Persistence of procoagulant surface expression on activated human platelets: involvement of apoptosis and aminophospholipid translocase activity

Abstract

Summary. Background: Activated platelets express a procoagulant surface when the asymmetric distribution of membrane phospholipids is scrambled, leading to phosphatidylserine (PS) exposure. PS expression, associated with apoptosis in nucleated cells, would be expected to be reversed by aminophospholipid translocase (APLT) activity. Objective: To determine whether the procoagulant surface of activated platelets persists after it forms; to examine whether PS expression on platelets is associated with loss of mitochondrial inner membrane potential (DW m ), a hallmark of apoptosis; and to investigate the role of APLT in persistence of PS expression. Methods: Platelets were stimulated with thrombin, collagen, a combination of both, or the Ca 2+ -ionophore A23187. Up to 4 h after activation, procoagulant surface expression was measured by annexin A5 binding by flow cytometry and by a prothrombinase assay. Flow cytometry was also used to measure PS expression concurrently with DW m collapse, using CMXRos. APLT activity in annexin A5-negative and -positive platelets was measured flow cytometrically as the percent of 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphatidylserine (NBD-PS) translocated from the outer to the inner membrane leaflet. Results and conclusions: Procoagulant surface expression on activated platelets persisted in vitro for at least 4 h; if such persistence occurs in vivo, there are important implications for the propagation of thrombosis. With the physiological stimuli, only 10-20% of the activated platelets expressed PS on their surface, and of these, only a portion exhibited DW m collapse, indicating that PS expression can be associated with platelet apoptosis, but can also occur independently. APLT activity was very low in the PS-expressing platelet subpopulation for up to 4 h after activation, indicating that the persistence of a procoagulant surface may be attributed, at least in part, to this reduced APLT activity.

Phosphatidylserine exposure and other apoptotic‐like events in Bernard‐Soulier syndrome platelets

“…It has become recognized that PS exposure on platelets can occur as a result of platelet activation or via apoptosis [12][13][14][15][16]31]. Dissipation of DC m is an early apoptotic event that occurs before PS exposure in nucleated cells; in platelets, it occurs rapidly upon stimulation with agonists, addi- tion of hydrophobic local anesthetics, or application of shear stress, but it is not associated with the PS exposure that occurs on platelets directly stimulated to be apoptotic with the BH3 mimetic ABT-737 [12][13][14]16,58,59].…”

“…This was measured as decreased TMRM fluorescence in platelets from controls and from BSS P2 (P1 was not available for these investigations). As we have previously shown [12,13], a significant proportion of control PSexposing platelets, whether resting or activated, exhibited Figure 1. Immunoblotting of PAR4 in control platelets (C1, C2) and platelets from BSS patient 1 (P1) and patient 2 (P2), using a specific goat polyclonal anti-PAR4 antibody [36].…”

“…In addition to PS exposure, activated platelets undergo membrane blebbing (microparticle (MP) formation), cell shrinkage, and extension of filopodia. As well, there is depolarization of mitochondrial inner membrane potential (DC m ), an early apop-totic event that precedes PS exposure, and activation and translocation of Bcl-2 family members and activation of caspase-9 and -3 [11][12][13][14][15][16].…”

Analysis of procoagulant phosphatidylserine‐exposing platelets by imaging flow cytometry

“…Blood was collected into acid‐citrate‐dextrose anticoagulant; washed platelets, prepared as described previously,13, 35 were suspended in Tyrode solution (137 mmol/L NaCl, 2.7 mmol/L KCl, 11.9 mmol/L NaHCO 3 , 0.42 mmol/L NaH 2 PO 4 , 1 mmol/L MgCl 2 , 2 mmol/L CaCl 2, and 5.5 mmol/L glucose) with 5 mmol/L HEPES, 0.35% BSA (fraction V) (MP Biomedicals, LLC, Santa Ana, CA) and apyrase (prepared from potatoes by a slight modification of the method of Molnar and Lorand,36 and used as described elsewhere35, 37), pH7.4. The platelet count was adjusted to 1 × 10 9 /mL and platelets were stimulated with a combination of the physiological agonists thrombin (1 U/mL) (Haematologic Technologies Inc., Essex Junction, VT) and collagen (10 μg/mL) (Horm, native type I collagen fibrils from equine tendon) (Takeda, Munich, Germany), or the nonphysiological calcium ionophore A23187 (3 μmol/L) (Sigma‐Aldrich, St. Louis, MO) that directly increases intracellular Ca 2+ concentrations, as described previously 13…”

“…A critical role has emerged for the procoagulant platelet subpopulation in regulating both normal hemostasis and pathological thrombus formation 1, 3. Indeed, we have previously demonstrated that PS exposure on activated platelets persists in vitro,13 and in vivo,14 and that blocking the procoagulant surface inhibits platelet‐mediated events in experimentally induced arterial thrombosis 15…”