We earlier reported that the soluble form of the CD40 ligand (sCD40L), is involved in thrombosis by stabilizing platelet thrombi. In this article, we have determined the mechanism by which this protein affects platelet biology. Addition of sCD40L to washed platelets was found to activate the receptor function of ␣IIb␤3 as measured by the induction of fibrinogen binding and the formation of platelet microparticles. Mutation in the KGD sequence (D117E) of sCD40L, the ␣IIb␤3-binding domain in the N terminus of the protein resulted in a loss of the platelet-stimulatory activity of this protein. Integrilin, a ␣IIb␤3 antagonist, but not an antibody to CD40 that blocked the ligand-binding activity, inhibited these platelet-stimulatory events. CD40 ؊/؊ platelets bound fibrinogen and formed microparticles similar to WT platelets, again indicating that CD40 is not involved in sCD40L-induced platelet activation. Exposure of platelets to sCD40L, but not D117E-sCD40L-coated surfaces, induced platelet thrombi formation under arterial shear rate. sCD40L-induced platelet stimulation resulted in the phosphorylation of tyrosine-759 in the cytoplasmic domain of ␤3. Platelets from the diYF mouse strain, expressing ␤3 in which both cytoplasmic tyrosines are mutated to phenylalanine, were defective in sCD40L-induced platelet stimulation. These data indicate that sCD40L is a primary platelet agonist and that platelet stimulation is induced by the binding of the KGD domain of sCD40L to ␣IIb␤3, triggering outside-in signaling by tyrosine phosphorylation of ␤3.

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The present study was designed to identify novel membrane proteins that signal during platelet aggregation. Because one putative mechanism for signaling by a membrane protein involves phosphorylation, we used oligonucleotide-based microarray analyses and mass spectrometric proteomics techniques to specifically discover membrane proteins and also identify those proteins that become phosphorylated on tyrosine, threonine, or serine residues upon platelet aggregation. Surprisingly, both techniques converged to identify a novel membrane protein we have termed PEAR1 (platelet endothelial aggregation receptor 1). Sequence analysis of PEAR1 predicts a type-1 membrane protein, 15 extracellular epidermal growth factor-like repeats, and multiple cytoplasmic tyrosines. Analysis of the tissue distribution of PEAR1 showed that it was most highly expressed in platelets and endothelial cells. Upon platelet aggregation induced by physiological agonists, PEAR1 became phosphorylated on tyrosine (Tyr-925), and serine (Ser-953 and Ser-1029) residues. PEAR1 tyrosine phosphorylation was blocked by eptifibatide, an ␣ IIb ␤ 3 antagonist, which inhibits platelet aggregation. Immune clustering of PEAR1 resulted in PEAR1 phosphorylation. Aggregation-induced PEAR1 tyrosine phosphorylation lead to the subsequent association with the ShcB adaptor protein. Platelet proximity induced by centrifugation also induced PEAR1 tyrosine phosphorylation, a reaction not inhibited by eptifibatide. These data suggest that PEAR1 is a novel platelet receptor that signals secondary to ␣ IIb ␤ 3 -mediated platelet-platelet contacts.Platelet aggregation during arterial thrombosis results in ischemic complications precipitating in acute myocardial infarction and stroke. Platelet aggregation is known to be mediated by signaling events initiated by primary platelet agonists such as thrombin, ADP, and collagen, which induce a conformational change in the platelet integrin ␣ IIb ␤ 3 , allowing it to bind soluble fibrinogen and von Willebrand factor, resulting in platelet cross-linking. Platelet-platelet contacts during aggregation subsequently initiate secondary signaling events. Aggregation-induced signaling can result in multiple platelet secondary signaling events such as calcium mobilization, protein tyrosine phosphorylations, cytoskeletal rearrangements, and the release of platelet-dense bodies and ␣-granules. Aggregation-induced signaling is key to the formation of stable aggregates, particularly when aggregation is induced by low concentrations of one or more primary agonists. Platelet activation also causes the release of ADP from dense bodies and the generation of thromboxane A2, both of which induce further platelet stimulation.Several mediators of aggregation-induced signals have been identified. One is ␣ IIb ␤ 3 itself, which becomes tyrosine-phosphorylated and also associates with numerous signaling and cytoskeletal proteins following platelet activation, allowing fibrinogen and/or von Willebrand factor binding and platelet aggregation. The importance o...

