Platelet apoptosis by cold-induced glycoprotein Ibα clustering.

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.

Mlck1a is expressed in zebrafish thrombocytes and is an essential component of thrombus formation.

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.

Insulin inhibits tissue factor expression in monocytes.

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.

Metabolic energy reduction by glucose deprivation and low gas exchange preserves platelet function after 48 h storage at 4 degrees C.

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.

Platelet signaling induced by lipoproteins.

Cardiovascular disease is the main cause of death and disability in the Western society. Lipoproteins play an important role in the development of this disease and affect different cell types involved in atherosclerosis and thrombosis. Based on their density, five classes of lipoproteins have been identified which all influence cells via distinct mechanisms. Modification turns lipoproteins into atherogenic particles with a prominent role in atherogenesis. The interaction of lipoproteins with platelets has been under investigation for a number of years. Especially the role of LDL in platelet signaling has been studied intensively as platelets of hypercholesterolemic patients are hyperreactive and show hyperaggregability in vitro and enhanced activity in vivo, suggesting that LDL enhances platelet responsiveness. Several signaling pathways induced by LDL have been revealed in vitro, such as signaling via p38 mitogen-activated protein kinase (p38MAPK) and p125 focal adhesion kinase (p125FAK). HDL opposes the activating properties of LDL on platelets, whereas the effects of chylomicrons, VLDL or IDL on platelet function are controversial. Modification of lipoproteins is associated with the generation of new constituents with new signaling properties. In particular, the platelet-activating properties of lysophosphatidic acid, which is a constituent of atherosclerotic plaques and is generated upon oxidation of LDL, have been investigated intensively. This review provides a summary of the activation of signaling pathways after platelet-lipoprotein interactions, with special emphasis on the role of these interactions in the development of thrombosis and atherosclerosis.

The phospholipid composition and cholesterol content of platelet-derived microparticles: a comparison with platelet membrane fractions.

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 = 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 = 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 plasma- and 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.

Adhesive surface determines raft composition in platelets adhered under flow.

Adhesion to von Willebrand factor (VWF) induces platelet spreading, whereas adhesion to collagen induces aggregation. Here we report that cholesterol-rich domains (CRDs) or rafts play a critical role in clustering of receptors that control these responses. Platelets adhered to VWF and collagen show CRDs concentrated in filopodia which contain both the VWF receptor glycoprotein (GP) Ibalpha and the collagen receptor GPVI. Biochemical analysis of CRDs shows a threefold enrichment of GPIbalpha (but not GPVI) in VWF-adhered platelets and a fourfold enrichment of GPVI (but not GPIbalpha) in collagen-adhered platelets. Depletion of cholesterol (i) leaves the initial adhesion unchanged, (ii) inhibits spreading on VWF and aggregate formation on collagen, (iii) leaves filopodia formation intact, and (iv) reduces the localization in filopodia of GPIbalpha but not of GPVI. These data show that the adhesive substrate determines the composition of CRDs, and that cholesterol is crucial for redistribution of GPIbalpha but not of GPVI.

A tripeptide mimetic of von Willebrand factor residues 981-983 enhances platelet adhesion to fibrinogen by signaling through integrin alpha(IIb)beta3.

BACKGROUND: RGD is a major recognition sequence for ligands of platelet alpha(IIb)beta3. OBJECTIVE AND METHODS: To identify potential binding sites for alpha(IIb)beta3 apart from RGD, we screened phage display libraries by blocking the enrichment of RGD-containing phages with a GRGDS peptide and identified a novel integrin recognition tripeptide sequence, VPW. RESULTS: Platelets adhered to an immobilized cyclic VPW containing peptide in a alpha(IIb)beta3-dependent manner; platelets and alpha(IIb)beta3-expressing CHO cells adhered faster to immobilized alpha(IIb)beta3-ligands in the presence of soluble VPW. In platelets adhering to fibrinogen, VPW accelerated the activation of the tyrosine kinase Syk which controls cytoskeletal rearrangements. In alpha(IIb)beta3-expressing CHO cells, VPW induced a faster formation of stress fibers. Sequence alignment positioned VPW to V980-P981-W982 in the von Willebrand factor (vWf) A-3 domain. In blood from a vWf-deficient individual, VPW increased platelet adhesion to fibrinogen but not to collagen under flow and rescued the impaired adhesion to vWf deficient in A-3. CONCLUSION: These data reveal a VPW sequence that contributes to alpha(IIb)beta3 activation in in vitro experiments. Whether the V980-P981-W982 sequence in vWf shows similar properties under in vivo conditions remains to be established.

IRS-1 and vascular complications in diabetes mellitus.

The expected explosive increase in the number of patients with diabetes mellitus will increase the stress on health care. Treatment is focused on preventing vascular complications associated with the disorder. In order to develop better treatment regimens, the field of research has made a great effort in understanding this disorder. This chapter summarizes the current views on the insulin signaling pathway with emphasis on intracellular signaling events associated with insulin resistance, which lead to the prothrombotic condition in the vasculature of patience with diabetes mellitus.

