11 protein z

Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice http://gateway.ut.ovid.com/gw1/ovidweb.cgi

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Editors: Colman, Robert W.; Clowes, Alexander W.;

Goldhaber, Samuel Z.; Marder, Victor J.; George, James N.

Title: Hemostasis and Thrombosis: Basic Principles and

Clinical Practice, 5th Edition

Copyright ©2006 Lippincott Williams & Wilkins

> Table of Contents > Part I - Basic Principles of Hemostasis and

Thrombosis > Section A - Coagulation and its Regulation > Chapter 11 -

Protein Z and Protein Z–Dependent Protease Inhibitor

Chapter 11

Protein Z and Protein Z–DependentProtease Inhibitor

George J. Broze Jr.

In 1977, Prowse and Esnouf identified an additional vitamin

K–dependent protein circulating in bovine plasma and named it

protein Z (PZ) because it was the last of the vitamin K–dependent

proteins to elute during anion exchange chromatography (1). PZ

serves as a cofactor for the inhibition of factor Xa by another

plasma protein called protein Z–dependent protease inhibitor (ZPI)

(2). ZPI is a member of the serpin superfamily of proteinase

inhibitors and not only inhibits factor Xa in a PZ–dependent

fashion, but also inhibits factor XIa in the absence of PZ (3,4). The

physiologic importance of the regulation of coagulation by PZ and

ZPI is not yet clear and is the focus of ongoing research.

PROTEIN Z

StructureThe human counterpart to bovine PZ was isolated in 1984 (5).

Mature human PZ is a 62,000–molecular weight, 360–amino acid,

single-chain glycoprotein whose structure is very similar to the

other vitamin K–dependent proteins, factors VII, IX, X, and protein

C (see Fig. 11-1) (5,6,7). A prepro-leader sequence directs the

vitamin K–dependent γ-Carboxylation of 13 glutamic acid residues


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within an N-terminal γ-carboxyglutamic acid (Gla) domain that is

followed by two epidermal growth factor (EGF)–like domains and a

C-terminal pseudocatalytic domain. PZ contains five potential

N-linked glycosylation sites (Fig. 11-1). In the first EGF-like

domain, a disaccharide or trisaccharide is attached to Ser53, and

Asp64 is probably a β hydroxyaspartic acid residue (8,9). Two

“extra” cysteine residues are present in PZ, but it is not

determined whether they form a disulfide bond. In contrast to

other coagulation factors that are serine proteinase zymogens, in

PZ, the region around the typical “activation site” is absent and

the histidine and serine residues of the canonical catalytic triad

have been replaced with lysine and aspartic acid residues,

respectively (7,9). The active site aspartic acid residue is

conserved. Therefore, like protein S, PZ does not serve a

proteolytic function.

The PZ gene is at chromosome 13q34, the location where the

genes for factor VII and factor X reside side by side (10). It spans

14 kb and consists of nine exons, including an alternatively spliced

exon that inserts a unique peptide of 22 amino acids in the

prepro-leader sequence of PZ. The exon–intron organization of the

PZ gene is identical to that of factors VII, IX, X, and protein C,

indicating that these genes were derived from a common ancestor

during evolution (Fig. 11-1) (10). Several polymorphisms have

been identified in the PZ gene, including one in the promoter

(a-13g) and one in exon 8 that leads to Arg255His replacement in

the encoded protein (11). A polymorphism in intron A (g103a) and

a polymorphism in intron F (g79a), which is in a high degree of

linkage disequilibrium with the a-13g and Arg255His

polymorphisms, are associated with reduced plasma levels of PZ

(12,13).

PropertiesThe range of PZ plasma levels in normal individuals is very broad

(95% interval of 32% to 168% of the mean) and appears to be

influenced predominantly by heritable factors (13,14,15). Reported

mean concentrations of PZ in adult plasmas have varied from 1.2

to 2.9 µg per mL, but the reason for this discrepancy is not

obvious. PZ circulates in plasma complexed with ZPI (see


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subsequent text).

