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Early ischemic ventricular fibrillation (VF) in the setting of an acute myocardial infarction (AMI) due to thrombotic coronary occlusion remains a major health problem. Several animal studies have shown that platelet-dense granule contents released during thrombus formation can induce arrhythmias. We hypothesize that the platelet release reaction is involved in the predisposition to early ischemic VF. A case-control study was performed in patients who survived VF during a first AMI ("cases," n = 26) and in patients with one previous AMI without arrhythmias ("controls," n = 24). All patients were on aspirin 100 mg OD. Baseline platelet activation was assessed with flow cytometry. Response to activation was assessed with aggregometry, flow cytometry and PFA-100 analysis. Differences in platelet contents and content release were assessed by labeling platelet-dense granules with mepacrine and by measuring serotonin and ADP/ATP content. Patient and infarct characteristics and baseline platelet function tests were similar between groups. The mean time from event was 4.9 (±3.2) years among cases and 4.7 (±2.7) years among controls. Dense granule release was similar in cases versus controls. Platelet serotonin content in cases was higher than in controls (611 ± 118 ng/10E(9) platelets vs. 536 ± 141 ng/10(9), p = 0.048). Even years after the event, elevations in the platelet dense granule contents between VF survivors and controls may be detected. These preliminary findings shed new light on the pathophysiological mechanisms underlying ischemic VF, as platelet-dense granules may contain mediators of early ischemic VF risk.

Initial platelet arrest at the exposed arterial vessel wall is mediated through glycoprotein Ibα binding to the A1 domain of von Willebrand factor. This interaction occurs at sites of elevated shear force, and strengthens upon increasing hydrodynamic drag. The increased interaction requires shear-dependent exposure of the von Willebrand factor A1 domain, but the contribution of glycoprotein Ibα remains ill defined. We have previously found that glycoprotein Ibα forms clusters upon platelet cooling and hypothesized that such a property enhances the interaction with von Willebrand factor under physiological conditions. We analyzed the distribution of glycoprotein Ibα with Förster resonance energy transfer using time-gated fluorescence lifetime imaging microscopy. Perfusion at a shear rate of 1,600 s(-1) induced glycoprotein Ibα clusters on platelets adhered to von Willebrand factor, while clustering did not require von Willebrand factor contact at 10,000 s(-1). Shear-induced clustering was reversible, not accompanied by granule release or αIIbβ3 activation and improved glycoprotein Ibα-dependent platelet interaction with von Willebrand factor. Clustering required glycoprotein Ibα translocation to lipid rafts and critically depended on arachidonic acid-mediated binding of 14-3-3ζ to its cytoplasmic tail. This newly identified mechanism emphasizes the ability of platelets to respond to mechanical force and provides new insights into how changes in hemodynamics influence arterial thrombus formation.

Background: Storing platelets for transfusion at room temperature increases the risk of microbial infection and decreases platelet functionality, leading to out-date discard rates of up to 20%. Cold storage may be a better alternative, but this treatment leads to rapid platelet clearance after transfusion, initiated by changes in glycoprotein Ibα, the receptor for von Willebrand factor., Design and Methods: We examined the change in glycoprotein Ibα distribution using Förster resonance energy transfer by time-gated fluorescence lifetime imaging microscopy., Results: Cold storage induced deglycosylation of glycoprotein Ibα ectodomain, exposing N-acetyl-D-glucosamine residues, which sequestered with GM1 gangliosides in lipid rafts. Raft-associated glycoprotein Ibα formed clusters upon binding of 14-3-3ζ adaptor proteins to its cytoplasmic tail, a process accompanied by mitochondrial injury and phosphatidyl serine exposure. Cold storage left glycoprotein Ibα surface expression unchanged and although glycoprotein V decreased, the fall did not affect glycoprotein Ibα clustering. Prevention of glycoprotein Ibα clustering by blockade of deglycosylation and 14-3-3ζ translocation increased the survival of cold-stored platelets to above the levels of platelets stored at room temperature without compromising hemostatic functions., Conclusions: We conclude that glycoprotein Ibα translocates to lipid rafts upon cold-induced deglycosylation and forms clusters by associating with 14-3-3ζ. Interference with these steps provides a means to enable cold storage of platelet concentrates in the near future.

