Article

hematology

Hemostasis in the Neonate

Marilyn J. MancoJohnson, MD*

Author Disclosure
Dr Manco-Johnson
did not disclose any
financial relationships
relevant to this

Objectives

After completing this article, readers should be able to:

1. Delineate the components essential for hemostasis that are at or above adult values in
healthy term and preterm neonates.
2. Describe the coagulation components that characteristically show quantitative or
qualitative differences in healthy term infants compared with the healthy adult.
3. Interpret screening clotting test values in newborn infants.
4. Interpret concentrations of specific clotting proteins relative to the gestational and
postnatal age of the infant.

article.

Abstract
The coagulation system is finely tuned to arrest bleeding at the site of vascular injury
and quickly remove clots that obstruct blood flow. In the fetus, components of the
coagulation system show unique developmentally regulated patterns and times for
maturation to normal adult protein quantities and functions. In addition, several
coagulation proteins contribute to cellular proliferation and differentiation uniquely
during fetal life. In spite of this, results of most screening tests of hemostasis vary
modestly from adult normal values in the healthy term infant, and both hemorrhage
and thrombosis are rare in the well infant.

Introduction
To understand the unique features of fetal and neonatal hemostasis, it is essential to
understand coagulation physiology. Coagulation must be regulated carefully to allow
rapid and effective activation sufficient to prevent excessive blood loss from the site of
injury, yet protect against uncontrolled formation of occlusive fibrin clots in the systemic
circulation. To achieve this requirement, coagulation activation is limited in time and space
to sites of vascular injury.

Physiology of Coagulation
The kinetics of coagulation complex formation and activities are physiologic only on cell
surfaces where the phospholipid (PL) bilayer concentrates complexes, substrates, and
activators sufficiently. In fluids, such as plasma, coagulation reactions are 1,000-fold slower
than on PL surfaces and are ineffective. The critical regulator of all coagulation processes
is thrombin, an enzyme formed by cleavage of a small peptide from its inactive precursor
(known as a zymogen), prothrombin (Figure). The critical coagulation protein is fibrinogen, a contractile protein that, following cleavage by thrombin, forms long polymeric
protein strands. Fibrin strands are made durable by side-to-side cross-linkage by factor XIII
(FXIII) following the activation of FXIII by thrombin. Stable cross-linked clots contract to
form a tight seal that prevents excessive blood loss while fibroblastic proliferation, also
stimulated by thrombin, restores tissue integrity and initiates scar formation. Eventually,
the fibrin clot no longer is needed, and by about 10 days following formation, fibrin is lysed
by the fibrinolytic system to restore and maintain vascular patency.
Thrombin formation is highly regulated. Thrombin predominantly is activated by the
action of a complex formed from a transmembrane protein, tissue factor (TF), with
activated factor VII (FVIIa) or zymogen factor VII (FVII). Under steady-state conditions,
*Professor of Pediatrics, University of Colorado, Denver, and the Childrens Hospital, Denver, Colo.
NeoReviews Vol.9 No.3 March 2008 e119

hematology

hemostasis

prothrombin to thrombin. The initial coagulation cascade initiated by

TF generates a small amount of
thrombin that has several activities:
1) platelet activation, thus recruiting a large volume of activation surface; activation of factor VIII
(FVIII) and factor V (FV) that
serve as scaffoldlike cofactors in two
parallel complexes for the activations of FX by activated factor IX
(FIXa) and thrombin by FXa,
respectively; 2) binding to the endothelial cell receptor, thrombomodulin, to form an activation
complex for the critical regulatory
protein C that dampens the rapid
activation by FVIIIa and FVa;
3) activation of FXIII to cross-link
the fibrin clot; 4) activation of the
thrombin activatable fibrinolytic inhibitor that allows the clot sufficient stability for hemostasis and
wound healing before activating fibrinolysis; and 5) induction of the
systemic inflammatory response
via activation of cellular proteaseactivated receptor receptors.
Primary, or initial, hemostasis, is
mediated through platelet adhesion
and activation. Platelets adhere to
damaged endothelium via the glycoprotein Ib/IX receptor. Small
amounts of thrombin stimulate
platelets to activate, with formation
of cytoplasmic pseudopods, translocation of granules containing
prothrombotic and vasoconstricFigure. Schematic diagram of the coagulation cascade. Reprinted with permission from
tive products to the surface, granuManco-Johnson M, et al. Neoreviews. 2000;1:e191-e195.
lar release, formation of the glycoprotein (GP) IIbIIIa receptor, and
no TF is exposed to the circulation. TF is produced in
cross-linkage of platelets through the GP IIbIIIa recepcells not exposed to the circulation, such as subendothetor via fibrinogen, fibronectin, thrombospondin, and
lial cell pericytes, fibroblasts, and smooth muscle cells as
the von Willebrand factor (VWF). The activated platewell as in monocytes. Both FVII and a small amount of
let contributes phospholipid surface for the activation
FVIIa (approximately 0.1% of FVII) circulate in the
of more thrombin.
plasma. When TF is exposed following endothelial cell
The activation of FX by FIXa and FVIIIa augments
damage or is expressed on the surface of activated cells,
the rate of thrombin generation 1,000-fold. Individuals
the TF-FVIIa/FVII complex forms rapidly. This comwho have hemophilia A (lacking FVIII) and hemophilia
plex rapidly activates factor X (FX) to activated factor X
B (lacking FIX) have normal initiation of thrombin gen(FXa). FXa, in complex with its cofactor factor V, cleaves
eration and rarely bleed spontaneously, but they are
e120 NeoReviews Vol.9 No.3 March 2008

