LpMab-12 Established by CasMab Technology Specifically Detects Sialylated O-Glycan on Thr52 of Platelet Aggregation-Stimulating Domain of Human Podoplanin

Podoplanin (PDPN), also known as Aggrus, possesses three tandem repeat of platelet aggregation-stimulating (PLAG) domains in its N-terminus. Among the PLAG domains, sialylated O-glycan on Thr52 of PLAG3 is essential for the binding to C-type lectin-like receptor-2 (CLEC-2) and the platelet-aggregating activity of human PDPN (hPDPN). Although various anti-hPDPN monoclonal antibodies (mAbs) have been generated, no specific mAb has been reported to target the epitope containing glycosylated Thr52. We recently established CasMab technology to develop mAbs against glycosylated membrane proteins. Herein, we report the development of a novel anti-glycopeptide mAb (GpMab), LpMab-12. LpMab-12 detected endogenous hPDPN by flow cytometry. Immunohistochemical analyses also showed that hPDPN-expressing lymphatic endothelial and cancer cells were clearly labeled by LpMab-12. The minimal epitope of LpMab-12 was identified as Asp49–Pro53 of hPDPN. Furthermore, LpMab-12 reacted with the synthetic glycopeptide of hPDPN, corresponding to 38–54 amino acids (hpp3854: ₃₈-EGGVAMPGAEDDVVTPG-₅₄), which carries α2–6 sialylated N-acetyl-_D-galactosamine (GalNAc) on Thr52. LpMab-12 did not recognize non-sialylated GalNAc-attached glycopeptide, indicating that sialylated GalNAc on Thr52 is necessary for the binding of LpMab-12 to hPDPN. Thus, LpMab-12 could serve as a new diagnostic tool for determining whether hPDPN possesses the sialylation on Thr52, a site-specific post-translational modification critical for the hPDPN association with CLEC-2.     

Functional sialylated O-glycan to platelet aggregation on Aggrus (T1alpha/Podoplanin) molecules expressed in Chinese hamster ovary cells

Aggrus, also called T1alpha and podoplanin, is a novel platelet aggregation-inducing factor that is expressed in various carcinoma cells. Aggrus/T1alpha/podoplanin is known to be expressed in lung type I alveolar cells or lymphatic endothelial cells. However, its physiological role has not been clarified. To assess the attribution of glycosylation to Aggrus platelet aggregation activity, recombinant molecules were stably expressed in a series of Chinese hamster ovary (CHO) cell mutants, N-glycan-deficient Lec1, CMP-sialic acid transporter-deficient Lec2, and UDP-galactose transporter-deficient Lec8. A new anti-human Aggrus monoclonal antibody, YM-1, was established to detect the expression of human Aggrus on these CHO cell mutants. Aggrus on Lec1 cells induced platelet aggregation, but those on Lec2 and Lec8 cells did not. Further, the glycans on Aggrus were analyzed by lectin blotting. Aggrus expressed in CHO and Lec1 cells showed Wheat-germ agglutinin, Jacalin, and Vicia villosa lectin bindings. Lectin blotting results indicated that sialylated core 1 structures, sialic acid plus Galbeta1,3GalNAc-Ser/Thr, were critical for the platelet aggregation activity. This oligosaccharide structure is known as tumor-associated antigen, which is potentially related to the metastasis process of cancer cells.     

Involvement of the snake toxin receptor CLEC-2, in podoplanin-mediated platelet activation, by cancer cells

