阅读说明：本技术 用于治疗和预防微血栓形成的纤溶酶原 (Plasminogen for the treatment and prevention of microthrombosis ) 是由 S·T·基西希 R·威尔茨 H·R·格尔丁 M·马楚尔 H-J·安德斯 C·X·史 C· 于 2020-01-24 设计创作，主要内容包括：本发明涉及用于预防或治疗患者血栓形成事件方法中的纤溶酶原,其中患者患有微血栓或具有患微血栓的风险。(The present invention relates to plasminogen for use in a method for the prevention or treatment of a thrombotic event in a patient, wherein the patient has or is at risk of having a microthrombus.)

1. Plasminogen for use in the prevention or treatment of a thrombotic event in a patient, wherein the patient has or is at risk of having a microthrombus with a diameter of less than 1 mm.

2. The plasminogen of claim 1, wherein plasminogen is Glu-plasminogen.

3. The plasminogen of claim 1, wherein the patient has an acquired Glu-plasminogen deficiency caused by increased Glu-plasminogen consumption, decreased Glu-plasmin protoplasm synthesis, or both.

4. The plasminogen according to any of claims 1 to 3, wherein plasminogen is Glu-plasminogen and the patient has at least one ischemic area which can cause necrosis of at least part of a tissue without administration of Glu-plasminogen to said patient.

5. The plasminogen according to any of claims 1 to 4, wherein said plasminogen has no proteolytic activity.

6. The plasminogen according to any of claims 1, 2, 4 and 5, wherein said patient has an acquired plasminogen-deficiency.

7. The plasminogen of claim 6, wherein said acquired plasminogen-deficiency is caused by an increased plasminogen consumption.

8. The plasminogen according to any of claims 1 and 5 to 7, wherein said plasminogen is Lys-plasminogen, or a combination of Glu-plasminogen and Lys-plasminogen and one or more other plasminogen derivatives.

9. The plasminogen according to any of claims 1 to 8, wherein a patient has or is at risk of having a microthrombus, and said microthrombus is at risk of causing thrombosis or embolism in a large blood vessel.

10. The plasminogen according to any of claims 1 to 9, wherein the patient has or is at risk of having a pathological state selected from the group consisting of: lipoprotein (a) -anemia, iron deficiency, vitamin D deficiency, vitamin K deficiency, vitamin H deficiency, anemia, homocysteinemia, protein Z deficiency, embolism, stroke, myocardial infarction, epistaxis, menorrhagia, von willebrand syndrome, moroneguent glaucard, liver dysfunction, antiphospholipid syndrome, migraine, thyroid dysfunction, abortion, failure of a lytic therapy by plasminogen activator, or a combination of two or more thereof, caused by stenosis of arteries, veins, arterioles, venules, or spasm of arteries, veins, venules.

11. The plasminogen according to any of claims 1 to 10, wherein said patient's plasminogen blood level is lower than that of the whole population of the same species.

12. The plasminogen of claim 11, wherein said low blood level of plasminogen is caused by one or more causes selected from the group consisting of: high physiological or pathological consumption of plasminogen, high clearance rate of plasminogen, low expression rate of plasminogen, or the presence of high levels of one or more inhibitors of plasminogen.

13. The plasminogen of claim 12, wherein said low blood level of plasminogen is caused by a high physiological or pathological consumption of plasminogen.

14. The plasminogen according to any of claims 1 to 13, wherein the level of plasminogen in the patient's blood is determined and, in case the determined level of plasminogen is at least 10% (mol/mol) lower than the average value of the whole population of the same species, a sufficient amount of plasminogen is administered to the patient to prevent or treat a thrombotic event.

15. The plasminogen according to any of claims 1 to 14, wherein the microthrombus is a microthrombus of capillaries.

16. The plasminogen according to any of claims 1 to 15, wherein a patient suffers from at least one ischemic area, which can lead to at least partial tissue necrosis without administration of plasminogen to said patient.

17. The plasminogen according to any of claims 1 to 16, wherein said patient has more than one thrombotic event.

18. The plasminogen according to any of claims 1 to 17, wherein a patient suffers from at least one ischemic area, can cause at least partial tissue necrosis without administration of plasminogen to said patient, and suffers from more than one thrombotic event.

