The lipoprotein-associated coagulation inhibitor (LACI) is present in vivo in at least three different pools: sequestered in platelets, associated with plasma lipoproteins, and released into plasma by intravenous heparin, possibly from vascular endothelium. In this study we have purified the heparin-relesable form of LACI from post-heparin plasma and show that it is structurally different from lipoprotein LACI. The purification scheme uses heparin-agarose chromatography, immunoaffinity chromatography, and size-exclusion chromatography and results in a 185,000-fold purification with a 33% yield. Heparin- releasable LACI (HRL), as analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, under reducing conditions, appears as a major band at 40 Kd and a minor band at 36 Kd. Immunoblot analysis suggests that the 36-Kd band arises from carboxyl-terminus proteolysis that occurs during the purification. HRL has a specific activity similar to that of HepG2 or lipoprotein LACI. HRL and lipoprotein LACI combine with lipoproteins in vitro while purified HepG2 LACI does not. I125-labeled HRL, injected into a rabbit, is cleared more slowly than I125-labeled HepG2 LACI, which may be due to attachment to lipoproteins in vivo. Preliminary evidence suggests that HRL is associated with vascular endothelium, possibly by attachment to glycosaminoglycans.

Tissue factor (TF) is an integral membrane glycoprotein that is believed to be the physiologic initiator of the blood coagulation cascade. Disruption of the mouse tissue factor gene leads to embryonic lethality between days E9.5-E11.5 of gestation. On E9.5, TF(-/-) embryos appear indistinguishable from their TF(+/+) and TF(+/-) littermates. By E10.5, TF(-/-) embryos are severely growth retarded, appear nearly bloodless, and are in most cases dead. Initial observations suggest that TF(-/-) embryos are dying of circulatory failure. Approximately 15% of the TF(-/-) embryos survive beyond E10.5, but none complete gestation. Heterozygotes appear normal and free of bleeding complications.

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type protease inhibitor that binds to and inactivates factor Xa directly, and in a factor Xa-dependent fashion inhibits the factor VIIa/tissue factor catalytic complex. TFPI is a slow, tight-binding, competitive, and reversible inhibitor of factor Xa, in which the formation of an initial encounter complex between TFPI and factor Xa is followed by slow isomerization to a final, tightened complex. Wild-type recombinant TFPI (rTFPI), expressed in mouse C127 cells, separates into two forms on heparin-agarose chromatography that elute at 0.3 mol/L and 0.6 mol/L NaCl. Western blot analysis shows that both forms contain the N- terminus of full-length TFPI, but only rTFPI(0.6) is recognized by an antibody directed against the C-terminus. rTFPI(0.3) and rTFPI(0.6) inhibit factor Xa with 1:1 stoichiometry and inhibit factor VIIa/tissue factor equally in an endpoint-type assay. However, rTFPI(0.6) is a more potent inhibitor than rTFPI(0.3) of coagulation in normal plasma induced by either factor Xa or tissue factor. The initial inhibition of factor Xa (less than 5 seconds) produced by rTFPI(0.6) is several-fold greater than that produced by rTFPI(0.3), presumably reflecting a lower Ki of the immediate encounter complex between factor Xa and TFPI. The differential effect of these forms of TFPI on tissue factor-induced coagulation in normal plasma appears to be directly related to their ability to inhibit factor Xa. To confirm the role of the C-terminal region of TFPI in optimal factor Xa inhibition, a carboxy-terminal mutant of rTFPI, which is truncated after leucine 252 and thus lacks the basic sequence K T K R K R K K Q R V K (residues 254–265), was expressed in C127 cells. This form of rTFPI elutes from heparin-agarose at 0.28 mol/L NaCl and inhibits factor Xa at a rate that is slower than rTFPI(0.3). The Ki(final)s for factor Xa inhibition by rTFPI(0.6), rTFPI(0.3), and rTFPI1–252 are 3.1 +/- 0.6, 19.6 +/- 0.8, and 19.6 +/- 3.0 pmol/L, respectively.

In contrast to the other well-studied vitamin K-dependent proteins that circulate in plasma, protein Z antigen is much more variable. The concentration in plasmas collected in EDTA from 455 normal, healthy donors is normally distributed with a mean of 2.9 micrograms/mL (46 nmol/L) and a SD of 1.0 microgram/mL (95% interval of 32% to 168% of the mean). No significant correlation to age or sex could be detected. In comparison, the concentration of protein C antigen measured with the same type of assay on the same 455 samples has a log normal distribution with a mean of 4.0 micrograms/mL (65 nmol/L) and a 95% interval of 70% to 138% of the mean. Also in marked contrast to other plasma vitamin K-dependent proteins, the total protein Z antigen level is extremely low in patients on stable warfarin therapy (range 1% to 16% of normal). Moreover, even though greater than 95% of the antigen in normal plasmas adsorbs to barium citrate (a crude reflection of the presence of gamma-carboxyglutamic acid (Gla) residues), in the patients taking warfarin almost all of the small amount of the antigen failed to adsorb, suggesting that virtually no protein Z had its full complement of Gla residues. Total protein C antigen in the same 25 patients averaged 53% of normal (34% to 72%) and 54% (average) of the total remaining antigen still adsorbed to barium citrate. The concentration of protein Z antigen in the plasma of a normal individual given a loading dose of warfarin fell at an initial rate of approximately 20% a day, indicating a plasma half-life (t1/2) of 2 to 3 days.