WO2010018203A1

WO2010018203A1 - Role of the coagulation system in type i diabetes and need for intervention

Info

Publication number: WO2010018203A1
Application number: PCT/EP2009/060465
Authority: WO
Inventors: Georg Hess; Andrea Horsch; Dietmar Zdunek
Original assignee: F. Hoffmann-La Roche Ag; Roche Diagnostics Gmbh
Priority date: 2008-08-14
Filing date: 2009-08-13
Publication date: 2010-02-18

Abstract

The present invention relates to a method for predicting or assessing the risk of a type 1 diabetes patient to suffer from a cardiovascular event and/orterminal renal failure and/or death. The method is based on the determination of the amount of D-dimer and optionally plasminogen-inhibitor-activator (PAI) in a sample of a subject suffering from type 1 diabetes. Moreover, the present invention pertains to a method for predicting the risk of a cardiovascular event and/or terminal renal failure and/or mortality for a subject suffering from type 1 diabetes based on the determination of the amount of D-dimer and optionally PAI in a sample of the said subject. Also encompassed by the present invention are devices and kits for carrying out the aforementioned methods.

Description

Role of the coagulation system in type I diabetes and need for intervention

The present invention relates to a method for predicting or assessing the risk of a type 1 diabetes patient to suffer from a cardiovascular event and/or terminal renal failure and/or death. The method is based on the determination of the amount of D-dimer and optionally plasminogen-inhibitor-activator (PAI) in a sample of a subject suffering from type 1 diabetes. Moreover, the present invention pertains to a method for predicting the risk of a cardiovascular event, terminal renal failure and/or mortality for a subject suffering from type 1 diabetes based on the determination of the amount of D-dimer and optionally PAI in a sample of the said subject. Also encompassed by the present invention are devices and kits for carrying out the aforementioned methods.

An aim of modern medicine is to provide personalized or individualized treatment regimens. Those are treatment regimens which take into account a patient's individual needs or risks. Personalized or individual treatment regimens shall be also taken into account for emergency measures. Specifically, in the case of acute cardiovascular events, a decision for a certain treatment regimen must be made, usually, within a short period of time. Cardiovascular complications, particularly heart diseases, are the leading cause of morbidity and mortality in the Western hemisphere. Cardiovascular complications can remain asymptomatic for long periods of time. However, they may have severe consequences once an acute cardiovascular event, such as myocardial infarction, as a cause of the cardiovascular complication occurs.

There are two main categories of diabetes mellitus type 1 and type 2, which can be distinguished by a combination of features known to the person skilled in the art.

In type 1 diabetes (previously called iuvenile-onset or insulin-dependent), insulin production is absent because of autoimmune pancreatic beta-cell destruction possibly triggered by environmental exposure in genetically susceptible people. Destruction progresses subclinically over months or years until beta-cell mass decreases to the point that insulin concentrations are no longer adequate to control plasma glucose levels. The type 1 diabetes generally develops in childhood or adolescence and until recently was the most common form diagnosed before age 30; however, it can also develop in adults. In type 2 diabetes (previously called adult-onset or non-insulin-dependent), insulin secretion is inadequate. Often insulin levels are very high, especially early in the disease, but peripheral insulin resistance and increased hepatic production of glucose make insulin levels inadequate to normalize plasma glucose levels. Insulin production then falls, further exacerbating hyperglycemia. The disease generally develops in adults and becomes more common with age. Plasma glucose levels reach higher levels after eating in older than in younger adults, especially after high carbohydrate loads, and take longer to return to normal, in part because of increased accumulation of visceral and abdominal fat and decreased muscle mass.

Chronic kidney disease may result from any cause of renal dysfunction of sufficient magnitude. The most common cause in the US is diabetic nephropathy, followed by hypertensive nephroangiosclerosis and various primary and secondary glomerulopathies. A chronic kidney disease (chronic renal failure) is long-standing, progressive deterioration of renal function. Symptoms develop slowly and include anorexia, nausea, vomiting, stomatitis, dysgeusia, nocturia, lassitude, fatigue, proritus, decreased mental accuity, muscle twitches and cramps, water retention, undernutrition, ulceration and bleeding, peripheral neuropathies, and seizures. Diagnosis is based on laboratory testing of renal function, sometimes followed by renal biopsy.

The conventional diagnostic techniques for cardiovascular complications and their prediction include electrocardiographic and echo car dio graphic measurements, analysis of symptoms and previous medical history of the patient, such as chest pain, and analysis of some clinical parameters. Recently, these conventional techniques have been further strengthened by the analysis of biomarkers and, in particular, by the analysis of the levels for cardiac Troponins in blood samples of emergency patients. Moreover, natriuretic peptides are also described as suitable biomarkers for diagnosing cardiovascular complications.

Myocardial dysfunction is a general term, describing several pathological states of the heart muscle (myocard). A myocardial dysfunction may be a temporary pathological state (caused by e.g. ischemia, toxic substances, alcohol), contrary to heart failure. Myocardial dysfunction may disappear after removing the underlying cause. A symptomless myocardial dysfunction may, however, also develop into heart failure (which has to be treated in a therapy). Because the symptoms set on gradually, the patient often is unable to recognize his/her worsening condition. As a consequence, necessary treatment that could slow down the course of the disease can not be administered. A myocardial dysfunction may, however, also be a heart failure, a chronic heart failure, even a severe chronic heart failure.

Patients having heart failure may also develop an acute cardiac disorder, in general an acute coronary syndrome. ACS covers the states of unstable angina pectoris UAP and acute myocardial infarction MI.

MI is classified as belonging to coronary heart diseases CHD and is often preceded by other events also classified as belonging to CHD, like unstable angina pectoris UAP.

Symptomatic for UAP is chest pain which is relieved by sublingual administration of nitroglycerine. UAP is caused by a partial occlusion of the coronary vessels leading to hypoxemia and myocardial ischemia. In case the occlusion is too severe or total, a myocardial necrosis (which is the pathological state underlying myocardial infarction) results. MI may occur without obvious symptoms, i.e. the subject does not show any discomfort, and the MI is not preceded by stable or unstable angina pectoris.

UAP, however, is a symptomatic event preceding MI. A CHD in a subject may also occur symptomless, i.e. the subject may not feel uncomfortable and exhibit any signs of CHD like shortness of breath, chest pain or others known to the person skilled in the art. The subject, however, may be pathological and suffer from a malfunction of his coronary vessels which may result in a MI and/or congestive heart failure CHF, meaning the heart does not have the capacity to perform as required in order to ensure the necessary provision of blood to the subject's body. This may result in severe complications, one example of which is cardiac death.

An acute myocardial infarction is caused by an occlusion of a heart coronary vessel, resulting in the death of a region of various size of the heart muscle tissue. The death of the myocard causes an elevation of Troponin T (a heart-specific molecule) or Troponin I, which can be detected in serum/plasma. Furthermore, the death of the myocard is connected with a loss of the pump function of the heart, resulting in an elevated level of natriuretic peptides.

The occlusion of a heart coronary vessel leading to MI is usually caused by the formation of a thrombus in an atherosclerotic vessel. Atheromatous plaque lining the vessel may become unstable and rupture or split. This exposes thrombogenic material which causes the activation of blood platelets and initiates the coagulation cascade. As a result of this process a blood clot, the thrombus, forms, which interferes with the blood flow in the respective vessel. As a consequence, parts of the myocard are not adequately supplied with oxygen. It is important to note that even atheromatous plaque of small size that in itself does not significantly obstruct the blood flow through the coronary vessel can rupture and result in thrombosis.

