Changes in the distribution of platelet membrane proteins revealed by energy transfer

The redistribution of platelet membrane proteins in response to platelet activation was studied. To investigate this process we prepared a variety of platelet ligands, including di- and tetrameric concanavalin A, IgG, thrombin, wheat-germ agglutinin and other lectins. These ligands were conjugated either with acceptor (rhodamine isothiocyanate) or donor (fluoresceine isothiocyanate) fluorophore. Platelets exposed to various combinations of ligand species were stimulated with different aggregating agents, and changes in sensitized fluorescence emission or donor quenching were recorded. Energy transfer was observed with thrombin, dimeric concanavalin A after addition of thrombin and various combinations of dimeric concanavalin A with other membrane ligands. The preincubation of platelets with colchicine prevented energy transfer between appropriate ligand pairs and platelet activator. Our studies show that nonradiative energy transfer can be used to analyze redistribution of membrane receptor sites in platelets.     

Role of the membrane concanavalin A binding site in platelet-fibrin interactions

Concanavalin A was employed to study the role of platelet membrane glycoproteins in platelet-fibrin interactions during clot formation. A rheological technique was used to study the interactions, measuring the clot rigidity and platelet contractile force simultaneously during the formation of network structure. Concanavalin A lowered the clot rigidity and contractile force of a platelet-rich plasma clot by a small extent. Plasma glycoproteins probably compete with platelet membranes for concanavalin A binding in platelet-rich plasma. Both native concanavalin A (tetrameric) and succinyl concanavalin A (dimeric) lowered the clot rigidity and contractile force of a washed platelet-fibrin clot dramatically, almost down to those values found for fibrin clots. Inhibition studies with alpha-methyl-D-mannoside indicated that the concanavalin A effects were specific for the concanavalin A binding capacity to platelets. The effects of native concanavalin A on platelet-fibrin clots were only partially reversible, while the succinyl concanavalin A effects were completely reversible. The observed concanavalin A effects are probably mainly due to concanavalin A binding to platelet membrane glycoproteins. The concanavalin A binding site appears to play an important role in the fibrin binding to platelets.     

Intracellular calcium mobilization is triggered by clustering of membrane glycoproteins in concanavalin A-stimulated platelets

Stimulation of human platelets with concanavalin A resulted in a significant increase in the concentration of cytoplasmic free Ca²⁺. This effect was due to two different processes: Ca²⁺ mobilization from internal stores and Ca²⁺ influx from the extracellular medium. Kinetic analysis revealed that the release of Ca²⁺ from internal storage sites occurred sooner than the opening of plasma membrane Ca²⁺ channels. The ability of concanavalin A to induce a sustained increase in cytoplasmic Ca²⁺ concentration was antagonized and reversed by methyl ∝-D -mannopyranoside, demonstrating that it was promoted by the interaction of the lectin with cell surface glycoproteins. Succinyl–concanavalin A, a dimeric derivative of the lectin, that does not promote patching/capping of the receptor, was able to bind to the platelet surface, and antagonized the effects of native concanavalin A. In addition, succinyl–concanavalin A, per se, was unable to induce Ca²⁺ mobilization in human platelets. Therefore, the action of the native concanavalin A was mediated by receptor clustering events. Concanavalin A mobilized Ca²⁺ from the same internal stores from which Ca²⁺ was mobilized in response to strong platelet agonists, such as thrombin and arachidonic acid. However, while thrombin was ineffective in inducing Ca²⁺ release after stimulation of platelets with Con A, Con A was able to cause a full discharge of Ca²⁺ from internal stores even in platelets previously stimulated with thrombin. These results demonstrate for the first time that the clustering of specific membrane glycoproteins can trigger platelet activation. The physiological implications during platelet aggregation are discussed.     