Glycoprotein (GP) V is a major substrate cleaved by the protease thrombin during thrombin-induced platelet activation. Previous analysis of platelets from GP V-null mice suggested a role for GP V as a negative modulator of platelet activation by thrombin. We now report the mechanism by which thrombin activates GP V ؊͞؊ platelets. We show that proteolytically inactive forms of thrombin induce robust stimulatory responses in GP V null mouse platelets, via the platelet GP Ib-IX-V complex. Because proteolytically inactive thrombin can activate wild-type mouse and human platelets after treatment with thrombin to cleave GP V, this mechanism is involved in thrombin-induced platelet aggregation. Platelet activation through GP Ib-IX depends on ADP secretion, and specific inhibitors demonstrate that the recently cloned P2Y 12 ADP receptor (Gi-coupled ADP receptor) is involved in this pathway, and that the P2Y1 receptor (G q-coupled ADP receptor) may play a less significant role. Thrombosis was generated in GP V null mice only in response to catalytically inactive thrombin, whereas thrombosis occurred in both genotypes (wild type and GP V null) in response to active thrombin. These data support a thrombin receptor function for the platelet membrane GP Ib-IX-V complex, and describe a novel thrombin signaling mechanism involving an initiating proteolytic event followed by stimulation of the GP Ib-IX via thrombin acting as a ligand, resulting in platelet activation.G lycoprotein (GP) Ib-IX-V is a major complex on the platelet surface, second only to ␣⌱⌱b␤3. This complex consists of several subunits: GP Ib␣, GP Ib␤, GP IX, and GP V in the ratio of 2:2:2:1. Absence of GP Ib-IX-V results in a severe bleeding disorder known as Bernard Soulier syndrome characterized by giant platelets and impaired von Willebrand factor (vWf) binding (1). GP Ib␣ is a receptor for vWf, and the GP Ib-IX-V complex is critical for platelet adhesion under arterial shear conditions (2). A role for GP Ib-IX-V in platelet activation has been proposed on the basis of observations that the signaling molecule 14-3-3 (3, 4) is associated with the complex, and that phosphorylation of pp72 syk occurs upon vWf binding to GP Ib␣ (5). In fact, Zaffran et al. (6) recently showed that in heterologous Chinese hamster ovary (CHO) cells expressing both ␣⌱⌱b␤3 and GP Ib-IX, inside-out activation of ␣⌱⌱b␤3 could occur upon vWf adhesion.The GP Ib␣ subunit also has a thrombin binding site on the extracellular domain that overlaps the vWf binding domain (7). Additionally, the complex has a platelet-specific thrombin substrate, GP V, that is cleaved very early during thrombin-induced platelet aggregation (8). Platelets from Bernard Soulier syndrome patients show an impaired response to thrombin (9), and antibodies that block thrombin binding to GP Ib␣ also partially inhibit platelet responses to thrombin (9). More recently, thrombin binding to GP Ib␣ has been shown to enhance platelet procoagulant activity (10). However, the physiological significance of this interaction ha...

UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) catalyzes the initial step in the synthesis of the mannose 6-phosphate determinant required for efficient intracellular targeting of newly synthesized lysosomal hydrolases to the lysosome. The enzyme was partially purified ϳ30,000-fold by chromatography of solubilized membrane proteins from lactating bovine mammary glands on DEAE-Sepharose, reactive green 19-agarose, and Superose 6. The partially purified enzyme was used to generate a panel of murine monoclonal antibodies. The antiGlcNAc-phosphotransferase monoclonal antibody PT18 was coupled to a solid support and used to immunopurify the enzyme ϳ480,000-fold to apparent homogeneity with an overall yield of 29%. The purified enzyme has a specific activity of 10-12 mol of GlcNAc phosphate transferred per h/mg using 100 mM ␣-methylmannoside as acceptor.The subunit structure of the enzyme was determined using a combination of analytical gel filtration chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino-terminal sequencing. The data indicate that bovine GlcNAc-phosphotransferase is a 540,000-Da complex composed of disulfide-linked homodimers of 166,000-and 51,000-Da subunits and two identical, noncovalently associated 56,000-Da subunits.The trafficking of most lysosomal hydrolases in higher eucaryotes is mediated by a mannose 6-phosphate-dependent pathway. Asparagine-linked oligosaccharides on newly synthesized lysosomal hydrolases are sequentially modified by two enzymes to generate a mannose 6-phosphate recognition marker. The initial and determining step in the biosynthesis of the mannose 6-phosphate recognition marker is catalyzed by the enzyme UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase).

