhibitor-1 is 32 mg:104 AU; the ratio of the freeze-dried plasma to coagulation factor II is 1 unit:32 mg; The preparation 6 comprises the following components in parts by mass: freeze-dried plasma, 64 parts of coagulation factor II, 67 parts of fibrinogen, 1.4 parts of coagulation factor V, 1.8 parts of coagulation factor X, 6.2 parts of antithrombin III, 2.8 parts of plasminogen, plasminogen activator inhibitor-1 and 0.208 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and plasminogen activator inhibitor-1 is 64 mg:2084 AU; The ratio of freeze-dried plasma to coagulation factor II is 1 unit: 64 mg; The preparation 7 comprises the following components in parts by mass: freeze-dried plasma, 32 parts of coagulation factor II, 134 parts of fibrinogen, 0.7 parts of coagulation factor V, 0.9 parts of coagulation factor X, 12.4 parts of antithrombin III, 5.6 parts of plasminogen, plasminogen activator inhibitor-1 and 0.448 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and plasminogen activator inhibitor-1 is 32 mg:416 AU; the ratio of the freeze-dried plasma to coagulation factor II is 1 unit:32 mg; The preparation 8 comprises the following components in parts by mass: freeze-dried plasma, 32 parts of coagulation factor II, 33.5 parts of fibrinogen, 0.7 parts of coagulation factor V, 0.9 parts of coagulation factor X, 3.1 parts of antithrombin III, 0 parts of plasminogen, plasminogen activator inhibitor-1 and 0.448 parts of von Willebrand factor; the mass enzyme activity ratio of the coagulation factor II and plasminogen activator inhibitor-1 is 32 mg:416 AU; the ratio of the freeze-dried plasma to coagulation factor II is 1 unit:32 mg; The freeze-dried plasma in the preparations 5 to 8 is freeze-dried plasma from rabbits.

3. The coagulation-fibrinolysis high-balance freeze-dried plasma according to claim 2, characterized in that: The raw material for preparing the freeze-dried plasma in the preparation 1 is fresh human plasma or human platelet-rich plasma; the raw material for preparing the freeze-dried plasma in the preparation 5 is fresh rabbit plasma or rabbit platelet-rich plasma.

4. The coagulation-fibrinolysis high-balance freeze-dried plasma according to claim 2, characterized in that: The raw material for preparing the freeze-dried plasma in Preparations 2 to 4 is fresh human plasma; the raw material for preparing the freeze-dried plasma in Preparations 6 to 8 is fresh rabbit plasma.

5. The method for preparing the coagulation-fibrinolysis high-balance freeze-dried plasma according to any one of claims 1 to 4, characterized in that: include: The components are mixed in proportion to obtain the coagulation-fibrinolysis high-balance freeze-dried plasma.

6. The preparation method according to claim 5, characterized in that: The method for preparing the freeze-dried plasma in the high-balance coagulation-fibrinolysis freeze-dried plasma comprises the following steps: Plasma, ascorbic acid, and glycine are mixed to obtain primary plasma; The primary plasma was pre-frozen at -40°C for 2 hours, then evacuated to below 30 Pa, and sublimated and dried at -40°C for 10 hours; After sublimation drying, the freeze-dried plasma was dried at -35°C for 6 hours to keep the water content of the freeze-dried plasma within 3%, thereby obtaining the freeze-dried plasma.

7. The preparation method according to claim 6, characterized in that: In the primary plasma, the concentration of ascorbic acid is 2-4 mmol / L, and the concentration of glycine is 60 mmol / L.

8. Use of the high-balanced coagulation-fibrinolysis freeze-dried plasma according to any one of claims 1 to 4 or the high-balanced coagulation-fibrinolysis freeze-dried plasma prepared by the preparation method according to any one of claims 5 to 7 in the preparation of hemostatic products.

9. The use according to claim 8, characterized in that: The hemostatic product is a hemostatic resuscitation product.

10. The use according to claim 9, characterized in that: The hemostatic resuscitation products include one or more hemostatic resuscitation products for general severe trauma, severe trauma mainly involving the liver, severe trauma mainly involving severe craniocerebral trauma, and severe trauma combined with seawater immersion.

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