EFFECT OF ATRIAL NATRIURETIC POLYPEPTIDES ON PLATELET FUNCTION

1987 ◽  
Author(s):  
T Asaji ◽  
E Murakami ◽  
N Takekoshi ◽  
S Matsui ◽  
T Imaoka
Keyword(s):  
Protein Phosphorylation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Cyclic Nucleotides ◽  
Human Platelet ◽  
Early Stage ◽  
Platelet Rich Plasma ◽  
Heart Insufficiency ◽  
32P Incorporation ◽  
Atrial Natriuretic

Atrial natriuretic polypeptides (ANP) have been shown to possess a potent diuretic and natriuretic activity, and medicated to patients with heart insufficiency as a drug to be mediated by cGMPaccumulation in glomeruli. A existence of receptors for ANP have recently beenreported in human platelet. But, whether ANP has a direct effect on platelet function remains to be known.Single stimulation of ANP in any concentration did not induce aggregation in neither platelet rich plasma, nor washed platelets. Also no effect of pretreatment with ANP was observed against aggregation triggered by known mediators of platelet activation (Thrombin, ADP, Epinephrine, Collagen) using platelet rich plasma and washed platelets.Therefore, biochemical parameters such as cyclic nucleotides (cAMP, cGMP), phosphatidylinositol hydrolysis and protein phosphorylation, leading to the early stage of platelet activation were examined to investigate the effect of ANP in receptor linked transducing mechanism. Neither cyclic nucleotides accumulation nor [32 P] phosphatidic acid production were detected in platelets treated with ANP. ANP caused a small increase of 32P incorporation into M 30K protein, but no change on the level of phosphorylation of 47K, 20K protein (Imaoka, T. and Haslam, R.J., J.Biol.Chem.258,11404, 1983) was observed.These results clearly suggested thatANP binding with membrane receptor was not linked with adenylate cyclase, ganulate cyclase and phosphatidylinositol phosphate turnover in human platelet, maybe because of too few numbers of ANP receptor. Mechanism of 30K protein phosphorylation and Ca++ mobilization are important subjects for future study, (supported by MESC of Japan)

scholarly journals ADP causes subsecond changes in protein phosphorylation of platelets

Blood ◽  
1987 ◽  
Vol 70 (2) ◽  
pp. 511-515
Author(s):  
DJ Carty ◽  
DL Freas ◽  
AR Gear
Keyword(s):  
Protein Phosphorylation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Onset Time ◽  
Biochemical Changes ◽  
Cyclooxygenase Inhibitor ◽  
Platelet Response ◽  
Calcium Fluxes

We developed a general quenched-flow approach to study platelet function as early as 0.3 seconds after stimulation. Phosphorylation of 20- and 47-kiloDalton (kD) proteins was analyzed during the first 5 seconds of platelet response to ADP from 0.5 to 10.0 mumol/L and compared with the progress of aggregation. The onset time for aggregation and phosphorylation of both proteins was less than 1 second; 20-K phosphorylation was increased greater than 200% and 47-K phosphorylation was increased 50%. The ADP sensitivity of 20-K phosphorylation was greater than that of 47-K phosphorylation (P less than .025), and of that of aggregation (P less than .01), with Ka values of 0.7, 1.0, and 1.2 mumol/L of ADP, respectively. The cyclooxygenase inhibitor indomethacin had no effect on aggregation, but inhibited both phosphorylations. Its inhibition of 20-K phosphorylation was greater than that of 47-K phosphorylation. Platelet activation by ADP thus induced biochemical changes well before 1 second. The quenched- flow approach may help to reveal relationships between phospholipase activation, calcium fluxes, and protein phosphorylation during these early periods of platelet activation.

