Proteome Analysis of Urinary Biomarkers in Acute Hypercoagulable State Rat Model.

Thrombotic diseases are usually preceded by a hypercoagulable state in the body. This study aimed to screen potential urinary biomarkers for hypercoagulable state based on proteome analysis. Wistar rats were administered with the hemostatic agent etamsylate to establish hypercoagulable state. Urine samples were collected for proteome analysis. We found 20 proteins with levels more than 1.5-fold in difference between control rats and model rats. We searched human homologs of 20 rat proteins and identified 13 human proteins. Of the 13 human homologous proteins, nine were members of human core urinary proteome. Human homologous proteins of differential proteins were highly expressed in 31 human tissues, especially in the kidneys followed by digestive system and reproductive system. Surprisingly, we did not identify known coagulation factors as differential proteins in the urine of model rats. Hypercoagulable state of the body may not involve direct changes in coagulation factors but causes the changes upstream of the coagulation cascade system. Common differential urinary proteins between different hypercoagulable states suggest some common pathways in the formation of hypercoagulable states and may serve as potential biomarkers for the prevention and treatment of thrombotic diseases.

Urinary proteome analysis of acute hypercoagulable state in rat model induced by ε-aminocaproic acid.

The hypercoagulable state occurs in a group of prothrombotic disorders associated with an increased risk for thromboembolic events, but it is difficult to diagnose due to the lack of available biomarkers. This study aimed to investigate systematic changes of urinary proteome in acute hypercoagulable state induced by certain antifibrinolytics. To reduce the effects of both genetic and environmental factors on the urinary proteome, we used a rat model of acute hypercoagulable state induced by an antifibrinolytic agent ε-aminocaproic acid, resembling human hypercoagulable state. Urine samples were collected during acute hypercoagulable state for analysis by liquid chromatography-tandem mass spectrometry (LCMS/MS). Of 65 significantly changed proteins in acute hypercoagulable state, 38 proteins had human orthologs, and 18 proteins were identified as stable in normal human urine. None of the identified proteins have been found to be clotting factors, but 4 proteins are known to be involved in the regulation of blood coagulation factors. Two proteins were verified as the markers associated with acute hypercoagulable state by Western blot analysis. In addition, four common differential urinary proteins have been found in acute hypercoagulable state induced by another antifibrinolytics tranexamic acid. These four proteins are potential biomarkers for early diagnosis of hypercoagulable state to prevent the development of thrombotic diseases.

Dynamic changes of urinary proteins in a rat model of acute hypercoagulable state induced by tranexamic acid.

The hypercoagulable state leads to the development of thrombotic diseases, but it is difficult to diagnose due to the lack of available biomarkers. This study aimed to investigate systematic changes of the urinary proteome in the acute hypercoagulable state. A rat model of the acute hypercoagulable state was induced by an antifibrinolytic agent tranexamic acid and urine samples were collected for proteomic analysis by liquid chromatography-tandem mass spectrometry. A total of 28 differential proteins were detected in the urinary proteome of the model rats, of which 12 had been previously considered as candidate biomarkers such as myoglobin, and 10 had been considered stable in healthy human urine. Of the 28 differentially expressed proteins 18 had counterparts in humans. Of these 18 proteins, 10 were members of the human core urinary proteome distributed in a variety of human tissues but concentrated in the urinary and digestive systems. Fumarylacetoacetase was verified as a potential marker of the acute hypercoagulable state by Western blot analysis. In conclusion, urine proteome analysis is a powerful approach to identify potential biomarkers of acute hypercoagulable state.

Collection and preservation of urinary proteins, using a fluff pulp diaper.

Change is the most fundamental property of a biomarker. In contrast to the blood, which is under homeostatic controls, urine reflects changes in the body earlier and is more sensitive, thus making it a better biomarker source. Moreover, drawing blood from infants and toddlers is difficult and not tolerated well. For patients limited by language, communicating their chief complaint is difficult. Thus, monitoring biomarkers in urine can provide valuable clues for the diagnosis of diseases, especially pediatric diseases. Collecting urine from young children and some adult patients is more challenging than collecting it from healthy adults. Here, we propose a method that uses a fluff pulp diaper to collect urine. Urinary proteins are then eluted and adsorbed onto a piece of nitrocellulose membrane, which can be dried and stored in a vacuum bag. SDS-PAGE and LC-MS/MS analysis indicated that this method is reproducible, and similar proteins were identified as those obtained by an acetone precipitation method. With this simple and economical method, it is possible to collect and preserve urine samples from infants, toddlers, and patients with special needs, even for large-scale biomarker studies.