Podoplanin (aggrus), a transmembrane sialoglycoprotein, is involved in tumor cell-induced platelet aggregation, tumor metastasis, and lymphatic vessel formation. However, the mechanism by which podoplanin induces these cellular processes including its receptor has not been elucidated to date. Podoplanin induced platelet aggregation with a long lag phase, which is dependent upon Src and phospholipase Cgamma2 activation. However, it does not bind to glycoprotein VI. This mode of platelet activation was reminiscent of the snake toxin rhodocytin, the receptor of which has been identified by us as a novel platelet activation receptor, C-type lectin-like receptor 2 (CLEC-2) (Suzuki-Inoue, K., Fuller, G. L., Garcia, A., Eble, J. A., Pohlmann, S., Inoue, O., Gartner, T. K., Hughan, S. C., Pearce, A. C., Laing, G. D., Theakston, R. D., Schweighoffer, E., Zitzmann, N., Morita, T., Tybulewicz, V. L., Ozaki, Y., and Watson, S. P. (2006) Blood 107, 542-549). Therefore, we sought to evaluate whether CLEC-2 serves as a physiological counterpart for podoplanin. Association between CLEC-2 and podoplanin was confirmed by flow cytometry. Furthermore, their association was dependent on sialic acid on O-glycans of podoplanin. Recombinant CLEC-2 inhibited platelet aggregation induced by podoplanin-expressing tumor cells or lymphatic endothelial cells, suggesting that CLEC-2 is responsible for platelet aggregation induced by endogenously expressed podoplanin on the cell surfaces. These findings suggest that CLEC-2 is a physiological target protein of podoplanin and imply that it is involved in podoplanin-induced platelet aggregation, tumor metastasis, and other cellular responses related to podoplanin.     

Renal cells activate the platelet receptor CLEC-2 through podoplanin

We have recently shown that the C-type lectin-like receptor, CLEC-2, is expressed on platelets and that it mediates powerful platelet aggregation by the snake venom toxin rhodocytin. In addition, we have provided indirect evidence for an endogenous ligand for CLEC-2 in renal cells expressing HIV-1. This putative ligand facilitates transmission of HIV through its incorporation into the viral envelope and binding to CLEC-2 on platelets. The aim of the present study was to identify the ligand on these cells which binds to CLEC-2 on platelets. Recombinant CLEC-2 exhibits specific binding to HEK-293T (human embryonic kidney) cells in which the HIV can be grown. Furthermore, HEK-293T cells activate both platelets and CLEC-2-transfected DT-40 B-cells. The transmembrane protein podoplanin was identified on HEK-293T cells and was demonstrated to mediate both binding of HEK-293T cells to CLEC-2 and HEK-293T cell activation of CLEC-2-transfected DT-40 B-cells. Podoplanin is expressed on renal cells (podocytes). Furthermore, a direct interaction between CLEC-2 and podoplanin was confirmed using surface plasmon resonance and was shown to be independent of glycosylation of CLEC-2. The interaction has an affinity of 24.5+/-3.7 microM. The present study identifies podoplanin as a ligand for CLEC-2 on renal cells.     

Induction of podoplanin by transforming growth factor-beta in human fibrosarcoma

Podoplanin/aggrus is increased in tumors and its expression was associated with tumor malignancy. Podoplanin on cancer cells serves as a platelet-aggregating factor, which is associated with the metastatic potential. However, regulators of podoplanin remain to be determined. Transforming growth factor-beta (TGF-beta) regulates many physiological events, including tumorigenesis. Here, we found that TGF-beta induced podoplanin in human fibrosarcoma HT1080 cells and enhanced the platelet-aggregating-ability of HT1080. TGF-beta type I receptor inhibitor (SB431542) and short hairpin RNAs for Smad4 inhibited the podoplanin induction by TGF-beta. These results suggest that TGF-beta is a physiological regulator of podoplanin in tumor cells.     

The transmembrane domain of podoplanin is required for its association with lipid rafts and the induction of epithelial-mesenchymal transition

Podoplanin is a transmembrane glycoprotein that is upregulated in cancer and was reported to induce an epithelial-mesenchymal transition (EMT) in MDCK cells. The promotion of EMT was dependent on podoplanin binding to ERM (ezrin, radixin, moesin) proteins through its cytoplasmic (CT) domain, which led to RhoA-associated kinase (ROCK)-dependent ERM phosphorylation. Using detergent-resistant membrane (DRM) assays, as well as transmembrane (TM) interactions and ganglioside GM1 binding, we present evidence supporting the localization of podoplanin in raft platforms important for cell signalling. Podoplanin mutant constructs harbouring a heterologous TM region or lacking the CT tail were unable to associate with DRMs, stimulate ERM phosphorylation and promote EMT or cell migration. Similar effects were observed upon disruption of a GXXXG motif within the TM domain, which is involved in podoplanin self-assembly. In contrast, deletion of the extracellular (EC) domain did not affect podoplanin DRM association. Together, these data suggest that both the CT and TM domains are required for podoplanin localization in raft platforms, and that this association appears to be necessary for podoplanin-mediated EMT and cell migration.     

