Abstract
Activation of human platelets by complement proteins, C5b-9, thrombin plus collagen, or a Ca2+ ionophore results in surface exposure of phosphatidylserine (PS), accompanied by the expression of membrane catalytic activity for the tenase (VIIaIXa) and prothrombinase (VaXa) coagulation enzyme complexes. The mechanism underlying this surface exposure of PS upon platelet activation remains unresolved. Using fluorescent derivatives of PS (NBD-PS), we have investigated how the transmembrane migration of PS is related to microvesiculation of the platelet plasma membrane and to fusion of storage granules with the plasma membrane. Gel-filtered platelets were incubated with NBD-PS, allowing 90 +/- 10% of the incorporated NBD-PS to accumulate into the inner leaflet of the plasma membrane. Migration of NBD-PS from the inner leaflet to the plasma membrane surface was monitored by time-based flow cytometry, and correlated with the appearance of platelet microparticles and alpha-granule secretion. Platelet activation by C5b-9 or the Ca2+ ionophore, A23187, increased surface exposure of NBD-PS, due to acceleration of the apparent rate of migration from inner to outer plasma membrane leaflets. The onset of this accelerated migration of NBD-PS to the surface coincided with the onset of plasma membrane vesiculation, and the NBD-PS that partitioned into the membrane of the shed microparticle was also rapidly exposed to the surface (t1/2 < 2 min). In addition to a temporal correlation, microparticle formation and the surface exposure of inner leaflet NBD-PS showed a similar requirement for Ca2+. These results demonstrate that agonist-induced microvesiculation of the platelet plasma membrane is accompanied by accelerated migration of a PS analogue from the inner leaflet to the surface of the shed microparticle membrane, suggesting the mechanism by which induction of platelet microparticle formation exposes catalytic surface for tenase and prothrombinase assembly.
Highlights
(PS),accompanied by the expressionof membrane cat- inner leaflet (Chap et al, 1977; Bevers et al, 1982; Zwaal and alytic activity for the tena(sVeIIIaIXa) and prothrom- Bevers, 1983).The maintenanceof this asymmetric distribubinase (VaXa) coagulationenzyme complexes
In order to provide a red fluorescence signal for fluorescence-gated flow cytosure of PS with decreased expression of membrane procoag- metry, plasma membrane glycoprotein Ib was labeled by ulant activity habseen observed fopr latelets and erythrocytes sequential incubation in the dark (10 min, room temperature) with 2
For flow cytometric measurement of bound fluorescein 5-isothiocyanate (FITC)"S12, the red fluorescence signal for fluorescence gating was provided by plasma membrane labeling with diIC16(3), using methods previously described (Gilbert et al, 1991)
Summary
2 0 4 aliquots of the platelet suspensions were withdrawn and pipetted into 20 p1 of Buffer 2 containing either 0 or 20 mg/ml albumin. A-Granule Secretion-Secretion from platelet storage granuleswas quantitated by surface expressionof the a-granule membrane protein P-selectin,quantitated by binding of FITC-labeled monoclonalantibody S12. Fluorescence-gated Flow Cytornetry-Samples were analyzed in a Becton-Dickinson FACScanflow cytometer (Mountain View, CA). Time (min) for biotin-W5 (detectedasphycoerythrinfluorescence)or for diIC16(3).For time-based measurements,data acquisition and storage was automated to minimize sampling delay, and samples were analyzed at 15-sintervals. Five thousand events from each sample were analyzed for forwardand right angle lighstcatter and for fluorescence due to NBD-lipid (or FITC-S12)using a 530/30 nm bandpass filter. Age of the total membrane-associated NBD derived from the NBD Data are of a single experiment representative of three experiments fluorescence of matched samples incubated without albumin
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