Demonstration of 125I-labelled thrombin binding platelet proteins by use of crossed immunoelectrophoresis and autoradiography

A possible receptor for thrombin on the platelet membrane has been identified. Whole platelets were treated with 125I-labelled thrombin followed by washing of the platelets, solubilization in Triton X-100, crossed immunoelectrophoresis and autoradiography. A heavily labelled antigen which migrated slightly more slowly than albumin was observed. No corresponding arc was seen on the same immunoplate when stained with Coomassie brilliant blue, indicating that the antigen possessed weak antigenic properties and/or was present in very small amounts. When 125I-labelled thrombin that had been inactivated by phenylmethylsulphonyl fluoride was used, no such labelled arc was seen. The radiolabelled immunoprecipitate does not represent any of the antigens identified hitherto in the immunoelectrophoretic patterns obtained with platelets or platelet material. The electrophoretic mobility of the antigen was influenced neither by neuraminidase treatment of the platelets prior to the 125I-labelled thrombin exposure nor by inclusion of concanavalin A, wheat-germ lectin or lentil lectin in the gel during the first-dimension electrophoresis. This suggests that the antigen does not represent a glycoprotein. Upon subcellular fractionation the radioactively labelled arc was observed in the cytosol fraction following crossed immunoelectrophoresis and autoradiography. Analysis of the secreted proteins after induction of the release reaction with 125I-labelled thrombin revealed labelling of immunoprecipitates representing thrombospondin, albumin and the 'line' form of platelet factor 4. This confirms that stable complexes of 125I-labelled thrombin and platelet proteins can exist in the presence of Triton X-100 and during electrophoresis.     

Distinct but critical roles for integrin alphaIIbbeta3 in platelet lamellipodia formation on fibrinogen, collagen-related peptide and thrombin

Integrins are the major receptor type known to facilitate cell adhesion and lamellipodia formation on extracellular matrix proteins. However, collagen-related peptide and thrombin have recently been shown to mediate platelet lamellipodia formation when presented as immobilized surfaces. The aims of this study were to establish if there exists a role for the platelet integrin alpha(IIb)beta(3) in this response; and if so, whether signalling from the integrin is required for lamellipodia formation on these surfaces. Real-time analysis was used to compare platelet morphological changes on surfaces of fibrinogen, collagen-related peptide or thrombin in the presence of various pharmacological inhibitors and platelets from 'knockout' mice. We demonstrate that collagen-related peptide and thrombin stimulate distinct patterns of platelet lamellipodia formation and elevation of intracellular Ca(2+) to that induced by the integrin alpha(IIb)beta(3) ligand, fibrinogen. Nevertheless, lamellipodia formation on collagen-related peptide and thrombin is dependent upon engagement of alpha(IIb)beta(3), consistent with release of alpha(IIb)beta(3) ligand(s) from platelet granules. However, the requirement for signalling by the integrin on fibrinogen can be bypassed by the addition of thrombin to the solution. These observations reveal a critical role for alpha(IIb)beta(3) in forming lamellipodia on collagen-related peptide and thrombin which is dependent on its ability to function as an adhesive receptor but not necessarily on its ability to signal. These results suggest that integrins may play an important role in lamellipodia formation triggered by nonintegrin ligands in platelets and possibly in other cell types.     

Demonstration of 125I-labelled thrombin binding platelet proteins by use of crossed immunoelectrophoresis and autoradiography

A possible receptor for thrombin on the platelet membrane has been identified. Whole platelets were treated with ¹²⁵I-labelled thrombin followed by washing of the platelets, solubilization in Triton X-100, crossed immunoelectrophoresis and autoradiography. A heavily labelled antigen which migrated slightly more slowly than albumin was observed. No corresponding arc was seen on the same immunoplate when stained with Coomassie brilliant blue, indicating that the antigen possessed weak antigenic properties and/or was present in very small amounts. When ¹²⁵I-labelled thrombin that had been inactivated by phenylmethylsulphonyl fluoride was used, no such labelled arc was seen. The radiolabelled immunoprecipitate does not represent any of the antigens identified hitherto in the immunoelectrophoretic patterns obtained with platelets or platelet material. The electrophoretic mobility of the antigen was influenced neither by neuraminidase treatment of the platelets prior to the ¹²⁵I-labelled thrombin exposure nor by inclusion of concanavalin A, wheat-germ lectin or lentil lectin in the gel during the first-dimension electrophoresis. This suggests that the antigen does not represent a glycoprotein. Upon subcellular fractionation the radioactively labelled arc was observed in the cytosol fraction following crossed immunoelectrophoresis and autoradiography. Analysis of the secreted proteins after induction of the release reaction with ¹²⁵I-labelled thrombin revealed labelling of immunoprecipitates representing thrombospondin, albumin and the ‘line’ form of platelet factor 4. This confirms that stable complexes of ¹²⁵I-labelled thrombin and platelet proteins can exist in the presence of Triton X-100 and during electrophoresis.     