19. The plasminogen according to any of claims 1 to 18, wherein the thrombotic event is caused by an infarction of any typical cause in the pathogenesis of infarction.

20. The plasminogen according to any of claims 1 to 19, wherein said thrombotic event is caused by the rupture of atherosclerotic plaques containing cholesterol crystals resulting from hypercholesterolemia.

21. The plasminogen according to any of claims 1 to 20, wherein the thrombotic event is caused by both infarction of any typical cause in the pathogenesis of infarction and rupture of atherosclerotic plaques containing cholesterol crystals resulting from hypercholesterolemia.

22. The plasminogen according to any of claims 1 to 21, wherein the thrombotic event results in an infarction.

23. The plasminogen according to any of claims 1 to 22, wherein said patient is administered plasminogen at least once with a plasminogen dose in the range of 0.01 to 100mg/kg body weight or in the range of 0.01 to 1mg/kg body weight.

24. The plasminogen according to any of claims 1 to 23, wherein plasminogen is administered to a patient at least once within 24 hours after a thrombotic event has occurred, or within one week before a high risk event of thrombosis will be experienced, such as surgery, or periodically when there is a risk of a thrombotic event occurring.

25. The plasminogen according to any of claims 1 to 24, wherein the plasminogen is administered to a patient according to one of the following dosing regimens:

(A) the plasminogen is administered to the patient once daily, preferably intravenously, in particular in a single dose of 0.01 to 1mg/kg body weight, over a period of three or more days;

(B) the plasminogen is administered to the patient once daily over a period of three or more days, preferably intra-arterially, in particular intra-arterially in a single dose of 0.01 to 1mg/kg body weight.

(C) Administering plasminogen to a patient once daily for three days or more, preferably intracranial administration, in particular intracranial administration at a single dose of 0.01 to 1mg/kg body weight;

(D) plasminogen is administered to the patient once every two days for three days or more, preferably intramuscularly, in particular in a single dose of 0.05 to 10mg/kg body weight.

(E) Administering plasminogen to a patient once a week for three weeks or more, preferably subcutaneously, in particular in a single dose of 0.1 to 100mg/kg body weight; or

(F) The patient is administered plasminogen once daily for 3 to 7 days, followed by every second day for 3 or more days, or weekly for 3 or more weeks.

26. The plasminogen according to any of claims 1 to 25, wherein the patient is administered with a dose suitable to replace no more than 15%, no more than 10% or no more than 5% of the amount of normal plasminogen. Plasma chamber

27. The plasminogen for use in any of claims 1 to 26, wherein said patient is:

(a) at least once during the treatment period with a plasminogen dose of 0.01 to 100mg/kg body weight; subsequently (b) determining in step (i) the level of plasminogen in the blood of the patient, and if the determined level of plasminogen is at least 10% (mol/mol) lower than the average level found in the whole population for the same species, administering in a further step (ii) a sufficient amount of plasminogen to the patient to prevent or treat a thrombotic event, and optionally (c) steps (i) and (ii) are repeated as long as the level of plasminogen determined in step (i) is at least 10% (mol/mol) lower than the average level found in the same population for the same species.

28. The plasminogen according to any of claims 1 to 27, wherein said patient suffers from deep vein thrombosis, pelvic vein thrombosis, pulmonary embolism, infarction of any organ, retinal vein occlusion, Disseminated Intravascular Coagulation (DIC), Thrombotic Thrombocytopenic Purpura (TTP), a vascular disease that occurs simultaneously with thrombotic events in the capillary flow pathway, in particular diabetic vascular disease, thrombophlebitis or a combination of two or more thereof.

29. The plasminogen according to any of claims 1 to 28, wherein said patient is at risk of developing a thrombotic event because said patient has undergone surgery, in particular bypass surgery, or has had an endovascular prosthesis due to suffering from atherosclerosis or arterial stenosis.

30. The plasminogen according to any of claims 1 to 29, wherein said patient suffers from Disseminated Intravascular Coagulation (DIC), acute kidney injury/failure (AKI), sepsis or a combination of two or more thereof.

Brief Description of Drawings

FIG. 1 shows the reduction in Glomerular Filtration Rate (GFR) expressed as a percentage change relative to untreated mice in mice given 10mg/kg cholesterol (CC group); mice were administered 10mg/kg cholesterol, followed 4 hours after cholesterol administration by 132 μ L of a composition comprising 65 μ g/mL Glu-plasminogen (CC + Glu-P group); and mice were given 132. mu.L of a composition comprising 65. mu.g/mL of Glu-plasminogen (PBS + Glu-P group), all after 24 hours.

