Regulation of blood platelet function by the AGC family of protein kinases

Upon vascular injury, platelets aggregate at the site of blood vessel injury to form a hemostatic plug maintaining the physiological integrity of the vascular system. Platelets respond to a variety of extracellular stimuli to undergo a rapid aggregation response, releasing active granule contents and leading to a rapidly growing thrombus. During the adhesion, activation, and aggregation of platelets at an injured site, the endothelium responds by limiting the size and growth of the hemostatic plug or thrombus, or even reversing platelet reactivity. These responses are defined as endothelial thromboregulation. There are three primary (and functionally independent) pathways during the early stages of thromboregulation by which the endothelium controls platelet reactivity (1) nitric oxide (NO); (2) prostacyclin (PGI₂ ); and (3) the ectonucleotidase CD39. NO and PGI2 stimulate signalling cascades that result in the activation of the AGC family of Ser/Thr protein kinases (PKA, PKG and PKC). Once activated these kinase blunt platelet function through the phosphorylation of signalling proteins requested for activation. In this study, the role of AGC family kinases and their signaling cascades in regulating platelet function was assessed. The experimental data produced during this study demonstrate new insights in to the regulation of these kinases in platelets. More specifically it was found that1. Peroxynitrite, a derivative of NO, regulated platelet function and particularly cytoskeletal rearrangement through PKC-dependent phosphorylation of VASPSer²³⁹⁄¹⁵⁷2. NO-mediated signalling in platelets had a requirement for PKC.3. Multiple forms of PKA are present in platelets, which are differentially localised.4. The potential regulation of platelet function by PKA is mediated through Akinase anchoring proteins.5. Lipid rafts may play an important role in platelet regulation by NO and PKG.In summary, this studies present insights of the factors regulating AGC kinases in blood platelets

Targeting cAMP Signalling to Combat Cardiovascular Diseases Platelet myosin light chain phosphatase: keeping it together

Abstract MLCP (myosin light chain phosphatase) regulates platelet function through its ability to control myosin IIa phosphorylation. Recent evidence suggests that MLCP is a de facto target for signalling events stimulated by cAMP. In the present mini-review, we discuss the mechanisms by which cAMP signalling maintains MLCP in an active state to control platelet contractile machinery

Author Correction: Effect of induced hypoglycemia on inflammation and oxidative stress in type 2 diabetes and control subjects (Scientific Reports, (2020), 10, 1, (4750), 10.1038/s41598-020-61531-z)

© 2020, The Author(s). The original version of this Article contained a typographical error in the spelling of the author Johannes Graumann, which was incorrectly given as Johannes Grauman. This has now been corrected in the PDF and HTML versions of the Article, and in the accompanying Supplementary Information file

Effect of induced hypoglycemia on inflammation and oxidative stress in type 2 diabetes and control subjects

Intensive diabetes control has been associated with increased mortality in type 2 diabetes (T2DM); this has been suggested to be due to increased hypoglycemia. We measured hypoglycemia-induced changes in endothelial parameters, oxidative stress markers and inflammation at baseline and after a 24-hour period in type 2 diabetic (T2DM) subjects versus age-matched controls. Case-control study: 10 T2DM and 8 control subjects. Blood glucose was reduced from 5 (90 mg/dl) to hypoglycemic levels of 2.8 mmol/L (50 mg/dl) for 1 hour by incremental hyperinsulinemic clamps using baseline and 24 hour samples. Measures of endothelial parameters, oxidative stress and inflammation at baseline and at 24-hours post hypoglycemia were performed: proteomic (Somalogic) analysis for inflammatory markers complemented by C-reactive protein (hsCRP) measurement, and proteomic markers and urinary isoprostanes for oxidative measures, together with endothelial function. Between baseline and 24 -hours after hypoglycemia, 15 of 140 inflammatory proteins differed in T2DM whilst only 1 of 140 differed in controls; all returned to baseline at 24-hours. However, elevated hsCRP levels were seen at 24-hours in T2DM (2.4 mg/L (1.2–5.4) vs. 3.9 mg/L (1.8–6.1), Baseline vs 24-hours, P < 0.05). In patients with T2DM, between baseline and 24-hour after hypoglycemia, only one of 15 oxidative stress proteins differed and this was not seen in controls. An increase (P = 0.016) from baseline (73.4 ng/mL) to 24 hours after hypoglycemia (91.7 ng/mL) was seen for urinary isoprostanes. Hypoglycemia resulted in inflammatory and oxidative stress markers being elevated in T2DM subjects but not controls 24-hours after the event

