Wm.E. Hathaway

Vitamin K in infancy.

Vitamin K is required for carboxylation of a number of calcium-binding proteins including the coagulation factors II, VII, IX and X; the anticoagulant proteins C and S; other proteins in kidney, dentin and lung and osteocalcin in bone. When there is deficiency of vitamin K, inactive, uncarboxylated forms of the clotting proteins (descarboxy-proteins or PIVKAs Proteins formed In Vitamin K Absence) circulate and are a much more sensitive measure of deficiency than the prothrombin time. There are two natural sources of vitamin K, plants and bacteria. Plants produce phylloquinone, also called vitamin K1. Bacteria produce a range of related compounds, the menaquinones or vitamins K2. The naturally occurring K vitamins are fat soluble and absorption from the small intestine is dependant on the presence of bile salts and pancreatic lipases. Dietary vitamin K~ is certainly the main, if not the only, source in human infants; contrary to what is commonly taught, colonic absorption of bacterially-produced menaquinones is unproven. The maternal:fetal gradient for vitamin K1 across the placenta may be as high as 40: 1, which explains the very low cord blood concentrations. The infant who is formula fed from birth is at an immediate advantage regarding vitamin K (though perhaps in no other way) because he can drink until satisfied and the milk contains a high concentration of the vitamin (about 50 gg/1 in most formulae); by contrast, the breast fed baby has a meagre supply of milk in the early days and the vitamin K content is only about 2 ~tg/1. Delay in establishing feeding, especially by breast, may precipitate vitamin K deficiency in the early days of life and it is the breast

Anticoagulant therapy by continuous heparinization in newborn and older infants

In order to evaluate the metabolism and anticoagulant effect of heparin in the newborn infant and young child, 15 babies were monitored during continuous intravenous heparinization for documented large vessel thromboses. Infants with the most significant thromboses had the highest clearance rates for heparin. Plasma heparin levels in the therapeutic range (0.3 to 0.5 U/ml) and clinical resolution of the thrombi were associated with heparin doses of 16 to 35 U/kg/hour (mean = 27 U/kg/hour). A micro whole blood clotting time was evaluated and shown to be a useful guide to heparin effect in these infants. With the exception of excessive oozing from puncture sites in one infant, no complications of heparin therapy were noted. The newborn infant appears to require a larger amount of heparin than adults in order to achieve adequate heparinization.

Vitamin K deficiency in the newborn infant: Prevalence and perinatal risk factors

The prevalence of vitamin K deficiency in newborn infants and the influence of perinatal risk factors were studied prospectively in 934 infants. A noncarboxylated prothrombin assay to detect proteins induced in vitamin K absence (PIVKA-II) was used to determine the presence of vitamin K deficiency; of 934 cord blood samples assayed, 2.9% were positive for PIVKA-II (0.015 to 0.15 U/ml). All infants found to have detectable PIVKA-II were born at term. The number of infants positive for PIVKA-II was greater in the group small for gestational age (7.4%) than in those appropriate (2.7%) or large (3.1%) for gestational age. Nine categories of perinatal risk groups were defined: however, the majority of infants who were PIVKA-II positive (63%) were normal. All infants received prophylactic vitamin K, and no infant with PIVKA-II in the cord sample subsequently had clinical bleeding. In two patients the rate of 50% disappearance of PIVKA-II after vitamin K administration approximated 70 hours. Two PIVKA-II positive patients with active bleeding or disseminated intravascular coagulation had an accelerated disappearance of 20 to 40 hours. The long disappearance time of PIVKA-II in a steady state may allow detection of vitamin K deficiency despite administration of vitamin K. The majority of cases of neonatal vitamin K deficiency occurred in normal newborn infants. Therefore, all infants should receive prophylactic vitamin K at birth.

Influence of factor VIII on overall coagulability and fibrinolytic potential of haemophilic plasma as measured by global assay: Monitoring in haemophilia A

Summary.  The objectives of the present study were to evaluate the analytical sensitivity of the recently developed Clot Formation and Lysis (CloFAL) global assay for factor VIII (FVIII) deficiency, both in vitro and ex vivo, to determine whether this global assay is influenced by FVIII inhibitors, and to investigate the coagulative response to FVIII replacement in haemophilia A patients using the CloFAL assay in comparison with FVIII activity. Among adults and children alike, the CloFAL assay coagulation index (CI) was significantly decreased in FVIII‐deficient vs. healthy subjects (adults median CI: 2% vs. 94% respectively; children median CI: 3% vs. 63%; P < 0.001 for each), and correlated significantly with activated partial thromboplastin time‐based FVIII activity across all individuals (r = 0.78; P < 0.001). The CloFAL assay was analytically sensitive to deficient FVIII activity and also influenced by the presence of von Willebrand factor. Severe haemophilia A patients without inhibitory antibodies to FVIII showed considerable heterogeneity in CloFAL assay waveforms, despite a uniformly diminished CI of 0–1%. During FVIII infusion half‐life studies in patients with severe haemophilia A, the CloFAL assay demonstrated a marked rise in coagulability 30 min following infusion, with progressive decrease in coagulability towards baseline over the ensuing 48‐h period. In each case, the profile of coagulative response to FVIII infusion as determined by CloFAL assay CI differed qualitatively from that measured by FVIII activity. These findings indicate that the CloFAL assay may be useful as an adjunctive test to FVIII activity measurements in the therapeutic monitoring of haemophilia A.

