PLASMINOGEN FOR TREATING AND PREVENTING MICROTHROMBOSIS
20220072110 · 2022-03-10
Inventors
- Stephan T. KIESSIG (Wiesloch, DE)
- Ricarda WELZ (Mauer, DE)
- Hanne Rieke GERDING (Mannheim, DE)
- Marc MAZUR (Weinheim, DE)
- Hans Joachim ANDERS (Muenchen, DE)
- Chongxu SHI (Muenchen, DE)
- Christoph SCHIMMELPFENNIG (Leipzig, DE)
- Satish Kumar DEVARAPU (Muenchen, DE)
Cpc classification
International classification
Abstract
The present invention relates to plasminogen for use in a method for preventing or treating a thrombotic event in a patient, wherein the patient is at risk of developing or is suffering from microthrombi.
Claims
1-30. (canceled)
31. A method for preventing or treating a thrombotic event in a patient, wherein the patient is administered with a sufficient amount of plasminogen, and wherein the patient is at risk of developing or is suffering from microthrombi having diameters of less than 1 mm.
32. The method of claim 31, wherein the plasminogen is Glu-plasminogen.
33. The method of claim 32, wherein the patient bears an acquired Glu-plasminogen deficiency caused by increased Glu-plasminogen consumption, decreased biosynthesis of Glu-plasminogen, or a combination of both.
34. The method of claim 31, wherein the plasminogen is Glu-plasminogen and the patient suffers from at least one ischemic region that would cause necrosis of at least a part of a tissue without administration of the Glu-plasminogen to the patient.
35. The method of claim 31, wherein the plasminogen has no proteolytic activity.
36. The method of claim 31, wherein the patient bears an acquired plasminogen deficiency.
37. The method of claim 36, wherein the acquired plasminogen deficiency is caused by increased plasminogen consumption.
38. The method of claim 31, wherein the plasminogen is Lys-plasminogen or a combination of Glu-plasminogen and Lys-plasminogen or a combination of Glu-plasminogen and Lys-plasminogen and one or more other plasminogen derivatives.
39. The method of claim 31, wherein the patient is at risk of developing or is suffering from microthrombi resulting in a thrombosis or embolization of the large blood vessels.
40. The method of claim 31, wherein the patient is at risk of developing or is suffering from a pathological state selected from the group consisting of stenosis of arteria, veins, arterioles, venules, capillaries or from spasms in arteria, veins, arterioles, venules, capillaries resulting in diseases like lipoprotein(a)-anemia, iron deficiency, vitamin D deficiency, vitamin K deficiency, vitamin H deficiency, anemia, homocysteinaemia, protein Z deficiency, emboly, stroke, myocardial infarction, epistaxis, hypermenorrhea, Von Willebrand Syndrome, Morbus Meulengracht, a liver dysfunction, antiphospholipid-syndrome, migraine, a thyroid dysfunction, abortion, therapy failures in lysis therapy using activators for plasminogen and a combination of two or more thereof.
41. The method of claim 31, wherein the patient has a lower blood level of plasminogen than the average blood level of plasminogen found throughout a population of the same species.
42. The method of claim 41, wherein the lower blood level of plasminogen is caused by one or more reasons selected from the group consisting of high physiologic or pathologic consumption of plasminogen, a high elimination rate of plasminogen, a low expression rate of plasminogen, and the presence of high levels of one or more inhibitors of plasminogen.
43. The method of claim 42, wherein the lower blood level of plasminogen is caused by high physiologic or pathologic consumption of plasminogen.
44. The method of claim 31, wherein the level of plasminogen in the patient's blood is determined and, the patient is administered with a sufficient amount of plasminogen to prevent or treat a thrombotic event if the determined level of plasminogen is at least 10% (mol/mol) lower in comparison to the average level of plasminogen found throughout population of the same species.
45. The method of claim 31, wherein the microthrombi are microthrombi of capillaries.
46. The method of claim 31, wherein the patient suffers from at least one ischemic region that would cause necrosis of at least a part of a tissue without administration of plasminogen to the patient.
47. The method of claim 31, wherein the patient suffers from more than one thrombotic event.
48. The method of claim 31, wherein the patient suffers from at least one ischemic region that would cause necrosis of at least a part of a tissue without the administration of plasminogen to the patient, and at least one thrombotic event.