Acute hypertriglyceridemia induces platelet hyperactivity that is not attenuated by insulin in polycystic ovary syndrome.

Atherothrombosis is associated with platelet hyperactivity. Hypertriglyceridemia and insulin resistance (IR) are features of polycystic ovary syndrome (PCOS). The effect of induced hypertriglyceridemia on IR and platelet function was examined in young women with PCOS. Following overnight fasting, 13 PCOS and 12 healthy women were infused with saline or 20% intralipid for 5 hours on separate days. Insulin sensitivity was measured using a hyperinsulinemic euglycaemic clamp in the final 2 hours of each infusion. Platelet responses to adenosine diphosphate (ADP) and prostacyclin (PGI2) were measured by flow cytometric analysis of platelet fibrinogen binding and P-selectin expression using whole blood taken during each infusion (at 2 hours) and at the end of each clamp. Lipid infusion increased triglycerides and reduced insulin sensitivity in both controls (median, interquartile range ) (5.25 [3.3, 6.48] versus 2.60 [0.88, 3.88] mg kg(-1) min(-1), P<0.001) and PCOS (3.15 [2.94, 3.85] versus 1.06 [0.72, 1.43] mg kg(-1) min(-1), P<0.001). Platelet activation by ADP was enhanced and ability to suppress platelet activation by PGI2 diminished during lipid infusion in both groups when compared to saline. Importantly, insulin infusion decreased lipid-induced platelet hyperactivity by decreasing their response to 1 μmol/L ADP (78.7% [67.9, 82.3] versus 62.8% [51.8, 73.3], P=0.02) and increasing sensitivity to 0.01 μmol/L PGI2 (67.6% [39.5, 83.8] versus 40.9% [23.8, 60.9], P=0.01) in controls, but not in PCOS. Acute hypertriglyceridemia induced IR, and increased platelet activation in both groups that was not reversed by insulin in PCOS subjects compared to controls. This suggests that platelet hyperactivity induced by acute hypertriglyceridemia and IR could contribute athero-thrombotic risk. www.isrctn.org. Unique Identifier: ISRCTN42448814

Regulation of blood platelet function by the AGC family of protein kinases

Upon vascular injury, platelets aggregate at the site of blood vessel injury to form a hemostatic plug maintaining the physiological integrity of the vascular system. Platelets respond to a variety of extracellular stimuli to undergo a rapid aggregation response, releasing active granule contents and leading to a rapidly growing thrombus. During the adhesion, activation, and aggregation of platelets at an injured site, the endothelium responds by limiting the size and growth of the hemostatic plug or thrombus, or even reversing platelet reactivity. These responses are defined as endothelial thromboregulation. There are three primary (and functionally independent) pathways during the early stages of thromboregulation by which the endothelium controls platelet reactivity (1) nitric oxide (NO); (2) prostacyclin (PGI₂ ); and (3) the ectonucleotidase CD39. NO and PGI2 stimulate signalling cascades that result in the activation of the AGC family of Ser/Thr protein kinases (PKA, PKG and PKC). Once activated these kinase blunt platelet function through the phosphorylation of signalling proteins requested for activation. In this study, the role of AGC family kinases and their signaling cascades in regulating platelet function was assessed. The experimental data produced during this study demonstrate new insights in to the regulation of these kinases in platelets. More specifically it was found that 1. Peroxynitrite, a derivative of NO, regulated platelet function and particularly cytoskeletal rearrangement through PKC-dependent phosphorylation of VASPSer²³⁹⁄¹⁵⁷ 2. NO-mediated signalling in platelets had a requirement for PKC. 3. Multiple forms of PKA are present in platelets, which are differentially localised. 4. The potential regulation of platelet function by PKA is mediated through Akinase anchoring proteins. 5. Lipid rafts may play an important role in platelet regulation by NO and PKG. In summary, this studies present insights of the factors regulating AGC kinases in blood platelets