hematology

unable to propagate the hemostatic response following

trauma.
The tissue factor pathway inhibitor (TFPI) inhibits
complexes of TF, FVIIa, and FX. Antithrombin is a
critical regulatory protein that inhibits activated factors
XI, X, IX, and thrombin. Heparin cofactor II is an
ancillary inhibitor of thrombin. The protein C system,
including protein C, protein S, thrombomodulin, and
the endothelial cell protein C receptor, is critical to
inactivation of the activated forms of the cofactors V and
VIII.

Characteristics Unique to the Fetal and

Neonatal Hemostatic System
Murine models of coagulation deficiencies have generated key observations regarding critical requirements of coagulation proteins in embryonic and fetal
development. In these models, total deletion of genes
for antithrombin, TF, TFPI, FV, and prothrombin are
lethal. Results of gene knock-out experiments support
a critical requirement for thrombin generation and
regulation. In contrast, deletion of genes encoding
proteins important in thrombin propagation (eg,
FVIII and FIX) or fibrinolysis (plasminogen, plasminogen activator, or antiplasmin) do not result in excess
fetal mortality.
Certain coagulation proteins, such as TF and thrombomodulin, have a unique fetal distribution. Whereas TF
distribution is limited to the neuroepithelium, vascular
cells, and monocytes in adults, high concentrations can
be detected widely in early development, including in the
skeletal muscle, pancreatic, ectodermal, and endodermal
tissues. TF serves key functions in tissue proliferation and
differentiation that are unique to the embryo and fetus.
The distribution of thrombomodulin expression parallels
that of thrombin. At 24 weeks gestation, plasma thrombomodulin is three times the concentration later found
in healthy adults.
Coagulation proteins that achieve at least the lower
limit of the normal adult range by term birth include
FVIII, FV, and FXIII (Table). Plasma concentrations of
fibrinogen and platelets should be normal at birth, even
in extremely preterm infants. Levels of VWF and alpha2-macroglobulin are increased at term birth compared
with healthy adults. In contrast, plasma concentrations of
the vitamin K-dependent proteinsfactors II, IX, and X
and proteins C and S can be detected in fetal plasma by
18 weeks gestation but do not increase substantially
until near term gestation. Factor VII, which functions
with TF, is a notable exception that achieves the lower

hemostasis

Proteins Involved in
Maintaining Hemostasis
Table.

Protein

Adult Level Present

At Term Birth

XIII
XII
XI
X
IX
VIII
von Willebrand
VII
V
Prothrombin
Fibrinogen
Tissue factor pathway inhibitor
Protein C
Protein S
Antithrombin
Alpha-2-macroglobulin
Heparin cofactor II
Plasminogen
Alpha-2-antiplasmin
Tissue plasminogen activator
Plasminogen activator inhibitor-1

Yes
No
No
No
No
May be higher
May be higher
No
Yes
No
Yes
No
No
No
No
Higher
No
No
Yes
No
Yes

