Table1_Heparin-induced thrombocytopenia associated with low-molecular-weight heparin: clinical feature analysis of cases and pharmacovigilance assessment of the FAERS database.docx

Background: Unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) are commonly used anticoagulants for the management of arterial and venous thromboses. However, it is crucial to be aware that LMWH can, in rare cases, lead to a dangerous complication known as heparin-induced thrombocytopenia (HIT). The objective of this study was to evaluate the pharmacovigilance and clinical features of HIT associated with LMWH, as well as identify treatment strategies and risk factors to facilitate prompt management.Methods: We extracted adverse event report data from the FDA Adverse Event Reporting System (FAERS) database for pharmacovigilance assessment. Case reports on LMWH-induced thrombocytopenia dated up to 20 March 2023 were collected for retrospective analysis.Results: Significantly elevated reporting rates of HIT were shown in adverse event (AE) data of LMWHs in the FAERS database, while tinzaparin had a higher proportional reporting ratio (PRR) and reporting odds ratio (ROR) than other LMWHs, indicating a greater likelihood of HIT. Case report analysis indicated that a total of 43 patients showed evidence of LMWH-induced thrombocytopenia with a median onset time of 8 days. Almost half of the events were caused by enoxaparin. LMWHs were mainly prescribed for the treatment of embolism and thromboprophylaxis of joint operation. Patients with a history of diabetes or surgery appeared to be more susceptible to HIT. Clinical symptoms were mostly presented as thrombus, skin lesion, and dyspnea. Almost 90% of the patients experienced a platelet reduction of more than 50% and had a Warkentin 4T score of more than 6, indicating a high likelihood of HIT. In all patients, LMWHs that were determined to be the cause were promptly withdrawn. Following the discontinuation of LMWHs, almost all patients were given alternative anticoagulants and eventually achieved recovery.Conclusion: LMWH-induced thrombocytopenia is rare but serious, with increased risk in patients with diabetes or a surgical history. Prompt recognition and management are crucial for the safe use of LMWHs.</p

Exogenous S100A8 regulate autophagy through RAGE receptor.

<p>K562 cells were transfected with control shRNA, RAGE shRNA or TLR4 shRNA for 48 hours, and then treated with S100A8 protein (1 µg/ml) for 24 hours. LC3, p62, RAGE and TLR4 were assayed by Western blot. All data were representatives of 3 independent experiments.</p

Overexpression of S100A8 increased the resistance of leukemia cells to chemotherapy.

<p>(<b>A</b>) K562 cells were transfected with control pLPCX or pLPCX-S100A8 plasmids. Protein level of S100A8 was assayed by Western blot. (<b>B</b>) K562 cells transfected with control pLPCX or pLPCX-S100A8 plasmids were treated with ADR (1 µg/mL) or VCR (1 µg/mL) for 24 hours. Apoptosis was analyzed by measuring Annexin V–positive cells with flow cytometry (n = 3;^*<i>P</i><0.05). (<b>C</b>) K562 cells were treated as B, LC3-I/II and BECN1 levels were assayed by Western blot analysis. <b>UT,</b> untreated group of K562 cells transfected with S100A8 plasmids. <b>Control,</b> K562 cells were transfected with control pLPCX plasmids. (<b>D</b>) K562 cells were transfected with pLPCX control or pLPCX -S100A8 cDNA for 48 hours and then treated with ADR (1 µg/mL) for 24 hours in the presence or absence of bafilomycin A1 (Baf; 100 nmol/L). The protein levels of LC3 and p62 were assayed by Western blot. (<b>E</b>) K562 cells were transfected pLPCX or pLPCX-S100A8 cDNA with or without the indicated shRNA for 48 hours. Protein levels of S100A8, PI3KC3, BECN1, Atg7, LC3, and p62 were assayed by Western blots. (<b>F</b>) K562 cells transfected with control pLPCX or pLPCX-S100A8 cDNA were subjected to TEM analysis. Autophagosomes were highlighted by arrows. (<b>G</b>) K562 cells transfected with PLPCX-S100A8 cDNA were treated with bafilomycin A1 (Baf; 100 nmol/L) or 3-methyladenine (3-MA; 10 mmo/L) for 12 hours. LC3 were assayed by Western blot. <b>Control,</b> K562 cells were transfected with control pLPCX. (<b>H</b>) K562 cells transfected the indicated shRNA were treated with ADR (1 µg/mL) and VCR (1 µg/mL) for 24 hours. Cell viability was analyzed by MTT assay (n = 3;^*<i>P</i><0.05). NS, not significant.</p

ULK1-mAtg13 regulated the fomation of S100A8-BECN1 complex formation in leukemia cells.

