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Background-Postoperative thromboembolic stroke affects 2% to 3% of patients undergoing carotid endarterectomy (CEA) and is preceded by 1 to 2 hours of increasing cerebral embolization. Previous work has demonstrated that high rates of postoperative embolization are associated with increased platelet reactivity to adenosine 5Ј-diphosphate (ADP). Our hypothesis was that preoperative administration of the platelet ADP antagonist clopidogrel could reduce postoperative embolization. Methods and Results-One hundred CEA patients on routine aspirin therapy (150 mg) were randomized to 75 mg clopidogrel (nϭ46) or placebo (nϭ54) the night before surgery. Platelet response to ADP was assessed by whole-blood flow cytometry. The number of emboli detected by transcranial Doppler within 3 hours of CEA was independently quantified. Time taken from flow restoration to skin closure was used as an indirect measure of the time to secure hemostasis. In comparison with placebo, clopidogrel produced a small (8.8%) but significant reduction in the platelet response to ADP (PϽ0.05) while conferring a 10-fold reduction in the relative risk of those patients having Ͼ20 emboli in the postoperative period (odds ratio, 10.23; 95% CI, 1.3 to 83.3; Pϭ0.01, Fisher's exact test). However, in the clopidogrel-treated patients, the time from flow restoration to skin closure (an indirect marker of hemostasis) was significantly increased (Pϭ0.04, Fisher's exact test), although there was no increase in bleeding complications or blood transfusions. Conclusions-This is the first study to show that a CEA patient's postoperative thromboembolic potential can be significantly reduced by targeted preoperative antiplatelet therapy without increasing the risk of bleeding complications.

Background Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have now examined the role of connexins in platelets, blood cells that circulate in isolation, but upon tissue injury adhere to each other and the vessel wall to prevent blood loss and facilitate wound repair. Methods and Results We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses prior to platelet-platelet contact, and reduced laser induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion and clot retraction indicating an important role for Cx37 hemichannels and gap junctions in platelet thrombus function. Conclusions Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of haemostasis and thrombosis and represent potential therapeutic targets.

Nest construction is taxonomically widespread, yet our understanding of adaptive intraspecific variation in nest design remains poor. Nest characteristics are expected to vary adaptively in response to predictable variation in spring temperatures over large spatial scales, yet such variation in nest design remains largely overlooked, particularly amongst open-cup-nesting birds. Here, we systematically examined the effects of latitudinal variation in spring temperatures and precipitation on the morphology, volume, composition, and insulatory properties of open-cup-nesting Common Blackbirds’ Turdus merula nests to test the hypothesis that birds living in cooler environments at more northerly latitudes would build better insulated nests than conspecifics living in warmer environments at more southerly latitudes. As spring temperatures increased with decreasing latitude, the external diameter of nests decreased. However, as nest wall thickness also decreased, there was no variation in the diameter of the internal nest cups. Only the mass of dry grasses within nests decreased with warmer temperatures at lower latitudes. The insulatory properties of nests declined with warmer temperatures at lower latitudes and nests containing greater amounts of dry grasses had higher insulatory properties. The insulatory properties of nests decreased with warmer temperatures at lower latitudes, via changes in morphology (wall thickness) and composition (dry grasses). Meanwhile, spring precipitation did not vary with latitude, and none of the nest characteristics varied with spring precipitation. This suggests that Common Blackbirds nesting at higher latitudes were building nests with thicker walls in order to counteract the cooler temperatures. We have provided evidence that the nest construction behavior of open-cup-nesting birds systematically varies in response to large-scale spatial variation in spring temperatures.

Mean platelet volume (MPV) and platelet count (PLT) are highly heritable and tightly regulated traits. We performed a genomewide association study for MPV and identified one SNP, rs342293, as having highly significant and reproducible association with MPV (per-G allele effect 0.016 ؎ 0.001 log fL; P < 1.08 ؋ 10 ؊24 ) and PLT (per-G effect ؊4.55 ؎ 0.80 10 9 /L; P < 7.19 ؋ 10 ؊8 ) in 8586 healthy subjects. Whole-genome expression analysis in the 1-MB region showed a significant association with platelet transcript levels for PIK3CG (n ‫؍‬ 35; P ‫؍‬ .047). The G allele at rs342293 was also associated with decreased binding of annexin V to platelets activated with collagen-related peptide (n ‫؍‬ 84; P ‫؍‬ .003). The region 7q22.3 identifies the first QTL influencing platelet volume, counts, and function in healthy subjects. Notably, the association signal maps to a chromosome region implicated in myeloid malignancies, indicating this site as an important regulatory site for hematopoiesis. The identification of loci regulating MPV by this and other studies will increase our insight in the processes of megakaryopoiesis and proplatelet formation, and it may aid the identification of genes that are somatically mutated in essential thrombocytosis. (Blood. 2009; 113:3831-3837) IntroductionPlatelets are anucleate blood cell fragments that play a key role in maintaining primary hemostasis and in wound healing. Mean platelet volume (MPV) and platelet count (PLT) are tightly regulated and inversely correlated in the healthy population. Increased MPV represents a strong, independent predictor of postevent outcome in coronary disease and myocardial infarction, 1,2 and changes of either parameter outside the normal ranges are routinely used to ascertain and manage a large number of clinical conditions. Studies in rodents, primates, and twins have confirmed that blood cell quantitative traits, such as MPV and PLT, have high heritability levels. [3][4][5] Genome-wide association studies that used dense genotyping in thousands of subjects have greatly improved the resolution of such complex polygenic traits in humans. 6,7 We therefore reasoned that it should be possible to identify novel quantitative trait loci (QTLs) for MPV and PLT by a similar approach. The identification of such loci will provide new insights in the complex cellular processes of megakaryopoiesis and platelet formation. In addition, it may contribute to our understanding of premalignant conditions such as polycythemia vera and particularly essential thrombocytosis (ET), in which somatically acquired mutations in the JAK2 gene explain only a fraction of cases. 8 Megakaryocytes (MKs), the platelet precursor cells, originate from pluripotent hematopoietic stem cells (HSCs) in a stepwise process of fate determination and proliferation controlled by the cytokine thrombopoietin 9 and several extracellular matrix proteins (ECMPs). [10][11][12] After migration of the MK progenitors from the HSC niche to the bone marrow, platelets are formed via the tightly regulated pro...