Ca2+ ionophore A23187 and thrombin inhibit the pertussis-toxin-induced ADP-ribosylation of the alpha-subunit of the inhibitory guanine-nucleotide-binding protein and other proteins in human platelets

Inhibitory guanine-nucleotide-binding proteins (Gi proteins) are substrates for pertussis toxin and the decreased pertussis-toxin-dependent ADP ribosylation of Gi proteins upon prior specific hormonal stimulation of cells is thought to reflect the receptor-mediated activation of Gi proteins, leading to their subsequent dissociation into alpha i and beta/gamma subunits. In the present study, the effect of various platelet stimuli on the subsequent pertussis-toxin-dependent ADP ribosylation of the alpha subunit of Gi (Gi alpha) in saponized platelets and platelet membranes were studied. Stimulation of intact platelets with the Ca(2+)-ionophore A23187 or thrombin, but not phorbol 12,13-dibutyrate, decreased the subsequent pertussis-toxin-dependent ADP ribosylation of Gi alpha in saponin-permeabilized platelets in a time-dependent and dose-dependent manner. Thrombin was more effective than A23187. Parallel measurements of Ca2+ mobilization and pertussis-toxin-dependent ADP ribosylation of Gi alpha in platelets showed that Ca2+ mobilization could only partly account for the decrease in pertussis-toxin-dependent ADP ribosylation in platelets stimulated by thrombin. When the ADP-ribosylation reaction was carried out in platelet membranes, a decrease in ADP ribosylation was still observed after stimulation of platelets with thrombin, but not with A23187. In addition to Gi alpha, two other proteins were found to be ADP ribosylated by pertussis toxin; their ADP ribosylation was also decreased after A23187 and thrombin stimulation of platelets. The results indicate that Ca2+ mobilization can decrease the pertussis-toxin-dependent ADP ribosylation of Gi alpha in saponized platelets; the decrease of pertussis-toxin-dependent ADP ribosylation of Gi alpha after thrombin stimulation of platelets can only, in part, be explained by Ca2+ mobilization and involves additional mechanisms; the decrease in pertussis-toxin-dependent ADP ribosylation after A23187 and thrombin stimulation is not confined to G1 alpha and involves other proteins. We conclude that the decrease in pertussis-toxin-dependent ADP ribosylation of Gi in thrombin-stimulated platelets might not be solely caused by a specific structural change, such as dissociation of Gi. It is likely that A23187 and thrombin stimulation of platelets generates substances which interfere with the ADP-ribosylating activity of pertussis toxin.     

A molecular model for singlet/singlet energy transfer of monovalent ligand/receptor interactions

A stochastic model is described that predicts the degree of singlet/singlet energy transfer in complexes formed between monovalent ligands and monovalent receptors. The modeling approach is intended to serve as an analytical tool for approximating the level of fluorescence quenching that can be expected to occur in fluorescently labeled monovalent ligands and receptors that are bound together in complexes. This approach has utility in areas such as modeling protein/protein interactions and designing fluorescence energy transfer assays.Using the crystallographic data for papain (monovalent ligand ) and concanavalin A (monovalent receptor ) along with a molecular graphics computational package the ligand and receptor were docked together to form a ligand/receptor complex. The intermolecular distances between the lysine resides of the ligand and receptor were then estimated, receptor complex was calculated assuming a value for the characteristic length R(0) of the donor/acceptor pair. Results from the stochastic model were used to calculate the level of fluorescence quenching one would expect for a resonance energy transfer competition assay based on the monovalent ligand/pair.Three key assumptions were made during the model development. First, all lysine resides for the ligand and receptor were equally reactive with the dye molecules so the stoichiometry of the donor and acceptor chromophores was governed by a binomial distribution. Second, the dye molecules were located at the alpha-carbon position for each reactive lysine residue. Finally, in the energy transfer competition assay, it was assumed that equilibrium existed between the ligand, receptor, and competing hapten at all times. Based on these assumptions, results are presented that indicate the maximum energy transfer for the monovalent papain/concanavalin. A complex is strongly dependent on the number of acceptor chromophores and on the value of R(0). Results are also presented on the approximate level of fluorescence quenching that may occur in a competition assay based on the papin/pConA complex. Lastly, a strategy is discussed for maximizing the dynamic range and linearity of energy transfer assays by optimizing several key design variables.     

Platelet glycoprotein Ib: a zinc-dependent binding protein for the heavy chain of high-molecular-weight kininogen.

