Pertussis toxin can activate human platelets. Comparative effects of holotoxin and its ADP-ribosylating S1 subunit

The activation of phospholipase C in human platelets is coupled to agonist receptors via guanine nucleotide-binding protein(s), and prior treatment of permeabilized platelets with GTP gamma S, GDP beta S, or pertussis toxin modifies platelet responses to agonists. Pertussis toxin is thought to act primarily as an uncoupler of Gi from cell receptors due to its ADP-ribosylating activity. However, we have found that pertussis toxin by itself can act as an agonist for intact or permeabilized platelets. Though believed to lack receptors for pertussis toxin, intact platelets, when incubated with the toxin (5-20 micrograms/ml), undergo aggregation and accumulate inositol trisphosphate and phosphatidic acid. Treatment of platelets with aspirin, incubation in the presence of creatine phosphate/creatine phosphokinase, or omission of Ca2+ and fibrinogen do not affect toxin-mediated phospholipase C activation. These effects are not observed with the ADP-ribosylating S1 monomer of toxin in intact or permeabilized platelets. Further, modification of the holotoxin with N-ethylmaleimide eliminates the toxin's ADP-ribosylating activity but does not affect its promotion of platelet aggregation and phospholipase C activation. Therefore, the activating effect of holotoxin is separable from its ADP-ribosylating activity and does not depend either upon cyclooxygenase or the ADP that may be released during platelet activation. Given the combined potentially stimulatory and inhibitory effects of pertussis holotoxin, we suggest caution in interpretation of results with this material.     

Human blood platelets possess specific binding sites for C1q

Although platelet interactions with C1q are implied by the inhibitory effect of C1q on collagen-induced platelet aggregation, specific receptors have not as yet been identified. To address the question of platelet receptors for free C1q, direct radioligand binding studies were performed by using human blood platelets and purified, 125I-labeled C1q, and a monoclonal antibody (II1/D1) (IgM, lambda) directed against C1q receptors on peripheral blood leukocytes. Washed platelets bound both purified 125I-labeled C1q and II1/D1 in a specific and saturable manner under physiologic ionic strength conditions. At equilibrium, approximately 4000 molecules of C1q bound per platelet with an apparent dissociation constant of 3.5 X 10(-7) M. Maximum C1q binding was achieved in 5 min and correlated well with inhibition of collagen-induced platelet aggregation. Equilibrium binding of 125I-labeled II1/D1 to washed platelets required an incubation period of 15 to 30 min and II1/D1 concentrations approaching 50 micrograms/ml. Approximately 2000 molecules of II1/D1 bound per platelet, with an apparent dissociation constant of 2.8 X 10(-8) M. II1/D1 binding could be inhibited by the collagenous tail of C1q (c-C1q), suggesting that platelet receptors for these ligands are either the same or in close proximity. The data demonstrate that human blood platelets possess specific and saturable binding sites for free C1q that may function as collagen receptors, and may antigenically resemble C1q receptors on peripheral blood leukocytes.     

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.     

The 650-kDa 12(S)-hydroxyeicosatetraenoic acid binding complex: occurrence in human platelets, identification of hsp90 as a constituent, and binding properties of its 50-kDa subunit.

A cytosolic 650-kDa complex which binds 12(S)-hydroxy-5,8,10, 14-eicosatetraenoic acid (12(S)-HETE) with high affinity and specificity has been found in various cell lines but not until now in platelet cytosol. After incubation of human platelets with 12(S)-[3H]HETE, a labeled cytosolic 650-kDa complex was isolated. As previously shown for the binding complex in Lewis lung carcinoma (LLC) cells, ATP treatment transformed the platelet complex into a 50-kDa ligand-binding subunit. These results are of interest for two reasons: (a) 12(S)-HETE is a major arachidonic acid metabolite in platelets, and (b) platelets contain large amounts of the cell adhesion molecule GpIIb/IIIa, the activation of which is regulated by 12(S)-HETE. Hsp90 was found to be a component of the 12(S)-HETE binding complex in Lewis lung carcinoma cells, and the 50-kDa ligand-binding subunit itself bound 12(S)-HETE with high affinity. Competition experiments showed that 12(R)-HETE, 15-deoxy-Delta12, 14-prostaglandin J2, and 5(S)-HETE had lower affinity for the 50-kDa subunit than 12(S)-HETE. The 12(S)-HETE binding protein appears to be distinct from known members of the steroid hormone receptor superfamily of nuclear receptors.     