As described above, cardiovascular complications as well as renal failure often develop gradually and patients may not show symptoms for a long time. Such patients do not receive appropriate treatment early in the course of the disease where therapeutic interventions may stop or even reverse the development of cardiovascular complications. Thus, it is highly desirable to identify patients with an increased risk of said diseases as early as possible in order to take preventive measures. Even if such measures are not possible, a reliable and simple prediction of the risk of a patient of suffering cardiovascular allows for close monitoring of high-risk patients so that these patients can receive appropriate treatment at the onset of said diseases.

The technical problem underlying the present invention can be seen as the provision of means and methods for complying with the aforementioned needs.

The technical problem is solved by the embodiments characterized in the claims and herein below.

Accordingly, the present invention relates to a method for predicting if a diabetes type 1 patient will suffer from a cardiovascular complication and/or terminal renal failure and/or death, the method comprising a) determining the amount of D-dimer; b) optionally determining the amount of a PAI; and c) comparing the amount of D-dimer and optionally of PAI to reference amounts, and establishing a prediction.

The method of the present invention, preferably, is an in vitro method. Moreover, it may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate to sample pre-treatments or evaluation of the results obtained by the method. The method of the present invention may be also used for monitoring, confirmation, and subclassification of a type 1 diabetes patient in respect to the risk of suffering a cardiovascular complication and/or death. The method may be carried out manually or assisted by automation. Preferably, steps (a), (b) and/or (c) may in total or in part be assisted by automation, e.g., by a suitable robotic and sensory equipment for the determination in step (a) and (b) or a computer-implemented comparison in step (c).

The term "predicting" as used herein refers to assessing the probability according to which a type 1 diabetes patient will suffer from a cardiovascular complication and/or terminal renal failure and/or death (i.e. mortality) within a defined time window (predictive window) in the future. The mortality may be caused by the cardiovascular complication and/or the renal failure. The predictive window is an interval in which the subject will develop one or more of the said complications according to the predicted probability. The predictive window may be the entire remaining lifespan of the subject upon analysis by the method of the present invention. Preferably, however, the predictive window is an interval of one month, six months or one, two, three, four, five or ten years after appearance of the cardiovascular complication (more preferably and precisely, after the sample to be analyzed by the method of the present invention has been obtained). As will be understood by those skilled in the art, such an assessment is usually not intended to be correct for 100% of the subjects to be analyzed. The term, however, requires that the assessment will be valid for a statistically significant portion of the subjects to be analyzed. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test, etc.. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %. The p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the probability envisaged by the present invention allows that the prediction will be correct for at least 60%, at least 70%, at least 80%, or at least 90% of the subjects of a given cohort.

The term "patient" or "subject" as used herein relates to animals, preferably mammals, and, more preferably, humans.

It is envisaged in accordance with the aforementioned method of the present invention that the subject shall suffer from type 1 diabetes. The subject thus exhibits the signs of diabetes which are known to the person skilled in the art and which have, partly, been laid out beforehand, see introductory part. More details can be found e.g. under www.merck.com/mmpe/index.html. Years of poorly controlled diabetes lead to multiple, primarily vascular complications that may affect both small (microvascular) and large (macrovascular) vessels. Microvascular disease underlies the three most common and devastating manifestations of diabetes mellitus: retinopathy, nephropathy, and neuropathy.

Diabetic nephropathy is a leading cause of chronic renal failure. It is characterized by thickening of the glomerula basement membrane, mesangial expansion, and glomerula sclerosis. These changes cause glomerula hypertension and progressive decline. Systemic hypertension may accelerate progression. The disease is usually asymptomatic until a nephrotic syndrome or renal failure develops.

Macrovascular disease (large-vessel atherosclerosis) is a result of the hyperinsulinemia, dyslipidemia, and hyperglycemia characteristics of diabetes. Manifestations are angina pectoris and myocardial infarction, transient ischemic attacks and strokes, and peripheral arterial disease. Diabetic cardiomyopathy is thought to result from many factors, including epicardial atherosclerosis, hypertension and left ventricular hypertrophy, microvascular disease, endothelial and autonomic dysfunction, obesity, and metabolic disturbances. Patients develop heart failure due to impairment in left ventricular systolic and diastolic function and are more likely to develop heart failure after myocardial infarction.

Chronic renal failure can be roughly categorized as diminished renal reserve, renal insufficiency, or renal failure (end-stage renal disease). Initially, as renal tissue loses function, there are few abnormabilities because the remaining tissue increases its performance. Decreased renal function interferes with the kidneys' abilities to maintain fluid and electrolyc homeostasis.

The diagnosis of renal failure includes the determination of serum creatinin levels. When creatinin levels rise, chronic renal failure is usually first suspected. The initial step is to determine whether the renal failure is acute, chronic, or acute superimposed on chronic (i.e. an acute disease that further compromises renal function in a patient with chronic renal failure). The cause of renal failure is also determined. Sometimes determing a duration of renal failure helps determine the cause. Testing includes urine analysis with examination of the urinary sediment, electrolytes, urea nitrogen, and creatinin, phosphate, calcium. Sometimes specific serologic tests inhibit to determine the cause. Urine analysis findings depend on the nature of the underlying disorder, but broad or especially waxy casts often are prominent in advanced renal failure of any cause. An ultrasound examination of the kidneys is usually helpful in evaluating for obstructive uropathy and in distinguishing acute from chronic renal failure based on kidney size. Except in certain conditions, patients with chronic renal failure have small shrunken kidneys with thinned, hyperechoic cortex. Obtaining a precise diagnosis becomes increasingly difficult as renal function reaches values close to those of end-stage renal disease. The definite diagnostic tool is renal biopsy, but it is not recommended when ultrasonography indicates small, or fϊbrotic kidneys.

Progression of chronic renal failure is predicted in most cases by the degree of proteinuria. Patients with nephrotic-range proteinuria usually have a poorer prognosis and progress to renal failure more rapidly. Progression may occur even if the underlying disorder is not active. Hypertension is associated with more rapid progression as well.

The term "sample" refers to a sample of a body fluid, to a sample of separated cells or to a sample from a tissue or an organ. Samples of body fluids can be obtained by well known techniques and include, preferably, samples of blood, plasma, serum, or urine, more preferably, samples of blood, plasma or serum. Tissue or organ samples may be obtained from any tissue or organ by, e.g., biopsy. Separated cells may be obtained from the body fluids or the tissues or organs by separating techniques such as centrifugation or cell sorting. Preferably, cell-, tissue- or organ samples are obtained from those cells, tissues or organs which express or produce the peptides referred to herein.

Plasminogen-activator-inhibitor (PAI) is a protein that participates in the regulation of blood clotting. Plasminogen is activated to plasmin by the proteases tissue plasminogen activator (tPA) and urokinase. By proteolytic cleavage of fibrin plasmin dissolves blood clots. PAI inhibits both tissue plasminogen activator and urokinase, thus preventing the activation of plasminogen. PAI is thought to play a role in several biological processes dependent on plasminogen or plasmin activity including wound healing, atherosclerosis, metabolic disturbances such as obesity and insulin resistance, tumor angiogenesis, chronic stress, bone remodeling, asthma, rheumatoid arthritis, fibrosis, glomerulonephritis and sepsis (Lijnen 2005, "Pleiotropic functions of plasminogen activator inhibitor-1" Journal of Thrombosis and Haemostasis 3: 35-45). Preferably, PAI-I has an amino acid sequence corresponding to the mRNA sequence described in 1986 by Ginsburg et al. "cDNA cloning of human plasminogen activator-inhibitor from endothelial cells" J. Clin. Invest. 78(6): 1673-1680.