Thrombospondin-1 induces platelet activation through CD36-dependent inhibition of the cAMP/protein kinase A signaling cascade

Cyclic adenosine monophosphate (cAMP)-dependent signaling modulates platelet function at sites of vascular injury. Here we show that thrombospondin-1 (TSP-1) prevents cAMP/protein kinase A (PKA) signaling through a CD36-dependent mechanism. Prostaglandin E 1 (PGE 1 ) induced a robust inhibition of both platelet aggregation and platelet arrest under physiologic conditions of flow. Exogenous TSP-1 reduced significantly PGE 1 -mediated inhibition of both platelet aggregation and platelet arrest. TSP-1 prevented PGE 1 -stimulated cAMP accrual and phosphorylation of PKA substrates, through a mechanism requiring phosphodiesterase3A. TSP-1 also inhibited VASP phosphorylation stimulated by the nonhydrolyzable cAMP analog, 8-bromocAMP, indicating that it may regulate cAMPmediated activation of PKA. The inhibitory effect of TSP-1 on cAMP signaling could be reproduced with a peptide possessing a CD36 binding sequence of TSP-1, while the effects of TSP-1 were prevented by a CD36 blocking antibody. TSP-1 and the CD36 binding peptide induced phosphorylation of Src kinases, p38 and JNK. More-over, inhibition of Src kinases blocked TSP-1-mediated regulation of cAMP concentrations and the phosphorylation of VASP, indicating that TSP-1 modulated the cAMP/PKA signaling events through a tyrosine kinase-dependent pathway downstream of CD36. These data reveal a new role for TSP-1 in promoting platelet aggregation through modulation of the cAMP-PKA signaling pathway. © 2010 by The American Society of Hematology

Globular adiponectin increases cGMP formation in blood platelets independently of nitric oxide

Background: Platelet-derived nitric oxide (NO) has been shown to play conflicting roles in platelet function, although it is accepted that NO mediates its actions through soluble guanylyl cyclase (sGC). This confusion concerning the roles of platelet NO may have arisen because of an uncharacterized mechanism for activation of sGC. Objectives: To examine the ability of the novel platelet agonist globular adiponectin (gAd) to stimulate the NO-independent cGMP-protein kinase G (PKG) signaling cascade. Methods: We used three independent markers of NO signaling, [ 3 H]l-citrulline production, cGMP accrual, and immunoblotting of vasodilator-stimulated phosphoprotein (VASP), to examine the NO signaling cascade in response to gAd. Results: gAd increased platelet cGMP formation, resulting in a dose- and time-dependent increase in phospho-VASP 157/239 . Phosphorylation of VASP in response to gAd was mediated by both protein kinase A and PKG. Importantly, cGMP formation occurred in the absence of NO synthase (NOS) activation and in the presence of NOS inhibitors. Indeed, inhibition of the NOS signaling cascade had no influence on gAd-mediated platelet aggregation. Exploration of the mechanism demonstrated that NO-independent cGMP formation, phosphorylation of VASP and association of sGCα 1 with heat shock protein-90 induced by gAd were blocked under conditions that inhibited Src kinases, implying a tyrosine kinase-dependent mechanism. Indeed, sGCα1 was reversibly tyrosine phosphorylated in response to gAd, collagen, and collagen-related peptide, an effect that required Src kinases and downstream Ca 2+ mobilization. Conclusions: These data demonstrate activation of the platelet cGMP signaling cascade by a novel tyrosine kinase-dependent mechanism in the absence of NO. © 2008 International Society on Thrombosis and Haemostasis

