Measurement of Fibrinogen Concentrations in Suspensions of Washed Rabbit and Human Platelets by Radioimmunoassays

Harfenist, EJ; Jl, Wrana; Packham, MA; Mustard, J. F.

doi:10.1055/s-0038-1661247

Cited by 16 publications

(7 citation statements)

References 28 publications

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“…There are a number of circumstances in which ADP stimulation of human platelets causes thromboxane formation and secretion of granule contents, confounding the interpretation of studies with human platelets. Since rabbit platelets do not form thromboxanes and undergo very little secretion ofgranule contents when stimulated with ADP [27], our results are only comparable with studies with human platelets aggregated by ADP under conditions in which thromboxane formation and secretion of granule contents do not occur to an appreciable extent.…”

Section: Increase Due To Stimulationcontrasting

confidence: 83%

“…1P3 is formed by thrombin-stimulated platelets [20][21][22][23], and has been shown to release Ca21 from intracellular membranes of platelets [24] and to induce secretion of granule contents from permeabilized platelets [25,26]. In contrast, stimulation of rabbit platelets with ADP, which induces very little secretion of granule contents [27], is not associated with the formation of IP3 [23]. In the present study, we have examined the effect of ethanol, at a concentration that is physiologically tolerable, on ADPor thrombin-induced aggregation, secretion of granule contents, and production of inositol phosphates by rabbit platelets, to determine whether the inhibitory effect of ethanol on platelet function is associated with an effect of ethanol on the production of IP3.…”

Section: Introductionmentioning

confidence: 99%

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Thrombin-induced inositol trisphosphate production by rabbit platelets is inhibited by ethanol

Rand

Vickers²,

Kinlough-Rathbone³

et al. 1988

Biochemical Journal

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Ethanol has an inhibitory effect on some platelet functions, but the mechanisms by which it exerts this effect are not known. Using suspensions of washed platelets, we observed that ethanol (1-9 mg/ml) did not affect the aggregation of rabbit platelets stimulated with ADP (0.5-10 microM). When platelets were prelabelled with 5-hydroxy[14C]tryptamine, aggregation and secretion of granule contents in response to thrombin (0.01-0.10 unit/ml) were not inhibited by ethanol, but these responses to thrombin at lower concentrations (less than 0.01 unit/ml) were inhibited by ethanol (2-4 mg/ml). Platelets were prelabelled with [3H]inositol so that increases in inositol phosphates upon stimulation could be assessed by measuring the amount of label in these compounds. ADP-induced increases in IP (inositol phosphate) and IP2 (inositol bisphosphate) were not affected by ethanol. IP3 (inositol trisphosphate) was not changed by ADP or ethanol. Although ethanol did not affect the increases in IP, IP2 and IP3 caused by stimulation of platelets with thrombin at concentrations greater than 0.01 unit/ml, ethanol did inhibit the increases observed at 2 and 3 min in these inositol phosphates caused by lower concentrations of thrombin (less than 0.01 unit/ml). Since ADP did not cause formation of IP3 in rabbit platelets, and since no thromboxane B2 was detected in platelets stimulated with the lower concentrations of thrombin, it is unlikely that the inhibitory effect of ethanol in IP3 formation was due to effects on further stimulation of platelets by released ADP or by thromboxane A2. Ethanol may inhibit platelet responses to thrombin by inhibiting the production of the second messenger, IP3.

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Section: Increase Due To Stimulationcontrasting

confidence: 83%

Section: Introductionmentioning

confidence: 99%

Thrombin-induced inositol trisphosphate production by rabbit platelets is inhibited by ethanol

Rand

Vickers²,

Kinlough-Rathbone³

et al. 1988

Biochemical Journal

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“…In contrast, ADP-stimulated washed rabbit platelets aggregate extensively in the absence of added fibrinogen, even in the absence of the release of the contents of the amine storage granules, although the extent of aggregation is slightly increased by the addition of fibrinogen (9,10). We have shown that insufficient plasma fibrinogen remains associated with washed platelets to support ADP-induced aggregation (9). However, rabbit platelets secrete more fibrinogen (8.2 Jlg/10 9 platelets) into the suspending medium upon ADP stimulation than do human platelets (1.0 Jlg/10 9 platelets), suggesting that secreted fibrinogen may be available to support aggregation, thus explaining the lack of requirement for exogenous fibrinogen (9).…”

Section: Introductionmentioning

confidence: 85%

“…This pattern of fibrinogen binding and dissociation is common to both human and rabbit platelets (3)(4)(5)(6)(7). Washed human platelets fail to aggregate, or aggregate to only a slight extent, when stimulated with ADP in the absence of added fibrinogen under conditions that minimize the release of granule contents (8,9). In contrast, ADP-stimulated washed rabbit platelets aggregate extensively in the absence of added fibrinogen, even in the absence of the release of the contents of the amine storage granules, although the extent of aggregation is slightly increased by the addition of fibrinogen (9,10).…”

