PPACK-thrombin inhibits thrombin-induced platelet aggregation and cytoplasmic acidification but does not inhibit platelet shape change

Blood ◽  
1990 ◽  
Vol 75 (10) ◽  
pp. 1989-1990 ◽  
Author(s):  
NJ Greco ◽  
TE Jr Tenner ◽  
NN Tandon ◽  
GA Jamieson
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Shape Change ◽  
Equilibrium Mixture ◽  
High Concentration ◽  
Chloromethyl Ketone ◽  
Molar Excess ◽  
Thrombin Activity ◽  
Cytoplasmic Acidification ◽  
Platelet Shape

Abstract We have re-evaluated the previously reported ability of TLCK-thrombin (N alpha-tosyl-L-lysine chloromethyl ketone-treated alpha-thrombin) and PPACK-thrombin (D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone- treated alpha-thrombin) to inhibit alpha-thrombin-induced platelet activation (Harmon JT, Jamieson GA: J Biol Chem 261:15928, 1986; and Harmon JT, Jamieson GA: Biochemistry 27:2151, 1988). Despite several cycles of derivatization with TLCK (10,000-fold molar excess), preparations of TLCK-thrombin have been found to contain about 4% residual alpha-thrombin activity, suggesting that these preparations are an equilibrium mixture of TLCK-thrombin and alpha-thrombin and cannot be used for evaluating competition between these two agents. In contrast, alpha-thrombin activity was completely inhibited by PPACK at 15-fold molar excess. PPACK-thrombin, free of unreacted PPACK and devoid of residual alpha-thrombin activity, did not markedly affect platelet shape change at concentrations as high as 1 mumol/L, but inhibited aggregation and secretion in intact platelets activated with the minimal concentration of alpha-thrombin causing a full response (0.3 to 0.5 nmol/L) and yielded a 50% inhibition constant (IC50) for inhibition of aggregation by PPACK-thrombin of 110 nmol/L. This inhibition was specific for alpha-thrombin-induced platelet activation, and no inhibition was seen with activation induced by ADP, collagen, epinephrine, ristocetin, or arachidonate. At these low alpha-thrombin concentrations (approximately 0.4 nmol/L), a persistent cytoplasmic acidification was observed of -0.062 +/- 0.016 pH units, although alkalinization was observed at higher alpha-thrombin concentrations (greater than 1 nmol/L). While inhibition of aggregation and secretion occurred when alpha-thrombin and PPACK-thrombin were added simultaneously, inhibition of cytoplasmic acidification and of the elevation of cytoplasmic [Ca2+] induced by low concentrations of alpha- thrombin (0.4 nmol/L) occurred only if platelets were preincubated with PPACK-thrombin for 5 minutes before the addition of alpha-thrombin. In platelets treated with Serratia marcescens protease to remove glycoprotein lb (GPlb), alpha-thrombin-induced shape change was attenuated but persisted in the presence of a high concentration (2 mumol/L) of PPACK-thrombin, although aggregation and secretion were inhibited, as seen in intact platelets. The IC50 value for inhibition of aggregation by PPACK-thrombin was approximately 1 mumol/L at the higher alpha-thrombin concentrations (5 nmol/L) required for full activation in this case. These results suggest that PPACK-thrombin may be a useful probe of platelet function since it specifically blocks platelet aggregation and secretion induced by alpha-thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)

PPACK-thrombin inhibits thrombin-induced platelet aggregation and cytoplasmic acidification but does not inhibit platelet shape change

Blood ◽  
1990 ◽  
Vol 75 (10) ◽  
pp. 1989-1990
Author(s):  
NJ Greco ◽  
TE Jr Tenner ◽  
NN Tandon ◽  
GA Jamieson
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Shape Change ◽  
Equilibrium Mixture ◽  
High Concentration ◽  
Chloromethyl Ketone ◽  
Molar Excess ◽  
Thrombin Activity ◽  
Cytoplasmic Acidification ◽  
Platelet Shape