Binding of fibrinogen molecules to pig platelets and their membranes

Following addition of ADP, 125I-labelled fibrinogen binds specifically to pig platelets. This binding is completely inhibited by the unlabelled fibrinogen. Quantitative analysis indicates the presence of 12,400-25,000 molecules of fibrinogen which can be bound with an association constant of 5 . 10(8) M-1 to platelets. Fibrinogen receptors were found to be active in the isolated platelet membranes as well. Quantitative analysis of the saturable binding of fibrinogen to the platelet membranes showed that these receptors react with the same affinity with fibrinogen molecules. In contrast to the intact platelets, the platelet membranes can specifically bind fibrinogen in the absence of ADP. We conclude that a specific receptor for fibrinogen is exposed on the surface as a result of cell damage which is the first step of the platelet membrane isolation.     

Influence of sodium on the alpha 2-adrenergic receptor system of human platelets. Role for intraplatelet sodium in receptor binding

The affinity of many types of membrane receptors for agonists is decreased by Na+ in radioligand binding experiments. We studied the alpha 2-adrenergic receptor of human platelets to determine whether Na+ acts at an intracellular or extracellular location. The Na+ content of intact platelets in an isotonic saline buffer was 38 nmol/10(8) platelets. This increased to 138 nmol/10(8) platelets with the Na+-selective ionophore monensin and decreased to 13 nmol/10(8) platelets with incubation in a Na+-free buffer. Epinephrine-induced platelet aggregation was increased by the addition of monensin and was decreased in the Na+-free buffer, while thrombin-induced aggregation was unaltered by either condition. Monensin, gramicidin, and ouabain (which all increased intraplatelet Na+) caused a 2-3-fold increase in the Kd of epinephrine (in competition with [3H]yohimbine) for alpha 2-adrenergic receptors on intact platelets. Conversely, incubation in a Na+-free buffer (which decreased intraplatelet Na+) decreased the Kd of the receptors for epinephrine 2-3-fold. These experiments suggest that changes in intracellular Na+ alter epinephrine binding. Control studies eliminated several alternative explanations for the effect of monensin on epinephrine binding: 1) monensin altered epinephrine binding only with intact platelets and not with platelet membranes; 2) although monensin depolarized platelets (assessed by [3H]methyltriphenylphosphonium uptake), other depolarizing conditions did not change epinephrine binding; 3) although monensin may increase intracellular pH (by exchanging Na+ for H+) such an increase in pH decreased the Kd of alpha 2-receptors on platelet membranes for epinephrine, an effect opposite to that produced by monensin in intact platelets. We conclude that alterations in the intracellular concentration of Na+ may change the affinity of platelet alpha 2-receptors for epinephrine. These results suggest a key role for intracellular Na+ in modulating binding at cell surface receptors in vivo.     

Lipid rafts orchestrate signaling by the platelet receptor glycoprotein VI

The platelet collagen receptor glycoprotein VI (GPVI) couples to the immune receptor adaptor Fc receptor gamma-chain (FcRgamma) and signals using many of the same intracellular signaling molecules as immune receptors. Studies of immune receptor signaling have revealed a critical role for specialized areas of the cell membrane known as lipid rafts, which are enriched in essential signaling molecules. However, the role of lipid rafts in signaling in nonimmune cells such as platelets remains poorly defined. This study shows that GPVI-FcRgamma does not constitutively associate with rafts, but is recruited to lipid rafts following receptor stimulation in both GPVI-expressing RBL-2H3 cells and human platelets. FcRgamma is required for GPVI association with lipid rafts, as mutant GPVI receptors that do not couple to FcRgamma were unable to associate with lipid rafts after receptor clustering. Following GPVI stimulation in platelets, virtually all phosphorylated FcRgamma was found in lipid rafts, but inhibition of FcRgamma phosphorylation did not block receptor association with lipid rafts. This work demonstrates that lipid rafts orchestrate GPVI receptor signaling in platelets in a manner analogous to immune cell receptors and supports a model of GPVI signaling in which FcRgamma phosphorylation is controlled by ligand-dependent association with lipid rafts.     

