L-arginine supplementation and thromboxane synthase inhibition increases cerebral blood flow in experimental cerebral malaria

Cerebral malaria pathogenesis involves vascular dysfunction with low nitric oxide (NO) bioavailability, vasoconstriction and impaired vasodilation, leading to ischemia, tissue hypoxia and ultimately death. Cerebral blood flow (CBF) involves NO and other pathways, including arachidonic acid (AA)-derived metabolites. Here we show that mice with experimental cerebral malaria (ECM) by P. berghei ANKA showed marked decreases in CBF (as assessed by laser speckle contrast imaging - LSCI) and that administration of L-arginine supplementation (50 mg/kg) and/or of the thromboxane synthase inhibitor Ozagrel (100 mg/kg) induced immediate increases in CBF. L-arginine in combination with artesunate (32 mg/kg) induced immediate reversal of brain ischemia in the short-term (1 hour), but the effect subsided after 3 and 6 hours. Neither L-arginine nor Ozagrel reversed blood brain barrier breakdown. Mice with ECM showed brain levels of selected AA-derived metabolites with a vasoconstrictor profile, with increased levels of 8-isoprostanes, 20-HETE and 14,15-DHET, whereas mice infected with a non-ECM-inducing strain of P. berghei (NK65) showed a vasodilator profile, with normal levels of 20-HETE and 14,15-DHET and increased levels of PGE2. L-arginine is capable of partially reversing cerebral ischemia and AA metabolites may play a role in the cerebrovascular dysfunction in ECM.

miR-34b-3p May Promote Antiplatelet Efficiency of Aspirin by Inhibiting Thromboxane Synthase Expression

Aspirin has been widely used for the prevention of cardiovascular diseases, but its antiplatelet efficiency varies between individuals. The present study aimed to evaluate response to aspirin based on gene profiles as well as potential regulating pathways using human blood samples and cell lines. Platelet function in patients 50 years or older with coronary artery disease on 100 mg/day aspirin was measured by light transmission aggregometry (LTA) of arachidonic acid (AA)-induced platelet aggregation. The expression of eight candidate genes—PTGS1/COX1, PLA2G4A, PLA2G6, PLA2G7, TBXAS1, TBXA2R, PTGIR, and ITGA2B—and the ingredients involved in AA metabolism were analyzed. Our data showed that the expressions of thromboxane A synthase 1 (TBXAS1), thromboxane synthase (TXS), and thromboxane B2 (TXB2) were increased in the upper quartile of platelet aggregation (LTA-AA_Q4) group compared with the lower quartile of platelet aggregation (LTA-AA_Q1) group. Our bioinformatics analysis suggested that TBXAS1 was targeted by miR-34b-3p via binding to its 3′-UTR, which was subsequently verified experimentally. Although overexpression of miR-34b-3p exhibited no apparent effect on cell proliferation, inhibition of miR-34b-3p promoted megakaryocyte viability. Our data demonstrated that the expression of TBXAS1 was higher in the aspirin hyporesponsiveness group than that in the hyperresponsiveness group, suggesting that high expression of TBXAS1 may be associated with aspirin hyporesponsiveness. miR-34b-3p may regulate the platelet and aspirin response by suppressing TBXAS1 expression and megakaryocyte proliferation.

Direct Molecular Fishing of New Protein Partners for Human Thromboxane Synthase

Thromboxane synthase (TBXAS1) catalyzes the isomerization reaction of prostaglandin H2 producing thromboxane A2, the autocrine and paracrine factor in many cell types. A high activity and metastability by these arachidonic acid derivatives suggests the existence of supramolecular structures that are involved in the regulation of the biosynthesis and directed translocation of thromboxane to the receptor. The objective of this study was to identify TBXAS1 protein partners from human liver tissue lysate using a complex approach based on the direct molecular fishing technique, LC-MS/MS protein identification, and protein-protein interaction validation by surface plasmon resonance (SPR). As a result, 12 potential TBXAS1 protein partners were identified, including the components regulating cytoskeleton organization (BBIP1 and ANKMY1), components of the coagulation cascade of human blood (SERPINA1, SERPINA3, APOH, FGA, and FN1), and the enzyme involved in the metabolism of xenobiotics and endogenous bioregulators (CYP2E1). SPR validation on the Biacore 3000 biosensor confirmed the effectiveness of the interaction between CYP2E1 (the enzyme that converts prostaglandin H2 to 12-HHT/thromboxane A2 proantagonist) and TBXAS1 (Kd = (4.3 0.4) 10-7 M). Importantly, the TBXAS1CYP2E1 complex formation increases fivefold in the presence of isatin (indole-2,3-dione, a low-molecular nonpeptide endogenous bioregulator, a product of CYP2E1). These results suggest that the interaction between these hemoproteins is important in the regulation of the biosynthesis of eicosanoids.

Chronic Pancytopenia and Increased Bone Density Due to TBXAS1 Deficiency

Non-dysmorphic 32-year old Caucasian female developed malaise, fluctuating body aches and progressive transfusion-dependent pancytopenia with peripheral blood morphology consistent with myelodysplastic syndrome (MDS). Reportedly, she had an episode of "low blood counts" of unclear etiology 23 years prior. Multiple attempts at bone marrow biopsy were unsuccessful due to technical difficulties with penetrating the bone, necessitating surgical biopsy by an orthopedic surgeon. Bone densitometry revealed increased bone density with Z-score of 4.5, consistent with mild thickening of long bone diaphysis noted via x-ray. Strikingly, the bone marrow biopsy revealed haphazard ossification, immature cartilage formation and thickened trabeculae replacing hematopoiesis within the marrow cavity. Next-generation sequencing revealed two novel biallelic mutations within the thromboxane synthase gene TBXAS1 (c.266T>C; c.989T>C). Both mutations affected conserved amino acid residues and were predicted to be disruptive to the protein function by two independent bioinformatics algorithms (Provean and PolyPhen-2). Thromboxane cascade regulates pathways of inflammation, coagulation and osteoclast activation, suggesting a mechanistic explanation of how thromboxane synthase malfunction causes abnormal bone remodeling and bone marrow failure. TBXAS1 disruption had been described in a rare autosomal recessive syndrome Ghosal hematodiaphyseal dysplasia (GHDD) associated with increased bone density and bone marrow failure. GHDD due to TBXAS1 mutations was genetically confirmed only in four families of Northern African and Middle Eastern descent (Nat Genet 2008; 40: 284-286). To our knowledge, this is the first genetically validated case of TBXAS1-/- Ghosal syndrome in the Caucasian population. Consistent with the well-established role of thromboxane in platelet function, our patient's platelets displayed impaired aggregation in response to arachidonic acid and structural abnormalities by transmission electron microscopy, revealing a functional platelet defect. Steroid therapy rendered the patient transfusion-independent and alleviated chronic pain and inflammation. A long-term follow-up will be needed to determine whether steroid treatment will result in stable improvement in hematopoiesis and decreased bone density, similar to other GHDD patients. This case illustrates how establishing the diagnosis of an underlying rare inherited bone marrow failure syndrome allows personalized, evidence-based treatment with lower risk of morbidity compared to cytotoxic chemotherapy and hematopoietic stem cell transplantation. Figure Figure. Disclosures No relevant conflicts of interest to declare.