scholarly journals Influence of γ-Secretase Inhibitor 24-Diamino-5-Phenylthiazole DAPT on Platelet Activation

2016 ◽  
Vol 38 (2) ◽  
pp. 726-736 ◽  
Author(s):  
Guoxing Liu ◽  
Guilai Liu ◽  
Madhumita Chatterjee ◽  
Anja T. Umbach ◽  
Hong Chen ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Function ◽  
Blood Platelets ◽  
Annexin V ◽  
Negative Regulator ◽  
Ros Generation ◽  
Integrin Activation ◽  
Forward Scatter ◽  
Secretase Inhibitor ◽  
Αiibβ3 Integrin

Background/Aims: DAPT (24-diamino-5-phenylthiazole) inhibits γ-secretase, which cleaves the signaling molecule CD44, a negative regulator of platelet activation and apoptosis. CD44 is a co-receptor for macrophage migration inhibitory factor (MIF) an anti-apoptotic pro-inflammatory cytokine expressed and released from blood platelets. Whether DAPT influences platelet function, remained, however, elusive. Activators of platelets include collagen related peptide (CRP). The present study thus explored whether DAPT modifies the stimulating effect of CRP on platelet function. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to DAPT (10 µM). Flow cytometry was employed to estimate Orai1 abundance with specific antibodies, cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, generation of reactive oxygen species (ROS) from DCFDA fluorescence, mitochondrial transmembrane potential from TMRE fluorescence, phospholipid scrambling of the cell membrane from annexin-V-binding, relative platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. Results: Exposure of platelets to 2-5 µg/ml CRP was followed by significant increase of Orai1 abundance, [Ca2+]i, and P-selectin abundance, as well as by αIIbβ3 integrin activation, ROS generation, mitochondrial depolarization, enhanced annexin-V-binding, decreased cell volume, and aggregation. All CRP induced effects were significantly blunted in the presence of DAPT. Conclusions: The γ-secretase inhibitor DAPT counteracts agonist induced platelet activation, apoptosis and aggregation.

scholarly journals Temsirolimus Sensitive Stimulation of Platelet Activity, Apoptosis and Aggregation by Collagen Related Peptide

2017 ◽  
Vol 42 (3) ◽  
pp. 1252-1263 ◽  
Author(s):  
Hang Cao ◽  
Rosi Bissinger ◽  
Anja T. Umbach ◽  
Meinrad Gawaz ◽  
Florian Lang
Keyword(s):  
Platelet Activation ◽  
Hippocampal Neurons ◽  
Blood Platelets ◽  
Annexin V ◽  
Caspase Activity ◽  
Integrin Activation ◽  
Forward Scatter ◽  
Related Peptide ◽  
Αiibβ3 Integrin ◽  
Stimulation Of

Background/Aims: The mammalian target of rapamycin (mTOR) inhibitor temsirolimus stimulates apoptosis of tumor cells and is thus therapeutically used for the treatment of diverse malignancies. On the other hand, temsirolimus has been shown to protect against apoptosis of hippocampal neurons. Similar to nucleated cells, blood platelets may enter suicidal death characterized by cell shrinkage and cell membrane scrambling. Platelet apoptosis is frequently preceded by Ca2+ entry, degranulation, integrin activation and stimulation of caspases. Those events could be triggered by collagen related peptide (CRP). The present study explored whether treatment of platelets with temsirolimus modifies platelet activation, caspase activity, platelet shrinkage, and phosphatidylserine abundance. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to temsirolimus (40 µg/ml) without or with additional CRP (2 µg/ ml or 5 µg/ml) treatment. Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fuorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding and relative platelet volume from forward scatter. Results: In the absence of CRP, the administration of temsirolimus (40 µg/ml) significantly decreased [Ca2+]i, but did not significantly modify P-selectin abundance, activated αIIbβ3 integrin, annexin-V-binding, cell volume, caspase activity and aggregation. Exposure of platelets to CRP was followed by significant increase of [Ca2+]i, P-selectin abundance, αIIbβ3 integrin activity, annexin-V-binding, ROS, caspase activity and aggregation, effects significantly blunted in the presence of temsirolimus. CRP further decreased forward scatter, an effect again significantly blunted by temsirolimus. Conclusions: Temsirolimus is a powerful inhibitor of platelet activation and suicidal platelet death.

