American Journal of Hematology 32:273-278 (1989)

Newborn’s Fibrinolytic Mechanism: Components and Plasmin Generation

James J. Corrigan, Jr., Jeffrey J. Sleeth, Monette Jeter, and Charles D. Lox Department of Pediatrics and Children’s Research Center, University of Arizona Health Sciences Center, Tucson, (J.J.C., J.J.S., M.J.);

Department of Obstetrics and Gynecology, Texas Tech University Health Sciences Center, Lubbock (C.D.L.)

Plasminogen activity and antigen, tissue-type plasminogen activator (tPA) activity and antigen, plasminogen activator inhibitor (PAl) activity, and plasmin generation rates were determined in 32 normal newborn plasmas and 25 normal adult plasmas. The newborns showed reduced levels of plasminogen activity and antigen and tPA antigen, and activity, normal levels of PA1 activity, and slower plasmin generation rates. The slower generation was shown to be due to the hypoplasminogenemia. The in vitro plasmin generation studies also showed that the newborn needed 11 times the usual concentration of uro- kinase and 5 times the usual concentration of tPA to achieve the minimal activation rate of the adult.

Key words: plasminogen activity and antigen, tPA antigen, hypoplasminogenemia, uro- kinase, fibrin formation

INTRODUCTION

The fibrinolytic mechanism of the normal human new- born is poorly understood. The reported studies suggest that this mechanism is not fully developed at the time of birth [ 1-31 and that the balance between fibrin formation and plasmin activity favors fibrin formation in the new- born 141. Numerous investigators have reported that the normal newborn’s plasma contains 50% to 75% less plasminogen activity and protein than adult plasma [3,4,6,7]. This hypoplasminogenemia remains until the infant is about 6 months of age [8-lo]. Also, the new- born’s plasminogen may be dysfunctional [4,11,12]. The level of the natural inhibitor of plasmin (a2-antiplasmin) in the newborn has been reported to be similar to that of the adult [5,6,8,10]. The other components of the fibri- nolytic mechanism, however, have not been extensively studied.

The availability of purified components of the fibrino- lytic mechanism, monoclonal antibodies, and specific chromogenic substrates allows for in vitro investigations that employ small samples of plasma. By using these reagents in a microtiter system we determined the plasma levels of tissue-type plasminogen activator (tPA) activity and antigen, and plasminogen activator inhibitor (PAI). In addition, plasmin generation rates of newborn and adult plasma were analyzed using two different sub-

strates, casein and a chromagen. The results show that the newborn has marked hypoplasminogenemia, reduced tPA antigen, reduced tPA activity, but similar levels of PA1 when compared to adults. Furthermore, the plasmin generation studies suggest that the slower response noted in the newborn is concentration dependent and is prob- ably not due to a dysfunctional plasminogen.

MATERIALS AND METHODS Materials

(S225 1) (Kabi Diagnostica, Stockholm, Sweden)

ucts, St. Louis)

(Calbiochem-Behring Corporation, La Jolla, CA)

1. H-D-Val-Leu-Lys-pNA chromogenic substrate

2. Human rtPA (two chain) (In Vitron Research Prod-

3. Anti-human plasminogen antibody made in rabbits

Received for publication April 17, 1989; accepted July 20, 1989.

Address reprint requests to Dr. J.J . Corrigan, Department of Pediat- rics, University of Arizona Health Sciences Center, Tucson, AZ 85724.

This work was supported in part by an Institutional Research Grant (2S07 RR05675-21) and by the Hematology Research Fund, Univer- sity of Arizona.

0 1989 Alan R. Liss, Inc.

274 Corrigan et al.

ring Diagnostics, Somerville, NJ)

Kit (American Diagnostica, Inc., Greenwich, CT)

(American Diagnostica, Inc., Greenwich, CT)

4. M-Partigen (plasminogen radial diffusion kit) (Beh-

5. Imubind-5. Tissue plasminogen activator ELISA

6. Spectrolyse tPA Activity and Inhibitor Assay Kit

7 . Urokinase (Abbott Labs, Chicago) 8. Casein (Sigma, St. Louis)

Methods

Blood samples were obtained from 25 normal resting adults by venipuncture within 1 minute following the use of a loose-fitting tourniquet [ 131 and from the umbilical cord at the time of delivery of 32 normal term newborns [ 141. The blood samples were processed as follows: nine volumes of whole blood were anticoagulated with one volume of 3.1 % sodium citrate [ 141; and one volume of an acetate buffer (pH 3.9) was added to one volume of whole blood and then anticoagulated with 3.1 % sodium citrate [ 13,151. The blood samples were centrifuged at 1,500g for 20 minutes at 4°C. The platelet poor plasma was quick frozen and stored at -40°C. The citrated plasma (non-acetate treated) was used for determination of plasminogen, tPA antigen, PA1 activity; and plasmin generation. The acetate-treated citrated plasma was used for tPA activity assays.

