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Keywords:

  • apoptosis;
  • Bcl-2;
  • megakaryocyte;
  • platelet

Abstract

  1. Top of page
  2. Abstract
  3. The intrinsic apoptosis pathway
  4. Apoptosis and platelet production
  5. Apoptosis, platelet life and platelet death
  6. Acknowledgments
  7. Disclosure of Conflict of Interests
  8. References

Summary.  In recent years, it has become increasingly apparent that the production of platelets and their subsequent life span in the circulation are regulated, at least in part, by apoptotic mechanisms. There is also evidence implicating the apoptotic machinery in the regulation of platelet functional responses. This review examines the role of the intrinsic apoptosis pathway, regulated by the Bcl-2 family of proteins, in platelet biology.


The intrinsic apoptosis pathway

  1. Top of page
  2. Abstract
  3. The intrinsic apoptosis pathway
  4. Apoptosis and platelet production
  5. Apoptosis, platelet life and platelet death
  6. Acknowledgments
  7. Disclosure of Conflict of Interests
  8. References

Apoptosis, a form of programmed cell death, plays a fundamental role in development, tissue homeostasis and host defense [1]. Two apoptotic pathways exist: the extrinsic, and the intrinsic. Once activated, both pathways eventually converge on caspases, the cysteinyl aspartate proteases responsible for cellular demolition and engulfment.

The extrinsic pathway is triggered by activation of death receptors on the cell surface. In contrast, the status of the intrinsic pathway is governed by the interplay between pro- and anti-apoptic members of the Bcl-2 family (Fig. 1). Five major anti-apoptic or ‘pro-survival’ proteins have been characterized: Bcl-2, Bcl-w, Bcl-xL, Mcl-1 and A1. They keep cell death in check by restraining the pro-apoptotic Bcl-2 proteins Bak and Bax, which are the final arbiters of cell death. When activated, Bak and Bax trigger mitochondrial damage, allowing the release of cytochrome c and other apoptogenic factors. It is this step that seals the fate of an individual cell – clonogenic survival beyond this point is not possible. A third subset of Bcl-2 proteins, the so-called BH3-only members of the family, sense and transmit pro-death signals to Bak and Bax. Precisely how they achieve this outcome is a subject of ongoing debate [2,3]. However, the general paradigm that Bcl-2 pro-survival proteins restrain pro-apoptotic Bak and Bax to prevent apoptosis, with BH3-only family members acting to override the pro-survivals and initiate the death program, is well established in a broad range of nucleated cells.

image

Figure 1.  The intrinsic apoptosis pathway. In healthy cells, pro-survival Bcl-2 family proteins (Bcl-2, Bcl-w, Bcl-xL, Mcl-1 and A1) keep pro-death Bak and Bax in check. Apoptotic signals such as DNA damage trigger the BH3-only proteins. These mediate activation of Bak and Bax, either directly or via inhibition of pro-survivals. Bak and Bax then initiate mitochondrial damage, cytochrome c release, and caspase activation.

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Apoptosis and platelet production

  1. Top of page
  2. Abstract
  3. The intrinsic apoptosis pathway
  4. Apoptosis and platelet production
  5. Apoptosis, platelet life and platelet death
  6. Acknowledgments
  7. Disclosure of Conflict of Interests
  8. References

The megakaryocyte is unique: to fulfil its raison d’être, i.e. the production of platelets, it must die. Radley and Haller [4] were the first to link the ultrastructural changes observed in ‘senescent’ megakaryocytes with those reported of apoptotic cells [5]. Characterized by compacted chromatin, it was suggested that these ‘naked’ nuclei – thought to represent megakaryocytes post platelet release – were degenerating prior to engulfment by macrophages. The observation that late-stage megakaryocytes grown in culture exhibited several hallmarks of apoptosis led to the suggestion that ‘maximal platelet production and megakaryocyte apoptosis are closely related events’ [6].

