Papers by Simone Schoenwaelder
Journal of Biological Chemistry, Dec 1, 1994
Integrins promote cell-substratum and cell-cell adhesion by acting as transmembrane linker molecu... more Integrins promote cell-substratum and cell-cell adhesion by acting as transmembrane linker molecules between extracellular adhesion proteins and the actin-rich cytoskeleton. The integrin alpha IIb beta 3 (platelet glycoprotein IIb/IIIa) is essential for platelet spreading, aggregation, fibrin clot retraction, and for the transduction of extracellular signals. We examined the effect of the specific tyrosine kinase inhibitor herbimycin A on integrin and cytoskeletal-mediated events in thrombin-stimulated platelets. Incubation of washed platelets for 24 h with herbimycin A (5 microM) abolished the thrombin-stimulated cytoskeletal enzyme activity of pp60c-src in parallel with a reduction in the tyrosine phosphorylation of multiple platelet proteins, as assessed with anti-phosphotyrosine immunoblots. However, thrombin-induced activation of protein kinase C and the production of thromboxane A2 were not altered by herbimycin A. Despite the absence of cytoskeletal pp60c-src enzyme activity, platelet shape change, aggregation, and serotonin release were unaltered following platelet stimulation with thrombin (0.05-1.0 unit/ml). Herbimycin A-treated platelets also demonstrated normal platelet aggregation in response to collagen (5 micrograms/ml), ionophore A23187 (2 microM), and ADP/adrenaline (10 microM each). However, the ability of herbimycin A-treated platelets to retract fibrin gels was significantly reduced. This defect in clot retraction was associated with reduced incorporation of integrin alpha IIb beta 3 into the cytoskeletal fraction of thrombin-aggregated platelets. Our studies suggest that tyrosine kinases in platelets regulate the cytoskeletal attachment of alpha IIb beta 3, as an essential process for the transmission of cellular contractile forces to fibrin polymers.
Blood, Dec 3, 2015
Platelet lifespan is limited to 10 days in humans and 5 days in mice. The intrinsic apoptosis pat... more Platelet lifespan is limited to 10 days in humans and 5 days in mice. The intrinsic apoptosis pathway regulates the survival of platelets, where the pro-survival protein Bcl-xLrestrains the essential death mediators Bak and Bax (Mason et al., Cell 2007). Hence, platelet lifespan and platelet counts in mice are increased in the absence of Bak and Bak/Bax. While platelet production in mice is normal in the absence of intrinsic apoptosis (Josefsson et al., J Exp Med 2011), the function of these long-lived platelets has not been investigated. In the current study we examined the functional outcome of extended platelet survival. We found that washed platelets from mice with a constitutive deletion of Bak and a platelet-specific deletion of Bax (Bak-/-BaxPf4Δ/Pf4Δ) were fully resistant to apoptosis induced by the BH3-mimetic ABT-737, as demonstrated by lack of phosphatidylserine exposure (binding of AnnexinV) and unaltered mitochondrial membrane potential. Tail bleeding times into 37°C saline, were extended in the absence of either Bak alone or both Bak and Bax. Furthermore, the electrolytic thrombosis model showed that despite normal time to arterial occlusion, the thrombi formed in Bak-/- BaxPf4Δ/Pf4Δ mice were unstable, a trend also observed in Bak-/-Baxfl/fl mice. The formation of stable thrombi is dependent on the release of secondary agonists, such as ADP and Thromboxane, from activated platelets. To investigate potential defects in platelet signaling pathways in the absence of Bax and/or Bak, we performed in vitro platelet activation assays. Flow cytometric measurements revealed that activation of the PAR4 receptor (by PAR4-AP) or GPVI (by convulxin) led to reduced integrin activation (JON/A) and degranulation (P-selectin exposure) in the absence of Bak and Bak/Bax, while loss of Bax alone had no effect. In contrast, the response to activation with ADP, which does not induce granule release, was similar in platelets from all genotypes. Similarly, platelet aggregation in response to intermediate concentrations of PAR4-AP was severely reduced in the absence of Bak and Bak/Bax, but normal in response to ADP. We next investigated if abnormal degranulation in response to agonists could explain the aggregation defect. Platelet aggregation was performed with PAR4-AP and the platelet supernatants were collected after centrifugation. Dense granule release (ATP and serotonin) and alpha granule release (PF4) were significantly reduced from platelets deficient in Bak, Bak/Bax, but not Bax alone. Untreated resting platelets of all genotypes contained similar amount of granular proteins (ATP, serotonin and PF4). Hence, altered granule content was not the reason behind the abnormality. We next