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Aggregation and/or oxygenated products of arachidonic acid are not required for collagen-induced deacylation of phosphatidylcholine in human platelets.

ABSTRACT: In the present study the effects of collagen on platelet aggregation and arachidonic acid (AA) mobilization, specifically from phosphatidylcholine (PC), were investigated in the presence and absence of BW755C, a selective inhibitor of cyclo-oxygenase and lipoxygenases. The inhibition of cyclo-oxygenase and lipoxygenase(s) by BW755C (75 microM) resulted in severe impairment in collagen-induced platelet aggregation. In the presence of BW755C, the aggregation response amounted to 14, 26, 37 and 49% of the corresponding controls (without BW755C) at 10, 25, 50 and 100 micrograms of collagen respectively. On the contrary, the amount of AA released from PC, which ranged from 3.5 to 8.6 nmol/10(9) platelets, in response to the action of collagen (10-100 micrograms) remained unaffected by the presence of BW755C. Substantial amounts of non-esterified AA were detected in the free fatty acid fractions obtained from collagen-stimulated platelets in the presence as well as in the absence of BW755C. However, the presence of BW755C caused a greater accumulation of free AA (mass) and this ranged from 4 to 16 nmol, depending upon the amount of collagen. In addition, small increases in free stearic and oleic acids were observed in collagen-stimulated platelets as compared with unstimulated platelets. The amount of AA lost from PC represented 67, 80, 49 and 52% of the free AA obtained at 10, 25, 50 and 100 micrograms of collagen respectively. Our results adhesion of platelets to collagen fibres may be responsible for much of the AA release from PC Furthermore, these results demonstrate that aggregation and/or cyclo-oxygenase/lipoxygenase metabolites are not obligatory for the release of AA from PC in collagen-stimulated human platelets.

SUBMITTER: Piche LA

PROVIDER: S-EPMC1133401 | biostudies-other | 1989 Oct

REPOSITORIES: biostudies-other

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Project description:Cytosolic Ca2+ levels and arachidonate liberation were investigated in platelets loaded with the fluorescent Ca2+ indicator dye fura-2, and labelled with [3H]arachidonate. Fura-2 was used in preference to quin2 because the latter interfered with [3H]arachidonate labelling of phospholipids. From a resting free Ca2+ level of around 100 nM, ionomycin (10-200 nM) evoked an instantaneous, concentration-dependent increase in cytosolic Ca2+ that only resulted in [3H]arachidonate liberation (up to 4-fold over control) at Ca2+ levels greater than 1 microM. Addition of collagen (10 micrograms/ml) evoked an elevation in Ca2+ up to 461 +/- 133 nM. These changes in Ca2+ were accompanied by a 2-4-fold elevation in [3H]arachidonate with depletion of [3H]phosphatidylcholine by 17 +/- 4% and [3H]phosphatidylinositol by 41 +/- 7%. Indomethacin (10 microM) reduced the elevation in Ca2+ by collagen to 115 +/- 18 nM but did not significantly inhibit the 2-4-fold increase in [3H]arachidonate. [3H]Phosphatidylcholine and [3H]phosphatidylinositol were decreased by 9 +/- 7% and 10 +/- 6%, respectively, with collagen in the presence of indomethacin. Stimulation of phosphoinositide turnover by collagen in the presence and absence of indomethacin was indicated by [32P]phosphatidate formation in cells prelabelled with [32P]Pi. This phosphatidate formation was decreased (75%) by the presence of indomethacin. In the presence of indomethacin, phorbol myristate acetate (20 nM) alone or in combination with ionomycin (30 nM) failed to stimulate arachidonate liberation despite a marked stimulation of aggregation. These results indicate that, whereas ionomycin requires Ca2+ in the microM range for arachidonate liberation, collagen, notably in the presence of indomethacin, does so at basal Ca2+ levels. The mechanisms underlying the regulation of arachidonate release by collagen are not clear, but do not appear to involve activation of protein kinase C, or an elevation of cytosolic free Ca2+.

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Aggregation and/or oxygenated products of arachidonic acid are not required for collagen-induced deacylation of phosphatidylcholine in human platelets.

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