We have re-evaluated the previously reported ability of TLCK-thrombin (N alpha-tosyl-L-lysine chloromethyl ketone-treated alpha-thrombin) and PPACK-thrombin (D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone- treated alpha-thrombin) to inhibit alpha-thrombin-induced platelet activation (Harmon JT, Jamieson GA: J Biol Chem 261:15928, 1986; and Harmon JT, Jamieson GA: Biochemistry 27:2151, 1988). Despite several cycles of derivatization with TLCK (10,000-fold molar excess), preparations of TLCK-thrombin have been found to contain about 4% residual alpha-thrombin activity, suggesting that these preparations are an equilibrium mixture of TLCK-thrombin and alpha-thrombin and cannot be used for evaluating competition between these two agents. In contrast, alpha-thrombin activity was completely inhibited by PPACK at 15-fold molar excess. PPACK-thrombin, free of unreacted PPACK and devoid of residual alpha-thrombin activity, did not markedly affect platelet shape change at concentrations as high as 1 mumol/L, but inhibited aggregation and secretion in intact platelets activated with the minimal concentration of alpha-thrombin causing a full response (0.3 to 0.5 nmol/L) and yielded a 50% inhibition constant (IC50) for inhibition of aggregation by PPACK-thrombin of 110 nmol/L. This inhibition was specific for alpha-thrombin-induced platelet activation, and no inhibition was seen with activation induced by ADP, collagen, epinephrine, ristocetin, or arachidonate. At these low alpha-thrombin concentrations (approximately 0.4 nmol/L), a persistent cytoplasmic acidification was observed of -0.062 +/- 0.016 pH units, although alkalinization was observed at higher alpha-thrombin concentrations (greater than 1 nmol/L). While inhibition of aggregation and secretion occurred when alpha-thrombin and PPACK-thrombin were added simultaneously, inhibition of cytoplasmic acidification and of the elevation of cytoplasmic [Ca2+] induced by low concentrations of alpha- thrombin (0.4 nmol/L) occurred only if platelets were preincubated with PPACK-thrombin for 5 minutes before the addition of alpha-thrombin. In platelets treated with Serratia marcescens protease to remove glycoprotein lb (GPlb), alpha-thrombin-induced shape change was attenuated but persisted in the presence of a high concentration (2 mumol/L) of PPACK-thrombin, although aggregation and secretion were inhibited, as seen in intact platelets. The IC50 value for inhibition of aggregation by PPACK-thrombin was approximately 1 mumol/L at the higher alpha-thrombin concentrations (5 nmol/L) required for full activation in this case. These results suggest that PPACK-thrombin may be a useful probe of platelet function since it specifically blocks platelet aggregation and secretion induced by alpha-thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition by sodium nitroprusside or PGE1 of tyrosine phosphorylation induced in platelets by thrombin or ADP

1992 ◽  
Vol 262 (3) ◽  
pp. C701-C707 ◽  
Author(s):  
A. Oda ◽  
B. J. Druker ◽  
M. Smith ◽  
E. W. Salzman
Keyword(s):  
Sodium Nitroprusside ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Shape Change ◽  
High Concentration ◽  
Serotonin Secretion ◽  
Granule Secretion ◽  
Camp And Cgmp ◽  
Platelet Shape ◽  
Platelet Functions