D-dimer is a small molecule that is present in the blood after a blood clot is degraded by fibrinolysis. The D-dimer is a well known marker for blood coagulation and, e.g., is useful for ruling out deep vein thrombosis and pulmonary embolism (for a review see Stein et al. (2004), Annals of Internal Medicine, 140(8), 589-602). During blot clot formation, two D elements of two fibrinogen molecules are cross-linked by factor XIII. The D-dimer is a degradation product that is generated when insoluble fibrin is degraded by plasmin. Plasmin degrades the fibrin mesh but is not capable to break down the cross-links between two different D elements. Accordingly, the term "D-dimer", preferably, relates to fibrin degradation products that comprise cross-link bonds between two D elements. It is to be understood that said degradation products, preferably, represent a heterogeneous class of molecules comprising cross-linked D-dimers since said molecules can occur in a wide range of molecular weights and can contain various numbers of the said motif (see e.g. Lippi and Guidi, 2004, Clin Chem 50: 2150-2152). Thus, when the amount of D-dimer is determined, not only the amount of molecules that consist of two cross-linked D elements is determined but also the amount of larger molecules that contain one or more D-dimer motifs.

D-dimer and PAI used herein encompass also variants of the aforementioned specific D- dimer and PAI polypeptides. Such variants have at least the same essential biological and immunological properties as the specific D-dimer and PAI polypeptides. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification, e.g., by ELISA assays using polyclonal or monoclonal antibodies specifically recognizing the said D-dimer and PAI polypeptides. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and/or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the amino sequence of the specific D-dimer and PAI polypeptides. The degree of identity between two amino acid sequences can be determined by algorithms well known in the art. Preferably, the degree of identity is to be determined by comparing two optimally aligned sequences over a comparison window, where the fragment of amino acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. MoI. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad Sci. (USA) 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by visual inspection. Given that two sequences have been identified for comparison, GAP and BESTFIT are preferably employed to determine their optimal alignment and, thus, the degree of identity. Preferably, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. Variants referred to above may be allelic variants or any other species specific homologs, paralogs, or orthologs. Moreover, the variants referred to herein include fragments or subunits of the specific D-dimer and PAI polypeptides or the aforementioned types of variants as long as these fragments have the essential immunological and biological properties as referred to above. Such fragments may be, e.g., degradation products of the D-dimer and PAI peptides. Further included are variants which differ due to posttranslational modifications such as phosphorylation or myristylation.

Determining the amount of D-dimer and PAI or any other peptide or polypeptide referred to in this specification relates to measuring the amount or concentration, preferably semi- quantitatively or quantitatively. Measuring can be done directly or indirectly. Direct measuring relates to measuring the amount or concentration of the peptide or polypeptide based on a signal which is obtained from the peptide or polypeptide itself and the intensity of which directly correlates with the number of molecules of the peptide present in the sample. Such a signal - sometimes referred to herein as intensity signal - may be obtained, e.g., by measuring an intensity value of a specific physical or chemical property of the peptide or polypeptide. Indirect measuring includes measuring of a signal obtained from a secondary component (i.e. a component not being the peptide or polypeptide itself) or a biological read out system, e.g., measurable cellular responses, ligands, labels, or enzymatic reaction products.

In accordance with the present invention, determining the amount of a peptide or polypeptide can be achieved by all known means for determining the amount of a peptide in a sample. Said means comprise immunoassay devices and methods which may utilize labelled molecules in various sandwich, competition, or other assay formats. Said assays will develop a signal which is indicative for the presence or absence of the peptide or polypeptide. Moreover, the signal strength can, preferably, be correlated directly or indirectly (e.g. reverse- proportional) to the amount of polypeptide present in a sample. Further suitable methods comprise measuring a physical or chemical property specific for the peptide or polypeptide such as its precise molecular mass or NMR spectrum. Said methods comprise, preferably, biosensors, optical devices coupled to immunoassays, biochips, analytical devices such as mass- spectrometers, NMR- analyzers, or chromatography devices. Further, methods include micro-plate ELISA-based methods, fully-automated or robotic immunoassays (available for example on Elecsys™ analyzers), CBA (an enzymatic Cobalt Binding Assay, available for example on Roche-Hitachi™ analyzers), and latex agglutination assays (available for example on Roche-Hitachi™ analyzers).

Preferably, determining the amount of a peptide or polypeptide comprises the steps of (a) contacting a cell capable of eliciting a cellular response the intensity of which is indicative of the amount of the peptide or polypeptide with the said peptide or polypeptide for an adequate period of time, (b) measuring the cellular response. For measuring cellular responses, the sample or processed sample is, preferably, added to a cell culture and an internal or external cellular response is measured. The cellular response may include the measurable expression of a reporter gene or the secretion of a substance, e.g. a peptide, polypeptide, or a small molecule. The expression or substance shall generate an intensity signal which correlates to the amount of the peptide or polypeptide.

Also preferably, determining the amount of a peptide or polypeptide comprises the step of measuring a specific intensity signal obtainable from the peptide or polypeptide in the sample. As described above, such a signal may be the signal intensity observed at an m/z variable specific for the peptide or polypeptide observed in mass spectra or a NMR spectrum specific for the peptide or polypeptide.

Determining the amount of a peptide or polypeptide, preferably, comprises the steps of (a) contacting the peptide with a specific ligand, (b) (optionally) removing non-bound ligand, (c) measuring the amount of bound ligand. The bound ligand will generate an intensity signal. Binding according to the present invention includes both covalent and non-covalent binding. A ligand according to the present invention can be any compound, e.g., a peptide, polypeptide, nucleic acid, or small molecule, binding to the peptide or polypeptide described herein. Preferred ligands include antibodies, nucleic acids, peptides or polypeptides such as receptors or binding partners for the peptide or polypeptide and fragments thereof comprising the binding domains for the peptides, and aptamers, e.g. nucleic acid or peptide aptamers. Methods to prepare such ligands are well-known in the art. For example, identification and production of suitable antibodies or aptamers is also offered by commercial suppliers. The person skilled in the art is familiar with methods to develop derivatives of such ligands with higher affinity or specificity. For example, random mutations can be introduced into the nucleic acids, peptides or polypeptides. These derivatives can then be tested for binding according to screening procedures known in the art, e.g. phage display. Antibodies as referred to herein include both polyclonal and monoclonal antibodies, as well as fragments thereof, such as Fv, Fab and F(ab)₂ fragments that are capable of binding antigen or hapten. The present invention also includes single chain antibodies and humanized hybrid antibodies wherein amino acid sequences of a non- human donor antibody exhibiting a desired antigen-specificity are combined with sequences of a human acceptor antibody. The donor sequences will usually include at least the antigen-binding amino acid residues of the donor but may comprise other structurally and/or functionally relevant amino acid residues of the donor antibody as well. Such hybrids can be prepared by several methods well known in the art. Preferably, the ligand or agent binds specifically to the peptide or polypeptide. Specific binding according to the present invention means that the ligand or agent should not bind substantially to ("cross- react" with) another peptide, polypeptide or substance present in the sample to be analyzed. Preferably, the specifically bound peptide or polypeptide should be bound with at least 3 times higher, more preferably at least 10 times higher and even more preferably at least 50 times higher affinity than any other relevant peptide or polypeptide. Non- specific binding may be tolerable, if it can still be distinguished and measured unequivocally, e.g. according to its size on a Western Blot, or by its relatively higher abundance in the sample. Binding of the ligand can be measured by any method known in the art. Preferably, said method is semi-quantitative or quantitative. Suitable methods are described in the following.

First, binding of a ligand may be measured directly, e.g. by NMR or surface plasmon resonance.

