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.

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 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.

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.

scholarly journals Membrane glycoproteins and platelet cytoskeleton in immune complex- induced platelet activation

Blood ◽  
1993 ◽  
Vol 81 (6) ◽  
pp. 1505-1512 ◽  
Author(s):  
J Kang ◽  
C Cabral ◽  
L Kushner ◽  
EW Salzman
Keyword(s):  
Platelet Activation ◽  
Immune Complexes ◽  
Actin Polymerization ◽  
Cytochalasin B ◽  
Specific Marker ◽  
Platelet Response ◽  
Membrane Glycoproteins ◽  
Filamentous Actin ◽  
Serotonin Secretion ◽  
Platelet Binding

To clarify the mechanism of platelet activation by immune complexes and the possible involvement of surface glycoproteins (GPs), we studied platelet activation induced by heat-aggregated IgG (HAG). We examined the effects of monoclonal antibodies (MoAbs) against GPIb, GPIIb/IIIa, and the Fc receptor on resting platelets and on platelets stimulated by HAG. HAG increased the cytosolic ionized calcium concentration ([Ca2+]i) and stimulated protein (P47 and P20) phosphorylation, phosphatidic acid (PA) synthesis, serotonin secretion, and platelet aggregation. IV.3, an anti-Fc gamma RII receptor MoAb, inhibited HAG binding to platelets and all subsequent platelet responses. Like IV.3, MoAbs against GPIIb/IIIa (Tab, 10E5, AP-3) or GPIb (AP-1, 6D1) strongly inhibited platelet activation by HAG. However, while anti-GPIIb/IIIa MoAbs inhibited binding of IV.3 and HAG to platelets, anti-GPIb MoAbs had little effect on platelet binding of IV.3 or HAG. These observations suggest a close topographical and functional association of GPIIb/IIIa with Fc gamma RII in the platelet response to HAG. Cytochalasin B, an inhibitor of actin polymerization, also inhibited platelet activation but not HAG or IV.3 binding. Measurement of the fluorescence of 7-nitrobenz-2-oxa-1,3-(NBD)-phallacidin, a specific marker for filamentous actin (F-actin), showed that both cytochalasin B and AP-1 blocked the increase of F-actin induced by HAG. The common effects of anti-GPIb MoAbs and of cytochalasin B suggest that unlike the activity of GPIIb/IIIa, the ability of anti-GPIb to inhibit the activation of platelets by immune complexes is associated with perturbation of the cytoskeleton.

scholarly journals Membrane glycoproteins and platelet cytoskeleton in immune complex- induced platelet activation

Blood ◽  
1993 ◽  
Vol 81 (6) ◽  
pp. 1505-1512 ◽  
Author(s):  
J Kang ◽  
C Cabral ◽  
L Kushner ◽  
EW Salzman
Keyword(s):  
Platelet Activation ◽  
Immune Complexes ◽  
Actin Polymerization ◽  
Cytochalasin B ◽  
Specific Marker ◽  
Platelet Response ◽  
Membrane Glycoproteins ◽  
Filamentous Actin ◽  
Serotonin Secretion ◽  
Platelet Binding

Abstract To clarify the mechanism of platelet activation by immune complexes and the possible involvement of surface glycoproteins (GPs), we studied platelet activation induced by heat-aggregated IgG (HAG). We examined the effects of monoclonal antibodies (MoAbs) against GPIb, GPIIb/IIIa, and the Fc receptor on resting platelets and on platelets stimulated by HAG. HAG increased the cytosolic ionized calcium concentration ([Ca2+]i) and stimulated protein (P47 and P20) phosphorylation, phosphatidic acid (PA) synthesis, serotonin secretion, and platelet aggregation. IV.3, an anti-Fc gamma RII receptor MoAb, inhibited HAG binding to platelets and all subsequent platelet responses. Like IV.3, MoAbs against GPIIb/IIIa (Tab, 10E5, AP-3) or GPIb (AP-1, 6D1) strongly inhibited platelet activation by HAG. However, while anti-GPIIb/IIIa MoAbs inhibited binding of IV.3 and HAG to platelets, anti-GPIb MoAbs had little effect on platelet binding of IV.3 or HAG. These observations suggest a close topographical and functional association of GPIIb/IIIa with Fc gamma RII in the platelet response to HAG. Cytochalasin B, an inhibitor of actin polymerization, also inhibited platelet activation but not HAG or IV.3 binding. Measurement of the fluorescence of 7-nitrobenz-2-oxa-1,3-(NBD)-phallacidin, a specific marker for filamentous actin (F-actin), showed that both cytochalasin B and AP-1 blocked the increase of F-actin induced by HAG. The common effects of anti-GPIb MoAbs and of cytochalasin B suggest that unlike the activity of GPIIb/IIIa, the ability of anti-GPIb to inhibit the activation of platelets by immune complexes is associated with perturbation of the cytoskeleton.

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).

The influence of lysophosphatidic acid on platelet protein phosphorylation

1987 ◽  
Vol 65 (7) ◽  
pp. 642-650 ◽  
Author(s):  
Jon. M. Gerrard ◽  
Laura L. Beattie ◽  
Jan M. McCrae ◽  
Sandra Singhroy
Keyword(s):  
Platelet Aggregation ◽  
Protein Phosphorylation ◽  
Lysophosphatidic Acid ◽  
Calcium Ionophore ◽  
Actin Binding ◽  
Time Points ◽  
Platelet Protein ◽  
Serotonin Secretion ◽  
Mlc Phosphorylation ◽  
Induced Aggregation

Lysophosphatidic acid (LPA) is a lysophospholipid that is produced during thrombin stimulation of platelets, which can promote platelet aggregation. The mechanism of the effect of LPA was explored in normal platelets and in platelets from a patient with a storage pool deficiency (SPD). A comparison with other lysophospholipids showed that only LPA exerted significant effects to cause or potentiate platelet aggregation. Aspirin, an inhibitor of prostaglandin endoperoxide synthetase, had little effect on LPA-induced aggregation, but completely blocked LPA-induced serotonin secretion. LPA also promoted phosphorylation of myosin light chain (MLC), a 47 kilodalton (kDa) protein, and actin-binding protein. Aspirin significantly inhibited the phosphorylation of the 47-kDa and actin-binding proteins at 3–8 min after the addition of LPA, but had no effect on protein phosphorylation within the 1st min and had no significant effect on MLC phosphorylation. In SPD platelets, aspirin partially inhibited both aggregation and phosphorylation of the 47-kDa protein (< 30% inhibition) and MLC (< 40% inhibition) at time points of 1 min or less. The addition of ADP to SPD platelets enhanced the LPA response in platelets either pretreated or not pretreated with aspirin. Studies with SPD platelets indicate that thromboxane and secreted ADP contribute to, but are not necessary for, LPA-induced aggregation and phosphorylation. A23187 (a calcium ionophore) and LPA showed some selectivity to promote MLC as opposed to the 47-kDa protein phosphorylation, particularly at low concentrations of agonists and at earlier time points. The protein phosphorylation changes seen are consistent with a role for MLC phosphorylation in the granule centralization promoted with LPA.

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