Summary. Background: Evidence suggests the wide variation in platelet response within the population is genetically controlled. Unraveling the complex relationship between sequence variation and platelet phenotype requires accurate and reproducible measurement of platelet response. Objective: To develop a methodology suitable for measuring signaling pathway-specific platelet phenotype, to use this to measure platelet response in a large cohort, and to demonstrate the effect size of sequence variation in a relevant model gene. Methods: Three established platelet assays were evaluated: mobilization of [Ca 2+ ] i , aggregometry and flow cytometry, each in response to adenosine 5¢-diphosphate (ADP) or the glycoprotein (GP) VI-specific crosslinked collagen-related peptide (CRP). Flow cytometric measurement of fibrinogen binding and P-selectin expression in response to a single, intermediate dose of each agonist gave the best combination of reproducibility and inter-individual variability and was used to measure the platelet response in 506 healthy volunteers. Pathway specificity was ensured by blocking the main subsidiary signaling pathways. Results: Individuals were identified who were hypo-or hyper-responders for both pathways, or who had differential responses to the two agonists, or between outcomes. 89 individuals, retested three months later using the same methodology, showed high concordance between the two visits in all four assays (r 2 = 0.872, 0.868, 0.766 and 0.549); all subjects retaining their phenotype at recall.The effect of sequence variation at the GP6 locus accounted for 35% of the variation in the CRP-XL response. Conclusion: Genotyping-phenotype association studies in a well-characterized, large cohort provides a powerful strategy to measure the effect of sequence variation in genes regulating the platelet response.

SummaryThe thiol isomerase enzymes protein disulphide isomerase (PDI) and endoplasmic reticulum protein 5 (ERp5) are released by resting and activated platelets. These re-associate with the cell surface where they modulate a range of platelet responses including adhesion, secretion and aggregation. Recent studies suggest the existence of yet uncharacterised platelet thiol isomerase proteins. This study aimed to identify which other thiol isomerase enzymes are present in human platelets. Through the use of immunoblotting, flow cytometry, cell-surface biotinylation and gene array analysis, we report the presence of five additional thiol isomerases in human and mouse platelets and megakaryocytes, namely; ERp57, ERp72, ERp44, ERp29 and TMX3. ERp72, ERp57, ERp44 and ERp29 are released by platelets and relocate to the cell surface following platelet activation. The transmembrane thiol isomerase TMX3 was also detected on the platelet surface but does not increase following activation. Extracellular PDI is also implicated in the regulation of coagulation by the modulation of tissue factor activity. ERp57 was identified within platelet-derived microparticle fractions, suggesting that ERp57 may also be involved in the regulation of coagulation as well as platelet function. These data collectively implicate the expanding family of platelet-surface thiol isomerases in the regulation of haemostasis.

The role of mechanosensitive (MS) Ca2+-permeable ion channels in platelets is unclear, despite the importance of shear stress in platelet function and life-threatening thrombus formation. We therefore sought to investigate the expression and functional relevance of MS channels in human platelets. The effect of shear stress on Ca2+ entry in human platelets and Meg-01 megakaryocytic cells loaded with Fluo-3 was examined by confocal microscopy. Cells were attached to glass coverslips within flow chambers that allowed applications of physiological and pathological shear stress. Arterial shear (1002.6 s−1) induced a sustained increase in [Ca2+]i in Meg-01 cells and enhanced the frequency of repetitive Ca2+ transients by 80% in platelets. These Ca2+ increases were abrogated by the MS channel inhibitor Grammostola spatulata mechanotoxin 4 (GsMTx-4) or by chelation of extracellular Ca2+. Thrombus formation was studied on collagen-coated surfaces using DiOC6-stained platelets. In addition, [Ca2+]i and functional responses of washed platelet suspensions were studied with Fura-2 and light transmission aggregometry, respectively. Thrombus size was reduced 50% by GsMTx-4, independently of P2X1 receptors. In contrast, GsMTx-4 had no effect on collagen-induced aggregation or on Ca2+ influx via TRPC6 or Orai1 channels and caused only a minor inhibition of P2X1-dependent Ca2+ entry. The Piezo1 agonist, Yoda1, potentiated shear-dependent platelet Ca2+ transients by 170%. Piezo1 mRNA transcripts and protein were detected with quantitative RT-PCR and Western blotting, respectively, in both platelets and Meg-01 cells. We conclude that platelets and Meg-01 cells express the MS cation channel Piezo1, which may contribute to Ca2+ entry and thrombus formation under arterial shear.