Background: Cold storage of platelets reduces bacterial growth and preserves their hemostatic properties better than current procedures do. However, storage at 0°C induces [14-3-3ζ-glycoprotein Ibα] association, 14-3-3ζ release from phospho-Bad, Bad activation and apoptosis., Design and Methods: We investigated whether arachidonic acid, which also binds 14-3-3ζ, contributes to coldinduced apoptosis., Results: Cold storage activated P38-mitogen-activated protein kinase and released arachidonic acid, which accumulated due to cold inactivation of cyclooxygenase-1/thromboxane synthase. Accumulated arachidonic acid released 14-3-3ζ from phospho-Bad and decreased the mitochondrial membrane potential, which are steps in the induction of apoptosis. Addition of arachidonic acid did the same and its depletion made platelets resistant to cold-induced apoptosis. Incubation with biotin-arachidonic acid revealed formation of an [arachidonic acid-14-3-3ζ-glycoprotein Ibα] complex. Indomethacin promoted complex formation by accumulating arachidonic acid and released 14-3-3ζ from cyclo-oxygenase-1. Arachidonic acid depletion prevented the cold-induced reduction of platelet survival in mice., Conclusions: We conclude that cold storage induced apoptosis through an [arachidonic acid-14-3-3ζ-glycoprotein Ibα] complex, which released 14-3-3ζ from Bad in an arachidonic acid-dependent manner. Although arachidonic acid depletion reduced agonist-induced thromboxane A(2) formation and aggregation, arachidonic acid repletion restored these functions, opening ways to reduce apoptosis during storage without compromising hemostatic functions post-transfusion.

Background: In normal platelets, insulin inhibits agonist-induced Ca(2+) mobilization by raising cyclic AMP. Platelet from patients with type 2 diabetes are resistant to insulin and show increased Ca(2+) mobilization, aggregation and procoagulant activity. We searched for the cause of this insulin resistance., Design and Methods: Platelets, the megakaryocytic cell line CHRF-288-11 and primary megakaryocytes were incubated with adipokines and with plasma from individuals with a disturbed adipokine profile. Thrombin-induced Ca(2+) mobilization and signaling through the insulin receptor and insulin receptor substrate 1 were measured. Abnormalities induced by adipokines were compared with abnormalities found in platelets from patients with type 2 diabetes., Results: Resistin, leptin, plasminogen activator inhibitor-1 and retinol binding protein 4 left platelets unchanged but induced insulin resistance in CHRF-288-11 cells. Interleukin-6, tumor necrosis factor-α and visfatin had no effect. These results were confirmed in primary megakaryocytes. Contact with adipokines for 2 hours disturbed insulin receptor substrate 1 Ser(307)-phosphorylation, while contact for 72 hours caused insulin receptor substrate 1 degradation. Plasma with a disturbed adipokine profile also made CHRF-288-11 cells insulin-resistant. Platelets from patients with type 2 diabetes showed decreased insulin receptor substrate 1 expression., Conclusions: Adipokines resistin, leptin, plasminogen activator-1 and retinol binding protein 4 disturb insulin receptor substrate 1 activity and expression in megakaryocytes. This might be a cause of the insulin resistance observed in platelets from patients with type 2 diabetes.

The main responses of P2Y(1) ligation are platelet shape change and transient aggregation while P2Y(12) ligation amplifies P2Y(1)-induced aggregation and accelerates aggregation, secretion and thromboxane A(2) production induced by other agonist-receptor complexes. We searched for new targets of P2Y signalling using micro-arrays with 144 peptides representing known phosphosites of protein tyrosine kinases. ADP induced phosphorylation of peptides representing surface receptors, second messenger enzymes and cytoskeletal proteins. Strong phosphorylation was found in peptides representing Ephrin-receptor family members. Blockade of P2Y(1/12) inhibited phosphorylation of EphA4- and EphB1-peptides on micro-arrays. The EphA2/4 inhibitor 2,5-dimethylpyrrolyl benzoic acid derivative interfered with P2Y(1/12)-induced EphA4 phosphorylation, left P2Y(1)-induced aggregation unchanged but inhibited with P2Y(12)-induced secretion, second phase aggregation and thrombus formation on collagen at 1600 s(-1). These results show that platelet EphA4 is an important intermediate in P2Y(12)-induced granule secretion.

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.

