Dataset Information

Membrane Ballooning in Aggregated Platelets is Synchronised and Mediates a Surge in Microvesiculation.

ABSTRACT: Human platelet transformation into balloons is part of the haemostatic response and thrombus architecture. Here we reveal that in aggregates of platelets in plasma, ballooning in multiple platelets occurs in a synchronised manner. This suggests a mechanism of coordination between cells, previously unrecognised. We aimed to understand this mechanism, and how it may contribute to thrombus development. Using spinning-disc confocal microscopy we visualised membrane ballooning in human platelet aggregates adherent to collagen-coated surfaces. Within an aggregate, multiple platelets undergo ballooning in a synchronised fashion, dependent upon extracellular calcium, in a manner that followed peak cytosolic calcium levels in the aggregate. Synchrony was observed in platelets within but not between aggregates, suggesting a level of intra-thrombus communication. Blocking phosphatidylserine, inhibiting thrombin or blocking PAR1 receptor, largely prevented synchrony without blocking ballooning itself. In contrast, inhibition of connexins, P2Y12, P2Y1 or thromboxane formation had no effect on synchrony or ballooning. Importantly, synchronised ballooning was closely followed by a surge in microvesicle formation, which was absent when synchrony was blocked. Our data demonstrate that the mechanism underlying synchronised membrane ballooning requires thrombin generation acting effectively in a positive feedback loop, mediating a subsequent surge in procoagulant activity and microvesicle release.

SUBMITTER: Agbani EO

PROVIDER: S-EPMC5459805 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Membrane Ballooning in Aggregated Platelets is Synchronised and Mediates a Surge in Microvesiculation.

Agbani Ejaife O EO Williams Christopher M CM Hers Ingeborg I Poole Alastair W AW

Scientific reports 20170605 1

Human platelet transformation into balloons is part of the haemostatic response and thrombus architecture. Here we reveal that in aggregates of platelets in plasma, ballooning in multiple platelets occurs in a synchronised manner. This suggests a mechanism of coordination between cells, previously unrecognised. We aimed to understand this mechanism, and how it may contribute to thrombus development. Using spinning-disc confocal microscopy we visualised membrane ballooning in human platelet aggre ...[more]

PMID: 28584295

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Project description:Membrane glycoproteins that mediate platelet-platelet interactions were investigated by identifying those associated with the cytoskeletal structures from aggregated platelets. The cytoskeletal structures from washed platelets, thrombin-activated platelets (platelets incubated with thrombin in the presence of mM EDTA to prevent aggregation) and thrombin- aggregated platelets (platelets activated in the presence of mM Ca(++) were prepared by first treating platelet suspensions with 1 percent Triton X-100 and 5 mM EGTA and then isolating the insoluble residue by centrifugation. The readily identifiable structures in electron micrographs of the residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue by SDS gel electrophoresis showed that it consisted primarily of three proteins: actin (mol wt = 43,000), myosin (mol wt = 200,000) and a high molecular weight polypeptide (mol wt = 255,000) which had properties indentical to actin-binding protein (filamin). When platelets are activated with thrombin in the presence of EDTA to prevent aggregation, there was a marked increase in the amount of insoluble precipitate in the subsequent Triton extraction. Transmission electron microscopy showed that this residue not only contained the random array of actin filaments as seen above, but also organized structures from individual platelets which appeared as balls of electron-dense filamentous material approximately 1mum in diameter. SDS polyacrylamide gel analysis of the Triton residue of activated platelets showed that this preparation contained more actin, myosin and actin-binding protein than that from washed platelets plus polypeptides with mol wt of 56,000 and 90,000 and other minor polypeptides. Thus, thrombin activation appeared to increase polymerization of actin in association with other cytoskeletal proteins into structures that are observable after Triton extraction. The cytoskeletal structures from thrombin-aggregated platelets were similar to those from thrombin-activated platelets, except that the structural elements from individual platelets remained aggregated rather than randomly dispersed in the actin filaments. This suggested that the membrane components that mediate the direct interaction of platelets were in Triton residue from aggregated platelets. Only a small percentage of the membrane surface proteins and glycoproteins were found in the cytoskeletal structures from either washed platelets or thrombin-activated platelets. In contrast, the aggregated cytoskeletal structures from thrombin-aggregated platelets contained membrane glycoproteins IIb (26 percent of the total in pre-extracted platelets) and III (14 percent), suggesting that one or both of these glycoproteins participate in the direct interaction of platelets during aggregation.

Dataset Information

Membrane Ballooning in Aggregated Platelets is Synchronised and Mediates a Surge in Microvesiculation.

Publications

Membrane Ballooning in Aggregated Platelets is Synchronised and Mediates a Surge in Microvesiculation.

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