The accumulation of blood borne tissue factor within a blood clot is thought to be important for normal fibrin generation and may cause propagation of the blood clot into an occlusive thrombus. Tissue factor pathway inhibitor (TFPI), the primary inhibitor of tissue factor activity, is present in platelets where it may have an important role in down regulating the activity of circulating tissue factor. Western blot analysis of gel filtered platelet lysates demonstrates the presence of full-length, 43 kDa TFPI. The lower molecular weight, C-terminally truncated forms of TFPI present in plasma are not present in platelet lysates suggesting that full length TFPI is selectively adsorbed from plasma or that TFPI is synthesized within megakaryocytes. Real time PCR analysis of cDNA produced from highly purified platelet mRNA demonstrates transcripts for both TFPI and TFPI-beta, a truncated form of TFPI produced by alternative splicing. TFPI mRNA is about 3000-fold more abundant than TFPI-beta mRNA. TFPI is not released from platelets following stimulation with the thrombin receptor activating peptide SFLLRN (TRAP) indicating that it is not stored as a soluble alpha-granule protein. Instead, it appears to be associated with platelet membranes. It remains largely insoluble following lysis of platelets by repeated freeze thaw or exposure to hypotonic buffer. There is little to no expression of TFPI on the surface of quiescent platelets as assessed by flow cytometry. TRAP stimulated platelets have surface TFPI that is detected by flow cytometry, but the amount varies between experiments. TFPI is consistently detectable by flow cytometry on the surface of coated-platelets that are produced by stimulation with thrombin/convulxin or thrombin/calcium ionophore. Platelet TFPI activity was measured in eight individuals by determining the rate of factor X activation by factor VIIa/tissue factor. Comparison of quiescent and coated-platelets demonstrates a significant increase in the amount of TFPI activity on the surface the coated-platelets (11.0+/−0.3 vs. 38.7+/−17.2 pmole/10 million platelets; p<0.005), but no significant increase in platelets activated with 20 or 200 micromolar TRAP. Since TFPI associates with the endothelial surface through a GPI-anchor, coated-platelets were treated with phospahtidyl inositol specific phospholipase C (PIPLC) to determine if it would remove TFPI. In experiments where PIPLC removed 56% of the CD59 from the platelet surface, only 22% of the TFPI was removed indicating that if platelet TFPI is GPI-anchored, it is expressed in a manner that is relatively protected from PIPLC. Confocal and electron microscopy studies demonstrate that TFPI is distributed throughout the cytoplasm of quiescent platelets without localization to granules suggesting that it may associate with the platelet cytoskeleton.