49. The method of claim 31, wherein the thrombotic event is caused by an infarction or wherein the thrombotic event results in an infarction.
50. The method of claim 31, wherein the thrombotic event is caused by the burst of an atherosclerotic plaque containing cholesterol crystals caused by hypercholesterolemia.
51. The method of claim 31, wherein the thrombotic event is caused by an infarction, and by the burst of an atherosclerotic plaque containing cholesterol crystals caused by hypercholesterolemia.
52. The method of claim 31, wherein the thrombotic event causes an infarction.
53. The method of claim 31, wherein the patient is administered with the plasminogen at least once with a dose of plasminogen in the range of 0.01 to 100 mg/kg body weight or in the range of 0.01 to 1 mg/kg body weight.
54. The method of claim 31, wherein the patient is administered with the plasminogen at least once within 24 hours after the occurrence of a thrombotic event, within one week before being subjected to an event with a high risk of developing a thrombotic event or on a regular basis when the patient is at risk of developing a thrombotic event.
55. The method of claim 54, wherein the event with a high risk of developing a thrombotic event is a surgery.
56. The method of claim 31, wherein the patient is administered with the plasminogen according to one of the following administration schemes: (A) the patient is administered the plasminogen intravenously once per day for at least three days; (B) the patient is administered the plasminogen intraarterially once per day for at least three days; (C) the patient is administered the plasminogen intracranially once per day for at least three days; (D) the patient is administered the plasminogen intramuscularly once every two days for at least three days; (E) the patient is administered with plasminogen subcutaneously once every week for at least three weeks; or (F) the patient is administered with plasminogen once per day for three to seven days and is subsequently administered once every two days for at least three days or once per week for at least three weeks.
57. The method of claim 31, wherein the patient is administered with a dose of the plasminogen suitable to replace not more than 15%, not more than 10%, or not more than 5%, of the normal plasminogen amount in the plasma compartment of the blood.
58. The method of claim 31, wherein: (a) the patient is administered with a dose of the plasminogen in the range of 0.01 to 100 mg/kg body weight during the treatment period; and subsequently (b) (i) the level of plasminogen in the patient's blood is determined and, (ii) the patient is administered with a sufficient amount of plasminogen to prevent or treat a thrombotic event if the determined level of plasminogen is at least 10% (mol/mol) lower in comparison to the average level found throughout a population of the same species.
59. The method of claim 31, wherein the patient suffers from deep vein thrombosis, pelvic vein thrombosis, pulmonary embolism, an infarction of any organ, retinal vein occlusion, disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura (TTP), an angiopathy coincidence with thrombotic events in capillary flow path, diabetic angiopathy, thrombophlebitis, or a combination of two or more thereof.
60. The method of claim 31, wherein the patient is at risk of developing a thrombotic event due to atherosclerosis or stenosis of arteries, having been subjected to a surgery, or having an implanted blood vessel endoprosthesis.
61. The method of claim 31, wherein the patient suffers from disseminated intravascular coagulation (DIC), acute kidney/renal injury (AKI), sepsis, or a combination of two or more thereof.
62. The method of claim 58, wherein steps (i) and (ii) are conducted repeatedly as long as the level of plasminogen determined in step (i) is at least 10% (mol/mol) lower in comparison to the average level found throughout the population of the same species.
Description
BRIEF DESCRIPTION OF THE FIGURES
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EXAMPLES
[0135] Preparation of Glu-Plasminogen Preparations
[0136] Glu-plasminogen was prepared as described in Experimental Example 1 of WO 2018/162754 with a purity of >95% (w/w) based on the total protein content. The human Glu-plasminogen preparation contained 1256 μg/mL of Glu-plasminogen (as determined by enzyme-linked immunosorbent assay, ELISA).
[0137] The total protein content of said preparation was 1259 μg/mL (as determined by Bradford protein assay). Accordingly, the purity of Glu-plasminogen was found to be >99.7% by weight, based on total protein content. The high purity was also confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
[0138] Proteolytic activity of the Glu-plasminogen (as determined by a standardized S-2288 (Chromogenix) proteolytic activity assay, referred to the total protein content, units/1.0 g/L of total protein content) was found below the detection limit.
