All-Trans Retinoic Acid Impairs Platelet Function and Thrombus Formation and Inhibits Protein Kinase CßI/δ Phosphorylation

Abstract

All-trans retinoic acid (ATRA) is widely used for induction of complete remission in patients with acute promyelocytic leukemia (APL). ATRA also regulates protein kinase C (PKC) activity. Therapeutic use of ATRA reportedly interferes with hemostatic function in APL patients, including effects on coagulation or other vascular cells, although effects of ATRA on platelets remain unclear. This study aims to investigate the effect of therapeutic-relevant doses of ATRA on platelet function. Human platelets were preincubated with ATRA (0–20 μM) for 1 hour at 37°C, followed by analysis of aggregation, granule secretion, receptor expression by flow cytometry, platelet spreading, or clot retraction. Additionally, ATRA (10 mg/kg) was injected intraperitoneally into mice and tail bleeding time and arterial thrombus formation were evaluated. ATRA inhibited platelet aggregation and adenosine triphosphate release induced by collagen (5 μg/mL) or thrombin (0.05 U/mL) in a dose-dependent manner without affecting P-selectin expression or surface levels of glycoprotein (GP) Ibα, GPVI, or αIIbβ3. ATRA-treated platelets demonstrated reduced spreading on immobilized fibrinogen or collagen and reduced thrombin-induced clot retraction together with reduced phosphorylation of Syk and PLCγ2. In addition, ATRA-treated mice displayed significantly impaired hemostasis and arterial thrombus formation in vivo. Further, in platelets stimulated with either collagen-related peptide or thrombin, ATRA selectively inhibited phosphorylation of PKCßI (Ser661) and PKCδ (Thr505), but not PKCα or PKCßII phosphorylation (Thr638/641). In conclusion, ATRA inhibits platelet function and thrombus formation, possibly involving direct or indirect inhibition of PKCßI/δ, indicating that ATRA might be beneficial for the treatment of thrombotic or cardiovascular diseases.

Chemical Modulators of Fibrinogen Production and Their Impact on Venous Thrombosis

“…RA reduced platelet activation in dietinduced atherosclerosis in rabbits, 44 and inhibited platelet aggregation, spreading, dense-granule secretion, and clot retraction in vitro. 45 While we cannot exclude a contribution of the increased number of thrombocytes, possible direct effects of RA include thrombocyte responses to injury, or even an increased production of other key coagulation factors through the same mechanism as behind increased fibrinogen production (mediated by RA and the retinoid X nuclear receptors 36 ), our data showed a trend toward increased thrombocyte binding after RA treatment that correlated with increased fibrinogen.…”

In Silico Analysis to Identify Molecular Targets for Chemicals of Concern: The Case Study of Flocoumafen, an Anticoagulant Pesticide

“…Interestingly, flocoumafen has been found to actively accumulate in the liver (Huckle et al 1988), where this pesticide is frequently found in wildlife (Alabau et al 2020). The extensive relationship among retinoic acid homeostasis, obesity, insulin resistance, and coagulation (Mody 2017; Luo et al 2019) clearly gives flocoumafen an important role as an environmental pollutant involved in metabolic syndrome—a systems biology finding that deserves special attention.…”

Matrine Impairs Platelet Function and Thrombosis and Inhibits ROS Production

“…Platelet aggregation was conducted in the presence of fibrinogen (0.5 mg/ml) as described previously (Luo et al, 2019;Wei G. et al, 2020). After matrine treatment, platelet aggregation induced by CRP (0.1 μg/ml) or thrombin (0.04 U/ml) was analyzed in a Lumi-Aggregometer Model 700 (Chrono-log Corporation, Havertown, PA, United States) at 37°C with stirring (1,000 rpm).…”

“…Platelet α-granule secretion was assessed via measuring P-selectin expression and integrin αIIbβ3 activation was evaluated via detecting the activation-dependent binding of PAC-1 to platelet αIIbβ3 by flow cytometry as described previously (Luo et al, 2019;Wei G. et al, 2020). Briefly, after matrine treatment, platelets were stimulated with CRP (0.1 or 2 μg/ml) or thrombin (0.04 or 0.5 U/ml) in the presence of PE-conjugated anti-P-selectin antibody or FITC-conjugated PAC-1 antibody followed by measuring P-selectin expression or PAC-1 binding by flow cytometry using FITC-conjugated mouse anti-human CD41a to set the platelet gate in the Forward Scatter and Side Scatter.…”

“…Thrombin-mediated clot retraction was initiated after addition of thrombin (1 U/ml) to washed platelets in the presence of 2 mM Ca 2+ and 0.5 mg/ml fibrinogen as described previously (Luo et al, 2019;Wei G. et al, 2020).…”