Epitope mapping of anti-mouse podoplanin monoclonal antibody PMab-1

Mouse podoplanin (mPDPN) is a type I transmembrane sialoglycoprotein, which is expressed on lymphatic endothelial cells, podocytes of the kidney, and type I alveolar cells of the lung. mPDPN is known as a platelet aggregation-inducing factor and possesses four platelet aggregation-stimulating (PLAG) domains: PLAG1, PLAG2, and PLAG3 in the N-terminus and PLAG4 in the middle of the mPDPN protein. mPDPN overexpression in cancers has been reportedly associated with hematogenous metastasis through interaction with the C-type lectin-like receptor 2 of platelets. We previously reported a rat anti-mPDPN monoclonal antibody clone PMab-1, which was developed by immunizing the PLAG2 and PLAG3 domains of mPDPN. PMab-1 is very useful in flow cytometry, western blot, and immunohistochemical analyses to detect both normal cells and cancers. However, the binding epitope of PMab-1 remains to be clarified. In the present study, flow cytometry, enzyme-linked immunosorbent assay, and immunohistochemical analyses were utilized to investigate the epitope of PMab-1. The results revealed that the critical epitope of PMab-1 is Asp39 and Met41 of mPDPN. These findings can be applied to the production of more functional anti-mPDPN monoclonal antibodies.     

Identification of a karyopherin alpha 2 recognition site in PLAG1, which functions as a nuclear localization signal

The activation of the pleomorphic adenoma gene 1 (PLAG1) is the most frequent gain-of-function mutation found in pleomorphic adenomas of the salivary glands. To gain more insight into the regulation of PLAG1 function, we searched for PLAG1-interacting proteins. Using the yeast two-hybrid system, we identified karyopherin alpha2 as a PLAG1-interacting protein. Physical interaction between PLAG1 and karyopherin alpha2 was confirmed by an in vitro glutathione S-transferase pull-down assay. Karyopherin alpha2 escorts proteins into the nucleus via interaction with a nuclear localization sequence (NLS) composed of short stretches of basic amino acids. Two putative NLSs were identified in PLAG1. The predicted NLS1 (KRKR) was essential for physical interaction with karyopherin alpha2 in glutathione S-transferase pull-down assay, and its mutation resulted in decreased nuclear import of PLAG1. Moreover, NLS1 was able to drive the nuclear import of the cytoplasmic protein beta-galactosidase. In contrast, predicted NLS2 of PLAG1 (KPRK) was not involved in karyopherin alpha2 binding nor in its nuclear import. The residual nuclear import of PLAG1 after mutation of the NLS1 was assigned to the zinc finger domain of PLAG1. These observations indicate that the nuclear import of PLAG1 is governed by its zinc finger domain and by NLS1, a karyopherin alpha2 recognition site.     

Direct demonstration of involvement of the adaptor protein ShcA in the regulation of Ca2+-induced platelet aggregation

Platelet aggregation is mediated by conformational change of integrin alpha(IIb)beta(3). Tyrosine-phosphorylation of cytoplasmic domain of beta(3) upon platelet activation has been demonstrated to play a critical role in this process. Recently, the adaptor protein ShcA has been shown to bind to the tyrosine-phosphorylated beta(3), while it remains open whether ShcA plays any role in platelet aggregation. Here, we show that ShcA bound to tyrosine-phosphorylated beta(3)-tail peptide through its phosphotyrosine-binding domain in vitro. Then, we examined the involvement of ShcA in platelet aggregation by a previously established in vitro assay using platelets permeabilized with streptolysin-O, where exogenous addition of platelet cytosol is required for reconstitution of the Ca(2+)-induced aggregation. When ShcA was specifically depleted with anti-ShcA antibody from the cytosol, this ShcA-depleted cytosol lost the aggregation-supporting activity, which was rescued by addition of purified recombinant ShcA. Thus, ShcA is essential for the Ca(2+)-induced platelet aggregation.     