Sudden cardiac death remains one of the most prevalent modes of death and is mainly caused by ventricular fibrillation (VF) in the setting of acute ischemia resulting from coronary thrombi. Animal experiments have shown that platelet activation may increase susceptibility of ischemic myocardium to VF, but the mechanism is unknown. In the present study, we evaluated the effects of activated blood platelet products (ABPPs) on electrophysiological properties and intracellular Ca(2+) (Ca(2+)(i)) homeostasis. Platelets were collected from healthy volunteers. After activation, their secreted ABPPs were added to superfusion solutions. Rabbit ventricular myocytes were freshly isolated, and membrane potentials and Ca(2+)(i) were recorded using patch-clamp methodology and indo-1 fluorescence measurements, respectively. ABPPs prolonged action potential duration and induced early and delayed afterdepolarizations. ABPPs increased L-type Ca(2+) current (I(Ca,L)) density, but left densities of sodium current, inward rectifier K(+) current, transient outward K(+) current, and rapid component of the delayed rectifier K(+) current unchanged. ABPPs did not affect kinetics or (in)activation properties of membrane currents. ABPPs increased systolic Ca(2+)(i), Ca(2+)(i) transient amplitude, and sarcoplasmic reticulum Ca(2+) content. ABPPs did not affect the Na(+)-Ca(2+) exchange current (I(NCX)) in Ca(2+)-buffered conditions. Products secreted from activated human platelets induce changes in I(Ca,L) and Ca(2+)(i), which result in action potential prolongation and the occurrence of early and delayed afterdepolarizations in rabbit myocytes. These changes may trigger and support reentrant arrhythmias in ischemia models of coronary thrombosis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objective: Scavenger receptor BI (SR-BI) is a cell surface receptor that promotes the selective uptake of cholesteryl esters from high-density lipoprotein (HDL) by the liver. In mice, SR-BI deficiency results in increased plasma HDL cholesterol levels and enhanced susceptibility to atherosclerosis. The aim of this study was to investigate the role of SR-BI deficiency on platelet function., Methods and Results: SR-BI-deficient mice were thrombocytopenic, and their platelets were abnormally large, probably because of an increased cholesterol content. The FeCl(3) acute injury model to study arterial thrombosis susceptibility showed that SR-BI wild-type mice developed total arterial occlusion after 24±2 minutes. In SR-BI-deficient mice, however, the time to occlusion was reduced to 13±1 minutes (P=0.02). Correspondingly, in SR-BI-deficient mice, platelets circulated in an activated state and showed increased adherence to immobilized fibrinogen. In contrast, platelet-specific disruption of SR-BI by bone marrow transplantation in wild-type mice did not alter plasma cholesterol levels or affect platelet count, size, cholesterol content, or reactivity, suggesting that changes in plasma cholesterol levels were responsible for the altered responsiveness of platelets in SR-BI-deficient mice., Conclusions: The function of SR-BI in HDL cholesterol homeostasis and prevention of atherosclerosis is indirectly also essential for maintaining normal platelet function and prevention of thrombosis.

Background: We have shown that 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose (α-PGG), an orally effective hypoglycemic small molecule, binds to insulin receptors and activates insulin-mediated glucose transport. Insulin has been shown to bind to its receptors on platelets and inhibit platelet activation. In this study we tested our hypothesis that if insulin possesses anti-platelet properties then insulin mimetic small molecules should mimic antiplatelet actions of insulin., Principal Findings: Incubation of human platelets with insulin or α-PGG induced phosphorylation of insulin receptors and IRS-1 and blocked ADP or collagen induced aggregation. Pre-treatment of platelets with α-PGG inhibited thrombin-induced release of P-selectin, secretion of ATP and aggregation. Addition of ADP or thrombin to platelets significantly decreased the basal cyclic AMP levels. Pre-incubation of platelets with α-PGG blocked ADP or thrombin induced decrease in platelet cyclic AMP levels but did not alter the basal or PGE(1) induced increase in cAMP levels. Addition of α-PGG to platelets blocked agonist induced rise in platelet cytosolic calcium and phosphorylation of Akt. Administration of α-PGG (20 mg kg(-1)) to wild type mice blocked ex vivo platelet aggregation induced by ADP or collagen., Conclusions: These data suggest that α-PGG inhibits platelet activation, at least in part, by inducing phosphorylation of insulin receptors leading to inhibition of agonist induced: (a) decrease in cyclic AMP; (b) rise in cytosolic calcium; and (c) phosphorylation of Akt. These findings taken together with our earlier reports that α-PGG mimics insulin signaling suggest that inhibition of platelet activation by α-PGG mimics antiplatelet actions of insulin.

