homestasis en el recien nacido

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Hemostasisin the NeonateMarilyn J. Manco-

Johnson, MD*

Author Disclosure

Dr Manco-Johnson

did not disclose any

financial relationships

relevant to this

article.

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.

Abstract

The coagulation system is finely tuned to arrest bleeding at the site of vascular injuryand 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.

IntroductionTo understand the unique features of fetal and neonatal hemostasis, it is essential to

understand coagulation physiology. Coagulation must be regulated carefully to allowrapid 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 CoagulationThe 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 fibrino-gen, 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.

Article hematology

NeoReviews Vol.9 No.3 March 2008 e119


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no TF is exposed to the circulation. TF is produced in

cells not exposed to the circulation, such as subendothe-

lial cell pericytes, fibroblasts, and smooth muscle cells as

well as in monocytes. Both FVII and a small amount of

FVIIa (approximately 0.1% of FVII) circulate in the

plasma. When TF is exposed following endothelial cell

damage or is expressed on the surface of activated cells,

the TF-FVIIa/FVII complex forms rapidly. This com-

plex rapidly activates factor X (FX) to activated factor X

(FXa). FXa, in complex with its cofactor factor V, cleaves

prothrombin to thrombin. The ini-

tial coagulation cascade initiated by

TF generates a small amount of

thrombin that has several activities:

1) platelet activation, thus recruit-

ing a large volume of activation sur-

face; activation of factor VIII

(FVIII) and factor V (FV) that

serve as scaffoldlike cofactors in two

parallel complexes for the activa-

tions of FX by activated factor IX

(FIXa) and thrombin by FXa,

respectively; 2) binding to the en-

dothelial cell receptor, thrombo-

modulin, to form an activationcomplex 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 in-

hibitor that allows the clot suffi-

cient stability for hemostasis and

wound healing before activating fi-

brinolysis; and 5) induction of the

systemic inflammatory response

via activation of cellular protease-activated receptor receptors.

Primary, or initial, hemostasis, is

mediated through platelet adhesion

and activation. Platelets adhere to

damaged endothelium via the gly-

coprotein Ib/IX receptor. Small

amounts of thrombin stimulate

platelets to activate, with formation

of cytoplasmic pseudopods, trans-

location of granules containing

prothrombotic and vasoconstric-

tive products to the surface, granu-lar release, formation of the glyco-

protein (GP) IIbIIIa receptor, and

cross-linkage of platelets through the GP IIbIIIa recep-

tor via fibrinogen, fibronectin, thrombospondin, and

the von Willebrand factor (VWF). The activated plate-

let contributes phospholipid surface for the activation

of more thrombin.

The activation of FX by FIXa and FVIIIa augments

the rate of thrombin generation 1,000-fold. Individuals

who have hemophilia A (lacking FVIII) and hemophilia

B (lacking FIX) have normal initiation of thrombin gen-

eration and rarely bleed spontaneously, but they are

Figure. Schematic diagram of the coagulation cascade. Reprinted with permission from

Manco-Johnson M, et al. Neoreviews. 2000;1:e191-e195.

hematology hemostasis

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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 SystemMurine models of coagulation deficiencies have gen-

erated key observations regarding critical require-

ments 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, plasmin-

ogen activator, or antiplasmin) do not result in excess

fetal mortality.Certain coagulation proteins, such as TF and throm-

bomodulin, 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 throm-

bomodulin 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 alpha-

2-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 Scan 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

end of the adult normal range by term gestation. VitaminK-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 pu-

berty. The contact factors, prekallikrein, high-molecular

weight kininogen, FXII, and FXI, also display delayed

maturation, achieving the normal adult range by approx-

imately 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 chil-

dren and adults that is correlated with increased cir-

culating 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 trans-

lucent than that of a healthy adult, has decreased fibril

Table. Proteins Involved in

Maintaining Hemostasis

ProteinAdult Level PresentAt Term Birth

XIII YesXII NoXI NoX NoIX No

VIII May be highervon Willebrand May be higher

VII NoV YesProthrombin NoFibrinogen YesTissue factor pathway inhibitor NoProtein C NoProtein S NoAntithrombin NoAlpha-2-macroglobulin HigherHeparin cofactor II NoPlasminogen NoAlpha-2-antiplasmin YesTissue plasminogen activator NoPlasminogen activator inhibitor-1 Yes




