Prochemerin Cleavage By Factor XIa Links Adipogenesis, Inflammation and Coagulation

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. Cleavage of chem163S at Lys158 generates chem158K, which has low bioactivity, with subsequent cleavage by the basic carboxypeptidases in plasma, carboxypeptidase N (CPN) or thrombin-activatable CPB2 (also known as thrombin-activatable fibrinolysis inhibitor or TAFI), to produce chem157S, the most active form of chemerin. Inactivation of chem157S to chem155A and smaller forms of chemerin occurs by further proteolytic cleavages.

We have developed specific ELISAs for chem163S, 158K, 157S and 155A. Using these chemerin-specific ELISAs, we found that most of the chemerin in normal human plasma is inactive chem163S, while chem158K is the dominant form in synovial fluids of arthritis patients. To identify the physiological activator of chemerin in plasma, we screened proteases from the coagulation, fibrinolytic and complement pathways. The proteases were incubated with purified chem163S protein in buffer and the resultant products were analyzed by MALDI-TOF mass spectrometry. The results showed that Factor XIa (FXIa) can cleave chem163S, generating a novel chemerin form, chem162R, as an intermediate product, and chem158K, as the final product. We compared the kinetics of FXIa cleavage of chem163S and FIX, the established physiological FXIa substrate. Peptides from chem163S and FIX containing the FXIa cleavage site were subjected to FXIa cleavage followed by HPLC analysis. The catalytic efficiency (k_cat/K_M) of FXIa cleavage of FIX was ~3-fold more efficient than chemerin cleavage, but the hydrolysis of chem163S still occurred at a physiologically relevant rate. By substituting Arg¹⁶² with Ala in chemerin protein/peptides, we found that processing at Arg¹⁶² was not required for cleavage at Lys¹⁵⁸ or regulation of chemerin bioactivity.

We further investigated FXIa cleavage of chem163 in plasma triggered by kaolin to activate FXI. FXI activity was measured by inclusion of a specific chromogenic substrate, and chemerin levels were measured by the chemerin-specific ELISAs. Contact phase activation of human platelet-poor plasma led to generation of FXIa and cleavage of chem163S, which was abolished in FXI-depleted plasma. Platelets markedly enhanced FXI activation and chem163S cleavage in platelet-rich plasma comparing to platelet-poor plasma. Following contact activation, 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 to a matched control group (90.69±9.6 ng/mL vs. 58.11±2.51 ng/mL, n=8; P<0.01) and inversely correlated with the plasma FXI levels. FXIa, generated upon contact phase activation, cleaves chem163S to generate chem158K, which can be further processed to the most active chemerin form, thus providing a molecular link between coagulation, inflammation, innate immunity and adipogenesis.