explored if platelet age was a factor behind the observed functional differences. To be able to directly compare platelet function in Bak/Bax deficient mice and wild-type controls, we synchronized platelet age to ~3 days in all genotypes. Platelets were depleted in vivo by injection of anti-platelet serum (APS). Newly generated platelets were collected at 72 h post injection, a time-point were platelet counts had returned to normal. Remarkably, synchronized platelet age normalized PAR4-AP and convulxin dependent integrin activation (JON/A) and degranulation (P-selectin exposure) in the absence of Bak and Bak/Bax to control levels. Similarly, the platelet aggregation and release defects were rescued. Lastly we investigated if synchronizing platelet age would revert the hemostatic defect of Bak/Bax mice in vivo. We determined tail bleeding times using mice, which were either untreated or depleted of platelets 72 h prior to the experiment. Strikingly, synchronization of platelet age to 3 days rescued the hemostatic defect in Bak-/-BaxPf4Δ/Pf4Δ mice. We conclude that extended platelet survival leads to platelet exhaustion, with reduced ability to mobilize granular release. Our studies suggest that, in the context of blood bank storage, extending platelet survival times by pharmacologically inhibiting apoptosis may result in a hemostatically compromised product. Disclosures No relevant conflicts of interest to declare.
Amyloidogenic protein aggregation impairs cell function and is a hallmark of many chronic degener... more Amyloidogenic protein aggregation impairs cell function and is a hallmark of many chronic degenerative disorders. Protein aggregation is also a major event during acute injury, however, unlike amyloidogenesis, the process of injury-induced protein aggregation remains largely undefined. To provide this insight, we profiled the insoluble proteome of several cell types after acute injury. These experiments show that the disulfide-driven process of nucleocytoplasmic coagulation (NCC) is the main form of injury-induced protein aggregation. NCC is mechanistically distinct from amyloidogenesis, but still broadly impairs cell function by promoting the aggregation of hundreds of abundant and essential intracellular proteins. A small proportion of the intracellular proteome resists NCC and is instead released from necrotic cells. Notably, the physico-chemical properties of NCC-resistant proteins are contrary to those of NCC-sensitive proteins. These observations challenge the dogma that liberation of constituents during necrosis is anarchic. Rather, inherent physico-chemical features including cysteine content, hydrophobicity and intrinsic disorder, determine whether a protein is released from necrotic cells. Furthermore, as half of the identified NCC-resistant proteins are known autoantigens, we propose that physico-chemical properties that control NCC also affect immune tolerance and other host responses important for the restoration of homeostasis after necrotic injury.
Journal of Biological Chemistry, May 1, 1999
Activation of the thiol protease calpain results in proteolysis of focal adhesion-associated prot... more Activation of the thiol protease calpain results in proteolysis of focal adhesion-associated proteins and severing of cytoskeletal-integrin links. We employed a commonly used inhibitor of calpain, calpeptin, to examine a role for this protease in the reorganization of the cytoskeleton under a variety of conditions. Calpeptin induced stress fiber formation in both forskolin-treated REF-52 fibroblasts and serum-starved Swiss 3T3 fibroblasts. Surprisingly, calpeptin was the only calpain inhibitor of several tested with the ability to induce these effects, suggesting that calpeptin may act on targets besides calpain. Here we show that calpeptin inhibits tyrosine phosphatases, enhancing tyrosine phosphorylation particularly of paxillin. Calpeptin preferentially inhibits membrane-associated phosphatase activity. Consistent with this observation, in vitro phosphatase assays using purified glutathione S-transferase fusion proteins demonstrated a preference for the transmembrane protein-tyrosine phosphatase-␣ over the cytosolic protein-tyrosine phosphatase-1B. Furthermore, unlike wide spectrum inhibitors of tyrosine phosphatases such as pervanadate, calpeptin appeared to inhibit a subset of phosphatases. Calpeptin-induced assembly of stress fibers was inhibited by botulinum toxin C3, indicating that calpeptin is acting on a phosphatase upstream of the small GTPase Rho, a protein that controls stress fiber and focal adhesion assembly. Not only does this work reveal that calpeptin is an inhibitor of proteintyrosine phosphatases, but it suggests that calpeptin will be a valuable tool to identify the phosphatase activity upstream of Rho.