Since then, a number of studies have supported the idea that the intrinsic apoptosis pathway is involved in platelet shedding. Overexpression of Bcl-2 in the hematopoietic compartment of mice [7], or deletion of the BH3-only protein Bim [8] caused mild thrombocytopenia. Megakaryocytes overexpressing Bcl-xL exhibited abnormal ultrastructure and a reduced ability to produce pro-platelet extensions in vitro [9]. This suggested that inhibition of the intrinsic pathway, and its downstream effectors – caspases – might interfere with platelet production.

Two groups have provided evidence that caspases may indeed be required by megakaryocytes. De Botton et al. [10] found active forms of caspase-3 and caspase-9 in cultured human megakaryocytes, and observed cleavage of substrates such as gelsolin in cells that had begun to shed platelets. Incubation with caspase inhibitors could block pro-platelet extension. Similarly, Clarke et al. [11] found that pro-platelet formation and the production of platelets by the human megakaryocytic cell line MEG-01 could be abrogated by caspase inhibition. Active caspases exhibited a discrete punctate distribution, and were localized to the cell body of pro-platelet-bearing megakaryocytes, but not the pro-platelets themselves. Despite the in vitro nature of the studies, and concerns about the specificity of the inhibitors used [12], the idea that caspases may facilitate the cytoskeletally intensive rearrangements needed for platelet shedding is an appealing one.

If megakaryocytes do undergo apoptosis as part of the platelet shedding process, it would appear to be a specialized form. De Botton et al. [10] could not detect DNA fragmentation, a hallmark of apoptotic cells, in megakaryocytes containing activated caspase-3. They did however observe cytochrome c in the cytosol, indicating permeabilization of the outer mitochondrial membrane. How might a megakaryocyte undergo apoptosis and yet produce functional platelets containing intact mitochondria? One possibility is that only a subset of the mitochondria are damaged; it may be that megakaryocytes can sequester those organelles destined for platelets. Intriguingly, a human variant of cytochrome c with enhanced pro-apoptotic activity, but normal redox function, caused thrombocytopenia [13]. The latter was attributed to premature platelet-shedding by megakaryocytes, which were seen releasing platelets into the intramedullary bone marrow space. This would suggest that the apoptotic machinery in megakaryocytes is subject to not only precise spatial, but also temporal, control, and that any perturbation, even one resulting in more ‘efficient’ apoptosis, can have a significant effect on platelet production.

How megakaryocyte apoptosis is regulated, and how important a role it plays in the generation of platelets, remain open questions. Are Bak and/or Bax the key mediators? Lindsten et al. [14] reported mild thrombocytopenia in mice lacking both proteins, but no analysis of the megakaryocyte lineage was presented. Work in our laboratory suggests there may indeed be a defect in platelet production in the absence of Bak and Bax (unpublished observation). Assuming that Bax and Bax are the gatekeepers of megakaryocyte apoptosis, what restrains them until the point at which they are activated? It seems likely that one or more pro-survival Bcl-2 family proteins might fulfil this role.

Apoptosis, platelet life and platelet death

  1. Top of page
  2. Abstract
  3. The intrinsic apoptosis pathway
  4. Apoptosis and platelet production
  5. Apoptosis, platelet life and platelet death
  6. Acknowledgments
  7. Disclosure of Conflict of Interests
  8. References

Once shed by megakaryocytes into the circulation, platelets encounter either one of the two fates: consumption in a hematostatic process, or clearance by the reticuloendothelial system. Barring the former, platelet life span is approximately 10 days in humans [15], 5 days in mice [16,17]. It appears that the intrinsic apoptosis pathway plays a major role in regulating this finite existence.

