Prothrombotic phenotype of Z deficiency

Zheng-Feng Yin*, Zhong-Fu Huang*, Jisong Cui†, Ryan Fiehler*, Nina Lasky*, David Ginsburg†, and George J. Broze, Jr.*‡

*Division of Hematology, Barnes-Jewish Hospital at Washington University School of Medicine, 216 South Kingshighway Boulevard, St. Louis, MO 63110; and †Howard Hughes Medical Institute and Departments of Human Genetics and Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109

Edited by Philip W. Majerus, Washington University School of Medicine, St. Louis, MO, and approved April 11, 2000 (received for review February 25, 2000) Protein Z (PZ) is a -dependent plasma protein whose that the role of PZ, like that of PC or PS, is to limit the function has been uncertain. The structure of PZ is very similar to response. that of the coagulation-related factors VII, IX, and X and PC, but PZ differs from these other in that it is not the zymogen of Methods a . We have shown recently that PZ forms a calcium Materials. Hepes, EDTA, barium chloride, sodium oxalate, BSA, ion-dependent complex with activated at phospholipid and rabbit brain cephalin were from Sigma. Affigel-10 was surfaces and that this interaction leads to the inhibition of acti- purchased from Bio-Rad. vated factor X activity through, in part, the action of a previously unidentified plasma protein named PZ-dependent protease inhib- Proteins. PZ and ZPI were purified from human plasma as itor. Herein, we report that the presence of PZ dampens the described (10). Prothrombin, factor X, and factor IXa were coagulation response in human plasma and that concomitant PZ products of Enzyme Research Laboratories (South Bend, IN). deficiency dramatically increases the severity of the prothrombotic phenotype of factor VLeiden mice. The results indicate that PZ plays Barium-Adsorbed Plasma. Pooled normal citrated plasma (George a physiologically important role in the regulation of coagulation. King Biomedical, Overland Park, KS) was barium-salt-adsorbed by adding 1.0 M BaCl2 to a final concentration of 0.1 M, stirring itamin K is required for the posttranslational formation of on ice for 30 min, and centrifuging at 10,000 ϫ g for 20 min at Va unique , ␥-carboxyglutamic acid, which is 4°C. The plasma supernatant was dialyzed against 0.1 M NaCl͞ present in a number of plasma proteins that are involved in 0.02 M Hepes, pH 7.4͞0.01 M sodium oxalate overnight at 4°C. hemostasis: prothrombin; factors VII, IX, and X; (PC); Barium sulfate (100 mg͞ml) was added with mixing (4°C); the and (PS). ␥-Carboxyglutamic acid-mediated calcium mixture was centrifuged at 10,000 ϫ g for 20 min; and the ion binding in these proteins is necessary for their association supernatant plasma was frozen (Ϫ70°C) in small aliquots. Based with phospholipid surfaces and is critical for their hemostatic on immunoassay, the concentration of PZ in the barium ad- function (1, 2). Prothrombin and factors VII, IX, and X are sorbed plasma was Ͻ3% (Ͻ1.2 nM) of the starting plasma. involved in the coagulant response leading to the generation of at sites of injury, whereas PC and PS play roles in an Coagulation Assays. Thrombin and factor Xa functional assays antithrombotic pathway that limits coagulation by inactivating were performed as described (12). Samples were obtained from the important coagulation cofactors Va and VIIIa (3). Protein Z the soluble phase after vortexing of reactions containing (PZ) is a 62-kDa, vitamin K-dependent plasma protein whose clots, diluted in 0.1 M NaCl͞0.02 M Hepes, pH 7.4 with 1.0 structure is similar to that of factors VII, IX, and X and PC. In mg͞ml BSA (factor Xa assay) or the same buffer with 5 mM contrast to these other proteins, however, PZ is not the zymogen EDTA (thrombin assay), and tested immediately. of a serine protease, because it lacks the His and Ser residues of Factor VLeiden (FVLeiden) and PZ -Deleted Mice. The production of the catalytic triad (4, 5). The rate of association (kassn ϭ 3.36 ϫ Ϫ5 Ϫ1 Ϫ1 Ϫ1 FVLeiden mice will be described in a separate publicaiton (un- 10 s ⅐M ) and rate of dissociation (kdssn ϭ 0.057 s )ofPZ with͞from phospholipids are significantly slower, and the plasma published work). To produce PZ gene-deleted mice, the full- level of ␥-carboxylated PZ during chronic therapy length murine PZ cDNA was isolated from a mouse liver cDNA (Յ1%) is dramatically lower than the rates and levels of the other library (lambda ZAP, Stratagene) with a PCR-generated probe vitamin K-dependent proteins (6, 7). Despite its initial isolation corresponding to codons 59–472 of the human PZ cDNA several years ago (8, 9), the physiological function of PZ has not sequence. Based on the intron–exon boundaries in the human been determined. PZ gene, a cDNA fragment predicted to encompass exon 2 of the We have shown recently that PZ forms a calcium ion-dependent mouse PZ gene was used to screen a P1 genomic DNA library complex with factor Xa at phospholipid surfaces (10). This PZ– made from the 129͞Sv mouse strain (Genome Systems, St. factor Xa interaction reduces the rate of factor Xa inhibition by Louis). A 10.5-kilobase (kb) KpnI fragment of the isolated but dramatically enhances the inhibition of factor Xa murine PZ gene that spanned exons 2–6 of the gene was produced by a previously unidentified plasma protein named subcloned for restriction enzyme mapping, partial DNA se- PZ-dependent protease inhibitor (ZPI; ref. 10). ZPI is a 72-kDa quencing, and construction of the targeting vector (see Fig. 2). member of the superfamily of proteinase inhibitors that A 1.3-kb fragment of PZ DNA extending from the KpnI site in intron 1 to the BglI site in exon 2 was inserted into the multiple contains a tyrosine as the P1 residue at its reactive center (10, 11). In systems that use purified components, ZPI produces rapid Ͻ inhibition of factor Xa (t1͞2 10 s) that requires the presence of PZ, This paper was submitted directly (Track II) to the PNAS office. calcium ions, and phospholipids (10). Initial studies of the inhibitory Abbreviations: PZ, PC, and PS, proteins Z, C, and S; factor na, activated factor n; ZPI, spectrum of ZPI have shown that it does not affect the catalytic PZ-dependent protease inhibitor; FV, ; kb, kilobase; En, embryonic day n. activity of thrombin, meizothrombin, factor VIIa, factor IXa, ‡To whom reprint requests should be addressed. E-mail: [email protected]. activated PC, tissue-plasminogen activator, -plasminogen The publication costs of this article were defrayed in part by page charge payment. This activator, , trypsin, leukocyte elastase, chymotrypsin, or article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. cathepsin G (11). §1734 solely to indicate this fact. Additional in vitro and in vivo experiments were undertaken to Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.120081897. determine the physiological relevance of PZ. The results show Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.120081897