Circulation, Nov 24, 2009
Background-Individuals with diabetes mellitus have an increased risk of cardiovascular disease an... more Background-Individuals with diabetes mellitus have an increased risk of cardiovascular disease and exhibit platelet hyperreactivity, increasing their resistance to antithrombotic therapies such as aspirin and clopidogrel. Reconstituted high-density lipoprotein (rHDL) has short-term beneficial effects on atherosclerotic plaques, but whether it can effectively reduce the reactivity of diabetic platelets is not known. Methods and Results-Individuals with type 2 diabetes mellitus were infused with placebo or rHDL (CSL-111; 20 mg • kg Ϫ1 • h Ϫ1) for 4 hours, resulting in an Ϸ1.4-fold increase in plasma HDL cholesterol levels. rHDL infusion was associated with a Ͼ50% reduction in the ex vivo platelet aggregation response to multiple agonists, an effect that persisted in washed platelets. In vitro studies in platelets from healthy individuals revealed that the inhibitory effects of rHDL on platelet function were time and dose dependent and resulted in a widespread attenuation of platelet function and a 50% reduction in thrombus formation under flow. These effects could be recapitulated, in part, by the isolated phospholipid component of rHDL, which enhanced efflux of cholesterol from platelets and reduced lipid raft assembly. In contrast, the apolipoprotein AI component of rHDL had minimal effect on platelet function, cholesterol efflux, or lipid raft assembly. Conclusion-These findings suggest that rHDL therapy is highly effective at inhibiting the heightened reactivity of diabetic platelets, partly through reducing the cholesterol content of platelet membranes. These properties, combined with the known short-term beneficial effects of rHDL on atherosclerotic lesions, suggest that rHDL infusions may be an effective approach to reduce atherothrombotic complications in diabetic individuals.
This thesis was scanned from the print manuscript for digital preservation and is copyright the a... more This thesis was scanned from the print manuscript for digital preservation and is copyright the author. Researchers can access this thesis by asking their local university, institution or public library to make a request on their behalf. Monash staff and postgraduate students can use the link in the References field.
Journal of Thrombosis and Haemostasis, Oct 1, 2012
Platelets, 2000
Efficient platelet adhesion and aggregation at sites of vascular injury requires the synergistic ... more Efficient platelet adhesion and aggregation at sites of vascular injury requires the synergistic contribution of multiple adhesion receptors. The initial adhesion of platelets to subendothelial matrix proteins involves GPIb/V/IX and one or more platelet integrins, including integrin alpha IIb beta 3, alpha 2 beta 1, alpha 5 beta 1 and possibly alpha 6 beta 1. In contrast, platelet-platelet adhesion (platelet cohesion or aggregation) is mediated exclusively by GPIb/V/IX and integrin alpha IIb beta 3. Integrin alpha IIb beta 3 is a remarkable receptor that not only stabilizes platelet-vessel wall and platelet-platelet adhesion contacts, but also transduces signals necessary for a range of other functional responses. These signals are linked to cytoskeletal reorganization and platelet spreading, membrane vesiculation and fibrin clot formation, and tension development on a fibrin clot leading to clot retraction. This diverse functional role of integrin alpha IIb beta 3 is reflected by its ability to induce the activation of a broad range of signaling enzymes that are involved in membrane phospholipid metabolism, protein phosphorylation, calcium mobilization and activation of small GTPases. An important calcium-dependent signaling enzyme involved in integrin alpha IIb beta 3 outside-in signaling is the thiol protease, calpain. This enzyme proteolyses a number of key structural and signaling proteins involved in cytoskeletal remodeling and platelet activation. These proteolytic events appear to play a potentially important role in modulating the adhesive and signaling function of integrin alpha IIb beta 3.
Molecular Cancer Research, Jul 1, 2011
Medulloblastoma is the most common malignant brain tumor in children and is associated with a poo... more Medulloblastoma is the most common malignant brain tumor in children and is associated with a poor outcome. We were interested in gaining further insight into the potential of targeting the human kinome as a novel approach to sensitize medulloblastoma to chemotherapeutic agents. A library of small interfering RNA (siRNA) was used to downregulate the known human protein and lipid kinases in medulloblastoma cell lines. The analysis of cell proliferation, in the presence or absence of a low dose of cisplatin after siRNA transfection, identified new protein and lipid kinases involved in medulloblastoma chemoresistance. PLK1 (polo-like kinase 1) was identified as a kinase involved in proliferation in medulloblastoma cell lines. Moreover, a set of 6 genes comprising ATR, LYK5, MPP2, PIK3CG, PIK4CA, and WNK4 were identified as contributing to both cell proliferation and resistance to cisplatin treatment in medulloblastoma cells. An analysis of the expression of the 6 target genes in primary medulloblastoma tumor samples and cell lines revealed overexpression of LYK5 and PIK3CG. The results of the siRNA screen were validated by target inhibition with specific pharmacological inhibitors. A pharmacological inhibitor of p110 (encoded by PIK3CG) impaired cell proliferation in medulloblastoma cell lines and sensitized the cells to cisplatin treatment. Together, our data show that the p110 phosphoinositide 3-kinase isoform is a novel target for combinatorial therapies in medulloblastoma.