[0139] The human Glu-plasminogen preparation contained only a negligible endotoxin level of <<1 EU/mL (as determined in a Limulus Amebocyte Lysate (LAL) endosafe endochrome assay according to European Pharmacopeia (version 5.0) chapter 2.6.14), and <0.35 g/L IgG, <0.05 g/L IgA and <0.35 g/L IgM (each determined in a nephelometric assay). Albumin (as determined by a polychromatic endpoint determination) and Lys-plasminogen (as determined by Western Blot) were not detectable.
[0140] In a further test of bioactivity, the concentration of the human Glu-plasminogen was set to 200 μg/mL and was then activated to plasmin. This corresponding to a concentration range naturally occurring in blood. The proteolytic activity of the obtained plasmin solution was determined by means of a para-nitrophenol-labeled (pNP-labeled) peptide substrate of plasmin. It was found that proteolytic plasmin activity was in a range of 109% in comparison to the activity naturally occurring in blood plasma plasmin proteolytic activity which was normalized to be 100%. Therefore, it was found that the human Glu-plasminogen was fully bioactive and could be converted in fully active plasmin.
Example 1
[0141] Animal Model (Induction of Microthrombi in Mice by Cholesterol Crystals)
[0142] Triggering the Formation of Microthrombi in the Kidney
[0143] Mice were administered with cholesterol (CC) by means of an injection of 10 mg/kg, 100 μL/mouse in a blood vessel leading to the kidney. The time point of injection was considered as time point zero (0 hours). It was found that cholesterol leads to the formation of clots in smaller vessels in the kidney, in particular in kidney capillaries.
[0144] Treatment with Plasminogen (PLG)
[0145] Glu-plasminogen was prepared as described in Experimental Example 1 of WO 2018/162754 with a purity of >95% (w/w). The properties were those as described above. Some of the mice remained untreated. Those which were treated were administered with an intravenous (i.v.) injection of 132 μL/mouse of a composition containing 65 μg/mL Glu-plasminogen in phosphate buffered saline 4 hours after cholesterol administration. The Injection of phosphate buffered saline after 4 hours of cholesterol administration was used as a control group, with no treatment. As an additional control group, the injection of PBS instead of CC and the injection of 132 μL/mouse of a composition containing 65 μg/mL Glu-plasminogen in phosphate buffered saline was analyzed after 4 hours.
[0146] Readout
[0147] 24 hours after cholesterol administration, the gglomerular filtration rate (GFR) was determined. Further, the infarct size in kidney was determined by means of staining with triphenyl tetrazolium chloride (TTC) of the kidney tissue. Further, the sacrificed mice were examined histologically, e.g., by means of determining the score the tubular injury (PAS), the endothelial injury (CD31) and the neutrophil immunocyte filtration.
[0148] Results and Discussion
[0149] The quantitative results are depicted as
[0150] However, administration of (Glu-)plasminogen effectively prevented necrosis of the tissue (cf.
[0151] Necrosis was reduced by the half in comparison to the necrosis found when only cholesterol is administered. Thus, (Glu-)plasminogen effectively reduced infarct size.
[0152] These results show that the administration of (Glu-)plasminogen effectively treats and prevents a patient suffering from (micro)thrombi.
[0153] The (Glu-)plasminogen produced according to this invention has surprisingly a high and excellent fibrinolytic activity in (micro)thrombotic events. Without being bound to this theory, it is assumed that (Glu-)plasminogen resolves existing microthrombi and can be used in the prophylaxis of micro- and/or macrothrombotic events. Such (micro)thrombotic events are often causal in infarctions such as, e.g., myocardial infarctions, strokes as well at kidney infarctions, a retinal vein occlusion, thrombotic thrombocytopenic purpura, etc.
Example 2
[0154] Clinical Situations with an Acquired Plasminogen (PLG) Deficiency Caused by Plasminogen (PLG) Consumption.
[0155] In general, an acquired deficiency of plasminogen (PLG) could be expected in conditions causing an increased consumption of plasminogen (PLG), in particular Glu-plasminogen. This could be probably found in any kind of thrombotic events which have a longer history such as in atherosclerosis. The damage of the blood vessels' inside, the intima, could last for a longer time period. This will first lead to a permanent but slight activation of the coagulation system permanently converting fibrinogen into fibrin. As long as no clot occluding the whole blood vessels' diameter is formed, the blood flow is still intact. Such clot may permanently activate the fibrinolytic system. Here, plasminogen (PLG) (e.g., Glu-plasminogen) will be transformed into plasmin and, therefore, the plasminogen (PLG) (e.g., Glu-plasminogen) is consumed. Presently, there are two therapeutic approaches: first, until no occluding clot is formed, the inhibition of the coagulation such as, e.g., by vitamin K antagonists, is sufficient. When the clot is formed (which may result in, e.g., a myocardial infarction, a stroke etc.), until now, only thrombolytic therapy is an option. This is typically performed by activating plasminogen (PLG) by tPA, uPA or streptokinase.
