- Email: [email protected]
Hemostasis and Platelet Dysfunction in Asphyxiated Neonates
Hemostasis and Platelet Dysfunction in Asphyxiated Neonates Mary E. Bauman, RN, BA, MN, NP, Po-Yin Cheung, MBBS, PhD, FRCP (Can&Edin), and M. Patricia Massicotte, MD, FRCPC, MHSc Hemostasis is the balance between bleeding and clotting and includes coagulation and fibrinolysis with platelet interactions. Despite developmental hemostasis that describes the major differences between neonates and older children and adults, neonates do not have increased bleeding or clotting unless clinical situations disturb the ‘‘balance.’’ Perinatal asphyxia alters the balance of hemostasis, resulting in abnormalities that may result in bleeding and thrombosis. The following discussion will describe normal hemostasis, laboratory measures of hemostasis, developmental hemostasis, and the effects of asphyxia on hemostasis. (J Pediatr 2011;158:e35-9).
Physiologic Hemostasis
I
ncreased knowledge about hemostasis (coagulation and fibrinolysis) has resulted in a paradigm shift in the description from the classical model of coagulation (cascade or waterfall)1,2 to a cell-based model.3,4 The ‘‘new’’ model incorporates the important cellular and protein interactions of coagulation which occur in vivo, rendering the relatively independent intrinsic and extrinsic pathways of the cascade model as artificial descriptions of the in vivo process. Cell-based coagulation has three phases: initiation, amplification and propagation (Figure 1). Mechanisms responsible for the regulation of thrombin and fibrin generation: termination, elimination, and stabilization are also an integral part of the model (Figure 2). Healthy platelets participate in the balance of hemostasis through the following activities: activation, adhesion, secretion of active substances, and aggregation. Collagen exposure and von Willebrand factor (vWF) released by damaged blood vessels result in platelet adhesion.5 In high shear stress conditions, such as arteries and microvasculature, high molecular weight vWF mediates platelet adhesion by binding to collagen and the nonactivated platelet through the platelet receptor glycoprotein (GP) Ib-IX. In low shear stress conditions (large veins), platelet adhesion occurs through direct interaction via GP VI with collagen. Platelets secrete active substances from intracellular granules which result in: (1) enhanced platelet adhesion and aggregation by adenosine diphosphate, vWF, fibrinogen and thrombospondin; (2) facilitated coagulation by factor V and fibrinogen; (3) increased vascular tone and contraction by serotonin; (4) increased cell proliferation and migration by platelet derived growth factor; and (5) increased aggregation through the binding of fibrinogen to the receptor GP IIb-IIIa on activated platelets.6
Developmental Hemostasis Neonates have a number of differences in hemostasis and fibrinolysis compared with adults that affect the incidence, treatment, and long-term outcome of thrombosis.7-10 Coagulation factors do not cross the placental barrier but are synthesized independently by the fetus. At birth the levels of contact factors (XII, X, high molecular–weight kininogen) and vitamin K–dependent factors are decreased to about 50% of adult values until approximately 6 months of age.7-9 Thrombin generation is decreased 30% to 50% as compared with adult levels.11,12 Although premature neonates have decreased levels of similar procoagulant factors compared with term neonates, there are no interventional studies determining the safety and efficacy of plasma transfusions in healthy or ill infants to overcome this physiological hypocoagulability. Plasma concentrations of the naturally occurring anticoagulant proteins (inhibitors of coagulation) are present in varying amounts with decreased levels of protein C, S and antithrombin and increased levels of a2-macroglobulin. The fibrinolytic system is down-regulated with decreased levels of plasminogen (50% of adult levels), increased plasminogen activator inhibitor and decreased levels of tissue plasminogen activator.7-9 Normal neonates are vitamin K deficient and accordingly have reduced levels of vitamin K–dependent clotting factors. To facilitate the synthesis of these factors, vitamin K should be administered shortly after birth.13 Prophylactic vitamin K is given for vitamin K deficiency bleeding in neonates. Platelet number and function are equally critical to balanced hemostasis. Platelet count in healthy neonates is elevated in the immediate postnatal period14 then decreases to adult levels. In contrast, lower platelet counts are found in premature neonates compared with term neFrom the Department of Pediatrics, University of Alberta, onates.14 Platelet function in neonates is hyporeactive compared with older Edmonton, Alberta, Canada