Plasminogen activity was determined by a caseinolytic method [16] and by a method using the chromogenic substrate S225 1 (H-D-Val-Leu-Lys-pNA) [ 17,181. Plas- minogen protein (antigen) was determined by radial im- munodiffusion using a commercial plate (M-Partigen) .

Plasmin generation experiments used casein or the chromogen S2251 as substrates. In these studies the plasma samples were acidified with 116 N HC1 and al- lowed to incubate for 15 minutes. The samples were then neutralized with an equal volume of 1/6 N NaOH [ 161. Plasmin generation rates were determined by a casein- olytic method [16] and by the use of the S2251 by the technique described by Scharrer et al. [19]. In the latter method a constant amount of S2251 is added to a fixed volume of plasma (or diluted plasma). Various concen- trations of either urokinase or tPA were added, and ab- sorbance was read at 405 nM over time in a spectropho- tometer.

tPA antigen was determined by an enzyme-linked im- munosorbent assay using a commercial kit (Imubind-5). tPA activity and PA1 activity were determined by a chro- mogenic substrate S2251 assay using a commercial kit (Spectrolyse tPA Activity and Inhibitor Assay Kit) [ 15,20-221.

The data are shown as mean, standard deviation, and range. Statistical analysis was by use of a t test between groups. A P value of less than .05 was considered a

TABLE 1. Plasminogen, Tissue Plasminogen Activator (tPA) and Plasminogen Activator Inhibitor (PAI) Levels in Newborns*

Plasminogen Activity (Cuiml)

Mean SD Range (No. 1

Mean SD Range

Antigen (pgiml)

(No.) Tissue plasminogen activator

Activity IUiml) Mean SD Range (No.)

Mean SD Range (No.)

Antigen (ngiml)

Plasminogen activator inhibitor Activity (IUiml)

Mean SD Range (No.)

Component Newborn Adult P

-

1.3 3.5 'C.001 0.3 0.5

0.6-2.0 2.5-4.5 (32) (25)

30 120 <:.001

20-40 100-200 5.6 13.0

(32) (25 )

0.022 0.05 ':. 05 0.037 0.060

0-0. I7 0-0. I4 (32) (25)

2.3 3.5 ': .05 1.9 1.8

0.4-8.8 3-10 (32) (25 )

1 .s I .4 X . 0 5 0.68 0.52

0.5 -2.7 0.7-2 .O (32) ( 2 3

*Comparison of newborns with adults. ( ) = No. of patients. SD =

standard deviation.

significant change. The study was approved by the In- stitutional Review Boards of each institution.

RESULTS Plasminogen, Tissue-Type Plasminogen Activator (tPA), and Plasminogen Activator Inhibitor (PAI) Plasma Levels

The plasma levels of plasminogen, tPA, and PA1 in 32 normal newborns and 25 normal adults are shown in Table I. Plasminogen activity (caseinolytic method) and antigen levels were low in the newborn ( P < .001 com- pared to adults). The activity was 63% lower and the antigen was 75% lower than the adult levels. Using the ratio of casein unit of activity to microgram of protein per ml plasma the newborn was 1 :23 and the adult was 1 :34.

tPA activity was 48% lower in the newborn. However, there was a wide range of values for both groups with the medians being 0.006 for the newborn and 0.01 for the adult. tPA antigen levels were significantly less in the newborn being 34% and 47% less using the mean (2.3 vs. 3.5) and median (1.6 vs. 1.8), respectively, in the

Newborn’s Fibrinolytic Mechanism 275

E 1.0

5 0.0 > c 0 . 5 y I I I I I I I I I I I Adult I I , .-

Newborn 0.2 -

0 .5 1 1.5 2 2.5 3 3.5 4 Time (hours)

Fig. 1. Plasmin generation, caseinolytic method. Casein units per ml plasma over 4 hours using urokinase as the activator. The results in the adult (upper panel) and the new- born (lower panel). (Plasma was treated with acid to remove inhibitor effect.)

comparison. PA1 activity levels in the newborns were similar to the adult plasma levels.