Second, if the ligand also serves as a substrate of an enzymatic activity of the peptide or polypeptide of interest, an enzymatic reaction product may be measured (e.g. the amount of a protease can be measured by measuring the amount of cleaved substrate, e.g. on a

Western Blot). Alternatively, the ligand may exhibit enzymatic properties itself and the

"ligand/peptide or polypeptide" complex or the ligand which was bound by the peptide or polypeptide, respectively, may be contacted with a suitable substrate allowing detection by the generation of an intensity signal. For measurement of enzymatic reaction products, preferably the amount of substrate is saturating. The substrate may also be labeled with a detectable label prior to the reaction. Preferably, the sample is contacted with the substrate for an adequate period of time. An adequate period of time refers to the time necessary for an detectable, preferably measurable, amount of product to be produced. Instead of measuring the amount of product, the time necessary for appearance of a given (e.g. detectable) amount of product can be measured.

Third, the ligand may be coupled covalently or non-covalently to a label allowing detection and measurement of the ligand. Labelling may be done by direct or indirect methods. Direct labeling involves coupling of the label directly (covalently or non-covalently) to the ligand. Indirect labeling involves binding (covalently or non-covalently) of a secondary ligand to the first ligand. The secondary ligand should specifically bind to the first ligand. Said secondary ligand may be coupled with a suitable label and/or be the target (receptor) of tertiary ligand binding to the secondary ligand. The use of secondary, tertiary or even higher order ligands is often used to increase the signal. Suitable secondary and higher order ligands may include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (Vector Laboratories, Inc.). The ligand or substrate may also be "tagged" with one or more tags as known in the art. Such tags may then be targets for higher order ligands. Suitable tags include biotin, digoxygenin, His-Tag, Glutathion-S- Transferase, FLAG, GFP, myc-tag, influenza A virus haemagglutinin (HA), maltose binding protein, and the like. In the case of a peptide or polypeptide, the tag is preferably at the N-terminus and/or C-terminus. Suitable labels are any labels detectable by an appropriate detection method. Typical labels include gold particles, latex beads, acridan ester, luminol, ruthenium, enzymatically active labels, radioactive labels, magnetic labels ("e.g. magnetic beads", including paramagnetic and superparamagnetic labels), and fluorescent labels. Enzymatically active labels include e.g. horseradish peroxidase, alkaline phosphatase, beta-Galactosidase, Luciferase, and derivatives thereof. Suitable substrates for detection include di-amino-benzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, NBT- BCIP (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate, available as ready-made stock solution from Roche Diagnostics), CDP-Star™ (Amersham Biosciences), ECF™ (Amersham Biosciences). A suitable enzyme-substrate combination may result in a colored reaction product, fluorescence or chemo luminescence, which can be measured according to methods known in the art (e.g. using a light-sensitive film or a suitable camera system). As for measuring the enzymatic reaction, the criteria given above apply analogously. Typical fluorescent labels include fluorescent proteins (such as GFP and its derivatives), Cy3, Cy5, Texas Red, Fluorescein, and the Alexa dyes (e.g. Alexa 568). Further fluorescent labels are available e.g. from Molecular Probes (Oregon). Also the use of quantum dots as fluorescent labels is contemplated. Typical radioactive labels include ³⁵S, ¹²⁵I, ³²P, ³³P and the like. A radioactive label can be detected by any method known and appropriate, e.g. a light-sensitive film or a phosphor imager. Suitable measurement methods according the present invention also include precipitation (particularly immunoprecipitation), electrochemiluminescence (electro-generated chemiluminescence), RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), sandwich enzyme immune tests, electrochemiluminescence sandwich immunoassays (ECLIA), dissociation-enhanced lanthanide fluoro immuno assay (DELFIA), scintillation proximity assay (SPA), turbidimetry, nephelometry, latex- enhanced turbidimetry or nephelometry, or solid phase immune tests. Further methods known in the art (such as gel electrophoresis, 2D gel electrophoresis, SDS polyacrylamid gel electrophoresis (SDS-PAGE), Western Blotting, and mass spectrometry), can be used alone or in combination with labeling or other detection methods as described above.

The amount of a peptide or polypeptide may be, also preferably, determined as follows: (a) contacting a solid support comprising a ligand for the peptide or polypeptide as specified above with a sample comprising the peptide or polypeptide and (b) measuring the amount peptide or polypeptide which is bound to the support. The ligand, preferably chosen from the group consisting of nucleic acids, peptides, polypeptides, antibodies and aptamers, is preferably present on a solid support in immobilized form. Materials for manufacturing solid supports are well known in the art and include, inter alia, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracytes, wells and walls of reaction trays, plastic tubes etc. The ligand or agent may be bound to many different carriers. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble or insoluble for the purposes of the invention. Suitable methods for fixing/immobilizing said ligand are well known and include, but are not limited to ionic, hydrophobic, covalent interactions and the like. It is also contemplated to use "suspension arrays" as arrays according to the present invention (Nolan 2002, Trends Biotechnol. 20(l):9-12). In such suspension arrays, the carrier, e.g. a microbead or microsphere, is present in suspension. The array consists of different microbeads or microspheres, possibly labeled, carrying different ligands. Methods of producing such arrays, for example based on solid-phase chemistry and photo-labile protective groups, are generally known (US 5,744,305).

The term "amount" as used herein encompasses the absolute amount of a polypeptide or peptide, the relative amount or concentration of the said polypeptide or peptide as well as any value or parameter which correlates thereto or can be derived therefrom. Such values or parameters comprise intensity signal values from all specific physical or chemical properties obtained from the said peptides by direct measurements, e.g., intensity values in mass spectra or NMR spectra. Moreover, encompassed are all values or parameters which are obtained by indirect measurements specified elsewhere in this description, e.g., response levels determined from biological read out systems in response to the peptides or intensity signals obtained from specifically bound ligands. It is to be understood that values correlating to the aforementioned amounts or parameters can also be obtained by all standard mathematical operations.

The term "comparing" as used herein encompasses comparing the amount of the peptide or polypeptide comprised by the sample to be analyzed with an amount of a suitable reference source specified elsewhere in this description. It is to be understood that comparing as used herein refers to a comparison of corresponding parameters or values, e.g., an absolute amount is compared to an absolute reference amount while a concentration is compared to a reference concentration or an intensity signal obtained from a test sample is compared to the same type of intensity signal of a reference sample. The comparison referred to in step (c) of the method of the present invention may be carried out manually or computer assisted. For a computer assisted comparison, the value of the determined amount may be compared to values corresponding to suitable references which are stored in a database by a computer program. The computer program may further evaluate the result of the comparison, i.e. automatically provide the desired assessment in a suitable output format. Based on the comparison of the amount determined in step a) and the reference amount, it is possible to predict the risk of the subject of suffering of one or more of the complications referred to herein. Therefore, the reference amount is to be chosen so that either a difference or a similarity in the compared amounts allows identifying those diabetes type 1 patients which are at risk of suffering of one or more of the complications referred to herein, and which are not.

Accordingly, the term "reference amount" as used herein refers to an amount which allows predicting whether a diabetes type 1 patients is at risk of suffering from a cardiovascular complication and/or terminal renal failure and/or death. Accordingly, the reference may either be derived from (i) a type 1 diabetes patient known to have suffered from one or more of the said complications, or (ii) a type 1 diabetes patient known to have not suffered from the said complications. Moreover, the reference amount may define a threshold amount, whereby an amount larger than the threshold shall be indicative for a subject at risk to develop one or more of the said complications while an amount lower than the threshold amount shall be an indicator for a subject not at risk to develop the said complications. The reference amount applicable for an individual subject may vary depending on various physiological parameters such as age, gender, or subpopulation, as well as on the means used for the determination of the polypeptide or peptide referred to herein. A suitable reference amount may be determined by the method of the present invention from a reference sample to be analyzed together, i.e. simultaneously or subsequently, with the test sample. A preferred reference amount serving as a threshold may be derived from the upper limit of normal (ULN), i.e. the upper limit of the physiological amount to be found in a population of apparently healthy subjects. The ULN for a given population of subjects can be determined by various well known techniques. A suitable technique may be to determine the median of the population for the peptide or polypeptide amounts to be determined in the method of the present invention.

