Altering the Alignment of the Active Site of Factor VIIa by Raising Tissue Factor above the Phospholipid Surface.
Authors: Kerestes Emily M. , Morrissey James H. 
Published: October 2019

Tissue factor (TF), an integral membrane protein, is the protein cofactor for coagulation factor VIIa (VIIa). The membrane-bound enzyme complex of TF and VIIa (TF:VIIa) initiates coagulation by activating factor X to Xa via limited proteolysis. Free VIIa has limited enzymatic activity, but when in complex with TF, its activity -- particularly its ability to cleave macromolecular substrates -- dramatically increases. VIIa in complex with truncated, soluble TF (sTF; consisting of the isolated extracellular domain) has much lower procoagulant activity than VIIa in complex with membrane-anchored TF in a suitable phospholipid membrane. When TF binds to VIIa, it re-aligns the active site of VIIa relative to the membrane surface, and it is hypothesized that one of the essential functions of protein cofactors in the clotting cascade is to position the active sites of their cognate proteases the proper distance and orientation above the membrane surface for optimal attack on the scissile bonds of membrane-bound protein substrates. We are testing how this alignment contributes to catalysis by altering the distance of TF above the phospholipid surface, thereby altering the position of VIIa’s active site above the membrane. We created a number of chimeras between the extracellular domain of TF and portions of the integral membrane protein, P-selectin, in order to raise TF varying distances above the phospholipid surface. TFCR9 is the shortest chimera, containing the 9th consensus repeat domain (CR) and transmembrane domain of P-selectin. TFCR8.9 contains the 8th and 9th CR domains as well as the transmembrane domain of P-selectin. TFLCR8.9 is identical to TFCR8.9 except that a flexible linker of four glycines and a serine residue repeated three times, (G4S)3, separates the TF and P-selectin sequences. All of the chimeras allosterically activated VIIa to the same extent that sTF or TF did (measured by cleavage of small peptidyl-amide substrates) confirming that the chimeras are properly folded. VIIa bound to the chimeras with Kd values ranging from 2.6 to 4.2 nM, which is equivalent to its interaction with sTF (Kd, 2-5 nM) rather than with TF in phospholipid membranes (Kd < 50 pM). This also confirms that the chimeras are properly folded and strongly suggests that binding interactions do not take place between VIIa and the membrane surface when VIIa binds to these chimeras. The chimera:VIIa complexes had reduced ability to activate X compared to TF:VIIa complexes, and the reduction was greater with the longer chimera, TFCR8.9, demonstrating a three-fold reduction in activity compared to TF. The chimeras had greatly reduced procoagulant activity compared to TF, with only 6-10% the specific activity of TF. Interestingly, the chimeras had significantly higher procoagulant activity if the chimera:VIIa complexes were pre-assembled before adding plasma, suggesting that the chimeras may have a defect in capturing VII and/or supporting its conversion to VIIa. These results imply that maintaining the proper distance between TF and the membrane surface is important not only for TF:VIIa to activate macromolecular substrates, but possibly also for TF to capture and bind membrane-bound VII and/or VIIa.