[0156] The use of one of the three drugs requires the presence of the targeted enzyme plasminogen (PLG). In case of the described acquired plasminogen (PLG) deficiency, the target for the lysis therapy is, however, not present anymore. It was surprisingly found that in this case and in the any other acquired plasminogen (PLG) (e.g., Glu-plasminogen) deficiencies, the substitution of this protein may be supportive in the lifesaving treatment of those patients.
[0157] When thrombi are formed, plasminogen (PLG) (e.g., Glu-plasminogen) from plasma will be consumed due to the local activation. The higher the amount of fibrin located either in a single large macrothrombus or in many locations in microthrombi, especially during an event exacerbating during a longer time, the higher the consumption of plasminogen (PLG) typically is. This may result in transient acquired plasminogen (PLG) deficiency, tipping the balance to a hypofibrinolytic and thus prothrombotic state (cf.
[0158] Thus it was found that the cause of thrombotic disease may be either in hypercoagulation or in hypofibrinolysis. The current treatments focus on (hyper-) coagulation only by using heparin, warfarin, or F.X antagonists.
[0159] It was surprisingly found that the consumption of plasminogen (PLG) leads to a hypofibrinolysis which can be named also a transient acquired plasminogen (PLG) (e.g., Glu-plasminogen) deficiency. Thus, it was found that that the treatment of an acquired transient plasminogen (PLG) (e.g., Glu-plasminogen) deficiency is able to resolve thrombotic disease. Diagnostic studies in diseases with a high prevalence and a life-threatening state provided evidence for this finding.
Example 2.1
[0160] Acute Kidney Injury/Failure (AKI)
[0161] The kidney is one of organs involved in multi organ failures also named “Multiple organ dysfunction syndrome (MODS)”.
[0162] This is defined as the presence of altered organ function in a patient who is acutely ill and in whom homeostasis cannot be maintained 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 cause MODS, including trauma, burns, and severe haemorrhagic shock. multiple organ dysfunctions, the extreme end of the continuum, are incremental degrees of physiologic derangements in individual organs (i.e., processes rather than events). Alteration in organ function can vary widely, ranging from a mild degree of organ dysfunction to frank organ failure (see multiple organ failure of sepsis, systemic inflammatory response syndrome (SIRS), toxic shock syndrome, and septic thrombophlebitis).
[0163] In an acute kidney failure, the microcirculation in the kidney is reduced in a way that no urine is produced any more. This allows an objective evaluation of the kidney function and their recovery.
[0164] In patients with an acute kidney injury/failure (AKI) and a pathogenesis over a longer time period, plasminogen (PLG) levels were found to be rather low in comparison to patients without a vascular genesis. A study focusing on this, was carried out together with Department of Clinical Chemistry (IKC) of the University Hospital Mannheim. Until March 2018, citrated plasma samples from 77 patients were collected, stored at below −40° C. All samples were measured batch-wise for plasminogen (PLG) and alpha-2-antiplasmin on a BCS XP Analyser (SIEMENS Healthcare) in a DIN EN ISO 15189 certified lab. A group of 53 healthy plasma donors were used as controls under the same conditions. The results were evaluated using u-test according to Mann-Whitney.
[0165] In the first validation study of patients with acute intrarenal kidney failure were selected at the Department of Clinical Chemistry (IKC), Mannheim, followed by criteria of acute kidney injury network (AKIN). Key parameters: creatinine raise up to 1.5-times the standard value in combination with the reduction of urine production.