GP vWF
Glycoprotein von Willebrand factor
Please see the Author Disclosures at the end of this article. 0022-3476/$ - see front matter. Copyright ª 2011 Mosby Inc. All rights reserved. 10.1016/j.jpeds.2010.11.011
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Figure 1. Cell-based model of coagulation. In this evolving structural-functional scheme, coagulation occurs in three phases: initiation, amplification and propagation. TF, Tissue factor.3
children and adults. The platelet function was evaluated by ex vivo studies by use of aggregometry and flow cytometry of umbilical cord blood collected at delivery. These studies demonstrate decreased activation responses including aggregation, granule secretion, calcium flux, fibrinogen receptor expression, and thromboxane A2 production to the following agonists: thrombin, adenosine diphosphate, epinephrine, collagen, and U46619 (a stable mimetic of thromboxane A2). There were no differences between cord and peripheral blood platelets.15 The platelet response to agonists in premature neonates is further decreased.16,17 Abnormal activity of the signal-transducing protein (Gq) may be partially responsible for in vitro hyporeactivity of the neonatal platelet.16-18 Despite platelet hyporeactivity, global hemostasis measures demonstrate efficient hemostasis and hemostatic profiles similar to adults.19 Interestingly, there is little evidence to support that platelet activation/hyporeactivity occur as a result of the birthing process.20 Despite these physiological differences of constituent proteins and inhibitors of coagulation and platelet properties, the ‘‘balance’’ of hemostasis is maintained in healthy term and premature neonates.
Laboratory Measures of Hemostasis Coagulation The most common tests used to measure in vivo coagulation are based on the cascade model of coagulation. These include the activated partial thromboplastin time and the international normalized ratio, a translation of the prothrombin time as an attempt to standardize prothrombin time results internationally, accounting for dife36
ferent analyzers and thromboplastin reagents used in testing. The activated partial thromboplastin time and international normalized ratio measure factors II, VIII, X, XI, and XII of the intrinsic pathway, and the vitamin K–dependent factors (II, VII, IX, X) of the extrinsic pathway, respectively. Importantly, neither of these tests includes cellular components (red blood cells, platelets, white blood cells), which are integral for in vivo coagulation to occur and thus are not a comprehensive measure of hemostasis. As a result, global measures of hemostasis, which include cellular components in the test medium (eg, thromboelastography), are being investigated and proposed as more appropriate measures of hemostasis. Thromboelastography uses activated whole blood to measure coagulation (formation of a clot), as well as fibrinolysis (clot degradation). Well-designed studies are required to evaluate the validity, accuracy, and application of this measure in neonates. Platelet Function The evaluation of platelet function (eg, aggregation, adhesion) is important, but the definitive method to use to measure function is controversial21 because the platelet number correlates weakly with the bleeding tendency. Bleeding time, which is a composite assessment of platelet and vascular factors in hemostasis, is rarely performed because it is not predictive of clinical outcomes. Measuring the function of washed platelets is not practical in neonates. Thus, common tests of platelet function include the evaluation of aggregatory responses to agonists (eg, ADP, collagen and thrombin) using whole blood. Biochemical (eg, plasma thromboxane and P-selectin levels, Bauman, Cheung, and Massicotte
February 2011
SUPPLEMENT
Figure 2. Regulation of coagulation. The dotted lines indicate inhibition of thrombin (IIa) formation or activity by tissue factor pathway inhibitor, antithrombin III, and activated protein C in the presence of protein S. Center, Plasmin, generated from plasminogen by tissue-plasminogen activator and urokinase plasminogen activator, breaks fibrin into degradation products. Right, Factor XIIIa, thrombin-activatable fibrinolysis inhibitor, plasminogen activator inhibitor type 1, and a 2-antiplasmin promote fibrin stability. TFPI, tissue factor pathway inhibitor; ATIII, antithrombin III; APC, activated protein C; PS, protein S; PL1, Plasmin; PLG, plasminogen; t-PA, tissue-plasminogen activator; u-PA, urokinase plasminogen activator; FDP, fibrin degradation product; TAFIa, factor XIIIa thrombin-activatable fibrinolysis inhibitor; PAI-1, plasminogen activator inhibitor type 1.
CD62 by flow cytometry) and morphologic (electron microscopy) examinations will provide additional information about some platelet abnormalities.