Upon platelet activation, numerous proteins are known to be tyrosine phosphorylated. To investigate the mechanisms of the regulation of tyrosine phosphorylation and its physiological significance, the effects on tyrosine phosphorylation of agents that elevate the platelet level of the cyclic nucleotides cAMP and cGMP were examined in aspirin-treated gel-filtered platelets by Western blotting with a specific antiphosphotyrosine antibody. The effects of these agents on other aspects of platelet activation, i.e., aggregation, secretion, and elevation of the concentration of cytosolic ionized calcium ([Ca2+]i), were also examined in parallel experiments. Tyrosine phosphorylation in platelets activated by alpha-thrombin (1 nM) was inhibited by prostaglandin (PG) E1 (2 microM) or by sodium nitroprusside (100 microM). Elevation of [Ca2+]i, aggregation, and serotonin secretion was also strongly inhibited. On the other hand, a higher concentration of alpha-thrombin (10 nM) induced tyrosine phosphorylation of the same proteins, elevation of [Ca2+]i, platelet aggregation, and serotonin secretion, irrespective of pretreatment of platelets by either PGE1 or sodium nitroprusside. Inhibition by sodium nitroprusside of tyrosine phosphorylation induced by alpha-thrombin (1 nM) was accompanied by an increased concentration of cGMP. 8-BrcGMP (2 mM) also inhibited tyrosine phosphorylation and aggregation, although less than sodium nitroprusside. ADP (20 microM) induced platelet shape change and tyrosine phosphorylation of only a few proteins; these effects were also inhibited by either PGE1 or sodium nitroprusside. Thus tyrosine phosphorylation in platelets can be inhibited by elevation of either cAMP or cGMP, an effect that is overcome by a high concentration of thrombin, resulting in granule secretion and aggregation. Some of the proteins that are tyrosine phosphorylated may be important in the regulation of platelet functions.

scholarly journals The Effect of Tirofiban on Fibrinogen/Agonist-Induced Platelet Shape Change and Aggregation

2008 ◽  
Vol 14 (3) ◽  
pp. 295-302 ◽  
Author(s):  
I. Anita Jagroop ◽  
Dimitri P. Mikhailidis
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Adenosine Diphosphate ◽  
Vascular Events ◽  
Spontaneous Platelet Aggregation ◽  
Induced Aggregation ◽  
Platelet Shape

There is evidence linking raised plasma fibrinogen (fib) and platelet hyperactivity with vascular events. One way to inhibit platelets is to block the platelet membrane glycoprotein (GP) IIb/IIIa receptor, which binds circulating fib or von Willebrand factor and cross-links platelets at the final common pathway to platelet aggregation. Tirofiban is a potent and specific fib receptor antagonist, used in the treatment of unstable angina. The authors assessed the effect of tirofiban on spontaneous platelet aggregation (SPA), fib-induced, serotonin (5HT)-induced, and adenosine diphosphate (ADP)-induced aggregation in whole blood by calculating the percentage free platelet count. These various agonists were used alone and in combination. The authors also measured the effect of tirofiban on agonists-induced (ADP, 5HT) platelet shape change (PSC). The effect of fib on PSC was also evaluated in platelet-rich plasma using a high-resolution (0.07 fL) channelyzer. Tirofiban significantly inhibited SPA, fib (2, 4, 8 g/L), ADP, ADP + fib combination, and 5HT-induced aggregation. Tirofiban had no effect on agonist-induced PSC. There was no apparent change in platelet volume with fib. In conclusion, tirofiban does not appear to have an effect on PSC, an early phase of platelet activation. Tirofiban seems to be a nonspecific and an effective inhibitor of platelet aggregation (a later phase of platelet activation) in whole blood. The clinical significance of these findings remains to be established.

Light Scattering and Platelet Aggregation

1975 ◽  
Author(s):  
J. F. Stoltz ◽  
A. Larcan ◽  
J. F. Batoz ◽  
F. Streif
Keyword(s):  
Experimental Study ◽  
Light Scattering ◽  
Platelet Aggregation ◽  
Shape Change ◽  
Light Variation ◽  
Aggregation Kinetics ◽  
Specific Absorption ◽  
Nephelometric Method ◽  
Platelet Concentration ◽  
Platelet Shape

After a short recall of the theories concerning nephelometry and light scattering, the authors develop their experimental study, which is divided into three parts :- platelets absorption i.e. wave lengths- light variation curves transmitted or scattered according to platelet concentration- aggregation by nephelometric method and by light scattering.These experiments allow the authors to conclude that platelets do not present a specific absorption; that the variations of the light transmitted or scattered is exponential in function of the platelet concentration and that the aggregation test affords essentially a measurement of the decrease of the number of free platelets in the medium.Besides, they observe that the measures of aggregation kinetics, or the problem of platelet shape change are not specific and should be investigated with the help of other methods.This work was supported by D. R. M. E. grant (Biological Section).