Plasmin Generation Experiments

Since large volumes of plasma were needed for these experiments, the samples were pooled (24 newborns and 18 adult plasma samples). For the following studies the plasma pooled samples were acidified with 1/6 HC1 and incubated for 15 minutes at 37°C (to remove the inhibitor effect) then neutralized with an equal volume of 1/6 N NaOH [ 161. To these treated samples was added a sub- strate (casein or S225 1) and plasminogen activator (uro- kinase or tPA) [16,19].

Caseinolytic method. An excess of activator (uroki- nase, 200 IU/ml) was added to undiluted treated plasma containing casein as the substrate and incubated at 37°C for 4 hours. The reaction was stopped at 30 minutes, 1 , 2, 3 , and 4 hours with TCA (trichloracetic acid). The amount of casein lysed was determined by the method of Alkjaersig et al. [16]. The results are displayed on Figure 1 as casein units (vertical axis) over time (horizontal axis). The plasmin generation in the adult plasma was maximal by 1 hour with no futher change over the sub- sequent 3 hours. The newborn’s plasma was slower to achieve maximal activation in that the peak did not occur until 3 hours. The lower final casein units lysed per ml of plasma for the newborn (1.2 /CU/ml compared to 3.0 CU/ml for the adult) was due to the lower plasminongen concentration in the newborn sample. When the peak

‘1 UK (25 units) TPA (1250 ng)

7

0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 Time (minutes)

Fig. 2. Plasmin generation, chromogenic method. Uroki- nase (UK) or tissue-type plasminogen activator (tPA) was added to undiluted acid-treated plasma containing 5-2251. The change in absorbance over time is shown.

results were expressed as casein units per microgram of plasminogen antigen it was found that the newborn was 0.04 CU/pg and the adult 0.025 CU/pg.

Chromogenic method. The method of Scharrer et al. [19] was used in these experiments. The reagents con- sisted of pooled adult and newborn treated plasma (un- diluted was called 100%); two-chain tPA (50 pg, 100 pg, and 200 pg/ml); urokinase (1,000 IU, 2,000 IU, and 4,000 IU/ml); chromogenic substrate S2251 (3 mM); and microtiter plates. The typical reaction mixture contained 50 p l of plasma (or diluted plasma); 50 pl of S2251; and 25 pl of activator in each well of an A/2 spectropho- tometer plate. When tPA was used as the activator 2 pl of soluble fibrin complex was also added (Desafib, American Diagnostica). The absorbance values were read at 405 nM on a Titertex spectrophotometer every minute for at least 15 minutes. The results are displayed as either optical density units (absorbance) or as slopes over time using optical density units.

Figure 2 shows the results for plasmin generation over time for adult and newborn undiluted plasma using uro- kinase (final concentration of 25 units) and tPA (final concentration of 1,250 ng). The newborn was slower to generate plasmin activity as shown by the diversion of the lines. With urokinase, the final absorbance unit of 0.65 was reached at 15 minutes by the adult and 35 minutes by the newborn. With tPA, the final absorbance unit of 1.3 was reached at 15 minutes in the adult and 25 minutes in the newborn plasma.

Figures 3 and 4 show the effect of increasing the con- centration of activator in undiluted adult and newborn plasma. In these experiments urokinase (25 to 1,000 IU final concentration) or tPA (1,250 to 5,000 ng final con-

276 Corrigan et al.

0 - O 7 l Adult

100 500 1000

Urokinase (units)

Fig. 3. Plasmin generation. Effect of increasing concentra- tions of urokinase.

1250 2500 5000 TPA (ng)

Fig. 4. Plasmin generation. Effect of increasing concentra- tions of tissue-type plasminogen activator (tPA).

centration) was added to adult and newborn plasma con- taining S2251. Absorbance was read at 0 time and at minute intervals for 15 minutes. The slope of the line was calculated for absorbance change over 15 minutes. Both figures show that by increasing the concentration of activator, plasmin activity increases, but the newborn always remained lower than the adult. The newborn's plasma needed 275 units of urokinase to achieve the minimal adult activation seen with 25 units, and 5,600 ng of tPA to achieve the minimal adult activation seen with 1,250 ng.