Possible Roles for Protein Phosphorylation in Platelet Responses

1979 ◽  
Author(s):  
R.J. Haslam ◽  
J.E.B. Fox ◽  
S.E. Salama ◽  
J.A. Lynham
Keyword(s):  
Cyclic Amp ◽  
Protein Phosphorylation ◽  
Protein Kinases ◽  
Platelet Function ◽  
Subcellular Fractionation ◽  
Human Platelets ◽  
Type I ◽  
Sds Page ◽  
Dependent Protein ◽  
32P Incorporation

The relationships between the phosphorylation of specific platelet polypeptides and platelet function were studied using washed human platelets labelled by preincubation with [32p] Pi. Platelet polypeptides were separated by SDS-PAGE and 32P incorporation into them determined by autoradiography. Whereas induction of platelet aggregation alone did not affect protein phosphorylation, induction of the release reaction increased 3P incorporation into several polypeptides (P75,P47,P40,P27,P20,P19), including the P-light chain of platelet myosin (P20). These changes were inhibited by drugs that blocked Ca2 movements and may be due to activation of Ca2+-dependent protein kinases. Compounds that inhibited platelet function by increasing cyclic AMP (e.g. PCE1) also suppressed these reactions but, in addition, increased phosphorylation of other polypeptides (P50,P49,P36,P24,P22). Type I and Type II cyclic AMP-dependent protein kinases were present in platelets and may mediate Che latter effects of cyclic AMP. Subcellular fractionation of 32p-labelled platelets that had been exposed to PCE1 showed that P24 was present in membranes that could take up Ca2+ by an ATP-dependent mechanism. Membranes from PCE1-treated platelets took up Ca2+ more rapidly than control membranes. Thus, the cyclic AMP-dependent phosphorylation of P24 may stimulate the removal of Ca2+ from platelet cytosol and suppress Ca2+-dependent phosphorylation reactions necessary for release of granule constituents.

Effect of Lidoflazine (R 7904) on Human Platelet Function in Vitro

1972 ◽  
Vol 28 (02) ◽  
pp. 228-236 ◽  
Author(s):  
F De Clerck
Keyword(s):  
Platelet Function ◽  
Mode Of Action ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Serotonin Release ◽  
Clot Retraction ◽  
Plasma Coagulation ◽  
Release Reaction ◽  
Platelet Release

SummaryThe effect of lidoflazine and of cinnarizine on human platelet function in vitro was compared to that of dipyridamole.Pre-incubation for 30 min at 37° C of platelet rich plasma with lidoflazine or with dipyridamole 5 ×10–4 M resulted in an appreciable inhibition of collagen aggregation in particular and to a lesser extent of ADP aggregation; cinnarizine was marginally active only.Clot retraction was inhibited by lidoflazine and by dipyridamole. Experiments on biphasic ADP aggregation and C14-serotonin release during aggregation show that lidoflazine reduces the platelet release reaction.The possible mode of action of the compound is discussed.Plasma coagulation and PF – 3 availability were not affected.

Studies on the Effect of Platelet Inhibitors on Platelet Adhesion to Collagen and Collagen-Induced Human Platelet Activation

1985 ◽  
Vol 53 (03) ◽  
pp. 337-342 ◽  
Author(s):  
S Krishnamurthi ◽  
V V Kakkar
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Adhesion ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Antiplatelet Agents ◽  
Calmodulin Antagonist ◽  
Release Reaction ◽  
Low Concentrations