The response to thromboxane A2 analogues in human platelets. Discrimination of two binding sites linked to distinct effector systems

Thromboxane A2 (TXA2) induces platelet shape change, secretion, and aggregation. Using a novel TXA2/prostaglandin endoperoxide receptor antagonist, [1r-[1 alpha(Z),2 beta,3 beta,5 alpha]]-(+)-7-[5-[[(1,1'- biphenyl)-4-yl]methoxy]-3-hydroxy-2-(1-piperidinyl) cyclopentyl]-4-heptenoic acid hydrochloride (GR32191), we demonstrate that these responses are mediated by at least two receptor-effector systems. GR32191 non-competitively inhibited platelet aggregation to the TXA2 mimetics, (15S)-hydroxy-11,9-(epoxymethano) prostadienoic acid (U46619) and [1S-(1 alpha,2 beta(5Z),3 alpha (1E,-3S), 4 alpha)]-7-[3-(3-hydroxy-4-(p-iodophenoxy)-1-butenyl)7- oxabicyclo[2.2.1]hept-2yl]-5-heptenoic acid by binding irreversibly to a TXA2/prostaglandin endoperoxide receptor. Dissociation of [3H]GR32191 from human platelets demonstrated two specific binding sites, one which was rapidly dissociating and a site to which binding was essentially irreversible. Stimulation by U46619 of platelets incubated with GR32191 and subsequently washed to expose the reversible binding site failed to aggregate or to secrete [3H]5-hydroxy-tryptamine; formation of inositol phosphates and activation of protein kinase C were markedly suppressed. In contrast, platelet shape change and calcium stimulation remained at 90% of control. Furthermore, stimulation of the reversible binding site with U46619 induced aggregation in the presence of ADP, demonstrating its functional importance in amplifying the response to other agonists. These data suggest that TXA2 mediates platelet activation through at least two receptor-effector systems; one linked to phospholipase C activation, resulting in platelet aggregation and secretion and a second site mediating an increase in cytosolic calcium and platelet shape change.     

Functional analysis of a recombinant glycoprotein Ia/IIa (Integrin alpha(2)beta(1)) I domain that inhibits platelet adhesion to collagen and endothelial matrix under flow conditions

The interaction of platelets with collagen plays an important role in primary hemostasis. Glycoprotein Ia/IIa (GPIa/IIa, integrin alpha(2)beta(1)) is a major platelet receptor for collagen. The binding site for collagen has been mapped to the I domain within the alpha(2) subunit (GPIa). In order to assess the role of the alpha(2)-I domain structure in GPIa/IIa binding to collagen, a recombinant I domain (amino acids 126-337) was expressed in Escherichia coli. The alpha(2)-I protein bound human types I and III collagen in a saturable and divalent cation-dependent manner and was blocked by the alpha(2)beta(1) function blocking antibody 6F1. The alpha(2)-I protein inhibited collagen-induced platelet aggregation (IC(50) = 600 nM). Unexpectedly, 6F1, an antibody that fails to inhibit platelet aggregation in platelet-rich plasma, blocked the inhibitory effect of the alpha(2)-I protein. The alpha(2)-I protein was able to prevent platelet adhesion to a collagen surface exposed to flowing blood under low shear stress. Interestingly, it inhibited platelet adhesion to extracellular matrix at high shear stress. These results, taken together, provide firm evidence that GPIa/IIa directly mediates the first contact of platelets with collagen under both stirring and flow conditions.     

O-Methylated and sulfoconjugated catecholamines: differential activities at human platelet alpha 2-adrenoceptors.

The physiological effects of the sulfoconjugates of epinephrine, norepinephrine, and the 3-O-methylated catecholamines, metanephrine, normetanephrine, and methoxytyramine were examined with regard to their alpha 2-adrenoceptor binding properties and aggregation activity in human platelets. Sulfoconjugation of catecholamines resulted in the loss of both their competitive potency for [3H]yohimbine binding and their influence on platelet aggregation. O-Methyl substituted catecholamines showed attenuation of their alpha 2-adrenoceptor binding affinities when compared with those of the corresponding non-esterified amines. Unlike the free amine epinephrine, which stimulated platelet aggregation, the O-methylated catecholamine derivatives inhibited aggregation. Inhibition was dose-dependent and restricted to the alpha 2-adrenoceptor mediated aggregation response stimulated by epinephrine (1 microM) or potentiated by subthreshold concentrations of epinephrine (30-300 nM) in the presence of subaggregatory doses of vasopressin (10-30 nM). Collagen- and ADP-induced platelet aggregation was not affected. The hydrophilic beta-antagonist CGP 12177 displayed no effects. However, high concentrations (0.1 mM) of both isomers of the strongly lipophilic beta-adrenoceptor antagonist propranolol inhibited the actions of all aggregators by stabilizing the membrane. Such a nonspecific membrane interaction of the methylated catecholamines could be excluded because of their low lipid solubility calculated in a n-octanol-phosphate buffer system at pH 7.4. We suggest therefore that methylated catecholamines are biological alpha 2-adrenoceptor antagonists acting on alpha 2-adrenoceptor stimulated reactions of human platelets. Whether this receptor antagonism is relevant to other human tissues needs clarification. Sulfated catecholamines, however, are wholly ineffective at this receptor site and may constitute a pathway to control the concentration of the active free catecholamines.     

