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.

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

Abstract 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.

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.

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

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.

Pretreatment with Oral Anticoagulants Decreases Platelet Activation in Patients Before and After Percutaneous Coronary Intervention

2002 ◽  
Vol 88 (12) ◽  
pp. 924-930 ◽  
Author(s):  
Wim Gerritsen ◽  
Fred Haas ◽  
Johannes Kelder ◽  
Freek Verheugt ◽  
H. W. Plokker ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Function ◽  
Coronary Angioplasty ◽  
Oral Anticoagulants ◽  
Coronary Intervention ◽  
Median Number ◽  
Platelet Response ◽  
Before And After ◽  
Group A ◽  
Group B

SummaryPlatelet activation plays a major role in acute vessel closure after coronary angioplasty. In the randomized Balloon Angioplasty and Anticoagulation Study (BAAS), pretreatment with oral anticoagulants in addition to aspirin resulted in a 47% reduction of acute complications as compared with aspirin alone. This result may suggest a direct effect of oral anticoagulants on platelet activation.Patients were randomized to aspirin alone (group A, n = 26) or to aspirin plus oral anticoagulants started one week before angioplasty (group B, n = 26). Platelet response tests were performed 1 hour before (baseline) and 1 hour after intervention and on day 1. Platelet activation was measured by flow cytometry, as the number of antibody-positive platelets per 10,000 counted. Platelet function was evaluated with use of the PFA-100® analyzer. In group B, the median number of P-selectin-positive platelets was lower before (28 vs. 54, P = 0.018) and after (13 vs. 24, P = 0.377) angioplasty than in group A. Also the median decrease in the number of P-selectin-positive platelets during angioplasty was lower in group B (Δ = 4) than in group A (Δ = 30, P = 0.022). No further significant change was observed in platelet activation on day 1 in the two groups. The ability of platelets to become stimulated as measured with the PFA-100® analyzer was not affected by oral anticoagulants.Pretreatment with oral anticoagulants resulted in less activated platelets before and after coronary angioplasty, which is in agreement with its clinical effect of reducing procedural complications. Platelet function was not affected by oral anticoagulants.

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)

The role of CD40 in CD40L- and antibody-mediated platelet activation

2005 ◽  
Vol 93 (06) ◽  
pp. 1137-1146 ◽  
Author(s):  
Ali Amirkhosravi ◽  
Todd Meyer ◽  
Farooq Siddiqui ◽  
Sarfraz Ahmad ◽  
Jamie Walker ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Function ◽  
Immune Complexes ◽  
Molar Ratio ◽  
Dependent Manner ◽  
Platelet Response ◽  
Initial Finding ◽  
Platelet Surface ◽  
Primary Hemostasis

SummaryOur initial finding that CD40– and CD40 ligand (CD40L)-deficient mice displayed prolonged tail bleeding and platelet function analyzer (PFA-100) closure times prompted us to further investigate the role of the CD40-CD40L dyad in primary hemostasis and platelet function. Recombinant human soluble CD40L (rhs CD40L), chemical cross-linking of which suggested a trimeric structure of the protein in solution, activated platelets in a CD40-dependent manner as evidenced by increased CD62P expression. CD40 monoclonal antibody (mAb) M3, which completely blocked rhs CD40L-induced platelet activation, also prolonged PFA-100 closure times of normal human blood. In contrast, CD40 mAb G28–5 showed less potential in blocking rhs CD40L-induced CD62P expression and did not affect PFA-100 closure times. However, when added to the platelets after rhs CD40L, G28–5 significantly enhanced the platelet response by causing clustering of, and signaling through, FcγRII. Similarly, higher order multimeric immune complexes formed at a 1/3 molar ratio of M90, a CD40L mAb, to rhs CD40L induced strong FcγRII-mediated platelet activation when translocated to the platelet surface in a CD40-dependent manner, including the induction of morphological shape changes, fibrinogen binding, platelet aggregation, dense granule release, microparticle generation and monocyte-platelet-conjugate formation. The results suggest that CD40 may play a role in primary hemostasis and platelet biology by two independent mechanisms: First, by functioning as a primary signaling receptor for CD40L and, second, by serving as a docking molecule for CD40L immune complexes. The latter would also provide a potential mechanistic explanation for the unexpected high incidence of CD40L mAb-associated thrombotic events in recent human and animal studies.Parts of this work have been presented on the 46th Annual Meeting of the American Society of Hematology (San Diego, 2004).

scholarly journals Influence of γ-Secretase Inhibitor 24-Diamino-5-Phenylthiazole DAPT on Platelet Activation

2016 ◽  
Vol 38 (2) ◽  
pp. 726-736 ◽  
Author(s):  
Guoxing Liu ◽  
Guilai Liu ◽  
Madhumita Chatterjee ◽  
Anja T. Umbach ◽  
Hong Chen ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Function ◽  
Blood Platelets ◽  
Annexin V ◽  
Negative Regulator ◽  
Ros Generation ◽  
Integrin Activation ◽  
Forward Scatter ◽  
Secretase Inhibitor ◽  
Αiibβ3 Integrin