Prolonged platelet preservation by transient metabolic suppression.

BACKGROUND: In this study whether metabolic suppression can be used to preserve platelet (PLT) function during prolonged storage was investigated. STUDY DESIGN AND METHODS: Washed human PLTs were incubated without glucose and with antimycin A to block energy generation. Metabolic suppressed PLTs (MSPs) were stored for 72 hours at different temperatures to find the optimal storage temperature. Controls were incubated with 5 mmol per L glucose and stored at 22 and 4 degrees C. RESULTS: Following metabolic recovery with glucose, MSPs stored at 37, 22, and 4 degrees C showed an increase in basal P-selectin expression (PSE) reaching greater than 40 percent after about 2, 20, and 48 hours; a decrease in thrombin receptor-activating peptide SFLLRN (TRAP)-induced PSE inversely related to the increase in basal PSE; and a decrease in TRAP-induced aggregation reaching less than 30 percent after about 4, 24, and more than 72 hours. When compared with control suspensions, MSPs stored at 4 degrees C better preserved a low basal PSE and in addition showed a better adhesion to surface coated-von Willebrand factor and fibrinogen in a flow chamber. CONCLUSION: Metabolic suppression before storage at 4 degrees C contributes to better preservation of PLT function.

Alcohol and polyphenolic grape extract inhibit platelet adhesion in flowing blood.

BACKGROUND: Moderate and prolonged alcohol consumption has been associated with decreased cardiovascular morbidity and mortality. Inhibition of platelet function in suspension attributes to these effects. Whether alcohol, red wine, or polyphenolic grape extracts (PGE) inhibit platelet adhesion is not known. We investigated platelet adhesion to fibrinogen and collagen in whole blood under standardised flow. MATERIALS AND METHODS: Before perfusion was started, citrated whole blood from 95 volunteers was preincubated for five min with different alcohol concentrations, unfractioned red wine and PGE. Then, blood was perfused in a single-passage flow chamber over coverslips coated with human fibrinogen or collagen type III at shear rates of 300 s(-1) and 1600 s(-1). RESULTS: Alcohol inhibited platelet adhesion to human fibrinogen at high shear rate (concentrations > or = 0.15 per thousand) and low shear rate (only at a concentration of 4.8 per thousand), whereas red wine (concentrations > or = 0.15 per thousand) inhibited platelet adhesion to human fibrinogen at both shear rates. In contrast, PGE (concentrations > or = 0.0225 g L(-1)) inhibited platelet adhesion to human fibrinogen only at low shear rate. None of these incubations affected adhesion to collagen. CONCLUSIONS: Alcohol, red wine and PGE inhibit adhesion to fibrinogen but not to collagen. This inhibition might contribute to the cardioprotective effects of moderate alcohol consumption.

Involvement of the beta3 E749ATSTFTN756 region in stabilizing integrin alphaIIbbeta3-ligand interaction.

Platelet integrin alphaIIbbeta3 must be activated via intracellular mechanisms before it binds soluble ligands, and it is thought to be activated at its extracellular site by surface-bound ligands. Integrin activation is associated with rearrangement of the cytoskeleton and phosphorylation of proteins that become localized in focal contacts. In these processes, the cytoplasmic tail of the beta-subunit plays a central role. We introduced peptides homologous to the E749ATSTFTN756 domain (E-N peptide) and the T755NITYRGT762 domain (T-T peptide) of beta3 in streptolysin O-permeabilized platelets and analyzed the initial interaction with soluble fibronectin, fibrinogen and PAC-1 after stimulation with thrombin. E-N peptide left the initial binding of fibronectin intact but interfered with stable receptor occupancy. E-N peptide also inhibited fibrinogen binding, thereby reducing the formation of large aggregates. Strikingly, E-N peptide did not disturb the binding of PAC-1, which is known to reflect activation of the integrin. E-N peptide also inhibited tyrosine phosphorylation of focal adhesion kinase, a response known to be dependent on alphaIIbbeta3. T-T peptide did not affect these processes. In a model for outside-in integrin activation, E-N peptide disrupted the binding of CHO cells expressing alphaIIbbeta3 to surface-bound ligand. Again, T-T peptide had no effect. We conclude that the E749ATSTFTN756 region of the beta3-tail stabilizes the binding of soluble and surface-bound ligand to integrin alphaIIbbeta3 via a mechanism that involves the phosphorylation of FAK.

Cytoplasmic regions of the beta3 subunit of integrin alphaIIbbeta3 involved in platelet adhesion on fibrinogen under flow conditions.

