CPI-17 Contributes to Calcium-Independent ADP-Induced Platelet Shape Change through P2Y1-Gq-PKC Pathways.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3644-3644
Author(s):  
Todd M. Getz ◽  
Kamala Bhavaraju ◽  
Satya P. Kunapuli
Keyword(s):  
Platelet Activation ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Shape Change ◽  
Rho Kinase ◽  
Human Platelets ◽  
Inhibitory Protein ◽  
Pkc Inhibitors ◽  
Pkc Activation ◽  
Platelet Shape

Abstract The initial event in platelet activation is the reorganization of the cytoskeleton causing the platelets to change from a discoid to a spiculated spheroid shape. Platelet shape change is primarily regulated by the phosphorylation of myosin light chain kinase. We have shown that this process is mediated through both calcium-dependent and calcium-independent Rho kinase pathways. CPI-17, a Protein kinase C (PKC) phosphorylated inhibitory protein of myosin light chain phosphatase, has been shown to have a role in platelet shape change downstream of thrombin-induced platelet activation. CPI-17 is a 17 kDa protein expressed in human platelets shown to inhibit myosin light chain phosphotase activity via PKCs. In this study we examined the role of CPI-17 in ADP-induced shape change and phosphorylation of CPI-17, downstream of the Gq coupled, P2Y1, and the Gi coupled, P2Y12 receptors. CPI-17 phosphorylation occurred upon activation of platelets with 2MeSADP. This phosphorylation was abolished in the presence of the P2Y1 receptor antagonist, MRS-2179. These results indicated that Gq signaling is important for platelet shape change and phosphorylation of CPI-17. In the presence of the calcium chelator, BAPTA, platelets changed shape in response to 2MeSADP; CPI-17 phosphorylation, however, was unaffected by BAPTA treatment under these conditions. However, CPI-17 phosphorylation was inhibited in the presence of the pan PKC inhibitors. These results indicate that CPI-17 phosphorylation occurs downstream of PKC activation. In the presence of BAPTA, treatment with PKC inhibitors decreased platelet shape change possibly due to reduced CPI-17 phosphorylation. The shape change caused by p160ROCK downstream of G12/13 pathways was unaffected by pan PKC inhibitors, but abolished by p160ROCK inhibitors H1152 or Y27632. Platelets incubated with BAPTA, pan PKC inhibitors, and p160ROCK inhibitor H1152, abolished ADP-induced platelet shape change and CPI-17 phosphorylation. In conclusion, ADP-induced platelet shape change occurs through a Gq-mediated, calcium-independent signaling pathway regulated by CPI-17 phosphorylation via PKC activation.

scholarly journals Trypsin causes platelet activation independently of known protease-activated receptors

2013 ◽  
Vol 110 (12) ◽  
pp. 1241-1249 ◽  
Author(s):  
Yingying Mao ◽  
Satya P. Kunapuli
Keyword(s):  
Intracellular Calcium ◽  
Platelet Activation ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Shape Change ◽  
Signalling Pathway ◽  
Protease Activated Receptors ◽  
Similar Shape ◽  
High Concentrations ◽  
Platelet Shape

SummaryTo identify a physiological agonist of PAR3, we used PAR4 null murine platelets, which were known to express only PAR3. In this study, we tested several proteases and found that trypsin, but not heat-inactivated trypsin, activated PAR4 null murine platelets. Even at high concentrations, trypsin caused shape change without increasing intracellular calcium levels in PAR4 null murine platelets. Consistent with this result, the Gq inhibitor YM-254890 had no effect on trypsin-induced shape change. However, trypsin-induced platelet shape change was abolished by either p160ROCK inhibitor, Y27632 or H1152. Furthermore, trypsin caused phosphorylation of myosin light chain (Thr18), but not Akt or Erk. Surprisingly, trypsin caused a similar shape change in PAR4-desensitised PAR3 null murine platelets as in PAR4 null murine platelets, indicating that trypsin did not activate PAR3 to cause shape change. More interestingly, the Src family kinase (SFK) inhibitor PP2 abolished trypsin-induced, but not AYPGKF-induced, shape change. Hence, trypsin activated a novel signalling pathway through RhoA/p160ROCK and was regulated by SFKs. In conclusion, our study demonstrates a novel protease signalling pathway in platelets that is independent of PARs. This protease-induced novel signalling pathway regulates platelet shape change through SFKs and p160ROCK.

