Binding of selected mAbs to (A) purified GPIb-IX, (B) washed human platelets, (C) unconjugated and (D) ovalbumin-conjugated shedding-site peptide
Binding of selected mAbs to (A) purified GPIb-IX, (B) washed human platelets, (C) unconjugated and (D) ovalbumin-conjugated shedding-site peptide. as a widely used shedding inhibitor GM6001 in both constitutive and induced GPIb shedding in human platelets. It does not identify mouse GPIb. Nor will it inhibit shedding NSC87877 of other platelet receptors. Finally, 5G6 binding displays no detectable effect on platelet activation and aggregation. == Conclusion == 5G6 specifically inhibits GPIb shedding with no detectable effect on platelet functions. The method of substrate-specific shedding inhibition by macromolecular binding of the shedding cleavage site can be applicable to many other transmembrane receptors undergoing ectodomain shedding. Keywords:Glycoprotein Ib; Glycocalicin; Platelet transfusion; ADAM proteins; Antibodies, Monoclonal == Introduction == Glycoprotein (GP)Ib is usually abundantly expressed around the platelet surface. GPIb is the platelet receptor for von Willebrand factor (VWF) and other ligands in blood circulation [1]. In addition to its function in mediating ligand-induced platelet activation during main hemostasis, GPIb plays an important role in thrombosis, thrombocytopenia, inflammation and other disease says [2,3]. GPIb is usually constantly proteolyzed in circulating platelets, with its extracellular domain name, also known as glycocalicin, released into the plasma [4]. This process, later named ectodomain shedding, has been also reported to occur in platelets activated by chemical or physiological agonists, such as -thrombin, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7, a calmodulin inhibitor that sequesters calmodulin from binding to its ligands), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, a drug that damages the mitochondria and induces apoptosis), and phorbol 12-myristate-13-acetate (PMA) [58]. ADAM17 is the physiological sheddase for GPIb [6]. Broad-spectrum metalloproteinase inhibitors, such as hydroxamic acid-based GM6001 that chelates the zinc ion required for the metalloproteinase activity, strongly inhibit W7- and CCCP-induced GPIb shedding [7,8]. Recombinant ADAM17 cleaves GPIb-based peptides at the Gly464-Val465 peptide bond, suggesting it as the shedding cleavage site in GPIb [8]. Despite abundant information obtained over the past few years, the biological significance of GPIb shedding Rabbit polyclonal to ADAM20 remains to be defined. Recently it was suggested that GPIb shedding plays a critical role in clearance of damaged platelets. Like human platelets, mouse platelets that had been storedin vitroor treated with CCCP to simulate cell damage were observed to shed a significant amount of GPIb, and they were cleared rapidly upon infusion [7]. Incubation of these platelets with GM6001, or a small-molecule inhibitor of p38 MAPK that is required for ADAM17 activity, blocked shedding of GPIb and improved the post-transfusion recovery and survival of these platelets [7,9]. These results suggest that blocking GPIb shedding can hamper the clearance of stored platelets. However, ADAM17 has broad substrate specificity [10,11]. With a relatively shallow substrate-binding groove uncovered on the surface of its NSC87877 catalytic domain and the ability to adapt the binding pocket to the shape of a substrate or an inhibitor, ADAM17 can identify and cleave a substrate with an extended backbone conformation that is not strictly dependent on any particular side chain [12,13]. ADAM17 has NSC87877 been shown to cleave physiologically GPIb, TNF- and many other substrates including GPV [14]. Thus, the evidence reported so far cannot rule out the possibility that a receptor around the platelet surface other than GPIb that is also a shedding substrate is the cause for platelet clearance. To definitively determine whether GPIb shedding is actually the trigger for platelet clearance or merely an inconsequential indication for damaged and to-be-cleared platelets, a reagent that specifically inhibits shedding of GPIb but not other receptors will be required. In the present study we statement novel anti-GPIb monoclonal antibodies (mAbs) that specifically inhibit shedding of human GPIb in platelets. == Materials and methods == == Materials and animals == Immunization of C57BL mice and production of monoclonal antibodies against GPIb were NSC87877 carried out by Green Mountain Antibodies (Burlington, VT). CCCP, L-cysteine and BSA were from Sigma-Aldrich (St. Louis, MO). GM6001, W7 and PMA were from Calbiochem (La Jolla, CA). The anti-GPV mAb SW16 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Biotinylated antibody was prepared using sulfo-NHS-biotin (Thermo Scientific, Rockford, IL) and following manufacturers training. Transgenic IL4Tg and hTg mice have been described [15]. All animal procedures have been performed in accordance with institutional guidelines and approval. == Preparation of washed human platelets == Human whole blood was obtained from healthy.