Platelet adhesion to surface-bound fibrinogen depends on integrin alphaIIbbeta3. In the present study, we investigated the role of the regions 749EATSTFT756N and 755TNITYRG762T of the beta3 cytoplasmic tail in the regulation of platelet adhesion under flow conditions, by introducing peptide mimetics in platelets. Introduction of peptide EATSTFTN (E-N) increased surface coverage by 35%, an effect caused by 25% more adhesion. In contrast, peptide TNITYRGT (T-T) decreased surface coverage by 16%, as a result of 25% less adhesion. An S-->P substitution in the E-N peptide, thereby mimicking a mutation in Glanzmann's thrombasthenia, abolished the effect of E-N. A suboptimal concentration of cytochalasin D is known to enhance ligand binding to alphaIIbbeta3 in platelet suspensions. Under flow, cytochalasin D (1 micro mol L-1) induced 50% more platelet adhesion, with a strong reduction in platelet spreading. Both peptides opposed the increase in adhesion by cytochalasin D and partly (E-N) and completely (T-T) restored platelet spreading. Thus, the 749EATSTFT756N and 755TNITYRG762T regions of beta3 contribute to the regulation of alphaIIbbeta3 anchorage to the cytoskeleton and platelet spreading to an adhesive surface.

Concentration of rafts in platelet filopodia correlates with recruitment of c-Src and CD63 to these domains.

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.

Low-density lipoprotein and its effect on human blood platelets.

Events leading to hyperactivity of human blood platelets are accompanied by an enhanced risk of atherosclerosis and arterial thrombosis. Lipoprotein disorders affect platelet functions, and hypersensitive platelets are observed in various stages of hyperlipidemia. Low-density lipoprotein (LDL), a circulating complex of lipids and proteins that is increased in hypercholesterolemia, enhances platelet function and increases sensitivity of platelets to several naturally occurring agonists. LDL sensitizes platelets via binding of apoB-100 to a receptor on the platelet membrane and via transfer of lipids to the platelet membrane. The receptor that mediates binding of LDL to the platelet and initiates subsequent intracellular signaling cascades has not yet been identified. Modification of native LDL generates a platelet-activating particle, and this interaction might contribute to the development of the atherosclerotic plaque. Lysophosphatidic acid is formed upon mild oxidation of LDL and is responsible for subsequent platelet activation induced by the modified LDL particle. Thus, LDL changes the functions of platelets via a broad spectrum of interactions.

Red wine and red wine polyphenolic compounds but not alcohol inhibit ADP-induced platelet aggregation.

Background: Moderate alcohol consumption reduces the risk of cardiovascular diseases, especially coronary heart disease (CHD). Because of the presence of polyphenols in red wine, this type of beverage may be superior to other alcoholic drinks in the prevention of CHD. Inhibition of platelet aggregation is thought to be one of the mechanisms underlying this favorable effect. The present study analyzes the direct effect of alcohol and red wine polyphenols on platelet aggregation. Methods: Unfractionated red wine, a red wine polyphenolic extract, and alcohol were added in different concentrations to a standardized quantity of blood platelets 2 min before aggregation was induced by different concentrations of ADP. Aggregation was measured in an aggregometer and results were compared to a control 0.9% NaCl solution. Results: Alcohol in concentrations up to 0.24 percent did not inhibit platelet aggregation in vitro initiated with ADP The polyphenolic red wine extract inhibited aggregation dose-dependently and significantly from concentrations of 45 mg/l ( [Formula: see text] ) or more. Red wine only inhibited platelet aggregation at very high concentrations ( approximately 0.24 and 0.48 alcohol%). Conclusions: Consumption of red wine has an inhibitory effect on platelet aggregation, which is caused by the polyphenolic compounds in the wine. Alcohol itself does not have a direct inhibitory effect within a range up to 0.24 percent. Since this effect is only observed at very high concentrations, it is unlikely to be of clinical relevance in a moderate drinking pattern. The results do not exclude platelet inhibition by wine in vivo. However, this must be related to metabolic changes rather than to direct blockade.

Platelet activation by the apoB/E receptor-binding domain of LDL.

Low density lipoprotein (LDL) increases the sensitivity of human platelets for agonists by activating p38MAPK. Antibody 4G3 disturbs apoB100 binding to the classical apoB/E receptor and inhibits LDL-induced p38MAPK activation, whereas an antibody against a distal domain on apoB 100 has no effect. Peptide RLTRKRGLKLA mimics the binding domain of apoB 100 called the B-site and activates platelet p38MAPK. Activation by B-site peptide is dose-dependent, transient and followed by desensitization, in accordance with receptor-mediated signalling. A scrambled peptide and a partially homologous peptide RKLRKRLLRDA mimicking the apoB/E receptor binding site of apoE in high density lipoprotein (HDL) also activate p38MAPK albeit 40% weaker, but an uncharged peptide lacks p38MAPK activating capacity. LDL and B-site peptide bind to the same binding sites and initiate similar signalling to p38MAPK and cytosolic phospholipase A2. Thus, LDL and to a lesser extent HDL activate platelets via specific domains in the protein moiety that recognize receptors of the LDL receptor family.