end of the adult normal range by term gestation. Vitamin

K-dependent factors show variable postnatal maturation,
ranging from free protein S, which exceeds the normal
adult range by 3 months, to prothrombin and protein C,
which do not achieve the normal adult range until puberty. The contact factors, prekallikrein, high-molecular
weight kininogen, FXII, and FXI, also display delayed
maturation, achieving the normal adult range by approximately 6 months of age.
Functional clotting and fibrinolytic activities can be
detected in embryonic plasma by 8 weeks of gestation.
Plasma of preterm infants displays a more rapid
rate of thrombin generation relative to healthy children and adults that is correlated with increased circulating TF. The total amount of thrombin generated,
however, is decreased, consistent with the lower fetal and
neonatal concentrations of prothrombin. Following
birth, human umbilical cord endothelial cells activated by
interleukin-1 (IL-1) exhibit twice as much TF activity as
do adult saphenous vein endothelial cells; the amounts of
TF mRNA expressed in response to IL-1 are equal.
A few coagulation proteins exhibit unique fetal forms.
The plasma clot of the fetus and neonate is more translucent than that of a healthy adult, has decreased fibril
NeoReviews Vol.9 No.3 March 2008 e121

hematology

hemostasis

length, and has prolonged time to clotting at negative

pH. Fetal fibrinogen contains twice the content of organically bound phosphorus, increased sialic acid, and
decreased N-alanine in the A-alpha chain. Fetal fibrinogen has a more negative charge, accelerated plasma clearance, and a prolonged thrombin time. Fibrinogen transitions to the adult form by 3 weeks after birth. VWF also
circulates in a fetal form that is characterized by ultra
large-molecular weight multimers, similar to those found
in endothelial cell cytoplasm or in the plasma of patients
who have thrombotic thrombocytopenia purpura. Although it appears logical for the ultra large neonatal
VWF multimers to result from a physiologic deficiency
of the metalloproteinase ADAMTS 13, responsible for
cleavage of VWF multimers following secretion into the
plasma, objective evidence does not support deficient
ADAMTS 13 activity in cord blood. The newborn has a
low plasma concentration of plasminogen, and the fetal
form of the plasminogen molecule exhibits 20% active
site expression following activation by urokinase compared with the adult molecule. However, deficient concentration and enzyme activation of fetal plasminogen is
compensated by a larger functional plasminogen compartment due to very low concentrations of the plasminogen binding protein, histidine-rich glycoprotein, slower
inactivation of fetal plasmin by antiplasmin, and more
rapid in vitro kinetics of fibrinolysis at lower concentrations of tissue plasminogen activator.
Finally, the vitamin K system exhibits unique fetal
characteristics. A tenfold gradient of vitamin K is determined between the maternal and fetal circulation. Vitamin K is necessary for a posttranslational modification of
vitamin K-dependent zymogen proteins in which carboxylation at the gamma position of 9 to 12 glutamic
acid residues located near the NH2 terminus, resulting in
gamma-carboxyglutamic acid (Gla), confers to modified
proteins the capacity for calcium-mediated binding to
phospholipid surfaces that is critical for coagulation activations. The vitamin K cycle includes the enzymes carboxylase, reductase, and vitamin K-epoxide reductase as
well as nicotinamide adenine dinucleotide phosphate.
Other Gla-containing proteins are found in bone, cartilage, dentin, kidney, pancreas, spleen, lung, testes, liver,
and placenta. Three percent of otherwise healthy term
infants show evidence of noncarboxylated prothrombin
in cord blood. Without postnatal supplementation of
vitamin K, approximately 1 in 1,000 infants develops
clinical signs of bleeding and 1 in 10,000 infants suffers
life-threatening hemorrhagic disease.
e122 NeoReviews Vol.9 No.3 March 2008

Results of Coagulation Tests in Healthy Term

and Preterm Infants
The activated partial thromboplastin time (PTT) of the
newborn is prolonged, primarily due to physiologically
low concentrations of the contact factors. The PTT
prolongation is inversely related to gestational age. The
PTT may not achieve adult normal values until 6 months
of age and is not prolonged more than a few seconds in
healthy term infants, but may be greatly prolonged in
healthy extremely preterm infants. Despite decreased
concentrations of many of the vitamin K-dependent clotting factors, the prothrombin time (PT) generally is
within 3 seconds of the upper limit of the adult normal
range in preterm infants and is almost normal in term
infants. The PT may remain slightly prolonged over the
first postnatal week in spite of vitamin K replacement.
The thrombin time is prolonged by about 30% in term
and preterm infants and does not achieve adult normal
values until 3 weeks of postnatal age. Fibrinogen concentrations and platelet counts should be normal, even in
extremely preterm infants. Fibrinogen concentrations
below 100 mg/dL (2.94 mcmol/L) and platelet counts
less than 100103/mcL (100109/L) always are indicative of a pathologic process.
Results of whole blood clotting tests, such as the
thromboelastogram, suggest increased clotting in term
infants, with shorter times to initiation and propagation
of clotting as well as higher maximal amplitude and
greater angle of clot formation. The increased hematocrit
of the term infant contributes to increased whole blood
coagulability and is accentuated by polycythemia.
Healthy preterm infants show even more robust coagulability on whole blood clotting tests. Tests of platelet
adhesion and aggregation, including the template bleeding time and the platelet function analyzer (PFA-100),
have shorter results in newborns than in children and
adults. Tests of plasma coagulability, in contrast, show
decreased size and delayed formation of plasma clots in
both term and preterm infants. Plasma thrombin generation assays show thrombin generation in preterm
plasma that is more rapid in onset, but decreased in
quantity compared with more mature infants, children,
and adults. Fibrinolysis, as tested on the euglobulin clot
lysis time, shows shorter lysis times at birth (ie, increased
fibrinolysis) in comparison with normal adult values.
FVIII concentration is within the adult normal range
at birth, allowing accurate diagnosis of hemophilia A.
However, FIX can be as low as 15 U/dL at birth, making
the distinction between normal and mild hemophilia B
often impossible to determine with certitude. Similarly,
sick newborns, particularly sick preterm infants, often