Regulation of phosphorylation of tau by cyclin-dependent kinase 5 and glycogen synthase kinase-3 at substrate level

Microtubule associated protein tau, which is expressed in six alternatively spliced molecular isoforms in human brain, is abnormally hyperphosphorylated in Alzheimer disease and related tauopathies. Here, we show (i) that GSK-3alpha and neither GSK-3beta nor cdk5 can phosphorylate tau at Ser262 and phosphorylation at Ser235 by cdk5 primes phosphorylation at Thr231 by GSK-3alpha/beta; (ii) that tau isoforms with two N-terminal inserts (tau4L, tau3L) are phosphorylated by cdk5 plus GSK-3 at Thr231 markedly more than isoforms lacking these inserts (tau4, tau3); and (iii) that Thr231 is phosphorylated approximately 50% more in free tau than in microtubule-bound tau, and the phosphorylation at this site results in the dissociation of tau from microtubules. These findings suggest that the phosphorylation of tau at Thr231 and Ser262 by cdk5 plus GSK-3, which inhibits its normal biological activity, is regulated both by its amino terminal inserts and its physical state.     

Essential in vivo roles of the platelet activation receptor CLEC-2 in tumour metastasis, lymphangiogenesis and thrombus formation

We have recently identified C-type lectin-like receptor 2 (CLEC-2) as a receptor for the platelet activating snake venom rhodocytin. CLEC-2 elicits powerful platelet activation signals in conjunction with single YxxL motif in its cytoplasmic tail, Src, Syk kinases, and phospholipase Cγ2. An endogenous ligand of CLEC-2 has been identified as podoplanin, which is a membrane protein of tumour cells and facilitates tumour metastasis by inducing platelet activation. Studies of CLEC-2-deficient mice have revealed several physiological roles of CLEC-2. Podoplanin is also expressed in lymphatic endothelial cells. In the developmental stages, when the primary lymph sac is derived from the cardinal vein, podoplanin activates platelets in lymphatic endothelial cells, which facilitates blood/lymphatic vessel separation. Moreover, CLEC-2 is involved in thrombus stabilization under flow conditions in part through homophilic interactions. The absence of CLEC-2 does not significantly increase bleeding tendency, implying that CLEC-2 may be a good target protein for anti-platelet drugs in addition to anti-metastatic drugs.     

Inhibition of tumor cell-induced platelet aggregation using a novel anti-podoplanin antibody reacting with its platelet-aggregation-stimulating domain

The mucin-type sialoglycoprotein, podoplanin (aggrus), is a platelet-aggregating factor on cancer cells. We previously described up-regulated expression of podoplanin in malignant astrocytic tumors including glioblastoma. Its expression was associated with tumor malignancy. In the present study, we investigated podoplanin expression and platelet-aggregating activities of glioblastoma cell lines. First, we established a highly reactive anti-podoplanin antibody, NZ-1, which inhibits podoplanin-induced platelet aggregation completely. Of 15 glioblastoma cell lines, LN319 highly expressed podoplanin and induced platelet aggregation. Glycan profiling using a lectin microarray showed that podoplanin on LN319 possesses sialic acid, which is important in podoplanin-induced platelet aggregation. Interestingly, NZ-1 neutralized platelet aggregation by LN319. These results suggest that podoplanin is a main reason for platelet aggregation induced by LN319. We infer that NZ-1 is useful to determine whether platelet aggregation is podoplanin-specific or not. Furthermore, podoplanin might become a therapeutic target of glioblastoma for antibody-based therapy.     

