Activation of protein kinase C by phorbol ester increases red blood cell scramblase activity and external phosphatidylserine

Barber, Latorya A.; Palascak, Mary B.; Qi, Xiaoyang; Joiner, Clinton H.; Franco, Robert S.

doi:10.1111/ejh.12506

Cited by 5 publications

(4 citation statements)

References 26 publications

(33 reference statements)

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“…Chelerythrine chloride and calphostin C have been used to inhibit the PKCα. Chelerythrine chloride is an inhibitor of the kinase domain of PKCα, whereas calphostin C blocks the PMA-and diacylglycerol (DAG)-binding site of the PKCα [41,42,44,45].…”

Section: Inhibition Of the Pkcαmentioning

confidence: 99%

Novel Insights in the Regulation of Phosphatidylserine Exposure in Human Red Blood Cells

Wesseling

Wagner-Britz²,

Nguyen³

et al. 2016

Cell Physiol Biochem

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Background/Aims: In previous publications we were able to demonstrate the exposure of phosphatidylserine (PS) in the outer membrane leaflet after activation of red blood cells (RBCs) by lysophosphatidic acid (LPA), phorbol-12 myristate-13acetate (PMA), or 4-bromo-A23187 (A23187). It has been concluded that three different mechanisms are responsible for the PS exposure in human RBCs: (i) Ca²⁺-stimulated scramblase activation (and flippase inhibition) by A23187, LPA, and PMA; (ii) PKCα activation by LPA and PMA; and (iii) enhanced lipid flip flop caused by LPA. Further studies aimed to elucidate interconnections between the increased Ca²⁺ content, scramblase- and PKCα-activation. In addition, the role of the Ca²⁺-activated K⁺ channel (Gardos channel) activity in the process of PS exposure needs to be investigated. Methods: The intracellular Ca²⁺ content and the PS exposure of RBCs have been investigated after treatment with LPA (2.5 µM), PMA (6 µM), or A23187 (2 µM). Fluo-4 and annexin V-FITC has been used to detect intracellular Ca²⁺ content and PS exposure, respectively. Both parameters (Ca²⁺ content, PS exposure) were studied using flow cytometry. Inhibitors of the scramblase, the PKCα, and the Gardos channel have been applied. Results: The percentage of RBCs showing PS exposure after activation with LPA, PMA, or A23187 is significantly reduced after inhibition of the scramblase using the specific inhibitor R5421 as well as after the inhibition of the PKCα using chelerythrine chloride or calphostin C. The inhibitory effect is more pronounced when the scramblase and the PKCα are inhibited simultaneously. Additionally, the inhibition of the Gardos channel using charybdotoxin resulted in a significant reduction of the percentage of RBCs showing PS exposure under all conditions measured. Similar results were obtained when the Gardos channel activity was suppressed by increased extracellular K⁺ content. Conclusion: PS exposure is mediated by the Ca²⁺-dependent scramblase but also by PKCα activated by LPA and PMA in a Ca²⁺-dependent and a Ca²⁺-independent manner. Furthermore, we hypothesize that a hyperpolarisation of RBCs caused by the opening of the Gardos channel is essential for the scramblase activity as well as for a fraction of the LPA-induced Ca²⁺ entry.

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Section: Inhibition Of the Pkcαmentioning

confidence: 99%

Novel Insights in the Regulation of Phosphatidylserine Exposure in Human Red Blood Cells

Wesseling

Wagner-Britz²,

Nguyen³

et al. 2016

Cell Physiol Biochem

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“…Supporting this, it has been reported that PKC activation can regulate scramblase activity and increase PS exposure. 30,31 In addition, PKC activation can alter the phosphorylation of cytoskeletal proteins, protein 4.1, 4.9 and adducin, which are involved in the regulation of membrane properties critical for functions and morphological changes of RBCs. 24,32 Paclitaxel is generally given through intravenous infusion at 135 to 175 mg/m 2 body surface area for 3 hours for therapeutic use, 33 which could be converted into 3.6 to 4.7 mg/kg in human and 22.5 to 29.1 mg/kg in rats.…”