British Journal of Haematology, Nov 1, 2009
Considerable progress has been made over the last two decades in delineating the key molecular ev... more Considerable progress has been made over the last two decades in delineating the key molecular events regulating the haemostatic function of platelets. Much of this new insight has been derived from the study of mouse models, in which the expression or structure of one or more platelet proteins has been genetically altered. Despite these advances on the research front, clinical progress in diagnosing patients with unexplained surgical bleeding or recurrent haemorrhage from mucocutaneous sites has been comparatively limited. There is a dearth of literature available to help physicians integrate and apply the burgeoning knowledge on platelet biology to diagnosing patients with atypical or unexplained platelet dysfunction. The purpose of this review is to summarise the major primary platelet disorders relevant to pathological bleeding in humans (excluding those primarily due to thrombocytopenia or acquired functional disorders), with a focus on lesions identified in mouse models that could represent candidate molecules for study in patients with impaired platelet function.
Nature Communications, Jul 15, 2014
Toll-like receptor 4 (TLR4) is activated by bacterial lipopolysaccharide (LPS) to mount innate im... more Toll-like receptor 4 (TLR4) is activated by bacterial lipopolysaccharide (LPS) to mount innate immune responses. The TLR4-induced release of pro-and anti-inflammatory cytokines generates robust inflammatory responses, which must then be restrained to avoid disease. New mechanisms for the critical regulation of TLR-induced cytokine responses are still emerging. Here we find TLR4 complexes localized in LPS-induced dorsal ruffles on the surface of macrophages. We discover that the small GTPase Rab8a is enriched in these ruffles and recruits phosphatidylinositol 3-kinase (PI3Kg) as an effector by interacting directly through its Ras-binding domain. Rab8a and PI3Kg function to regulate Akt signalling generated by surface TLR4. Rab8a and PI3Kg do not affect TLR4 endocytosis, but instead regulate mammalian target of rapamycin signalling as a mechanism for biasing the cytokine profile to constrain inflammation in innate immunity.
Journal of Biological Chemistry, Oct 1, 1994
The cytoskeleton participates in the coordinated regulation of intracellular signaling molecules,... more The cytoskeleton participates in the coordinated regulation of intracellular signaling molecules, following agonist stimulation of cells. We have demonstrated that von Willebrand factor (vWF) induced the cytoskeletal association and activation of phosphatidylinositol 3-kinase (PtdIns 3-kinase) in human platelets. The activation of PtdIns 3-kinase coincided with the tyrosine phosphorylation of multiple platelet proteins, as assessed by anti-phosphotyrosine immunoblotting. One of these tyrosine-phosphorylated proteins, pp60c-src, became specifically enriched in the cytoskeletal fraction of vWF-stimulated platelets. The vWF-stimulated cytoskeletal association of PtdIns 3-kinase and pp60c-src required platelet stirring and aggregation, was specifically blocked by an anti-GPIb monoclonal antibody, and was not observed in platelets lacking the glycoprotein Ib/IX complex (Bernard-Soulier syndrome). Pretreatment of normal platelets with 5 mM EDTA (37 degrees C for 90 min) or RGDS (2 mM), which disrupts the binding of various adhesive proteins to platelet integrins and inhibits fibrinogen-mediated platelet aggregation, did not alter the vWF-stimulated activation and cytoskeletal association of PtdIns 3-kinase and pp60c-src. Pretreatment of platelets with acetylsalicylic acid (1 mM) completely abolished vWF-stimulated production of thromboxane A2, dense granule release, and the activation of protein kinase C, without altering the activation and cytoskeletal translocation of PtdIns 3-kinase and pp60c-src. Our results suggest that vWF binding to the platelet adhesion receptor glycoprotein Ib/IX can mediate activation and translocation of both tyrosine and lipid kinase(s) independent of other agonists.
