Developmental Hemostasis

Introduction

The presence of a complex hemostatic system is fundamental to all multicellular organisms with a blood circulatory system. Blood must remain in a fluid form to flow, and for survival, organisms must be able to stop that flow at sites of local injury, through the process of physiologic thrombus formation. However, under certain conditions, thrombus formation can become pathologic. In the mid-1800s Rudolf Virchow described a triad of factors that contributed to pathologic blood clotting: blood flow, blood vessel wall, and blood composition. The description is still relevant nowadays, and in fact is just as relevant to the development of bleeding as it is to abnormal clotting. Hemostasis involves complex interactions among the vascular endothelium, cellular elements within blood (particularly the platelets), and plasma proteins in the context of blood flow. Although developmental hemostasis is often discussed purely in terms of changes in plasma proteins, it is important to remember that all of the elements of the Virchow triad change with age. Furthermore, changes in plasma proteins are not restricted to the proteins that constitute the coagulation system but occur throughout the entire plasma proteome. Although hemostasis is a dynamic, evolving process that is age dependent and begins in utero, there is arguably no greater time of change in the hemostatic system than around the time of birth. Although evolving, the hemostatic system in healthy fetuses and infants must be considered physiologic. The evaluation of newborn infants for hemorrhagic or thrombotic complications presents unique problems that are not encountered in older children and adults. ^, For example, physiologic levels of many coagulation proteins in neonates are low, which makes the diagnoses of some inherited and acquired hemostatic problems difficult to establish. An understanding of developmental hemostasis in the broadest sense optimizes the prevention, diagnosis, and treatment of hemostatic problems during infancy and undoubtedly provides new insights into the pathophysiology of hemorrhagic and thrombotic complications for all ages.

The Coagulation System

Our understanding of the hemostatic physiology in neonates and infants is poor compared with knowledge of this subject in adults. This deficiency is due to several factors: in neonates and infants, multiple reference ranges are required because these patients have rapidly evolving systems ^, ; blood sampling in the young is technically difficult; only small blood samples can be obtained; microtechniques are required ; and greater variability in plasma concentrations of coagulation proteins necessitates the use of large patient numbers to establish normative data.

Coagulation proteins are independently synthesized by the fetus and do not cross the placenta. By 10 weeks gestational age, plasma concentrations of most coagulation proteins are measurable, and they continue to increase gradually in parallel with gestational age. A number of studies have described normal values for hemostatic functional assays in fetuses or neonates ( Table 111.1 ). Samples obtained during fetoscopy provide the best assessment of normal values for fetuses and, by extrapolation, very premature infants ( Tables 111.2 and 111.3 ). True reference ranges for extremely premature infants are not available because most of these infants have postnatal complications. Tables 111.4–111.7 provide reference ranges for coagulation proteins, inhibitors of coagulation, and components of the fibrinolytic system for premature (30 to 36 weeks gestational age) and term infants on day 1 after birth, as well as longitudinally during the first 6 months after birth. ^{, ,} Studies continue to demonstrate that samples obtained from umbilical cord blood may have considerable differences from samples obtained simultaneously from peripheral blood of the infant.

In addition to the multitude reported studies of functional assays, Attard and colleagues described the antigenic levels of coagulation proteins in neonates compared with adults and confirmed that 10 proteins are quantitatively significantly different. Further research is required to understand the role of posttranslational modifications and the correlations (or not) between quantitative levels and functional levels.

The factors controlling these changes remain unknown. Studies in mice have reported that, of the 41 microRNAs (miRNAs) that are overexpressed in newborn livers compared with adult livers, 21 have hemostatic messenger RNA (mRNA) as their targets. miRNAs are an abundant class of small noncoding RNAs that are negative regulators in a number of physiologic and pathologic processes. Teruel and colleagues proposed that the miRNAs might have negative regulatory effects on the hemostatic proteins during the newborn period. In a subsequent study, the same group showed a significant reduction of sialic acid content in neonatal antithrombin (AT) compared with adult AT in mice. The mRNA levels of St3gal3 and St3gal4, two sialyltransferases potentially involved in AT sialylation, were 85% lower in neonates in comparison with adults. In silico analysis of miRNAs overexpressed in neonates revealed that mir-200a might target these sialyltransferases.

Previous studies have measured mRNA levels for factor (F)VII, FVIII, FIX, FX, fibrinogen, AT, and protein C in hepatocytes from 5- to 10-week-old human embryos and fetuses and in those from adults. Embryonic-fetal transcripts and adult mRNAs are similar in size; the nucleotide sequences of mRNA for FIX and FX are identical. ^, However, the expression of mRNA was variable, with adult values existing for some coagulation proteins but decreased for others. Similar concentrations of prothrombin mRNA were found in the livers of newborn and adult rabbits ; however, another study reported lower prothrombin mRNA concentrations in sheep.

In terms of understanding the controlling mechanisms of plasma protein concentrations, one of the most interesting studies demonstrated that, even with a transplanted adult liver in situ, children maintain plasma levels of certain coagulation proteins at their expected age-specific levels. Thus the liver, despite being the site of production for most of the coagulation proteins and where control of levels may be exerted through miRNAs, is not the primary regulator of plasma levels. The authors hypothesized hormonal control, vascular endothelial control via an as-yet-unidentified mechanism, or control via variable clearance. However, whatever the mechanism, vascular endothelium seems a likely candidate as the primary regulator.

The variable results for coagulation screening tests reflect the use of cord blood samples rather than samples from infants, differing ethnic populations, or use of different reagents. ^, Reference ranges for prothrombin time and activated partial thromboplastin times will differ with each different reagent and analyzer system, often significantly. ^, The thrombin-clotting time performed in the absence of calcium is prolonged because of the presence of the “fetal” form of fibrinogen at birth. ^{, ,} For Tables 111.3 and 111.4 , the thrombin-clotting time was measured in the presence of calcium so that abnormal values secondary to the presence of heparin, as well as low levels of fibrinogen, could be detected.