scholarly journals Fibrinogen-independent aggregation and deaggregation of human platelets: studies in two afibrinogenemic patients

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 221-226 ◽  
Author(s):  
M Cattaneo ◽  
RL Kinlough-Rathbone ◽  
A Lecchi ◽  
C Bevilacqua ◽  
MA Packham ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Plasma Proteins ◽  
Platelet Rich Plasma ◽  
Platelet Activating Factor ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Human Fibrinogen ◽  
Control Subjects ◽  
Platelet Aggregates ◽  
And Control

Abstract Platelets from two afibrinogenemic patients were used to determine whether fibrinogen is essential for platelet aggregation and to examine whether released fibrinogen contributes to the stabilization of platelet aggregates when platelets have been induced to aggregate and release their granule contents by stimulation with thrombin. The addition of adenosine diphosphate (ADP) to platelet-rich plasma (PRP) or to suspensions of washed platelets from the afibrinogenemic patients caused the formation of small aggregates, which was either not inhibited or only slightly inhibited by the F(ab')2 fragments of an antibody to fibrinogen but was inhibited by an antibody (10E5) to glycoprotein IIb/IIIa. Thus there is a component of ADP-induced platelet aggregation that is not dependent on fibrinogen or other plasma proteins but is dependent on glycoprotein IIb/IIIa. There was little difference in the extent of aggregation and the release of granule contents of normal and afibrinogenemic platelets in response to the release-inducing agents collagen, platelet-activating factor (PAF), sodium arachidonate, or thrombin. With normal or afibrinogenemic platelets, aggregation by thrombin (0.2 U/mL or higher) was not inhibited by the F(ab')2 fragments of an antibody to human fibrinogen. Deaggregation by combinations of inhibitors of platelets aggregated by 1 U/mL thrombin showed no difference between platelets from afibrinogenemic and control subjects, indicating that released fibrinogen does not make a major contribution to the stabilization of platelet aggregates formed by thrombin stimulation.

scholarly journals Fibrinogen-independent aggregation and deaggregation of human platelets: studies in two afibrinogenemic patients

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 221-226
Author(s):  
M Cattaneo ◽  
RL Kinlough-Rathbone ◽  
A Lecchi ◽  
C Bevilacqua ◽  
MA Packham ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Plasma Proteins ◽  
Platelet Rich Plasma ◽  
Platelet Activating Factor ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Human Fibrinogen ◽  
Control Subjects ◽  
Platelet Aggregates ◽  
And Control

Platelets from two afibrinogenemic patients were used to determine whether fibrinogen is essential for platelet aggregation and to examine whether released fibrinogen contributes to the stabilization of platelet aggregates when platelets have been induced to aggregate and release their granule contents by stimulation with thrombin. The addition of adenosine diphosphate (ADP) to platelet-rich plasma (PRP) or to suspensions of washed platelets from the afibrinogenemic patients caused the formation of small aggregates, which was either not inhibited or only slightly inhibited by the F(ab')2 fragments of an antibody to fibrinogen but was inhibited by an antibody (10E5) to glycoprotein IIb/IIIa. Thus there is a component of ADP-induced platelet aggregation that is not dependent on fibrinogen or other plasma proteins but is dependent on glycoprotein IIb/IIIa. There was little difference in the extent of aggregation and the release of granule contents of normal and afibrinogenemic platelets in response to the release-inducing agents collagen, platelet-activating factor (PAF), sodium arachidonate, or thrombin. With normal or afibrinogenemic platelets, aggregation by thrombin (0.2 U/mL or higher) was not inhibited by the F(ab')2 fragments of an antibody to human fibrinogen. Deaggregation by combinations of inhibitors of platelets aggregated by 1 U/mL thrombin showed no difference between platelets from afibrinogenemic and control subjects, indicating that released fibrinogen does not make a major contribution to the stabilization of platelet aggregates formed by thrombin stimulation.