scholarly journals Mechanism of Action of Halofenate, A Platelet Inhibitor

1977 ◽  
Author(s):  
G. R. Favis ◽  
R. W. Colman
Keyword(s):  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Thiobarbituric Acid ◽  
Serotonin Release ◽  
Prostaglandin Synthesis ◽  
Second Phase ◽  
Change Curve ◽  
Cellulose Column ◽  
Platelet Shape

Halofenate (Hal) has previously been shown to inhibit epinephrine (Epi) and ADP induced platelet aggregation and C14-serotonin release. We further investigated the site of action of Hal by examining platelet shape change as a membrane event and malondialdehyde (MDA) formation as a measure of prostaglandin synthesis. Platelet-rich-plasma (PRP) with and without Hal wasdiluted in an EDTA buffer and examined in a spectrophotometer modified for stirring and maintained at 37°. ADP induced increase in absorbance was recorded and the velocity of the shape change curve was plotted against ADP concentration. MDA production was measured by the thiobarbituric acid assay and utilized a DEAE-52 cellulose column to concentrate the chromogen. Hal in pharmacologic concentrations (.96mM) had no effect on Epi induced primary aggregation or on ADP induced shape change. However, at higher than pharmacologic amounts (3.36mM), Hal did inhibit ADP induced shape change. Epi-induced MDA formation (.18μM-.33μM) normally occurs concomitant with the second phase of aggregation and serotonin release but was markedly decreased by Hal (.06μM-.085μM). This inhibition was not due to a direct effect on prostaglandin synthesis since sodium arachi-donate (1mM) caused secondary aggregation in PRP treated with Hal but not PRP treated with aspirin (4mM). Hal (.96mM) does not seem to inhibit platelet aggregation through an inhibition of ADP induced shape change or of Epi induced primary aggregation. Since Hal treated platelets respond to arachidonate, Hal must work at some earlier step than arachidonate induced prostaglandin synthesis. We suggest that this may be an alteration of the platelet membrane structure which makes ADP and Epi binding sites less accessible or which impairs arachidonic acid release by phospholipase. Decreased MDA formation and inhibition of aggregation would then be secondary to this membrane change.

Possible Differences In The Mode Of Action Of Ditazole And Non-Steroidal Anti-Inflammatory Drugs As Potential Antithrombotic Agents

1981 ◽  
Author(s):  
A K Sim ◽  
A P McCraw ◽  
L Caprino ◽  
F Antonetti ◽  
L Morelli
Keyword(s):  
Platelet Aggregation ◽  
Mode Of Action ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Dose Range ◽  
Oral Drug ◽  
Inflammatory Drugs ◽  
Anti Inflammatory ◽  
Induced Aggregation ◽  
Platelet Shape

Ditazole (4,5-diphenyl-2-diethanolamino-oxazole), a weak anti-inflammatory drug, has been shown to be a potent inhibitor of platelet aggregation, adhesiveness and bleeding time. Acetylsalicylic acid (ASA), dipyridamole and a combination of these two drugs induced a platelet shape change which was much shorter lasting than their effect on platelet aggregation. Conversely, similar doses of ditazole induced a potent shape change but no effect on aggregation. Ditazole has now been shown to reversibly antagonise thromboxane A2 (TXA2)-induced contraction of rabbit aortic strips at an optimal concentration of 25 μm in the perfusate. Separately, over a dose range of 50-400 mg/kg/p.o., TXA2 production was inhibited between 39% and 85% in spontaneously clotted rabbit blood. In addition, we have shown that TXA2 formation following arachidonic acid-induced aggregation of platelet-rich plasma (PRP) is similarly inhibited. Ditazole however did not inhibit prostacyclin (PGI2) production in rabbit aortic rings following oral drug administration over a dose range of 50-400 mg/kg. At 1000 and 2000 mg/kg PGI2 production was inhibited by 23% and 41% respectively. TXA2 and PGI2 levels were measured by radioimmunoassay of their stable derivatives TXB2 and 6-keto-PGF1α. It is suggested that the mode of action of ditazole may be more specific than the cyclo-oxygenase/PG-synthetase blocking activity of most other non-steroidal anti-inflammatory drugs.

