Coordinated Membrane Ballooning and Procoagulant-Spreading in Human Platelets
Background—Platelets are central to the process of haemostasis, rapidly aggregating at sites of blood vessel injury and acting as coagulation nidus sites. Upon interaction with sub-endothelial matrix platelets are transformed into balloon-like structures as part of the haemostatic response. It remains unclear, however, how and why platelets generate these structures. We set out to determine the physiological relevance and cellular and molecular mechanisms underlying platelet membrane ballooning.
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.
Conclusions—Ballooning and procoagulant-spreading of platelets are driven by fluid entry into the cells, and are important for amplification of localised coagulation in thrombosis.
- Membrane ballooning
- Fluorescent imaging
- High-resolution microscopy
- cell physiology
- Received December 19, 2014.
- Revision received July 21, 2015.
- Accepted July 30, 2015.