SummaryThe effect of pyridoxal 5’-phosphate (PALP) and trifluoperazine (TFPZ), the calmodulin antagonist, on in vitro platelet adhesion to collagen and collagen-induced platelet activation was studied using platelet-rich-plasma (PRP) or washed platelets (WPL). Platelet aggregation and [14C]-5HT release induced by “threshold” or low concentrations of collagen (0.6 μg/ ml) in PRP were completely abolished by PALP (24 mM), TFPZ (250 μM) as well as indomethacin (10 μM). At higher concentrations of collagen (10–15 μg/ml) in PRP and WPL, the use of stirred and unstirred platelets treated with collagen enabled a distinction to be made between aggregation and adhesion- mediated release reaction. Platelet aggregation and the aggregation-mediated release reaction induced by these concentrations of collagen in stirred platelets were completely abolished by PALP, TFPZ and indomethacin although neither adhesion to collagen nor the adhesion-mediated release reaction of unstirred platelets was significantly affected by these inhibitors. Interestingly, both adhesion and the adhesion-mediated release reaction were abolished by concentrations of PALP 10–40 fold higher than those required to abolish aggregation. Collagen-induced platelet aggregation, but not platelet adhesion, was inhibited in resuspended platelets pretreated with PALP and NaBH4 indicating a separation in the membrane sites involved in aggregation and adhesion. The results further emphasize the distinction between adhesion and aggregation-mediated events with regards to collagen with the latter being more susceptible to inhibition by antiplatelet agents such as PALP and TFPZ.

LPS Mediates the Activation of Platelet by Toll-Like Receptor 4.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3001-3001
Author(s):  
Liping Ma ◽  
Jing Wei ◽  
Jian-Xing Chang ◽  
Cheng Zhang ◽  
Zhi-Xin Pei ◽  
...  
Keyword(s):  
Western Blot ◽  
Platelet Activation ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Conflicts Of Interest ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Platelet Suspension

Abstract Abstract 3001 Poster Board II-978 Lipopolysaccsharide (LPS) is a principal outer membrane component of gram-negative bacteria. It initiates an inflammatory response to infection by activating Toll-like receptor-4 (TLR4) on various tissues or cells in host. Platelet contributes to the inflammation process through respond to invading pathogens, membrane adhesion molecule (CD62P, P-selectin) and CD40L on platelet are the indexes to determine platelet activation. The present experiment was designed to investigate the expression of Toll-like receptor 4 (TLR4) on platelet and to determine whether platelet TLR4 involves in platelet activation induced by Lipopolysaccsharide (LPS). Human platelet-rich plasma (PRP) and platelet suspension obtained from 15 healthy people were pretreated with a concentration of 0.2μg/ml of LPS in the presence or absence of thrombin (1 U/ml) for 1 hour. The expressions of TLR4,CD62P and CD40L on platelets were detected through flow cytometry, and platelet TLR4 was further determined by performing western blot analysis. The results show that the percentage of TLR4-positive platelet induced by thrombin was increased by 32.34% compared with the resting platelets (25.44%, P<0.01). TLR4 expression on platelets treated with LPS was remarkedly elevated in the presence or absent of thrombin. However, the expression level was much higher in presence of both than thrombin alone( 39.16%,P<0.01). Moreover, the similar results were found in Western blot analysis. Synchronously, the expressions of CD62P and CD40L on resting platelets were 6.39% and 2.45%, they were also markedly increased when treated with thrombin(42.68% and 14.80%) and LPS respectively, and the increase of CD62P was more significant when stimulated with both of LPS and thrombin(63.03%). Although anti-TLR4 antibody inhibited significantly the increases of TLR4, CD62P and CD40L on platelets induced by LPS, it didn't effect their increases induced by thrombin. The experiment results support the evidence that functional TLR4 can be expressed on human platelet. It may involve in platelet activation as an important mediator of LPS- induced CD62P and CD40L expressions on platelets. Disclosures: No relevant conflicts of interest to declare.

Effects of atrial natriuretic factor on human platelet function

Life Sciences ◽  
1985 ◽  
Vol 37 (15) ◽  
pp. 1395-1402 ◽  
Author(s):  
Raffaele De Caterina ◽  
Massimo Volpe ◽  
Steven A. Atlas ◽  
Babette B. Weksler
Keyword(s):  
Platelet Function ◽  
Atrial Natriuretic Factor ◽  
Human Platelet ◽  
Natriuretic Factor ◽  
Atrial Natriuretic

scholarly journals Thrombin causes subsecond changes in protein phosphorylation of platelets

