Platelets are the second most abundant cell type in blood and are essential for maintaining haemostasis. Their count and volume are tightly controlled within narrow physiological ranges, but there is only limited understanding of the molecular processes controlling both traits. Here we carried out a high-powered meta-analysis of genome-wide association studies (GWAS) in up to 66,867 individuals of European ancestry, followed by extensive biological and functional assessment. We identified 68 genomic loci reliably associated with platelet count and volume mapping to established and putative novel regulators of megakaryopoiesis and platelet formation. These genes show megakaryocyte-specific gene expression patterns and extensive network connectivity. Using gene silencing in Danio rerio and Drosophila melanogaster, we identified 11 of the genes as novel regulators of blood cell formation. Taken together, our findings advance understanding of novel gene functions controlling fate-determining events during megakaryopoiesis and platelet formation, providing a new example of successful translation of GWAS to function.

The interaction of blood platelets with collagen is generally considered to be of primary importance in the arrest of bleeding and to have a role in the pathogenesis of thrombosis and atherosclerosis. Following damage to the vascular endothelium, circulating platelets come into contact with exposed collagen fibrils in the subendothelium and spread along it; this is followed by the secretion of several biologically active substances and by aggregation of platelets. The glycoproteins of the platelet plasma membrane have an important role in the mechanisms underlying these processes. So far, two specific defects of platelet function in patients with a bleeding disorder are known to be associated with a glycoprotein defect and the study of these patients has contributed significantly to present concepts of platelet function. The glycoprotein (GP) IIB-III complex, absent or deleted in the aggregation-defective Glanzmann's thrombasthenia, has been identified as the platelet fibrinogen receptor. GPIb, which is absent in the adhesion-defective Bernard-Soulier syndrome, has been identified as the von Willebrand factor receptor on platelets. We now report a defect of the platelet plasma membrane glycoprotein composition in a patient whose platelets are totally unresponsive to collagen.

Ral is a ubiquitously expressed Ras-like small GTPase which is abundantly present in human platelets. The biological function of Ral and the signaling pathway in which Ral is involved are largely unknown. Here we describe a novel method to measure Ral activation utilizing the Ral binding domain of the putative Ral effector RLIP76 as an activation-specific probe. With this assay we investigated the signaling pathway that leads to Ral activation in human platelets. We found that Ral is rapidly activated after stimulation with various platelet agonists, including ␣-thrombin. In contrast, the platelet antagonist prostaglandin I 2 inhibited ␣-thrombininduced Ral activation. Activation of Ral by ␣-thrombin could be inhibited by depletion of intracellular Ca 2؉ , whereas the induction of intracellular Ca 2؉ resulted in the activation of Ral. Our results show that Ral can be activated by extracellular stimuli. Furthermore, we show that increased levels of intracellular Ca 2؉ are sufficient for Ral activation in platelets. This activation mechanism correlates with the activation mechanism of the small GTPase Rap1, a putative upstream regulator of Ral guanine nucleotide exchange factors.RalA and RalB are very similar small GTPases that have 55% sequence identity with Ras (6,7,41). Ral and Ras have comparable nucleotide binding characteristics and low intrinsic GTPase activity (19). The Ral proteins are ubiquitously expressed but are particularly abundant in brain, testes, and platelets (2, 21, 39). Ral becomes posttranslationally processed and is found in the plasma membrane (16) as well as in endocytotic vesicles (41) or synaptic vesicles (50). In platelets, Ral is a major GTP binding protein that is present in the plasma membrane and specifically in dense granules, a class of secretory organelles (29,41).Recently, a putative effector protein of RalA and RalB has been identified; it has been designated RLIP76 but is also termed RalBP1 or RIP1 (5,22,40). RLIP76 interacts with the active, GTP-bound form of Ral, both in the yeast two-hybrid system and in vitro. Interaction studies using the yeast twohybrid system and deletion mutants of RLIP76 demonstrate that the Ral binding region is located in the C-terminal region, between amino acids 403 and 499 (22). Interestingly, RLIP76 exhibits GTPase-activating protein (GAP) activity for the Rholike GTPase Cdc42, suggesting that Ral may be involved in the regulation of Cdc42 (5, 22, 40). Cdc42 plays a role in the organization of the actin cytoskeleton and the regulation of cytoskeletal polarity.Ral also interacts with phospholipase D1 (PLD1) (21, 28). The interaction between Ral and PLD1 is independent of the nucleotide binding of Ral and occurs via the N-terminal region of Ral (21,28). In platelets and other cells, PLD has been implicated in vesicle transport, regulation of the actin cytoskeleton, and generation of lysophosphatidic acid, which is secreted by platelets upon activation (14, 31).Different proteins that regulate the activity of Ral have been identified. A RalGAP with...