Background: The collagen receptor glycoprotein VI generates activating signals through an immunoreceptor tyrosine-based activating motif on the co-associated Fc receptor gamma chain. Leukocyte-associated immunoglobulin-like receptor-1 also ligates collagen but generates inhibitory signals through immunoreceptor tyrosine-based inhibitory motifs. Thus far, the cellular expression of glycoprotein VI and leukocyte-associated immunoglobulin-like receptor-1 appears mutually exclusive., Design and Methods: Using flow cytometry, we studied expression of collagen receptors on differentiating human megakaryocytes. CD34(+) cells were isolated from umbilical cord blood and matured to megakaryocytes in vitro. Freshly isolated bone marrow cells were used to study primary megakaryocytes. Upon cell sorting, cytospins were made to examine cytological characteristics of differentiation., Results: Megakaryocyte maturation is accompanied by up-regulation of glycoprotein VI and down-regulation of leukocyte-associated immunoglobulin-like receptor-1. Interestingly, both in cultures from hematopoietic stem cells and primary cells obtained directly from bone marrow, we identified a subset of morphologically distinct megakaryocytes which co-express glycoprotein VI and leukocyte-associated immunoglobulin-like receptor-1., Conclusions: This is the first report of a primary cell that co-expresses these collagen receptors with opposite signaling properties. Since megakaryocytes mature in the collagen-rich environment of the bone marrow, these findings may point to a role for leukocyte-associated immunoglobulin-like receptor-1 in the control of megakaryocyte maturation/migration.

Background: Cold-storage of platelets followed by rewarming induces changes in Glycoprotein (GP) Ibα-distribution indicative of receptor clustering and initiates thromboxane A(2) -formation. GPIbα is associated with 14-3-3 proteins, which contribute to GPIbα-signaling and in nucleated cells take part in apoptosis regulation., Objectives and Methods: We investigated whether GPIbα-clustering induces platelet apoptosis through 14-3-3 proteins during cold (4 h 0 °C)-rewarming (1 h 37 °C)., Results: During cold-rewarming, 14-3-3 proteins associate with GPIbα and dissociate from Bad inducing Bad-dephosphorylation and activation. This initiates pro-apoptosis changes in Bax/Bcl-x(L) and Bax-translocation to the mitochondria, inducing cytochrome c release. The result is activation of caspase-9, which triggers phosphatidylserine exposure and platelet phagocytosis by macrophages. Responses are prevented by N-acetyl-D-glucosamine (GN), which blocks GPIbα-clustering, and by O-sialoglycoprotein endopeptidase, which removes extracellular GPIbα., Conclusions: Cold-rewarming triggers apoptosis through a GN-sensitive GPIbα-change indicative of receptor clustering. Attempts to improve platelet transfusion by cold-storage should focus on prevention of the GPIbα-change., (© 2010 International Society on Thrombosis and Haemostasis.)

LAIR-1 (Leukocyte Associated Ig-like Receptor -1) is a collagen receptor that functions as an inhibitory receptor on immune cells. It has a soluble family member, LAIR-2, that also binds collagen and can interfere with LAIR-1/collagen interactions. Collagen is a main initiator for platelet adhesion and aggregation. Here, we explored the potential of soluble LAIR proteins to inhibit thrombus formation in vitro. LAIR-2/Fc but not LAIR-1/Fc inhibited collagen-induced platelet aggregation. In addition, LAIR-2/Fc also interfered with platelet adhesion to collagen at low shear rate (300 s(-1); IC(50) = 18 microg/ml) and high shear rate (1500 s(-1); IC(50) = 30 microg/ml). Additional experiments revealed that LAIR-2/Fc leaves interactions between collagen and alpha2beta1 unaffected, but efficiently prevents binding of collagen to Glycoprotein VI and von Willebrand factor. Thus, LAIR-2/Fc has the capacity to interfere with platelet-collagen interactions mediated by Glycoprotein VI and the VWF/Glycoprotein Ib axis.