<p>(<b>A-C</b>) K562 cells were transfected with S100A8 shRNA (A and B) or ULK1 shRNA (C) for 48 hours and then were treated with ADR (1 µg/mL) for 24 hours. Cells were then processed for immunoprecipitation (IP) or Western blotting (IB) as described in Materials and Methods. All data are representative of 3 experiments. (<b>D</b>) K562 cells transfected with S100A8 shRNA (A and B) or ULK1 shRNA (C) for 48 hours were treated with ADR (1 µg/mL) or VCR (1 µg/mL) for 24 hours. Apoptosis was analyzed by measuring Annexin V–positive cells with flow cytometry (n = 3;^*<i>P</i><0.05).</p

S100A8 regulated the chemotherapy–induced autophagy in leukemia cells.

<p>(<b>A and B)</b> K562 cells were transfected with control shRNA or S100A8 shRNA for 48 hours and then treated with ADR (1 µg/mL) and VCR (1 µg/mL) for 24 hours in the presence or absence of bafilomycin A1 (Baf; 100 nmol/L). The protein levels of LC3 and p62 were assayed by Western blot (<b>A</b>); LC3 puncta were analyzed by LC3 antibody or mRFP–GFP–LC3 (Magnification is 10×60 oil) (<b>B</b>) (n = 3;^*<i>P</i><0.05). (<b>C</b>) K562 cells were transfected with control shRNA or S100A8 shRNA for 48 hours and then treated with ADR (1 µg/mL) and VCR (1 µg/mL) for 24 hours. Autophagosome-like structures (indicated by the red arrows) were assayed by TEM (n = 3;^*<i>P</i><0.05). Bar = 2 µm. (<b>D and E</b>) K562/A02 cells were transfected with control shRNA or S100A8 shRNA for 48 hours. After pretreatment with rapamycin (Rap; 100 nmol/L) for 6 hours, cells were treated with ADR (1 µg/mL) for 24 hours. Apoptosis was analyzed by measuring Annexin V–positive cells with flow cytometry (<b>D</b>). Autophagy was analyzed by measuring LC3 puncta formation (<b>E</b>; n = 3;^*<i>P</i><0.05).</p

S100A8 was elevated in drug resistance leukemia cells and chemotherapy agents induced S100A8 expression in leukemia cells.

<p>(<b>A</b>) Protein level of S100A8 was analyzed by Western blotting in Jurkat, HL-60, K562 and MV4-11 cells (n = 3, ^*<i>P</i><0.05). (<b>B and C</b>) S100A8 mRNA level in leukemia cells was analyzed by real time RT-PCR (n = 3, ^*<i>P</i><0.05 versus Jurkat cells in <b>B</b> and ^*<i>P</i><0.05 versus HL-60 or K562 in <b>C</b>, Jurkat group set as 1). (<b>D</b>) Basal LC3-I/II level was analyzed by Western blotting in leukemia cells (n = 3, ^*<i>P</i><0.05 <i>versus</i> Jurkat cells ). (<b>E and F</b>) IC50 levels of adriamycin (ADR) in Jurkat, K562, HL-60, MV-4-11, K562/A02, and HL-60/ADR cells (n = 3, ^*<i>P</i><0.05 versus Jurkat cells in <b>E</b>, ^*<i>P</i><0.05 versus HL-60 or K562 in <b>F</b>). (<b>G</b>) Jurkat, HL-60, K562 and MV4-11 cells were treated with ADR (1 µg/ml), VCR (1 µg/ml) or As2O3 (5 µM) for 24 hours and S100A8 protein level was analyzed by Western blotting (n = 3, *P<0.05 vs. UT, untreated group). AU, arbitrary unit. (<b>H</b>) Jurkat, HL-60, K562 and MV4-11 cells were treated with ADR (1 µg/ml), vincristine (VCR, 1 µg/ml) or arsenic trioxide (As2O3, 5 µM) for 24 hours and S100A8 mRNA level was analyzed by real time RT-PCR (n = 3, *P<0.05 versus control group, control group set as 1).</p

Suppression of S100A8 sensitized drug resistance leukemia cells to chemotherapy.