FIG. 2 shows the reduction in Glomerular Filtration Rate (GFR) in microliters/minute of flow (basal group) in untreated mice; mice were given 10mg/kg cholesterol (group CC); mice were given 10mg/kg cholesterol, followed by 132 μ L of a composition comprising 65 μ g/mL Glu-plasminogen (group CC + Glu-P) 4 hours after cholesterol administration; and mice were administered 132. mu.L of a composition containing 65. mu.g/mL Glu-plasminogen (PBS + Glu-p), both after 24 hours.

FIG. 3 shows infarct size (CC group) of mice dosed with 10mg/kg cholesterol, expressed as a percentage of the whole kidney; mice were given 10mg/kg cholesterol, followed by 132 μ L of a composition comprising 65 μ g/mL Glu-plasminogen (group CC + Glu-P) 4 hours after cholesterol administration; and mice were given 132. mu.L of a composition containing 65. mu.g/mL Glu-plasminogen (PBS + Glu-p group), both after 24 hours.

Fig. 4 shows the percentage of diagnostic frequency of patients with reduced levels of Plasminogen (PLG) (n 600), Plasminogen (PLG) deficiency (black) and non-Plasminogen (PLG) deficiency (grey).

FIG. 5 shows alpha-2-antiplasmin (A2AP) activity in different patient groups, disease free/control population (Norm), lipoprotein (A) (LpA), Iron deficient (Iron), vitamin D deficient (VitD), vitamin K deficient (VitK), vitamin H deficient (VitH), anemia (Anaem), homocysteine levels (Hcys), protein Z deficient (PZ), thrombosis (Thromb), embolism (Emb), Stroke (Stroke), Myocardial Infarction (MI), epitaxy (Epist), menorrhagia (HM), von Willebrand syndrome (vWS), Morbus Meulenggraht (Meul), liver disease (Li), antiphospholipid disease (APL), migraine (Migr), thyroid disease (Tyr), abortion (Abort).

Fig. 6 shows Plasminogen (PLG) activity in different patient groups (abbreviations identical to those used in fig. 5).

FIG. 7 shows the ratio of activity of alpha-2-antiplasmin to Plasminogen (PLG) (A2AP/PLG) in different patient groups (abbreviations same as used in FIG. 5).

FIG. 8 shows Plasminogen (PLG) activity of a control group (CP) compared to a group of acute kidney injury/failure patients (AKI).

FIG. 9 shows α -2-antiplasmin (A2AP) activity of control group (CP) compared to a group of acute kidney injury/failure patients (AKI).

FIG. 10 shows the ratio of alpha-2-antiplasmin: Plasminogen (PLG) (A2AP/PLG) activity in the control group (CP) compared to a group of acute kidney injury/failure patients (AKI).

FIG. 11 shows Plasminogen (PLG) activity of a Control Population (CP) compared to a panel of disseminated intravascular coagulation patients (DIC).

FIG. 12 shows the alpha-2-antiplasmin (A2AP) activity of the Control Population (CP) compared to a panel of disseminated intravascular coagulation patients (DIC).

FIG. 13 shows the ratio of alpha-2-antiplasmin: Plasminogen (PLG) (A2AP/PLG) activity in the Control Population (CP) compared to a panel of disseminated intravascular coagulation patients (DIC).

FIG. 14 shows Plasminogen (PLG) activity of control group (CP) compared to a group of Sepsis patients (Sepsis).

FIG. 15 shows α -2-antiplasmin (A2AP) activity of control group (CP) compared to a group of Sepsis patients (Sepsis).

FIG. 16 shows the ratio of alpha-2-antiplasmin: Plasminogen (PLG) (A2AP/PLG) activity in the control group (CP) compared to a group of septic patients (Sepsis).

FIG. 17 is a summary of diagnostic studies of acquired Plasminogen (PLG) deficiency. Here, the percentage of Plasminogen (PLG) activity was measured. It was found that 40% of acute kidney/kidney injury (AKI) patients and 62% of Disseminated Intravascular Coagulation (DIC) patients exhibited a statistically significant Plasminogen (PLG) deficiency compared to the Control Population (CP).

