Background: Classic platelet aggregation in citrated plasma is typically irreversible. However, in the presence of physiological extracellular calcium concentration, platelet aggregates are often unstable. The mechanisms of this are not clear.

Aims: We hypothesized that low extracellular calcium induces depletion of intracellular calcium stores (ICS) known to activate calcium-independent phospholipase A2 (iPLA2) producing additional TxA2.

Methods: Fresh whole blood of healthy volunteers was used for all experiments. Platelet aggregation was performed using turbidimetry for platelet-rich plasma (citrated or hirudinated) or washed platelets in Tyrode’s buffer with or without 2 mM CaCl₂. ICS calcium level was measured using Fluo-5N. Structure of aggregates was analyzed by confocal fluorescent microscopy and flow cytometry.

Results: Reversible platelet aggregation was observed in the presence calcium in hirudinated platelet-rich plasma or washed platelet suspension in response to ADP or a combination of adrenalin and serotonin. At low extracellular calcium, the aggregation was irreversible and became reversible upon inhibition of TxA2 synthesis by aspirin or iPLA2 inhibitor bromoenol lactone. Analysis of small aggregates and single platelets by flow cytometry and of large aggregates by confocal microscopy demonstrated rapid depletion of ICS in response to activation only without extracellular calcium. Without stirring, no aggregates formed in response to activation and the ICS depletion did not occur. Depletion of ICS by thapsigargin (an inhibitor of ICS repletion) caused irreversible platelet aggregation even without ADP. Additional destabilization of aggregates in presence of extracellular calcium might occur due to the presence of non-adhesive procoagulant platelets. Approximately 1% of platelets within aggregates became procoаgulant when stimulated by ADP in the presence of calcium and stirring; none were observed without stirring.

Conclusions: Without extracellular calcium platelet aggregates formed in response to ADP under stirring are stabilized by TxA2 synthesized by iPLA2 activated by the ICS depletion. The study was supported by the Russian Science Foundation grant 17-74-20045.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/physiological-extracellular-calcium-levels-impairs-platelet-aggregation-as-a-result-of-ipla2-inhibition-due-to-lack-of-calcium-stores-depletion/