Objective: Patients with type 2 diabetes have an increased risk of cardiovascular disease and show abnormalities in the coagulation cascade. We investigated whether increased synthesis of tissue factor (TF) by platelets could contribute to the hypercoagulant state., Research Design and Methods: Platelets from type 2 diabetic patients and matched control subjects were adhered to different surface-coated proteins, and TF premRNA splicing, TF protein, and TF procoagulant activity were measured., Results: Different adhesive proteins induced different levels of TF synthesis. A mimetic of active clopidogrel metabolite (AR-C69931 MX) reduced TF synthesis by 56 +/- 10%, an aspirin-like inhibitor (indomethacin) by 82 +/- 9%, and the combination by 96 +/- 2%, indicating that ADP release and thromboxane A(2) production followed by activation of P2Y12 and thromboxane receptors mediate surface-induced TF synthesis. Interference with intracellular pathways revealed inhibition by agents that raise cAMP and interfere with phosphatidylinositol 3-kinase/protein kinase B. Insulin is known to raise cAMP in platelets and inhibited collagen III-induced TF premRNA splicing and reduced TF activity by 35 +/- 5 and 47 +/- 5% at 1 and 100 nmol/l. Inhibition by insulin was reduced in type 2 diabetes platelets resulting in an approximately 1.6-fold higher TF synthesis than in matched control subjects., Conclusions: We characterized the extra- and intracellular mechanisms that couple surface activation to TF synthesis in adhering platelets. In healthy individuals, TF synthesis is inhibited by insulin, but in patients with type 2 diabetes inhibition is impaired. This leads to the novel finding that platelets from type 2 diabetic patients produce more TF than platelets from matched control subjects.

Incubation at 0 degrees C is known to expose b- N -acetyl-D-glucosamine residues on glycoprotein (GP) Ibalpha inducing receptor clustering and alpha(M)beta(2)-mediated platelet destruction by macrophages. Here we show that incubation at 0/37 degrees C (4 hours at 0 degrees C, followed by 1 hour at 37 degrees C to mimic cold-storage and post-transfusion conditions) triggers a conformational change in the N -terminal flank (NTF, amino acids, aa 1-35) but not in aa 36-282 of GPIbalpha as detected by antibody binding. Addition of the sugar N -acetyl-D-glucosamine (GN) inhibits responses induced by 0/37 degrees C. Incubation at 0 degrees C shifts GPIbalpha from the membrane skeleton to the cytoskeleton. Different GPIbalpha conformations have little effect on VWF/ristocetin-induced aggregation, but arrest of NTF change by GN interferes with agglutination and spreading on a VWF-coated surface under flow. Strikingly, incubation at 0/37 degrees C initiates thromboxane A(2) formation through a von Willebrand factor (VWF)-independent and GPIbalpha-dependent mechanism, as confirmed in VWF- and GPIbalpha-deficient platelets. We conclude that the NTF change induced by 0/37 degrees C incubation reflects clustering of GPIbalpha supports VWF/ristocetin-induced agglutination and spreading and is sufficient to initiate platelet activation in the absence of VWF.

Background: We have used the advantages of the zebrafish model system to demonstrate which of the vertebrate myosin light chain kinase (MLCK) genes is expressed in thrombocytes and important for thrombus formation., Methods and Results: Here we report that Mlck1a is an essential component of thrombus formation. Phylogenetic data revealed four zebrafish orthologous for three human MLCK genes. To investigate expression of the zebrafish mlck genes in thrombocytes we compared GFP-tagged platelets with other cells by microarray analysis, and showed that mlck1a expression was 4.5-fold enriched in platelets. Furthermore, mlck1a mRNA and mRNA for the platelet-specific cd41 co-localized in thrombi. Expression of other mlck subtypes was lower in GFP-tagged platelets (mlck1b; 0.77-fold enriched) and absent in thrombi (mlck1b, -2, -3). To investigate the role of Mlck1a in thrombus formation, we knocked down mlck1a using two morpholinos. This resulted in impaired morphology changes of platelets adhering on fibrinogen. In a thrombosis model, in which thrombocytes adhere to the vessel wall damaged by laser irradiation, thrombus formation was slowed down in mlck1a-deficient embryos., Conclusion: We conclude that Mlck1a is the subtype of MLCK that contributes to platelet shape change and thrombus formation.