The reference amount with respect to all-cause mortality defining a threshold amount for D-dimer, as referred to in accordance with the present invention is, preferably 0.8, 1.0, 1.08, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.52, 2.7 or 2.9 μg FEU/ml, more preferably 2.52 μg FEU/ml or 1.08 μg FEU/ml, most preferably 1.08 μg FEU/ml (FEU: fibrinogen equivalent units).

An amount of D-dimer larger than the reference amount is, more preferably, indicative for a subject being at risk of developing one or more of the said complications.

The reference amount with respect to terminal renal failure defining a threshold amount for D-dimer, as referred to in accordance with the present invention is, preferably, 0.3, 0.4, 0.52, 0.6, 0.7 or 0.8 μg FEU/ml, more preferably 0.52 μg FEU/ml (FEU: fibrinogen equivalent units).

An amount of D-dimer smaller than the reference amount is, more preferably, indicative for a subject not being at risk of developing one or more of the said complications.

The reference amount with respect to combined fatal and non-fatal cardiovascular events defining a threshold amount for D-dimer, as referred to in accordance with the present invention is, preferably, 1.8, 2.0, 2.2, 2.52, 2.7 or 2.9 μg FEU/ml, more preferably 2.52 μg FEU/ml (FEU: fibrinogen equivalent units).

The reference amount with respect to non-fatal cardiovascular events defining a threshold amount for D-dimer, as referred to in accordance with the present invention is, preferably, 1.8, 2.0, 2.2, 2.52, 2.7 or 2.9 μg FEU/ml, more preferably 2.52 μg FEU/ml (FEU: fibrinogen equivalent units).

The reference amount with respect to fatal cardiovascular events defining a threshold amount for D-dimer, as referred to in accordance with the present invention is, preferably, 0.8, 1.0, 1.08, 1.3, 1.6, 1.9, 2.1, 2.3, 2.52, 2.7 or 2.9 μg FEU/ml, more preferably 1.08 μg FEU/ml or 2.52 μg FEU/ml, most preferably 2.52 μg FEU/ml (FEU: fibrinogen equivalent units).

An amount of D-dimer smaller than the reference amount is, preferably, indicative for a subject not being at risk of developing one or more of the said complications. Conversely, an amount of D-dimer larger than the reference amount indicates an elevated risk with respect to the diseases and disorders referred to above.

The reference amount with respect to all-cause mortality defining a threshold amount for PAI as referred to in accordance with the present invention is, preferably, 1.8, 2.0, 2.25, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.1, 4.3 or 4.5 U/ml, more preferably 4.1 U/ml or 2.25 U/ml, most preferably 2.25 U/ml.

An amount of PAI larger than the reference amount is, more preferably, indicative for a subject being at risk of developing one or more of the said complications.

The reference amount with respect to terminal renal failure defining a threshold amount for PAI as referred to in accordance with the present invention is, preferably, 0.7, 0.9, 1.15, 1.3 or 1.5 U/ml, more preferably 1.15 U/ml.

An amount of PAI smaller than the reference amount is, more preferably, indicative for a subject not being at risk of developing one or more of the said complications.

The reference amount with respect to combined fatal and non-fatal cardiovascular events defining a threshold amount for PAI as referred to in accordance with the present invention is, preferably, 1.8, 2.0, 2.25, 2.4 or 2.6 U/ml, more preferably 2.25 U/ml.

An amount of PAI smaller than the reference amount is, more preferably, indicative for a subject not being at risk of developing one or more of the said complications. The reference amount with respect to non- fatal cardiovascular events defining a threshold amount for PAI as referred to in accordance with the present invention is, preferably, 0.7, 0.9, 1.15, 1.3, 1.5, 3.3, 3.5, 3.7, 3.9, 4.1, 4.3, 4.5 or 4.7, more preferably 1.15 U/ml or 4.1 U/ml, most preferably 4.1 U/ml.

The reference amount with respect to fatal cardiovascular events defining a threshold amount for PAI as referred to in accordance with the present invention is, preferably, 3.4, 3.6, 3.8, 4.0, 4.1, 4.3, 4.5 or 4.7 U/ml, more preferably, 4.1 U/ml.

An amount of PAI smaller than the reference amount is, preferably, indicative for a subject not being at risk of developing one or more of the said complications. Conversely, an amount of PAI larger than the reference amount indicates an elevated risk with respect to the diseases and disorders referred to above.

Advantageously, it has been found in the study underlying the present invention that D- dimer and optionally PAI are reliable prognostic biomarkers for predicting the risk of a type 1 diabetes patient to suffer from cardiovascular complications and/or terminal renal failure and/or death. Thanks to the present invention, a risk stratification can be easily performed, allowing to initiate medical, physical or dietary treatments of the patient, including adapting the patient's lifestyle. In case the patients' risk turns out to be non existent or low, a time and/or cost intensive or, as the case may be, dangerous therapy can be avoided. Thus, the method of the present invention will be beneficial for the health system in that resources will be saved. It is to be understood that according to the method of the present invention described herein above and below, means for the determination of the amount of D-dimer and optionally PAI can be used for the manufacture of a diagnostic composition for identifying a subject being susceptible for a cardiac intervention.

The term "cardiovascular complication", preferably, refers to an acute cardiovascular event. Preferred acute cardiovascular events are stroke or acute coronary syndromes (ACS). ACS patients can show unstable angina pectoris (UAP) or myocardial infarction (MI). MI can be an ST-elevation MI (STEMI) or a non-ST-elevated MI (NSTEMI). The occurring of an ACS can be followed by a left ventricular dysfunction (LVD) and symptoms of heart failure. The term "mortality" as used herein relates to any kind of mortality, in particular mortality which is caused by the said acute cardiovascular events, e.g., as a result of myocardial (re- )infarction or heart failure.

The present invention, furthermore, relates to a method of assessing the risk of a diabetes type 1 patient to suffer from cardiovascular complications and/or terminal renal failure and/or death, the method comprising

a) determining the amount of D-dimer in a sample of a diabetes type 1 patient; and optionally b) determining the amount of PAI in a sample of a diabetes type 1 patient; and c) comparing the amount of D-dimer and optionally PAI determined in steps a) and b) to reference amounts, thereby assessing the said risk.

The term "assessing the risk" as used herein means estimating the probability whether a subject will in the future suffer from a cardiovascular complication and/or terminal renal failure and/or death, or not. As will be understood by those skilled in the art, the assessment underlying the invention is usually not intended to be correct for all (i.e. 100%) of the subjects to be identified. The term, however, requires that a statistically significant portion of subjects can be identified (e.g. a cohort in a cohort study). Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %. The p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. More preferably, at least 60%, at least 70%, at least 80% or at least 90% of the subjects of a population can be properly identified by the method of the present invention.

The expression "assessing the risk of suffering from a complication/mortality" as used herein means that the subject (i.e. a type 1 diabetes patient) to be analyzed by the method of the present invention is allocated either into the group of subjects of a population having a low risk for the said complications or mortality, or into a group of subjects having an elevated or a highly elevated risk. Risk assessment is performed with apparently healthy individuals, i.e. with individuals who suffer from type 1 diabetes but do not show symptoms of those complications for which the risk assessment is performed. A low risk as referred to in accordance with the present invention means that the risk of complication/mortality within a predetermined predictive window may be slightly elevated with respect to individuals not suffering from type 1 diabetes, but is lower than for 75 % of those individuals who suffer from type 1 diabetes.

An elevated risk as referred to in accordance with the present invention means that the risk of complication/mortality within a predetermined predictive window is elevated significantly for a subj ect with re sp ect to individuals with low risk o f complication/mortality.

