Chemerin Bioactivity Is Regulated by Factor XIa: A Novel Interface Linking Between Coagulation, Hemostasis and Immunity
Authors: Yamaguchi Yasuto , Morser John , Leung Lawrence L. 
Published: October 2019

Abstract 2258

Proteolytic modulation of chemokines by extracellular proteases, either released upon leukocyte degranulation or activated by the coagulation and fibrinolytic cascades, plays an important role in inflammation and immunity. Chemerin is a novel chemoattractant involved in innate and adaptive immunity and adipogenic differentiation via its receptor, CMKLR1, expressed on dendritic cells, macrophages and adipocytes. Chemerin circulates as an inactive precursor (chem163S) in blood whose bioactivity is regulated through proteolytic processing at its C-terminus. The most potent form of chemerin (chem157S) can be generated from chem163S in vitro by elastase directly or by sequential cleavages by plasmin to chem158K and then plasma carboxypeptidase-2 (CPB2, also termed thrombin-activatable fibrinolysis inhibitor TAFIa) or CPN to chem157S. Thus proteolytic processing is essential for chemerin activation. We have recently developed specific ELISAs for chem163S, 158K and 157S and showed that chem158K is present in plasma at a low level compared to chem163S. In contrast chem158K is the dominant form in synovial fluids of patients with inflammatory arthritis, suggesting that cleavage of chem163S at position 158K is a major step in the activation of chemerin in vivo. Although chem158K is present in vivo, it is unknown if it is generated by plasmin with a second cleavage by CPB2 or CPN to generate the active isoform, chem157S, as CPB2 inhibits plasmin generation. In this study we screened a panel of 21 proteases involved in coagulation, fibrinolysis and inflammation to determine which one could cleave the chemerin precursor chem163S to modulate its bioactivity. We carried out in vitro enzyme assays using 100 nM enzyme and 10 μM chem163S as a substrate, determining the results by SDS-PAGE and mass spectroscopy. Among the 21 proteases tested, cathepsin G, elastase, tryptase, plasmin, chymase, protease 3 and factor XIa could cleave chem163S. Mass spectroscopy identified tryptase, plasmin and factor XIa as enzymes that can digest chem163S to generate chem158K. Based on our previous observation that platelets are a rich cellular source of chemerin and release partially cleaved chemerin upon thrombin activation, we focused on factor XIa and compared its potency to plasmin. Factor XIa converted chem163S into chem158K in a dose-dependent manner whereas incubation with high concentrations of plasmin (300 nM) resulted in production of a non-specific smaller fragment, showing that the specificity of factor XIa was better than that of plasmin. Time-course studies showed that factor XIa and plasmin cleaved chem163S at comparable rates. Finally, we found that the fraction of chem158K to levels of total chemerin as determined by ELISA in factor XI-deficient plasma was lower than that in factor II-, IX-, X-deficient plasmas as well as normal pooled plasma. These data demonstrated that factor XIa could be the enzyme responsible for the first step of chemerin activation in the plasma compartment, ultimately leading to downstream inflammatory responses and provide a novel molecular link between coagulation, hemostasis and immunity.

Disclosures:

No relevant conflicts of interest to declare.