Regulation of blood platelet function by the AGC family of protein kinases

Upon vascular injury, platelets aggregate at the site of blood vessel injury to form a hemostatic plug maintaining the physiological integrity of the vascular system. Platelets respond to a variety of extracellular stimuli to undergo a rapid aggregation response, releasing active granule contents and leading to a rapidly growing thrombus. During the adhesion, activation, and aggregation of platelets at an injured site, the endothelium responds by limiting the size and growth of the hemostatic plug or thrombus, or even reversing platelet reactivity. These responses are defined as endothelial thromboregulation. There are three primary (and functionally independent) pathways during the early stages of thromboregulation by which the endothelium controls platelet reactivity (1) nitric oxide (NO); (2) prostacyclin (PGI2 ); and (3) the ectonucleotidase CD39. NO and PGI2 stimulate signalling cascades that result in the activation of the AGC family of Ser/Thr protein kinases (PKA, PKG and PKC). Once activated these kinase blunt platelet function through the phosphorylation of signalling proteins requested for activation. In this study, the role of AGC family kinases and their signaling cascades in regulating platelet function was assessed. The experimental data produced during this study demonstrate new insights in to the regulation of these kinases in platelets. More specifically it was found that 1. Peroxynitrite, a derivative of NO, regulated platelet function and particularly cytoskeletal rearrangement through PKC-dependent phosphorylation of VASPSer239/157 2. NO-mediated signalling in platelets had a requirement for PKC. 3. Multiple forms of PKA are present in platelets, which are differentially localised. 4. The potential regulation of platelet function by PKA is mediated through Akinase anchoring proteins. 5. Lipid rafts may play an important role in platelet regulation by NO and PKG. In summary, this studies present insights of the factors regulating AGC kinases in blood platelets.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Regulation of blood platelet function by the AGC family of protein kinases

Upon vascular injury, platelets aggregate at the site of blood vessel injury to form a hemostatic plug maintaining the physiological integrity of the vascular system. Platelets respond to a variety of extracellular stimuli to undergo a rapid aggregation response, releasing active granule contents and leading to a rapidly growing thrombus. During the adhesion, activation, and aggregation of platelets at an injured site, the endothelium responds by limiting the size and growth of the hemostatic plug or thrombus, or even reversing platelet reactivity. These responses are defined as endothelial thromboregulation. There are three primary (and functionally independent) pathways during the early stages of thromboregulation by which the endothelium controls platelet reactivity (1) nitric oxide (NO); (2) prostacyclin (PGI2 ); and (3) the ectonucleotidase CD39. NO and PGI2 stimulate signalling cascades that result in the activation of the AGC family of Ser/Thr protein kinases (PKA, PKG and PKC). Once activated these kinase blunt platelet function through the phosphorylation of signalling proteins requested for activation. In this study, the role of AGC family kinases and their signaling cascades in regulating platelet function was assessed. The experimental data produced during this study demonstrate new insights in to the regulation of these kinases in platelets. More specifically it was found that 1. Peroxynitrite, a derivative of NO, regulated platelet function and particularly cytoskeletal rearrangement through PKC-dependent phosphorylation of VASPSer239/157 2. NO-mediated signalling in platelets had a requirement for PKC. 3. Multiple forms of PKA are present in platelets, which are differentially localised. 4. The potential regulation of platelet function by PKA is mediated through Akinase anchoring proteins. 5. Lipid rafts may play an important role in platelet regulation by NO and PKG. In summary, this studies present insights of the factors regulating AGC kinases in blood platelets.EThOS - Electronic Theses Online ServiceGBUnited Kingdo