Asphyxia and Hemostasis Asphyxia occurs in one to six per 1000 live births,22 but there are few studies published that describe hematologic sequelae. Both large volumes of blood required for studies and challenges with parental consent in neonates provide barriers to large prospective studies. However, the following hematologic and hemostatic sequelae have been described, including altered cellular components of whole blood, certain proteins, and increased activation of coagulation and platelets. From a coagulation perspective, moderate thrombocytopenia is reported in asphyxiated neonates although statistical significance is inconsistent among publications.23,24 Destructive thrombocytopenia occurs in 22% of critically ill neonates, most of whom experienced birth asphyxia and had platelet counts decreasing to <100 109/L. Increased platelet turnover is demonstrated by decreased platelet survival and increased mean platelet volume, with normal overall marrow cellularity, compared with normal controls.25 Indeed, after platelet infusion in neonates, there is an initial increase in platelet count; however, the response is poorer than predicted, and the platelet count declines to pretransfusion levels Hemostasis and Platelet Dysfunction in Asphyxiated Neonates
within 24 to 48 hours after infusion.25 Thus increased destruction of platelets is described as the most important mechanism of thrombocytopenia in asphyxia. Thromboxane levels are increased in asphyxiated neonates and this indicates platelet activation.26 In a study of in vitro behavior of cord blood platelets, under acidotic conditions the plasma proteins surrounding platelets were less negative and less repellant of each other compared with neutral conditions.27 This altered physiological property of platelets in respiratory acidosis may be an additional factor contributing to hemorrhagic and thrombotic complications in neonates with asphyxia. Although the effect of hypoxia on platelet function requires further investigation, the reoxygenation process contributes to platelet activation and aggregatory dysfunction during the recovery of asphyxiated neonates.28 Furthermore, the oxygen concentration used in the resuscitation affects ex vivo platelet function in asphyxiated newborn piglets with significantly more activation (higher thromboxane levels) and worsened platelet aggregatory response toward collagen stimulation when 100% oxygen was used, compared with room-air.28,29 Thrombopoietin is a growth factor responsible for stimulation, production, and maturation of megakaryocytes and is a major regulator of platelet counts. Thrombopoietin is increased during hypoxia.30 Platelet count negatively correlated with thrombopoietin levels on days 1, 3, and 7 of life in asphyxiated neonates.30 Interestingly, thrombopoietin levels e37
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on day 7 correlated with the clinical severity of asphyxia; however, neither platelet count nor thrombopoietin levels were predictors of death.30 Asphyxiated neonates have evidence of disseminated intravascular coagulation. Levels of factor XIII are markedly lower in the high-risk neonates with the lowest values coinciding with lowest Apgar scored.31 Plasma levels of thrombinantithrombin complexes, D-dimer, fibrinogen, fibrin degradation products, and soluble fibrin monomer complexes are also reported to be higher in the asphyxiated neonates correlating to disseminated intravascular coagulation.32-34 Although not all neonates with respiratory distress have experienced asphyxia, thrombin inhibition may be altered and was abnormal in both term and premature neonates with respiratory distress.34 As mentioned above, hemostatic complications (disseminated intravascular coagulopathy, thrombocytopenia, and thromboembolism) are related to the activated-depressed coagulation pathways and platelet function. Thus the best approach to hemostatic complications of asphyxiated neonates is to prevent the asphyxiating event. Supportive management including blood products transfusion with packed red blood cells, platelets, fresh frozen plasma, and cryoprecipitate is commonly practiced in many neonatal intensive care units. Although there are no generally accepted guidelines for transfusion in neonates, especially for those with asphyxia, the effects of concurrent therapies may also affect our decision-making. For example, even though therapeutic hypothermia has not been associated with an increased incidence of hemostatic complications,35 hypothermia may impair coagulation36 and accelerate microvascular thrombosis formation.37 Similar theoretical controversies exist in the administration of inhaled nitric oxide, which inhibits platelet function38 and may affect the development of intraventricular hemorrhage in premature neonates. The infusion of dobutamine at a high dose (20 mg/kg/min) will aggravate platelet aggregatory dysfunction.39 On the other hand, the hemostatic disturbance can also complicate the recovery and affect the clinical management of these critically ill neonates. The development of venous thrombosis in vital organs may affect the outcome, and the use of heparin or thrombolytic agents to treat thromboses needs to be considered with hematology consultation.40 It is uncertain whether the hemostatic disturbance may contribute to the patency of vascular access. Further studies are needed to examine the hemostatic problem and its management in asphyxiated neonates. Neonates have a different physiologic hemostasis compared with older children and adults. In healthy neonates the balance of hemostasis is maintained without increased risk for hemorrhage or thrombosis except in high-risk situations such as asphyxia. Despite the paucity of evidence, the asphyxiated neonate appears to have a disturbed balance in hemostasis potentially predisposing them to hemorrhage or thrombosis. Further prospective laboratory studies with clear definitions of asphyxia are needed to characterize the abnormalities in hemostasis. Prospective epidemiologic studies e38
Vol. 158, No. 2, Suppl. 1 evaluating outcomes will provide the necessary background data for intervention studies. n
Author Disclosures Mary E. Bauman, RN, NP, Po-Yin Cheung, MBBS, PhD, FRCP (Can & Edin), and M. Patricia Massicotte, MD, FRCPC, have no financial arrangement or affiliation with a corporate organization or a manufacturer of a product discussed in this supplement. Reprint requests: M. Patricia Massicotte, MD, FRCPC, MHSc, Department of Pediatrics, University of Alberta, Stollery Children’s Hospital, 8440 112th St, WMC 4H2.11, Edmonton, Alberta, Canada T6G 2B7. E-mail: patti. [email protected].