Platelet response to activation varies widely between individuals but shows interindividual consistency and strong heritability. The genetic basis of this variation has not been properly explored. We therefore systematically measured the effect on function of sequence variation in 97 candidate genes in the collagen and adenosine-diphosphate (ADP) signaling pathways. Resequencing of the genes in 48 European DNA samples nearly doubled the number of known single nucleotide polymorphisms (SNPs) and informed the selection of 1327 SNPs for genotyping in 500 healthy Northern European subjects with known platelet responses to collagen-related peptide (CRP-XL) and ADP. This identified 17 novel associations with platelet function (P < .005) accounting for approximately 46% of the variation in response. Further investigations with platelets of known genotype explored the mechanisms behind some of the associations. SNPs in PEAR1 associated with increased platelet response to CRP-XL and increased PEAR1 protein expression after platelet degranulation. The minor allele of a 3' untranslated region (UTR) SNP (rs2769668) in VAV3 was associated with higher protein expression (P = .03) and increased P-selectin exposure after ADP activation (P = .004). Furthermore the minor allele of the intronic SNP rs17786144 in ITPR1 modified Ca(2+) levels after activation with ADP (P < .004). These data provide novel insights into key hubs within platelet signaling networks.

The serotonin transporter is implicated in the uptake of the vasoconstrictor serotonin from the circulation into the platelets, where 95% of all blood serotonin is stored and released in response to vascular injury. In vivo studies indicated that platelet-derived serotonin mediates liver regeneration after partial hepatectomy. We have recently generated serotonin transporter knockout rats and demonstrated that their platelets were almost completely depleted of serotonin. Here we show that these rats exhibit impaired hemostasis and contain about 1-6% of wild-type serotonin levels in the blood. Despite the marked reduction of serotonin levels in blood and platelets, efficient liver regeneration and collagen-induced platelet aggregation occur in rats lacking the serotonin transporter. These results provide evidence that liver regeneration is not dependent on the release of serotonin from platelets. Our findings indicate that very low levels of serotonin in blood are sufficient for liver regeneration.

Objective: The sensitivity of platelets to aggregating agents increases when low-density lipoprotein (LDL) binds to apolipoprotein E receptor 2' (apoER2'), triggering activation of p38MAPK and formation of thromboxane A2. LDL signaling is terminated by PECAM-1 through recruitment and activation of the Ser/Thr protein phosphatase PP2A, but platelets remain unresponsive to LDL when PECAM-1 activation disappears. We report a second mechanism that halts LDL signaling and in addition lowers platelet responsiveness to aggregating agents., Methods and Results: After a first stimulation with LDL, platelets remain unresponsive to LDL for 60 minutes, despite normal apoER2' activation by a second dose of LDL. A possible cause is persistent activation of the tyrosine phosphatases SHP-1 and SHP-2, which may not only block a second activation of p38MAPK, PECAM-1, and PP2A by LDL but also seem to reduce aggregation by TRAP, collagen, and ADP., Conclusion: These findings reveal that p38MAPK phosphorylation and platelet activation by LDL are suppressed by two mechanisms: (1) short activation of PECAM-1/PP2A, and (2) prolonged activation of SHP-1 and SHP-2. Activation of SHP-1 and SHP-2 is accompanied by reduced responsiveness to aggregating agents, which--if present in vivo--would make LDL an aggregation inhibitor during prolonged contact with platelets.

Objectives: Platelets from healthy subjects are inhibited by insulin but type 2 diabetes mellitus (T2DM) platelets have become insulin-resistant, which might explain their hyperactivity. In the present study we investigated whether monocytes are responsive to insulin., Methods and Results: LPS-induced tissue factor (TF) upregulation was measured in human monocytes and monocytic THP-1 cells in a factor Xa generation assay. Insulin (0.1-100 nmol L(-1)) induced a dose-dependent inhibition in both cell types and in monocytes 100 nmol L(-1) insulin inhibited cytosolic, membrane-bound and microparticle TF by 32 +/- 2, 27 +/- 3 and 52 +/- 4% (n = 3). Insulin induced Tyr phosphorylation of the insulin receptor (INS-R) and formation of an INS-R - G(i)alpha(2) complex, suggesting interference with LPS-induced cAMP control. Indeed, insulin interfered with LPS-induced cAMP decrease and TF upregulation in a manner similar to an inhibitor of G(i) (pertussis toxin) and agents that raise cAMP (iloprost, forskolin, IBMX) reduced TF upregulation. Although LPS failed to raise cytosolic Ca(2+), quenching of Ca(2+) increases (BAPTA-AM) reduced and induction of Ca(2+) entry (ionophore, P2X7 activation) enhanced upregulation of TF mRNA and procoagulant activity. Insulin interfered with MCP-1-induced Ca(2+) mobilization but not with ATP-induced Ca(2+) rises., Conclusions: Insulin inhibits TF expression in monocytes and monocyte-derived microparticles through interference with G(i)alpha(2)-mediated cAMP suppression, which attenuates Ca(2+)-mediated TF synthesis.