[0166] Study-Outline Acute Kidney Injury/Failure (AKI) [0167] Parameters: Alpha-2-antiplasmin (A2-AP), plasminogen (PLG), activity Ratio R=A2-AP/plasminogen (PLG) in blood samples, used for the standard diagnostic as well [0168] 77 patients with acute kidney injury/failure (AKI) [0169] 53 control population (healthy blood donors) (CP)
[0170] Results were analyzed by Mann-Whitney test and showed: [0171] A significant (**) acquired plasminogen (PLG) deficiency in AKI patients [0172] A significant (**) acquired alpha-2-antiplasmin deficiency in AKI patients [0173] No significant difference of the (activity-based) ratio (A2AP/PLG)
TABLE-US-00001 TABLE 1 Summary of results of plasminogen (PLG) activity in AKI patients versus control Table analyzed Data Column A CP-plasminogen (PLG) (control group) Column B AKI-Plasminogen (PLG) (group of AKI patients) Mann-Whitney test: P value (Gaussian approximation) 0.0031 P value summary ** Are medians significantly Yes different? (P < 0.05) One- or two-tailed P value? Two-tailed Sum of ranks in column A, B 4096, 4420 Mann-Whitney U 1417
[0174] The Mann-Whitney test of acute kidney injury/failure (AKI) showed a significant difference of plasminogen (PLG) activity. P=0.0031 in patients (Ptx-77) and control group (CP-53). The results are shown in
TABLE-US-00002 TABLE 2 Summary of results of alpha-2-antiplasmin activity in AKI patients versus control group Table Analyzed Data Column A CP-alpha-2-antiplasmin (control group) Column B AKI-alpha-2-antiplasmin (group of AKI patients) Mann-Whitney test: P value (Gaussian approximation) 0.0011 P value summary ** Are medians significantly Yes different? (P < 0.05) One- or two-tailed P value? Two-tailed Sum of ranks in column A, B 4160, 4355 Mann-Whitney U 1352
[0175] The Mann-Whitney test of acute kidney failure showed a significant difference of alpha-2-antiplasmin Activity. P=0.0011 in patients (Ptx-77) and control group (CP-53). The results are shown in
TABLE-US-00003 TABLE 3 Summary of results of ratio of alpha-2-antiplasmin to plasminogen (PLG) in AKI patients versus control group Table Analyzed Data Column A CP-ratio (control group) Column B AKI-ratio (group of AKI patients) Mann-Whitney test: P value (Gaussian approximation) 0.1241 P value summary Ns Are medians significantly No different? (P < 0.05) One- or two-tailed P value? Two-tailed Sum of ranks in column A, B 3147, 5369 Mann-Whitney U 1716
[0176] The Mann-Whitney test of acute kidney failure showed no significant difference of the (activity-based) ratio (A2AP/PLG; P=0.1241) in patients (Ptx-77) and in the control group (CP-53). The results are shown in
Example 2.2
[0177] Disseminated Intravascular Coagulation (DIC)
[0178] The validation study of patients with disseminated intravascular (micro-)coagulation (DIC) was carried out by the Department of Clinical Chemistry (IKC), Mannheim. Patients with DIC were identified by D-dimers levels and internal criteria.
[0179] Study-Outline-DIC: [0180] Parameters: alpha-2-antiplasmin (A2-AP), plasminogen (PLG) activity. D-dimers, ratio R=A2-AP/plasminogen (PLG) in blood samples, used for the standard diagnostic as well [0181] 13 Patients with DIC (DIC) [0182] 53 control population (CP)
[0183] Results were analyzed by Mann-Whitney test and showed: [0184] A significant (**) acquired plasminogen (PLG) deficiency in patients with DIC [0185] No acquired alpha-2-antiplasmin deficiency in patients with DIC [0186] A significant (***) difference of the (activity-based) ratio (A2AP/PLG)->increased inhibition of fibrinolysis
TABLE-US-00004 TABLE 4 Summary of results of plasminogen (PLG) activity in DIC patients versus control group Table Analyzed PLG Column A CP-plasminogen (PLG) (control group) Column B DIC-plasminogen (PLG) (group of DIC patients) Mann-Whitney test P value (Gaussian approximation) 0.0013 P value summary ** Are medians significantly Yes different? (P < 0.05) One- or two-tailed P value? Two-tailed Sum of ranks in column A, B 2256, 519 Mann-Whitney U 288.0
[0187] The Mann-Whitney test of DIC showed a significant difference of plasminogen (PLG) activity. P=0.0013 in patients (Ptx-13) and control group (CP-53). The results are depicted in
TABLE-US-00005 TABLE 5 Summary of results of alpha-2-antiplasmin activity in DIC patients versus control group Table Analyzed A2-AP Column A CP-alpha-2-antiplasmin (control group) Column B DIC-alpha-2-antiplasmin (group of DIC patients) Mann-Whitney test P value (Gaussian approximation) 0.0730 P value summary ns Are medians significantly No different? (P < 0.05) One- or two-tailed P value? Two-tailed Sum of ranks in column A, B 2138, 637.5 Mann-Whitney U 406.5
