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Delayed targeting of CD39 to activated platelet GPIIb/IIIa via a single-chain antibody: breaking the link between antithrombotic potency and bleeding?

ABSTRACT: The ecto-nucleoside triphosphate diphosphohydrolase CD39 represents a promising antithrombotic therapeutic. It degrades adenosine 5'-diphosphate (ADP), a main platelet activating/recruiting agent. We hypothesized that delayed enrichment of CD39 on developing thrombi will allow for a low and safe systemic concentration and thus avoid bleeding. We use a single-chain antibody (scFv, specific for activated GPIIb/IIIa) for targeting CD39. This should allow delayed enrichment on growing thrombi but not on the initial sealing layer of platelets, which do not yet express activated GPIIb/IIIa. CD39 was recombinantly fused to an activated GPIIb/IIIa-specific scFv (targ-CD39) and a nonfunctional scFv (non-targ-CD39). Targ-CD39 was more effective at preventing ADP-induced platelet activation than non-targ-CD39. In a mouse carotid artery thrombosis model, non-targ-CD39, although protective against vessel occlusion, was associated with significant bleeding on tail transection. In contrast, targ-CD39 concentrated at the thrombus site; hence, a dose ?10 times less of CD39 prevented vessel occlusion to a similar extent as high-dose non-targ-CD39, without prolonged bleeding time. An equimolar dose of non-targ-CD39 at this low concentration was ineffective at preventing vessel occlusion. Thus, delayed targeting of CD39 via scFv to activated platelets provides strong antithrombotic potency and yet prevents bleeding and thereby promotes CD39 toward clinical use.

SUBMITTER: Hohmann JD

PROVIDER: S-EPMC3630824 | biostudies-other | 2013 Apr

REPOSITORIES: biostudies-other

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Delayed targeting of CD39 to activated platelet GPIIb/IIIa via a single-chain antibody: breaking the link between antithrombotic potency and bleeding?

Hohmann Jan David JD Wang Xiaowei X Krajewski Stefanie S Selan Carly C Haller Carolyn A CA Straub Andreas A Chaikof Elliot L EL Nandurkar Harshal H HH Hagemeyer Christoph E CE Peter Karlheinz K

Blood 20130204 16

The ecto-nucleoside triphosphate diphosphohydrolase CD39 represents a promising antithrombotic therapeutic. It degrades adenosine 5'-diphosphate (ADP), a main platelet activating/recruiting agent. We hypothesized that delayed enrichment of CD39 on developing thrombi will allow for a low and safe systemic concentration and thus avoid bleeding. We use a single-chain antibody (scFv, specific for activated GPIIb/IIIa) for targeting CD39. This should allow delayed enrichment on growing thrombi but no ...[more]