Several studies have highlighted a specific role for membrane cholesterol domains in platelet signalling. Upon adhesion to von Willebrand factor (VWF) or collagen, cholesterol-rich domains (CRDs) accumulate in filopodial extensions and selectively harbour counterpart receptors (GPIb and GPVI) and associated signalling molecules. In the present study we have addressed the role of membrane cholesterol in Ca(2+) signalling and secretion during the interaction of platelets with VWF and collagen. VWF/ristocetin-induced platelet aggregation was delayed after treatment with methyl beta-cyclodextrin (mbCD), but the maximal aggregation response was not affected. Platelet spreading but not adhesion to immobilised VWF under flow was attenuated by cholesterol removal, and accompanied by moderate lowering in the spiking Ca(2+) response. On the other hand, platelet interaction with collagen was quite sensitive to cholesterol depletion. Platelet aggregation decreased after treatment with mbCD, and Ca(2+) responses were decreased, both under static and flow conditions. Cholesterol depletion affected the secondary feedback activation via release of thromboxane A(2) and ADP. The collagen-induced secretion of alpha granules and surface translocation of P-selectin and CD63 was also critically affected by cholesterol depletion. Confocal microscopy showed localization of p-Tyr at sites of contact with substrate and other platelets, where also CRDs accumulate. Our data thus reveal a more critical role for membrane cholesterol in collagen-induced than in VWF-induced Ca(2+) signalling, and furthermore support the concept that secondary activation responses are dependent on intact CRDs.

There is a strong correlation between the level of plasma low-density lipoprotein (LDL) and death by cardiovascular disease (CVD). As a main carrier of cholesterol, a high low-density lipoprotein concentration stimulates atherogenesis by its capacity to become oxidized and to become endocytosed by macrophages in the vessel wall forming cholesterol-rich plaques that are sites for arterial occlusion. New evidence points at a second role of low-density lipoprotein in increasing cardiovascular disease-risk. Contact with low-density lipoprotein induces platelet hypersensitivity to agonists that initiate platelet functions thereby enhancing adhesion, aggregation and secretion of granule contents. The signalling pathways that mediate the priming of platelets by native and oxidized low-density lipoprotein have now been characterized.

Objective: The interaction of platelets with low density lipoprotein (LDL) contributes to the development of cardiovascular disease. Platelets are activated by native LDL (nLDL) through apoE Receptor 2' (apoER2')-mediated signaling to p38(MAPK) and by oxidized LDL (oxLDL) through lysophosphatidic acid (LPA) signaling to Rho A and Ca2+. Here we report a new mechanism for platelet activation by oxLDL., Methods and Results: Oxidation of nLDL increases p38(MAPK) activation through a mechanism that is (1) independent of LPA, and (2) unlike nLDL-signaling not desensitized by prolonged platelet-LDL contact or inhibited by receptor-associated protein or chondroitinase ABC. Antibodies against scavenger receptors CD36 and SR-A alone fail to block p38(MAPK) activation by oxLDL but combined blockade inhibits p38(MAPK) by >40% and platelet adhesion to fibrinogen under flow by >60%. Mouse platelets deficient in either CD36 or SR-A show normal p38(MAPK) activation by oxLDL but combined deficiency of CD36 and SR-A disrupts oxLDL-induced activation of p38(MAPK) by >70%., Conclusion: These findings reveal a novel platelet-activating pathway stimulated by oxLDL that is initiated by the combined action of CD36 and SR-A.

Metabolic studies have revealed a gradual impairment in platelet integrity during storage, a process termed the platelet storage lesion. Recent evidence shows that stored platelets also lose signaling responses to physiological agonists with impaired integrin activation, secretion, and aggregation of the cells. On the other hand, storage leads to a gain in platelet activation properties, such as release of microparticles and appearance of surface epitopes for their clearance by macrophages. New techniques for measuring flow-induced thrombus formation and platelet-dependent coagulation provide evidence that the hemostatic activity of platelets decreases during storage. Besides pharmacological inhibition, novel storage strategies, like metabolic suppression, should be considered to better preserve platelet functionality while limiting the expression of clearance markers. Understanding the changes that occur in association with the platelet storage lesion and the use of updated storage methods will help to generate platelets for transfusion with optimal hemostatic function and a long circulation time after transfusion.

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 alpha-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, and Western 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 neutralizes VEGF 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 alpha-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, platelet VEGF 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.

