Background: Timely secretion of dense granules by activated platelets is essential for normal hemostasis, however, the underlying kinetics remain poorly understood.

Aims: Define quantitative framework for dense granule secretion and the role of intracellular calcium spikes in activated platelets.

Methods: Our flow-chamber-based microscopy approach enables simultaneous monitoring of dense granule release and calcium oscillations in platelets attached to fibrinogen-coated surface.

Results: Upon thrombin activation, individual dense granules within each cell are secreted in several distinct sets, rather than randomly. Spatially separated granules are often released concurrently and show no correlation with plasma membrane proximity, ruling out that such temporally coordinated secretions are caused by heterogeneous intracellular environment. The secretion events occur invariably within 1s of a calcium spike, implying that elevated intracellular calcium triggers release of a subset of dense granules. Strikingly, there are significantly more calcium spikes than the secretion events in each activated platelet, suggesting that the calcium-dependent triggering mechanism is probabilistic, rather than deterministic in nature. With increasing thrombin concentration, the frequency of calcium spikes increases, but probability to observe a secretion event in response to a calcium spike remains unchanged. Thrombin also increases the number of granules released during one event, indicating that calcium-independent mechanisms control secretion of individual granules within one set. The cooperative nature of granule secretion is supported by the observed direct enhancement of granule release by supernatant collected from activated platelets and results of mathematical modeling. Such activation could not be recapitulated by the separate addition of ADP or ATP, suggesting the contribution from other secreted components or their synergistic activity.

Scheme of dense granule secretion in single platelets

Conclusions: Live observation of single thrombin-activated platelets reveals unexpected complexity of dense granules secretion, which exhibits cooperative behavior that is regulated by calcium-dependent and calcium-independent mechanisms. Future work should examine the kinetics and regulation of granule release in a growing thrombus.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/dense-granules-are-released-cooperatively-in-activated-platelets/