scholarly journals The effect of aggregation and release on platelet prothrombin- converting activity

Blood ◽  
1979 ◽  
Vol 54 (3) ◽  
pp. 659-672 ◽  
Author(s):  
AC Cox ◽  
P Inyangetor ◽  
CT Esmon ◽  
BN White
Keyword(s):  
Platelet Aggregation ◽  
Prostaglandin E1 ◽  
Dense Body ◽  
Shape Change ◽  
Factor Xa ◽  
Dense Bodies ◽  
Activated Platelets ◽  
Induced Aggregation ◽  
Body Secretion ◽  
Platelet Shape

Abstract Platelets provide a procoagulant activity for the conversion of prothrombin to thrombin during normal hemostatis. This activity designated as platelet prothrombin-converting activity (PPCA) was monitored as rate of thrombin production in a two-stage assay using gel- filtered bovine platelets, factor Xa, and prothrombin. Expression of PPCA was not associated with ADP-induced release or platelet shape change but was associated with aggregation. Release of the contents of dense bodies, measured by release of 14C-5-hydroxytryptamine, was not required for expression of PPCA during platelet aggregation. During the PPCA assay, 5-hydroxytrypamine was released, but only after onset of thrombin production. Furthermore, the release of 5-hydroxytryptamine was retarded during the assay by the addition of 2 mM theophylline and 100 nM prostaglandin E1 without a comparable reduction in PPCA. In addition, 125I-factor-Xa was bound in greater amounts to platelets (aspirin-treated) after ADP-induced aggregation (without detectable release) than to unactivated control platelets. Finally, the PPCA of the ADP-activated platelets was saturated with respect to factors Xa and Va at less than 1 nM concentrations, indicating that the aggregation induced by ADP leads to the exposure of specific procoagulant sites by some process other than dense body secretion.

Roles of SLP-76, phosphoinositide 3-kinase, and gelsolin in the platelet shape changes initiated by the collagen receptor GPVI/FcRγ-chain complex

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3786-3792 ◽  
Author(s):  
Hervé Falet ◽  
Kurt L. Barkalow ◽  
Vadim I. Pivniouk ◽  
Michael J. Barnes ◽  
Raif S. Geha ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Shape Change ◽  
Chain Complex ◽  
Actin Assembly ◽  
Coated Surface ◽  
Phosphoinositide 3 Kinase ◽  
Collagen Receptor ◽  
Shape Changes ◽  
Platelet Shape

Abstract How platelet shape change initiated by a collagen-related peptide (CRP) specific for the GPVI/FcRγ-chain complex (GPVI/FcRγ-chain) is coupled to SLP-76, phosphoinositide (PI) 3-kinase, and gelsolin is reported. As shown by video microscopy, platelets rapidly round and grow dynamic filopodial projections that rotate around the periphery of the cell after they contact a CRP-coated surface. Lamellae subsequently spread between the projections. All the actin-driven shape changes require SLP-76 expression. SLP-76 is essential for the Ca++mobilization induced by CRP, whereas PI 3-kinase only modulates it. The extension of lamellae requires net actin assembly and an exposure of actin filament barbed ends downstream of PI 3-kinase. Gelsolin expression is also required for the extension of lamellae, but not for the formation of filopodia. Altogether, the data describe the role of SLP-76 in the platelet activation initiated by GPVI/FcRγ-chain and the roles of PI 3-kinase and gelsolin in lamellae spreading.

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.

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