Summary. Background: The signal(s) for removal of senescent platelets from the circulation are not fully understood; phosphatidylserine (PS) expression on platelets and another marker of apoptosis, loss of mitochondrial inner membrane potential (DY m ), have been implicated in platelet clearance. Objective: To investigate whether shortened platelet survival and steady-state platelet senescence are associated with increased surface exposure of PS and DY m collapse. Methods: Survival of in-vitro biotinylated rabbit platelets treated with thrombin or Ca 2+ -ionophore A23187 was tracked by flow cytometry after injection. Steady-state platelet senescence was investigated by infusing biotin to label a platelet cohort. PS expression and DY m of in-vitro biotinylated platelets and of the aging platelet cohort biotinylated in-vivo were measured by flow cytometry using annexin V-FLUOS and the DY m -sensitive dye CMXRos, respectively. Results: Although PS expression, DY m and survival of thrombin-degranulated platelets were similar to those of control platelets, increasing concentrations of A23187 caused increased surface exposure of PS and progressive shortening of platelet survival; only onesixth of PS-expressing platelets also exhibited DY m loss. The cohort of senescent, biotinylated platelets remaining in the circulation at 96 h had increased exposure of PS and collapsed DY m ; of the 17% of PS-expressing platelets, one-third did not exhibit DY m loss. There was also an increase in platelets with collapsed DY m but not expressing PS. Conclusions: Platelets with shortened survival and senescent platelets have increased surface exposure of PS, that may be involved in their clearance. PS expression can occur independently of DY m collapse and conversely, in aged platelets, DY m loss can occur independently of PS expression.

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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.

Summary. Background:  Shielding of procoagulant phosphatidylserine (PS) with annexin A5 attenuates thrombosis, but annexin A5 (35.7 kDa) is rapidly cleared from the circulation. In contrast, Diannexin, a 73.1 kDa homodimer of annexin A5, has an extended half‐life. Objectives:  To quantify the affinity of Diannexin for PS, examine its interaction with activated platelets and determine its effects on platelet‐mediated events during thrombus formation. Methods:  The affinities of Diannexin and annexin A5 for PS‐containing lipid bilayers were compared using surface plasmon resonance, and binding to activated platelets was assessed by flow cytometry. Calibrated automated thrombography and thromboelastography were employed to study the effects of Diannexin on thrombin generation and platelet‐fibrin clot formation, respectively, whereas intravital videomicroscopy was used to examine its effect on platelet accumulation and activation after laser‐induced injury to murine cremaster arterioles, and a tail tip bleeding model was used to explore its effects on hemostasis. Results:  Diannexin and annexin A5 bind PS with KD values of 0.6 and 5 nm, respectively, and both bind to the same subpopulation of PS‐exposing platelets. Diannexin inhibited thrombin generation and platelet‐fibrin clot formation in vitro at 10 nm (P < 0.05–0.001 compared with control), and reduced platelet accumulation at 1 μg g−1 (P < 0.05) and activation at 0.25 μg g−1 (P < 0.001) in experimentally induced arterial thrombi in mice while increasing blood loss at 1 μg g−1 (P < 0.01). Conclusions:  Diannexin binds to PS with high affinity and is a potent inhibitor of platelet‐mediated events during thrombus formation.

Background:To avoid radioisotopic labeling and permit comparison of the survival of two platelet populations concurrently in one animal, we compared simultaneous recoveries and survival times of homologous rabbit platelets labeled in vitro with the lipophilic dyes PKH26 (red fluorescing) and PKH67 (green fluorescing) and with two levels of biotin (low, 1 g/ml; high, 10 g/ml). Methods: Blood samples were drawn up to 96 h postinfusion and analyzed by flow cytometry. Biotin-labeled samples were incubated with phycoerythrin-streptavidin before analysis. Results: Recovery of PKH26-labeled platelets at 1 h was lower (37.5%) than that of PKH67-labeled platelets (47.3%; P Ͻ 0.001). Platelet survival times were 62.4 and 61.9 h. Recoveries at 1 h of platelets labeled with two levels of biotin were similar (86.6% and 84.6%) and greater