cAMP signaling regulates platelet myosin light chain (MLC) phosphorylation and shape change through targeting the RhoA-Rho kinase-MLC phosphatase signaling pathway

Blood ◽  
2013 ◽  
Vol 122 (20) ◽  
pp. 3533-3545 ◽  
Author(s):  
Ahmed Aburima ◽  
Katie S. Wraith ◽  
Zaher Raslan ◽  
Robert Law ◽  
Simbarashe Magwenzi ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Shape Change ◽  
Rho Kinase ◽  
Camp Signaling ◽  
Key Points ◽  
Rhoa Kinase ◽  
Mlc Phosphorylation ◽  
Kinase A ◽  
Platelet Shape

Key Points Protein kinase A (PKA) phosphorylates RhoA on serine188 to inhibit RhoA membrane translocation and RhoA kinase (ROCK) signaling. Inhibition of RhoA/ROCK2 promotes myosin light chain (MLC) phosphatase activity, which prevents the phosphorylation of MLC and platelet shape change.

scholarly journals Maturation and the role of PKC-mediated contractility in ovine cerebral arteries

2009 ◽  
Vol 297 (6) ◽  
pp. H2242-H2252 ◽  
Author(s):  
Ravi Goyal ◽  
Ashwani Mittal ◽  
Nina Chu ◽  
Lijun Shi ◽  
Lubo Zhang ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Contractile Response ◽  
Cerebral Arteries ◽  
Age Groups ◽  
Rho Kinase ◽  
Immunoblot Analysis ◽  
Mek Inhibitor ◽  
Inhibitory Protein ◽  
Regulatory Myosin Light Chain

Ca2+-independent pathways such as protein kinase C (PKC), extracellular-regulated kinases 1 and 2 (ERK1/2), and Rho kinase 1 and 2 (ROCK1/2) play important roles in modulating cerebral vascular tone. Because the roles of these kinases vary with maturational age, we tested the hypothesis that PKC differentially regulates the Ca2+-independent pathways and their effects on cerebral arterial contractility with development. We simultaneously examined the responses of arterial tension and intracellular Ca2+ concentration and used Western immunoblot analysis to measure ERK1/2, RhoA, 20 kDa regulatory myosin light chain (MLC20), PKC-potentiated inhibitory protein of 17 kDa (CPI-17), and caldesmon. Phorbol 12,13-dibutyrate (PDBu)-mediated PKC activation produced a robust contractile response, which was increased a further 20 to 30% by U-0126 (MEK inhibitor) in cerebral arteries of both age groups. Of interest, in the fetal cerebral arteries, PDBu leads to an increased phosphorylation of ERK2 compared with ERK1, whereas in adult arteries, we observed an increased phosphorylation of ERK1 compared with ERK2. Also, in the present study, RhoA/ROCK played a significant role in the PDBu-mediated contractility of fetal cerebral arteries, whereas in adult cerebral arteries, CPI-17 and caldesmon had a significantly greater role compared with the fetus. PDBu also led to an increased MLC20 phosphorylation, a response blunted by the inhibition of myosin light chain kinase only in the fetus. Overall, the present study demonstrates an important maturational shift from RhoA/ROCK-mediated to CPI-17/caldesmon-mediated PKC-induced contractile response in ovine cerebral arteries.

Evidence for Ca2+-independent phosphorylation of human platelet myosin

1987 ◽  
Author(s):  
J L Daniel ◽  
M Rigmaiden
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Myosin Light Chain ◽  
Shape Change ◽  
Human Platelets ◽  
Myosin Phosphorylation ◽  
Kinase C ◽  
Synthetic Inhibitor ◽  
Measurable Change ◽  
Platelet Shape