scholarly journals Effect of Bexarotene on Platelet Activation and Apoptosis

2017 ◽  
Vol 42 (2) ◽  
pp. 838-847 ◽  
Author(s):  
Hang Cao ◽  
Rosi Bissinger ◽  
Anja T. Umbach ◽  
Meinrad Gawaz ◽  
Florian Lang
Keyword(s):  
Cell Volume ◽  
Platelet Activation ◽  
Blood Platelets ◽  
Annexin V ◽  
Additional Treatment ◽  
Caspase Activity ◽  
Integrin Activation ◽  
Suicidal Death ◽  
Αiibβ3 Integrin ◽  
Binding Cell

Background/Aims: The retinoid X receptor (RXRs) stimulator Bexarotene ((4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl] benzoic acid) is used for the treatment of several malignancies. Bexarotene is at least in part effective by stimulation of apoptosis of tumor cells. Moreover, Bexarotene triggers eryptosis, the suicidal death of erythrocytes. Similar to erythrocytes, blood platelets lack nuclei but are nevertheless able to enter an apoptosis-like phenotype, characterized by caspase activation, cell shrinkage and cell membrane scrambling with phospha-tidylserine translocation to the cell surface. Platelet apoptosis is triggered by increase of cytosolic Ca2+-activity ([Ca2+]i), which further leads to degranulation and integrin activation. Platelet activation and apoptosis could be elicited by thrombin or collagen related peptide (CRP). The present study explored whether treatment of platelets with bexarotene modifies platelet activation and apoptosis following exposure to thrombin or CRP. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to bexarotene (6 µg/ml) without or with an additional treatment with thrombin (0.01 U/ml) or CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, and relative platelet volume from forward scatter. Results: In the absence of thrombin or CRP, the administration of bexarotene slightly but significantly increased [Ca2+]i, but did not significantly modify P-selectin abundance, activated αIIbβ3 integrin, annexin-V-binding, cell volume, or caspase activity. Exposure of platelets to thrombin or CRP was followed by significant increase of [Ca2+]i, P-selectin abundance, active αIIbβ3 integrin, annexin-V-binding, and caspase activity. The effects of thrombin on [Ca2+]i, annexin-V-binding, cell volume, and caspase activity as well as the effects of CRP on [Ca2+]i, P-selectin abundance, activated αIIbβ3 integrin, annexin-V-binding, cell volume, and caspase activity were significantly augmented in the presence of bexarotene. Conclusions: Bexarotene sensitizes blood platelets for thrombin and/or CRP induced activation and apoptosis.

scholarly journals Effect of Lysosomotropic Polyamineoxidase Inhibitor MDL-72527 on Platelet Activation

2016 ◽  
Vol 38 (5) ◽  
pp. 1695-1702 ◽  
Author(s):  
Guoxing Liu ◽  
Hang Cao ◽  
Guilai Liu ◽  
David Heinzmann ◽  
Hong Chen ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Annexin V ◽  
Oxidase Inhibitor ◽  
Wild Type ◽  
Integrin Activation ◽  
Forward Scatter ◽  
Mdl 72527 ◽  
Phospholipid Scrambling ◽  
Subsequent Activation ◽  
Αiibβ3 Integrin