References 1. Davie EW, Ratnoff OD. Waterfall Sequence For Intrinsic Blood Clotting. Science 1964;145:1310-2. 2. Macfarlane RG. An Enzyme Cascade In The Blood Clotting Mechanism, And It’s Function As A Biochemical Amplifier. Nature 1964; 202:498-9. 3. Becker RC. Cell-based models of coagulation: a paradigm in evolution. J Thromb Thrombolysis 2005;20:65-8. 4. Hoffman M, Monroe DM 3rd. A cell-based model of hemostasis. Thromb Haemost 2001;85:958-65. 5. Ruggeri ZM. Von Willebrand factor, platelets and endothelial cell interactions. J Thromb Haemost 2003;1:1335-42. 6. Rendu F, Brohard-Bohn B. The platelet release reaction: Granules’ constituents, secretion and functions. Platelets 2001;12:261-73. 7. Andrew M, Paes B, Milner R, Johnston M, Mitchell L, Tollefsen DM, et al. Development of the human coagulation system in the healthy premature infant. Blood 1988;72:1651-7. 8. Andrew M, Paes B, Milner R, Johnston M, Mitchell L, Tollefsen DM, et al. Development of the human coagulation system in the full-term infant. Blood 1987;70:165-72. 9. Andrew M, Vegh P, Johnston M, Bowker J, Ofosu F, Mitchell L. Maturation of the hemostatic system during childhood. Blood 1992;80:19982005. 10. Kuhle S, Massicotte PM. Maturation of the coagulation system during childhood. Progress in Pediatric Cardiology 2005;21:3-7. 11. Andrew M, Mitchell L, Vegh P, Ofosu F. Thrombin regulation in children differs from adults in the absence and presence of heparin. Thromb Haemost 1994;72:836-42. 12. Andrew M, Schmidt B, Mitchell L, Paes B, Ofosu F. Thrombin generation in newborn plasma is critically dependent on the concentration of prothrombin. Thromb Haemost 1990;63:27-30. 13. Puckett RM, Offringa M. Prophylactic vitamin K for vitamin K deficiency bleeding in neonates. Cochrane database of systematic reviews (Online) 2000. 14. Patrick CH, Lazarchick J, Stubbs T, Pittard WB. Mean platelet volume and platelet distribution width in the neonate. Am J Pediatr Hematol Oncol 1987;9:130-2. 15. Sitaru AG, Holzhauer S, Speer CP, Singer D, Obergfell A, Walter U, et al. Neonatal platelets from cord blood and peripheral blood. Platelets 2005; 16:203-10. 16. Israels SJ, Cheang T, Roberston C, McMillan-Ward EM, McNicol A. Impaired signal transduction in neonatal platelets. Pediatric Research 1999; 45:687-91. 17. Israels SJ, Rand ML, Michelson AD. Neonatal platelet function. Semin Thromb Hemost 2003;29:363-72. 18. Michelson AD. Platelet function in the newborn. Seminars in Thrombosis and Hemostasis 1998;24:507-12.
