Abstract 113: Monocytes Embedded in Plasma Clots Synthesize Coagulation Factors: Balancing Procoagulant and Anticoagulant Signals

Abstract

Objective: Previous studies have demonstrated that vascular cells acutely regulate fibrin clot structure and stability. However, the mechanisms by which fibrin clots push vascular cells, in particular leukocytes, to synthesize pro-and anti-coagulant proteins is unknown. Here, we characterize monocyte-derived pro-and anti-coagulant activity in clots. Methods: Human monocytes were isolated from whole blood using CD14 microbeads. In parallel, cell-free plasma was isolated and clot formation induced in the presence of monocytes by the addition of tissue factor and calcium. RNA was isolated using RNAzol. Cell surface tissue factor (TF), tissue factor pathway inhibitor (TFPI), urokinase plasminogen activator (uPA) and its receptor (uPAR) as well as plasminogen activator inhibitor-1 (PAI-1) was measured by ELISA. TF activity was measured using a chromogenic factor Xa assay. Results: Monocytes embedded within plasma clots transcribed mRNA for uPAR (2.3-fold), uPA (9.5-fold), TF (9.2-fold), TFPI, and PAI-1 (6.2-fold) in a time-dependent manner. Consistent with this transcriptional response, increased levels of TF, TFPI, and uPAR protein were visually detected on the monocyte surface 18-hours after plasma clot formation. Accumulation of TF, TFPI and uPAR protein was blocked by actinomycin D and cycloheximide, indicating protein synthesis was dependent on transcription of new mRNA. TF-activity was similarly increased (8.9-fold) on the surface monocytes (i.e., increase in plasma-clot versus suspension monocytes). Lastly, active PAI-1 (11.1-fold increase), but not uPA, increased in the culture supernatant over time. Conclusions: Plasma clot formation induced robust increases in coagulation-related mRNAs and their corresponding proteins. The balance between the transcription of pro- and anti-coagulant mRNAs and their subsequent translation into bioactive protein likely plays key roles in clot development, stability and resolution.