Protein Z-dependent protease inhibitor deficiency produces a more severe murine phenotype than protein Z deficiency

Abstract

Protein Z (PZ) is a plasma vitamin K-dependent protein that functions as a cofactor to dramatically enhance the inhibition of coagulation factor Xa by the serpin, protein Z-dependent protease inhibitor (ZPI). In vitro, ZPI not only inhibits factor Xa in a calcium ion-, phospholipid-, and PZ-dependent fashion, but also directly inhibits coagulation factor XIa. In murine gene-deletion models, PZ and ZPI deficiency enhances thrombosis following arterial injury and increases mortality from pulmonary thromboembolism following collagen/epinephrine infusion. On a factor V(Leiden) genetic background, ZPI deficiency produces a significantly more severe phenotype than PZ deficiency, implying that factor XIa inhibition by ZPI is physiologically relevant. The studies in mice suggest that human PZ and ZPI deficiency would be associated with a modest thrombotic risk with ZPI deficiency producing a more severe phenotype.

Figure 1

ZPI gene disruption in mice. (A) The targeting construct (middle) contains a PGK-Neo cassette that replaces DNA fragment between the Pst 1 site in intron A and the EcoRV site in exon 2, thereby removing the ZPI DNA that encodes the signal peptide and N-terminus of ZPI, and inducing a frame-shift mutation. An HSV-TK cassette was added at the 3′-end of the construct to permit negative selection. The targeting vector was linearized with Not 1 and introduced into 129/Sv-derived RW4 ES cells by electroporation and stable transfectants were selected using G418 and gancyclovir. The predicted product of homologous recombination is shown at the bottom. The position of the 673 bp hybridization probe used to detect successful gene targeting is also depicted. (B) Southern blot analysis. Genomic DNA prepared from tail biopsies was analyzed by restriction digestion with Xba1 and hybridization with the probe. (C) Western blot analysis of ZPI in mouse plasma. SDS-PAGE (10%) and Western blotting of mouse plasma (5 μL of a 1:10 dilution) with rabbit antihuman ZPI polyclonal antibodies.

Figure 2

PZ and ZPI mice in thrombosis models. (A) Collagen/epinephrine-induced (270 μg/27 μg/kg) mortality from pulmonary thromboembolism with respiratory arrest by 5 minutes. (B) FeCl₃-induced (5%) carotid artery occlusion at 40 minutes.

Figure 3

Hemorrhage and intravascular coagulation in E13.5 FV(λ/λ)/ZPI(−/−) embryos. A-E, FV(λ/λ)/ZPI(−/−) embryos; F, FV(λ/λ)/ZPI(+/+) embryo. A,B, hematoxylin-eosin staining; C-F, antifibrinogen/fibrin staining (red) developed using AEC with hematoxylin counterstaining. (A) Intracranial hemorrhage (×5). (B) Intra-abdominal hemorrhage (×5). (C-E) Intravascular fibrin in the vessels of the brain (C, ×40), lung (D, ×40), and liver (E, ×40). In addition to the intravascular thrombosis, note disturbed cellular architecture and parenchymal fibrinogen/fibrin staining in the liver of the FV(λ/λ)/ZPI(−/−) embryo (E) compared with the liver of the FV(λ/λ)/ZPI(+/+) embryo (F).