Subcellular Localozation of TFPI in Human Umbilical Vein Endothelial Cells (HUVEC)
Tissue factor pathway inhibitor (TFPI) is a serine protease inhibitor which regulates tissue factor (TF) induced blood coagulation by inhibiting the action of factor VIIa-TF complexes and factor Xa. In plasma, more than 80 % of the TFPI is bound to lipoproteins, 10 -20 % is carrier free, whereas platelets contains a small amount of TFPI. A major pool is probably assosiated with the vascular endothelium (1). This pool may have a crucial regulatory function because the endothelial surface serves as a binding site for factor VII/VIIa, mediating the activation of the extrinsic coagulation system.The normal distribution of TFPI within human umbilical vein endothelial cells (HUVEC) was examined on ultrathin cryosections labeled with specific antibodies and colloidal gold probes (2,3). Label was seen associated with the cell surface and with many intracellular structures. The surface label was patchy, the signal intensity varied between cells and between different parts of the same cell. Intracellular TFPI was found in many compartments including the Golgi complex (fig.l) a finding that was expected as these cells are the main site of TFPI synthesis, and endocytic organelles. Anti TFPI-antibodies were seen over organelles that also contained 3-(2,4-Dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP), a compound that accumulates in organelles with low lumenal pH. DAMP was visualized using anti-DNP antibodies (fig.2). Structures that contained exclusively TFPI or DAMP were also seen, suggesting that TFPI did not enter all endocytic organelles and was not exclusively localized to low pH compartments. The TFPI-antibodies also colocalized with specific markers of endosomes (MPR) (fig.3) and lysosomes (LAMP-1) (fig.4)(4). TFPI colocalized with both, demonstrating the appearence of TFPI also in these endocytic compartments.