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February 2011 19. Cvirn G, Kutschera J, Wagner T, Ferstl U, Vrecko K, Hallstrom S, et al. Collagen/endogenous thrombin-induced platelet aggregation in cord versus adult whole blood. Neonatology 2009;95:187-92. 20. Grosshaupt B, Muntean W, Sedlmayr P. Hyporeactivity of neonatal platelets is not caused by preactivation during birth. Eur J Pediatr 1997;156:944-8. 21. Michelson AD. Methods for the measurement of platelet function. Am J Cardiol 2009;103:20A-6. 22. World-Health-Organization. Child Health and Development: Health of the Newborn. In: World Health Organization; 1991; Geneva, Switzerland: World Health Organization; 1991. 23. Nako Y, Tomomasa T, Morikawa A. Plasma thrombomodulin level in newborn infants with and without perinatal asphyxia.[see comment]. Acta Paediatr 1997;86:91-5. 24. Phelan JP, Kirkendall C, Korst LM, Martin GI. Nucleated red blood cell and platelet counts in asphyxiated neonates sufficient to result in permanent neurologic impairment. J Matern Fetal Neonatal Med 2007;20:377-80. 25. Castle V, Andrew M, Kelton J, Giron D, Johnston M, Carter C. Frequency and mechanism of neonatal thrombocytopenia. J Pediatr 1986; 108:749-55. 26. Brus F, van Oeveren W, Okken A, Oetomo SB. Number and activation of circulating polymorphonuclear leukocytes and platelets are associated with neonatal respiratory distress syndrome severity. Pediatrics 1997; 99:672-80. 27. Kosztolanyi G, Jobst K. Electrokinetic behaviour of cord blood platelets in acidosis: an in vitro study. Biol Neonate 1979;35:255-7. 28. Cheung P-Y, Stevens JP, Haase E, Stang L, Bigam DL, Etches W, et al. Platelet dysfunction in asphyxiated newborn piglets resuscitated with 21% and 100% oxygen. Pediatr Res 2006;59:636-40. 29. Postma S, Emara M, Obaid L, Johnson ST, Bigam DL, Cheung P-Y. Temporal platelet aggregatory function in hypoxic newborn piglets reoxygenated with 18%, 21%, and 100% oxygen. Shock 2007;27:448-54.
Hemostasis and Platelet Dysfunction in Asphyxiated Neonates
SUPPLEMENT 30. Aly H, El Beshlawy A, Badrawi N, Mohsen L, Mansour E, Ramy N, et al. Thrombopoietin level is increased in the serum of asphyxiated neonates: a prospective controlled study. J Perinatol 2005;25:320-4. 31. Mautone A, Giordano P, Montagna O, Quercia M, Altomare M, De Mattia D. Coagulation and fibrinolytic systems in the ill preterm newborn. Acta Paediatr 1997;86:1100-4. 32. Castle V, Coates G, Mitchell LG, O’Brodovich H, Andrew M. The effect of hypoxia on platelet survival and site of sequestration in the newborn rabbit. Thromb Haemost 1988;59:45-8. 33. Fichera A, Pratico G, Sciacca F. [Neonatal hypoxia and hemocoagulative changes]. Pediatr Med Chir 1989;11:643-7. 34. Suzuki S, Morishita S. Hypercoagulability and DIC in high-risk infants. Semin Thromb Hemost 1998;24:463-6. 35. Azzopardi DV, Strohm B, Edwards AD, Dyet L, Halliday H, Jaszczak E, et al. Moderate hypothermia to treat perinatal asphyxial encephalopathy. NEJM 2009;361:1349-58. 36. Dirkmann D, Hanke AA, Gorlinger K, Peters J. Hypothermia and acidosis synergistically impair coagulation in human whole blood. Anesth Analg 2008;106:1627-32. 37. Lindenblatt N, Menger MD, Klar E, Vollmar B. Sustained hypothermia accelerates microvascular thrombus formation in mice. Am J Physiol Heart Circ Physiol 2005;289:H2680-7. 38. Cheung PY, Salas E, Etches PC, Phillipos E, Schulz R, Radomski MW. Inhaled nitric oxide and inhibition of platelet aggregation in critically ill neonates. Lancet 1998;351:1181-2. 39. Al-Salam Z, Emara M, Wehlage S, Abozaid S, Johnson ST, Bigam DL, et al. The effect of dobutamine on platelet aggregatory function in newborn piglets with hypoxia and reoxygenation. Shock 2008;30:293-8. 40. Monagle P, Chan A, Massicotte P, Chalmers E, Michelson AD. Antithrombotic therapy in neonates and children: the Eighth ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest 2008;133: 645S-87.