Similar to other coagulation factors, the liver appears to be the

major source of PZ. The level of PZ is reduced in individuals with

severe liver disease and is low in newborn infants (16,17). Oral

contraceptive use substantially increases PZ levels (18). Plasma PZ

is reportedly increased with chronic hemodialysis and reduced in

the nephrotic syndrome (19,20). Whether PZ behaves as a

negative acute-phase reactant is controversial (19,21).

Immunoreactive PZ has been detected in atherosclerotic plaques

(22).

In contrast to other plasma vitamin K–dependent proteins, the

coumarin class of oral anticoagulants dramatically affects levels of

PZ. For example, in patients on stable warfarin therapy, levels of

antigenic and γ-carboxylated PZ are 8% ± 4% and 1% ± 2%

respectively, in comparison to levels of antigenic and γ

carboxylated protein C of 53 ± 8 and 28 + 6 (14). The interaction

of PZ with phospholipid vesicles also differs distinctively from that

of the other vitamin K–dependent coagulation factors. Although the

ultimate binding affinity of PZ is comparable to that of the other

proteins, its association (3.4 10-5 per second M) and dissociation

(0.06 per second) rate constants are markedly slower (23).

Despite its isolation, the physiologic function of PZ remained an

enigma for many years. Bovine PZ was shown to interact with

diisopropylphosphoryl (DIP)-inactivated thrombin (Kd = 0.15 µM)

and mediate the binding of DIP-thrombin to phospholipids (24).

Human PZ, however, binds thrombin poorly (Kd = 8.9 µM) and has

a minimal impact on thrombin's association with phospholipids

(25). Additional studies showed that the enhanced binding of

thrombin to bovine PZ requires the 36–amino acid C-terminal

extension present in bovine but absent in human PZ (25).

Thrombin cleavage of bovine PZ at Arg365 releases this C-terminal

peptide (25,26).

Subsequently, it was noted that the procoagulant activity of factor

Xa in a one-stage plasma coagulation assay was reduced if factor

Xa was first incubated with PZ (2). This inhibitory effect of PZ

required the presence of phospholipids


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and Ca2+ ions and was time dependent, apparently reflecting the

slow association of PZ with phospholipids (23). PZ that was

proteolytically cleaved at Arg43, thereby separating its Gla domain

from the remainder of the molecule, lacked inhibitory activity (2).

These results suggested that an interaction between factor Xa and

PZ occurs at the phospholipid surface, and additional studies

showed that the inhibitory effect of PZ on factor Xa activity in the

one-stage coagulation assay was due, at least in part, to a plasma

ZPI that recognizes the factor Xa–PZ complex (2).


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FIGURE 11-1. Amino acid sequence of protein Z. Disulfide

bonds have been placed by analogy to other vitamin

K–dependent proteins. Cysteine residues at positions 131 and

233 are not present in the homologous vitamin K–dependent

proteins. Solid diamonds indicate potential N-linked

glycosylation sites. The solid circle denotes site of a

disaccharide or trisaccharide linked to Ser53. Shaded residues

are sites of amino acids involved in the catalytic triad of serine

proteases. Dashed lines indicate intron–exon boundaries (27).

γ,γ carboxylated acid; β, potential β-hydroxyaspartic acid at

residue 64. (Modified from Ichinose A, Davie E. The blood

coagulation factors: their cDNAs, genes, and expression. In:

Colman R, Hirsh J, Marder V, et al., eds. Hemostasis and

thrombosis: basic principles and clinical practice. Philadelphia,

PA: JB Lippincott Co, 1994:19–54, with permission.)

PROTEIN Z–DEPENDENT PROTEASEINHIBITOR

StructureZPI was isolated from human plasma in 1998 and shown to be a

previously unidentified, 72,000–molecular weight, single-chain

glycoprotein (2). 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 (3). ATGs 1 to 4 are followed by short,

open reading frames, whereas ATG5 and ATG6 1 to 4 are in an

uninterrupted open reading frame that includes the encoded ZPI

protein. In vitro experiments show that ATG6 is sufficient for the

expression of rZPI in cultured Chinese hamster ovary (CHO) cells.