Examples

Preparation of Glu-plasminogen formulations

Glu-plasminogen was prepared as described in example 1 of WO2018/162754 and was more than 95% (w/w) pure based on total protein content. The human Glu-plasminogen formulation contained 1256. mu.g/mL Glu-plasminogen (as determined by enzyme-linked immunosorbent assay, ELISA).

The total protein content of the preparation was 1259 μ g/mL (determined by Bradford protein assay). Thus, Glu-plasminogen is > 99.7% pure by weight based on total protein content. The high purity was also confirmed by the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

The proteolytic activity of Glu-plasminogen is below the limit of detection, measured using the standardized S-2288(Chromogenix) proteolytic activity assay, measured as total protein content, in units of 1.0g/L total protein content.

The human Glu-plasminogen preparation contains only negligible endotoxin levels below 1EU/mL (determined by the safe endophromosomal assay in Limulus Amoebocyte Lysate (LAL) according to european pharmacopoeia (5.0 edition) chapter 2.6.14), and <0.35g/L IgG, <0.05g/L IgA and <0.35g/L IgM (all determined by turbidimetric assays). Albumin (determined by multicolor end-point) and lysine-plasminogen (determined by western blot) were not detectable.

In a further bioactivity test, the concentration of human Glu-plasminogen was set at 200. mu.g/mL and then activated to plasmin. This corresponds to the range of plasmin concentrations naturally present in the blood. The proteolytic activity of the plasmin solution obtained was determined by plasmin-p-nitrophenol-labeled (pNP-labeled) peptide substrate. As a result, it was found that the proteolytic activity of the preparation was in the range of 109% compared to the proteolytic activity of plasmin naturally present in plasma (considered as 100%). Thus, human Glu-plasminogen is shown to have full biological activity and can be converted to fully active plasmin.

Example 1

Animal model (Cholesterol crystal induced mouse microthrombus)

Triggering the formation of microthrombosis in the kidney

Each mouse was injected with 100 microliters of 10mg/kg cholesterol (CC) in the blood vessels leading to the kidneys. The time point of injection was considered to be time point zero (0 hours). It has been found that cholesterol can lead to the formation of blood clots in the smaller blood vessels of the kidney, particularly in the renal capillaries.

Plasminogen (PLG) treatment

Glu-plasminogen was prepared as described in example 1 of WO2018/162754, with a purity > 95% (w/w) and the properties as described above. Some mice that had not received treatment were retained. 4 hours after cholesterol administration, each treated mouse was injected intravenously (i.v.) with 132 μ L of a composition of 65 μ g/mL Glu-plasminogen (in phosphate buffered saline). Phosphate buffered saline was also injected 4 hours after cholesterol administration as an untreated control group. As an additional control group, PBS was injected instead of CC and 132. mu.L of a composition of 65. mu.g/mL Glu-plasminogen (in phosphate buffered saline) was injected 4 hours later.

Measurement data

Glomerular Filtration Rate (GFR) was measured 24 hours after cholesterol administration. In addition, infarct size in the kidney was determined by staining with triphenyltetrazolium chloride (TTC) of kidney tissue. In addition, sacrificed mice are examined histologically, e.g., by determining the score of renal tubular injury (PAS), endothelial injury (CD31), and neutrophil immune cell filtration.

Results and discussion

The quantitative results are shown in FIGS. 1 to 3. Administration of cholesterol was found to cause microthrombus. These were also confirmed in histological observation of mouse kidney after 24 hours. These microthromboes were found to have a significant effect on Glomerular Filtration Rate (GFR) (see FIGS. 1 and 2, which are samples including cholesterol (CC)) and resulted in more than half (50%) of renal tissue necrosis (see FIG. 3, which is a sample containing only cholesterol (CC)). The use of (Glu-) plasminogen alone had no significant effect on Glomerular Filtration Rate (GFR) (see FIGS. 1 and 2, right). It also did not restore the effect of microthrombosis on Glomerular Filtration Rate (GFR) caused by cholesterol administration (see FIGS. 1 and 2, samples including cholesterol (CC) and (Glu-) plasminogen (Glu-P)).