Influence of factor IX on overall plasma coagulability and fibrinolytic potential as measured by global assay: monitoring in haemophilia B

Summary.  We sought to determine the influence of factor IX (FIX) deficiency upon overall coagulative and fibrinolytic capacities in plasma using the clot formation and lysis (CloFAL) assay, and to investigate the role of this global assay as an adjunctive monitoring tool in haemophilia B. CloFAL assay parameters were measured in vitro in platelet‐poor plasma in relation to FIX activity and antigen (FIX:Ag), and were determined ex vivo among FIX‐deficient patients (n = 41) in comparison to healthy individuals (n = 48). Supplementation of FIX‐deficient plasma with FIX in vitro demonstrated a non‐linear concentration dependence of FIX upon overall plasma coagulability. Ex vivo, coagulability was significantly decreased in FIX‐deficient vs. healthy subjects among adults [median coagulation index (CI): 4% vs. 104% respectively; P < 0.001] and children (median CI: 9% vs. 63%; P < 0.001). Fibrinolytic capacity was increased in adult FIX‐deficient vs. healthy subjects (median fibrinolytic index: 216% vs. 125%, respectively, P < 0.001), and was supported by a trend in shortened euglobulin lysis time (ELT). Severe haemophilia B patients showed heterogeneity in aberrant CloFAL assay waveforms, influenced partly by FIX:Ag levels. Patients with relatively preserved FIX:Ag (i.e. dysfunctional FIX) exhibited a shorter time to maximal amplitude in clot formation than those with type I deficiency. During patient treatment monitoring, markedly hypocoagulable CloFAL assay waveforms normalized following 100% correction with infused FIX. The CloFAL global assay detects FIX deficiency, demonstrates differences in coagulability between dysfunctional FIX and type I deficiency, and appears useful as an adjunctive test to routine FIX measurement in monitoring haemophilia B treatment.

Immunoelectrophoretic studies of prekallikrein in human plasma

Using a rabbit anti-human prekallikrein antibody crossed immunoelectrophoresis (CIE) and Laurell rocket antigen determinations were done in plasma of subjects with Fletcher (prekallikrein, PKA), Fitzgerald (high molecular weight kininogen), Hageman (XII), and PTA (XI) deficiencies as well as in patients with activation of coagulation (intravascular coagulation syndromes). Abnormal CIE patterns were seen in the Fletcher and Fitzgerald deficient plasmas and also in some of the patients with intravascular coagulation. In vitro studies of plasma treated with thrombin, plasmin, and contact activating agents indicated that abnormal CIE patterns and increased PKA antigen levels were indicative of activation of the Hageman factor dependent pathway and not the result of plasma clotting by thrombin. In vivo activation of the Hageman factor dependent pathway frequently results in an abnormal CIE and a low PKA antigen level.

941 PREVALENCE OF VITAMIN K DEFICIENCY IN NEWBORN INFANTS: INFLUENCE OF PERINATAL RISK FACTORS

Measurement of noncarboxylated prothrombin (PIVKA-II assay) was used to study 559 infants for vitamin K (vit K) deficiency. This test, performed by immunoelectrophoresis of plasma before and after BaCO3 precipitation, is sensitive to 0.03 u/ml PIVKA-II, has an intra- and inter-test coefficient of variation of 3-5%, and is negative in liver disease. Catagories of infants studied and results are as follows. As part of an ongoing study of 1,000 cord bloods, 534 cords were assayed and 2.6% were PIVKA-II positive (0.03-0.15 u/ml). The PT of the PIVKA-II positive samples ranged from 11.5-23 seconds and correlated inversely with prothrombin activities of 10-55%. SGA infants were at increased risk for K deficiency (14%); however, other groups were not (preterm, post-term, infants with fetal distress, and infants of mothers with hypertension or third trimester infection), Interestingly, of 16 infants born to mothers on chronic anticonvulsant therapy, only one showed PIVKA-II. Eight breast-fed infants who were given neonatal vit K did not develop a deficiency by two months of age. A K deficient infant given 1 mg vit K and then fed entirely by parenteral nutrition without supplemental vit K showed absence of PIVKA-II for five weeks.In summary, 2.6% of all newborns may be vit K deficient at birth. While most K deficient infants were born of normal pregnancies and deliveries, SGA infants were more likely to be PIVKA positive. Vit K is stored in newborns to an appreciable degree.

835 PURIFICATION, BIOCHEMICAL CHARACTERIZATION AND FUNCTION OF FETAL AT-III

Heparin clearance in the newborn is more rapid than in the normal adult and anticoagulation with heparin is clinically difficult to achieve. Circulating levels of antithrombin III (AT-III) are physiologically low in term and preterm infants and minor differences in the function of AT-III in the newborn would have significant implications for the treatment of thrombosis. AT-III was isolated by heparin affinity chromatography from adult venous and newborn umbilical cord blood. The proteins thus purified were compared by SDS-PAGE, rocket immunoelectrophoresis, protein concentration by microbiuret relative to optical density at 280 nm, heparin cofactor specific activity, progressive neutralization of thrombin and factor Xa at 37° C, and pH related anti-thrombin kinetics. Finally, the reaction rates of thrombin neutralization relative to the concentration of heparin present were measured. The structural evaluations revealed a fetal AT-III molecule of molecular weight, charge and electrophoretic migration indistinguishable from that of normal adult. The functional studies showed that, on an equimolar basis, the rates of thrombin and Xa interactions with fetal AT-III were as rapid as those with adult AT-III. The catalytic rates of various concentrations of heparin were also normal. These findings suggest that “heparin resistance” in the newborn is the result of a quantitative deficiency of a normally functioning protein and support considerations for replacement therapy with AT-III.