Ticlopidine Facilitates the Deaggregation of Human Platelets Aggregated by Thrombin

1994 ◽  
Vol 71 (01) ◽  
pp. 091-094 ◽  
Author(s):  
M Cattaneo ◽  
B Akkawat ◽  
R L Kinlough-Rathbone ◽  
M A Packham ◽  
C Cimminiello ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Platelet Aggregation ◽  
Prostaglandin E1 ◽  
Human Platelet ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Before And After ◽  
Normal Human ◽  
Platelet Aggregates ◽  
Induced Aggregation

SummaryNormal human platelets aggregated by thrombin undergo the release reaction and are not readily deaggregated by the combination of inhibitors hirudin, prostaglandin E1 (PGE1) and chymotrypsin. Released adenosine diphosphate (ADP) plays an important role in the stabilization of thrombin-induced human platelet aggregates. Since ticlopidine inhibits the platelet responses to ADP, we studied thrombin-induced aggregation and deaggregation of 14C-serotonin-labeled platelets from 12 patients with cardiovascular disease before and 7 days after the oral administration of ticlopidine, 250 mg b.i.d. Before and after ticlopidine, platelets stimulated with 1 U/ml thrombin aggregated, released about 80–90% 14C-serotinin and did not deaggregate spontaneously within 5 min from stimulation. Before ticlopidine, hirudin (5× the activity of thrombin) and PGE1 (10 μmol/1) plus chymotrypsin (10 U/ml) or plasmin (0.06 U/ml), added at the peak of platelet aggregation, caused slight or no platelet deaggregation. After ticlopidine, the extent of platelet deaggregation caused by the same inhibitors was significantly greater than before ticlopidine. The addition of ADP (10 μmol/1) to platelet suspensions 5 s after thrombin did not prevent the deaggregation of ticlopidine-treated platelets. Thus, ticlopidine facilitates the deaggregation of thrombin-induced human platelet aggregates, most probably because it inhibits the effects of ADP on platelets.

Effects of Lead Acetate on Platelet Aggregation, Ultrastructure and Serotonin Release

1971 ◽  
Vol 26 (03) ◽  
pp. 455-466 ◽  
Author(s):  
R. B Davis ◽  
G. C Holtz
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Lead Acetate ◽  
Platelet Rich Plasma ◽  
Adenosine Diphosphate ◽  
Blood Platelet ◽  
Human Platelets ◽  
Serotonin Release ◽  
Aggregation Of Platelets

SummaryThe effects of lead on blood platelet function and ultrastructure have been investigated. Lead acetate was injected intravenously in 27 rats and was added to rat and human platelet rich plasma in vitro. In vitro studies showed that concentrations of 2.5 × 10-3 M lead acetate reduced or blocked aggregation of rat and human platelets by adenosine diphosphate, collagen, and thrombin. Radioactive serotonin release from human platelets was inhibited by 10-4 M lead acetate. One hour after the injection of lead, platelet aggregation by thrombin was reduced, but platelet aggregation by adenosine diphosphate and collagen showed little change. Three days after lead, aggregation of platelets by collagen and thrombin was blocked and aggregation by adenosine diphosphate reduced. Thrombocytopenia was present 4 days after intravenous lead acetate. Electron micrographs of platelets showed that the mean number of mitochondria per platelet was increased, whereas alpha granules were reduced. Dense bodies were not significantly changed. Lead acetate affects platelet function in concentrations reported in human bone marrow in lead poisoning, and may relate to the binding of free sulfhydryl groups by lead.

Inhibition of Thrombin-, Epinephrine- and Collagen-Induced Platelet Aggregation by α1-Acid Glycoprotein

1979 ◽  
Author(s):  
P. Andersen ◽  
C. Eika
Keyword(s):  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Adenosine Diphosphate ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Clotting Time ◽  
Acid Glycoprotein ◽  
High Concentrations ◽  
Spontaneous Platelet Aggregation ◽  
Induced Aggregation

α1-Acid glycoprotein (α1,-acid GP) isolated from human plasma was found to inhibit thrombin-induced aggregation of washed human platelets (0.05 NIH U/ml final conc.), and inhibition was complete with physiological concentrations of α1-acid GP (1.0-1.5 g/1 final conc.). The inhibitory effect seemed to occur immediately on thrombin addition, thus similar to the effect of heparin previously observed. As opposed to heparin, however, α1-acid GP did not affect spontaneous platelet aggregation. Furthermore, α1-acid GP (in optimal cone.) reduced the combined inhibitory effect of heparin and antithrombin III on thrombin-induced platelet aggregation, thus consistent with the previous findings using heparin thrombin clotting time.Snyder and Coodley (1976) found α1-acid GP to inhibit platelet aggregation induced by epinephrine and adenosine diphosphate in platelet-rich plasma. As we also found α1-acid GP to inhibit collagen-induced platelet aggregation, α1-acid GP may possibly act as an inhibitor of the release reaction though fairly high concentrations (10 mg/ml final cone.) was needed for complete inhibition.