Northern analysis suggests that the liver is the major site of ZPI

synthesis (3). The predicted 423 residue amino acid sequence of

mature ZPI is 25% to 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


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(rasp-1) (28). Therefore, ZPI is likely the human homolog of rat

rasp-1, which was identified as a gene whose transcription is

increased following subtotal hepatectomy in rats (28). Alignment of

the amino acid sequence of ZPI with those of other serpins predicts

that Tyr387 is the P1 residue at the reactive center of the ZPI

molecule (see Fig. 11-2). Consistent with this notion,

rZPI(Tyr387Ala), an altered form of ZPI in which tyrosine 387 has

been changed to alanine, lacks PZ-dependent factor Xa inhibitory

activity (3).

FIGURE 11-2. Carboxy-terminal sequences of protein

Z–dependent protease inhibitor (ZPI) and other serpins.

Diamond denotes P1-P1′ cleavage site. Residues identical to

ZPI are indicated in bold type. Rasp-1, rat

regeneration-associated serpin protein; α1 AT, α1-trypsin; AT,

antithrombin; HC-II, heparin cofactor-II; and PN-1, protease

nexin-1.

PropertiesAlthough less marked than PZ, plasma levels of ZPI also span a

broad range (95% interval 46% to 154% of the mean) with a mean

concentration of ZPI of approximately 4.0 µg per mL (9). PZ and

ZPI form a complex and in pooled normal plasma, which contains

excess ZPI, all the PZ appears to be bound to ZPI (29). Therefore,

an early report that found a t1/2 of 2 to 3 days for PZ in plasma

was likely studying the clearance of the PZ–ZPI complex (14). The


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plasma level of ZPI is related to the level of PZ: Oral contraceptive

use raises both PZ (approximately 35%) and ZPI (approximately

17%) levels, whereas warfarin treatment reduces PZ

(approximately 92%) and ZPI (approximately 53%) levels (18).

This interrelation of plasma concentrations of PZ and ZPI might be

explained if the rate of clearance of the PZ–ZPI complex differs

from that of PZ or ZPI alone. Alternatively, the synthesis,

secretion, or extraplasma localization of one of these proteins may

be affected by the presence of the other.

PROTEIN Z AND PROTEIN Z–DEPENDENTPROTEASE INHIBITOR FUNCTION

Factor Xa InhibitionIn the presence of phospholipids and Ca2+ the rate of factor Xa

inhibition by ZPI is enhanced greater than 1,000-fold (t1/2<10

seconds vs. 210 minutes) by preincubation of factor Xa with PZ

(3,4). Indirect evidence strongly suggests that the inhibitory

process involves the formation of a stoichiometric complex of

factor Xa-ZPI-PZ at the phospholipid surface (3,4). Heparin does

not affect ZPI-mediated inhibition of factor Xa in the presence of

PZ. The combination of PZ and ZPI dramatically delays the

initiation and reduces the ultimate rate of thrombin generation in

mixtures containing prothrombin, factor V, phospholipids, and Ca2+

(4). In similar mixtures containing factor Va, however, PZ and ZPI

do not inhibit thrombin generation. Therefore, the anti–factor Xa

action of PZ and ZPI presumably must precede the activation of

factor V and the formation of the prothrombinase complex. With

coagulation induced in plasma by factor IXa, the presence of PZ

delays the onset and the extent of thrombin production (30).

PZ is not the only protein that has been shown to function as a

cofactor to enhance the inhibitory activity of a serpin toward an

enzyme. Thrombomodulin increases the rate of thrombin inhibition

by protein C inhibitor approximately 140-fold (31). This effect of

thrombomodulin reportedly depends primarily on the interaction

between thrombin and thrombomodulin. Vitronectin increases the

rate of thrombin inhibition by plasminogen activator inhibitor-1

(PAI-1) approximately 200-fold (32). Vitronectin appears to


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produce this enhancement by both binding PAI-1, thereby inducing

a conformational change at its reactive center, and through a

protein–protein interaction with thrombin. Similarly, the cofactor

action of PZ presumably involves its ability both to bind and to

bring ZPI to the phospholipid surface, as well as its ability to

interact with factor Xa at this surface (2,29).