However, administration of (Glu-) plasminogen effectively prevented tissue necrosis (see FIG. 3, samples including cholesterol (CC) and (Glu-) plasminogen (Glu-P)). Necrosis was reduced by half compared to that which occurred when cholesterol alone was administered. Therefore, (Glu-) plasminogen effectively reduces infarct size.

These results indicate that administration of (Glu-) plasminogen is effective in the treatment and prevention of patients suffering from (micro) thrombosis.

The (Glu-) plasminogen produced according to the present invention surprisingly has a high and excellent fibrinolytic activity in the event of (micro) thrombosis. It is possible, without being bound by this theory, that (Glu-) plasminogen can solubilize existing microthromboses and can be used to prevent the occurrence of microthrombotic and/or macrothrombotic events. Such (micro) thrombotic events are often the cause of infarctions, such as myocardial infarction, stroke and renal infarction, retinal vein occlusion, thrombotic thrombocytopenic purpura, and the like.

Example 2

Clinical manifestations of acquired Plasminogen (PLG) deficiency caused by the consumption of Plasminogen (PLG).

In general, when Plasminogen (PLG), in particular Glu-plasminogen consumption, is increased, an acquired plasminogen deficiency (PLG) may occur. This may occur in any type of thrombotic event with a longer history, such as in atherosclerosis. Inside the vessel, damage to the intima may persist for a longer period of time. This will first result in a sustained but slight activation of the coagulation system, continuously converting fibrinogen to fibrin. Blood flow can still occur as long as no clot is formed that can occlude the entire vessel diameter. Such clots may continuously activate the fibrinolytic system. In this way, Plasminogen (PLG) (e.g., Glu-plasminogen) will be converted to plasmin, and thus Plasminogen (PLG) (e.g., Glu-plasminogen) will be consumed. Currently, there are two treatment methods: first, inhibition of coagulation (e.g., by vitamin K antagonists) is sufficient before an occlusive clot is not formed. When a clot has formed (which may lead to, for example, myocardial infarction, stroke, etc.), to date, the only option has been thrombolytic therapy. This is typically achieved by activating Plasminogen (PLG) by tPA, uPA or streptokinase.

The presence of the targeted enzyme Plasminogen (PLG) is required for use with each of the three drugs mentioned above. However, in the case of the above-mentioned acquired Plasminogen (PLG) deficiency, the target of thrombolytic therapy is no longer present. It was surprisingly found that in this case and in any other acquired Plasminogen (PLG) (e.g. Glu-plasminogen) deficiency, alternative treatment of this protein helps to save the lives of these patients.

When a thrombus forms, Plasminogen (PLG) (e.g., Glu-plasminogen) in the plasma will be consumed due to local activation. The higher the amount of fibrin in a single large thrombus or a plurality of microthrombosis, especially if the condition deteriorates over a longer period of time, the higher the consumption of Plasminogen (PLG) is generally. This may lead to a temporary acquired Plasminogen (PLG) deficiency, tilting the equilibrium to a low fibrinolytic state, leading to a quasi-thrombotic state (see fig. 4). Reduced levels of Plasminogen (PLG) have been shown in a variety of contexts, including septic liver disease myocardial infarction, argentine hemorrhagic fever, as well as after L-asparaginase treatment, thrombolytic therapy and surgery. Because of the important role of proteins in fibrinolysis, a reduced level of Plasminogen (PLG) may compromise the body's ability to degrade fibrin, thereby making it susceptible to thrombotic/thrombotic disease.

Thus, the cause of thrombotic disease may be either a hypercoagulable state or the dissolution of low fibrin. Current treatments are directed only to (hyper) coagulation by using heparin, warfarin or F.X antagonists.

It has surprisingly been found that the consumption of Plasminogen (PLG) results in a decrease in fibrinolysis, which may also be referred to as transient acquired Plasminogen (PLG) (e.g., Glu-plasminogen) deficiency. Accordingly, the present invention finds that treatment of transient acquired Plasminogen (PLG) (e.g., Glu-plasminogen) deficiency can address thrombotic diseases. Diagnostic studies of diseases with high prevalence and life-threatening states provide evidence for this finding.

