The glycoprotein Ib complex of human blood platelets

Human glycoprotein Ib (GPIb) is a major integral membrane protein on human blood platelets responsible for the initial attachment of platelets to the exposed vascular subendothelium. In this report we describe an isolation method for a 'GPIb complex' as well as for its individual components. A three-step procedure involving Triton X-114 phase-partition, affinity chromatography on wheat germ agglutinin and ion-exchange chromatography on DEAE-Sephacel yielded milligram quantities of purified GPIb complex. The single components of the complex were further purified by gel filtration on AcA34 in 0.1% sodium dodecyl sulfate. As well as GPIb, the complex contains GP17, actin-binding protein, actin and a series of unidentified proteins with different molecular masses. In contrast to GPIb alpha, which is very rich in O-linked oligosaccharides, sugar analysis revealed that GPIb beta and GP17 seem to have only N-linked chains of the lactosamine type. The C-terminal alpha-chain remnant, which probably spans the plasma membrane, was identified and isolated for the first time. Western blotting with polyclonal rabbit anti-GPIb antibodies and silver-staining of one- or two-dimensional dodecyl sulfate/polyacrylamide gels revealed that it has an apparent molecular mass of 20 kDa and is linked to GPIb beta by a disulfide bridge close to the membrane. The thrombin-binding site on GPIb is located near the N-terminus on a 40-kDa fragment of GPIb alpha. A disulfide bridge in the N-terminal region is not essential for thrombin binding to GPIb.     

Platelet glycoprotein Ib alpha binds to thrombin anion-binding exosite II inducing allosteric changes in the activity of thrombin

The glycoprotein (GP) Ib-IX complex is a platelet surface receptor that binds thrombin as one of its ligands, although the biological significance of thrombin interaction remains unclear. In this study we have used several approaches to investigate the GPIb alpha-thrombin interaction in more detail and to study its effect on the thrombin-induced elaboration of fibrin. We found that both glycocalicin and the amino-terminal fragment of GPIb alpha reduced the release of fibrinopeptide A from fibrinogen by about 50% by a noncompetitive allosteric mechanism. Similarly, GPIb alpha caused in thrombin an allosteric reduction in the rate of turnover of the small peptide substrate d-Phe-Pro-Arg-pNA. The K(d) for the glycocalicin-thrombin interaction was 1 microm at physiological ionic strength but was highly salt-dependent, decreasing to 0.19 microm at 100 mm NaCl (Gamma(salt) = -4.2). The salt dependence was characteristic of other thrombin ligands that bind to exosite II of this enzyme, and we confirmed this as the GPIb alpha-binding site on thrombin by using thrombin mutants and by competition binding studies. R68E or R70E mutations in exosite I of thrombin had little effect on its interaction with GPIb alpha. Both the allosteric inhibition of fibrinogen turnover caused by GPIb alpha binding to these mutants, and the K(d) values for their interactions with GPIb alpha were similar to those of wild-type thrombin. In contrast, R89E and K248E mutations in exosite II of thrombin markedly increased the K(d) values for the interactions of these thrombin mutants with GPIb alpha by 10- and 25-fold, respectively. Finally, we demonstrated that low molecular weight heparin (which binds to thrombin exosite II) but not hirugen (residues 54-65 of hirudin, which binds to exosite I of thrombin) inhibited thrombin binding to GPIb alpha. These data demonstrate that GPIb alpha binds to thrombin exosite II and in so doing causes a conformational change in the active site of thrombin by an allosteric mechanism that alters the accessibility of both its natural substrate, fibrinogen, and the small peptidyl substrate d-Phe-Pro-Arg-pNA.     