A highly elevated risk as referred to in accordance with the present invention means that the risk of complication/mortality within a predetermined predictive window is even more elevated significantly for a subject with respect to individuals with low risk of complication/mortality or those with an elevated risk of complication/mortality.

All cause mortality

Preferably, according to a risk stratification with D-dimer an individual with a low risk of suffering from all cause mortality has a risk of 9 %, 1 1 %, 13 %, 15 % or 18 % to die within the predictive window. More preferably, the risk is 13 %. A serum level of D-dimer of less than 0.52 μg FEU/ml is an indicator of a low risk of mortality.

Preferably, an elevated risk of suffering from all cause mortality is a risk that is elevated by at least 20 %, 40 %, 60 % 80 % or 100 % as compared to the individuals with low risk. More preferably, an elevated risk of suffering from all cause mortality is a risk that is elevated by at least 40 % as compared to the individuals with low risk. A serum level of D- dimer equal to or greater than 0.52 μg FEU/ml is an indicator of an elevated risk of mortality.

Preferably, a highly elevated risk of suffering from all cause mortality is a risk that is elevated by at least 80 %, 100 %, 130 %, 150 % or 180 % as compared to the individuals with low risk. More preferably the risk is 130 %. A serum level of D-dimer equal to or greater than 2.52 μg FEU/ml is an indicator of a highly elevated risk of mortality.

Preferably, according to a risk stratification with PAI an individual with a low risk of suffering from all cause mortality has a risk of 6%, 10 %, 14 %, 18 % or 22 % to die within the predictive window. More preferably, the risk is 14 %. A serum level of PAI of less than 1.15 U/ml FEU/ml is an indicator of a low risk of mortality. Preferably, an elevated risk of suffering from all cause mortality is a risk that is elevated by at least 20%, 30 %, 40 % or 60 % as compared to the individuals with low risk. More preferably, an elevated risk of suffering from all cause mortality is a risk that is elevated by a at least 30 % as compared to the individuals with low risk. A serum level of PAI equal to or greater than 1.15 U/ml is an indicator of an elevated risk of mortality.

Preferably, a highly elevated risk of suffering from all cause mortality is a risk that is elevated by at least 70 %, 80 %, 90 %, 100 %, 120 % or 140 % as compared to the individuals with low risk. More preferably, a highly elevated risk of suffering from all cause mortality is a risk that is elevated by at least 100 % as compared to the individuals with low risk. More preferably, a highly elevated risk of suffering from all cause mortality is a risk that is elevated by at least 100 % as compared to the individuals with low risk. A serum level of PAI equal to or greater than 4.1 U/ml is an indicator of a highly elevated risk o f mortality.

Terminal renal failure

If the risk of a patient for suffering terminal renal failure is assessed, the method of the present invention is used, preferably, to exclude an increased risk. Exclusion of an elevated risk as referred to in accordance with the present invention means that the risk of complication/mortality within a predetermined predictive window is decreased significantly for a subject with respect to the average risk for complication/mortality in a population of subjects.

Preferably, a serum level of D-dimer below 0.52 μg FEU/ml excludes an increased risk of all cause mortality.

Preferably, a serum level of PAI below 1.15 U/ml excludes an increased risk of all cause mortality. Combined fatal and non-fatal cardiovascular events Preferably, according to a risk stratification with D-dimer an individual with a low risk of suffering from combined fatal and non-fatal cardiovascular events has a risk of 10 %, 14 %, 18 %, 22 % or 26 % to suffer from a fatal or non- fatal cardiovascular event within the predictive window. More preferably, the risk is 18 %. Preferably, a serum level of D-dimer of less than 0.4, 0.52 or 0.6 μg FEU/ml, more preferably of less 0.52 μg FEU/ml is an indicator of a low risk of a fatal or non- fatal cardiovascular event. Preferably, an elevated risk of suffering from a fatal or non- fatal cardiovascular event is a risk that is elevated by at least 60 %, 80 %, 100 %, 120 % or 140 % as compared to the individuals with low risk. More preferably, an elevated risk of suffering from a fatal or non- fatal cardiovascular event is a risk that is elevated by at least 100 % as compared to the individuals with low risk. Preferably, a serum level of D-dimer equal to or greater than 2.00, 2.25, 2.52, 2.75 or 3.00 μg FEU/ml, more preferably of more than 2.52 μg FEU/ml is an indicator of an elevated risk of fatal and non- fatal cardiovascular events.

Preferably, according to a risk stratification with PAI an individual with a low risk of suffering from combined fatal and non-fatal cardiovascular events has a risk of 10 %, 15%, 20 %, 25 % or 30 % to suffer from a fatal or non-fatal cardiovascular event within the predictive window. More preferably, the risk is 20 %. Preferably, a serum level of PAI of less than 0.90, 1.00, 1.10, 1.15, 1.25 or 1.5 U/ml, more preferably of less than 1.15 U/ml is an indicator of a low risk of a fatal or non- fatal cardiovascular event.

Preferably, an elevated risk of suffering from a fatal or non- fatal cardiovascular event is a risk that is elevated by at least 15 %, 20 %, 27 % or 35 % as compared to the individuals with low risk. More preferably, the risk is 27 %. Preferably, a serum level of PAI equal to or greater than 1.80, 2.00, 2.25, 2.50 or 2.75 U/ml , more preferably equal to or greater than 2.25 U/ml is an indicator of an elevated risk of fatal and non-fatal cardiovascular events. Preferably, a highly elevated risk of suffering from a fatal or non-fatal cardiovascular event is a risk that is elevated by at least 36 % as compared to the individuals with low risk. Preferably, a serum level of PAI equal to or greater than 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.1, 4.3, 4.6 or 5.0 U/ml, more preferably equal to or greater than 4.1 U/ml is an indicator of a highly elevated risk of fatal and non- fatal cardiovascular events.

Non-fatal cardiovascular events

Preferably, according to a risk stratification with D-dimer an individual with a low risk of suffering from non- fatal cardiovascular events has a risk of 6 %, 10 %, 13 %, 16 % or 20 % to suffer from a non-fatal cardiovascular event within the predictive window. More preferably, the risk is 13 %. Preferably, a serum level of D-dimer of less than 0.4, 0.52 or 0.6 μg FEU/ml, more preferably of less 0.52 μg FEU/ml is an indicator of a low risk of a non-fatal cardiovascular event.

Preferably, an elevated risk of suffering from a non- fatal cardiovascular event is a risk that is elevated by at least 50 %, 60 %, 70 %, 86 %, 100 % or 130 % as compared to the individuals with low risk. More preferably, an elevated risk of suffering from a non-fatal cardiovascular event is a risk that is elevated by at least 86 % as compared to the individuals with low risk. Preferably, a serum level of D-dimer equal to or greater than 2.00, 2.25, 2.52, 2.75 or 3.00 μg FEU/ml, more preferably of more than 2.52 μg FEU/ml is is an indicator of an elevated risk of a non- fatal cardiovascular event.

Preferably, according to a risk stratification with PAI an individual with a low risk of suffering from combined fatal and non-fatal cardiovascular events has a risk of 6 %, 9 %, 11 % or 15 % to suffer from a fatal or non- fatal cardiovascular event within the predictive window. More preferably, the risk is 11 %. Preferably, a serum level of PAI of less than 0.90, 1.00, 1.10, 1.15, 1.25 or 1.5 U/ml, more preferably of less than 1.15 U/ml is an indicator of a low risk of a non- fatal cardiovascular event.

Preferably, an elevated risk of suffering from a fatal or non- fatal cardiovascular event is a risk that is elevated by at least 30 %, 40 %, 54 %, 65 % or 80 % as compared to the individuals with low risk. More preferably, an elevated risk of suffering from a fatal or non-fatal cardiovascular event is a risk that is elevated by at least 54 % as compared to the individuals with low risk. Preferably, a serum level of PAI equal to or greater than 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.1, 4.3, 4.6 or 5.0 U/ml, more preferably equal to or greater than 4.1 U/ml is an indicator of an elevated risk of non- fatal cardiovascular events.