Domains 3 and 5 of high-molecular-weight kininogen (HK) have been shown to bind to platelets in a zinc-dependent reaction. However, the platelet-binding proteins responsible for this interaction have not been identified. We have focused on the platelet-binding site for the heavy chain (domain 3), which we approached using a domain 3-derived peptide ligand and isolated binding proteins by affinity chromatography. The domain 3-derived peptide, thrombin, HK, factor XII, as well as antibody to glycocalicin (the N-terminal portion of the alpha chain of GPIb) recognized a protein at 74 kD. We also isolated the thrombin receptor (PAR 1) at 45 kD, however, none of the above-mentioned ligands bound to this protein. Isolation of platelet membrane proteins using a monoclonal anti-glycocalicin antibody column revealed the same HK binding protein at 74 kD, which was reactive with anti-GPIb and represents a GPIb fragment. By photoaffinity labeling, HK interacted with membrane GPIb, which was then isolated in native form (135 kD) along with gC1qR, a ligand for the HK light chain. Finally, (125)I-HK binding to platelets was significantly inhibited by the anti-GPIb antibody. These results suggest that the GPIb alpha chain, a known thrombin binding protein, is also one of the zinc-dependent platelet membrane binding sites for HK domain 3.     

On the susceptibility of human platelets to aggregation by concanavalin A and the effect of this lectin on their response to ADP

Concanavalin A aggregated gel-filtered platetes in 0.9% NaCl solution signifying cross-bridging by the lectin. Aggregation of these platelets by concanavalin A was temperature dependent; it did not occur at 0–4 °C unless the platelets were previously trypsinized. The level of aggregation of trypsinized platelets by concanavalin A at 0–4°C was similar to that of untreated platelets at 37°C. It is suggested that trypsin facilitates platelet aggregation by concanavalin A at 0–4°C by causing a configurational change in membrane glycoproteins which orientates concanavalin A receptor sites into positions that favour lectin cross-bridging. Concanavalin A failed to aggregate platelets in plasma. Radioisotope studies showed that the amount of [³H]concanavalin A which combined with platelets in plasma was extremely low compared with gel-filtered platelets in saline. The aggregation of Ehrlich ascites cells by concanavalin A was considerably reduced when platelet-free plasma was added to the medium suggesting that it was due to the presence of concanavalin A-reactive components in the plasma.Concanavalin A inhibited the ADP-induced aggregation of platelets suspended in plasma or in a salts solution supplemented with calcium and fibrinogen, although the inhibitory effect was more conspicuous in the latter case. The results suggests that concanavalin A produces its inhibitory effect on ADP-induced platelet aggregation by interacting with membrane glycoproteins, and this further suggests their involvement in aggregation.     

Assembly of the platelet prothrombinase complex is linked to vesiculation of the platelet plasma membrane. Studies in Scott syndrome: an isolated defect in platelet procoagulant activity

Activation of human platelets by complement proteins C5b-9 is accompanied by the release of small plasma membrane vesicles (microparticles) that are highly enriched in binding sites for coagulation factor Va and exhibit prothrombinase activity. We have now examined whether assembly of the prothrombinase enzyme complex (factors VaXa) is directly linked to the process of microparticle formation. Gel-filtered platelets were incubated without stirring with various agonists at 37 degrees C, and the functional expression of cell surface receptors on platelets and on shed microparticles was analyzed using specific monoclonal antibodies and fluorescence-gated flow cytometry. In addition to the C5b-9 proteins, thrombin, collagen, and the calcium ionophore A23187 were each found to induce formation of platelet microparticles that incorporated plasma membrane glycoproteins GP Ib, IIb, and IIIa. These microparticles were enriched in binding sites for factor Va, and their formation paralleled the expression of catalytic surface for the prothrombinase enzyme complex. Little or no microparticle release or prothrombinase activity were observed when platelets were stimulated with epinephrine and ADP, despite exposure of platelet fibrinogen receptors by these agonists. When platelets were exposed to thrombin plus collagen, the shed microparticles contained activated GP IIb-IIIa complexes that bound fibrinogen. By contrast, GP IIb-IIIa incorporated into C5b-9 induced microparticles did not express fibrinogen receptor function. Platelets from a patient with an isolated defect in inducible procoagulant activity (Scott syndrome) were found to be markedly impaired in their capacity to generate microparticles in response to all platelet activators, and this was accompanied by a comparable decrease in the number and function of inducible factor Va receptors. Taken together, these data indicate that the exposure of the platelet factor Va receptor is directly coupled to plasma membrane vesiculation and that this event can be dissociated from other activation-dependent platelet responses. Since a catalytic membrane surface is required for optimal thrombin generation, platelet microparticle formation may play a role in the normal hemostatic response to vascular injury.     