Bioactive amide of prostaglandin E1 and ethanolamine plasmalogen analog of platelet-activating factor inhibits several pathways of human platelet aggregation

The influence of an amide of prostaglandin E1 and ethanolamine plasmalogen platelet-activating factor analog 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phospho-(N-11alpha, 15alpha-dioxy-9-keto-13-prostenoyl)ethanolamine (PGE1-PPAF) on platelet-activating factor (PAF)-, ADP-, and thrombin-induced human platelet aggregation has been studied. It was found that PGE1-PPAF inhibits the PAF-, ADP-, and thrombin-induced platelet aggregation in platelet-rich plasma. 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine inhibited PAF-induced aggregation up to 50% but had no influence on platelet aggregation induced by ADP or thrombin. The ethanolamine plasmalogen analog of PAF 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phospho-(N-palmitoyl)ethanolami ne, having a palmitoyl residue instead of PGE1, did not inhibit platelet aggregation induced by PAF, ADP, or thrombin. We propose that inhibition of human platelet aggregation by PGE1-PPAF is mediated by its action on platelet PAF-receptors and the adenylate cyclase system.     

The F2-isoprostane, 8-epi-prostaglandin F2 alpha, a potent agonist of the vascular thromboxane/endoperoxide receptor, is a platelet thromboxane/endoperoxide receptor antagonist.

F2-isoprostanes are a recently discovered series of prostaglandin (PG)F2-like compounds that are produced in vivo in humans by nonenzymatic free radical catalyzed peroxidation of arachidonic acid. One of the compounds that can be produced in abundance by this mechanism is 8-epi-PGF2 alpha. 8-epi-PGF2 alpha is a potent vasoconstrictor in the rat, an effect that has been shown to be mediated via interaction with vascular thromboxane (TxA2)/endoperoxide (PGH2) receptors. In an effort to further understand the biological properties of this prostanoid in relation to its ability to interact with TxA2/PGH2 receptors, we examined its effects on human and rat platelets. At concentrations of 10(-6) M and 10(-5) M, 8-epi-PGF2 alpha induced only a shape change in human platelets and at higher concentrations (10(-4) M) induced reversible but not irreversible aggregation. Both the shape change and reversible aggregation were unaffected by indomethacin but were inhibited by the TxA2/PGH2 receptor antagonist SQ29548. Conversely, 8-epi-PGF2 alpha inhibited platelet aggregation induced by the TxA2/PGH2 receptor agonists U46619 (10(-6) M) and IBOP (3.3 x 10(-7) M) with an IC50 of 1.6 x 10(-6) M and 1.8 x 10(-6) M, respectively. 8-epi-PGF2 alpha also inhibited platelet aggregation induced by arachidonic acid. Similarly, in rat platelets, 8-epi-PGF2 alpha alone induced only modest reversible aggregation but completely inhibited U46619-induced aggregation.     

A MUC1 tandem repeat reporter protein produced in CHO-K1 cells has sialylated core 1 O-glycans and becomes more densely glycosylated if coexpressed with polypeptide-GalNAc-T4 transferase

A recombinant mucin O-glycosylation reporter protein, containing 1.7 tandem repeats (TRs) from the transmembrane mucin MUC1, was constructed. The reporter protein, MUC1(1.7TR)-IgG2a, was produced in CHO-K1 cells to study the glycosylation of the MUC1 TR and the in vivo role of polypeptide-GalNAc-T4 glycosyltransferase. N-terminal sequencing of MUC1(1.7TR)-IgG2a showed that all five potential O-glycosylation sites within the TR were used, with an average density of 4.5 glycans per repeat. The least occupied site was Thr in the PDTR motif, where 75% of the molecules were glycosylated, compared to 88-97% at the other sites. This glycan density was confirmed by an alternative liquid chromatography-mass spectrometry (LC-MS) based approach. The O-linked oligosaccharides were released from MUC1(1.7TR)-IgG2a and analyzed by nano-LC-MS and LC-MS/MS. Four oligosaccharides were present, NeuAcalpha2-3Galbeta1-3GalNAcol, NeuAcalpha2-3Galbeta1-3(NeuAcalpha2-6)GalNAcol, Galbeta1-3(NeuAcalpha2-6)GalNAcol, and Galbeta1-3GalNAcol, the two first being most abundant. Coexpression of the human polypeptide-GalNAc-T4 transferase with MUC1(1.7TR)-IgG2a increased the glycan occupancy at Thr in PDTR, Ser in VTSA, and Ser in GSTA, supporting the function of GalNAc-T4 proposed from previous in vitro studies. The expression of GalNAc-T4 with a mutation in the first lectin domain (alpha) had no glycosylation effect on PDTR and GSTA but surprisingly gave a dominant negative effect with a decreased glycosylation to around 50% at the Ser in VTSA. The results show that introduction of glycosyltransferases can specifically alter the sites for O-glycosylation in vivo.     