Characterization of a novel focal adhesion kinase inhibitor in human platelets

Focal adhesion kinase (FAK) is activated in human platelets downstream of integrins, e.g. α_IIbβ₃, and other adhesion receptors e.g. GPVI. Mice in which platelets lack FAK have been shown to exhibit extended bleeding times and their platelets have been shown to display decreased spreading on fibrinogen-coated surfaces. Recently, a novel FAK inhibitor (PF-573,228) has become available, its selectivity for FAK shown in vitro and in cell lines. We determined the effect of this inhibitor on platelet function and signaling pathways. Like murine platelets lacking FAK, we found that PF-573,228 was effective at blocking human platelet spreading on fibrinogen-coated surfaces but did not affect the initial adhesion. We also found a reduced spreading on CRP-coated surfaces. Further analysis of the morphology of platelets adhered to these surfaces showed the defect in spreading occurred at the transition from filopodia to lamellipodia. Similar to that seen with murine neutrophils lacking FAK, we also observed an unexpected defect in intracellular calcium release in human platelets pre-treated with PF-573,228 which correlated with impaired dense granule secretion and aggregation. The aggregation defect could be partially rescued by addition of ADP, normally secreted from dense granules, suggesting that PF-573,228 has effects on FAK downstream of α_IIbβ₃ and elsewhere. Our data show that PF-573,228 is a useful tool for analysis of FAK function in cells and reveal that in human platelets FAK may regulate a rise in cell calcium and platelet spreading.     

Hydrodynamic effects and receptor interactions of platelets and their aggregates in linear shear flow.

We have modeled platelet aggregation in a linear shear flow by accounting for two body collision hydrodynamics, platelet activation and receptor biology. Considering platelets and their aggregates as unequal-sized spheres with DLVO interactions (psi(platelet) = -15 mV, Hamaker constant = 10(-19) J), detailed hydrodynamics provided the flow field around the colliding platelets. Trajectory calculations were performed to obtain the far upstream cross-sectional area and the particle flux through this area provided the collision frequency. Only a fraction of platelets brought together by a shearing fluid flow were held together if successfully bound by fibrinogen cross-bridging GPIIb/IIIa receptors on the platelet surfaces. This fraction was calculated by modeling receptor-mediated aggregation using the formalism of Bell (Bell, G. I. 1979. A theoretical model for adhesion between cells mediated by multivalent ligands. Cell Biophys. 1:133-147) where the forward rate of bond formation dictated aggregation during collision and was estimated from the diffusional limited rate of lateral association of receptors multiplied by an effectiveness factor, eta, to give an apparent rate. For a value of eta = 0.0178, we calculated the overall efficiency (including both receptor binding and hydrodynamics effects) for equal-sized platelets with 50,000 receptors/platelet to be 0.206 for G = 41.9 s(-1), 0.05 for G = 335 s(-1), and 0.0086 for G = 1920 s(-1), values which are in agreement with efficiencies determined from initial platelet singlet consumption rates in flow through a tube. From our analysis, we predict that bond formation proceeds at a rate of approximately 0.1925 bonds/microm2 per ms, which is approximately 50-fold slower than the diffusion limited rate of association. This value of eta is also consistent with a colloidal stability of unactivated platelets at low shear rates. Fibrinogen was calculated to mediate aggregation quite efficiently at low shear rates but not at high shear rates. Although secondary collisions (an orbitlike trajectory) form only a small fraction of the total number of collisions, they become important at high shear rates (>750 s(-1)), as these are the only collisions that provide enough time to result in successful aggregate formation mediated by fibrinogen. The overall method provides a hydrodynamic and receptor correction of the Smoluchowski collision kernel and gives a first estimate of eta for the fibrinogen-GPIIb/IIIa cross-bridging of platelets. We also predict that secondary collisions extend the shear rate range at which fibrinogen can mediate successful aggregation.     

