Matings of mice heterozygous for a protein C (PC) deficient allele, produced by targeted PC gene inactivation, yielded the expected Mendelian distribution of PC genotypes. Pups with a total deficiency of PC (PC Ϫ / Ϫ ), obtained at embryonic day (E) 17.5 and at birth, appeared to develop normally macroscopically, but possessed obvious signs of bleeding and thrombosis and did not survive beyond 24 h after delivery. Microscopic examination of tissues and blood vessels of E17.5 PC Ϫ / Ϫ mice revealed their normal development, but scattered microvascular thrombosis in the brain combined with focal necrosis in the liver was observed. In addition, bleeding was noted in the brain near sites of fibrin deposition. The severity of these pathologies was exaggerated in PC Ϫ / Ϫ neonates. Plasma clottable fibrinogen was not detectable in coagulation assays in PC Ϫ / Ϫ neonatal mice, suggestive of fibrinogen depletion and secondary consumptive coagulopathy. Thus, while total PC deficiency did not affect the anatomic development of the embryo, severe perinatal consumptive coagulopathy occurred in the brain and liver of PC Ϫ / Ϫ mice, suggesting that a total PC deficiency is inconsistent with short-term survival. ( J. Clin. Invest. 1998.

Blood coagulation in vivo is initiated by factor VII (FVII) binding to its cellular receptor tissue factor (TF). FVII is the only known ligand for TF, so it was expected that FVII-deficient embryos would have a similar phenotype to TF-deficient embryos, which have defective vitello-embryonic circulation and die around 9.5 days of gestation. Surprisingly, we find that FVII-deficient (FVII-/-) embryos developed normally. FVII-/- mice succumbed perinatally because of fatal haemorrhaging from normal blood vessels. At embryonic day 9.5, maternal-fetal transfer of FVII was undetectable and survival of embryos did not depend on TF-FVII-initiated fibrin formation. Thus, the TF-/- embryonic lethal and the FVII-/- survival-phenotypes suggest a role for TF during embryogenesis beyond fibrin formation.

The contributions to functional phospholipid (PL) binding of the cluster of amino acid side chains of human protein C (PC) comprising F4, L5, and L8 have been assessed by construction of mutants of PC and activated protein C (APC) designed wherein a hydrophilic side chain replaced the wild-type hydrophobic groups at these positions. The PL-dependent plasma-based anticoagulant activities of [F4Q]-r-APC and [L8Q]r-APC were severely reduced to 5% and < 2%, respectively, of wild-type r-APC. Activity losses of the mutants toward inactivation of coagulation factor VIII, measured in the complete in vitro tenase system, have also been observed. As evidenced through Ca(2+)-induced intrinsic fluorescence changes, both [F4Q]r-PC and [L8Q]r-PC were able to adopt Ca(2+)-dependent conformations that appeared similar to that of wtr-PC, ruling out shortcomings associated with such Ca(2+)-induced transitions as the basis for their anticoagulant activity losses. However, despite this, [L8Q]r-PC showed greatly defective macroscopic binding properties to PL vesicles, as did to a lesser extent [F4Q]r-PC. These findings were similar to those reported previously for [L5Q]r-PC/APC [Zhang, L., & Castellino, F. J. (1994) J. Biol. Chem. 269, 3590-3595]. We thus propose that the PL-dependent activity losses of these mutants are related to their suboptimal binding to PL or to their misorientation on the PL surface leading to poor alignment of the active sites of the r-APC mutants with the complementary cleavage sites on fVIII/fVIIIa and fV/fVa.(ABSTRACT TRUNCATED AT 250 WORDS)

We have previously proposed that a cluster of surface-exposed hydrophobic amino acids, viz., F4, L5, and L8, present at the amino-terminus of the Ca(2+)-bound form of gamma-carboxyglutamic acid domain (GD) of human protein C (PC), contributes a substantial portion of the total functional binding energy of PC and its activated form, APC, to acidic phospholipid (PL) vesicles. A deeper understanding of the importance of the hydrophobic nature of sequence position 5, and the particular relevance of leucine at that location, was sought by examination of the properties of a series of mutant proteins containing A5, V5, I5, and W5 as replacements for L5 in recombinant (r)-PC and APC. The Ca(2+)- and PL-dependent plasma-based anticoagulant activities of [L5A]r-APC, [L5V]r-APC, [L5I]r-APC, and [L5W]r-APC were determined to be approximately 28%, 51%, 98%, and 105%, respectively, of that of wild-type r-APC. A similar trend in activities of the mutant enzymes was observed in in vitro factor V/Va and factor VIII/VIIIa inactivation assays. Apparently normal Ca(2+)-dependent conformations were adopted by each of the mutant proteins, but the Ca(2+)-bound form of [L5A]r-PC was relatively the most defective of the mutants in its binding to PL. These results confirm the importance of the hydrophobic character at sequence position 5 as critical to the functional binding of PC to PL.