On the association of glycoprotein Ib and actin-binding protein in human platelets

Glycoprotein (GP) Ib was purified from lysates of human platelets prepared in the presence or absence of inhibitors of the endogenous calcium-activated neutral protease (CANP) by immunoaffinity chromatography, employing the GPIb-specific murine monoclonal antibody, AP1, coupled to Sepharose CL4B. When derived from lysates prepared in the presence of EDTA or leupeptin, the eluate from the AP1-affinity column contained a 240,000-260,000-mol-wt protein in addition to GPIb. In SDS PAGE, this protein was stained by Coomassie Blue R, but not by the periodic acid-Schiff reagent, and it was not labeled with 125I in intact platelets by the lactoperoxidase-catalyzed method. When derived from lysates prepared in the absence of CANP inhibitors, the eluate contained only GPIb and its proteolytic derivative, glycocalicin. A change in the electrophoretic mobility of GPIb consistent with its association with the 240,000-260,000-mol-wt protein was confirmed by crossed immunoelectrophoresis. By an immunoblot technique involving transfer of proteins eluted from the AP1-affinity column and separated by SDS PAGE onto a nitrocellulose membrane, the 240,000-260,000-mol-wt protein bound polyclonal goat antibody raised against rabbit macrophage actin-binding protein (ABP). On the basis of these results, we conclude the GPIb is tightly associated with ABP under conditions in which the endogenous CANP is inhibited, and that this apparent transmembrane complex of GPIb-ABP can be isolated in lysates of nonactivated human platelets.     

Identification of glycoprotein Ib beta as one of the major proteins phosphorylated during exposure of intact platelets to agents that activate cyclic AMP-dependent protein kinase

Platelet function is inhibited by prostaglandin E1, prostaglandin I2, or forskolin, agents that increase the intracellular concentration of cyclic AMP. The inhibition appears to result from cyclic AMP-stimulated phosphorylation of specific intracellular proteins. One of the major increases in phosphorylation occurs in a polypeptide of Mr = 24,000 (P24). In this study, an effort was made to identify P24. Platelets prelabeled with [32P]phosphate were incubated with prostaglandin E1, prostaglandin I2, or forskolin. Proteins that became phosphorylated were detected by autoradiography of sodium dodecyl sulfate-polyacrylamide gels. Several lines of evidence indicated that P24 was the beta-subunit of the plasma membrane glycoprotein (GP) Ib, a glycoprotein that is essential for the adhesion of platelets to damaged subendothelium, for the rapid response of platelets to thrombin, and for the attachment of the membrane skeleton to the cytoplasmic face of the plasma membrane. P24 co-migrated with GP Ib beta on reduced gels (Mr = 24,000) and also on nonreduced gels (when GP Ib beta is disulfide-linked to GP Ib alpha and migrates with Mr = 170,000). Like GP Ib beta, P24 was associated with actin filaments in Triton X-100 lysates. Like GP Ib beta, it was selectively associated with filaments of the membrane skeleton and was released from filaments when the Ca2+-dependent protease was active. Antibodies against GP Ib immunoprecipitated P24 from platelet lysates. Finally, exposure of Bernard-Soulier platelets (which lack GP Ib) to prostaglandin E1 resulted in phosphorylation of other polypeptides, but not of P24. These studies show that P24, one of the major polypeptides phosphorylated when platelets are exposed to agents that inhibit platelet function by increasing the concentration of cyclic AMP, is the beta-subunit of GP Ib.     

The glycocalicin portion of platelet glycoprotein Ib expresses both high and moderate affinity receptor sites for thrombin. A soluble radioreceptor assay for the interaction of thrombin with platelets

A soluble radioreceptor assay has been developed to characterize thrombin receptor activities of the human platelet membrane. 125I-Thrombin was added to platelet membranes solubilized in 1% Triton X-100, and thrombin bound to platelet receptors was separated from free thrombin by precipitation with wheat germ agglutinin (WGA) in the presence of alpha 1-acid glycoprotein as carrier. Both high affinity binding (Ki, 0.09 nM; R1, 0.30 pmol/mg protein) and moderate affinity binding (K2, 38 nM; R2, 72 pmol/mg protein) were detected in the detergent-solubilized membrane preparations and these binding parameters were in excellent agreement with values previously determined using intact platelets (Harmon, J. T., and Jamieson, G. A. (1985) Biochemistry 24, 58-64). Using the soluble radioreceptor assay, both high and moderate affinity binding was detected in highly purified preparations of glycoprotein Ib (GPIb) and glycocalicin, and the binding isotherms were identical with those of the crude detergent-solubilized membrane preparation. Treatment of detergent-solubilized membranes with increasing concentrations of a monospecific polyclonal antibody to glycocalicin resulted in the stepwise depletion of GPIb and concomitant reductions of thrombin binding activity. These results demonstrate that both high and moderate affinity binding of thrombin to platelets is completely expressed in the glycocalicin portion of GPIb.     