[0188] The Mann-Whitney test of DIC showed no significant difference of alpha-2-antiplasmin activity. P=0.073 in patients (Ptx-13) and control group (CP-53). The results are depicted in
TABLE-US-00006 TABLE 6 Summary of results of ratio of alpha-2-antiplasmin to plasminogen (PLG) in DIC patients versus control group Table Analyzed Ratio-DIC Column A CP-ratio (control group) Column B DIC-ratio (group of DIC patients) Mann-Whitney test P value (Gaussian approximation) <0.0001 P value summary *** Are medians significantly Yes different? (P < 0.05) One- or two-tailed P value? Two-tailed Sum of ranks in column A, B 1634, 1141 Mann-Whitney U 203.0
[0189] The Mann-Whitney test of DIC showed a significant increased (activity-based) (activity-based) ratio (A2AP/PLG; P<0.0001) in patients (Ptx-13) than in the control group (CP-53). The results are depicted in
[0190] In summary, in a study of 53 individuals of a (healthy) control population (CP), 77 patients with acute kidney/renal injury (AKI) and 21 patients with disseminated intravascular coagulation patients (DIC), 40% of patients with AKI and 62% of patients with DIC showed a statistically significant deficiency of plasminogen (PLG) in comparison to the control population (CP). Herein, the Mann-Whitney test showed a median interquartile range p>0.003 for CP vs. AKI and >0.001 for CP vs. DIC. The results are depicted in
Example 2.3
[0191] Sepsis
[0192] The validation study of patients with sepsis was carried out at the Department of Clinical Chemistry (IKC), Mannheim (identification by D-dimers and internal criteria).
[0193] Study-Outline-Sepsis: [0194] Parameters: alpha-2-antiplasmin (A2AP), plasminogen (PLG) activity, Ratio R=A2-AP/plasminogen (PLG), PCTP, DD in blood samples, used for the standard diagnostic as well. [0195] 9 sepsis patients (sepsis) [0196] 53 control population (CP)
[0197] Results were analyzed by Mann-Whitney test and showed: [0198] A significant (*) acquired Plasminogen (PLG) deficiency in sepsis patients [0199] No acquired alpha-2-antiplasmin deficiency in patients with sepsis [0200] No significant difference of the (activity-based) ratio (A2AP/PLG)
TABLE-US-00007 TABLE 7 Summary of results of plasminogen (PLG) activity in sepsis patients versus control group Table Analyzed Sepsis-PLG Column A CP-plasminogen (PLG) (control group) Column B Sepsis-plasminogen (PLG) (group of sepsis patients) Mann-Whitney test P value (Gaussian approximation) 0.0377 P value summary * Are medians significantly Yes different? (P < 0.05) One- or two-tailed P value? Two-tailed Sum of ranks in column A, B 1774, 179 Mann-Whitney U 134.0
[0201] The Mann-Whitney test showed a significant difference of plasminogen (PLG) activity. P=0.0377 in patients (Ptx-9) and control group (CP-53). The results are depicted in
TABLE-US-00008 TABLE 8 Summary of results of alpha-2-antiplasmin activity in sepsis patients versus control group Table Analyzed Sepsis-A2AP Column A CP-alpha-2-antiplasmin (control group) Column B Sepsis-Alpha-2-antiplasimin (group of sepsis patients) Mann-Whitney test P value (Gaussian approximation) 0.0704 P value summary ns Are medians significantly No different? (P < 0.05) One- or two-tailed P value? Two-tailed Sum of ranks in column A, B 1761, 192.5 Mann-Whitney U 147.5
[0202] The Mann-Whitney test showed no significant difference of alpha-2-antiplasmin activity: P=0.0704 in patients (Ptx-9) and control group (CP-53). The results are depicted in
TABLE-US-00009 TABLE 9 Summary of results of ratio of alpha-2-antiplasmin to plasminogen (PLG) in sepsis patients versus control group Table Analyzed Sepsis-Ratio Column A CP-ratio (control group) Column B Sepsis-ratio (group of sepsis patients) Mann-Whitney test P value (Gaussian approximation) 0.2182 P value summary ns Are medians significantly No different? (P < 0.05) One- or two-tailed P value? Two-tailed Sum of ranks in column A, B 1608, 345.5 Mann-Whitney U 176.5
[0203] The Mann-Whitney test showed a non-significant but increased (activity-based) ratio (A2AP/PLG; P=0.2182) in patients (Ptx-13) than in the control group (CP-53). The results are depicted in
Example 2.4
[0204] Control Population
[0205] Raw-data: measurement of plasminogen (PLG) and A2AP in the control population (CP), healthy plasma donors from a plasma center.