Background/Aims: The polyamine oxidase inhibitor MDL-72527 (N1,N4-bis(2,3-butadienyl)-1,4-butanediamine) were expected to increase the abundance of spermine, a powerful inhibitor of platelet activation. Nothing is known, however, on the sensitivity of platelet function and survival to MDL-72527 exposure. The present study thus explored whether MDL-72527 modifies function and survival of platelets without and with platelet activation by collagen related peptide (CRP). Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to MDL-72527 (100 µM) with or without subsequent activation with CRP (2-5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, generation of reactive oxygen species (ROS) from DCFDA fluorescence, phospholipid scrambling of the cell membrane from annexin-V-binding, platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. Results: In the absence of CRP, exposure of platelets to MDL-72527 did not significantly modify [Ca2+]i, P-selectin abundance, αIIbβ3 integrin abundance, ROS, annexin-V-binding, and forward scatter. The addition of 2-5 µg/ml CRP was followed by significant increase of [Ca2+]i, P-selectin abundance, αIIbβ3 integrin activation, ROS abundance, annexin-V-binding, and aggregation as well as a significant decrease of forward scatter, all effects significantly blunted or virtually abolished in the presence of MDL-72527. Conclusions: MDL-72527 is a powerful inhibitor of platelet activation, apoptosis and aggregation.

scholarly journals Garcinol A Novel Inhibitor of Platelet Activation and Apoptosis

Toxins ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 382 ◽  
Author(s):  
Hang Cao ◽  
Abdulla Al Mamun Bhuyan ◽  
Anja T. Umbach ◽  
Ke Ma ◽  
Oliver Borst ◽  
...  
Keyword(s):  
Blood Platelets ◽  
Annexin V ◽  
Caspase Activity ◽  
Platelet Activity ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Tumor Cell Apoptosis ◽  
Αiibβ3 Integrin ◽  
Binding Cell ◽  
Active Caspase

Garcinol, an anti-inflammatory and anti-carcinogenic polyisoprenylated benzophenone isolated from Garcinia plants, stimulates tumor cell apoptosis and suicidal erythrocyte death, but supports the survival of hepatocytes and neurons. The present study explored whether the substance influences platelet function and/or apoptosis. To this end, we exposed murine blood platelets to garcinol (33 µM, 30 min) without and with activation by collagen-related peptide (CRP) (2–5 µg/mL) or thrombin (0.01 U/mL); flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, relative platelet volume from forward scatter, and aggregation utilizing staining with CD9-APC and CD9-PE. As a result, in the absence of CRP and thrombin, the exposure of the platelets to garcinol did not significantly modify [Ca2+]i, P-selectin abundance, activated αIIbβ3 integrin, annexin-V-binding, cell volume, caspase activity, and aggregation. Exposure of platelets to CRP or thrombin was followed by a significant increase of [Ca2+]i, P-selectin abundance, αIIbβ3 integrin activity, annexin-V-binding, caspase activity, and aggregation, as well as significant cell shrinkage. All effects of CRP were strong and significant; those of thrombin were only partially and slightly blunted in the presence of garcinol. In conclusion, garcinol blunts CRP-induced platelet activity, apoptosis and aggregation.

scholarly journals Inhibition of Collagen Related Peptide Induced Platelet Activation and Apoptosis by Ceritinib

2018 ◽  
Vol 45 (4) ◽  
pp. 1707-1716 ◽  
Author(s):  
Hang Cao ◽  
Anja T. Umbach ◽  
Rosi Bissinger ◽  
Meinrad Gawaz ◽  
Florian Lang
Keyword(s):  
Platelet Activation ◽  
Blood Platelets ◽  
Annexin V ◽  
Caspase Activity ◽  
Platelet Surface ◽  
Anaplastic Lymphoma ◽  
Related Peptide ◽  
Platelet Apoptosis ◽  
Induced Apoptosis ◽  
Αiibβ3 Integrin