Structural and functional mapping of the thrombin domain involved in the binding to the platelet glycoprotein Ib.

The activation of human platelets by alpha-thrombin is mediated in part by cleavage of the protease-activated receptor (PAR) 1 and 4 and by the glycoprotein (Ib)alpha, (Gp(Ib)alpha), which binds with high affinity to alpha-thrombin. Recent studies have shown that the thrombin domain referred to as heparin binding site (HBS) is involved in the interaction with the platelet Gp(Ib)alpha. The HBS is rich in basic amino acids. To identify the key amino acid residues involved in the binding to Gp(Ib)alpha, we have performed alanine scanning mutagenesis of the basic HBS R93, R97, R101, R233, K236, K240, R233/K236/Q239, as well as of the neutral Q239 residues, located in different regions of the domain. For comparison, mutation at R67 within the fibrinogen recognition site (FRS) of thrombin was performed as well. Solid-phase binding experiments showed that the Kd of thrombin-GpIb interaction was reduced 22-fold for R93A, 8-fold for R97A, 13-fold for R101A, 29-fold for R233A, 21-fold for K236A, 5-fold for K240A, and 31-fold for the triple mutant R233A/K236A/Q239A, while the Q239A and R67A forms did not show any significant affinity change. The platelet activating capacity of these mutants was evaluated as well. Using gel-filtered platelets, the EC50 value of thrombin-induced aggregation was from 5- to 13-fold higher in the HBS mutants than in the WT form, and was linearly and positively correlated with the corresponding Kd values pertaining to thrombin binding to GpIb. Measurements of PAR-1 hydrolysis on the platelet membrane showed that the HBS mutants R233A, R101A, R93A, K236A, and R233/K236/Q239 forms had a reduction of the apparent kcat/Km value. These results are a consequence of a defective binding to GpIb, which is known to optimize the interaction with PAR-1 in situ. A confirm of this hypothesis came from the demonstration that the kcat/Km value pertaining to the hydrolysis by the HBS-mutated thrombins of the synthetic PAR-1 38-60 peptide in solution was similar to that one obtained with the WT form. In conclusion, these experiments provide a structural and functional mapping of the thrombin HBS subregions involved in the binding to the platelet Gp(Ib)alpha and in the cell activation.     

Cyclic AMP-dependent phosphorylation of glycoprotein Ib inhibits collagen-induced polymerization of actin in platelets

Platelet function is inhibited by agents such as prostaglandin E1 (PGE1) that elevate the cytoplasmic concentration of cyclic AMP. Inhibition presumably results from the cyclic AMP-stimulated phosphorylation of intracellular proteins. Polypeptides that become phosphorylated are actin-binding protein, P51 (Mr = 51,000), P36 (Mr = 36,000), P24 (Mr = 24,000), and P22 (Mr = 22,000). Recently, we identified P24 as the beta-chain of glycoprotein (GP) Ib, a component of the plasma membrane GP Ib.IX complex. The existence of Bernard-Soulier syndrome, a hereditary disorder in which platelets selectively lack the GP Ib.IX complex, enabled us to examine whether the phosphorylation of GP Ib beta (P24) is responsible for any of the inhibitory effects of elevated cyclic AMP on platelet function. Exposure of control platelets to PGE1 increased phosphorylation of actin-binding protein, P51, P36, GP Ib beta, and P22. Prostaglandin E1 induced the same phosphorylation reactions in Bernard-Soulier platelets, except that of GP Ib beta, which is absent. In control platelets, PGE1 inhibited collagen-induced phosphorylation of myosin light chain, phosphorylation of P47 (an unidentified Mr 47,000 cytoplasmic protein that is phosphorylated by protein kinase C in stimulated platelets), aggregation, and the secretion of granule contents. Despite the absence of GP Ib beta, PGE1 also inhibited these collagen-induced responses in Bernard-Soulier platelets. However, while PGE1 inhibited collagen-induced polymerization of actin in control platelets, it did not inhibit actin polymerization in Bernard-Soulier platelets. These results suggest that cyclic AMP-induced phosphorylation of GP Ib inhibits collagen-induced actin polymerization in platelets. Because actin polymerization is required for at least some of the functional responses of platelets to an agonist, phosphorylation of Gp Ib beta may be one way in which cyclic AMP inhibits platelet function.     