PMID: 23380744

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Project description:Platelet plasma membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa) form a Ca(2+)-dependent heterodimer. GPIIb/IIIa, which serves as the receptor for fibrinogen and other adhesive proteins at the surface of activated platelets. Using equilibrium dialysis measurements, it was established that both GPIIb and GPIIIa in solution have low-affinity Ca(2-)-binding sites (Kd0.2-0.3 mM), five in GPIIb and two in GPIIIa, and it was confirmed that only the alpha-chain of GPIIb (GPIIb alpha) binds Ca2+. Furthermore, Ca2+ binding was found with two CNBr fragments of GPIIb, GPIIb alpha-(1-285) and GPIIb alpha-(314-489), which carry three out of the four putative Ca(2+)-binding sites. GPIIb/IIIa in solution has a single high-affinity Ca(2+)-binding site (Kd1 80 +/- 30 nM at 21 degrees C), whose degree of saturation regulates the state of association of GPIIb and GPIIIa in the GPIIb/IIIa heterodimer at room temperature, and 3-4 medium-affinity Ca(2+)-binding sites (Kd2 40 +/- 15 microM at 21 degrees C). When GPIIb/IIIa was incorporated into liposomes, Kd1 decreased by an order of magnitude (9 +/- 3 nM at 21 degrees C) and reached the dissociation constant estimated for the high-affinity Ca(2+)-binding sites at the platelet surface [Brass & Shattil (1982) J. Biol. Chem. 257, 1400-1405], whereas Kd2 remained unchanged. The high-affinity Ca(2+)-binding site of GPIIb/IIIa in solution at 4 degrees C has almost the same affinity (Kd1 65 +/- 20 nM) as at 21 degrees C; however, at 37 degrees C, either its affinity decreases enough so as to become experimentally indistinguishable from the medium-affinity Ca(2+)-binding sites determined at this temperature (number of binding sites 3.9 +/- 1.2 mol of Ca2+/mol of GP, Kd 25 +/- 11 microM), or vanishes altogether. Studies on Ca(2+)-dependent dissociation of GPIIIb/IIIa at 37 degrees C in solution seem to support the former interpretation. Further work will be necessary to decide whether the dissociation of GPIIb/IIIa in the platelet membrane at 37 degrees C is regulated by the degree of saturation of the high-affinity Ca(2+)-binding site, as occurs in solution. It is suggested that the high-affinity Ca(2+)-binding site could be related to the putative GPIIIa-binding region in GPIIb (residues 558-747 of the alpha chain).

Project description:Human platelet glycoproteins IIb (GPIIb) and IIIa (GPIIIa) form the subunits of the Ca(2+)-dependent heterodimer GPIIb/IIIa, which belongs to the integrin family of phylogenetically related receptors mediating a wide variety of cell-cell and cell-substratum interactions. GPIIb/IIIa plays a central role in haemostasis as a receptor for fibrinogen and other adhesive proteins at the surface of activated platelets. The covalent structure of the subunits is largely known; however, the tertiary and quaternary structures of the heterodimer remain to be determined. To this end, our approach consisted of limited proteolysis of the isolated heterodimer with proteinases of different specificities, followed by protein-chemical and immunochemical analyses of the peptide fragments within each isolated proteolytic product. From the information obtained, we have drawn a rudimentary map which outlines the demarcation of compact domains and the subunit peptide stretches carrying the sequences putatively involved in intrachain, intrasubunit and intersubunit non-covalent connectivity in the heterodimer. Three compact domains have been well defined: one in the heavy (H) chain of GPIIb [GPIIbH-(600-700)], and two in GPIIIa, the N-terminal [GPIIIa-(1-52)] and the core [GPIIIa-(423-622)] domains. Between the latter two domains there is a proteolysis-susceptible region, which is partly involved in ligand binding [GPIIIa-(100-220)] and partly implicated as being in teh subunit interface of the heterodimer. Contrary to GPIIIa, GPIIbH is highly susceptible to proteolysis all along its sequence. Equally susceptible are the extracellular end of the transmembrane segment of both GPIIIa and the light (L) chain of GPIIb (GPIIbL), and the N-terminal end of GPIIbL. Three sequence stretches along the C-terminal half of GPIIbH, one sequence stretch in GPIIbL and three sequence stretches within the GPIIIa-(217-421) region were putatively involved in the subunit interface of the heterodimer. Most likely, the N-terminal end of GPIIbL is folded over the N- and C-terminal regions of GPIIbH, and the N-terminal end of GPIIbH is folded against the GPIIbH-(600-700) domain. This map of GPIIb/IIIa does not fit the current accommodation of the amino acid sequence of GPIIb and GPIIIa in the head/two-tails image of the heterodimer obtained by metal-rotary-shadowing electron microscopy.

Dataset Information

Delayed targeting of CD39 to activated platelet GPIIb/IIIa via a single-chain antibody: breaking the link between antithrombotic potency and bleeding?

Publications

Delayed targeting of CD39 to activated platelet GPIIb/IIIa via a single-chain antibody: breaking the link between antithrombotic potency and bleeding?

Similar Datasets

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