hematology

manifest protein C concentrations less than 10 U/dL,

and the diagnosis of genetic protein C deficiency versus
acquired or physiologic deficiency cannot be confirmed
for several weeks or months. In contrast, at birth, newborns have VWF concentrations that are higher than
healthy adults, and mild type 1 von Willebrand disease
cannot be excluded in the newborn period due to physiologic elevation.

Suggested Reading
Andrew M, Paes B, Johnston M. Development of the haemostatic
system in the neonate and young infant. Am J Pediatr Hematol
Oncol. 1990;12:95104
Andrew M, Paes B, Johnston M, et al. Development of the human
coagulation system in the healthy premature infant. Blood.
1988;72:16511657
Goldenberg NA, Hathaway WE, Jacobson L, Manco-Johnson MJ.
A new global assay of coagulation and fibrinolysis. Thromb Res.
2005;116:345356

hemostasis

Hathaway WE, Bonnar J. Physiology of coagulation in the fetus and

newborn infant. In: Hemostatic Disorders of the Pregnant
Woman and Newborn Infant. New York, NY: Elsevier Science
Publishing Company; 1987:5775
Manco-Johnson MJ. Development of hemostasis in the fetus.
Pediatr Res. 2005;115(suppl1):55 63
Manco-Johnson MJ, Jacobson LJ, Hacker MR, Townsend SF,
Murphy J, Hay WJR. Development of coagulation regulatory
proteins in the fetal and neonatal lamb. Pediatr Res. 2002;52:
580 588
Petaja J, Manco-Johnson MJ. Protein C pathway in infants and
children. Semin Thromb Hemost. 2003;29:349 362
Reverdiau-Moalic P, Delahousse B, Body G, Bardos P, Leroy J,
Gruel Y. Evolution of blood coagulation activators and inhibitors in the healthy human fetus. Blood. 1996;88:900 906
Reverdiau-Moalic P, Gruel Y, Delahousse B, et al. Comparative
study of the fibrinolytic system in human fetuses and pregnant
women. Thromb Res. 1991;61:489 499
Streif W, Paes B, Berry AM, Andreasen RB, Chan AC. Influence of
exogenous factor VIIa on thrombin generation in plasma of
full-term and pre-term newborns. Blood Coagul Fibrinolysis.
2000;11:349 57

NeoReviews Quiz
9. The critical regulator of the coagulation process is thrombin, which is derived by cleavage from its inactive
precursor prothrombin. Thrombin converts fibrinogen, the critical coagulation protein, into fibrin, which
forms long polymeric protein strands. Of the following, the initial activation of thrombin following
vascular endothelial cell damage occurs by the action of a complex formed by tissue factor with
coagulation protein factor:
A.
B.
C.
D.
E.

V.
VII.
VIII.
X.
XIII.

10. Murine models of coagulation deficiencies have generated key observations regarding critical requirements
of coagulation proteins during embryonic and fetal development. Of the following, the deletion of genes
for the coagulation proteins most likely to be lethal involves:
A.
B.
C.
D.
E.

Antiplasmin.
Factor VIII.
Factor IX.
Plasminogen.
Prothrombin.

11. Coagulation proteins in the developing fetus reach the normal adult range at variable times during
gestation. Of the following, the coagulation protein most delayed in its maturation during fetal
development is:
A.
B.
C.
D.
E.

Alpha 2-macroglobulin.
Factor VIII.
Fibrinogen.
Prothrombin.
von Willebrand factor.
NeoReviews Vol.9 No.3 March 2008 e123