Abstract Number: PB0258
Meeting: ISTH 2020 Congress
Background: Previously, we showed that histidine-rich glycoprotein (HRG) binds factor (F) XIIa with high affinity, inhibits FXII autoactivation and FXIIa-mediated activation of FXI, and attenuates ferric chloride-induced arterial thrombosis in mice. Therefore, HRG downregulates the contact pathway in vitro and in vivo.
Aims: To identify the domain on HRG responsible for contact pathway inhibition.
Methods: PCR-amplified HRG domain constructs (N-terminal [N1, N2, and N1N2], proline-rich regions [PRR1, PRR2], histidine-rich region [HRR], and C-terminal [COOH]) were subcloned into maltose-binding protein (MBP)-containing pMALc5e vectors and expressed in E. coli. Plasma-derived HRG and recombinant domain constructs were examined for their (a) affinities for FXII, FXIIa, and polyphosphate by surface plasmon resonance (SPR) and immunoassay, and their effects on (b) polyphosphate (70mer) mediated FXII autoactivation and (c) the activated partial thromboplastin time (APTT) in HRG-depleted human plasma.
Results: Using SPR, plasma-derived HRG and recombinant domain constructs bind FXIIa, but not FXII or β-FXII. HRR binds FXIIa with a Kd of 129 nM, similar to that of HRG (Kd 51 nM). By immunoassay, HRR and PRR2 bind immobilized biotin-polyphosphate with high affinity (Kd 12 nM and 26 nM, respectively), whereas HRG binds with a Kd of 152 nM. HRG and HRR inhibit polyphosphate-mediated FXII autoactivation by over 85% with IC50 values of 200 and 500 nM, respectively. PRR2 has an IC50 of 652 nM, whereas the remaining domains have significantly lower potencies (IC50 >800 nM). In the APTT, each of the constructs has little effect (prolongation < 2-fold), except HRR which, like HRG, significantly prolongs clotting in HRG-depleted plasma by 7-fold.
Conclusions: The interaction of HRG with FXIIa and polyphosphate is predominantly mediated by the HRR domain. Therefore, by downregulating the contact system, HRR is the domain responsible for the anticoagulant properties of HRG.
To cite this abstract in AMA style:Truong TK, Malik RA, Ajewole E, Fredenburgh JC, Stafford AR, Leslie BA, Madarati HM, Kretz CA, Weitz JI. Identification of the Histidine-rich Glycoprotein Domain Responsible for Contact Pathway Inhibition [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/identification-of-the-histidine-rich-glycoprotein-domain-responsible-for-contact-pathway-inhibition/. Accessed May 6, 2021.
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