6734–6738 ͉ PNAS ͉ June 6, 2000 ͉ vol. 97 ͉ no. 12 Downloaded by guest on September 25, 2021 cloning site of pBluescript II between KpnI and BglI. A 1.5-kb neomycin phosphotransferase (neo) cassette containing the phosphoglycerate kinase promoter, was introduced at the XhoI site of pBluescript II in the same orientation as the PZ DNA. A 1.8-kb herpes simplex virus–thymidine kinase cassette was added at the XbaI site of pBluescript II for negative selection. The targeting vector was completed by inserting a 5.9-kb AatII–BglI fragment of PZ DNA containing a portion of exon 2 and exons 3–6 at the SalI site of pBluescript II. The structure of the targeting construct was confirmed by PCR, restriction mapping, and sequencing of ligation junctions. The targeting vector was linearized and introduced into 129͞Sv-derived RW4 embryonic stem cells by electroporation, and stable transfectants were selected as described (13). Of 480 clones, 11 contained the expected allele by Southern analysis. Two independent stem cell clones were injected into C57BL͞6 blastocysts as described (13), and the resulting chimeric males were bred to C57BL͞6 females to generate initial F1 PZ(ϩ͞Ϫ) offspring. To produce FV(␭͞ ϩ)͞PZ(ϩ͞Ϫ) mice, PZ(Ϫ͞Ϫ) mice derived from F1 PZ(ϩ͞Ϫ) intercrosses were mated with homozygous FVLeiden [FV(␭͞␭)] mice on a mixed C57BL͞6 ϫ 129͞Sv genetic background. Animals from subsequent FV(␭͞ϩ)͞PZ(ϩ͞Ϫ) crosses were then used in various mating strategies to determine the effect of the PZ genotype on the survival of FVLeiden mice. Because of the effect of genetic background on the phenotype of FV(␭͞␭) mice (unpublished work), only the phenotypes of littermate mice were compared in these studies.

