Inner Mitochondrial Membrane Disruption Links Apoptotic and Agonist-Initiated Phosphatidylserine Externalization in Platelets

Abstract

Objective: Phosphatidylserine exposure mediates platelet procoagulant function and regulates platelet life span. Apoptotic, necrotic, and integrin-mediated mechanisms have been implicated as intracellular determinants of platelet phosphatidylserine exposure. Here, we investigate (1) the role of mitochondrial events in platelet phosphatidylserine exposure initiated by these distinct stimuli and (2) the cellular interactions of the procoagulant platelet in vitro and in vivo.

Approach and results: Key mitochondrial events were examined, including cytochrome c release and inner mitochondrial membrane (IMM) disruption. In both ABT-737 (apoptotic) and agonist (necrotic)-treated platelets, phosphatidylserine externalization was temporally correlated with IMM disruption. Agonist stimulation resulted in rapid cyclophilin D-dependent IMM disruption that coincided with phosphatidylserine exposure. ABT-737 treatment caused rapid cytochrome c release, eventually followed by caspase-dependent IMM disruption that again closely coincided with phosphatidylserine exposure. A nonmitochondrial and integrin-mediated mechanism has been implicated in the formation of a novel phosphatidylserine-externalizing platelet subpopulation. Using image cytometry, this subpopulation is demonstrated to be the result of the interaction of an aggregatory platelet and a procoagulant platelet rather than indicative of a novel intracellular mechanism regulating platelet phosphatidylserine externalization. Using electron microscopy, similar interactions between aggregatory and procoagulant platelets are demonstrated in vitro and in vivo within a mesenteric vein hemostatic thrombus.

Conclusions: Platelet phosphatidylserine externalization is closely associated with the mitochondrial event of IMM disruption identifying a common pathway in phosphatidylserine-externalizing platelets. The limited interaction of procoagulant platelets and integrin-active aggregatory platelets identifies a potential mechanism for procoagulant platelet retention within the hemostatic thrombus.

Keywords: blood platelet; cell death; cytosol; mitochondrial membrane; phosphatidylserine.

Figure 1. Assessment of inner and outer mitochondrial membrane integrity in apoptotic and agonist-stimulated platelets

A) Cytochrome c release and MOMP in ABT-737 treated platelets. Murine platelets were treated with ABT-737 (1 µM) as indicated and cytochrome c retention assessed in digitonin-permeabilized platelets. B) IMM disruption in ABT-737 treated murine platelets. A calcein-cobalt assay was used to assess IMM integrity in ABT-737 treated (1 µM) platelets at the indicated time points. C, D) Kinetic analysis of ABT-737-treated (C) and thrombin and convulxin stimulated (D) platelets. MOMP (dashed line - % with loss of cytochrome c), IMM disruption (gray line - % with loss of calcein-staining), and PSer externalization (solid line - % ann V⁺) were assessed. *p<0.01 versus unstim (time 0).

Figure 2. Mechanisms regulating mitochondrial membrane integrity and PSer externalization in apoptotic platelets

A, B) Kinetic analysis of Bax/Bak dKO (A) and caspase-inhibited (10 µM Q-VD-Oph 10 minutes pre-treated) (B) platelets following treatment with ABT-737 (1 µM). MOMP (dashed line - % with loss of cytochrome c, IMM disruption (gray line - % with loss of calcein-staining), and PSer externalization (solid line - % ann V⁺) were assessed. C) Quantification of IMM disruption, MOMP formation, and PSer externalization at 90 minutes with ABT-737 treatment in WT, Q-VD-Oph treated, or Bax/Bak double knock-out platelets. *p<0.01 versus unstim (time 0) (A, B) or wild type (C).

Figure 3. Mitochondrial swelling in apoptotic and agonist-stimulated platelets

A–E) Anti – CD41 (blue), MitoTracker (purple), and ann V (green) were used to stain human platelets treated/stimulated as indicated, which were visualized using confocal microscopy. Scale bar indicates 2 um. F) Relative mitochondrial area / platelet was assessed in ann V⁺ and ann V^– platelets (n≥3). *p<0.05 versus unstim.

Figure 4. Mechanisms regulating procoagulant platelet subpopulation formation in agonist-stimulated platelets

Murine platelets (2×10⁸/mL) were stimulated with thrombin (100 nM). PSer externalization was evaluated using ann V. Cytoplasmic calcium was evaluated using Fura Red (A, D); ΔΨ_m was assessed using TMRM (B, E); and integrin-activation state was interrogated using JON/A (C, F). Arrows are drawn to identify cytometric subpopulation i (classical procoagulant platelet) and cytometric subpopulation ii (integrin-dependent procoagulant platelet). (A–C) Dot plot of thrombin-stimulated platelets investigated with the indicated fluorescent markers. (D–F) Quantitation of the % of type i and type ii procoagulant platelets using the corresponding fluorescent marker in WT, Bax/Bak dKO, and CypD KO platelets. (n=4). *p<0.01 versus WT.

Figure 5. Cytometric characterization of the integrin-dependent procoagulant platelet subpopulation

A) FSC and SSC characteristics of classical (i) and integrin-dependent (ii) procoagulant platelet subpopulations among thrombin-stimulated platelets as defined using JON/A and ann V. Pop. i (ann V⁺, JON/A^low) (gray), pop. ii (ann V⁺, JON/A^high) (black), other platelets (red). B) Platelets were labeled with Cell tracker violet (CTV) or Cell tracker green (CTG) separately. Each stained platelets were mixed and stimulated with Thrombin to quantify platelets aggregates (CTV⁺/CTG⁺ events indicating platelet>1) *p<0.01 versus 0.1 × 10⁸ platelets. C) Platelets were labeled separately with CTV or CTG, mixed, and stimulated with thrombin. CTV, CTG, ann V and JON/A were utilized for analysis. The CTV⁺/CTG⁺ platelet subpopulation was indicated in green. D) Quantitation of visual analysis of stimulated murine platelets using image cytometry of individual cytometric events. 1000 images were analyzed per mouse. n=3. E) Representative events obtained using image cytometry.

Figure 6. Transmission electron microscopy of murine procoagulant platelets

A) Platelets obtained from WT or CypD KO mice were stimulated with thrombin and convulxin and analyzed by TEM at the indicated time points. Scale bar indicates 2 µm. B, C) Representative micrographs of an (B) electron-lucent procoagulant platelet (arrows in B and C) closely associated with a more electron-dense platelet, and of a (C) cross-section of the periphery of a hemostatic plug obtained following puncture injury of the mouse mesentery.