In the presence of phospholipid vesicles and calcium ions, protein Z (PZ) serves as a cofactor for the inhibition of coagulation factor Xa by a plasma protein called PZ-dependent protease inhibitor (ZPI). To further characterize ZPI, its cDNA has been isolated and cloned from a human liver cDNA library. The ZPI cDNA is 2.44 kb in length and has a relatively long 5' region (466 nt) that contains six potential ATG translation start codons. ATG's 1-4 are followed by short open reading frames, whereas ATG(5) and ATG(6) are in an uninterrupted open reading frame that includes the encoded ZPI protein. In vitro experiments show that ATG(6) is sufficient for the expression of rZPI in cultured Chinese hamster ovary cells. Northern analysis suggests the liver is a major site of ZPI synthesis. The predicted 423 residue amino acid sequence of the mature ZPI protein is 25-35% homologous with members of the serpin superfamily of protease inhibitors and is 78% identical to the amino acid sequence predicted by a previously described cDNA isolated from rat liver, regeneration-associated serpin protein-1 (rasp-1). Thus, ZPI is likely the human homologue of rat rasp-1. Alignment of the amino acid sequence of ZPI with those of other serpins predicts that Y387 is the P(1) residue at the reactive center of the ZPI molecule. Consistent with this notion, rZPI(Y387A), an altered form of ZPI in which tyrosine 387 has been changed to alanine, lacks PZ-dependent factor Xa inhibitory activity.

Human protein Z (PZ) is a 62,000-M r , vitamin K-dependent plasma protein whose structure is similar to coagulation factors VII, IX, X, protein C, and protein S, but whose function is not known. The procoagulant activity of factor Xa in a one-stage plasma coagulation assay is reduced when factor Xa is first incubated with PZ. This apparent inhibitory effect is time dependent, requires the presence of calcium ions and procoagulant phospholipids (rabbit brain cephalin), and appears predominantly related to the incubation period of PZ with cephalin. In serum the initial rate of inhibition of factor Xa with calcium ions and cephalin also is enhanced in the presence PZ. A PZ-dependent protease inhibitor (ZPI) has been isolated from plasma. ZPI is a 72,000-M r single-chain protein with an N-terminal amino acid sequence of LAPSPQSPEXXA (X ‫؍‬ indeterminate) and an estimated concentration in citrate-treated plasma of 1.0-1.6 g͞ml. In systems using purified components, the factor Xa inhibition produced by ZPI is rapid (>95% within 1 min by coagulation assay) and requires the presence of PZ, calcium ions, and cephalin. The inhibitory process appears to involve the formation of a factor Xa-PZ-ZPI complex at the phospholipid surface.Vitamin K is required for the posttranslational formation of ␥-carboxyglutamic acid (Gla), which is present in a number of plasma proteins that are involved in coagulation: prothrombin, factors VII, IX, and X, protein C, and protein S (1, 2). Gla-mediated calcium ion binding in these proteins is necessary for their association with phospholipid surfaces and is critical for their hemostatic function (3). In 1977, Prowse and Esnouf (4) identified an additional vitamin K-dependent protein circulating in bovine plasma and named it protein Z (PZ). Initially thought to represent a single-chain form of bovine factor X, bovine PZ was later identified as a discrete Glacontaining protein (5, 6). The human counterpart of bovine PZ was isolated in 1984 (7).Human PZ is a 62,000-M r glycoprotein that has a plasma half-life of Ϸ2.5 days (8). Plasma PZ levels in blood donors span a broad range with a mean concentration of 2.9 Ϯ 1.0 g͞ml in EDTA-anticoagulated samples (corresponding to Ϸ2.6 g͞ml in citrated plasma) (8). The N-terminal half of PZ is very homologous to those of factors VII, IX, and X, and contains a Gla-domain, two epidermal growth factor-like domains, and a region that connects to a homologue of the catalytic domains present in the serine protease zymogens. In the C-terminal domain of PZ, however, the region around the typical ''activation'' site is absent and the His and Ser residues of the catalytic triad are lacking (the Asp residue is conserved) (9, 10).McDonald et al. (11) recently reported that the kinetics of the binding of human and bovine PZ to phosphatidylcholine͞ phosphatidylserine (75%͞25%) vesicles is different from that of the other vitamin K-dependent coagulation factors. The k assn (10) and k dssn (s Ϫ1 ) rate constants are 1.95 and 0.0063 for bovine PZ and 3.36 and 0.057 ...

Protein Z (PZ) is a vitamin K-dependent plasma protein whose function has been uncertain. The structure of PZ is very similar to that of the coagulation-related factors VII, IX, and X and PC, but PZ differs from these other proteins in that it is not the zymogen of a serine protease. We have shown recently that PZ forms a calcium ion-dependent complex with activated factor X at phospholipid surfaces and that this interaction leads to the inhibition of activated factor X activity through, in part, the action of a previously unidentified plasma protein named PZ-dependent protease inhibitor. Herein, we report that the presence of PZ dampens the coagulation response in human plasma and that concomitant PZ deficiency dramatically increases the severity of the prothrombotic phenotype of factor VLeiden mice. The results indicate that PZ plays a physiologically important role in the regulation of coagulation.