Phosphorylation of platelet myosin is thought to be required for activation of the contractile events occurring during platelet activation. At present the only known mechanism for Onitiating myosin phosphorylation is through a Ca2+-calmodulin-dependent activation of myosin light chain kinase. However, our previous studies using the fluorescent Ca2+-indicator quin2 indicated that both platelet shape change and myosin phosphorylation could be induced in an EGTA-containing media in the absence of a measurable change in cytosolic free Ca2+ concentration (Hallam, Daniel, Kendrick-Jones & Rink. Biochem. J. 232 (1985) 373). In order to confirm this finding, we fyave investigated the regulation of myosin phosphorylation usin^+a preparation of electrically-permeabilized platelets and Ca2+ buffers to control the internal Ca2+ concentration. Fifty percent myosin phosphorylation was obtained at 700 nM Ca2+. When thrombin (5 U/ml) was added to this system, this curve shifted both to the left and upward; 50% myosin phosphorylation was obtained at 400 nM Ca2+.A synthetic inhibitor of protein kinase C, H7, had no effect on myosin phosphorylation in the absence of agonist but did inhibit the thrombin-induced shift to left suggesting that protein kinase C may modulate myosin phosphorylation. We also compared the effects of H7 agonist-induced myosin phosphorylation and shape change in control and an quin2 loaded platelets. Comparable inhibition of both phosphorylation and the rate of shape change was observed with both quin2 and H7. Addition of H7 to quin2-loaded platelets resulted in complete inhibition of both agonist-induced shape change and myosin phosphorylation. These results indicate that both protein kinase C and Ca2+-dependent reactions are involved in complete expression of myosin phosphorylation in human platelets.

scholarly journals Oxidized low-density lipoproteins induce rapid platelet activation and shape change through tyrosine kinase and Rho kinase–signaling pathways

Blood ◽  
2013 ◽  
Vol 122 (4) ◽  
pp. 580-589 ◽  
Author(s):  
Katie S. Wraith ◽  
Simbarashe Magwenzi ◽  
Ahmed Aburima ◽  
Yichuan Wen ◽  
David Leake ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Platelet Activation ◽  
Shape Change ◽  
Oxidized Ldl ◽  
Rapid Change ◽  
Rho Kinase ◽  
Low Density Lipoproteins ◽  
Low Density ◽  
Key Points ◽  
Platelet Shape

Key Points Oxidized LDL stimulates rapid change in platelet shape through ligation of CD36. Ligation of CD36 by oxidized LDL simultaneously activates tyrosine and Rho kinase–dependent signaling pathways.

EARLY (< 5 SEC) PHOSPHORYLATIONS OF PLATELET PROTEINS FOLLOWING ACTIVATION BY ADP AND ADRENALIN, SEPARATELY AND IN COMBINATION

1987 ◽  
Author(s):  
LR A Gear ◽  
D Freas ◽  
J D Carty
Keyword(s):  
Light Chain ◽  
Gel Electrophoresis ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Shape Change ◽  
Human Platelets ◽  
Single Particles ◽  
Specific Analysis ◽  
Mlc Phosphorylation

Understanding the earliest events (< 1 sec) in signal transduction of platelets is important, since there is evicenee that “shape change,” aggregation and secretion can all begin within this period. We have employed a guenched-flow approach to study these early events and found that thrombin can induce rapid phosphorylation of myosin light-chain kinase (20K) and a 47K protein (Blood, 67, 1738, 1986). To investigate the role of rapid phosphorylations in platelet activation, we have studied the influence of adrenalin and ADP during early (0.3 to 5 sec) stimulation. Aggregation in washed human platelets was assessed by following the loss of single particles and phosphorylation by analysing 32P-labeled proteins after gel electrophoresis. 15 µM adrenalin (without ADP) did not initiate significant aggregation or phosphorylation of myosin light chain (MLC). Phosphorylation of the 47K protein was increased by 20% at 5 sec. 0.5 µM ADP did not induce significant aggregation, but increased phosphorylation of MLC by 130% and the 47 protein by 20%. The combination of 0.5 µM ADP and 15 uM adrenalin induced significant aggregation by 0,3 sec (7.6%), which increased to 25.6% by 5 sec. Interestingly, MLC or 47K protein phosphorylation was not increased above control levels. However, the phosphorylation of four other proteins (77K, 102K, 140K and 185K), which previously had been very rapid (<1 sec) and reversible with 0.5 µM ADP alone, was now maintained, peaking at 3 sec. 10 µM ADP caused small sustained increases in phosphorylation of the same proteins. Adrenalin also caused rapid increases in the phosphorylation of 27K, 213 and 250K proteins. High levels of ADP (10 µM) only increased the 213 and 250K proteins; therefore the 27K protein appears adrenalin specific. Analysis of these early platelet phosphorylations will help understand how they are linked to initiation and maintenance of aggregation. Supported by NIH HL-27014.