PLATELET-ACTIVATING FACTOR (PAF)-INDUCED INTRACELLULAR Ca MOBILIZATION IN HUMAN PLATELETS.

1987 ◽  
Author(s):  
K Matsuno ◽  
F Katabami ◽  
M Koyama ◽  
K Abe ◽  
K Sakurada ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Light Transmission ◽  
Platelet Rich Plasma ◽  
Platelet Activating Factor ◽  
Human Platelets ◽  
Dependent Manner ◽  
Platelet Suspension ◽  
Fura 2 ◽  
Intracellular Ca ◽  
Specific Antagonist

PAF-induced intracellular Ca2+ mobilization and platelet aggregation were investigated in human platelets. Cytosolic free Ca2+ concentration ([Ca2+]cyt) was measured by using fluorescent 45Ca2+ probe quin2 and fura-2, and photoprotein aequorin. Ca2+ uptake was measured after stimulation by PAF. Platelet aggregation was studied by recording the change in light transmission with platelet rich plasma (PRP) or washed platelet suspension (WPS).These three Ca2+ -indicators could determine the elevation of [Ca2+ ]cyt that was stimulated by PAF in the presence of extra- cellular Ca2+ (quin2 method: 98.2nM to 289.7nM; fura-2 method: 102.OnM to 351.4nM; aequorin method: 4.1μM to 8.2μM). In the absence of extracellular Ca2+ , however, little elevation of [Ca2+ ]cyt was detected after stimulation by PAF. PAF could evoke the transient Ca2+ uptake.New PAF specific antagonist, ONO-6240 inhibited PAF-induced platelet aggregation at a concentration from lμM dose-dependently, whereas it didn’t inhibit collagen- and thrombin-induced platelet aggregation at a concentration of lOOyM. ONO-6240 inhibited PAF- induced increase in [Ca2+ ]cyt in a dose-dependent manner as deter mined by these Ca2+ -indicators, as well as platelet aggregation.These results suggest the increase in [Ca2+ ]cyt is responsible for platelet aggregation induced by PAF, and the increased Ca2+ is derived from external Ca2+ influx chiefly.

PLATELET AGGREGATION DYNAMICS TO ADENOSINE DIPHOSPHATE IN NON-STIRRED SUSPENSIONS: LONG-RANGEINTERACTIONS FOR HUMAN, BUT NOT RABBIT, PLATELETS

1987 ◽  
Author(s):  
K Longmire ◽  
M M Frojmovic
Keyword(s):  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Linear Regression Analysis ◽  
Time Lag ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Dense Granule ◽  
Second Phase ◽  
Particle Count ◽  
Induced Aggregation

