Methods and Results: Using 4D live-cell imaging and electron microscopy, we show that human platelets adherent to collagen are transformed into phosphatidylserine-exposing balloon-like structures with expansive macro/micro-vesiculate contact surfaces, by a process which we termed procoagulant-spreading. We reveal that ballooning is mechanistically and structurally distinct from membrane blebbing and involves disruption to the platelet microtubule cytoskeleton and inflation through fluid entry. Unlike blebbing, procoagulant ballooning is irreversible and a consequence of Na+, Cl- and water entry. Furthermore, membrane ballooning correlated with micro-particle generation. Inhibition of Na+, Cl- or water entry impaired ballooning, procoagulant-spreading, micro-particle generation and also diminished local thrombin generation. Human Scott syndrome platelets, which lack expression of Ano-6, also showed a marked reduction in membrane ballooning, consistent with a role for chloride entry in the process. Finally, blockade of water entry by acetazolamide attenuated ballooning in vitro, and markedly suppressed thrombus formation in vivo in a mouse model of thrombosis.
Conclusion: Ballooning and procoagulant-spreading of platelets are driven by fluid entry into the cells, and are important for amplification of localised coagulation in thrombosis.
Bibliographical noteDate of Acceptance: 30/07/2015
- membrane ballooning