Two potential pathways for PZ–dependent factor Xa inhibition by

ZPI are shown in Figure 11-3. On the left, PZ and

factor Xa first form a complex at the phospholipid surface, and this

complex is subsequently recognized by ZPI. On the right, a

preformed PZ–ZPI complex is directed to the phospholipid surface

by its PZ moiety and binds factor Xa. The final result of either

pathway is the formation of a Ca2+-dependent complex at the

phospholipid surface that contains PZ, factor Xa, and ZPI. Because

PZ circulates bound to ZPI, the pathway on the right presumably

reflects the inhibitory mechanism that occurs in the plasma milieu.

FIGURE 11-3. Two pathways for the inhibition of factor Xa by

protein Z–dependent protease inhibitor (ZPI) with protein Z

(PZ). On the left, ZPI binds to a preformed PZ–factor Xa


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complex at the phospholipid surface. On the right, the

circulating PZ–ZPI complex binds to factor Xa at the

phospholipid surface. Both pathways result in a final inhibitory

complex containing PZ-factor Xa-ZPI. PL, phospholipid surface;

Ca2+ denotes Ca2+ binding of factor Xa and PZ to the

phospholipid surface (33). (Reproduced from Broze G, Jr.

Protein Z and thrombosis. Lancet 2001;357:900–901, with

permission.)

Factor XIa InhibitionZPI also inactivates factor XIa in a reaction that does not require

the presence of PZ, phospholipids, or Ca2+ and that is not affected

by the presence of high-molecular-weight kininogen (4). Heparin

increases the rate and extent of factor XIa inhibition produced by

ZPI (4), whereas factor XIa inhibition by ZPI is reduced when ZPI

is bound to PZ (29). An apparent interaction between factor XIa

and ZPI can be detected in the plasma milieu (see subsequent

text), suggesting that ZPI competes effectively with other factor

XIa inhibitors (e.g., α1-antitrypsin, C1 esterase inhibitor,

antithrombin) and the substrate factor IX in plasma for the active

site of factor XIa.

Instability of Protein Z–DependentProtease Inhibitor–Proteinase ComplexesAs is typical for members of the serpin superfamily of proteinase

inhibitors, ZPI is proteolytically cleaved during its inhibition of

factor Xa and factor XIa with a reduction in its size from 72 kDa to

68 kDa. The N-terminal amino acid sequences of the peptides

(4.2-kDa) released from ZPI following its interaction with factor Xa

and factor XIa are identical, SMPPVIKVDRPF, and correspond to the

amino acid sequence in the ZPI molecule following Tyr387 (4).

Therefore, the reactive center of ZPI that is involved in its

inactivation of both factors Xa and XIa is Tyr387-Ser388 (P1-P1′).

The factor Xa–ZPI and factor XIa–ZPI inhibitory complexes,

however, are dramatically less stable than other protease–serpin


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complexes. In contrast to the thrombin–antithrombin interaction,

for example, the factor Xa–ZPI and factor XIa–ZPI complexes do

not survive sodium dodecyl (lauryl) surfate–polyacrylamide gel

electrophoresis (SDS-PAGE) but can be detected in the less

denaturing conditions of native-PAGE (without SDS) (4).

Dissociation of the thrombin–antithrombin complex is very slow

(approximately 2.5 × 10-6 per second) and appears to proceed

exclusively through the cleavage of antithrombin (34). Dissociation

of the factor Xa–ZPI complex is much more rapid (approximately

1.7 × 10-4 per second) and likely also occurs through the cleavage

of ZPI (4). In this regard, therefore, ZPI behaves as a very poor

substrate for the factor Xa-PZ-phospholipid Ca2+ complex and for

factor XIa. In view of the instability of the complexes of factor Xa

and factor XIa with ZPI, it seems likely that these proteinases

would ultimately be transferred from ZPI to alternative proteinase

inhibitors.