Glycoprotein Ib beta is the only phosphorylated major membrane glycoprotein in human platelets.

Platelets were metabolically labelled with 32P and the phosphoproteins examined by two-dimensional non-reduced/reduced gel electrophoresis and isoelectric-focusing/gel electrophoresis. Comparison with similar separations of surface-labelled platelets showed that the only major glycoprotein which is phosphorylated is the beta-subunit of glycoprotein Ib, indicating that this subunit contains a cytoplasmic segment. The identification was confirmed using immunoblotting with an antibody to the beta-subunit. Phosphoserine was the principal phosphorylation site, with some phosphothreonine, but phosphotyrosine was absent. No quantitative or qualitative differences could be detected in the phosphorylation of glycoprotein Ib beta from resting or activated platelets. These results exclude changes in phosphorylation of the major platelet membrane glycoproteins as a method of signal transmission by these receptors.     

Binding kinetics of PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to intact human platelets.

The binding of [3H]PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to intact human gel-filtered platelets was measured at 22 degrees C. Specific binding reached saturation within 15 min at high doses of [3H]PAF-acether (0.5-0.9 nM), whereas about 90 min were required when low doses (0.02-0.5 nM) were used. Above 1 nM, [3H]PAF-acether non-specific binding increased progressively, which together with the demonstration of a 3H-labelled metabolite suggested uptake and metabolism of [3H]PAF-acether. Equilibrium analysis revealed one class of specific receptors with a Ka of 18.86 +/- 4.82 X 10(9) M-1 and 242 +/- 64 binding sites per platelet. Non-equilibrium binding revealed a similar Ka (16.87 X 10(9) M-1). Specific binding became irreversible after prolonged incubation, a process that was enhanced at increasing concentrations of [3H]PAF-acether. Platelets made desensitized to PAF-acether by prior incubation with unlabelled PAF-acether failed to bind a second dose of PAF-acether (3H-labelled), suggesting that desensitization resulted from loss of available binding sites. Under the conditions of the binding studies, PAF-acether induced exposure of the fibrinogen receptor, aggregation (in a stirred suspension) and alterations in (poly)-phosphatidylinositides. These results suggest that PAF-acether initiates platelet responses via receptor-mediated processes.     

Binding of Par-4 to the actin cytoskeleton is essential for Par-4/Dlk-mediated apoptosis

Prostate apoptosis response-4 (Par-4) is a 38-kDa protein originally identified as a gene product upregulated in prostate cancer cells undergoing apoptosis. Cell death mediated by Par-4 and its interaction partner DAP like kinase (Dlk) is characterized by dramatic changes of the cytoskeleton. To uncover the role of the cytoskeleton in Par-4/Dlk-mediated apoptosis, we analyzed Par-4 for a direct association with cytoskeletal structures. Confocal fluorescence microscopy revealed that endogenous Par-4 is specifically associated with stress fibers in rat fibroblasts. In vitro cosedimentation analyses and in vivo FRET analyses showed that Par-4 directly binds to F-actin. Actin binding is mediated by the N-terminal 266 amino acids, but does not require the C-terminal region of Par-4 containing the leucine zipper and the death domain. Furthermore, the interaction of Par-4 with actin filaments leads to the formation of actin bundles in vitro and in vivo. In rat fibroblasts, this microfilament association is essential for the pro-apoptotic function of Par-4, since both disruption of the actin cytoskeleton by cytochalasin D treatment and overexpression of Par-4 constructs impaired in actin binding result in a significant decrease of apoptosis induction by Par-4 and Dlk. We propose a model, in which Par-4 recruits Dlk to stress fibers, leading to enhanced phosphorylation of the regulatory light chain of myosin II (MLC) and to the induction of apoptosis.     

