Keywords
cardiopulmonary - coagulation - neonate - serpin - TFPI
Introduction
Cardiac surgery is a common cause of severe bleeding in neonates.[1] Cardiopulmonary bypass (CPB) is routinely used to isolate and support the cardiopulmonary
system during complex cardiac procedures and places different stressors on neonatal
haemostasis than occur in adults undergoing CPB. These include a larger relative prime
volume, obligate use of donor blood and more extensive haemodilution of plasma proteins.[2] The neonatal haemostatic system also undergoes multifactorial change during CPB,
including alterations of platelet number and function,[3]
[4]
[5] dilution of plasma growth factors, such as thrombopoietin,[3] and alteration in plasma coagulation proteins.
The neonatal haemostatic system is altered during CPB by transfusion of blood products
collected from adults and teenagers to prime the bypass pump and treat surgical bleeding.
Maternal coagulation proteins do not cross the placenta,[6] and therefore, the plasma concentrations of neonatal coagulation proteins tend to
be lower than in adults.[7]
[8] Pro-coagulant factors II, VII, IX, X, XI and XII are particularly low at birth,
ranging from 30 to 50% of adults.[7]
[8] Anticoagulant plasma proteins, protein C, protein S, and anti-thrombin are also
below 50% of adult concentrations at birth,[7]
[8] producing some balance between the pro- and anticoagulant potential of neonatal
plasma.[9] Likewise, neonatal concentrations of proteins modulating plasma protease cascades
including C4b-binding protein and plasminogen are reduced.[7]
[8] Transfusion of blood products to prime the bypass pump and maintain haemostasis
shifts the neonatal circulation towards that of an adult, which alters the balance
between pro- and anticoagulant proteins in neonatal plasma. This balance is further
altered by heparin administration during CPB, and its subsequent reversal with protamine.
Exposure of blood to artificial materials in the bypass pump can cause contact activation
of plasma coagulation, complement and inflammatory cascades.[10]
[11] Tissue factor-dependent coagulation and inflammation, as a result of monocyte activation
and surgical trauma, also occur during CPB.[12]
[13]
[14]
[15] In addition, coagulation proteases can cross-activate other plasma proteolytic cascades
and vice versa.[16]
[17] Thus, the neonate undergoing CPB is susceptible to widespread and unregulated proteolysis
with consumption of plasma protease inhibitors that regulate blood coagulation, fibrinolysis,
complement and inflammation. Members of the serpin superfamily of protease inhibitors
undergo large and distinct conformational changes when they interact with proteolytic
enzymes.[18]
[19]
[20] These occur through structural transition of the active site loop as it partially
or fully inserts into a large β-sheet, altering thermodynamic stability of the serpin.[19] Serpin conformations include the virgin form, which is the active inhibitor and
is cleared slowly from plasma; the cleaved form, in which the reactive site loop is
proteolytically inactivated and also is cleared slowly from plasma;[19] and the protease-complexed form, which is an equimolar complex of the serpin and
a target protease and is more rapidly cleared from plasma.[19]
[21] We sought to utilize serpin conformational changes that occur during neonatal CPB
as ‘endogenous biosensors’ to detect activation of the different plasma proteolytic
cascades during the surgical procedure. To this end, we examined the concentration
and the conformational stability of the serpins anti-thrombin, antitrypsin, anti-chymotrypsin,
anti-plasmin and C1-inhibitor, as well as the Kunitz-type protease inhibitor, tissue
factor pathway inhibitor (TFPI), in plasma samples obtained at different time points
across the course of neonatal CPB surgery and correlated the measurements with bleeding
outcomes.
Materials and Methods
Study Design
Neonatal patients requiring cardiac surgery with CPB were enrolled after obtaining
informed parental consent. Exclusion criteria were birth weight under 2.5 kg, known
inherited haemostatic abnormality, extracorporeal membrane oxygenation at the time
of surgery or history of previous CPB. The study was approved by the Children's Hospital
of Wisconsin Institutional Review Board.
Sample Collection
Blood was collected from arterial lines that were infused with 1 unit/mL heparin at
1 to 2 mL/hour to prevent blood from clotting in the line. Before each blood sample
collection, the first 1 to 2 mL of blood was discarded to remove the heparin in the
line before drawing the test sample. Blood was collected into 3.2% sodium citrate
at four time points. The samples were as follows: baseline, admittance to the operating
room; on-CPB, following heparin administration and the start of CPB, aortic cross-clamp
removal and prior to platelet or cryoprecipitate transfusion; post-CPB, after patient
removal from CPB, completed rewarming and protamine administration; and post-op, upon
cardiac intensive care unit admission. Each sample was 0.5 mL whole blood which was
transported to the research laboratory, spun at 2,500 × g for 10 minutes at 25°C, and plasma stored at –80°C.