e39
Physiologic Hemostasis
I
ncreased knowledge about hemostasis (coagulation and fibrinolysis) has resulted in a paradigm shift in the description from the classical model of coagulation (cascade or waterfall)1,2 to a cell-based model.3,4 The ‘‘new’’ model incorporates the important cellular and protein interactions of coagulation which occur in vivo, rendering the relatively independent intrinsic and extrinsic pathways of the cascade model as artificial descriptions of the in vivo process. Cell-based coagulation has three phases: initiation, amplification and propagation (Figure 1). Mechanisms responsible for the regulation of thrombin and fibrin generation: termination, elimination, and stabilization are also an integral part of the model (Figure 2). Healthy platelets participate in the balance of hemostasis through the following activities: activation, adhesion, secretion of active substances, and aggregation. Collagen exposure and von Willebrand factor (vWF) released by damaged blood vessels result in platelet adhesion.5 In high shear stress conditions, such as arteries and microvasculature, high molecular weight vWF mediates platelet adhesion by binding to collagen and the nonactivated platelet through the platelet receptor glycoprotein (GP) Ib-IX. In low shear stress conditions (large veins), platelet adhesion occurs through direct interaction via GP VI with collagen. Platelets secrete active substances from intracellular granules which result in: (1) enhanced platelet adhesion and aggregation by adenosine diphosphate, vWF, fibrinogen and thrombospondin; (2) facilitated coagulation by factor V and fibrinogen; (3) increased vascular tone and contraction by serotonin; (4) increased cell proliferation and migration by platelet derived growth factor; and (5) increased aggregation through the binding of fibrinogen to the receptor GP IIb-IIIa on activated platelets.6
Developmental Hemostasis Neonates have a number of differences in hemostasis and fibrinolysis compared with adults that affect the incidence, treatment, and long-term outcome of thrombosis.7-10 Coagulation factors do not cross the placental barrier but are synthesized independently by the fetus. At birth the levels of contact factors (XII, X, high molecular–weight kininogen) and vitamin K–dependent factors are decreased to about 50% of adult values until approximately 6 months of age.7-9 Thrombin generation is decreased 30% to 50% as compared with adult levels.11,12 Although premature neonates have decreased levels of similar procoagulant factors compared with term neonates, there are no interventional studies determining the safety and efficacy of plasma transfusions in healthy or ill infants to overcome this physiological hypocoagulability. Plasma concentrations of the naturally occurring anticoagulant proteins (inhibitors of coagulation) are present in varying amounts with decreased levels of protein C, S and antithrombin and increased levels of a2-macroglobulin. The fibrinolytic system is down-regulated with decreased levels of plasminogen (50% of adult levels), increased plasminogen activator inhibitor and decreased levels of tissue plasminogen activator.7-9 Normal neonates are vitamin K deficient and accordingly have reduced levels of vitamin K–dependent clotting factors. To facilitate the synthesis of these factors, vitamin K should be administered shortly after birth.13 Prophylactic vitamin K is given for vitamin K deficiency bleeding in neonates. Platelet number and function are equally critical to balanced hemostasis. Platelet count in healthy neonates is elevated in the immediate postnatal period14 then decreases to adult levels. In contrast, lower platelet counts are found in premature neonates compared with term neFrom the Department of Pediatrics, University of Alberta, onates.14 Platelet function in neonates is hyporeactive compared with older Edmonton, Alberta, Canada
GP vWF
Glycoprotein von Willebrand factor
Please see the Author Disclosures at the end of this article. 0022-3476/$ - see front matter. Copyright ª 2011 Mosby Inc. All rights reserved. 10.1016/j.jpeds.2010.11.011
e35
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Vol. 158, No. 2, Suppl. 1
Figure 1. Cell-based model of coagulation. In this evolving structural-functional scheme, coagulation occurs in three phases: initiation, amplification and propagation. TF, Tissue factor.3
children and adults. The platelet function was evaluated by ex vivo studies by use of aggregometry and flow cytometry of umbilical cord blood collected at delivery. These studies demonstrate decreased activation responses including aggregation, granule secretion, calcium flux, fibrinogen receptor expression, and thromboxane A2 production to the following agonists: thrombin, adenosine diphosphate, epinephrine, collagen, and U46619 (a stable mimetic of thromboxane A2). There were no differences between cord and peripheral blood platelets.15 The platelet response to agonists in premature neonates is further decreased.16,17 Abnormal activity of the signal-transducing protein (Gq) may be partially responsible for in vitro hyporeactivity of the neonatal platelet.16-18 Despite platelet hyporeactivity, global hemostasis measures demonstrate efficient hemostasis and hemostatic profiles similar to adults.19 Interestingly, there is little evidence to support that platelet activation/hyporeactivity occur as a result of the birthing process.20 Despite these physiological differences of constituent proteins and inhibitors of coagulation and platelet properties, the ‘‘balance’’ of hemostasis is maintained in healthy term and premature neonates.