[0206] Reference ranges could be defined in the control population PLG: 90% to 144%; A2AP: 97% to 119%; and Ratio: 0.80 to 1.25.
TABLE-US-00010 TABLE 10 Normalized activity of plasminogen (PLG) and A2AP of controls (normalized to an average activity of 100%) Controls No. PLG A2AP Ratio 1 101.3 105.1 1.04 5 of 50 individuals => 1.26 2 94.4 97.3 1.03 3 107.3 97.1 0.90 4 103.7 107.3 1.03 5 103.8 108.9 1.05 6 100.4 105.9 1.05 7 96.3 101.4 1.05 8 81.6 103.3 1.27 9 95 94.9 1.00 10 147.9 114.1 0.77 11 120.8 113 0.94 12 122 108.3 0.89 13 116.5 98.5 0.85 14 99.6 110.5 1.11 15 100.9 98.3 0.97 16 120.1 101.5 0.85 17 107.6 108.7 1.01 18 112.4 118.9 1.06 19 96.7 106 1.10 20 123.4 117.4 0.95 21 100.9 107.4 1.06 22 91.3 97 1.06 23 123.4 117.4 0.95 24 100.9 107.4 1.06 25 91.3 97 1.06 26 141.1 112 0.79 27 110.9 108 0.97 28 145.5 115.9 0.80 29 100.1 106.7 1.07 30 100.7 107.3 1.07 31 149.5 118.9 0.80 32 130.4 118.9 0.91 33 95.7 97.9 1.02 34 108 107.8 1.00 35 91.1 115.2 1.26 36 141.3 118.9 0.84 37 111.3 105.6 0.95 38 126.3 106.5 0.84 39 97.2 112.6 1.16 40 112.3 112.3 1.00 41 89.3 108.8 1.22 42 97.5 97.4 1.00 43 107.6 106 0.99 44 95.1 96.9 1.02 45 117.3 118.9 1.01 46 94 93.7 1.00 47 128.2 113.7 0.89 48 85.9 108.5 1.26 49 106.4 106.7 1.00 50 123.7 103.4 0.84 51 133.4 111.5 0.84 52 115.4 107.4 0.93 53 118.6 118.3 1.00 Mean 110.06 107.52 0.99 Standard Dev 16.65 7.19 0.12 Min 81.60 93.70 0.77 Max 149.50 118.90 1.27 Range 67.90 25.20 0.49 MW + 2SD 143.36 121.89 1.23 MW − 2SD 76.76 93.14 0.75 VK 15.13% 6.69% 11.95% 80% percentile <89.57 <96.915 <0.80 120% percentile >144.84 >118.9 >1.25
Example 2.5
[0207] Diagnosis in Patients with Acquired Plasminogen (PLG) Deficiencies Caused by Plasminogen (PLG) Consumption
[0208] At Centrum für Blutgerinnungsstörungen and Transfusionsmedizin (CBT) Bonn, from a total of 6000 (from non-acute states from GP's offices (general practitioners' medical offices/family practices)) patients with measured plasminogen (PLG) levels, all levels with decreased plasminogen (PLG) levels were selected and analyzed (n=700 {11.6%}). The frequency of diagnosis among patients with a decreased plasminogen (PLG) level was analyzed. Many diagnostic hits occurred where a plasminogen (PLG) deficiency was not expectable. The results are shown in
TABLE-US-00011 TABLE 11 Results of diagnosis in different patient groups, correlations of different parameters Thrombl Prot C APC PAP PLG Quick n Time APTT Act Ratio ATIII AZAP Ratio PAI 1 Complex D-Dimer PLT CRP PLG 0.1007 −0.1078 −0.0032 0.2576 −0.0103 0.1813 0.2097 −0.8148 0.1018 −0.0949 −0.0035 0.2000 0.1793 Quick −0.0905 −0.2004 0.6040 0.1218 0.1539 0.2505 0.0537 −0.0438 −0.0387 0.0298 0.0357 −0.0497 Thrombl 0.0839 −0.0831 0.0635 −0.0483 −0.0368 0.0802 −0.0251 −0.1529 −0.0288 0.4293 −0.0754 n Time APTT −0.0864 −0.0025 −0.0559 −0.0167 −0.0063 0.0409 −0.1795 −0.0618 0.0515 0.0204 Prot C −0.0104 0.2444 0.3517 −0.0354 0.0922 −0.1784 −0.0185 0.0630 0.0373 Act. APC −0.0548 0.0509 0.0444 −0.0409 −0.3889 −0.0500 −0.0498 −0.0355 Ratio ATIII 0.4439 0.0512 −0.0878 −0.0624 −0.1363 0.2074 −0.1637 AZAP 0.3335 0.0394 0.0469 −0.1000 0.2259 −0.0136 Ratio −0.0721 0.1070 −0.0373 −0.0378 −0.1433 PAI 1 −0.2503 0.0517 −0.0330 0.1750 PAP −0.0218 −0.2029 0.1150 Complex D-Dimer −0.0869 0.2367 PLT 0.0359