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2000
Remodeling of filamentous actin into distinct arrangements is precisely controlled by members of ... more Remodeling of filamentous actin into distinct arrangements is precisely controlled by members of the Rho family of small GTPases [1]. A well characterized member of this family is RhoA, whose activation results in reorganization of the cytoskeleton into thick actin stress fibers terminating in integrin-rich focal adhesions [2]. Regulation of RhoA is required to maintain adhesion in stationary cells, but is also critical for cell spreading and migration [3]. Despite its biological importance, the signaling events leading to RhoA activation are not fully understood. Several independent studies have implicated tyrosine phosphorylation as a critical event upstream of RhoA [4]. Consistent with this, our recent studies have demonstrated the existence of a protein tyrosine phosphatase (PTPase), sensitive to the dipeptide aldehyde calpeptin, acting upstream of RhoA [5]. Here we identify the SH2 (Src homology region 2)containing PTPase Shp-2 as a calpeptin-sensitive PTPase, and show that calpeptin interferes with the catalytic activity of Shp-2 in vitro and with Shp-2 signaling in vivo. Finally, we show that perturbation of Shp-2 activity by a variety of genetic manipulations results in raised levels of active RhoA. Together, these studies identify Shp-2 as a PTPase acting upstream of RhoA to regulate its activity and contribute to the coordinated control of cell movement.
Carolina Digital Repository (University of North Carolina at Chapel Hill), 1999
Activation of the thiol protease calpain results in proteolysis of focal adhesion-associated prot... more Activation of the thiol protease calpain results in proteolysis of focal adhesion-associated proteins and severing of cytoskeletal-integrin links. We employed a commonly used inhibitor of calpain, calpeptin, to examine a role for this protease in the reorganization of the cytoskeleton under a variety of conditions. Calpeptin induced stress fiber formation in both forskolin-treated REF-52 fibroblasts and serum-starved Swiss 3T3 fibroblasts. Surprisingly, calpeptin was the only calpain inhibitor of several tested with the ability to induce these effects, suggesting that calpeptin may act on targets besides calpain. Here we show that calpeptin inhibits tyrosine phosphatases, enhancing tyrosine phosphorylation particularly of paxillin. Calpeptin preferentially inhibits membrane-associated phosphatase activity. Consistent with this observation, in vitro phosphatase assays using purified glutathione S-transferase fusion proteins demonstrated a preference for the transmembrane protein-tyrosine phosphatase-␣ over the cytosolic protein-tyrosine phosphatase-1B. Furthermore, unlike wide spectrum inhibitors of tyrosine phosphatases such as pervanadate, calpeptin appeared to inhibit a subset of phosphatases. Calpeptin-induced assembly of stress fibers was inhibited by botulinum toxin C3, indicating that calpeptin is acting on a phosphatase upstream of the small GTPase Rho, a protein that controls stress fiber and focal adhesion assembly. Not only does this work reveal that calpeptin is an inhibitor of proteintyrosine phosphatases, but it suggests that calpeptin will be a valuable tool to identify the phosphatase activity upstream of Rho.
Blood, Nov 16, 2008
Human platelets exhibit a circulating lifespan of ~10 days, mouse platelets ~5 days. This finite ... more Human platelets exhibit a circulating lifespan of ~10 days, mouse platelets ~5 days. This finite existence is circumscribed by members of the Bcl-2 family of proteins, which control the intrinsic apoptosis pathway. Pro-survival Bcl-xL is the critical regulator of platelet lifespan, functioning to keep pro-death Bak and Bax in check, thereby maintaining platelet viability. After 5–10 days in the circulation, platelets not consumed in hemostatic processes initiate a Bak and Bax-dependent cell death program and clearance from the bloodstream. Mutations in Bcl-xL reduce platelet lifespan in a dose-dependent fashion, while deletion of Bak and Bax extend it. Studies with the BH3 mimetic compound ABT-737, which inhibits pro-survival Bcl-xL, have shown that platelets induced to undergo cell death in vitro exhibit many of the hallmarks of apoptosis in nucleated cells, including mitochondrial damage, caspase activation and externalization of membrane phosphatidylserine (PS). Whether any of these features occur during physiological platelet clearance remains unclear. Certainly, mitochondrial damage can reduce the recovery of transfused platelets, but whether PS – which is known to promote the pro-coagulant activity of agonist-activated platelets – also acts as a clearance signal for dying platelets in vivo is yet to be established. Conversely, Bak and Bax may play a role in mediating PS exposure triggered by activation. Supporting the idea that there may be crosstalk between classical platelet signaling pathways and the intrinsic apoptosis pathway is recent evidence that platelet agonists can also activate caspases. Intriguingly, elements of the intrinsic pathway may also contribute to the generation of platelets by megakaryocytes. Several groups have demonstrated that megakaryocytes contain activated caspases and that their inhibition can block platelet shedding by cultured cells. Preliminary evidence we have generated suggests that Bcl-2 family proteins may be required for platelet production in vivo. Thus, it appears that there is much to be understood about the role of the intrinsic apoptosis pathway in the regulation of platelet biogenesis, function, and death.