Consumption of Protein Z–DependentProtease Inhibitor During CoagulationSerum produced from plasma in vitro by the induction of

coagulation with kaolin, phospholipids, and Ca2+ or tissue factor

and Ca2+, contains little ZPI functional activity. Western blot

analysis shows that during coagulation of plasma in vitro, ZPI is

proteolytically cleaved at its C-terminus with reduction in its

apparent molecular weight from 72 kDa to 68 kDa (4). Factor Xa,

in the presence of PZ, is responsible for the consumption of ZPI in

tissue factor–induced coagulation. Factor XIa also contributes,

however, when coagulation is initiated by direct contact activation

(e.g., kaolin) and relatively large concentrations of factor XIa are

generated (4).

PROTEIN Z/PROTEIN Z-DEPENDENTPROTEASE INHIBITOR AND CLINICALCOAGULATION

Protein Z and HemorrhageIt has been suggested that PZ deficiency is associated with a

hemorrhagic disorder, perhaps related to capillary fragility (35).


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Thirty-six individuals with bleeding disorders of unknown etiology

were studied. Many of these individuals had a positive

Rumpel-Leede test (83%) and a prolonged bleeding time (43%).

The mean PZ level in the patients was 54% (range 22% to 112%).

In additional studies, prothrombin complex concentrates, which

contain PZ, have been used to prevent perioperative hemorrhage in

individuals with a bleeding history and perceived PZ deficiency

(36,37). Two subsequent studies, however, have failed to detect a

relation between PZ deficiency and a bleeding tendency (38,39),

and it should be noted that 10% of apparently healthy individuals

(Red Cross blood donors) have PZ levels of less than 50% (14).

Further, PZ-null mice have normal bleeding times and do not have

a hemorrhagic phenotype (30,40). Therefore, a clear relation

between low levels of PZ and a hemorrhagic diathesis remains to

be established.

Protein Z and ThrombosisUnchallenged, PZ knockout mice do not express an obvious

phenotype. When combined with the homozygous factor

VLeiden(FVλ/λ) genotype, however, the genotype causes intrauterine

and perinatal thrombosis and an apparent consumptive

coagulopathy that leads to near absolute mortality (30). The

genetic combinations FVλ/λ/PZ+/- and FVλ/λ/PZ-/- also reduce the

survival of mice by greater than 50%. It should be noted that the

factor V genotype appears to produce a more severe thrombotic

phenotype in mice than the factor V genotype in humans.

Nevertheless, the results of the murine PZ × FV crosses strongly

suggest that PZ deficiency is a prothrombotic trait and are

consistent with human data showing that a combination of

prothrombotic traits significantly increases the risk of thrombosis.

Studies exploring the association between PZ levels and ischemic

stroke have produced conflicting results (13,41,42,43,44,45). In a

group of 169 young patients (mean age 33 years) without

hypertension or dyslipidemia, Vasse et al. found that low

convalescent PZ levels (<15th percentile) were associated with a

fourfold increased risk of stroke (41). Heeb et al. studying 154

older individuals (median age 58 years), reported that low PZ

(<15th percentile) was associated with an increased risk of stroke


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in men [odds ratio (OR), 3.6; 95% confidence intervals (CI), 1.5 to

4.3] and those aged above 58 years (OR, 2.6; 95% CI, 1.4 to 4.9)

(43). They (Heeb et al.) found that the risk of stroke associated

with a low PZ was most apparent in individuals without diabetes,

hypertension, or hypercholesterolemia, and other risk factors for

stroke.

In contrast, Kobelt et al. reported an association of high PZ levels

with stroke in a group of 125 patients (mean age 40 years) without

a history of venous thrombosis (42). After adjusting for possible

confounders (age, sex, hypertension, diabetes, smoking, body

mass index, hyperlipidemia, and fibrinogen), the risk of

stroke in individuals with PZ in the highest quartile (>150%)