Background/Aims: The anaplastic lymphoma (tyrosine) kinase (ALK) inhibitor ceritinib triggers apoptosis of tumor cells and eryptosis of erythrocytes. Blood platelets may similarly enter a state resembling apoptosis, which could be triggered by activation with collagen related peptide (CRP). CRP-induced platelet apoptosis is characterized by cell membrane scrambling with phosphatidylserine exposure to the platelet surface and cell shrinkage, preceded by externalization of Ca2+ channel Orai1, increase of cytosolic Ca2+-activity ([Ca2+]i), formation of reactive oxygen species (ROS), and caspase activation. The present study explored whether ceritinib triggers platelet apoptosis and/or modifies the CRP induced apoptosis. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to ceritinib (1.5 µg/ml) without or with 2.5 – 15 min pretreatment with CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, ROS abundance from 2’,7’-dichlorodihydrofluorescein diacetate fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. Results: In the absence of CRP, ceritinib slightly, but significantly decreased [Ca2+]i without significantly modifying the other measured parameters. CRP significantly increased [Ca2+]i, ROS abundance, P-selectin abundance, activated αIIbβ3 integrin, annexin-V-binding, caspase activity as well as aggregation and decreased cell volume, all effects significantly blunted in the presence of ceritinib. Conclusions: The present observations uncover a novel, unexpected effect of ceritinib, i.e. inhibition of CRP-induced platelet activation and apoptosis.

scholarly journals Involvement of Ca2+ Activated Cl- Channel Ano6 in Platelet Activation and Apoptosis

2015 ◽  
Vol 37 (5) ◽  
pp. 1934-1944 ◽  
Author(s):  
Guoxing Liu ◽  
Guilai Liu ◽  
Hong Chen ◽  
Oliver Borst ◽  
Meinrad Gawaz ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Blood Platelets ◽  
Annexin V ◽  
Bleeding Disorder ◽  
Integrin Activation ◽  
Cell Shrinkage ◽  
Platelet Surface ◽  
Cl Channel ◽  
Phosphatidylserine Exposure ◽  
Αiibβ3 Integrin

Background/Aims: The ubiquitously expressed Ca2+ Activated Cl- Channel Ano6 participates in the stimulation of cell membrane scrambling. Defective Ano6 underlies the Scott syndrome, an inherited bleeding disorder with impaired scrambling of plasma membrane phospholipids. At least in theory, the bleeding disorder of Scott syndrome may result from impaired platelet function. Activators of platelets include thrombin and collagen related peptide (CRP), which trigger increase of cytosolic Ca2+-activity ([Ca2+]i), production of reactive oxygen species (ROS), degranulation, integrin activation, as well as cell shrinkage and phospholipid scrambling of the cell membrane. The present study thus explored whether Ano6 modifies activation-induced alterations of cytosolic Ca2+-activity ([Ca2+]i), degranulation (P-selectin exposure), integrin activation, phosphatidylserine exposure on the platelet surface and platelet volume. Methods: Platelets from mice lacking Ano6 (ano6-/-) were compared to platelets from corresponding wild-type mice (ano6+/+). [Ca2+]i was estimated from Fluo-3 fluorescence, ROS from DCFDA fluorescence, degranulation from P-selectin abundance, integrin activation from αIIbβ3-integrin abundance, phosphatidylserine abundance from annexin-V-binding, and cell volume from forward scatter. Results: Platelet number in blood was slightly higher in ano6-/- mice than in ano6+/+ mice. Without activation [Ca2+]i and volume were similar in ano6-/- and ano6+/+ platelets as well as ROS abundance, P-selectin abundance, αIIbβ3 integrin activation, and phosphatidylserine exposure were negligible in both genotypes. Thrombin (0.01 U/ml) and CRP (2 or 5 µg/ml) increased [Ca2+]i, ROS abundance, platelet degranulation, αIIbβ3 integrin activation, and triggered annexin-V-binding as well as cell shrinkage, all effects less pronounced in ano6-/- than in ano6+/+ platelets. Conclusions: Genetic knockout of Ano6 blunts thrombin- and CRP-induced activation and apoptosis of blood platelets.

scholarly journals Inhibitory Effect of Afatinib on Platelet Activation and Apoptosis

2017 ◽  
Vol 43 (6) ◽  
pp. 2264-2276 ◽  
Author(s):  
Hang Cao ◽  
Abdulla Al Mamun Bhuyan ◽  
Anja T. Umbach ◽  
Rosi Bissinger ◽  
Meinrad Gawaz ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Caspase 3 ◽  
Kinase Inhibitor ◽  
Growth Factor Receptor ◽  
Annexin V ◽  
Subsequent Treatment ◽  
Caspase Activity ◽  
Forward Scatter ◽  
Platelet Surface ◽  
Αiibβ3 Integrin