Adult plasma was diluted 50% ( 1 volume plasma and 1 volume Tris buffer pH 7.4), and the experiment shown in Figure 2 was repeated using tPA as the activator. As noted in Figure 5 the diluted adult plasma gave results that were the same as undiluted newborn plasma.

DISCUSSION

The fibrinolytic data in this study showed that the normal term newborn had hypoplasminogenemia and re-

Adult vs Newborn Plasma + TPA

2 1 I

1.6 i h

1.4 cu cu &I 1.2 v

.4

.2 I

v

0 5 10 15 20 25 30 35 40 Time 1-37 (minutes)

Fig. 5. Plasmin generation. In this experiment adult plasma (acid treated) was diluted 50% and the experiment shown in Figure 2 was repeated. The activator was tissue-type plas- minogen activator (tPA).

duced tPA antigen and activity but similar PA1 activity when compared to the adult. Also, the plasmin genera- tion experiments suggested that the newborn had a slower response to exogenous urokinase and tPA. Hy- poplasminogenemia in the newborn has been reported by numerous investigators [2,3,5,8,10]. These earlier stud- ies showed that the hypoplasminogenemia was due to reduced synthesis and not to activation and consumption of the protein [2,8,9,10]. Whether a functional abnor- mality exists in newborn plasminogen is unclear. Studies that have employed either native plasma or plasminogen isolated by chromatography have suggested that there may be a dysfunctional plasminogen in the newborn [4,1 I , 121. However, these observations have not been confirmed, and one study reported that the plasmin gen- eration rates for newborns and adults were similar 1231. In our study, plasmin generation was clearly slower in the newborn as was seen by using casein as the substrate and urokinase as the activator (Fig. 1) and by using S- 225 1 chromogen as the substrate and urokinase or tPA as the activator (Fig. 2). However, when the adult plasma

sample was diluted 50%, the plasmin generation seen with tPA was identical with that of the newborn (Fig. 5) . Our study also showed that the proportion of plasmino- gen activity to immunoreactive protein (antigen) in the newborn was similar to the adult, and that the maximal casein units of activity per microgram of protein gener- ated by urokinase (Fig. 1) was likewise similar. These data suggest that the plasmin generation was dependent upon the plasminogen concentration and not indicative of a dysfunctional plasminogen in the newborn.

Mackinnon and associates reported that maternal plas- minogen activator inhibitor was high and significantly greater than what was found in the neonates [7]. Our results show that the newborn’s PA1 level is not different from the non-pregnant adult level. These findings sug- gest that PA1 is probably not transferred from the mother to the fetus.

The treatment of major thrombotic events in the new- born with urokinase and streptokinase has been reported, but their efficacy is controversial [ 3 ] . The majority of reports have indicated that no thrombolysis was pro- duced or that the response was delayed [24-321. Since the newborn has hypoplasminogenemia with normal adult levels of PA1 and cuz-antiplasmin, it could be ex- pected that the response to standard concentrations and doses of plasminogen activators would be blunted. The studies in our investigation using increased concentration of activators showed that it took 11 times the amount of urokinase and 5 times the amount of tPA in the newborn to achieve the activation seen in the adult (Figs. 3 , 4). This would suggest that thrombolytic therapy in the new- born will need a higher concentration of infused activator than what is used in older children and adults. There are no controlled studies in the newborn using thrombolytic agents and, when used, no uniform standard dosage that would confirm these in vitro observations. Future clini- cal studies, however, will need to evaluate the appropri- ate dose-response relationship for these activators in the newborn.

The data from our study in addition to those reported by others suggest that the newborn’s plasma fibrinolytic mechanism has the following characteristics when com- pared to the adult: reduced plasminogen activity and an- tigen; reduced tPA antigen, normal to reduced tPA ac- tivity, normal PA1 activity, normal cr2-antiplasmin, reduced histidine-rich glycoprotein [33], and slower plasmin generation. The evidence for a dysfunctional plasminogen is controversial. Nevertheless, as suggested by Suarez et al. [4], it appears that the newborn’s hemo- static mechanism is toward fibrin formation and is not balanced by a similiarly active fibrinolytic mechanism. The data also suggest that for thrombolytic therapy to be successful, a much higher concentration of activator will be necessary to induce a fibrinolytic state.

Newborn’s Fibrinolytic Mechanism 277

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