Fatal cardiovascular events

Preferably, according to a risk stratification with D-dimer an individual with a low risk of suffering from fatal cardiovascular events has a risk of 2.5 %, 5, 8 %, 12 % or 15 % to suffer from a fatal cardiovascular event within the predictive window. More preferably, the risk is 5 %. Preferably, a serum level of D-dimer of less than 0.4, 0.52 or 0.6 μg FEU/ml, more preferably of less 0.52 μg FEU/ml is an indicator of a low risk of a fatal cardiovascular event.

Preferably, an elevated risk of suffering from a non- fatal cardiovascular event is a risk that is elevated by at least 70 %, 80 %, 91 %, 100 %, 120 % or 140 % as compared to the individuals with low risk. More preferably, an elevated risk of suffering from a non-fatal cardiovascular event is a risk that is elevated by at least 91 % as compared to the individuals with low risk. Preferably, a serum level of D-dimer equal to or greater than

0.80, 0.90, 1.00, 1.08, 1.20 or 1.40 μg FEU/ml, more preferably equal to or greater than 1.08 μg FEU/ml is an indicator of an elevated risk of a cardiovascular event. Preferably, a highly elevated risk of suffering from a non-fatal cardiovascular event is a risk that is elevated by at least 80 % 100 %, 120 %, 133 %, 150 % or 180 % as compared to the individuals with low risk. More preferably, a highly elevated risk of suffering from a non- fatal cardiovascular event is a risk that is elevated by at least 133 % as compared to the individuals with low risk. Preferably, a serum level of D-dimer equal to or greater than 2.00, 2.25, 2.52, 2.75 or 3.00 μg FEU/ml, more preferably of more than 2.52 μg FEU/ml is an indicator of a highly elevated risk of a fatal cardiovascular event.

In principle, it has been found that D-dimer optionally in combination with PAI can be used for the manufacture of a diagnostic composition for predicting whether a type 1 diabetic patient is at risk of a complication/mortality.

The present invention further relates to a method of deciding on initiating a therapy in a diabetes type 1 patient being susceptible to suffer from a cardiovascular complication and/or renal failure and/or death, the method comprising a) determining the amount of D-dimer in a sample of a diabetes type 1 patient; and optionally b) determining the amount of PAI in a sample of the type I diabetes patient; c) comparing the amount of the peptides determined in steps a) and b) to a reference amount; d) deciding on said therapy.

Preferably, the said therapy to be selected for a subject by the method of the present invention said therapy is a drug-based therapy. More preferably, from at least one of the following groups are administered: ACE-inhibitors, AT-I receptor blockers, β-receptor blocking agents, aldosteron antagonists, antiplatelet drugs, anticoagulant drugs, statins, glycoprotein IIB/IIIA antagonists and inhibitors of factor Xa. Preferred ACE-inhibitors are captopril, enalapril, fosinopril, lisinopril, perindopril, quinapril, ramipril, or trandolapril. Preferred AT-I receptor blockers are candesartan, losartan, or valsartan. Preferred β- receptor blocking agents are bisoprolol, carvedilol, metoprolol or succinate. Preferred aldosterone antagonists are spironolacton or eplerenone. Preferred antiplatelet drugs are acetysalycylic acid, clopidogrel and ticlopidine. A preferred anticoagulant is warfarin. Preferred statins are Atorvastatin, Cerivastatin, Fluvastatin, Lovastatin, Pravastatin, Rosuvastatin and Simvastatin. Preferred glycoprotein IIB/IIIA antagonists are abciximab, eptifibatide and tirofϊban. Preferred inhibitors of factor Xa are tick anticoagulant peptide (TAP), antistatin, lefaxin and DX-9065. Preferred therapeutic options instead of or supplementary to pharmacotherapy are lifestyle changes, preferably cessation of smoking, limitation of alcohol intake, and increased physical activity.

Advantageously, by determining the amounts of D-dimer and optionally PAI in a sample of a subject suffering from type 1 diabetes, it can be decided whether a subject will be susceptible for a therapy as referred to above. Specifically, it is envisaged that a subject having amounts of D-dimer and optionally PAI larger than the reference amount will be suitable to be treated by the aforementioned therapy while a subject with D-dimer and optionally PAI lower than the reference amount will not benefit from the therapy.

Encompassed by the present invention is, further, a device adapted to carry out the methods of the present invention, comprising means for determining amounts of D-dimer and optionally PAI in a sample of the subject and means for comparing said amount to a reference amount, whereby a type 1 diabetes patient having a predisposition for the complications as specified beforehand is identified.

The term "device" as used herein relates to a system of means comprising at least the aforementioned means operatively linked to each other as to allow the prediction. Preferred means for determining the amounts of D-dimer and optionally PAI, and means for carrying out the comparison are disclosed above in connection with the method of the invention. How to link the means in an operating manner will depend on the type of means included into the device. For example, where means for automatically determining the amount of the peptides are applied, the data obtained by said automatically operating means can be processed by, e.g., a computer program in order to obtain the desired results. Preferably, the means are comprised by a single device in such a case. Said device may accordingly include an analyzing unit for the measurement of the amount of the peptides or polypeptides in an applied sample and a computer unit for processing the resulting data for the evaluation. Alternatively, where means such as test stripes are used for determining the amount of the peptides or polypeptides, the means for comparison may comprise control stripes or tables allocating the determined amount to a reference amount. The test stripes are, preferably, coupled to a ligand which specifically binds to the peptides or polypeptides referred to herein. The strip or device, preferably, comprises means for detection of the binding of said peptides or polypeptides to the said ligand. Preferred means for detection are disclosed in connection with embodiments relating to the method of the invention above. In such a case, the means are operatively linked in that the user of the system brings together the result of the determination of the amount and the diagnostic or prognostic value thereof due to the instructions and interpretations given in a manual. The means may appear as separate devices in such an embodiment and are, preferably, packaged together as a kit. The person skilled in the art will realize how to link the means without further ado. Preferred devices are those which can be applied without the particular knowledge of a specialized clinician, e.g., test stripes or electronic devices which merely require loading with a sample. The results may be given as output of raw data which need interpretation by the clinician. Preferably, the output of the device is, however, processed, i.e. evaluated, raw data the interpretation of which does not require a clinician. Further preferred devices comprise the analyzing units/devices (e.g., biosensors, arrays, solid supports coupled to ligands specifically recognizing the peptide, Plasmon surface resonance devices, NMR spectrometers, mass-spectrometers etc.) or evaluation units/devices referred to above in accordance with the method of the invention.

Accordingly, the present invention also relates to a device for predicting the risk of a diabetes type 1 patient to suffer from cardiovascular complications and/or terminal renal failure and/or death, comprising means for determining the amounts of D-dimer and optionally PAI in a sample of the subject and means for comparing said amount to a reference amount.

Further envisaged is a device for assessing the risk of a diabetes type 1 patient to suffer from cardiovascular complications and/or terminal renal failure and/or death, comprising means for determining the amounts of D-dimer and optionally PAI in a sample of the subject and means for comparing said amount to a reference amount.

The present invention also relates to a device for deciding on the administration of medicaments in a diabetes type 1 patient being susceptible to suffer from a cardiovascular complication and/or terminal renal failure and/or death, comprising means for determining the amounts of D-dimer and optionally PAI in a sample of the subject and means for comparing said amount to a reference amount.