Background/Aims: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor afatinib is used for the treatment of several malignancies. Afatinib is at least partially effective by triggering apoptosis of tumor cells. Platelets may similarly undergo apoptosis, which is characterized by caspase 3 activation, cell shrinkage and phosphatidylserine translocation. However, an effect of afatinib on platelets has never been reported. The present study explored whether treatment of platelets with afatinib modifies platelet activation and apoptosis in the absence and presence of platelet activators thrombin or collagen related peptide (CRP). Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to afatinib (18 µg/ml) without or with subsequent treatment with thrombin (0.005 U/ml or 0.01 U/ml) or CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate Orai1 abundance at the platelet surface with specific antibodies, cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. Results: In the absence of thrombin and CRP, the administration of afatinib (18 µg/ml) slightly, but significantly, increased [Ca2+]i and annexin-V-binding, but did not significantly modify Orai1 abundance, P-selectin abundance, activated αIIbβ3 integrin, cell volume, caspase activity and aggregation. Exposure of platelets to 0.005 U/ml or 0.01 U/ml thrombin or 2 µg/ml or 5 µg/ ml CRP was followed by a significant increase of Orai1 abundance, increase of [Ca2+]i, P-selectin abundance, αIIbβ3 integrin activity, annexin-V-binding, caspase activity, and aggregation, as well as a significant decrease of forward scatter, all effects significantly blunted (thrombin) or virtually abolished (CRP) by afatinib. Conclusions: Afatinib is a powerful inhibitor of platelet activation, platelet apoptosis and platelet aggregation.

scholarly journals In-depth PtdIns(3,4,5)P3 signalosome analysis identifies DAPP1 as a negative regulator of GPVI-driven platelet function

2017 ◽  
Vol 1 (14) ◽  
pp. 918-932 ◽  
Author(s):  
Tom N. Durrant ◽  
James L. Hutchinson ◽  
Kate J. Heesom ◽  
Karen E. Anderson ◽  
Len R. Stephens ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Function ◽  
Binding Proteins ◽  
Thrombus Formation ◽  
Negative Regulator ◽  
Valuable Resource ◽  
Future Studies ◽  
Glycoprotein Vi ◽  
Key Points ◽  
Depth Analysis

Key Points We present the first in-depth analysis of platelet PtdIns(3,4,5)P3-binding proteins, providing a valuable resource for future studies. The PtdIns(3,4,5)P3-binding protein, DAPP1, negatively regulates glycoprotein VI–driven platelet activation and thrombus formation.

Abstract 384: Interleukin-6 Mediates the Altered Platelet Function Associated with Experimental Colonic Inflammation

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Li-Sue S Yan
Keyword(s):  
Platelet Activation ◽  
Platelet Function ◽  
Interleukin 6 ◽  
Blood Platelets ◽  
Critical Role ◽  
Experimental Colitis ◽  
Membrane Expression ◽  
Colonic Inflammation ◽  
Dss Colitis ◽  
Chronic Infusion