Vanags et al. [18] were the first to report the presence of Bcl-2 family proteins in human platelets. Noting that platelet functional responses such as phosphatidylerine (PS) exposure are characteristic of dying nucleated cells, they proposed that the anucleate platelet might be capable of undergoing apoptosis. We and others recently demonstrated that this is indeed the case [19,20]. Apoptosis triggered by the intrinsic pathway circumscribes platelet life span in vivo. The critical regulator of platelet survival is Bcl-xL, which functions to keep Bak (and to a lesser extent Bax) in check [19]. Mutations in Bcl-xL caused dose-dependent reductions in platelet life span, whilst genetic deletion of Bak and Bax extended platelet life span to almost twice normal. Ex vivo treatment of platelets with the BH3 mimetic compound ABT-737, a potent inhibitor of Bcl-xL [21], triggered mitochondrial damage, caspase activation and membrane externalization of PS [19,20]. Administration of ABT-737 to mice and dogs caused rapid-onset thrombocytopenia, with dramatic decreases in circulating platelet count occurring within 2 h. This effect was mediated by Bak and Bax, as circulating platelets in Bak−/−Bax+/− mice were refractory to ABT-737 [19]. Thus, platelet life span is governed by the interplay between Bcl-2 family proteins. A number of questions remain. For example, it is unclear what triggers the apoptotic program. We have proposed the ‘molecular clock’ model, whereby simple degradation of Bcl-xL over time [22] leads to Bak activation [19], but it may be that some other signal, perhaps mediated by BH3-only proteins, activates apoptosis. Downstream of mitochondrial damage, the signal that facilitates clearance from the circulation also remains to be identified. Does PS exposure trigger the engulfment of platelets?

PS is the archetypal ‘eat me’ signal displayed by cells undergoing apoptosis. However, platelets also expose PS when activated by a range of physiologic agonists [23]. In this context, it facilitates assembly of the pro-thrombinase complex, which plays an essential role in generating thrombin [24]. Whether the apoptotic machinery is required for this type of PS exposure is unclear. What is the relationship between platelet activation and platelet apoptosis? Several studies have suggested that physiologic agonists can induce caspase activation [25–27], but there is some conflicting evidence [28]. Similarly, whilst several groups have reported caspase activation in response to the non-physiologic stimulus calcium ionophore [18,26,27,29], others have failed to detect it [28,30]. We have found that platelets deficient for Bak and Bax expose negligible PS when treated with ABT-737, but expose PS at wild type levels when stimulated with dual agonist or ionophore (manuscript submitted). Whilst this does not rule out caspase activation in response to agonist by some other means, it indicates that agonist-induced PS exposure is not mediated by the intrinsic apoptosis pathway. It also demonstrates that whilst ionophore might be capable of triggering apoptosis, the mechanism by which it drives PS exposure is not dependent on Bak and Bax.

There is clearly much to be understood about the role of the intrinsic pathway in platelets, and the role of mitochondria more broadly. An elegant recent study demonstrated that platelets lacking cyclophilin D, a protein required for mitochondrial permeability transition pore formation and subsequent loss of mitochondrial transmembrane potential (ΔΨm), exhibited impaired activation responses when treated with dual agonist, including an almost complete inability to expose PS [31]. This suggests an essential requirement for loss of ΔΨm in platelet activation. Interestingly, we have found that incubation of platelets with ABT-737 over a 3 h time period did not trigger loss of ΔΨm, indicating that the mitochondrial changes occurring in PS-positive apoptotic platelets are distinct from those occurring in agonist-stimulated platelets (manuscript submitted). It had previously been shown that uncoupling mitochondrial oxidative phosphorylation by treating platelets with carbonyl cyanide m-chlorophenylhydrazone (CCCP) resulted in a dramatic decrease in recovery post-transfusion [32]. Thus, mitochondria play a central role in mediating platelet behavior during life and death. Understanding how different signals are able to provoke differing mitochondrial outcomes and thereby direct appropriate platelet functional responses is likely to yield new insights into platelet biology.

Acknowledgments

  1. Top of page
  2. Abstract
  3. The intrinsic apoptosis pathway
  4. Apoptosis and platelet production
  5. Apoptosis, platelet life and platelet death
  6. Acknowledgments
  7. Disclosure of Conflict of Interests
  8. References

This work was supported by the Australian Research Council (QEII Fellowship), the National Health and Medical Research Council (Senior Research Fellowship, Project Grants 461247, 516725 and 575535) and the Sylvia and Charles Viertel Charitable Foundation (Senior Medical Research Fellowship).

References

  1. Top of page
  2. Abstract
  3. The intrinsic apoptosis pathway
  4. Apoptosis and platelet production
  5. Apoptosis, platelet life and platelet death
  6. Acknowledgments
  7. Disclosure of Conflict of Interests
  8. References