The simplest experimental approach for a theoretical description of platelet aggregation is based on kinetics of early multiplet formation (‹4 platelets per aggregate)occurring with diffusion-dependent particle collisions (no flow). The Smoluchowski theory was used to calculate collision efficiencies, αβ, from a linear plot of platelet particle count (Nt)−1 vs time (t) following addition of adenosine diphosphate (ADP) to citrated platelet-rich-plasma (PRP) for 7 human (H) and 2 rabbit (R) donors. A 0.1 ml sample of PRP was stirred with ADP for 0.5s, then immediately transferred to a 37°C bath for no-stir (diffusion) studies or further stirred with ADP for stir-induced aggregation studies. Samples were fixed with 0.5 ml 0.8% glutaraldehyde with particle count (Nt) determined with a resistive counter and % aggregation (PA) computed (reproducibility/sensitivity ‹ 5%). For stir conditions, R platelets were as sensitive and as rapidly aggregated by ADP (2-10 μM) as H platelets, with ∼ 1 s time lag for onset of PA. However, for no-stir conditions, linear regression analysis of data for ADP (5-10 μM) induced PA for H platelets for 0-30 s gave αβ = 7.5±4.6 (r = 0.9±0.05). Analysis at longer “diffusion” times showed a second phase (60-300 s) in some H donors with aB = 0.5±0.4 (4/9 donors), while R platelets showed only 1 phase with αβ = 0.65±0.15 (0-60 to 0-900 s) (r = 0.8±0.1). The ADP sensitivity ([ADP]½ corresponding to 50% of maximal changes) for the abnormally rapid PA in no stir H PRP for early times, measured over 0.4-100 μM range, was found to be ∼9 μM (5-17 μM range) and 3.5 μM (3-10 μM) for measurements respectively at 5-10 and 20-30s; these values were ∼ 3-8 × greater than lADPji measured for stirred suspensions for rate/extent of PA or rate of turbidometrically-measured macroaggregation (TA), while › [ADP] threshold for secondary aggregation in TA (10 H donors). These abnormally large aB values and their ADP sensitivity observed for human platelets are consistent with long-range interactions mediated by“chemotactic” agents released from the cells but distinct from normal dense granule release requiring macroaggregation, or by as yet uncharacterized membrane or polymetric bridges.

Diurnal variation in melatonin effect on adenosine triphosphate and serotonin release by human platelets

1990 ◽  
Vol 123 (4) ◽  
pp. 453-458 ◽  
Author(s):  
María de las M. Del Zar ◽  
Marta Martinuzzo ◽  
Daniel P. Cardinali ◽  
Luis O. Carreras ◽  
María I. Vacas
Keyword(s):  
Platelet Aggregation ◽  
Diurnal Variation ◽  
Adenosine Triphosphate ◽  
Platelet Rich Plasma ◽  
Atp Release ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Serotonin Release ◽  
Normal Volunteers ◽  
Dose Dependent

Abstract. The effect of the pineal hormone melatonin on adenosine diphosphate-induced human platelet aggregation and adenosine triphosphate release was assessed in platelet-rich plasma obtained from normal volunteers at 08.30 and 20.30 h. In 10−7-10−5 mol/l concentrations melatonin inhibited ADP-induced platelet aggregation only in the evening (p<0.05). ADP-induced ATP release, an index of platelet secretory processes, showed a generally greater, dose-dependent inhibition after adding melatonin (10−9-10−5 mol/l) at 20.30 h as compared with 08.30 h. The inhibitory activity of melatonin (10−9-10−5 mol/l) on [3H]serotonin release elicited by thrombin in washed human platelets obtained from normal volunteers was dose-dependent; the effect was generally greater at 20.30 h. The activity of the potent platelet anti-aggregating agent prostacyclin did not exhibit diurnal differences with respect to impairing ADP-induced platelet-rich plasma aggregation. These results indicate the existence of a diurnal variation of sensitivity to melatonin in human platelets.

Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

1998 ◽  
Vol 79 (01) ◽  
pp. 177-185 ◽  
Author(s):  
Ashia Siddiqua ◽  
Michael Wilkinson ◽  
Vijay Kakkar ◽  
Yatin Patel ◽  
Salman Rahman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Functional Characterization ◽  
Human Platelets ◽  
Western Blots ◽  
Activated Platelets ◽  
Reducing Conditions ◽  
Fibronectin Binding

SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

Phospholipase C from Clostridium Perfringens Induces Human Platelet Aggregation in Plasma

1988 ◽  
Vol 59 (02) ◽  
pp. 236-239 ◽  
Author(s):  
Giovanna Barzaghi ◽  
Chiara Cerletti ◽  
Giovanni de Gaetano
Keyword(s):  
Platelet Aggregation ◽  
Receptor Antagonist ◽  
Phospholipase C ◽  
Clostridium Perfringens ◽  
Human Platelet ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Platelet Activating Factor ◽  
Inhibitory Effect ◽  
Thromboxane Receptor Antagonist