versus the lowest quartile (<76%) was 2.5-fold (95% CI, 1.05 to

5.72). Lichy et al. analyzed the PZ intron F g79a polymorphism in

a group of 200 patients with stroke and found that the presence of

at least one “a” allele is associated with a reduced risk of stroke

(OR, 0.6; 95% CI, 0.4 to 0.95) after adjusting for age, sex,

hypertension, hypercholesterolemia, and family history (13). They

also noted a significant relation between the “a” allele and reduced

PZ plasma levels in healthy individuals, implying that lower levels

of PZ may protect against stroke. In this study of individuals from

southwest Germany, the genotypes of 30% (60/200) of the stroke

cases and 41% (81/199) of the healthy controls contained at least

one “a.” Interestingly, the prevalence of the intron F “a” allele in

an Italian population is 37%, whereas in an English population it

appears to be much lower, 20% (11,12). Because the intron F g79a

polymorphism is unlikely to directly affect PZ expression, its

association with PZ levels may reflect the effect of a separate

polymorphism with which it is in a high degree of linkage

disequilibrium.

Two additional studies detected no relation between stroke and

convalescent PZ levels (44,45). McQuillan et al. however, reported

that significantly higher PZ levels were found in plasma samples

from patients with stroke, taken within 7 days of the acute event

(45). In contrast, Fedi et al. found an association between low PZ

levels (<15th percentile) and the acute coronary syndrome (OR,

3.3; 95% CI, 1.1 to 9.7) that was increased further by concomitant


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smoking (OR, 9.5; 95% CI, 2.4 to 37.2) (46). Low plasma

concentrations of PZ have also been reported in ischemic colitis

(47).

In regard to venous thrombosis, one study did not find a relation

with low levels of PZ in a small cohort of patients (41), and

another study, in which PZ levels were not determined, failed to

detect a relation with polymorphisms within the PZ gene (11). A

report (48) that low PZ affects the age of onset and the severity of

thrombosis in patients with the factor VLeaden mutation was not

confirmed in a later study (18). Recent results from the Leiden

Thrombophilia Study (LETS) showed a modestly increased risk of

venous thrombosis with low PZ (<10th percentile) in men (OR, 2.4;

95% CI, 1.2 to 4.9) and older individuals (>55 years, OR, 3.3;

95% CI, 1.2 to 8.7) on subgroup analysis (18). These same

groups, men and older individuals, are those in which Heeb et al.

found an association between low PZ (<15th percentile) and

ischemic stroke (43). A mechanism, however, to explain why

younger women may be protected from the thrombotic risk

associated with low levels of PZ is lacking. In LETS, neither high

nor low concentrations of ZPI were related to venous thrombosis

(43).

Low levels (<5th percentile) of PZ are common in individuals with

antiphospholipid antibodies and are associated with the thrombotic

complications and fetal wastage of the antiphospholipid syndrome

(OR, 6.6; 95% CI, 2.3 to 19.4) (49,50,51). Reduced levels of PZ

(<15th percentile) are also associated with early miscarriage in the

absence of antiphospholipid antibodies (OR, 6.7; 95% CI, 3.1 to

14.8) and maternal anti-PZ antibodies are reportedly related to

early fetal death and other pregnancy complications (52,53,54).

In sum, available clinical data provide a conflicting picture of the

role of PZ (and ZPI) in thrombotic disease. That PZ and ZPI

produce potent inhibition of factor Xa suggests that deficiencies of

these proteins could be associated with a procoagulant state, and

the results of the studies in PZ-/- V mice appear to confirm this

notion (2,30). On the other hand, certain clinical studies report

that high levels of PZ predispose to stroke or that low levels of PZ

may protect from stroke (13,42). The biologic foundation for these


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latter results is not readily apparent. The frequently offered

explanation of effect of bovine PZ on the binding of inactive

thrombin to phospholipids does not hold for active thrombin or

human PZ (13,24,25,26,35,42). Finally, it must be noted that the

very broad range of the plasma levels of PZ and ZPI implies that

their plasma concentrations need not be maintained near their

population means for a critical physiologic purpose. This suggests

that isolated low or high plasma levels of these proteins are

unlikely to produce a dramatic pathologic effect and/or,

alternatively, that the physiologically important roles of PZ and ZPI

occur outside the plasma milieu. Only additional investigation will

clarify these issues.

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