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Prochemerin cleavage by factor XIa links coagulation and inflammation.

ABSTRACT: Chemerin is a chemoattractant and adipokine that circulates in blood as inactive prochemerin (chem163S). Chem163S is activated by a series of C-terminal proteolytic cleavages resulting in diverse chemerin forms with different levels of activity. We screened a panel of proteases in the coagulation, fibrinolytic, and inflammatory cascades to identify those that process prochemerin in plasma. Factor XIa (FXIa) cleaved chem163S, generating a novel chemerin form, chem162R, as an intermediate product, and chem158K, as the final product. Processing at Arg162 was not required for cleavage at Lys158 or regulation of chemerin bioactivity. Contact phase activation of human platelet-poor plasma by kaolin led to cleavage of chem163S, which was undetectable in FXI-depleted plasma and markedly enhanced in platelet-rich plasma (PRP). Contact phase activation by polyphosphate in PRP resulted in 75% cleavage of chem163S. This cleavage was partially inhibited by hirudin, which blocks thrombin activation of FXI. After activation of plasma, levels of the most potent form of chemerin, chem157S, as well as inactive chem155A, increased. Plasma levels of chem163S in FXI-deficient patients were significantly higher compared with a matched control group (91 ± 10 ng/mL vs 58 ± 3 ng/mL, n = 8; P < .01) and inversely correlated with the plasma FXI levels. Thus FXIa, generated on contact phase activation, cleaves chem163S to generate chem158K, which can be further processed to the most active chemerin form, providing a molecular link between coagulation and inflammation.

SUBMITTER: Ge X

PROVIDER: S-EPMC5774209 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Prochemerin cleavage by factor XIa links coagulation and inflammation.

Ge Xiaomei X Yamaguchi Yasuto Y Zhao Lei L Bury Loredana L Gresele Paolo P Berube Caroline C Leung Lawrence L LL Morser John J

Blood 20171120 3

Chemerin is a chemoattractant and adipokine that circulates in blood as inactive prochemerin (chem163S). Chem163S is activated by a series of C-terminal proteolytic cleavages resulting in diverse chemerin forms with different levels of activity. We screened a panel of proteases in the coagulation, fibrinolytic, and inflammatory cascades to identify those that process prochemerin in plasma. Factor XIa (FXIa) cleaved chem163S, generating a novel chemerin form, chem162R, as an intermediate product, ...[more]

PMID: 29158361

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Project description:Alpha-1 antitrypsin (AAT, also known as alpha-1 proteinase inhibitor or SERPINA1) is the most abundant member of the serpin superfamily found in human plasma. The naturally occurring variant AAT M358R, altered at the P1 position of the critical reactive center loop (RCL), is re-directed away from inhibition of AAT's chief natural target, neutrophil elastase, and toward accelerated inhibition of thrombin (FIIa), kallikrein (Kal), and other proteases such as factor XIa (FXIa). FXIa is an emerging target for the development of antithrombotic agents, since patients with FXI deficiency are protected from thromboembolic disease and do not exhibit a strong bleeding tendency. Previously, we used phage display, bacterial lysate screening, and combinatorial mutagenesis to identify AAT-RC, an engineered AAT M358R with additional changes between RCL positions P7-P3', CLEVEPR-STE [with changes bolded and the P1-P1' (R358-S359) reactive center shown as R-S]. AAT-RC was 279- and 16-fold more selective for FXIa/IIa or FXIa/Kal than AAT M358R; the increased selectivity came at a cost of a 2.3-fold decrease in the rate of FXIa inhibition and a 3.3-fold increase in the stoichiometry of inhibition (SI). Here, we asked which alterations in AAT-RC were most important for the observed increases in selectivity for FXIa inhibition. We back-mutated AAT-RC to AAT-RC-1 (P7-P3' FLEVEPRSTE), AAT-RC-2 (P7-P3' FLEAEPRSTE), and AAT RC-3 (P7-P3' FLEAIPR-STE). Proteins were expressed as cleavable, hexahistidine-tagged glutathione sulfotransferase fusion proteins in E. coli and purified by proteolytic elution from glutathione agarose, with polishing on nickel chelate agarose. Selectivity for FXIa over Kal of AAT-RC-1, -2, and -3 was 14, 21, and 2.3, respectively. AAT-RC-2 inhibited FXIa 31% more rapidly than AAT M358R, with the same SI, and enhanced selectivity for FXIa over Kal, FXa, FXIIa, activated protein C, and FIIa of 25-, 130-, 420-, 440-, and 470-fold, respectively. Structural modeling of the AAT-RC-2/FXIa encounter complex suggested that both E (Glu) substitutions at P3 and P3' may promote FXIa binding via hydrogen bonding to K192 in FXIa. AAT-RC-2 is the most selective and active AAT variant reported to date for FXIa inhibition and will be tested in animal models of thrombosis and bleeding.

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Prochemerin cleavage by factor XIa links coagulation and inflammation.

Publications

Prochemerin cleavage by factor XIa links coagulation and inflammation.

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