Surgical Haemostatic Management and Transfusion
Bypass pump prime (450 mL) was administered as fresh whole blood (< 2 days storage)
or a 1:1 ratio of packed red blood cells (PRBCs) and fresh frozen plasma (FFP) when
fresh whole blood was not available. All neonates received tranexamic acid as a 30
mg/kg loading dose, followed by 10 mg/kg/hour infusion to inhibit fibrinolysis during
surgery. In addition, 0.1 mg/mL tranexamic acid was added to the CPB prime. Heparin
was dosed to maintain the activated clot time above 480 seconds. An institutional
transfusion strategy for neonatal congenital cardiac surgery with CPB was applied
throughout the study. Immediately prior to termination of CPB, all neonates received
one-fourth unit of single donor apheresis platelets and 1 unit of cryoprecipitate.
After CPB, protamine, one-fourth unit of the same single donor apheresis platelets
and PRBCs were administered. Beyond this uniform protocol, neonates were transfused
to maintain haematocrit above 35%, platelet count over 100 × 10[3]/µL and fibrinogen above 200 mg/dL. Modified ultrafiltration was performed uniformly
in all neonates at the end of CPB.
Post-Operative Bleeding Definition
A standardized definition of post-operative bleeding was used as follows: total chest
tube output ≥ 84 mL/kg during the first 24 post-operative hours (≥ 3.5 mL/kg/hour
average); ≥ 7 mL/kg/hour chest tube output for ≥ 2 consecutive hours during the first
12 post-operative hours; and re-exploration for bleeding or cardiac tamponade physiology
during the first 24 post-operative hours.[22]
Fibrinogen, D-Dimer and Total Protein Assays
Fibrinogen was measured using the STA Fibrinogen 5 Kit and D-dimer was measured using
the STA Liatest D-Di (Diagnostica Stago, Inc, Parsippany, New Jersey, United States).
Plasma total protein was measured using the bicinchoninic acid protein assay (ThermoScientific,
Waltham, Massachusetts, United States).
Concentration of Plasma Protease Inhibitors
Enzyme-linked immunosorbent assay (ELISA) assays measured the plasma concentrations
of anti-thrombin, antitrypsin, anti-plasmin, C1-inhibitor and TFPI. Serpin assays
were performed as recommended by the manufacturer, C1-inhibitor and anti-plasmin (Sino
Biological, Beijing, China); and antitrypsin and anti-thrombin (Enzyme Research Laboratories,
South Bend, Indiana, United States). TFPI ELISA assays measuring total TFPI and TFPIα
were developed in our laboratory.[23]
[24] The results of the assays were recorded as % adult normal plasma, based on comparison
to the normal pooled plasma control tested on each plate.
Transverse Urea Gradient Gel Electrophoresis
Transverse urea gradient (TUG) gels containing 7% polyacrylamide and a continuous
horizontal linear 0 to 8 M urea gradient within an ammediol buffer system were performed
under non-denaturing conditions.[19]
[20] Each gel was loaded with 5 µL of plasma diluted into 145 µL sample buffer. Proteins
were visualized by Western blot.
Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis
Plasma TFPI ligand pull-down using factor Xa (FXa) beads were performed as previously
described.[25] Briefly, FXa beads was added to 50 μL of plasma, rotated at 4°C for 1 hour and washed
with phosphate-buffered saline (PBS) 0.05% Tween-20 followed by PBS. Washed beads
were boiled in sodium dodecyl sulphate (SDS) sample buffer and the supernatant was
loaded onto a 4 to 20% continuous gradient SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) gel.
Western Blotting
Proteins separated on the TUG or SDS-PAGE gels were transferred to polyvinylidene
difluoride (TUG) or nitrocellulose (SDS-PAGE) membrane at 400 mAmps for 1 hour. The
membranes were washed in PBS, blocked for 1 hour and incubated overnight at 4°C with
polyclonal antibodies against antitrypsin (Millipore, Temecula, California, United
States), anti-plasmin (Sino Biological), anti-chymotrypsin (Proteintech, Chicago,
Illinois, United States), anti-thrombin (Enzyme Research Laboratories) and C1-inhibitor
(Cedarlane, Burlington, North Carolina, United States), then washed and probed with
secondary antibodies for 1 hour at room temperature. Blots were imaged using a LI-COR
Odyssey Imaging System (Licor, Lincoln, Nebraska, United States).