Furthermore, the present invention encompasses a kit adapted to carry out the methods of the present invention, comprising means for determining the amounts of D-dimer and optionally PAI in a sample of the subject and means for comparing said amount to a reference amount, whereby a type 1 diabetes patient having a predisposition for the complications as specified beforehand is identified. The term "kit" as used herein refers to a collection of the aforementioned means, preferably, provided in separately or within a single container. The container, also preferably, comprises instructions for carrying out the method of the present invention.

The present invention pertains to a kit for predicting the risk of a diabetes type 1 patient to suffer from cardiovascular complications and/or terminal renal failure and/or death, comprising means for determining the amounts of D-dimer and optionally PAI in a sample of the subject and means for comparing said amount to a reference amount.

Also, the present invention relates to a kit for assessing the risk of a diabetes type 1 patient to suffer from cardiovascular complications and/or terminal renal failure and/or death, comprising means for determining the amounts of D-dimer and optionally PAI in a sample of the subject and means for comparing said amount to a reference amount.

Finally, the present invention relates to a kit for deciding on the administration of medicaments in a diabetes type 1 patient being susceptible to suffer from a cardiovascular complication and/or terminal renal failure and/or death, comprising means for determining the amounts of D-dimer and optionally PAI in a sample of the subject and means for comparing said amount to a reference amount.

All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

The figures merely illustrate the invention and are not intended to limit its scope in any way. Figures

Fig. 1 shows the time periods until patients suffer from death (all cause mortality) depending on the quartiles of the levels of D-dimer.

Fig. 2 shows the time periods until patients suffer from death (all cause mortality) depending on the quartiles of the levels of PAI.

Fig. 3 shows the time periods until patients suffer from terminal renal failure depending on the quartiles of the levels of D-dimer. Fig. 4 shows the time periods until patients suffer from terminal renal failure depending on the quartiles of the levels of PAI.

Example 1: The amounts of D-dimer and PAI were determined in serum samples of 891 patients suffering from type 1 diabetes by using the commercially available Elecsys Immunoassays from Roche Diagnostics, Germany. It was analyzed whether these markers correlate with mortality of any cause, terminal renal failure and non-fatal cardiovascular events in a follow-up period of twelve years. Of the 891 patients 178 patients died within the follow-up period (109 patients thereof due to cardiovascular disease). The results showed that subjects with increased levels of D-dimer and PAI are at elevated risk of suffering from an acute cardiovascular event.

The results of the study are summarized in the following table.

N = 891 patients

Patients per quartile: n=223

D-dimer (levels in μg FEU/ml; FEU: fibrinogen equivalent units)

25 -th percentile: < 0.52

50 Λt^mh percentile: 1.08

75 -t^mh percentile: 2.52

D-dimer all cause mortality (total n=178) terminal renal failure (total n=89)

l. Quartil n=29 (-16.3 %) n=7 (-7.9 %)

2. Quartil n=41 (-23.0 %) n=l 9 (~21.3 %)

3. Quartil n=41 (-23.0 %) n=34 (-38.2 %) 4. Quartil n=67 (-37.6 %) n=29 (-32.6 %)

Cardiovascular events

D-dimer fatal and non-fatal non-fatal events fatal events events (total n=211) (n=133) (n=78) 1. Quartil n=41 (- -19.4 %) n=29 -21.8 %) n=12 (- -15.4 %)

2. Quartil n=45 (- -21 .3 %) n=30 ⁽r -22. 6 %) n=15 (- -19 .2 %)

3. Quartil n=43 (- -20 .4 %) n=20 -15. 0 %) n=23 (- -29 .5 %)

4. Quartil n=82 (- -38 .9 %) n=54 -40. 6 %) n=28 (- -35 .9 %)

PAI (median of Quartiles in U/ml) 25^th percentile: < 1.15 percentile: 2.25

75 -th percentile: 4.1

PAI all cause mortality (total n=178) terminal renal failure (total n=89)

1. Quartil n=31 (- -17 .4 %) n=5 (-5.6 %)

2. Quartil n=41 (- -23 .0 %) n=34 (-38.2 %)

3. Quartil n=43 (- -24 .2 %) n=19 (~21.3 %)

4. Quartil n=63 (- -35 .4 %) n=31 (~34.8 %)

Cardiovascular events

PAI fatal and non- fatal non- fatal events fatal events events (total n=211) (n=133) (n=78)

1. Quartil n=45 (- -21 .3 %) n=24 (~18.0 %) n=21 (-26.9 %)

2. Quartil n=48 (- -22 .7 %) n=37 (-27.8 %) n=l l (-14.1 %)

3. Quartil n=57 (- -27 .0 %) n=35 (-26.3 %) n=22 (-28.2 %)

4. Quartil n=61 (- -28 .9 %) n=37 (-27.8 %) n=24 (-30.8 %)

Claims

1. A method for predicting if a diabetes type 1 patient will suffer from a cardiovascular complication and/or terminal renal failure and/or death, the method comprising a) determining the amount of D-dimer; and optionally b) determining the amount plasminogen activation inhibitor PAI; and b) comparing the amount of D-dimer and optionally of PAI to reference amounts, and establishing a prediction.

2. The method of claim 1, wherein the cardiovascular complication is a chronic cardiovascular disease or an acute cardiovascular complication.

3. The method of claim 1 or 2, wherein the cardiovascular complication is an acute cardiovascular complication.

4. The method of claim 3, wherein the acute cardiovascular complication is stroke, acute coronary syndromes (ACS), unstable angina pectoris (UAP), myocardial infarction (MI), ST-elevation MI (STEMI) and non-ST-elevated MI (NSTEMI).

5. The method of any of claims 1 to 4, wherein an amount of D-dimer larger than the reference amount is indicative for a subject susceptible to suffer from one or more of the said complications.

6. The method according to claim 5, wherein an amount of the second marker for blood coagulation larger than the reference amount is indicative for a subject susceptible to suffer from one or more of the said complications.

7. The method according to any of claims 5 or 6, wherein said reference amount for D- dimer is 1.08 μg FEU/ml.

8. The method of claim 6, wherein said reference amount for plasminogen-activator inhibitor is 2.25 U/ml.

9. The method of claim 1, wherein an amount of D-dimer smaller than the reference amount is indicative for a subject not being susceptible to suffer from terminal renal failure.

10. The method according to claim 9, wherein an amount of the second marker for blood coagulation smaller than the reference amount is indicative for a subject not being susceptible to suffer from one or more of the said complications.

11. The method according to any of claims 9 or 10, wherein said reference amount for D- dimer is 0.52 μg FEU/ml.

12. The method of claim 10, wherein said reference amount for plasminogen-activator inhibitor is 1.15 U/ml.

13. A method for assessing the risk of a diabetes type 1 patient to suffer from cardiovascular complications and/or death, the method comprising a) determining the amount of D-dimer in a sample of a diabetes type 1 patient; and optionally b) determining the amount of PAI; and c) comparing the amount of D-dimer and optionally PAI determined in steps a) and b) to reference amounts, thereby assessing the said risk.

14. A method for deciding on the administration of medicaments in a diabetes type 1 patient being susceptible to suffer from a cardiovascular complication and/or death, the method comprising a) determining the amount of D-dimer in a sample of a diabetes type 1 patient; and optionally b) determining the amount of PAI; c) comparing the amount of D-dimer and optionally of PAI determined in steps a) and b) to reference amounts, d) deciding on the said administration.

15. A device for predicting the risk of a diabetes type 1 patient to suffer from cardiovascular complications and/or death, comprising means for determining the amount of D-dimer and optionally PAI in a sample of the subject and means for comparing said amounts to a reference amount.

16. A kit for predicting the risk of a diabetes type 1 patient to suffer from cardiovascular complications and/or death, comprising means for determining the amount of D-dimer and optionally PAI in a sample of the subject and means for comparing said amounts to a reference amount.