Section: Introductionmentioning

confidence: 99%

“…Washed human platelets fail to aggregate, or aggregate to only a slight extent, when stimulated with ADP in the absence of added fibrinogen under conditions that minimize the release of granule contents (8,9). In contrast, ADP-stimulated washed rabbit platelets aggregate extensively in the absence of added fibrinogen, even in the absence of the release of the contents of the amine storage granules, although the extent of aggregation is slightly increased by the addition of fibrinogen (9,10). We have shown that insufficient plasma fibrinogen remains associated with washed platelets to support ADP-induced aggregation (9).…”

Section: Introductionmentioning

confidence: 99%

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Expression of Fibrinogen on the Surface of ADP-Stimulated Platelets: Comparison of Human and Rabbit Platelets

1988

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SummaryFibrinogen is a cofactor in the aggregation of human platelets and must be added to suspensions of washed human platelets forextensive aggregation to occur in response to ADR Although thepatterns of fibrinogen binding and dissociation during plateletaggregation and deaggregation are similar for human and rabbitplatelets, washed rabbit platelets aggregate extensively whenstimulated with ADP even in the absence of added fibrinogen. Todetermine whether secreted fibrinogen was present on the surfaceof ADP-stimulated platelets and available to support aggregation, the binding of 125I-F(ab’)2 fragments of anti-fibrinogen antibodiesto formaldehyde-fixed platelets was measured. Although nofibrinogen was detected on human platelets either before or aftertreatment with ADP, fibrinogen was expressed on the surface ofrabbit platelets after stimulation with ADP. However, thissecreted fibrinogen did not dissociate during deaggregation of theplatelets. Thus, the aggregation of rabbit platelets in the absenceof exogeneous fibrinogen may be supported by secreted fibrino-gen, but a fibrinogen-independent component has not beencompletely ruled out.

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Conditions influencing release of granule contents from human platelets in citrated plasma induced by ADP or the thrombin receptor activating peptide SFLLRN: Direct measurement of percent release of β-thromboglobulin and assessment by flow cytometry of P-selectin expression

Rand¹,

Perry²,

Packham³

et al. 1996

Am. J. Hematol.

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Contrary to a recent report [Rinder et al.: Blood 82:505, 19931, aspirin does inhibit the release of a-granule contents as well as inhibiting the release of dense granule contents by human platelets during ADP-induced aggregation in citrated platelet-rich plasma (PRP). Measurements were: percent release of 14C-serotonin from prelabeled platelets, radio-immunoassay of p-thromboglobulin (PTG), and expression on the platelet surface of the a-granule constituent, P-selectin, by flow cytometry. During the second phase of ADP-induced aggregation, 69.0 2 8.3% of pTG and 54.1 f 4.6% of 14C-serotonin were released (means 2 SEM, n = 13); aspirin treatment reduced these values to 6.0 f 1.2 and 1 .O 0.3%, respectively. In contrast, incubation of platelets with ADP without stirring caused only 6.7 & 1.7% release of pTG and 2.1 2 0.4% release of ''C-serotonin; these low values were not appreciably affected by aspirin. During ADP-induced primary aggrega-tion in PRP anticoagulated with FPRCH,CI (PPACK), only 4.7 2 0.9% release of PTG and no detectable release of 14C-serotonin occurred; aspirin had no effect. In both stirred and unstirred PRP, the thrombin receptor activating peptide, SFLLRN (50 pM), caused at least 75% release of the contents of both granules, which was partially inhibited by aspirin. Upon incubation of platelets with ADP (2-10 pM), the mean fluorescence intensity due to P-selectin was 4 4 % of that induced by SFLLRN. In this unstirred system used for flow cytometry, aspirin treatment caused no significant inhibition of P-selectin expression. Thus, under conditions in which ADP does not cause secondary aggregation (physiologi-cal Ca2+ concentration or unstirred citrated PRP) release of the contents of both types of granules is less than 7% and aspirin is not inhibitory; the P-selectin expression associated with this low percent release is also unaffected by aspirin. However, aspirin does strongly inhibit the extensive release of both a-granule and dense granule contents during ADP-induced secondary aggregation in citrated PRP.

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Measurement of Fibrinogen Concentrations in Suspensions of Washed Rabbit and Human Platelets by Radioimmunoassays

Cited by 16 publications

References 28 publications

Thrombin-induced inositol trisphosphate production by rabbit platelets is inhibited by ethanol

Thrombin-induced inositol trisphosphate production by rabbit platelets is inhibited by ethanol

Expression of Fibrinogen on the Surface of ADP-Stimulated Platelets: Comparison of Human and Rabbit Platelets

Conditions influencing release of granule contents from human platelets in citrated plasma induced by ADP or the thrombin receptor activating peptide SFLLRN: Direct measurement of percent release of β-thromboglobulin and assessment by flow cytometry of P-selectin expression

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