Inhibition of platelet adhesion and aggregation by a defined region (Gly-486-Lys-502) of high molecular weight kininogen

Proteolytic cleavage of single chain high molecular weight kininogen (HK) by kallikrein releases the short-lived vasodilator bradykinin and leaves behind two-chain high molecular weight kininogen (HKa). HKa and particularly its His-Gly-Lys-rich domain 5 have been previously reported to exert anti-adhesive properties by binding to the extracellular matrix protein vitronectin (VN). In this study the ability of HKa and domain 5 to interfere with platelet adhesion and aggregation was investigated. In a purified system HKa and particularly domain 5 but not HK inhibited the binding of VN to the alpha(IIb)beta(3) integrin, whereas the binding of fibrinogen to this integrin was not affected. The region Gly-486-Lys-502 from the carboxyl terminus of the domain 5 was identified as responsible for inhibition of the VN-alpha(IIb)beta(3)-integrin interaction, as this portion was also found to mediate kininogen binding to VN. Through these interactions, HKa, the isolated domain 5, and the peptide Gly-486-Lys-502 abrogated the alpha(IIb)beta(3)-integrin-dependent adhesion of human platelets to VN but not to fibrinogen. The codistribution of VN and HKa at sites of ex vivo platelet aggregation was demonstrated by transmission immune electron microscopy, indicating that the described interaction is likely to take place in vivo. Moreover, domain 5 and the peptide Gly-486-Lys-502 dose-dependently blocked platelet aggregation, resembling the inhibitory effect of monoclonal antibody 13H1 against multimeric VN. Finally, treatment of mice with isolated domain 5 resulted in a significantly prolonged tail bleeding time. Taken together, our data emphasize the inhibitory role of HK domain 5 on platelet adhesion and aggregation; new anti-thrombotic compounds may become available on the basis of peptide Gly-486-Lys-502 of HK domain 5.     

The Pro33 isoform of integrin beta3 enhances outside-in signaling in human platelets by regulating the activation of serine/threonine phosphatases

Integrin beta(3) is polymorphic at residue 33 (Leu(33) or Pro(33)), and the Pro(33)-positive platelets display enhanced aggregation, P-selectin secretion, and shorter bleeding times. Because outside-in signaling is critical for platelet function, we hypothesized that the Pro(33) variant provides a more efficient signaling than the Leu(33) isoform. When compared with Pro(33)-negative platelets, Pro(33)-positive platelets demonstrated significantly greater serine/threonine phosphorylation of extracellular signal-regulated kinase (ERK2) and myosin light chain (MLC) but not cytoplasmic phospholipase A2 upon thrombin-induced aggregation. Tyrosine phosphorylation of integrin beta(3) and the adaptor protein Shc was no different in the fibrinogen-engaged platelets from both genotypes. The addition of Integrilin (alpha(IIb)beta(3)-fibrinogen blocker) or okadaic acid (serine/threonine phosphatase inhibitor) dramatically enhanced ERK2 and MLC phosphorylation in the Pro(33)-negative platelets when compared with Pro(33)-positive platelets, suggesting that integrin engagement during platelet aggregation activates serine/threonine phosphatases. The phosphatase activity of myosin phosphatase (MP) that dephosphorylates MLC is inactivated by phosphorylation of the myosin binding subunit of MP at Thr(696), and aggregating Pro(33)-positive platelets exhibited an increased Thr(696) phosphorylation of MP. These studies highlight a role for the dephosphorylation events via the serine/threonine phosphatases during the integrin outside-in signaling mechanism, and the Leu(33) --> Pro polymorphism regulates this process. Furthermore, these findings support a mechanism whereby the reported enhanced alpha granule secretion in the Pro(33)-positive platelets could be mediated by an increased phosphorylation of MLC, which in turn is caused by an increased phosphorylation and subsequent inactivation of myosin phosphatase.