Localization of the domain of actin-binding protein that binds to membrane glycoprotein Ib and actin in human platelets

The Mr approximately 540,000 dimeric actin gelation protein, actin-binding protein (ABP), has previously been shown in human platelets to link actin to membrane glycoprotein Ib (GPIb) (Fox, J. E. B. (1985) J. Biol. Chem. 260, 11970-11977; Okita, J. R., Pidard, D., Newman, P. J., Montgomery, R. R., and Kunicki, T. J. (1985) J. Cell Biol. 100, 317-321). We have examined further the interaction between ABP and GPIb. Platelet extracts were depleted of ABP by precipitation with anti-ABP monoclonal antibodies (mAbs); in resulting precipitates, ABP monomer is complexed with GPIb in a 5:1 molar ratio. The ABP.GPIb complex is resistant to chaotropic solvents but dissociated by the ionic detergent, sodium dodecyl sulfate. Treatment of intact platelets with the ionophore A23187 activates a Ca2+-dependent protease which cleaves the Mr approximately 270,000 ABP subunit into three fragments of Mr 190,000, 100,000, and 90,000; the latter fragment is derived from the Mr 100,000 fragment. Anti-ABP mAbs coprecipitated GPIb with the Mr 100,000 and 90,000 fragments, but not with the Mr 190,000 fragment which contains the ABP self-association site. In the reciprocal experiment, anti-GPIb antibodies co-precipitated only the Mr 100,000 and 90,000 ABP fragments. Actin also co-precipitated with the Mr 100,000 and 90,000, but not with the Mr 190,000 ABP fragment. The anti-ABP mAb that precipitated the Mr 100,000-90,000 GPIb-binding ABP fragment recognizes a trypsin cleavage fragment of ABP that binds actin filaments in vitro. These findings establish that both the GPIb-binding site and actin-binding sites are in the same region of the ABP monomer. Because of the extended bipolar conformation of the ABP molecule, the data suggest that the GPIb.actin-binding region is located remote from the self-association, or dimerization, site of the ABP subunit.     

Addition of both platelets and thrombin in combination accelerates tumor cells to adhere to endothelial cells In vitro

Summary Platelets and coagulation are involved in the pathogenesis of blood-borne metastases. The aim of this study is to obtain more information about the mechanisms involved in the initial adhesion of tumor cells to endothelial cells. In short term experiments with tumor cells, suspended in the medium of cultured endothelial cells, we tested whether addition of both platelets and thrombin cause more tumor cell adhesion to endothelial cells, than when either platelets or thrombin are acting alone. HeLa cells or HT29 cells, prelabeled with radioactive ⁵¹Cr, human platelets, and thrombin were added to human endothelial cell cultures. Following 15 min of shaking at 37° C, the percentage of tumor cell adhesion was calculated. The percentages of adhering tumor cells with the presence of both platelets and thrombin were greatly increased compared to controls. Addition of hirudin 2 min before thrombin lowered the adhesion percentage of tumor cells. Hirudin added immediately before and 2 min after thrombin gave only minor effects. When the endothelium was treated with superoxide dismutase, catalase, and mannitol, the adhesion of tumor cells was lowered with catalase and superoxide dismutase. The cause of tumor cell-endothelial cell interaction is probably complex. Our results show that activated platelets enhance the tumor cell adhesion, and that generation of active oxygen species may be important in the initial phase of the interaction.     

The occupancy of glycoprotein IIb-IIIa complex modulates thrombin activation of human platelets

Platelet membrane glycoprotein (GP IIb-IIIa), besides its activity as adhesive protein receptor, displays a number of properties supporting its involvement in the mechanisms of transduction of the activation signal. Recently we have observed that GP IIb-IIIa ligands, mostly fibrinogen, inhibit Ca2+ movement and cytoskeleton reorganization caused by mild platelet activation. These findings led us to investigate the effect of GP IIb-IIIa ligands on agonist-induced platelet responses, with particular attention to the two major messenger generating systems, involving the activation of phospholipase C and the inhibition of cAMP production. In this paper we demonstrate that the occupancy of the major adhesive protein receptor on the platelet surface modulates the phosphatidylinositol cycle decreasing the amount of IP3, IP2 and IP produced after mild platelet activation as well as the pattern of protein phosphorylation. The platelet cAMP content of activated platelets was also affected and kept higher when evaluated under the same experimental conditions. Our data provide evidence for a role of fibrinogen binding in regulating the degree of activation of circulating platelets.     