Background/Aims: DAPT (24-diamino-5-phenylthiazole) inhibits γ-secretase, which cleaves the signaling molecule CD44, a negative regulator of platelet activation and apoptosis. CD44 is a co-receptor for macrophage migration inhibitory factor (MIF) an anti-apoptotic pro-inflammatory cytokine expressed and released from blood platelets. Whether DAPT influences platelet function, remained, however, elusive. Activators of platelets include collagen related peptide (CRP). The present study thus explored whether DAPT modifies the stimulating effect of CRP on platelet function. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to DAPT (10 µM). Flow cytometry was employed to estimate Orai1 abundance with specific antibodies, cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, generation of reactive oxygen species (ROS) from DCFDA fluorescence, mitochondrial transmembrane potential from TMRE fluorescence, phospholipid scrambling of the cell membrane from annexin-V-binding, relative platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. Results: Exposure of platelets to 2-5 µg/ml CRP was followed by significant increase of Orai1 abundance, [Ca2+]i, and P-selectin abundance, as well as by αIIbβ3 integrin activation, ROS generation, mitochondrial depolarization, enhanced annexin-V-binding, decreased cell volume, and aggregation. All CRP induced effects were significantly blunted in the presence of DAPT. Conclusions: The γ-secretase inhibitor DAPT counteracts agonist induced platelet activation, apoptosis and aggregation.

Abstract 39: Platelet Reactivity to Prostaglandin E2 is a Novel Modifiable Risk Factor for St-Elevation Myocardial Infarction

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Eitan A Friedman ◽  
Elias V Haddad ◽  
Valentinas Joksas ◽  
Shi Huang ◽  
Meng Xu ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Cardiovascular Risk ◽  
Platelet Activation ◽  
Platelet Reactivity ◽  
Platelet Rich Plasma ◽  
Coronary Intervention ◽  
Prostaglandin E ◽  
Platelet Response ◽  
Increased Risk ◽  
St Elevation

Background: Patients with lower thresholds for platelet activation are at increased risk for primary and recurrent myocardial infarction (MI) and overall cardiovascular (CV) mortality. We have demonstrated that there are two phenotypes of platelet response to Prostaglandin E 2 (PGE 2 ), such that it increases threshold for aggregation in 45% of individuals (inhibitory) and lowers threshold for aggregation in 55% (potentiating). As PGE 2 is present in atherosclerotic plaques, and its receptors are present on platelets, biologic variability in PGE 2 responses may have clinical implications. We hypothesized that patients with higher thresholds for platelet activation would have a lower risk of thrombotic CV events, specifically ST-Elevation MI (STEMI). Methods: 85 patients undergoing percutaneous coronary intervention for stable or unstable coronary disease were phenotyped for PGE 2 response. Platelet rich plasma was treated with various concentrations of U46,619 (thromboxane agonist) with or without PGE 2 100 nM, and phenotype determined by light aggregometry. Analysis of the maximum PGE 2 effect (maximum aggregation with PGE 2 minus maximum aggregation without it) was performed using linear and non-linear statistical methods. Results: Traditional cardiovascular risk factors were similar between groups. A higher percentage of patients with the potentiating phenotype had a history of STEMI than those with the inhibitory phenotype. Logistic regression using restricted cubic spline showed that the predicted probabilities of STEMI increased from 0.04 (at the strongest inhibitory phenotype) to 0.43 (at the median phenotype). The OR of phenotype at the median relative to that at the 10th quantile was 7.4 (95 % CI=1.6, 34.8). Conclusions: PGE 2 inhibitory phenotype confers a decreased lifetime risk of STEMI in individuals at high risk for CV events. We have previously shown that an EP3 receptor antagonist converts the potentiating to the inhibitory phenotype. Thus, the PGE2 phenotype may be a novel marker of cardiovascular risk that may also identify patients who would benefit from an EP3 antagonist.

scholarly journals microRNAs as Promising Biomarkers of Platelet Activity in Antiplatelet Therapy Monitoring

2020 ◽  
Vol 21 (10) ◽  
pp. 3477
Author(s):  
Teresa L. Krammer ◽  
Manuel Mayr ◽  
Matthias Hackl
Keyword(s):  
Antiplatelet Therapy ◽  
Platelet Activation ◽  
Platelet Function ◽  
Platelet Reactivity ◽  
Therapy Monitoring ◽  
Platelet Inhibition ◽  
High Morbidity ◽  
Platelet Function Tests ◽  
Function Tests ◽  
Plasma Mirna

Given the high morbidity and mortality of cardiovascular diseases (CVDs), novel biomarkers for platelet reactivity are urgently needed. Ischemic events in CVDs are causally linked to platelets, small anucleate cells important for hemostasis. The major side-effect of antiplatelet therapy are life-threatening bleeding events. Current platelet function tests are not sufficient in guiding treatment decisions. Platelets host a broad spectrum of microRNAs (miRNAs) and are a major source of cell-free miRNAs in the blood stream. Platelet-related miRNAs have been suggested as biomarkers of platelet activation and assessment of antiplatelet therapy responsiveness. Platelets release miRNAs upon activation, possibly leading to alterations of plasma miRNA levels in conjunction with CVD or inadequate platelet inhibition. Unlike current platelet function tests, which measure platelet activation ex vivo, signatures of platelet-related miRNAs potentially enable the assessment of in vivo platelet reactivity. Evidence suggests that some miRNAs are responsive to platelet inhibition, making them promising biomarker candidates. In this review, we explain the secretion of miRNAs upon platelet activation and discuss the potential use of platelet-related miRNAs as biomarkers for CVD and antiplatelet therapy monitoring, but also highlight remaining gaps in our knowledge and uncertainties regarding clinical utility. We also elaborate on technical issues and limitations concerning plasma miRNA quantification.

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