Background: Moderate and prolonged consumption of red wine is associated with decreased cardiovascular morbidity and mortality. Inhibition of platelet functions by ingredients in red wine is thought to be one of the causes. However, the molecular mechanism of this inhibition has remained unexplained., Materials and Methods: We measured aggregation, changes in cytosolic Ca(2+) and tyrosine phosphorylation of the inhibitory receptor platelet endothelial cell adhesion molecule-1 (PECAM-1) in platelets stimulated with thrombin receptor (PAR-1) activating peptide (TRAP) and ADP and investigated the effects of alcohol-free polyphenolic grape extract (PGE), alcohol, and the polyphenols catechin, epi-catechin, resveratrol, trans-resveratrol, and gallic acid., Results: Polyphenolic grape extract induced dose-dependent inhibition of TRAP-induced and ADP-induced platelet aggregation and Ca(2+) mobilization. Inhibition was accompanied by activation of PECAM-1. Apart from a slight inhibition by catechin, ethanol or other individual polyphenols failed to inhibit aggregation or activate PECAM-1., Conclusions: Red wine inhibits platelet functions through its PGE content, which stimulates the inhibitory receptor PECAM-1, thereby attenuating platelet activation.

Objective: Protein misfolding diseases result from the deposition of insoluble protein aggregates that often contain fibrils called amyloid. Amyloids are found in Alzheimer disease, atherosclerosis, diabetes mellitus, and systemic amyloidosis, which are diseases where platelet activation might be implicated., Methods and Results: We induced amyloid properties in 6 unrelated proteins and found that all induced platelet aggregation in contrast to fresh controls. Amyloid-induced platelet aggregation was independent of thromboxane A2 formation and ADP secretion but enhanced by feedback stimulation through these pathways. Treatments that raised cAMP (iloprost), sequestered Ca2+ (BAPTA-AM) or prevented amyloid-platelet interaction (sRAGE, tissue-type plasminogen activator [tPA]) induced almost complete inhibition. Modulation of the function of CD36 (CD36-/- mice), p38(MAPK) (SB203580), COX-1 (indomethacin), and glycoprotein Ib alpha (Nk-protease, 6D1 antibody) induced approximately 50% inhibition. Interference with fibrinogen binding (RGDS) revealed a major contribution of alphaIIb beta3-independent aggregation (agglutination)., Conclusions: Protein misfolding resulting in the appearance of amyloid induces platelet aggregation. Amyloid activates platelets through 2 pathways: one is through CD36, p38(MAPK), thromboxane A2-mediated induction of aggregation; the other is through glycoprotein Ib alpha-mediated aggregation and agglutination. The platelet stimulating properties of amyloid might explain the enhanced platelet activation observed in many diseases accompanied by the appearance of misfolded proteins with amyloid.

Introduction: We showed earlier that metabolically suppressed platelets (MSP) prepared by incubation in glucose-free, antimycin A medium at 37 degrees C better sustained storage at 4 degrees C than untreated controls at 22 degrees C. However, the use of the mitochondrial inhibitor antimycin A is incompatible with platelet transfusion., Objectives: The aim of this study was to investigate how energy-reduced (ER) platelets could be prepared in the absence of antimycin A., Study Design and Methods: Platelets in gas-impermeable bags in glucose-free medium were kept at 22 degrees C for 4 h to reduce energy stores and thereafter stored at 4 degrees C (ER22-4). Controls were energy-reduced platelets without prior incubation at 22 degrees C (ER4), and MSPs in test tubes and untreated platelets in gas-permeable bags with glucose and stored at 22 degrees C (C22) and 4 degrees C (C4)., Results: After 48 h storage, ER22-4 were superior to C22 with respect to pH preservation (6 x 4 +/- 0 x 4 vs. 5 x 0 +/- 0 x 4, n= 4), platelet count (800 +/- 225 vs. 650 +/- 150 x 10(9)), thrombin receptor-activating peptide-induced aggregation (50 +/- 15 vs. 10 +/- 5%) and glycoprotein (GP)Ib alpha expression (60 +/- 15% vs. 28 +/- 15). GPIb alpha expression was higher in ER22-4 than in ER4, indicating that energy suppression preserved GPIb alpha during cold storage., Conclusion: Metabolic suppression without the use of antimycin A could be mimicked by storage of platelets in glucose-free medium in gas-impermeable bags. Energy suppression preserved GPIb alpha expression during storage at 4 degrees C.