[0209] Table 11 describes the correlation coefficient from a linear regression R.sup.2=r. This was performed in the computer program Microsoft Excel (using the function “KORREL” in the German version) by correlating each two parameters with each other. This indicates whether there is a statistical relationship between two parameters or not. It was assumed that a correlation rates r of −0.5>r<0.5 indicate no or a weak correlation. Correlation rates r of −1.0 to −0.5 or 0.5 to 1.0 indicate a significant correlation. Thus, there was only a significant correlation for PLG with Ratio and for Quick with Pro C Act.
[0210] In Table 11, the following parameters are shown: PLG: plasminogen [IU/mL]; Quick [sec]; thrombin time [sec]; APTT: activated partial thromboplastine time [sec]; Prot C Act=protein C-activated [IU/mL]; APC ratio: activated protein C resistance [%]; ATIII=antithrombin III [%]; A2AP: alpha-2-antiplasmin [%]; ratio (A2AP/PLG), PAI-1: plasminogen activator inhibitor-1 [ng/mL], PAP complex: plasm iniogen-antiplasm in.complex; D-Dimer: D-dimer; PLT=platelets [×10.sup.6/μL]; CRP: C-reactive protein [μg/mL]
TABLE-US-00012 TABLE 12 Results of p-values in different patient groups Disease Abbreviation P-value n Lipoprotein(A) LpA <0.0001 119 Iron deficiency Iron <0.0001 55 Vitamin D deficiency VitD <0.0001 37 Vitamin K deficiency VitK <0.0001 21 Anemia Anaem <0.0001 56 Homocysteine level Hcys <0.0001 45 Protein Z deficiency PZ <0.0001 21 Thrombosis Thromb <0.0001 81 Embolism Emb <0.0001 86 Stroke Stroke <0.0005 9 Vitamin H deficiency VitH <0.0001 87 Myocardial infarction MI n.s. 3 Epitaxis Epist <0.0001 71 Hypermenorrhoe HM <0.0001 61 Von Willebrand syndrom vWS <0.0001 54 Morbus Meulengracht Meul <0.0001 21 Liver diseases Li <0.0001 29 Antiphospholipid diseases APL <0.0001 51 Migraine Migr <0.0001 29 Thyroid diseases Thyr <0.0001 41 Abortions Abort <0.0001 158 No/control population Norm n/A 55
[0211] In this study, plasminogen (PLG) was identified as an independent parameter. There was no correlation to any other parameter from the coagulation or fibrinolytic system except the weak correlation to the (activity-based) ratio (A2AP/PLG) (where plasminogen (PLG) is present. All samples were drawn from non-acute patients. Therefore, only a few patients with acute myocardial infarction were present in this study. The selected patient population (with low plasminogen (PLG) levels) showed a with normal A2AP levels.