PAR1-Induced Human Platelet Shape Change, Aggregation and Secretion Requires Gα13 Switch Region I Signaling.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1522-1522
Author(s):  
Jin-Sheng Huang ◽  
Lanlan Dong ◽  
Guy C. Le Breton
Keyword(s):  
G Protein ◽  
Platelet Activation ◽  
Platelet Function ◽  
Human Platelet ◽  
Shape Change ◽  
Human Platelets ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Rhoa Activation ◽  
Platelet Shape

Abstract While it is known that platelets possess multiple G protein signaling pathways that contribute to the different platelet functional responses, the relative participation of these individual pathways in platelet shape change, aggregation and secretion is not well characterized. To a large extent this is due to the lack of suitable reagents which selectively interfere with specific G protein signaling events, and which can be applied to the study of intact human platelets. With the exception of pepducins, which modulate receptor-G protein coupling (Kuliopulos, A. and Covic, L. Life Sciences 74, 255–262, 2003), the field has for the most part been limited to agents which interfere with different downstream kinases or other downstream effectors. However, the G protein pathways share many of these downstream targets, and consequently, it has been difficult to assign a specific platelet function to a certain G protein. In order to address this issue, it was reasoned that more direct information about specific G protein involvement in human platelet activation might be obtained by interfering with the initial G protein signal transduction events, rather than by interfering with the secondary downstream consequences of this transduction process. Based on this consideration, the present study used a specific Gα13 switch region I (SRI) peptide to investigate the involvement of Gα13 signaling in protease-activated receptor 1 (PAR1)-mediated human platelet function. Specifically, a myristoylated peptide representing the Gα13 SRI (Myr-G13SRIpep) was synthesized and evaluated for its effects on PAR1 activation. Initial studies using dot blot and mass spectrum analysis demonstrated that Myr-G13SRIpep, and its random sequence control (Myr-G13SRIRandom-pep), were equally taken up by intact human platelets. Radioligand binding experiments revealed that Myr-G13SRIpep did not interfere with PAR1-ligand interaction. Subsequent experiments demonstrated that G13SRIpep specifically bound to platelet p115Rho guanine nucleotide exchange factor (p115RhoGEF) and blocked PAR1-mediated RhoA activation. These results suggest a direct interaction of Gα13 SRI with p115RhoGEF, and indicate a possible mechanism for Myr-G13SRIpep inhibition of RhoA activation. Platelet function studies revealed that Myr-G13SRIpep inhibited PAR1-stimulated platelet shape change, aggregation and dense granule secretion in a dose-dependent manner. On the other hand, Myr-G13SRIpep did not inhibit platelet activation induced by ADP, A23187 or PAR4 activating peptide (AYPGKF). Taken together, these findings demonstrate that the inhibitory effects of Myr-G13SRIpep are limited to PAR1 signaling mechanisms and are not due to nonspecific effects on platelet function. These results also suggest a significant role for Gα13 SRI signaling in the process of PAR1-mediated human platelet activation. In additional studies it was found that Myr-G13SRIpep also inhibited low-dose thrombin-induced aggregation and PAR1-induced intraplatelet calcium mobilization. Collectively, these results provide evidence that: 1. interaction of Gα13 SRI with p115RhoGEF is required for G13-mediated RhoA activation in platelets; 2. signaling through the G13 pathway is critical for PAR1-mediated human platelet functional changes; 3. Gα13 SRI signaling is involved in low-dose thrombin-induced platelet aggregation as well as PAR1-mediated calcium mobilization; and 4. permeable peptides representing SRI of Gα-subunits should be a useful approach for studying individual G protein signaling pathways in intact cells.

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