Synergistic effects of lectins in the interaction of thrombin with human platelets

Several lectins have been studied for their effects on the interaction of thrombin with human platelets. Wheat germ agglutinin, concanavalin A and Ricinus communis lectin increased the number of high affinity sites for diisopropylphosphothrombin on washed platelets from 3000 to about 12 000 but the binding affinities were unchanged (Kd approx 4 nM). Two other lectins, Lens culinaris and Bandieria simplicifolia, were without effect. (2) Using formalinized platelets to avoid possible complications of the platelet release reaction, wheat germ agglutinin showed a marked increase (5-fold) in the binding of active thrombin, peanut agglutinin had no effect while Ricinus communis and :Bandieria simplicifolia showed marginal increases (2-fold). Thrombin binding was decreased to about one quarter with Lens culinaris, Phaseolus vulgaris and concanavalin A. (3) Wheat germ agglutinin caused a synergistic increase of platelet aggregation at low concentrations of thrombin (12.5 mU/ml) and ADP (1 microM), both in the absence and presence of added fibrinogen, but had no effect on ristocetin-induced aggregation.     

Differential effects of concanavalin A and succinyl concanavalin A on the macromolecular events of platelet activation

Concanavalin A is capable of activating platelets in a concentration-dependent manner as judged by [14C]serotonin secretion from prelabeled platelets. In contrast, succinyl concanavalin A does not induce platelet secretion. Concanavalin A treatment also results in a number of alterations in platelet macromolecules which are presumably associated with the process of platelet activation. These include the phosphorylation of 20 and 47 kDa platelet proteins, the increased polymerization and association of new proteins with the platelet cytoskeleton and the association of the platelet membrane glycoprotein IIb/III complex with the platelet cytoskeleton. Succinyl concanavalin A treatment results in none of these macromolecular events. This difference is observed despite the demonstration that both lectins bind to the platelet surface. Gel overlay experiments also indicate that concanavalin A and succinyl concanavalin A bind to the same receptors. These differences in the biological effects of concanavalin A and succinyl concanavalin A on platelets may be due to decreased receptor crosslinking by the succinylated derivative. The formation of multiple linked interactions between surface receptors may be an important event in the activation of platelets by concanavalin A.     

Membrane microenvironmental changes during activation of human blood platelets by thrombin. A study with a fluorescent probe.

Membrane microenvironmental changes associated with thrombin-induced platelet activation were followed by fluorescence intensity and polarization studies of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled human platelets. The labeling of washed platelets with DPH did not alter platelet intactness and morphology. In response to thrombin, DPH-labeled platelets exhibited reduced serotonin release, yet aggregation was barely inhibited. Shape change induced by thrombin or ADP was indistinguishable in control and in DPH-labeled platelets. During platelet aggregation induced by thrombin, fluorescence intensity increased by about 14%, which may indicate a more hydrophobic exposure of the probe. However, no change in fluorescence was detected during platelet shape change, induced either by thrombin in presence of EDTA or by ADP. Thrombin-activated platelets exhibited an increase in values of fluorescence polarization (P) during the stages of shape change and secretion, which further increased during aggregation. A similar pattern of increase in P values characterized platelet shape changes, caused either by thrombin in the presence of EDTA or by ADP. Changes in individual platelets are discernible from the alterations of the aggregating cells. These results may indicate that platelet activation is accompanied by an increase in rigidity of the membrane lipids. Functionally, the elevated "microviscosity" may reflect a primary role of membrane lipids in modulating the process of platelet activation or secondary transitions in lipids due to membrane events mediated by proteins.     