Patients with inflammatory bowel disease (IBD) are susceptible to microvascular thrombosis and thromboembolism. The increased incidence of thrombosis is accompanied by enhanced coagulation and abnormalities in platelet function. Clinical studies have also revealed alterations in platelet activation, enhanced platelet-leukocyte interaction, and elevated plasma levels of prothrombotic cytokines, such as IL-6. This study was directed towards determining whether: 1) experimental colitis, induced by 6 days of dextran sodium sulfate (DSS) ingestion, is associated with platelet activation and the formation of platelet-leukocyte aggregates (PLAs), 2) IL-6 deficiency alters these responses to DSS colitis, and 3) the platelet abnormalities observed in DSS mice can be recapitulated by chronic infusion of murine recombinant IL-6. Flow cytometry was used to characterize platelet function in heparin-anticoagulated whole blood. Platelets were identified by characteristic light scattering and membrane expression of CD41. Platelet activation was monitored using the expression of an activation epitope of GPIIb/IIIa integrin (with JON/A antibody). The combination of CD41, CD45.2, Gr-1, F4/80 and isotype control antibodies were used to detect and quantify aggregates of leukocytes, neutrophils and monocytes with platelets in control, wild type (WT) colitic, IL-6 -/- colitic, and WT mice implanted with IL-6 loaded Alzet osmotic minipumps (for 6 days). Our results indicate that DSS colitis is associated with increased numbers of activated platelets and the formation of aggregates of leukocytes (PLA), neutrophils (PNA) and monocytes (PMA) with platelets. These platelet responses to experimental colitis were largely undetected in IL-6 -/- mice. Chronic infusion (at a rate that yielded plasma IL-6 levels similar to those detected in DSS colitic mice) of IL-6 recapitulated the increased platelet activation and formation of PLA, PNA, and PMA observed in DSS-colitic mice. Collectively, these findings show that the altered platelet function detected in human IBD can be reproduced in an animal model of colonic inflammation and that interleukin-6 plays a critical role in the genesis of these platelet abnormalities in the setting of experimental IBD.

Incomplete Platelet Dense Granule Formation in Normal Neonates.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3210-3210
Author(s):  
Walter H. Kahr ◽  
Shoma Baidya ◽  
Animitra Das ◽  
Ayca Toprak ◽  
Hilary Christensen ◽  
...  
Keyword(s):  
Cord Blood ◽  
Thin Section ◽  
Platelet Activation ◽  
Platelet Function ◽  
Blood Platelets ◽  
Coagulation Factor ◽  
Dense Granule ◽  
Older Children ◽  
Granule Formation ◽  
Dense Granules

Abstract Platelets are important in maintaining hemostasis in newborns, where bleeding can arise from abnormal platelet function and/or thrombocytopenia. It is well established that plasma coagulation factor concentrations are lower in neonates compared to children and adults, but less is known about the development and function of neonatal platelets. It has been postulated that platelets from neonates, and to a greater extend from premature neonates, are dysfunctional due to low dense granule counts (Blood2006;108:331a), however, other studies have shown normal neonate platelet function. Our previous studies indicated a slightly decreased number of dense granules per platelet in neonates (Blood2005;106:4159–4156). We have now extended these studies to a larger cohort of 19 normal neonatal cord blood samples (gestational age 37.5–40 weeks) from planned Caesarean sections, which were analyzed under optimal sample handling conditions and compared to platelets from 10 children (age 8–10 years). 50 platelets from each subject were evaluated for dense granule content utilizing whole mount and thin section electron microscopy (EM) for the quantification of dense granules (detected via their electron-dense calcium content) and ultrastructural assessment. A subset of samples was tested via flow cytometry for P-selectin expression as a measure of platelet activation, and platelet structural integrity was also assessed using thin section EM. Our data revealed that platelets in neonatal cord blood had a mean dense granule count of 2.3 (SD=2.2) per platelet, compared to 4.4 dense granules per platelet (SD=2.7) in blood from older children; t-test comparisons showed the difference between these groups to be highly significant (P<0.001). Interestingly, 22% of cord blood platelets contained no measurable dense granules, whereas only 3% of platelets from older children where devoid of dense granules. We suspected that the mean dense granule counts of <1 per platelet in neonatal cord blood reported by others may have arisen due to high levels of platelet activation during sample acquisition or handling. In our samples platelet activation as measured by P-selectin expression was similar in both populations and did not exceed 7.5%, and platelet morphology as assessed by thin section EM was also comparable. Our studies confirm that neonatal cord blood platelets contain fewer recognizable dense granules than those found in older children. Two possible explanations for this observation are: normal numbers of dense granules are present in neonatal platelets, but a subset cannot be detected via EM owing to insufficient calcium uptake; there are fewer dense granules in neonatal platelets owing to peculiarities in the development of megakaryocytes, where recent studies have suggested that dense granules originate by an active transport mechanism and move into proplatelets. These possibilities point to the usefulness of studying fetal and neonatal megakaryopoiesis.

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