Platelets Contain Tissue Factor Pathway Inhibitor-2 Derived from Megakaryocytes and Inhibits Fibrinolysis [Protein Structure and Folding]

September 28th, 2014 by Vadivel, K., Ponnuraj, S.-M., Kumar, Y., Zaiss, A. K., Bunce, M. W., Camire, R. M., Wu, L., Evseenko, D., Herschman, H. R., Bajaj, M. S., Bajaj, S. P.

Tissue factor pathway inhibitor-2 (TFPI-2) is a homologue of TFPI-1 and contains three Kunitz-type domains and a basic C-terminus region. The NH2-terminal domain of TFPI-2 is the only inhibitory domain, and it inhibits plasma kallikrein, factor XIa and plasmin. However, plasma TFPI-2 levels are negligible (≤20 pM) in the context of influencing clotting or fibrinolysis. Here, we report that platelets contain significant amounts of TFPI-2 derived from megakaryocytes. We employed RT-PCR, Western blotting, immunohistochemistry and confocal microscopy to determine that platelets, MEG-01 megakaryoblastic cells, and bone marrow megakaryocytes contain TFPI-2. ELISA data reveal that TFPI-2 binds Factor V (FV) and partially B-domain deleted FV (FV-1033) with Kd ~9 nM, and FVa with Kd ~100 nM. Steady state analysis of surface plasmon resonance data reveal that TFPI-2 and TFPI-1 bind FV-1033 with Kd ~36-48 nM, and FVa with Kd ~252-456 nM. Further, TFPI-1 (but not TFPI-1161) competes with TFPI-2 in binding to FV. These data indicate that C-terminal basic region of TFPI-2 is similar to that of TFPI-1 and plays a role in binding to FV B-domain acidic-region. Using pull-down assays and Western blots, we show that TFPI-2 is associated with platelet FV/FVa. TFPI-2 (~7 nM) in plasma of women at the onset of labor is also, in part, associated with FV. Importantly, TFPI-2 in platelets and in plasma of pregnant women inhibits FXIa and tPA-induced clot fibrinolysis. In conclusion, TFPI-2 in platelets from normal or pregnant subjects, and plasma from pregnant women binds FV/Va and regulates intrinsic coagulation and fibrinolysis.