Example 2.1

Acute kidney injury/failure (AKI)

Multiple organ failure, also known as "multiple organ dysfunction syndrome" (MODS), has one of its organs involved in the kidney. This disease is defined as an acute disease in which the patient has altered organ function and is unable to maintain homeostasis without intervention. MODS may eventually lead to Multiple Organ Failure Syndrome (MOFS) and death. Acute Lung Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) are common manifestations of MODS or MOFS. However, other conditions besides sepsis can also lead to MODS, including trauma, burns and severe hemorrhagic shock. Multiple organ dysfunction, which is an extreme state, is a progressively more severe physiological disorder of an individual organ (i.e., a series of processes rather than a single event). The degree of organ function changes varies widely from mild organ dysfunction to overt organ failure (multiple organ failure such as sepsis, Systemic Inflammatory Response Syndrome (SIRS), toxic shock syndrome and septic thrombophlebitis).

In acute renal failure, microcirculation in the kidney decreases and urine is no longer produced. Thus, an objective assessment of renal function and its recovery is possible.

In patients with acute kidney injury/failure (AKI) and with a longer duration of onset Plasminogen (PLG) levels are considerably lower compared to patients without vascular disease. A study was conducted on this subject in collaboration with the clinical chemistry department (IKC) of the university of Mannheim Hospital. By 3 months 2018, citrate plasma samples from 77 patients were collected and stored below-40 ℃. Plasminogen (PLG) and a-2-antiplasmin were measured in batches for all samples in a DIN EN ISO 15189 certified laboratory with a BCS XP analyzer (SIEMENS Healthcare). A group of 53 healthy plasma was used as a control under the same conditions. The results were evaluated according to Mann-Whitney using u-test.

In the first confirmatory study of the mannheim clinical chemical system (IKC), patients with acute intrarenal failure were selected, followed by patients with the Acute Kidney Injury Network (AKIN). Key parameters are as follows: creatinine increased to 1.5 times the standard value while urine volume decreased.

Outline of research: acute kidney injury/failure (AKI)

Parameters: alpha-2-antiplasmin (a2-AP), Plasminogen (PLG), activity ratio R ═ a 2-AP/Plasminogen (PLG) in blood samples, which was also used for standard diagnosis

77 acute renal injury/failure (AKI) patients

53 control population (healthy blood donors) (CP)

The results were analyzed by the Mann-Whitney test and showed:

AKI patients present with significant (. star.) acquired Plasminogen (PLG) deficiency

AKI patients present with significant (. star.) acquired alpha-2-antiplasmin deficiency

The activity ratio of (A2AP/PLG) was not significantly different

TABLE 1 summary of results of Plasminogen (PLG) activity in AKI patients versus control groups

Table analysis	Data of
		Column A	CP-fiberProlysin (PLG) (control group)
Column B	AKI-Plasminogen (PLG) (AKI patient group)
		Mannhutney test:
p value (Gauss approximation)	0.0031
		Summary of P values	**
Is there a significant difference in median? (P)<0.05)	Is that
		Single or double tailed P values?	Double tail
Sum of scores in column A, B	4096,4420
		Manhutney U	1417

The Mannich test (Mann-Whitney test) of acute kidney injury/failure (AKI) showed significant differences in Plasminogen (PLG) activity. In patient (Ptx-77) and control (CP-53), P is 0.0031. The results are shown in FIG. 8.

TABLE 2 summary of results for alpha-2-antiplasmin Activity in AKI patients versus controls

Mann-Whitney test of acute renal failure showed significant differences in alpha-2-antiplasmin activity. In patient (Ptx-77) and control (CP-53), P is 0.0011. The results are shown in FIG. 9.

TABLE 3 summary of results for alpha-2-antiplasmin to Plasminogen (PLG) ratio in AKI patients versus controls

Table analysis	Data of
		Column A	CP ratio (control group)
Column B	AKI ratio (AKI patient group)
		Mannheim test
P value (Gauss approximation)	0.1241
		Summary of P values	**
Is there a significant difference in median? (P)<0.05)	Is not provided with
		Single or double tailed P values?	Double tail
Sum of scores in column A, B	3147,5369
		Manhutney U	1716

The Mann-Whitney test for acute renal failure showed no significant difference in the ratio of activity (A2 AP/PLG; P0.1241) between patient (Ptx-77) and control (CP-53). The results are shown in FIG. 10.

Example 2.2

Disseminated Intravascular Coagulation (DIC)

Validation studies of patients with disseminated intravascular (micro) coagulation (DIC) were performed by the mannheim clinical chemistry system (IKC).