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1738-1743 ◽  
Author(s):  
DJ Carty ◽  
F Spielberg ◽  
AR Gear
Keyword(s):  
Protein Phosphorylation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Onset Time ◽  
Biochemical Changes ◽  
Platelet Response ◽  
Calcium Fluxes ◽  
Serotonin Secretion ◽  
External Calcium

Abstract We have developed a general quenched-flow approach to study platelet function as early as 0.3 seconds after stimulation. Phosphorylation of 20- and 40-kd proteins has been analyzed during the first five seconds of platelet response to thrombin from 0.1 to 5.0 U/mL and compared with the progress of aggregation and serotonin secretion. The onset time for aggregation and phosphorylation of both proteins was less than one second, although with lowest (less than 0.5 U/mL) thrombin levels, a lag of up to 0.6 seconds occurred before 40K phosphorylation increased. The thrombin sensitivity of aggregation and 20K phosphorylation was approximately twice that of 40K phosphorylation, with Ka values of 0.51 and 0.53 v 1.10 U/mL, respectively. External calcium was necessary for maximal 20K phosphorylation, since EDTA inhibited this by 30%. The 40K phosphorylation was not affected by EDTA. Platelet activation by thrombin thus induced biochemical changes well before one second. The quenched-flow approach may help to reveal relationships between phospholipase activation, calcium fluxes, and protein phosphorylation during these early periods of platelet function.

Antiplatelet and Antithrombotic Activity of SL65.0472, a Mixed 5-HT1B/5-HT2A Receptor Antagonist

2001 ◽  
Vol 85 (03) ◽  
pp. 521-528 ◽  
Author(s):  
Janine Lorrain ◽  
Sylvette Lochot ◽  
Monique Delahaye ◽  
Alain Lalé ◽  
Pierre Savi ◽  
...  
Keyword(s):  
Receptor Antagonist ◽  
Platelet Activation ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Shape Change ◽  
Antithrombotic Activity ◽  
Thrombosis Model ◽  
Artery Thrombosis ◽  
Ic50 Values

SummaryThe antiplatelet and antithrombotic activity of SL65.0472 (7-fluoro-2-oxo-4-[2-[4-(thieno [3,2-c]pyrin-4-yl) piperazin-1-yl]ethyl]-1,2-dihydroquinoline-acetamide), a mixed 5-HT1B/5-HT2A receptor antagonist was investigated on 5HT-induced human platelet activation in vitro, and in rat, rabbit and canine platelet dependent thrombosis models. SL65.0472 inhibited 5-HT-induced platelet shape change in the presence of EDTA (IC50 values = 35, 69 and 225 nM in rabbit, rat and human platelet rich plasma (PRP)), and also inhibited aggregation induced in human PRP by 3-5 μM 5-HT + threshold concentrations of ADP (0.5-1 M) or collagen (0.3 g/ml) with mean IC50 values of 49 ± 13 and 48 ± 6 nM respectively. SL65.0472 inhibited thrombus formation when given both intravenously 5 min and orally 2 h prior to assembly of an arterio-venous (A-V) shunt in rats as from 0.1 and 0.3 mg/kg respectively. It was active in a rabbit A-V shunt model with significant decreases in thrombus weight as from 0.1 mg/kg i. v. and at 10 mg/kg p. o. The delay to occlusion in an electric current-induced rabbit femoral artery thrombosis model was increased by 251% (p <0.05) after 20 mg/kg p. o. SL65.0472 (30 μg/kg i. v.) virtually abolished coronary cyclic flow variations (7.2 ± 1.0/h to 0.6 ± 0.6/h, p <0.05) and increased minimum coronary blood flow (1.2 ± 0.8 ml/ min to 31.8 ± 8.4 ml/min, p <0.05) in a coronary artery thrombosis model in the anaesthetised dog. Finally, SL65.0472 significantly increased the amount of blood lost after rat tail transection at 3 mg/kg p. o. Thus the anti-5-HT2A component of SL65.0472 is reflected by its ability to inhibit 5-HT-induced platelet activation, and platelet-rich thrombus formation.

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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