<p><b>(A)</b> HL60/ADR and K562/A02 cells were transfected with control shRNA or S100A8 shRNA for 48 hours. Protein and mRNA level of S100A8 was assayed by Western blot and real time RT-PCR, respectively. (<b>B</b>) HL60/ADR and K562/A02 cells were transfected with control shRNA or S100A8 shRNA for 48 hours, then treated with adriamycin (ADR) and vincristine (VCR) for an additional 24 hours. Cell viability was analyzed by MTT. (<b>C and D</b>) HL60/ADR and K562/A02 cells were transfected with control shRNA or S100A8 shRNA for 48 hours, treated with ADR (12.5 µg/mL), VCR (12.5 µg/mL) for additional 24 hours. Apoptosis was analyzed by measuring positive percentage of Annexin V cells via flow cytometry (<b>C</b>; n = 3;^*<i>P</i><0.05); cleaved PARP was analyzed by Western blotting (<b>D</b>). (<b>E</b>) HL60/ADR and K562/A02 cells were transfected with control shRNA or S100A8 shRNA for 48 hours, and then treated with ADR (12.5 µg/mL), VCR (12.5 µg/mL) for additional 24 hours with or without ZVAD-FMK (20 µmol/L). Activation of caspase-3 was analyzed (n = 3;^*<i>P</i><0.05). (<b>F</b>) HL60/ADR cells were transfected with control shRNA or S100A8 shRNA (from Gene Pharma, China) for 48 hours and then treated with ADR (12.5 µg/mL), VCR (12.5 µg/mL) for 24 hours. S100A8 protein was determined by Western blot; Cell viability was analyzed by MTT; apoptosis was analyzed by flow cytometry (n = 3;^*<i>P</i><0.05).</p

Change in heart rates of rats 2 hr after administration of VB.

<p>After being administered equivalent volumes of distilled water, rats in the NC group did not exhibit any changes in heart rate. After being administered VB, rats in groups LT-2, MT-2, and HT-2 exhibited significant decreases in heart rate (<i>p</i><0.01).</p

Score plots for OPLS-DA analysis of ¹H NMR data for serum, myocardial extracts and liver extracts of NC and VB treated groups.

<p>Two independent analyses were performed to study the time (NC, LT-2, LT-6 and LT-12) (a, b and c) and dose (NC, LT-2, MT-2 and HT-2) (d, e and f) effects of VB-induced toxicity. (a, d): score plots for serum; (b, e): score plots for myocardial extracts; (c, f): score plots for liver extracts. Ellipses around sample points stood for the 75% confidence.</p

Fold change plots color-coded with <i>p</i>-values adjusted by Benjamini-Hochberg method.

<p>Fold change plots indicating significance of altered metabolites in serum (a-e), myocardial extracts (f-j) and liver extracts (k-o) of NC rats vs. LT-12 rats (a, f and k), NC rats vs. LT-6 rats (b, g and l), NC rats vs. LT-2 rats (c, h and m), NC rats vs. MT-2 rats (d, i and n) and NC rats vs. HT-2 rats (e, j and o) after VB treatment. The blue and red dashed lines represented variations of 20% and 100%, respectively. Metabolites abbreviation: 3-HB: 3-Hydroxybutyrate; Ace: acetate; Ala: alanine; Arg: arginine; Bet: betaine; Cr: creatine; Cho: choline; Cit: citrate; DMK: acetone; Fum: fumarate; Glc: glucose; Gle: glycerol; Gln: glutamine; Glu: glutamate; Gly: glycine; GSH: glutathione; Hyp: hypoxanthine; Ino: inosine; Lac: Lactate; Lys: lysine; LDL/VLDL: low-density-lipoproteins/ very-low-density lipoproteins; Leu/Ile: Leucine/Isoleucine; MeOH: methanol; NAGP: N-Acetyl Glycoproteins; Nia: niacinamide; Phe: phenylalanine; Suc: succinate; Tyr: tyrosine; Tau: taurine; Val: valine; Uri: Uridine; Xan: xanthine.</p