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length, and has prolonged time to clotting at negative

pH. Fetal fibrinogen contains twice the content of or-

ganically bound phosphorus, increased sialic acid, and

decreased N-alanine in the A-alpha chain. Fetal fibrino-

gen has a more negative charge, accelerated plasma clear-

ance, and a prolonged thrombin time. Fibrinogen tran-

sitions 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. Al-

though it appears logical for the ultra large neonatal

VWF multimers to result from a physiologic deficiency

of the metalloproteinase ADAMTS 13, responsible forcleavage 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 com-

pared with the adult molecule. However, deficient con-

centration and enzyme activation of fetal plasminogen is

compensated by a larger functional plasminogen com-

partment due to very low concentrations of the plasmin-

ogen binding protein, histidine-rich glycoprotein, slower

inactivation of fetal plasmin by antiplasmin, and morerapid in vitro kinetics of fibrinolysis at lower concentra-

tions of tissue plasminogen activator.

Finally, the vitamin K system exhibits unique fetal

characteristics. A tenfold gradient of vitamin K is deter-

mined between the maternal and fetal circulation. Vita-

min K is necessary for a posttranslational modification of

vitamin K-dependent zymogen proteins in which car-

boxylation 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 acti-

vations. The vitamin K cycle includes the enzymes car-

boxylase, reductase, and vitamin K-epoxide reductase as

well as nicotinamide adenine dinucleotide phosphate.

Other Gla-containing proteins are found in bone, carti-

lage, 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.

Results of Coagulation Tests in Healthy Termand Preterm InfantsThe 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 clot-

ting 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 thefirst 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 concen-

trations 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 indic-

ative 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 propagationof 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 coagu-

lability on whole blood clotting tests. Tests of platelet

adhesion and aggregation, including the template bleed-

ing 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 gener-ation 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


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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, new-

borns 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 phys-

iologic elevation.

Suggested ReadingAndrew M, Paes B, Johnston M. Development of the haemostatic

system in the neonate and young infant. Am J Pediatr HematolOncol.1990;12:95104

Andrew M, Paes B, Johnston M, et al. Development of the humancoagulation system in the healthy premature infant. Blood.

1988;72:16511657Goldenberg NA, Hathaway WE, Jacobson L, Manco-Johnson MJ.

A new global assay of coagulation and fibrinolysis.Thromb Res.2005;116:345356

Hathaway WE, Bonnar J. Physiology of coagulation in thefetusand

newborn infant. In: Hemostatic Disorders of the Pregnant

Woman and Newborn Infant. New York, NY: Elsevier SciencePublishing Company; 1987:5775

Manco-Johnson MJ. Development of hemostasis in the fetus.

Pediatr Res.2005;115(suppl1):5563

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;52:580588

Petaja J, Manco-Johnson MJ. Protein C pathway in infants andchildren.Semin Thromb Hemost.2003;29:349362

Reverdiau-Moalic P, Delahousse B, Body G, Bardos P, Leroy J,Gruel Y. Evolution of blood coagulation activators and inhibi-tors in the healthy human fetus.Blood.1996;88:900906

Reverdiau-Moalic P, Gruel Y, Delahousse B, et al. Comparative

study of the fibrinolytic system in human fetuses and pregnantwomen.Thromb Res.1991;61:489 499Streif W, Paes B, Berry AM, Andreasen RB, Chan AC. Influence of

exogenous factor VIIa on thrombin generation in plasma offull-term and pre-term newborns. Blood Coagul Fibrinolysis.2000;11:34957

NeoReviews Quiz

9. The critical regulator of the coagulation process is thrombin, which is derived by cleavage from its inactiveprecursor prothrombin. Thrombin converts fibrinogen, the critical coagulation protein, into fibrin, whichforms 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 withcoagulation protein factor:

A. V.B. VII.C. VIII.D. X.E. XIII.

10. Murine models of coagulation deficiencies have generated key observations regarding critical requirementsof coagulation proteins during embryonic and fetal development. Of the following, the deletion of genesfor the coagulation proteins mostlikely to be lethal involves:

A. Antiplasmin.

B. Factor VIII.C. Factor IX.D. Plasminogen.E. Prothrombin.

11. Coagulation proteins in the developing fetus reach the normal adult range at variable times duringgestation. Of the following, the coagulation protein mostdelayed in its maturation during fetaldevelopment is:

A. Alpha 2-macroglobulin.B. Factor VIII.C. Fibrinogen.D. Prothrombin.E. von Willebrand factor.



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