Platelet aggregation is initiated by the release of mediators as adenosine diphosphate (ADP) stored in platelet granules. Possible candidates for transport proteins mediating accumulation of these mediators in granules include multidrug resistance protein 4 (MRP4, ABCC4), a transport pump for cyclic nucleotides and nucleotide analogs. We investigated the expression of MRP4 in human platelets by immunoblotting, detecting a strong signal at 170 kDa. Immunofluorescence microscopy using 2 MRP4-specific antibodies revealed staining mainly in intracellular structures, which largely colocalized with the accumulation of mepacrine as marker for delta-granules and to a lower extent at the plasma membrane. IntroductionThe critical role played by platelets in hemostasis and thrombosis is related to their function as exocytotic cells that secrete effector molecules at the side of vascular injury. Platelets contain at least 3 types of intracellular granules, in which these mediators are stored and concentrated, known as alpha, dense, and lysosomal granules. 1 While alpha granules contain mainly polypeptides, as fibrinogen, von Willebrand factor, growth factors, and protease inhibitors, dense granules contain small molecules, specifically adenosine diphosphate (ADP), adenosine triphosphate (ATP), serotonin, and calcium. 2 Humans with defective dense granule exocytosis suffer from delta storage pool disease associated with a moderate bleeding tendency. The most severe delta storage pool disease is observed in Hermansky-Pudlak syndrome (HPS), a rare autosomal recessive disorder in which oculocutaneous albinism, bleeding, and lysosomal ceroid storage result from defects of melanosomes, platelet-dense granules, and lysosomes. [3][4][5] Little is known, however, about transport proteins mediating accumulation of the effector molecules in granules or their transport across the plasma membrane. Possible candidates include the multidrug resistance protein 4 (MRP4/ABCC4) and MRP5 (ABCC5). These belong to the C-branch of the human ATP-binding cassette (ABC) transporter superfamily, which consists of 12 members, 9 of which comprise the group of multidrug resistance proteins (MRP1-9; ABCC1-6 and ABCC10-12). 6,7 MRPs are integral membrane glycoproteins that mediate the primary active unidirectional export of organic anions from cells. Conjugates of lipophilic compounds with glutathione, glucuronate, and sulfate are preferred substrates of MRP1-3, [8][9][10] while cyclic purine nucleotides and nucleotide analogs have been identified as substrates for MRP4 and MRP5. 11-15 MRP5 mRNA has been detected in many tissues, 16,17 and the MRP5 protein could be localized in erythrocytes, 12 in smooth muscle cells of the genitourinary tract, 18 and in human heart cardiomyocytes, vascular endothelial, and smooth muscle cells. 19 MRP4 mRNA was detected in prostate, liver, testis, ovary, brain, kidney, and adrenal gland. 16,[20][21][22] Studies in membrane vesicles containing recombinant MRP4 indicate that it represents a transporter with a relatively br...

Summary. Background: The processes that govern the distribution of molecules between platelets and the microparticles (MP) they release are unknown. Certain proteins are sorted selectively into MP, but lipid sorting has not been studied. Objectives: To compare the phospholipid composition and cholesterol content of platelet-derived MP obtained with various stimuli with that of isolated platelet membrane fractions. Methods: Washed platelets from venous blood of healthy individuals (n ¼ 6) were stimulated with collagen, thrombin, collagen plus thrombin, or A23187. Platelet activation, MP release and antigen exposure were assessed by flow cytometry. MPs were isolated by differential centrifugation. Platelet plasma-, granule-and intracellular membranes were isolated from platelet concentrates (n ¼ 3; 10 donors each) by pressure homogenization and Percoll density gradient fractionation. The phospholipid composition and cholesterol content of MPs and membrane fractions were analyzed by high performance thin layer chromatography. Results: The phospholipid composition of MPs was intermediate compared with that of platelet plasmaand granule membranes, and differed significantly from that of intracellular membranes. There were small but significant differences in phospholipid composition between the MPs produced by the various agonists, which paralleled differences in P-selectin exposure in case of the physiological agonists collagen, thrombin, or collagen plus thrombin. The cholesterol content of MPs tended to be higher than that of the three-platelet membrane fractions. Conclusions: Regarding its phospholipid content, the MP membrane is a composite of the platelet plasma-and granule membranes, showing subtle differences depending on the platelet agonist. The higher cholesterol content of MPs suggests their enrichment in lipid rafts.