DIC patients were determined by D-dimer levels and internal criteria.

Study outline-DIC:

parameters: alpha-2-antiplasmin (A2-AP), Plasminogen (PLG) activity. D-dimer, ratio R ═ a 2-AP/Plasminogen (PLG) in blood samples, which was also used for standard diagnosis

13 DIC patients (DIC)

53 Control Population (CP)

The results were analyzed by the Mann-Whitney test, showing:

DIC patients present with significant (. prime.) acquired Plasminogen (PLG) deficiency

Absence of acquired alpha-2-antiplasmin deficiency in DIC patients

A2AP/PLG showed a significant difference in the ratio of activities (. apprxeq.) - > an increase in fibrinolysis inhibition

TABLE 4 summary of Plasminogen (PLG) activity results in DIC patients versus controls

Table analysis	PLG
		Column A	CP-Plasminogen (PLG) (control group)
Column B	DIC-Plasminogen (PLG) (DIC patient group)
		Mannheim test
P value (Gauss approximation)	0.0013
		Summary of P values	**
Is there a significant difference in median? (P)<0.05)	Is that
		Single or double tailed P values?	Double tail
Sum of scores in column A, B	2256,519
		Manhutney U	288.0

Mann-Whitney assay of DIC showed significant differences in Plasminogen (PLG) activity. In patient (Ptx-13) and control (CP-53), P is 0.0013. The results are shown in FIG. 11.

TABLE 5 summary of results for alpha-2-antiplasmin activity in DIC patients versus controls

Mann-Whitney examination of DIC showed no significant difference in α -2-antiplasminase activity. In patient (Ptx-13) and control (CP-53), P is 0.073. The results are shown in FIG. 12.

TABLE 6 results of the ratio of alpha-2-antiplasmin to Plasminogen (PLG) in DIC patients and controls

Table analysis	ratio-DIC
		Column A	CP ratio (control group)
Column B	DIC ratio (DIC patient group)
		Mannheim test
P value (high)Si Yuan)	<0.0001
		Summary of P values	***
Is there a significant difference in median? (P)<0.05)	Is that
		Single or double tailed P values?	Double tail
Sum of scores in column A, B	1634,1141
		Manhutney U	203.0

Mann-Whitney examination of DIC showed that the ratio of A2AP/PLG activity in patient (Ptx-13) was significantly increased (P <0.0001) compared to control (CP-53). The results are shown in FIG. 13.

In summary, in a study with 53 healthy human Control Populations (CP), 77 patients with acute renal/renal injury (AKI), and 21 patients with Disseminated Intravascular Coagulation (DIC), 40% of patients with AKI and 62% of patients with DIC showed a statistically significant Plasminogen (PLG) deficiency compared to the Control Populations (CP). Here, the Mann-Whitney test showed that the median quartile spacing p of CP and AKI was >0.003, while the median quartile spacing p of CP and DIC was > 0.001. The results are shown in FIG. 17.

Example 2.3

Septicemia

Validation studies in patients with sepsis were performed in the Mannheim clinical chemistry line (IKC) and identified based on D-dimers and internal standards.

Study outline-sepsis:

parameters: alpha-2-antiplasmin (A2AP), Plasminogen (PLG) activity,

The ratio R is a 2-AP/Plasminogen (PLG), PCTP, DD in blood samples, which are also used for standard diagnosis.

9 septicemia patients (sepsis)

53 Control Population (CP)

The results were analyzed by the Mann-Whitney test, showing:

patients with sepsis present with significant (. mu.) -acquired Plasminogen (PLG) deficiency

Absence of acquired alpha-2-antiplasmin deficiency in septicemia patients

No significant difference in the A2AP/PLG activity ratio

TABLE 7 summary of Plasminogen (PLG) activity results in septic patients versus controls

Table analysis

Table analysis	septicemia-PLG
		Column A	CP-Plasminogen (PLG) (control group)
Column B	sepsis-Plasminogen (PLG) (sepsis patient group)
		Mannheim test
P value (Gauss approximation)	0.0377
		Summary of P values	*
Is there a significant difference in median? (P)<0.05)	Is that
		Single or double tailed P values?	Double tail
Sum of scores in column A, B	1774,179
		Manhutney U	134.0

The Mann-Whitney test showed significant differences in Plasminogen (PLG) activity: p of patient (Ptx-9) and control (CP-53) was 0.0377. The results are shown in FIG. 14.