Laboratory Measures of Hemostasis Coagulation The most common tests used to measure in vivo coagulation are based on the cascade model of coagulation. These include the activated partial thromboplastin time and the international normalized ratio, a translation of the prothrombin time as an attempt to standardize prothrombin time results internationally, accounting for dife36
ferent analyzers and thromboplastin reagents used in testing. The activated partial thromboplastin time and international normalized ratio measure factors II, VIII, X, XI, and XII of the intrinsic pathway, and the vitamin K–dependent factors (II, VII, IX, X) of the extrinsic pathway, respectively. Importantly, neither of these tests includes cellular components (red blood cells, platelets, white blood cells), which are integral for in vivo coagulation to occur and thus are not a comprehensive measure of hemostasis. As a result, global measures of hemostasis, which include cellular components in the test medium (eg, thromboelastography), are being investigated and proposed as more appropriate measures of hemostasis. Thromboelastography uses activated whole blood to measure coagulation (formation of a clot), as well as fibrinolysis (clot degradation). Well-designed studies are required to evaluate the validity, accuracy, and application of this measure in neonates. Platelet Function The evaluation of platelet function (eg, aggregation, adhesion) is important, but the definitive method to use to measure function is controversial21 because the platelet number correlates weakly with the bleeding tendency. Bleeding time, which is a composite assessment of platelet and vascular factors in hemostasis, is rarely performed because it is not predictive of clinical outcomes. Measuring the function of washed platelets is not practical in neonates. Thus, common tests of platelet function include the evaluation of aggregatory responses to agonists (eg, ADP, collagen and thrombin) using whole blood. Biochemical (eg, plasma thromboxane and P-selectin levels, Bauman, Cheung, and Massicotte
February 2011
SUPPLEMENT
Figure 2. Regulation of coagulation. The dotted lines indicate inhibition of thrombin (IIa) formation or activity by tissue factor pathway inhibitor, antithrombin III, and activated protein C in the presence of protein S. Center, Plasmin, generated from plasminogen by tissue-plasminogen activator and urokinase plasminogen activator, breaks fibrin into degradation products. Right, Factor XIIIa, thrombin-activatable fibrinolysis inhibitor, plasminogen activator inhibitor type 1, and a 2-antiplasmin promote fibrin stability. TFPI, tissue factor pathway inhibitor; ATIII, antithrombin III; APC, activated protein C; PS, protein S; PL1, Plasmin; PLG, plasminogen; t-PA, tissue-plasminogen activator; u-PA, urokinase plasminogen activator; FDP, fibrin degradation product; TAFIa, factor XIIIa thrombin-activatable fibrinolysis inhibitor; PAI-1, plasminogen activator inhibitor type 1.
CD62 by flow cytometry) and morphologic (electron microscopy) examinations will provide additional information about some platelet abnormalities.
Asphyxia and Hemostasis Asphyxia occurs in one to six per 1000 live births,22 but there are few studies published that describe hematologic sequelae. Both large volumes of blood required for studies and challenges with parental consent in neonates provide barriers to large prospective studies. However, the following hematologic and hemostatic sequelae have been described, including altered cellular components of whole blood, certain proteins, and increased activation of coagulation and platelets. From a coagulation perspective, moderate thrombocytopenia is reported in asphyxiated neonates although statistical significance is inconsistent among publications.23,24 Destructive thrombocytopenia occurs in 22% of critically ill neonates, most of whom experienced birth asphyxia and had platelet counts decreasing to <100 109/L. Increased platelet turnover is demonstrated by decreased platelet survival and increased mean platelet volume, with normal overall marrow cellularity, compared with normal controls.25 Indeed, after platelet infusion in neonates, there is an initial increase in platelet count; however, the response is poorer than predicted, and the platelet count declines to pretransfusion levels Hemostasis and Platelet Dysfunction in Asphyxiated Neonates