Genotyping of Mice. DNA prepared from tail biopsies was used for Southern and PCR analysis. For Southern blotting, the hybrid- ization probe was a Ϸ0.5-kb XbaI–KpnI fragment of PZ genomic DNA just 5Ј of the region of homologous recombination (see Fig. 2). The PCR assay for the PZ gene used three primers derived from (i) a portion of exon 2 deleted in the mutant allele (5Ј-AAACAACGTTCTGCGGAGGTGGA-3Ј); (ii) intron 2 (5Ј-AACGAACTAGTTAGTCCTGAGACA-3Ј); and (iii) neo cassette (5Ј-ATTGCTGAAGAGCTTGGCGG-3Ј). The PCR product derived from the wild-type PZ allele is 214 bp; that derived from the mutant PZ allele is 449 bp. The PCR assay for Fig. 1. Regulation of coagulation by PZ. (A) Factor Xa activity. Factor IXa (0.5 the FVLeiden genotype used two primers: (i) intron 10, upstream of an inserted lox P sequence (5Ј-CCTCTGGACTCTGACTG- nM) was used to induce coagulation in mixtures containing rabbit brain phospholipids (cephalin, 15 ␮M); CaCl (10 mM); barium-adsorbed, oxalated, Ј Ј 2 CAG-3 ); and (ii) intron 10, downstream of the lox P (5 - pooled normal plasma (50% vol͞vol); and factor X (100 nM) with or without Ј TATTCTGGACTACAAGAGTGA-3 ). The PCR product de- PZ (30 nM). Phospholipids and CaCl2 with or without PZ were incubated 2 min

rived from the wild-type FV allele is 400 bp; that derived from before the addition of the remaining reagents, and the reaction was initiated MEDICAL SCIENCES the FVLeiden allele is 544 bp. by the addition of the plasma. (B) Thrombin generation. Reactions were the same as described for A, except prothrombin (700 nM) was also added to each Other Methods. Mouse blood for count and mixture. ■, ϪPZ; F, ϩPZ. assay was obtained from the retro-orbital plexus with a hepa- rinized capillary tube. Fibrinogen levels were determined with a modification of the microturbidimetric method (14). Pooled thrombin (to prevent significant thrombin generation), the fac- plasma from PZ(ϩ͞ϩ) mice (n ϭ 12) was used as standard and tor Xa activity produced after the induction of coagulation by assigned a fibrinogen level of 100%. Western blotting for mouse factor IXa was reduced dramatically in the presence of PZ (Fig. plasma PZ was performed as described with 1 ␮l of murine 1A). Thrombin generation was examined in the same system by plasma and rabbit polyclonal anti-human PZ antibodies (13). the addition of prothrombin as well as factor X to the plasma. In FV(␭͞␭) embryos with PZ(ϩ͞ϩ), PZ(ϩ͞Ϫ), and PZ(Ϫ͞Ϫ) the presence of PZ, thrombin generation was delayed signifi- genotypes (four each) were fixed in paraformaldehyde, embed- cantly, and the peak thrombin concentration was reduced Ͼ50% ded in paraffin, and sectioned at 6 ␮m. Three serial sections were (Fig. 1B). These in vitro results suggested that PZ could play a mounted per slide. Every fifth slide was stained with hematoxylin role in limiting coagulation. and eosin, and the following slide was analyzed after antifi- brin(ogen) immunohistochemical staining as described (13). PZ Gene Disruption in Mice. To investigate the in vivo consequences of PZ deficiency, the PZ gene was disrupted in mice. Successful Results gene targeting resulted in replacement of a 139-bp fragment of Effect of PZ on in Vitro Coagulation. In in vitro studies, the effect of exon 2, encoding a portion of the signal sequence and the N PZ on factor Xa activity and thrombin generation during coag- terminus of PZ, by a neo cassette (Fig. 2A). The expected ulation was determined by using pooled normal human plasma structure of the targeted PZ locus was confirmed by Southern that had been depleted of PZ (Ͻ3%) and other vitamin K- blot analysis, and PZ was not detectable in the plasma of dependent factors by barium salt adsorption (see Methods). In homozygous gene-disrupted mice by Western blotting (Fig. 2 B this artificial plasma replenished with factor X but not pro- and C).