Redistribution of the fibrinogen receptor of human platelets after surface activation

We investigated the whole cell distribution of the platelet membrane receptor for fibrinogen in surface-activated human platelets. Fibrinogen-labeled colloidal gold was used in conjunction with platelet whole mount preparations to visualize directly the fibrinogen receptor. Unstimulated platelets fail to bind fibrinogen, and binding was minimal in the stages of activation immediately following adhesion. The amount of fibrinogen bound per platelet increased rapidly during the shape changes associated with surface activation until 7,600 +/- 500 labels were present at saturation. Maximal binding of fibrinogen was followed by receptor redistribution. During the early stages of spreading, fibrinogen labels were uniformly distributed over the entire platelet surface, including pseudopodia, but the labels become progressively centralized as the spreading process continued. In well spread platelets, labels were found over the central regions, whereas peripheral areas were cleared of receptors. Receptor redistribution during spreading was accompanied by cytoskeletal reorganization such that a direct correlation was seen between the development of specific ultrastructural zones and the distribution of surface receptor sites suggesting a link between the surface receptors and the cytoskeleton. The association of fibrinogen receptors with contractile elements of the cytoskeleton, which permits coordinated receptor centralization, is important to the understanding of the role of fibrinogen in normal platelet aggregation and clot retraction.     

The clearance mechanism of chilled blood platelets

Platelet transfusion is a very common lifesaving medical procedure. Not widely known is the fact that platelets, unlike other blood cells, rapidly leave the circulation if refrigerated prior to transfusion. This peculiarity requires blood services to store platelets at room temperature, limiting platelet supplies for clinical needs. Here, we describe the mechanism of this clearance system, a longstanding mystery. Chilling platelets clusters their von Willebrand (vWf) receptors, eliciting recognition of mouse and human platelets by hepatic macrophage complement type 3 (CR3) receptors. CR3-expressing but not CR3-deficient mice exposed to cold rapidly decrease platelet counts. Cooling primes platelets for activation. We propose that platelets are thermosensors, primed at peripheral sites where most injuries occurred throughout evolution. Clearance prevents pathologic thrombosis by primed platelets. Chilled platelets bind vWf and function normally in vitro and ex vivo after transfusion into CR3-deficient mice. Therefore, GPIb modification might permit cold platelet storage.     

Mechanisms of interaction of a plasmalogenic analog of platelet-activating factor with human platelets.

The interaction of a plasmalogenic analog of platelet-activating factor (1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phosphocholine; 1-alkenyl-PAF) with human platelets was studied. 1-Alkenyl-PAF induced an increase in intracellular Ca2+ concentration and inhibition of adenylate cyclase at significantly higher concentrations than PAF. 1-Alkenyl-PAF inhibits PAF-induced platelet aggregation but has no effect on ADP- or thrombin-induced aggregation of human platelets. In contrast to PAF, 1-alkenyl-PAF increases [3H]PGE1 binding with human platelets. The properties of 1-alkenyl-PAF as an agonist or antagonist of PAF receptors apparently depend on its concentration in the cell medium. Under physiological conditions 1-alkenyl-PAF might be a natural PAF antagonist acting in the human cardiovascular system.     

Redox regulation of morphology,cell stiffness,and lectin-induced aggregation of human platelets

Redox regulation and carbohydrate recognition are potent molecular mechanisms which can contribute to platelet aggregation in response to various stimuli. The purpose of this study is to investigate the relationship between these mechanisms and to examine whether cell surface glycocalyx and cell stiffness of human platelets are sensitive to the redox potential formed by glutathione. To this end, human platelets were treated with different concentrations (0.05 μM to 6 mM) and ratios of reduced or oxidized glutathione (GSH or GSSG), and platelet morphological, mechanical, and functional properties were determined using conventional light microscopy, atomic force microscopy, and lectin-induced cell aggregation analysis. It was found that lowering the glutathione redox potential changed platelet morphology and increased platelet stiffness as well as modulated nonuniformly platelet aggregation in response to plant lectins with different carbohydrate-binding specificity including wheat germ agglutinin, Sambucus nigra agglutinin, and Canavalia ensiformis agglutinin. Extracellular redox potential and redox buffering capacity of the GSSG/2GSH couple were shown to control the availability of specific lectin-binding glycoligands on the cell surface, while the intracellular glutathione redox state affected the general functional ability of platelets to be aggregated independently of the type of lectins. Our data provide the first experimental evidence that glutathione as a redox molecule can affect the mechanical stiffness of human platelets and induce changes of the cell surface glycocalyx, which may represent a new mechanism of redox regulation of intercellular contacts.     