Thrombin activation of factor XI on activated platelets requires the interaction of factor XI and platelet glycoprotein Ib alpha with thrombin anion-binding exosites I and II,respectively

Activation of factor XI (FXI) by thrombin on stimulated platelets plays a physiological role in hemostasis, providing additional thrombin generation required in cases of severe hemostatic challenge. Using a collection of 53 thrombin mutants, we identified 16 mutants with <50% of the wild-type thrombin FXI-activating activity in the presence of dextran sulfate. These mutants mapped to anion-binding exosite (ABE) I, ABE-II, the Na+-binding site, and the 50-insertion loop. Only the ABE-II mutants showed reduced binding to dextran sulfate-linked agarose. Selected thrombin mutants in ABE-I (R68A, R70A, and R73A), ABE-II (R98A, R245A, and K248A), the 50-insertion loop (W50A), and the Na+-binding site (E229A and R233A) with <10% of the wild-type activity also showed a markedly reduced ability to activate FXI in the presence of stimulated platelets. The ABE-I, 50-insertion loop, and Na+-binding site mutants had impaired binding to FXI, but normal binding to glycocalicin, the soluble form of glycoprotein Ibalpha (GPIb alpha). In contrast, the ABE-II mutants were defective in binding to glycocalicin, but displayed normal binding to FXI. Our data support a quaternary complex model of thrombin activation of FXI on stimulated platelets. Thrombin bound to one GPIb alpha molecule, via ABE-II on its posterior surface, is properly oriented for its activation of FXI bound to a neighboring GPI alpha molecule, via ABE-I on its anterior surface. GPIb alpha plays a critical role in the co-localization of thrombin and FXI and the resultant efficient activation of FXI.     

Binding of thrombin to thrombomodulin accelerates inhibition of the enzyme by antithrombin III. Evidence for a heparin-independent mechanism

The endothelial cell surface provides a receptor for thrombin-designated thrombomodulin (TM) which regulates thrombin formation and the activity of the enzyme at the vessel wall surface by serving as a potent cofactor for the activation of protein C by thrombin. Heparin-like structures of the vessel wall have been proposed as another regulatory mechanism catalyzing the inhibition of thrombin by antithrombin III. In the present study, the interaction of antithrombin III with the thrombin-TM complex and its interference with heparin and polycations were investigated by using human components and TM isolated from the microvasculature of rabbit lung. Purified TM bound thrombin and acted as a cofactor for protein C activation. The addition of heparin (0.5 unit/mL) to the reaction mixture interfered neither with the binding of thrombin to TM nor with the activation of protein C. However, the polycations protamine (1 unit/mL) as well as polybrene (0.1 mg/mL) affected the thrombin-TM interaction. This was documented by an increase in the Michaelis constant from 8.3 microM for thrombin alone to 19.5 microM for thrombin-TM with the chromogenic substrate compound S-2238 in the presence of 1 unit/mL protamine. When the inhibition of thrombin by antithrombin III was determined, the second-order rate constant k2 = 8.4 X 10(3) M-1 s-1 increased about 8-fold in the presence of TM, implying an accelerative function of TM in this reaction. Although purified TM did not bind to antithrombin III-Sepharose, suggesting the absence of heparin-like structures within the receptor molecule, protamine reversed the accelerative effect of TM in the inhibition reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thrombomodulin blocks the ability of thrombin to activate platelets

When thrombin is complexed to the endothelial cell surface receptor thrombomodulin, it loses its procoagulant activities in that it no longer clots fibrinogen or activates factor V. Studies were initiated to determine if complex formation also blocks thrombin's other major procoagulant function, the activation of platelets. When bound to thrombomodulin, thrombin no longer induces platelets to either aggregate or release [14C] serotonin. Binding studies using 125I-labeled thrombin or diisopropyl phosphorothrombin indicate that the complex does not bind to the platelet. When thrombomodulin is added after thrombin has bound to the platelets, the thrombin rapidly redistributes onto the thrombomodulin. These data suggest that in addition to its other anticoagulant effects, thrombomodulin may also act to inhibit and/or reverse platelet activation by thrombin.     