Purpose: One of the key factors that promotes angiogenesis is vascular endothelial growth factor (VEGF). Platelets are the main source of VEGF in blood and contribute to angiogenesis by release of growth factors, including VEGF, from their a-granules on activation. The monoclonal antibody bevacizumab blocks VEGF in the blood of patients within hours after administration. Platelets are known to endocytose plasma proteins including immunoglobulins. We tested the hypothesis that platelets take up bevacizumab. Experimental Design: Fluorescence-activated cell sorting analysis, immunofluorescence imaging, andWestern blotting were used to study uptake and release of bevacizumab by platelets in vitro and in vivo. The angiogenic activity of platelets preincubated with bevacizumab was studied in endothelial proliferation assays. Finally, we determined whether treatment with bevacizumab neutralizesVEGF in platelets from cancer patients. Results: We found that platelets are able to take up bevacizumab. Activation of platelets preincubated with bevacizumab resulted in release of the antibody and release of VEGF neutralized by bevacizumab. Immunofluorescence microscopy revealed that FITC-labeled bevacizumab and P-selectin colocalize, indicating a-granule localization. In addition, bevacizumab uptake inhibited platelet-induced human endothelial cell proliferation. In in vivo rabbit experiments, FITC-labeled bevacizumab was present in platelets after 2 h and up to 2 weeks following i.v. administration. Finally, we found that platelets take up bevacizumab in patients receiving bevacizumab treatment. Within 8 h after bevacizumab administration, plateletVEGF was almost completely neutralized due to this uptake. Conclusion: These studies show that bevacizumab is taken up by platelets and may explain its clinical effect on wound healing and tumor growth.

The molecular mechanism that causes non-adhesive, discoid platelets to transform into sticky dendritic bodies that form blood clumps is a complex series of events. Recently it has become clear that lipid microdomains--also known as rafts--play a crucial role in this process. We have used a non-cytolytic derivative of perfringolysin-O, a cholesterol binding cytolysin, that binds selectively to cholesterol-rich membrane domains, combined with confocal- and immunoelectron microscopy to visualize cholesterol-raft dynamics during platelet adhesion. In resting platelets cholesterol was uniformly distributed on the cell surface and confined to distinct intracellular compartments (i.e. multivesicular bodies, dense granules, and the internal membranes of alpha-granules). Upon interaction with fibrinogen, cholesterol accumulated at the tips of filopodia and at the leading edge of spreading cells. Stimulation with thrombin receptor activating peptide (TRAP) resulted in a similar redistribution of cholesterol towards filopodia. The adhesion-dependent raft aggregation was accompanied by concentration of the tyrosine kinase c-Src and the tetraspanin CD63 in these domains, whereas glycoprotein Ib (GPIb) was not selectively targeted to the raft clusters. c-Src, the tetraspanin CD63, and GPIb were recovered in biochemically isolated low-density membrane fractions. Disruption of rafts by depleting membrane cholesterol had no effect on platelet shape change but inhibited platelet spreading on fibrinogen and TRAP-induced aggregation. Our results demonstrate that cholesterol rafts in platelets are dynamic entities in the membrane that co-cluster with the tyrosine kinase c-Src and the costimulatory molecule CD63 in specialized domains at the cell surface, thereby providing a possible mechanism in functioning as signaling centres.

Store-regulated Ca(2+) entry (SOCE) is an important mechanism of elevating cytosolic [Ca(2+)]i in platelets, though the Ca(2+) influx channels involved are still unclear. We screened human platelets and their precursor cells (human stem cells and megakaryocytes) for the presence of candidate influx channels, i.e., isoforms of the Trp family of proteins. Primary stem cells were cultured with thrombopoietin to allow differentiation into megakaryocytes. The undifferentiated stem cells (CD34(+)) showed mRNA expression of only a spliced variant Trp1A. Immature (CD61(+)/CD42b(low)) and mature (CD61(+)/CD42b(high)) megakaryocytes as well as platelets expressed in addition unspliced Trp1 as well as Trp4 (less abundant) and Trp6 isoforms. This unspliced isoform appeared to be specific for cells of the megakaryocyte/platelet lineage, since immature (CD14(+)/CD61(-)/CD42b(-)) and mature monocytes expressed only the Trp1A isoform. This conclusion was confirmed by the presence of Trp1A, 3, 4 and 6 transcripts in the immature megakaryocytic Dami cell line, and of Trp1, 1A, 4 and 6 transcripts in the more mature CHRF-288 cell line. The up-regulation of Trp1, 4 and 6 in the lineage from primary stem cells to mature megakaryocytes and platelets was accompanied by increased influx of extracellular Ca(2+) after pretreatment of the cells with thapsigargin or thrombin. Expression of new Trp isoforms in the differentiated cells is thus accompanied by increased SOCE.