Protein kinase C translocation in human blood platelets

Protein kinase C (PKC) activity and translocation in response to the phorbol ester, phorbol 12-myristate, 13-acetate (PMA), serotonin (5-HT) and thrombin was assessed in human platelets. Stimulation with PMA and 5-HT for 10 minutes or thrombin for 1 minute elicited platelet PKC translocation from cytosol to membrane. The catecholamines, norepinephrine or epinephrine at 10 microM concentrations did not induce redistribution of platelet PKC. Serotonin (0.5-100 microM) and the specific 5-HT2 receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (10-100 microM) but not the 5-HT1A or 5-HT1B agonists, (+/-) 8-hydroxy-dipropylamino-tetralin (8-OH-DPAT) or 5-methoxy-3-3-(1,2,3,6-tetrahydro-4-pyridin) 1H-indole succinate (RU 24969) induced dose-dependent PKC translocations. Serotonin-evoked PKC translocation was blocked by selective 5-HT2 receptor antagonists, ketanserin and spiroperidol. These results suggest that, in human platelets, PMA, thrombin and 5-HT can elicit PKC translocation from cytosol to membrane. Serotonin-induced PKC translocation in platelets is mediated via 5-HT2 receptors.     

Influence of dietary fatty acids on membrane fluidity and activation of rat platelets

The apparent steady-state fluorescence anisotropy of DPH- or TMA-DPH-labeled washed rat platelets is strongly affected by factors that also influence the turbidity by these platelet suspensions. Sonicated preparations from platelet lipids have a low turbidity and give anisotropy values which are hardly affected by the experimental conditions. We studied the effect of four high-fat diets on membrane fluidity, lipid composition and activation tendency of washed platelets. The diets contained 50 energy% of oils with different levels of saturated and (poly)unsaturated fatty acids. Only small diet-induced differences in DPH fluorescence anisotropy were found, which were comparable for intact platelets and platelet lipids. These differences were unrelated to the degree of saturation of the dietary fatty acids. Platelets from rats fed mainly saturated fatty acids differed significantly from other diet groups in a higher unsaturation degree of phospholipids and a lower cholesterol/phospholipid ratio, but this was not detected by DPH in terms of decreased anisotropy. These platelets aggregated less than other platelets in response to thrombin or collagen. The lower response to collagen persisted in indomethacin-treated platelets activated with the thromboxane A2 mimetic U46619, indicating a different sensitivity of these platelets for thromboxane A2. We conclude that in rat platelets: (a) the overall membrane fluidity and phospholipid unsaturation degree are subject to strong homeostatic control; (b) steady-state anisotropy with DPH or TMA-DPH label is inadequate to reveal subtile changes in lipid profile; (c) changes in platelet responsiveness to thrombin and thromboxane A2, rather than (plasma) membrane fluidity, determine the effect of dietary fatty acids on platelet aggregation.     

"Thrombin" receptor-directed ligand accounts for activation by thrombin of platelet phospholipase C and accumulation of 3-phosphorylated phosphoinositides.

Using three experimental approaches, we have addressed the questions of whether the presence of saturably bound thrombin plays a role in potentiating the activation of platelet phospholipase C (PLC) and/or accumulation of the 3-phosphorylated phosphoinositides (3-PPI), i.e. phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, and whether the generation of tethered ligand (Vu, T-K.H., Hung, D. T., Wheaton, V. I., and Coughlin, S. R. (1991) Cell 64, 1057-1068) by thrombin can account fully for thrombin's proteolytic effects in activating platelets, as gauged by the above parameters. We have 1) measured PLC activation or 3-PPI after we have exposed platelets to thrombin for various periods and either blocked thrombin's proteolytic activity without interrupting its binding or blocked both binding and proteolytic activity of thrombin; 2) attempted to potentiate 3-PPI accumulation, using combinations of protein kinase C stimulation, Ca2+ elevation, and saturating but proteolytically inactive thrombins; and 3) compared the activation of platelets by thrombin with activation by the "thrombin" receptor-directed peptide, SFLLRNPNDKYEPF (SFLL; a portion of the tethered ligand created by thrombin's proteolytic activity), and examined the effect of thrombin on this latter activation. We conclude that the initial and sustained effects of thrombin in stimulating PLC and the accumulation of 3-PPI are completely attributable to thrombin's proteolytic activity. Further, thrombin's effects in promoting these responses can be accounted for by the actions of SFLL peptide, and by implication, formation of tethered ligand.     