Elsevier eBooks, 2019
Abstract The discovery of serotonin in platelets in the 1950s heralded a new era of research in t... more Abstract The discovery of serotonin in platelets in the 1950s heralded a new era of research in the study of blood clot formation. Platelets, which represent the cellular components in the blood responsible for initiating blood clot formation, are responsible for maintaining a low concentration of plasma serotonin by sequestering and storing a remarkable concentration of serotonin. While platelets are vital for regulating circulating serotonin levels in the plasma, serotonin itself is suggested to play a fundamental role in both platelet function and blood clot formation. However, despite many studies, the role for serotonin in thrombus formation is still not completely elucidated. This chapter will explore a brief history of both platelets and serotonin and present a current understanding of the interactions between the two. Here we discuss the vital role of platelets in the regulation of circulating serotonin concentrations, the significant role of serotonin in platelets for the hemostatic and thrombotic responses, and the clinical implications of modulating this process in health and disease.
Blood, Dec 10, 2015
9. Haroche J, Cohen-Aubart F, Emile JF, et al. Reproducible and sustained efficacy of targeted th... more 9. Haroche J, Cohen-Aubart F, Emile JF, et al. Reproducible and sustained efficacy of targeted therapy with vemurafenib in patients with BRAF(V600E)-mutated Erdheim-Chester disease.
European journal of medicinal chemistry, Oct 1, 2016
A series of amino-substituted triazines were developed and examined for PI3Kβ inhibition and anti... more A series of amino-substituted triazines were developed and examined for PI3Kβ inhibition and anti-platelet function. Structural adaptations of a morpholine ring of the prototype pan-PI3K inhibitor ZSTK474 yielded PI3Kβ selective compounds, where the selectivity largely derives from an interaction with the non-conserved Asp862 residue, as shown by site directed mutagenesis. The most PI3Kβ selective inhibitor from the series was studied in detail through a series of in vitro and in vivo functional studies. MIPS-9922, 10 potently inhibited ADP-induced washed platelet aggregation. It also inhibited integrin αIIbβ3 activation and αIIbβ3 dependent platelet adhesion to immobilized vWF under high shear. It prevented arterial thrombus formation in the in vivo electrolytic mouse model of thrombosis without inducing prolonged bleeding or excess blood loss.
Blood, May 3, 2012
Apoptotic caspases, including caspase-9, are thought to facilitate platelet shedding by megakaryo... more Apoptotic caspases, including caspase-9, are thought to facilitate platelet shedding by megakaryocytes. They are known to be activated during platelet apoptosis, and have also been implicated in platelet hemostatic responses. However, the precise requirement for, and the regulation of, apoptotic caspases have never been defined in either megakaryocytes or platelets. To establish the role of caspases in platelet production and function, we generated mice lacking caspase-9 in their hematopoietic system. We demonstrate that both megakaryocytes and platelets possess a functional apoptotic caspase cascade downstream of Bcl-2 family-mediated mitochondrial damage. Caspase-9 is the initiator caspase, and its loss blocks effector caspase activation. Surprisingly, steady-state thrombopoiesis is unperturbed in the absence of caspase-9, indicating that the apoptotic caspase cascade is not required for platelet production. In platelets, loss of caspase-9 confers resistance to the BH3 mimetic ABT-737, blocking phospha-tidylserine (PS) exposure and delaying ABT-737-induced thrombocytopenia in vivo. Despite this, steady-state platelet lifespan is normal. Casp9 Ϫ/Ϫ platelets are fully capable of physiologic hemostatic responses and functional regulation of adhesive integrins in response to agonist. These studies demonstrate that the apoptotic caspase cascade is required for the efficient death of megakaryocytes and platelets, but is dispensable for their generation and function. (Blood. 2012;119(18):4283-4290)
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Papers by Simone Schoenwaelder