Induction of central signalling pathways and select functional effects in human platelets by β‐boswellic acid

Abstract

We have recently shown that in polymorphonuclear leukocytes, 11‐keto boswellic acids (KBAs) induce Ca2+ mobilisation and activation of mitogen‐activated protein kinases (MAPK). Here we addressed the effects of BAs on central signalling pathways in human platelets and on various platelet functions. We found that β‐BA (10 μM), the 11‐methylene analogue of KBA, caused a pronounced mobilisation of Ca2+ from internal stores and induced the phosphorylation of p38 MAPK, extracellular signal‐regulated kinase (ERK)2, and Akt. These effects of β‐BA were concentration dependent, and the magnitude of the responses was comparable to those obtained after platelet stimulation with thrombin or collagen. Based on inhibitor studies, β‐BA triggers Ca2+ mobilisation via the phospholipase (PL)C/inositol‐1,4,5‐trisphosphate pathway, and involves Src family kinase signalling. Investigation of platelet functions revealed that β‐BA (10 μM) strongly stimulates the platelet‐induced generation of thrombin in an ex‐vivo in‐vitro model, the liberation of arachidonic acid (AA), and induces platelet aggregation in a Ca2+‐dependent manner. In contrast to β‐BA, the 11‐keto‐BAs (KBA or AKBA) evoke only moderate Ca2+ mobilisation and activate p38 MAPK, but fail to induce phosphorylation of ERK2 or Akt, and do not cause aggregation or significant generation of thrombin. In summary, β‐BA potently induces Ca2+ mobilisation as well as the activation of pivotal protein kinases, and elicits functional platelet responses such as thrombin generation, liberation of AA, and aggregation. British Journal of Pharmacology (2005) 146, 514–524. doi:

Boswellic Acids Stimulate Arachidonic Acid Release and 12-Lipoxygenase Activity in Human Platelets Independent of Ca2+and Differentially Interact with Platelet-Type 12-Lipoxygenase

“…Platelets were freshly isolated from human venous blood of healthy adult donors (St. Markus Hospital, Frankfurt, Germany) as described previously (Poeckel et al, 2005). Washed platelets were finally resuspended in PBS, pH 7.4, and 1 mg/ml glucose (PG buffer) or in PBS, pH 7.4, and 1 mg/ml glucose plus 1 mM CaCl 2 .…”

“…The Sepharose beads were washed three times with binding buffer (HEPES, pH 7.4, 200 mM NaCl, and 1 mM EDTA), and precipitated proteins were finally separated and denatured by the addition of 2ϫ SDS-polyacrylamide gel electrophoresis (PAGE) sample loading buffer [SDS-b; 20 mM Tris/HCl, pH 8, 2 mM EDTA, 5% SDS (w/v), and 10% ␤-mercaptoethanol]. After boiling (95°C, 6 min), Sepharose beads were removed by centrifugation and proteins in the supernatant were analyzed by SDS-PAGE as described previously (Poeckel et al, 2005). Proteins were visualized by Western blotting (Poeckel et al, 2005) or Coomassie staining, respectively.…”

“…Thus, stimulating properties (Safayhi et al, 2000;Altmann et al, 2002Altmann et al, , 2004Poeckel et al, 2005) and inhibitory effects (Safayhi et al, 1992(Safayhi et al, , 1995Werz et al, 1998;Poeckel et al, 2006) of boswellic acids have been reported for these functions, depending on the cell type and the respective experimental settings. For example, for inhibition of 5-lipoxygenase by AKBA, IC 50 values in the range of 1.5 M (Safayhi et al, 1995) up to 50 M (Werz et al, 1997(Werz et al, , 1998 were determined, but also 5-lipoxygenase stimulatory effects in this concentration range were described previously (Safayhi et al, 2000;Altmann et al, 2004).…”

“…We observed recently that boswellic acids are capable of elevating the release of arachidonic acid in human isolated polymorphonuclear leukocytes (PMNL) (Altmann et al, 2004) and platelets (Poeckel et al, 2005). Platelets do not express 5-lipoxygenase but contain the closely related p12-LO that converts arachidonic acid to 12-hydro(pero)xyeicosatetraenoic acid [12-H(P)ETE] (Yoshimoto and Takahashi, 2002).…”

Inhibition of 5-Lipoxygenase Product Synthesis by Natural Compounds of Plant Origin

“…Although 5-LO seemingly possesses a specific PT-binding site, and BAs (i. e., AKBA 89) as well as 3-oxo-TA 90 directly inhibit 5-LO catalysis, it should be noted that PTs affect many signal transduction pathways (e. g., MAPK, Akt, Ca 2+ ) and effectors (i. e., reactive oxygen species) [157], [168], [169]. Hence, besides direct interference with 5-LO, these multiple (intra-)cellular targets and routes may be affected by PT that eventually governs inhibition of 5-LO product synthesis.…”