Yin et al. PNAS ͉ June 6, 2000 ͉ vol. 97 ͉ no. 12 ͉ 6735 Downloaded by guest on September 25, 2021 Fig. 2. Generation of PZ(Ϫ͞Ϫ) mice. (A) Gene targeting. The targeting construct (Top) contains a neo cassette (neo) that replaces a 139-bp portion of exon 2 and induces a frameshift mutation. A herpes simplex virus–thymidine kinase cassette (tk) was added at the 3Ј end of the mouse PZ DNA to permit negative selection. The predicted product of homologous recombination is shown (Bottom). The position of the Ϸ500-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 XbaI and hybridization with the probe. Expected DNA fragment sizes are 9.0 kb for the wild-type allele and 3.2 kb for the disrupted allele. (C) Western blot analysis of mouse plasma with rabbit anti-human PZ polyclonal antibodies.

DNA analysis of 303 progeny derived from PZ(ϩ͞Ϫ) inter- C57BL͞6 ϫ 129 genetic background present in these experi- crosses showed that PZ(Ϫ͞Ϫ) mice were born in expected ments, Ϸ20% of the FV(␭͞␭) mice die during the neonatal numbers: 23% PZ(ϩ͞ϩ), 54% PZ(ϩ͞Ϫ), and 23% PZ(Ϫ͞Ϫ). period with microvascular . Further, the growth and development of PZ null mice were The genotypes of 6-week-old mice derived from a variety of indistinguishable from those of their PZ(ϩ͞Ϫ) and PZ(ϩ͞ϩ) FVLeiden͞PZ mating strategies are shown in Table 1. The PZ(Ϫ͞Ϫ) littermates. Blood platelet counts (ϫ103) were 1,115 Ϯ 210, genotype markedly increases the mortality of FV(␭͞␭) mice, and 1,120 Ϯ 281, and 1,116 Ϯ 98, and fibrinogen levels (percentages) only a single FV(␭͞␭)͞PZ(Ϫ͞Ϫ) animal has survived to adulthood were 100 Ϯ 28, 102 Ϯ 33, and 93 Ϯ 29 in PZ(ϩ͞ϩ)(n ϭ 15), PZ(ϩ͞Ϫ)(n ϭ 14), and PZ(Ϫ͞Ϫ)(n ϭ 14) mice, respectively.