Section: Discussionmentioning

confidence: 99%

Pro-Coagulant and Pro-Thrombotic Effects of Paclitaxel Mediated by Red Blood Cells

et al. 2018

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Background Paclitaxel is one of the most widely used anti-cancer drugs, but numerous case reports of thrombotic events in the cancer patients using paclitaxel raise concern over its pro-thrombotic risk. Materials and Methods We investigated whether paclitaxel can elicit pro-thrombotic properties in red blood cells (RBCs) through phosphatidylserine (PS) exposure and microvesicle (MV) release. Results In freshly isolated human RBCs, paclitaxel induced thrombin generation through PS exposure and MV release, whereas either coagulation factors or platelets were unaffected. Paclitaxel-induced PS exposure in RBC was mediated by scramblase activation which was induced by calcium-independent protein kinase C (PKC)ζ activation. Paclitaxel also increased RBC-endothelial cell adhesion and RBC aggregate formation which can also contribute to thrombosis. Indeed, intravenous administration of paclitaxel to rats induced PS exposure and PKCζ activation in RBCs in vivo which ultimately promoted venous thrombus formation. Conclusion These results demonstrated that paclitaxel may elicit pro-thrombotic properties in RBCs through PS exposure and MV release, which can ultimately promote thrombus formation.

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“…Upon PMA treatment, PKC translocates to the RBC membrane, becomes activated, and phosphorylates a variety of RBC membrane proteins (Barber et al, 2015; Faquin et al, 1986; Manno et al, 2005; Palfrey & Waseem, 1985; Wagner‐Britz et al, 2013) (Figure 2). One of the major target is Protein 4.1 phosphorylated at serine (312 and 331) residues (Gauthier et al, 2011; Ling et al, 1988; Manno et al, 2005).…”

Section: Intracellular Signal Transduction Mechanismsmentioning

confidence: 99%

“…Ca 2+ entry(Wagner-Britz et al, 2013).Upon PMA treatment, PKC translocates to the RBC membrane, becomes activated, and phosphorylates a variety of RBC membrane proteins(Barber et al, 2015;Faquin et al, 1986;Manno et al, 2005;Palfrey & Waseem, 1985;Wagner-Britz et al, 2013) (Figure2). One of the major target is Protein 4.1 phosphorylated at serine (312 and 331) F I G U R E 2 A schematic illustration for PKC, NO synthesis, and cyclic GMP (cGMP) signaling under shear stress.…”

mentioning

confidence: 99%

Signaling mechanisms in red blood cells: A view through the protein phosphorylation and deformability

Cilek

Uğurel

Goksel

et al. 2023

Journal Cellular Physiology

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Intracellular signaling mechanisms in red blood cells (RBCs) involve various protein kinases and phosphatases and enable rapid adaptive responses to hypoxia, metabolic requirements, oxidative stress, or shear stress by regulating the physiological properties of the cell. Protein phosphorylation is a ubiquitous mechanism for intracellular signal transduction, volume regulation, and cytoskeletal organization in RBCs. Spectrin‐based cytoskeleton connects integral membrane proteins, band 3 and glycophorin C to junctional proteins, ankyrin and Protein 4.1. Phosphorylation leads to a conformational change in the protein structure, weakening the interactions between proteins in the cytoskeletal network that confers a more flexible nature for the RBC membrane. The structural organization of the membrane and the cytoskeleton determines RBC deformability that allows cells to change their ability to deform under shear stress to pass through narrow capillaries. The shear stress sensing mechanisms and oxygenation‐deoxygenation transitions regulate cell volume and mechanical properties of the membrane through the activation of ion transporters and specific phosphorylation events mediated by signal transduction. In this review, we summarize the roles of Protein kinase C, cAMP‐Protein kinase A, cGMP‐nitric oxide, RhoGTPase, and MAP/ERK pathways in the modulation of RBC deformability in both healthy and disease states. We emphasize that targeting signaling elements may be a therapeutic strategy for the treatment of hemoglobinopathies or channelopathies. We expect the present review will provide additional insights into RBC responses to shear stress and hypoxia via signaling mechanisms and shed light on the current and novel treatment options for pathophysiological conditions.

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Activation of protein kinase C by phorbol ester increases red blood cell scramblase activity and external phosphatidylserine

Cited by 5 publications

References 26 publications

Novel Insights in the Regulation of Phosphatidylserine Exposure in Human Red Blood Cells

Novel Insights in the Regulation of Phosphatidylserine Exposure in Human Red Blood Cells

Pro-Coagulant and Pro-Thrombotic Effects of Paclitaxel Mediated by Red Blood Cells

Signaling mechanisms in red blood cells: A view through the protein phosphorylation and deformability

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