Analysis of the Protein C System Under Flow and the Impact of Red Blood Cells

Introduction The rapid coagulation response to vascular injury is mediated by the formation of the extrinsictenase, intrinsictenase, and prothrombinase complexes. The prothrombotic response to injury is down-regulated by the presence of circulating active protease inhibitors as well as the protein C pathway. Protein C is activated by the thrombin-thrombomodulin complex; activated protein C (APC) then regulates thrombin generation by proteolytically inactivating factors Va and VIIIa, cofactors of the procoagulant prothrombinase and intrinsic tenasecomplexes, respectively. Previous reports have analyzed the biochemistry of the protein C system in closed systems. Our goal is to characterize the behavior of the protein C system under flow as well as the impact of circulating cells on the activation of protein C.

Methods Experiments were conducted in phospholipid (3:1 ratio of synthetic phosphatidylcholine and phosphatidylserine) coated capillaries containing rabbit thrombomodulin (TM) that were preloaded with α-thrombin (αIIa) or recombinant meizothrombin (rMZ). Protein C (PC) activation was evaluated under flow at pH 7.4 and 37°C in either a buffered solution containing 2 mM CaCl₂ and PC at its mean physiological concentration (65 nM) or in a mock blood mixture containing 60% of buffer containing 65 nM PC and 40% freshly prepared washed red blood cells; shear rates ranged from 100-1000 s^-1. Capillary effluents were collected and then assayed for APC levels using a modified aPTT assay. To establish whether PC activation is under dilutional or diffusional control, the steady state concentrations of APC achieved at different shears were normalized to the residence time of one capillary volume specific for each shear rate. The efficiency of PC activation was also analyzed by normalizing the amount of APC generated to the amount of PC present in the mixture (1.3 pmol PC in buffer only vs. 0.78 pmol of PC in mock blood trials).

Results At low shear rates (100 s^-1 and 250 s^-1) in the buffer only system the rMZ•TM complex generates 42-55% higher levels of APC than the αIIa•TM complex. Protein C activation by the αIIa-TM complex appears to be dilutionally controlled at shear rates ≥ 500 s^-1, while diffusionally controlled at lower shear rates (≤ 250 s^-1). The inclusion of red blood cells in the reaction system under flow resulted in a broader range of dilutional control (≥ 250 s^-1) compared to the buffer only system (≥ 500 s^-1). Normalization of the data to account for the differential amount of protein C present in a given volume indicate a two-fold greater efficiency of PC activation in the presence of red blood cells (14.7 ± 1.2 mol APC•mol^-1 PC•min^-1•cm^-2) compared to buffer alone (6.7 ± 0.6 mol APC•mol^-1 PC•min^-1•cm^-2).

Conclusions In the presence of catalytically inert red blood cells the activation of protein C is regulated by diffusion only at the lowest shear rates tested (100 s^-1). These data suggest that the dynamics and aggregation of red blood cell effects are shear dependent as red blood cells deform and migrate toward the center of the channel at increasing shear rates. We can hypothesize that at high shear rates (≥ 500 s^-1), when the levels of APC generated in the red blood cell system and buffer only system are similar, the excluded volume created by the red blood cells agglomerated at the center of the capillary leaves a cell-free region adjacent to the wall which is large enough to accommodate the space needed for surface catalysis (depletion zone). Indeed the adjustment of PC concentration for excluded volume in red blood cell solutions yields the same concentration of APC generated as in the buffer solution. However, at low shear rates (100 s^-1) the red blood cells do not create a distinct channel and the depletion zone extending from the capillary wall overlaps with red blood cells and maintains the diffusional control of the protein C system. These studies provide a foundation for studying the impact of circulating cells on the biochemistry of the coagulation cascade