Immunohistological analysis of the tissues from PZ(Ϫ͞Ϫ) mice Table 1. Progeny of FVLeiden͞PZ matings did not demonstrate vascular thrombosis or hepatic fibrin dep- Mating pairs Observed (%) Expected, % P* osition (not shown). Thus, in the absence of an additional challenge, murine PZ deficiency produces a grossly normal FV(␭͞␭)͞PZ(ϩ͞Ϫ) ϫ FV(␭͞␭)͞PZ(ϩ͞Ϫ) phenotype. To evaluate further the potential prothrombotic risk At 6 weeks associated with PZ deficiency, PZ gene-disrupted mice were FV(␭͞␭)͞PZ(ϩ͞ϩ) 40 (52) 25 crossbred with mice carrying the FVLeiden mutation. FV(␭͞␭)͞PZ(ϩ͞Ϫ) 37 (48) 50 Ͻ0.01 FV(␭͞␭)͞PZ(Ϫ͞Ϫ) 0 (0) 25 Ͻ0.01 Effect of PZ Deficiency on the FVLeiden Phenotype. In humans, an At E17.5–E18.5† altered form of coagulation FV, termed FVLeiden, is caused by a FV(␭͞␭)͞PZ(ϩ͞ϩ) 7 (21) 25 missense mutation that replaces Arg-506 in the FV molecule FV(␭͞␭)͞PZ(ϩ͞Ϫ) 18 (53) 50 with a glutamine residue (R506Q; ref. 15). In contrast to normal FV(␭͞␭)͞PZ(Ϫ͞Ϫ) 9 (26) 25 FV, the activated form of FVLeiden is resistant to inactivation by FV(␭͞␭)͞PZ(ϩ͞Ϫ) ϫ FV(ϩ͞ϩ)͞PZ(Ϫ͞Ϫ) (6 weeks) activated PC, and FVLeiden functions poorly as a cofactor for FV(␭͞ϩ)͞PZ(ϩ͞Ϫ) 28 (74) 50 activated PC-mediated inactivation of factor VIIIa (15–20). FV(␭͞ϩ)͞PZ(Ϫ͞Ϫ) 10 (26) 50 Ͻ0.01 These abnormalities lead to increased thrombin production and FV(␭͞␭)͞PZ(Ϫ͞Ϫ)‡ ϫ FV(␭͞␭)͞PZ(ϩ͞Ϫ) (6 weeks) a prothrombotic phenotype. Genetic heterozygosity for FVLeiden FV(␭͞␭)͞PZ(ϩ͞Ϫ) 16 (100) 50 [FV(␭͞ϩ)] is common in humans of European descent (Ϸ5%), FV(␭͞␭)͞PZ(Ϫ͞Ϫ) 0 (0) 50 Ͻ0.01 ␭ ϩ ␭ ␭ and the FV( ͞ ) and FV( ͞ ) genotypes have been shown to *Difference between observed and expected frequencies derived by the increase the risk of thrombosis by Ϸ5- and Ϸ25-fold, respectively confidence interval for binomial distributions. (21, 22). Mice homozygous for the FVLeiden mutation (R504Q, †En, embryonic day n. equivalent to the human R506Q mutation) express a strain- ‡Single FV(␭͞␭)͞PZ(Ϫ͞Ϫ) male, derived from a F(␭͞ϩ)͞PZ(ϩ͞Ϫ) intercross, specific thrombotic phenotype (unpublished work). In the mixed mated with five females.

6736 ͉ www.pnas.org Yin et al. Downloaded by guest on September 25, 2021 Fig. 3. E17.5 FV(␭͞␭) embryos with PZ(Ϫ͞Ϫ), PZ(ϩ͞Ϫ), and PZ(ϩ͞ϩ) genotypes.

thus far. The PZ(ϩ͞Ϫ) genotype also increases mortality in PZ(ϩ͞Ϫ) mice were lost in utero or during the perinatal period. To FV(␭͞␭) mice, because the ratio of FV(␭͞␭)͞PZ(ϩ͞ϩ)toFV(␭͞ determine whether the PZ genotype affected the intrauterine ␭)͞PZ(ϩ͞Ϫ) progeny derived from FV(␭͞␭)͞PZ(ϩ͞Ϫ) inter- survival of FV(␭͞␭) mice and to obtain appropriate tissues for crosses is 1:1, instead of the anticipated 1:2 (P Ͻ 0.01). Platelet histological analysis, embryos derived from FV(␭͞␭)͞PZ(ϩ͞Ϫ) counts (ϫ103) and fibrinogen levels (percentages) were similar in intercrosses were examined at late gestation. FV(␭͞␭)͞PZ(Ϫ͞Ϫ) FV(␭͞␭)͞PZ(ϩ͞ϩ) (832 Ϯ 183 and 87 Ϯ 24, respectively; n ϭ 19) and FV(␭͞␭)͞PZ(ϩ͞Ϫ) embryos were present at E17.5–E18.5 in and FV(␭͞␭)͞PZ(ϩ͞Ϫ) (891 Ϯ 212 and 89 Ϯ 21, respectively; n ϭ expected numbers, but eight of the nine FV(␭͞␭)͞PZ(Ϫ͞Ϫ) em- 19) animals. The survival of FV(␭͞ϩ)͞PZ(Ϫ͞Ϫ) mice is signifi- bryos showed obvious signs of hemorrhage during a visual inspec- tion (Table 1 and Fig. 3). Antifibrin(ogen) immunohistochemical cantly less than that of FV(␭͞ϩ)͞PZ(ϩ͞Ϫ)animals(P Ͻ 0.01). analysis of the FV(␭͞␭) embryos showed vascular thrombosis and Thus, the level of PZ affects the phenotype of heterozygous hepatic fibrin deposition, the scope of which was related directly to FVLeiden mice as well. the PZ genotype. [PZ(Ϫ͞Ϫ) Ͼ PZ(ϩ͞Ϫ) Ͼ PZ(ϩ͞ϩ); Fig. 4]. Of the nine pups found dead during the 6 weeks after birth, one had the FV(␭͞␭)͞PZ(Ϫ͞Ϫ) genotype; three had the FV(␭͞␭)͞ Discussion PZ(ϩ͞Ϫ) genotype; and five had the FV(␭͞␭)͞PZ(ϩ͞ϩ) geno- These in vitro and in vivo studies suggest that PZ plays an type, implying that many of the FV(␭͞␭)͞PZ(Ϫ͞Ϫ) and FV(␭͞␭)͞ important role in dampening coagulation. The cofactor effect of MEDICAL SCIENCES

