Coordinated Membrane Ballooning and Procoagulant Spreading in Human Platelets.
BACKGROUND
Platelets are central to the process of hemostasis, rapidly aggregating at sites of blood vessel injury and acting as coagulation nidus sites. On interaction with the subendothelial matrix, platelets are transformed into balloonlike structures as part of the hemostatic 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.


RESULTS
Using 4-dimensional live-cell imaging and electron microscopy, we show that human platelets adherent to collagen are transformed into phosphatidylserine-exposing balloonlike structures with expansive macro/microvesiculate contact surfaces, by a process that 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 microparticle generation. Inhibition of Na(+), Cl(-), or water entry impaired ballooning, procoagulant spreading, and microparticle generation, and it 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, the 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 the amplification of localized coagulation in thrombosis.
From School of Physiology & Pharmacology, University of Bristol, United Kingdom (E.O.A., M.T.J.v.d.B., E.B., C.M.W., I.H., A.W.P.; Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, The Netherlands (N.J.A.M., J.M.E.M.C., J.W.M.H.); and Welsh Blood Service and Arthur Bloom Haemophilia Centre, School of Medicine, Cardiff University, United Kingdom (P.W.C.). a.poole@bristol.ac.uk e.agbani@bristol.ac.uk.
Ejaife O
EO
Agbani
From School of Physiology & Pharmacology, University of Bristol, United Kingdom (E.O.A., M.T.J.v.d.B., E.B., C.M.W., I.H., A.W.P.; Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, The Netherlands (N.J.A.M., J.M.E.M.C., J.W.M.H.); and Welsh Blood Service and Arthur Bloom Haemophilia Centre, School of Medicine, Cardiff University, United Kingdom (P.W.C.).
Marion T J
MT
van den Bosch
From School of Physiology & Pharmacology, University of Bristol, United Kingdom (E.O.A., M.T.J.v.d.B., E.B., C.M.W., I.H., A.W.P.; Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, The Netherlands (N.J.A.M., J.M.E.M.C., J.W.M.H.); and Welsh Blood Service and Arthur Bloom Haemophilia Centre, School of Medicine, Cardiff University, United Kingdom (P.W.C.).
Ed
E
Brown
From School of Physiology & Pharmacology, University of Bristol, United Kingdom (E.O.A., M.T.J.v.d.B., E.B., C.M.W., I.H., A.W.P.; Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, The Netherlands (N.J.A.M., J.M.E.M.C., J.W.M.H.); and Welsh Blood Service and Arthur Bloom Haemophilia Centre, School of Medicine, Cardiff University, United Kingdom (P.W.C.).
Christopher M
CM
Williams
From School of Physiology & Pharmacology, University of Bristol, United Kingdom (E.O.A., M.T.J.v.d.B., E.B., C.M.W., I.H., A.W.P.; Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, The Netherlands (N.J.A.M., J.M.E.M.C., J.W.M.H.); and Welsh Blood Service and Arthur Bloom Haemophilia Centre, School of Medicine, Cardiff University, United Kingdom (P.W.C.).
Nadine J A
NJ
Mattheij
From School of Physiology & Pharmacology, University of Bristol, United Kingdom (E.O.A., M.T.J.v.d.B., E.B., C.M.W., I.H., A.W.P.; Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, The Netherlands (N.J.A.M., J.M.E.M.C., J.W.M.H.); and Welsh Blood Service and Arthur Bloom Haemophilia Centre, School of Medicine, Cardiff University, United Kingdom (P.W.C.).
Judith M E M
JM
Cosemans
From School of Physiology & Pharmacology, University of Bristol, United Kingdom (E.O.A., M.T.J.v.d.B., E.B., C.M.W., I.H., A.W.P.; Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, The Netherlands (N.J.A.M., J.M.E.M.C., J.W.M.H.); and Welsh Blood Service and Arthur Bloom Haemophilia Centre, School of Medicine, Cardiff University, United Kingdom (P.W.C.).
Peter W
PW
Collins
From School of Physiology & Pharmacology, University of Bristol, United Kingdom (E.O.A., M.T.J.v.d.B., E.B., C.M.W., I.H., A.W.P.; Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, The Netherlands (N.J.A.M., J.M.E.M.C., J.W.M.H.); and Welsh Blood Service and Arthur Bloom Haemophilia Centre, School of Medicine, Cardiff University, United Kingdom (P.W.C.).
Johan W M
JW
Heemskerk
From School of Physiology & Pharmacology, University of Bristol, United Kingdom (E.O.A., M.T.J.v.d.B., E.B., C.M.W., I.H., A.W.P.; Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, The Netherlands (N.J.A.M., J.M.E.M.C., J.W.M.H.); and Welsh Blood Service and Arthur Bloom Haemophilia Centre, School of Medicine, Cardiff University, United Kingdom (P.W.C.).
Ingeborg
I
Hers
From School of Physiology & Pharmacology, University of Bristol, United Kingdom (E.O.A., M.T.J.v.d.B., E.B., C.M.W., I.H., A.W.P.; Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, The Netherlands (N.J.A.M., J.M.E.M.C., J.W.M.H.); and Welsh Blood Service and Arthur Bloom Haemophilia Centre, School of Medicine, Cardiff University, United Kingdom (P.W.C.). a.poole@bristol.ac.uk e.agbani@bristol.ac.uk.
Alastair W
AW
Poole
D000086:Acetazolamide / Q000494:pharmacology; D000205:Actomyosin / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D000818:Animals; D000075368:Anoctamins; D001778:Blood Coagulation Disorders / Q000097:blood; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D002341:Carotid Artery Thrombosis / Q000097:blood / Q000139:chemically induced / Q000188:drug therapy; D002448:Cell Adhesion; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D048430:Cell Shape / Q000187:drug effects / Q000502:physiology; D048429:Cell Size / Q000187:drug effects; D055252:Cell-Derived Microparticles; D002712:Chlorides / Q000378:metabolism; D003094:Collagen; D015638:Cytochalasin D / Q000494:pharmacology; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008870:Microtubules / Q000187:drug effects; D049294:Phospholipid Transfer Proteins / Q000172:deficiency / Q000502:physiology; D011725:Pyridines / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism; D013917:Thrombin / Q000096:biosynthesis; D013927:Thrombosis / Q000517:prevention & control; D014867:Water / Q000378:metabolism
D000577:Amides; C555078:ANO6 protein, human; D000075368:Anoctamins; D002712:Chlorides; D006576:Heterocyclic Compounds, 4 or More Rings; D049294:Phospholipid Transfer Proteins; D011725:Pyridines; D014867:Water; C108830:Y 27632; C472645:blebbistatin; D015638:Cytochalasin D; D003094:Collagen; D000205:Actomyosin; D012964:Sodium; D013917:Thrombin; D000086:Acetazolamide
blood coagulation; blood platelets; cell-derived microparticles; collagen; fluorescent imaging; membrane ballooning; procoagulant-spreading