Platelet C1q receptor interactions with collagen- and C1q-coated surfaces

We recently described specific binding sites for C1q on human blood platelets. Structural similarities between the amino-terminal of C1q and collagen have suggested that receptors for both molecules on platelets might be the same. The present study thus compared the interaction of purified C1q receptors (C1qR) and whole platelets with collagen- and C1q-coated polystyrene surfaces. Surfaces coated with BSA or gelatin served as controls. Purified 125I-labeled C1qR recognized both C1q- and collagen-coated surfaces in a divalent, cation-independent manner. This adhesion was inhibited by polyclonal or monoclonal (II1/D1) anti-C1qR antibodies. Although C1qR adhered preferentially to C1q-coated surfaces, adhesion to bovine and human type I collagen, as well as to human type III and V collagen, was also noted. In parallel studies, 51Cr-labeled platelets bound equally well to collagen- or C1q-coated surfaces, albeit in a magnesium-dependent manner. Partial inhibition of platelet adhesion was observed in the presence of RGDS, despite the inability of RGDS to modify C1qR interaction with C1q or collagen. Moreover, anti C1qR antibodies selectively inhibited platelet adhesion to C1q-coated surfaces, whereas antibodies specific for the GPIa/IIa collagen receptor (6F1) preferentially inhibited platelet collagen interactions. These data support the presence of distinct platelet membrane C1qR, which may cross-react with collagen, and suggest that C1qR are necessary but not sufficient for platelet adhesion to C1q-coated surfaces. Additional divalent cation and/or RGD-sensitive binding sites may participate.     

GITR ligand provided by thrombopoietic cells inhibits NK cell antitumor activity

Thrombocytopenia inhibits tumor growth and especially metastasis in mice, whereas additional depletion of NK cells reverts this antimetastatic phenotype. It has therefore been speculated that platelets may protect hematogenously disseminating tumor cells from NK-dependent antitumor immunity. Tumor cells do not travel through the blood alone, but are rapidly coated by platelets, and this phenomenon has been proposed to shield disseminating tumor cells from NK-mediated lysis. However, the underlying mechanisms remain largely unclear. In this study, we show that megakaryocytes acquire expression of the TNF family member glucocorticoid-induced TNF-related ligand (GITRL) during differentiation, resulting in GITRL expression by platelets. Upon platelet activation, GITRL is upregulated on the platelet surface in parallel with the α-granular activation marker P-selectin. GITRL is also rapidly mobilized to the platelet surface following interaction with tumor cells, which results in platelet coating. Whereas GITRL, in the fashion of several other TNF family members, is capable of transducing reverse signals, no influence on platelet activation and function was observed upon GITRL triggering. However, platelet coating of tumor cells inhibited NK cell cytotoxicity and IFN-γ production that could partially be restored by blocking GITR on NK cells, thus indicating that platelet-derived GITRL mediates NK-inhibitory forward signaling via GITR. These data identify conferment of GITRL pseudoexpression to tumor cells by platelets as a mechanism by which platelets may alter tumor cell immunogenicity. Our data thus provide further evidence for the involvement of platelets in facilitating evasion of tumor cells from NK cell immune surveillance.     