Fig. 4. Antifibrin(ogen) staining of tissues from E17.5 embryos. (Upper) Thrombosis (arrows) of spinal (Left), chest wall (Center), and pulmonary (Right) vessels in a FV(␭͞␭)͞PZ(Ϫ͞Ϫ) embryo. (Lower) Hepatic fibrin deposition in FV(␭͞␭) embryos with PZ(Ϫ͞Ϫ)(Left), PZ(ϩ͞Ϫ)(Center), and PZ(ϩ͞ϩ)(Right) genotypes.

Yin et al. PNAS ͉ June 6, 2000 ͉ vol. 97 ͉ no. 12 ͉ 6737 Downloaded by guest on September 25, 2021 PZ for the inactivation of factor Xa by ZPI is presumably an thrombosis associated with PZ deficiency in humans remains to important part of this regulatory action of PZ, but additional, be determined. Based on the data from the murine gene-deletion ZPI-independent effects of PZ have not been excluded. Isolated models and the very broad range of PZ plasma levels present in PZ deficiency is apparently compatible with normal survival in apparently normal blood donors (7), this risk is likely to be mice. When combined with the homozygous FVLeiden genotype, modest. however, PZ deficiency causes intrauterine and perinatal throm- The oral warfarin interferes with the action of bosis and an apparent consumptive coagulopathy that leads to vitamin K, leading to the production of incompletely ␥-carbox- near absolute mortality. The genetic combinations FV(␭͞␭)͞ ylated, nonfunctional proteins. The reduction in functional ϩ Ϫ ␭ ϩ Ϫ Ϫ PZ( ͞ ) and FV( ͞ )͞PZ( ͞ ) produce smaller, although prothrombin and factor X levels seems to be responsible for the significant reductions in survival. The intensification of the antithrombotic effects of warfarin therapy (25). Levels of PC and thrombotic phenotype in FV mice produced by PZ defi- Leiden PS, however, are also reduced by warfarin treatment, and the ciency is consistent with recent human data showing that a combination of prothrombotic traits significantly increases the microvascular thrombosis that occurs with warfarin-induced skin risk of thrombosis (23) and underscores the multigenic nature of necrosis is thought to be due to a relative imbalance between . procoagulant and anticoagulant forces during the initiation of In contrast to pathway inhibitor (TFPI) null and warfarin therapy (26). PZ deficiency, like PC and PS deficiency, PC null mice (13, 24), which develop lethal disseminated intra- may increase the risk of this infrequent but serious complication vascular coagulation, homozygous PZ-deficient mice have an of warfarin therapy (27, 28). Finally, the levels of vitamin apparently normal phenotype, at least in the absence of a K-dependent plasma proteins are low during the perinatal thrombotic challenge. In this regard, they are similar to FV(␭͞ period in comparison to adult levels (29). It is conceivable that ϩ), TFPI(ϩ͞Ϫ), and PC(ϩ͞Ϫ) mice, which are also asymptom- relative deficiencies of endogenous PC (24), PS, and͞or PZ may atic in the unchallenged state (refs. 13 and 24; unpublished contribute to the partial neonatal mortality observed in mice work). Like the PZ(Ϫ͞Ϫ) genotype, the TFPI(ϩ͞Ϫ) genotype that carry the FV(␭͞␭) genotype as their sole defect. in combination with the FV(␭͞␭) genotype produces near absolute mortality (unpublished work). Thus, the thrombotic We thank Terri Lewis for secretarial assistance in the preparation of this risk associated with homozygous PZ deficiency seems similar to manuscript. This work was supported in part by National Institutes of that of heterozygous TFPI deficiency in the mouse. The risk of Health Grant HL-60782.

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