TABLE 8 summary of the results for alpha-2-antiplasmin activity in septic patients versus controls

The Mann-Whitney test showed no significant difference in α -2-antiplasmin activity: p-0.0704 for patient (Ptx-9) and control (CP-53). The results are shown in FIG. 15.

TABLE 9 summary of results for the ratio of alpha-2-antiplasmin to Plasminogen (PLG) in septic patients and controls

Table analysis	Sepsis-Ratio
		Column A	CP ratio (control group)
Column B	Sepsis ratio (sepsis patient group)
		Mannheim test
P value (Gauss approximation)	0.2182
		Summary of P values	Without significant difference (ns)
Is there a significant difference in median? (P)<0.05)	Is not provided with
		Single or double tailed P values?	Double tail
Sum of scores in column A, B	1608,345.5
		Manhutney U	176.5

The Mann-Whitney test showed that the A2AP/PLG activity ratio of the patient (Ptx-13) was increased but not significant compared to the control (CP-53) (P0.2182). The results are shown in FIG. 16.

Example 2.4

Control population

Raw data: measurement of Plasminogen (PLG) and A2AP in a healthy plasma donor Control Population (CP) from a plasmalogue center.

Reference range of PLG for control population: 90% to 144%; reference range of A2 AP: 97% to 119%; ratio of the two: 0.80 to 1.25.

TABLE 10 normalized Activity of Plasminogen (PLG) and A2AP (average activity normalized to 100%) of control population

Example 2.5

Diagnosis of patients with acquired plasminogen deficiency (PLG) due to consumption of Plasminogen (PLG) in the medical centre of Bonn's blood coagulation disorders and transfusionund transfusionis, CBT), Plasminogen (PLG) levels were measured on a total of 6000 patients (from general/family clinics) in a non-acute state, and patients in which all Plasminogen (PLG) levels were reduced were selected and analyzed (n 700, 11.6%). Analysis of the frequency of confirmed diagnosis of patients with reduced levels of Plasminogen (PLG) has shown that many are diagnosed in the presence of an unpredictable lack of Plasminogen (PLG). The results are shown in fig. 4 and table 11. A prospective study was conducted in CBT to demonstrate these issuesNow. At the same time, a parallel prospective study was also performed on acute patients with the same diagnosis.

Table 11 describes the correlation coefficient of the linear regression R2 ═ R. This is calculated in the computer program Microsoft Excel (using the "KORREL" function in german version) by correlating every two parameters with each other. The calculated value is the correlation rate, indicating whether there is a statistical correlation between the two parameters. If the correlation ratio r is-0.5 > r <0.5, no correlation or weak correlation is indicated. A correlation ratio r of from 1.0 to-0.5 or from 0.5 to 1.0 indicates significant correlation. Thus, it can be seen that there is only a significant correlation of PLG to Ratio and Quick to Pro C Act.

In table 11, the symbols represent the parameters: PLG ═ plasminogen [ IU/mL ]; quick ═ fast [ sec ]; thrombin time ═ thrombin time [ sec ]; APTT ═ activated partial thromboplastin time [ sec ]; prot C Act ═ protein C activation [ IU/mL ]; APC ratio ═ activated protein C resistance [% ]; ATIII ═ antithrombin III [% ]; a2AP ═ α -2-antiplasmin [% ]; ratio A2AP/PLG, PAI-1 plasminogen activator inhibitor-1 [ ng/mL ], PAP Complex plasminogen-antiplasmin Complex; D-Dimer; PLT ═ platelets [ x106/μ L ]; CRP ═ C-reactive protein [ μ g/mL ]

TABLE 12 p-value results for different patient groups

In this study, Plasminogen (PLG) was determined as an independent parameter. Apart from the weak correlation with the (activity-based) ratio (A2AP/PLG) in the presence of Plasminogen (PLG), it was not correlated with any other parameter of the coagulation or fibrinolytic system. Since all samples were from non-acute patients, there were only a few acute myocardial infarction patients in the study, and the selected patient population, which had low levels of Plasminogen (PLG), showed normal levels of A2 AP.