Inhibition of Sorbitol Dehydrogenase Increases Platelets Adhesion in Animals Mice with Fidarestat
Authors: Flores-Nascimento Mariane Cristina , Zapponi Kiara Cristina Senger , Mendes Camila , Ferreira Regiane , Siqueira Lucia Helena , Fornari Thais Arouca , Orsi Fernanda Loureiro de Andrade , Mazetto Bruna M , Annichino-Bizzacchi Joyce M 
Published: October 2019

Abstract
BACKGROUND: Deep vein thrombosis (DVT) is multi-causal disease associated to a high morbi-mortality due to complications as pulmonary embolism and post-thrombotic syndrome. The identification of factors enrolled on the disease is important to better understand its pathophysiology. As the role of the platelets on DVT is still not completely defined, we previously performed a proteomic study on platelets from 3 DVT patients and compared to results obtained from 1 sibling and 1 neighbor for each patient in order to minimize the genetic and environmental interferences. These high-selected patients presented spontaneous and recurrent episodes of proximal DVT and mentioned a familiar history of thrombotic diseases.
METHODS: platelets were washed, lysed, and their proteins were alkylated, reduced, precipitated with acetone and hydrolyzed by trypsin. 100mg of peptides were then separated by hydrophobicity using HPLC, and 8 fractions were obtained and directed to the LTQ-Orbitrap mass spectrometer. The proteins search was performed by Sorcerer-SEQUEST. In this analysis, sorbitol dehydrogenase (SORD) was present only in patients and absent in siblings and neighbors. In order to evaluate the SORD gene expression, qPCR was performed on platelets RNA from the same individuals applying specific primers after the evaluation of a minimal leukocyte contamination with specific primers to CD45. The influence of SORD on platelets adhesion was evaluated with the administration of a direct inhibitor (fidarestat) to 12 mice C57Bl6 by gavage at 10mg/kg and the results were compared to that obtained from 12 animals gavaged in a solution of 5% gum arabic. To the adhesion test, blood was obtained from cava veins in 3.8% sodium citrate and centrifuged at 100G/10’. Platelets were collected, pelleted and washed in Krebs buffer. Platelets at 1.2 x 108/μL were added to 96 wells plates pre-covered with fibrinogen 50μg/mL and their adhesion properties were evaluated with and without stimulation with 50mU of thrombin after incubation at 37°C for 30’. The percentage of adherent cells was calculated by comparing acid phosphatase activity to that of a standard curve with known numbers of platelets (0 – 100%) from the same group at 405nm. On the qPCR, the analyses were performed with fold change, and we considered significant results lower then 0.5x and higher then 1.5x. The Mann-Whitney test was applied to analyze the adhesion results, and P<0.05 were considered significant.
RESULTS: On qPCR, the gene expression of SORD was increased in patients (1x) in comparison to siblings (0.51x), but similar to neighbors (0.77x). The adhesion of platelets unstimulated was similar between both groups of animals (Median fidarestat= 41.6; 2.5-91.6; SD=25.9; median controls = 45; 9.8-91.3; SD=22.6; P=0.69), but after stimulation with thrombin, platelets from treated animals presented increased adhesion (median = 74.4; 42.9-98.8; SD=24) when compared to controls (median = 43.9; 13.6-48.2; SD=12.1; P=0.0148).
CONCLUSIONS: SORD was present only in patients with DVT and absent in healthy siblings and neighbors on the proteomic analysis. The increase of SORD gene expression in patients in comparison to all the controls can corroborate the involvement of this protein on DVT physiology. The inhibition of this protein with fidarestat promoted the increase of platelets adhesion to plates covered with fibrinogen only after stimulation with thrombin. Our hypothesis is that SORD may be a protective protein based on the increase of the gene expression, protein levels and adherent properties after thrombogenic stimuli that could be an attempt to decrease the platelet function and minimize the damage.

Disclosures
No relevant conflicts of interest to declare.