Hypoalbuminemia Increases Clot Fibrin Network Density and Reduces Fibrinolytic Susceptibility through a Paraoxonase 1 Dependent Mechanism

Introduction
Hypoalbuminemia is a predictor for thrombotic cardiovascular disease. Albumin is a negative acute phase reactant and may thus be a biomarker for thrombo-inflammation, but some data suggest a mechanistic role for albumin in fibrinolysis. The aim of this study was to investigate the mechanistic role of albumin in fibrinolysis. We hypothesized that albumin modifies clot structure and fibrinolytic susceptibility in two well-established rodent models of hypoalbuminemia and analbuminemia.

Methods
Blood or plasma from a hypoalbuminemic nephrotic syndrome rat model and an albumin knockout rat model were analyzed by thromboelastometry, clot lysis assays, fibrin clot turbidity, fibrin clot density, and plasma clot immunoblotting. Some studies were conducted without vs. with recombinant albumin repletion. We mined albumin and fibrin clot proteome datasets to identify proteins both carried by albumin and incorporated in clots, hypothesizing that albumin delivers proteins that alter fibrinolytic susceptibility to the forming clot.

Results
Albumin levels were proportional to fibrinolysis in a clot lysis assay in both nephrotic and albumin knockout rat plasma. Hypoalbuminemia accelerated fibrin clot formation in nephrotic rat plasma and fibrin clot network density was increased in hypoalbuminemic plasma clots from both nephrotic rats (p<0.01) and albumin knockout rats (p<0.001). Recombinant albumin repletion to healthy control concentrations corrected fibrin network density and fibrinolysis in both nephrotic rat and human plasma as well as in albumin knockout rat plasma (p<0.05). Paraoxonase 1 (PON1) enzyme was identified in both albumin and fibrin clot proteomes. Naringenin, a PON1-specific inhibitor decreased fibrin clot lysis in normal rat plasma in a dose-dependent manner (IC50= 1 uM; p<0.001), but did not alter clot lysis in albumin knockout plasma (p>0.9). Moreover, naringinen ameliorated the ability of recombinant albumin repletion to correct hypofibrinolysis (p<0.001 vs control).

Conclusions
Dense hypoalbuminemic clots demonstrated hypofibrinolysis, and albumin repletion corrected both clot density and hypofibrinolysis. PON1 inhibition decreased clot lysis in plasma with normal albumin levels and ameliorated the ability of albumin repletion to correct fibrinolysis in albumin knockout plasma. Collectively, these data suggest a mechanistic link between hypoalbuminemia and hypofibrinolysis and suggest that albumin indirectly regulates fibrinolysis via its effects on clot density, through a PON1 dependent mechanism.