The glycoprotein Ib-IX-V complex mediates localization of factor XI to lipid rafts on the platelet membrane

Factor XI binds to activated platelets where it is efficiently activated by thrombin. The factor XI receptor is the platelet membrane glycoprotein (GP) Ib-IX-V complex (Baglia, F. A., Badellino, K. O., Li, C. Q., Lopez, J. A., and Walsh, P. N. (2002) J. Biol. Chem. 277, 1662-1668), a significant fraction of which exists within lipid rafts on stimulated platelets (Shrimpton, C. N., Borthakur, G., Larrucea, S., Cruz, M. A., Dong, J. F., and Lopez, J. A. (2002) J. Exp. Med. 196, 1057-1066). Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids implicated in localizing membrane ligands and in cellular signaling. We now show that factor XI was localized to lipid rafts in activated platelets ( approximately 8% of total bound) but not in resting platelets. Optimal binding of factor XI to membrane rafts required prothrombin (and Ca2+) or high molecular weight kininogen (and Zn2+), which are required for factor XI binding to platelets. An antibody to GPIb (SZ-2) that disrupts factor XI binding to the GPIb-IX-V complex also disrupted factor XI-raft association. The isolated recombinant Apple 3 domain of factor XI, which mediates factor XI binding to platelets, also completely displaces factor XI from membrane rafts. To investigate the physiological relevance of the factor XI-raft association, the structural integrity of lipid rafts was disrupted by cholesterol depletion utilizing methyl-beta-cyclodextrin. Cholesterol depletion completely prevented FXI binding to lipid rafts, and initial rates of factor XI activation by thrombin on activated platelets were inhibited >85%. We conclude that factor XI is localized to GPIb in membrane rafts and that this association is important for promoting the activation of factor XI by thrombin on the platelet surface.     

Thrombin Stimulates Glucose Transport in Human Platelets via the Translocation of the Glucose Transporter GLUT-3 from α-Granules to the Cell Surface

Increased energy metabolism in the circulating blood platelet plays an essential role in platelet plug formation and clot retraction. This increased energy consumption is mainly due to enhanced anaerobic consumption of glucose via the glycolytic pathway. The aim of the present study was to determine the role of glucose transport as a potential rate-limiting step for human platelet glucose metabolism. We measured in isolated platelet preparations the effect of thrombin and ADP activation, on glucose transport (2-deoxyglucose uptake), and the cellular distribution of the platelet glucose transporter (GLUT), GLUT-3. Thrombin (0.5 U/ml) caused a pronounced shape change and secretion of most α-granules within 10 min. During that time glucose transport increased approximately threefold, concomitant with a similar increase in expression of GLUT-3 on the plasma membrane as observed by immunocytochemistry. A major shift in GLUT-3 labeling was observed from the α-granule membranes in resting platelets to the plasma membrane after thrombin treatment. ADP induced shape change but no significant α-granule secretion. Accordingly, ADP-treated platelets showed no increased glucose transport and no increased GLUT-3 labeling on the plasma membrane. These studies suggest that, in human blood platelets, increased energy metabolism may be precisely coupled to the platelet activation response by means of the translocation of GLUT-3 by regulated secretion of α-granules. Observations in megakaryocytes and platelets freshly fixed from blood confirmed the predominant GLUT-3 localization in α-granules in the isolated cells, except that even less GLUT-3 is present at the plasma membrane in the circulating cells (~15%), indicating that glucose uptake may be upregulated five to six times during in vivo activation of platelets.     

The involvement of cytoskeleton in the regulation of transbilayer movement of phospholipids in human blood platelets

Activation of human platelets by different activators resulted in a different extent of degradation of the cytoskeletal proteins actin-binding protein and myosin, as well as of the non-cytoskeletal protein P235. The highest extent of proteolysis was observed with Ca-ionophore A23187 and decreased on going from A23187 greater than collagen plus thrombin greater than collagen greater than thrombin = ADP. The same order of potency has been found previously ((1983) Biochim. Biophys. Acta 736, 57-66) for the ability of platelet activators to induce exposure of aminophospholipids in the outer leaflet of the platelet plasma membrane, and to stimulate platelets to become procoagulant. Degradation of cytoskeletal proteins as a result of platelet stimulation by collagen plus thrombin was prevented in the presence of dibutyryl cAMP or EDTA but not in the presence of aspirin. This also runs in parallel with platelet procoagulant activity. Moreover, platelets from a patient with a partial deficiency in platelet procoagulant activity revealed a diminished extent of degradation of cytoskeletal proteins upon platelet stimulation with collagen plus thrombin. It is concluded that alterations in cytoskeletal organization upon platelet stimulation may lead to alterations in the orientation of (amino)phospholipids in the plasma membrane, and may therefore play a regulatory role in the expression of platelet procoagulant activity.