within 24 to 48 hours after infusion.25 Thus increased destruction of platelets is described as the most important mechanism of thrombocytopenia in asphyxia. Thromboxane levels are increased in asphyxiated neonates and this indicates platelet activation.26 In a study of in vitro behavior of cord blood platelets, under acidotic conditions the plasma proteins surrounding platelets were less negative and less repellant of each other compared with neutral conditions.27 This altered physiological property of platelets in respiratory acidosis may be an additional factor contributing to hemorrhagic and thrombotic complications in neonates with asphyxia. Although the effect of hypoxia on platelet function requires further investigation, the reoxygenation process contributes to platelet activation and aggregatory dysfunction during the recovery of asphyxiated neonates.28 Furthermore, the oxygen concentration used in the resuscitation affects ex vivo platelet function in asphyxiated newborn piglets with significantly more activation (higher thromboxane levels) and worsened platelet aggregatory response toward collagen stimulation when 100% oxygen was used, compared with room-air.28,29 Thrombopoietin is a growth factor responsible for stimulation, production, and maturation of megakaryocytes and is a major regulator of platelet counts. Thrombopoietin is increased during hypoxia.30 Platelet count negatively correlated with thrombopoietin levels on days 1, 3, and 7 of life in asphyxiated neonates.30 Interestingly, thrombopoietin levels e37
THE JOURNAL OF PEDIATRICS
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on day 7 correlated with the clinical severity of asphyxia; however, neither platelet count nor thrombopoietin levels were predictors of death.30 Asphyxiated neonates have evidence of disseminated intravascular coagulation. Levels of factor XIII are markedly lower in the high-risk neonates with the lowest values coinciding with lowest Apgar scored.31 Plasma levels of thrombinantithrombin complexes, D-dimer, fibrinogen, fibrin degradation products, and soluble fibrin monomer complexes are also reported to be higher in the asphyxiated neonates correlating to disseminated intravascular coagulation.32-34 Although not all neonates with respiratory distress have experienced asphyxia, thrombin inhibition may be altered and was abnormal in both term and premature neonates with respiratory distress.34 As mentioned above, hemostatic complications (disseminated intravascular coagulopathy, thrombocytopenia, and thromboembolism) are related to the activated-depressed coagulation pathways and platelet function. Thus the best approach to hemostatic complications of asphyxiated neonates is to prevent the asphyxiating event. Supportive management including blood products transfusion with packed red blood cells, platelets, fresh frozen plasma, and cryoprecipitate is commonly practiced in many neonatal intensive care units. Although there are no generally accepted guidelines for transfusion in neonates, especially for those with asphyxia, the effects of concurrent therapies may also affect our decision-making. For example, even though therapeutic hypothermia has not been associated with an increased incidence of hemostatic complications,35 hypothermia may impair coagulation36 and accelerate microvascular thrombosis formation.37 Similar theoretical controversies exist in the administration of inhaled nitric oxide, which inhibits platelet function38 and may affect the development of intraventricular hemorrhage in premature neonates. The infusion of dobutamine at a high dose (20 mg/kg/min) will aggravate platelet aggregatory dysfunction.39 On the other hand, the hemostatic disturbance can also complicate the recovery and affect the clinical management of these critically ill neonates. The development of venous thrombosis in vital organs may affect the outcome, and the use of heparin or thrombolytic agents to treat thromboses needs to be considered with hematology consultation.40 It is uncertain whether the hemostatic disturbance may contribute to the patency of vascular access. Further studies are needed to examine the hemostatic problem and its management in asphyxiated neonates. Neonates have a different physiologic hemostasis compared with older children and adults. In healthy neonates the balance of hemostasis is maintained without increased risk for hemorrhage or thrombosis except in high-risk situations such as asphyxia. Despite the paucity of evidence, the asphyxiated neonate appears to have a disturbed balance in hemostasis potentially predisposing them to hemorrhage or thrombosis. Further prospective laboratory studies with clear definitions of asphyxia are needed to characterize the abnormalities in hemostasis. Prospective epidemiologic studies e38
Vol. 158, No. 2, Suppl. 1 evaluating outcomes will provide the necessary background data for intervention studies. n
Author Disclosures Mary E. Bauman, RN, NP, Po-Yin Cheung, MBBS, PhD, FRCP (Can & Edin), and M. Patricia Massicotte, MD, FRCPC, have no financial arrangement or affiliation with a corporate organization or a manufacturer of a product discussed in this supplement. Reprint requests: M. Patricia Massicotte, MD, FRCPC, MHSc, Department of Pediatrics, University of Alberta, Stollery Children’s Hospital, 8440 112th St, WMC 4H2.11, Edmonton, Alberta, Canada T6G 2B7. E-mail: patti. [email protected].
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