SummaryWe studied the aggregating effect of different concentrations of phospholipase C (PLC) (extracted from Clostridium perfringens) on human platelet-rich plasma (PRP). PRP was preincubated with PLC for 3 min at 37° C and the platelet aggregation was followed for 10 min. The threshold aggregating concentration (TAG) of PLC was 3-4 U/ml.We also studied the potentiation of PLC with other stimuli on platelet aggregation. Potentiating stimuli, such as arachidonic acid (AA), ADP. Platelet Activating Factor (PAF) and U-46619 (a stable analogue of cyclic endoperoxides) were all used at subthreshold concentrations. We also studied the possible inhibitory effect of aspirin, apyrase, TMQ, a prostaglandin endoper- oxide/thromboxane receptor antagonist and BN-52021, a PAF receptor antagonist. Only aspirin and apyrase were able to reduce aggregation induced by PLC alone and PLC + AA and PLC + ADP respectively. TMQ and BN-52021 were inactive. In ex vivo experiments oral aspirin (500 mg) partially inhibited platelet aggregation induced by PLC alone, PLC + AA and PLC + ADP 2 and 24 h after administration. Aspirin 20 mg for 7 days also reduced aggregation induced by PLC + AA.

Adenosine Diphosphate-Induced Aggregation of Human Platelets in Flow Through Tubes: III. Shear and Extrinsic Fibrinogen-Dependent Effects

1994 ◽  
Vol 71 (01) ◽  
pp. 078-090 ◽  
Author(s):  
H L Goldsmith ◽  
M M Frojmovic ◽  
Susan Braovac ◽  
Fiona McIntosh ◽  
T Wong
Keyword(s):  
Shear Rate ◽  
Single Cells ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Size Analysis ◽  
Fibrinogen Concentration ◽  
Human Fibrinogen ◽  
Shear Rates ◽  
Rate Dependent ◽  
Induced Aggregation

SummaryThe effect of shear rate and fibrinogen concentration on adenosine diphosphate-induced aggregation of suspensions of washed human platelets in Poiseuille flow at 23°C was studied using a previously described double infusion technique and resistive particle counter size analysis (1). Using suspensions of multiple-centrifuged and -washed cells in Tyrodes-albumin [3 × 105 μl−1; (17)] with [fibrinogen] from 0 to 1.2μM, the, rate and extent of aggregation with 0.7 μM ADP in Tyrodes-albumin were measured over a range of mean transit times from 0.2 to 43 s, and at mean tube shear rates, Ḡ, = 41.9, 335 and 1,335 s−1. As measured by the decrease in singlet concentration, aggregation at 1.2 μM fibrinogen increased with increasing Ḡ up to 1,335 s1, in contrast to that previously reported in citratcd plasma, in which aggregation reached a maximum at Ḡ = 335 s−1. Without added fibrinogen, there was no aggregation at Ḡ = 41.9 s1; at Ḡ = 335 s1, there was significant aggregation but with an initial lag time, aggregation increasing further at Ḡ = 1,335 s−1. Without added fibrinogen, aggregation was abolished at all Ḡ upon incubation with the hexapeptide GRGDSP, but was almost unaffected by addition of an F(ab’)2 fragment of an antibody to human fibrinogen. Aggregation in the absence of added fibrinogen was also observed at 37°C. The activation of the multiple-washed platelets was tested using flow cytometry with the fluorescently labelled monoclonal antibodies FITC-PAC1 and FITC-9F9. It was shown that 57% of single cells in unactivated PRT expressed maximal GPIIb-IIIa fibrinogen receptors (MoAb PAC1) and 54% expressed pre-bound fibrinogen (MoAb 9F9), with further increases on ADP activation. However, incubation with GRGDSP and the F(ab’)2 fragment did not inhibit the prebound fibrinogen. Moreover, relatively unactivated cells (8% expressing receptor, 14% prebound fibrinogen), prepared from acidified cPRP by single centrifugation with 50 nM of the stable prostacyclin derivative, ZK 36 374, and resuspension in Tyrodes-albumin at 5 × 104 μl−1, aggregated with 2 and 5 μM ADP at Ḡ = 335 and 1,335 s−1 in the absence of added fibrinogen. We therefore postulate that a protein such as von Willebrand factor, secreted during platelet isolation or in flow at sufficiently high shear rates, may yield the observed shear-rate dependent aggregation without fibrinogen.

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