Thrombin interaction with platelets. Influence of a platelet protease nexin

A fraction of the 125I-thrombin that binds to human platelets is taken into a sodium dodecyl sulfate-resistant 77 kDa complex with a platelet factor (Bennett, W. F., and Glenn, K. C. (1980) Cell 22, 621-627). Here we show that this platelet factor is in several respects similar to protease nexin I (PNI), a fibroblast thrombin inhibitor. The complexes are of the appropriate size, bind to Sepharose that has been derivatized with anti-PNI antibody, do not form when the thrombin active site has been blocked with diisopropylphosphofluoridate, and do not appear on platelets when heparin is present. However, the platelet factor does not bind urokinase, indicating that this "platelet PN" may be distinct from PNI. Following brief incubation with 125I-thrombin, platelet PN X 125I X thrombin complexes are found both associated with the platelets and free in the binding medium. 125I-Thrombin has a higher affinity for platelet PN than for platelet receptors. In 30-s binding incubations carried out with thrombin at concentrations below 0.3 nM, formation of the 77-kDa complex accounts for most of the platelet specific binding of 125I-thrombin. Subtracting this large contribution to 125I-thrombin-specific binding reveals that the reversible binding of 125I-thrombin to platelet receptors exhibits sigmoidal thrombin dose-dependence. Thrombin stimulation of platelet [14C]serotonin release exhibits similar thrombin dose dependence. These results indicate that platelets may possess a mechanism for suppressing their interaction with active thrombin at thrombin doses below 0.3 nM. It is possible that platelet PN carries out this function by capturing thrombin before thrombin binds to its signal-transmitting receptors.     

Platelet and vascular smooth muscle thromboxane A2/prostaglandin H2 receptors

Thromboxane A2 (TxA2) and prostaglandin H2 (PGH2) aggregate platelets and contract vascular smooth muscle. Inasmuch as both compounds produce the same effects and presumably through the same receptor, their receptors have been referred to as TxA2/PGH2 receptors. Pharmacological studies of stable agonists and antagonists of the TxA2/PGH2 receptors have shown different rank order potencies for these compounds in platelets compared with blood vessels. These studies have provided evidence to support the hypothesis that the platelet TxA2/PGH2 receptor is different from the one found in vascular tissue. The vascular receptor has been named [TxA2/PGH2]tau and the platelet receptor has been named [TxA2/PGH2]alpha. In the past few years several radiolabeled antagonists and agonists have been developed and used in radioligand-binding studies, primarily in platelets. One of these ligands, 125I-labeled PTA-OH, a TxA2/PGH2 receptor antagonist, has been extensively used to characterize the human platelet TxA2/PGH2-binding site. It has been found to have a Kd of approximately 20 nM and a Bmax of 2500 binding sites/platelet. Through the combination of pharmacological and biochemical approaches, it should be possible to characterize platelet and vascular TxA2/PGH2 receptors.     

Identification of lysophospholipid receptors in human platelets: the relation of two agonists, lysophosphatidic acid and sphingosine 1-phosphate

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (Sph-1-P) are known as structurally related bio-active lipids activating platelets through their respective receptors. Although the receptors for LPA and Sph-1-P have been recently identified in various cells, the identification and characterization of ones in platelets have been reported only preliminarily. In this report, we first investigated the distinct modes of LPA and Sph-1-P actions in platelet activation and found that LPA functioned as a much stronger agonist than Sph-1-P, and high concentrations of Sph-1-P specifically desensitized LPA-induced intracellular Ca(2+) mobilization. In order to identify the responsible receptors underlying these observations, we analyzed the LPA and Sph-1-P receptors which might be expressed in human platelets, by RT-PCR. We found for the first time that Edg2, 4, 6 and 7 mRNA are expressed in human platelets.     

Affinity isolation and characterization of immunoglobulin G Fc fragment-binding glycoprotein from human blood platelets.

Immune complexes bind to several eukaryotic cell types including human blood platelets through the Fc fragment of immunoglobulin (Ig) G. Utilizing immobilized Fc fragment of IgG enabled us to isolate from human blood platelets a glycoprotein of an apparent Mr = 255,000 which, upon reduction, dissociated into sub-units of an apparent Mr = 50,000. This Fc fragment-binding glycoprotein has an isoelectric point between pH 6.3 and 6.9 and is composed of 34% hydrophobic, 25% acidic, and 14% basic amino acids. The Fc fragment-binding glycoprotein was also isolated from human platelet membrane preparations and was unaffected by prior treatment of platelets with thrombin. Isolated Fc fragment-binding glycoprotein formed an in vitro complex with aggregated immunoglobulin G. These results suggest that the isolated Fc fragment-binding component may prove useful in studies concerning the functional role of glycoproteins as cellular receptors for the Fc fragment of IgG.