Platelet aggregation is a dynamic entity, capable of directing its own growth and stability via the activation of signaling cascades that lead to the expression and secretion of various secondary agonists. Here we show that the signaling pathways triggered during platelet aggregation include an intrinsic pro-thrombotic activity mediated by 2 homophilic adhesion molecules, CD84 and CD150 (

Lysosomal enzymes are targeted to the lysosome through binding to mannose 6-phosphate receptors because their glycans are modified with mannose 6-phosphate. This modification is catalyzed by UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase). Bovine GlcNAc-phosphotransferase was isolated using monoclonal antibody affinity chromatography, and an ␣ 2 ␤ 2 ␥ 2 -subunit structure was proposed. Although cDNA encoding the ␥-subunit has been described, cDNAs for the ␣-and ␤-subunits have not. Using partial amino acid sequences from the bovine ␣-and ␤-subunits, we have isolated a human cDNA that encodes both the ␣-and ␤-subunits. Both subunits contain a single predicted membrane-spanning domain. The ␣-and ␤-subunits appear to be generated by a proteolytic cleavage at the Lys 928 -Asp 929 bond. Transfection of 293T cells with the ␣/␤-subunitsprecursor cDNA with or without the ␥-subunit cDNA results in a 3.6-or 17-fold increase in GlcNAc-phosphotransferase activity in cell lysates, suggesting that the precursor cDNA contains the catalytic domain. The sequence lacks significant similarity with any described vertebrate enzyme except for two Notch-like repeats in the ␣-subunit. However, a 112-amino acid sequence is highly similar to a group of bacterial capsular polymerases (46% identity). A BAC clone containing the gene that spanned 85.3 kb and was composed of 21 exons was sequenced and localized to chromosome 12q23. We now report the cloning of both the cDNA and genomic DNA of the precursor of GlcNAc-phosphotransferase. The completion of cloning all three subunits of GlcNAc-phosphotransferase allows expression of recombinant enzyme and dissection of lysosomal targeting disorders.In higher eukaryotes most lysosomal hydrolases are targeted to the lysosome via a mannose 6-phosphate (M6P) 2 -dependent pathway.Before targeting, lysosomal enzymes are modified by the addition of M6P in a two-step reaction. In the first step UDP-N-acetylglucosaminelysosomal enzyme phosphotransferase (GlcNAc-phosphotransferase; EC 2.7.8.17) catalyzes the transfer of GlcNAc 1-phosphate from UDPGlcNAc to the terminal or penultimate mannose on high mannose-type glycans of lysosomal hydrolases (1-3). The second enzymatic step occurs in the trans-Golgi network, where the covering GlcNAc is removed by N-acetylglucosamine-1-phosphodiester ␣-N-acetylglucosaminidase (EC 3.1.4.45), which has the trivial name "uncovering enzyme" (4, 5). The lysosomal enzymes, now modified with M6P, bind to M6P receptors in the trans-Golgi network and are translocated to the endosome and subsequently to the lysosome. Recognition of lysosomal hydrolases by GlcNAc-phosphotransferase is the initiating step in lysosomal hydrolase trafficking. Lysosomal hydrolases that are known substrates for GlcNAc-phosphotransferase exhibit low K m values, whereas non-lysosomal glycoproteins bearing similar glycans have higher K m values (1, 3, 6). GlcNAc-phosphotransferase was isolated using monoclonal antibody affinity chromatography fr...

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