New Insights Into The Mechanisms Of Ferric Chloride-Induced Thrombosis: a Reductionist Microfluidic Approach

The ferric chloride (FeCl3) murine model of thrombosis is used extensively in hematology, in which application of ferric chloride (200 mM to 1M) to the adventitial side of mouse arteries results in occlusive thrombosis. This effect has historically been attributed to the denudation of endothelial cells by way of free iron-induced oxidative stress. Recently, a well-designed SEM analysis revealed that endothelial cells actually remain intact, and that erythrocytes adhere to the endothelium prior to platelets (Barr, et al, Blood, 2013). The same study found that washed red blood cells exposed to FeCl3 adhere to endothelial cells in vitro . These novel findings inspired us to comprehensively investigate the mechanisms of FeCl3-induced thrombosis via an in vitro reductionist approach: interrogating the effect of FeCl3 on individual blood components in the absence and presence of endothelial cells. To this end, a microfluidic channel was designed to approximately recreate the in vivo blood/FeCl3 interface ([Fig 1][1]). By using a microfluidic platform, we were able to tightly control ferric chloride influx; determine the shear rate in the “artery” ; visualize the effects of ferric chloride on isolated blood components in a controlled environment; and quantitatively track clot characteristics such as composition, size, and time to occlusion. We found that a wide range of ferric chloride concentrations causes aggregation of whole blood in our system, even in the absence of endothelial cells. In addition, FeCl3 causes “clotting” of plasma proteins and blood cells in a dose-dependent manner ([Fig 2A][2]). Once aggregation is initiated, however, FeCl3 concentration does not affect the time required for stable clot formation. Interestingly, at low concentrations, the presence of blood cells are the slowest to form clots while cell/plasma mixtures (platelet-rich plasma and whole blood) are the fastest, perhaps due to plasma proteins forming net-like structures that enhances cell binding and aggregation. ([Fig 2B][2]) ![Figure][3] ![Figure][3] This universal aggregation effect of FeCl3 led us to posit a charge-based hypothesis. Mechanistically, as Fe3+ ions bind negatively charged surfaces, such as cell membranes and plasma proteins, the intrinsic charge equilibrium of blood may shift, resulting in protein and cell aggregation. Indeed, erythrocyte suspensions exposed to Al3+, which is chemically similar to Fe3+, resulted in cell aggregation. In contrast, erythrocyte suspensions exposed to Cr3+, which forms substitutionally inert water complexes in solution and thus would not be expected to bind protein and cell surfaces, did not result in aggregation ([Fig 2C][2]). This suggests that inner-sphere complexation of Fe(III) to plasma proteins/endothelial or blood membranes may have a mechanistic role in FeCl3-induced thrombosis and further explains how the sheet-like protein aggregates could act as Fe3+ linkers that effectively enhances cell binding. However, a complete understanding of FeCl3-induced thrombosis requires the inclusion of endothelial cells. To that end, we used our previously developed technique to “endothelialized” microfluidic channels (Tsai, et al, JCI , 2012) ([Fig 3A][4]). We found that the effect of FeCl3 on endothelial cells is likewise concentration dependent: they remain viable at low FeCl3 concentrations (<50 mM) but have compromised cell membranes at higher concentrations ([Fig 3B][4]). However, the dead endothelial cells remain intact, corroborating the results of Barr et al. As shown in [Fig 3C][4], FeCl3-induced clotting of platelet-rich plasma nevertheless occurs when endothelial cells are alive. This would suggest that at varying apparent FeCl3 concentrations, the primary means of clotting will differ. If endothelial cells are dead, it is posited that a normal clotting cascade would proceed, in addition to the Fe3+ based aggregation of negatively charged blood components. Overall, our microfluidic studies reveal that FeCl3 has multiple dose-dependent effects involving aggregation of different components of blood and endothelial cells. In addition, we discovered a novel mechanism in which charge effects may have a role in FeCl3-induced thrombosis that is independent of biological ligand-receptor interactions. Further studies are needed to determine how these charge effects interact with the biological aspects of FeCl3-induced thrombosis and the implications for data interpretation when using this model system. ![Figure][3] Disclosures: No relevant conflicts of interest to declare. [1]: #F1 [2]: #F2 [3]: pending:yes [4]: #F3