Binding of cytochalasin B to platelets

The binding of cytochalasin B (CB) to human platelets and to isolated platelet cytosol and membranes has been analyzed with [3H]CB. High- and low affinity classes of saturable binding sites were associated with intact platelets. Binding at very low concentrations of CB (i.e., high-affinity binding) was partially prevented by 100 mM D-galactose or D-glucose and to a much lesser extent by L-glucose. Binding to platelet cytosol also involved two classes of sites with affinities and capacities similar to those observed with the whole cells. None of this binding, however, was affected by 100 mM D-galactose. Saturable binding to platelet membranes occurred at sites with a uniform binding affinity. Approximately 52% of this binding was prevented by 1 M D-galactose and another 15% by cytochalasin E (CE). We hypothesize that binding in the cytosol is to monomeric (low-affinity) and polymerized (high-affinity) actin, whereas membrane binding (high-affinity only) occurs primarily at sites involved with galactose transport.     

Binding of fibronectin to alpha-granule-deficient platelets

Most of the proposed functions for fibronectin involve its interaction with cells, yet the molecular nature of cellular fibronectin binding site(s) has remained obscure. Thrombin induces saturable platelet binding sites for plasma fibronectin and concurrently stimulates surface expression of a number of platelet alpha-granule constituents including thrombospondin and fibrin which are known to interact with fibronectin. To test the hypothesis that these (or other alpha-granule proteins) mediate plasma fibronectin binding, we used platelets of patients with the Gray Platelet Syndrome. These cells were deficient in thrombospondin, beta-thromboglobulin, platelet factor 4, fibronectin, and fibrinogen as measured in radioimmunoassay. They also had reduced von Willebrand factor content as judged by immunofluorescence. At plasma fibronectin inputs from 0.03 to 3 times the apparent kilodalton, these Gray platelets bound virtually identical quantities of fibronectin as normal cells. Thus, platelets containing 1,500 molecules of thrombospondin per platelet could bind more than 100,000 molecules of plasma fibronectin per cell following thrombin stimulation. These data preclude any simple model in which newly surface expressed thrombospondin (or other alpha-granule protein) functions as the major thrombin-stimulated plasma fibronectin receptor in this cell type.     

Differential hydrolysis of phospholipid molecular species during activation of human platelets with thrombin and collagen

Mass changes in the various molecular species of phospholipids were determined after stimulation of human platelets with thrombin and collagen. Upon stimulation, every molecular species of phosphatidylinositol and phosphatidylserine was equally hydrolyzed, whereas the molecular species of phosphatidylcholine and diacyl- and alkenylacylphosphatidylethanolamine containing arachidonic acid were selectively hydrolyzed. At low Ca2+ concentrations, which result from mobilization of intracellular Ca2+ stores, phosphatidylinositol, phosphatidylcholine, and diacylphosphatidylethanolamine were hydrolyzed after stimulation with thrombin, whereas only phosphatidylinositol was hydrolyzed with production of thromboxane B2 after stimulation with collagen. At high Ca2+ concentrations, phosphatidylcholine and diacylphosphatidylethanolamine were hydrolyzed after stimulation with collagen, and phosphatidylserine and alkenylacylphosphatidylethanolamine were degraded after stimulation with both thrombin and collagen. [1-14C]Arachidonic acid was heterogeneously incorporated into the individual molecular species of the various phospholipid classes, indicating that the determination of mass is essential for an accurate picture of phospholipid hydrolysis. The data reported here indicate that the Ca2+ concentration affects the differential degradation of phospholipid molecular species in activated human platelets.