Platelets Contain Tissue Factor Pathway Inhibitor-2 Derived from Megakaryocytes and Inhibits Fibrinolysis

• Published in: The Journal of biological chemistry Vol. 289; no. 45; pp. 31647 - 31661
• Main Authors: Vadivel, Kanagasabai, Ponnuraj, Sathya-Moorthy, Kumar, Yogesh, Zaiss, Anne K., Bunce, Matthew W., Camire, Rodney M., Wu, Ling, Evseenko, Denis, Herschman, Harvey R., Bajaj, Madhu S., Bajaj, S. Paul
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 07.11.2014; American Society for Biochemistry and Molecular Biology
• Subjects: Blood Coagulation; Blood Platelets - cytology; Blood Platelets - enzymology; Bone Marrow Cells - cytology; Female; Fetal Blood - enzymology; Fibrinolysis - physiology; Gene Expression Regulation; Glycoproteins - genetics; Glycoproteins - metabolism; Hemostasis; Humans; Ligands; Lipoproteins - genetics; Lipoproteins - metabolism; Megakaryocytes - cytology; Platelet Membrane Glycoprotein IIb - metabolism; Pregnancy; Protease Inhibitors - chemistry; Protein Binding; Protein Structure and Folding; Protein Structure, Tertiary; Surface Plasmon Resonance; Platelet; Protease Inhibitor; Hemostasis; Fibrinolysis; Plasmin
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.M114.569665

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 N-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 binds 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 bind 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 the C-terminal basic region of TFPI-2 is similar to that of TFPI-1 and plays a role in binding to the 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 tissue-type plasminogen activator-induced clot fibrinolysis. In conclusion, TFPI-2 in platelets from normal or pregnant subjects and in plasma from pregnant women binds FV/Va and regulates intrinsic coagulation and fibrinolysis.