Statistical Methods
To account for repeated measures within each subject, a generalized estimating equation
with an autoregressive covariance structure was used to compare bleeders and non-bleeders.
Either a normal distribution with identity link function or a gamma distribution with
log link function was specified depending on the outcome. A Bonferroni step-down (Holm)
correction method was used to adjust for multiple comparisons. A p-value of < 0.05 was considered as statistically significant. SAS 9.4 was used for
the analyses.
Results
Study Population
This prospective observational study enrolled 44 term neonates < 30 days of age (median:
7.0 days) undergoing surgical repair of congenital heart defects with CPB. There were
20 females and 24 males with median weight of 3.3 kg. The cohort had a mean Risk Adjusted
Classification for Congenital Heart Surgery-1 (RACHS-1) score of 4, reflecting high
complexity of the cardiac procedures.
Post-Operative Bleeding Assessment
Of the 44 neonates, 16 (36%) met the criteria for post-operative bleeding. Population
comparisons were performed between the non-bleeder and bleeder groups ([Table 1]). There were no differences found in age at surgery, birth weight or height or total
time on CPB. There were no difference in RACHS-1 score, and the number of each procedure
type by group is presented in [Table 1]. There were no differences in baseline platelet count, haematocrit, pro-thrombin
time, activated partial thromboplastin time, time on CBP, heparin level while on CPB
or the initial protamine administered to reverse heparin between groups. The bleeder
group experienced more chest tube output in the first 24 hours following surgery (294
vs. 115 mL). The bleeder group was given approximately twofold (330 vs. 152 mL) more
average total combined volume of blood products (FFP, PRBC, platelets, cryoprecipitate)
during surgery than was the non-bleeder group. This difference was largely accounted
for by greater amount of PRBC (p = 0.0009) and FFP (p = 0.002) transfused in the bleeder group, while amounts of platelet and cryoprecipitate
were equal between groups ([Table 1]).
Table 1
Demographics, surgical procedures, clinical data and transfusion of blood products
in the non-bleeder and bleeder groups
|
Non-bleeder
|
Bleeder
|
|
Gender
|
|
|
p = 0.28
|
Male
|
17
|
7
|
|
Female
|
11
|
9
|
|
Age at surgery (d)
|
7.82 ± 4.24
|
7.0 ± 5.44
|
p = 0.47
|
Birth weight (kg)
|
3.42 ± 0.44
|
3.25 ± 0.49
|
p = 0.25
|
Birth height (cm)
|
50.66 ± 3.12
|
49.34 ± 2.16
|
p = 0.14
|
Race
|
|
|
p = 0.086
|
White
|
23
|
9
|
|
Non-white
|
5
|
7
|
|
Surgical procedure type
|
|
|
|
RACHS-1
|
3.82 ± 2.04
|
4.93 ± 1.33
|
p = 0.25
|
Norwood
|
11
|
9
|
|
ASO ± VSD
|
8
|
0
|
|
IAA ± VSD
|
1
|
2
|
|
TOF
|
1
|
2
|
|
Central shunt
|
1
|
1
|
|
TAPVR
|
1
|
1
|
|
VSD
|
2
|
0
|
|
AP window
|
1
|
0
|
|
DORV
|
1
|
0
|
|
Truncus arteriosus
|
0
|
1
|
|
ASD
|
1
|
0
|
|
Clinical data
|
|
|
|
Baseline platelet count (103/µL)
|
291.36 ± 125.74
|
244.73 ± 118.13
|
p = 0.24
|
Baseline haematocrit
|
42.82 ± 6.03
|
42.45 ± 6.14
|
p = 0.11
|
Baseline PT
|
16.30 ± 1.44
|
15.59 ± 0.83
|
p = 0.087
|
Baseline aPTT
|
63.56 ± 40.44
|
47.04 ± 8.54
|
p = 0.062
|
CPB time (min)
|
196.18 ± 74.71
|
244.56 ± 163.85
|
p = 0.55
|
Heparin level on CPB (IU/mL)
|
3.32 ± 1.06
|
3.5 ± 1.4
|
p = 0.50
|
Initial protamine administered
|
24.89 ± 12.51
|
23.67 ± 9.5
|
p = 0.93
|
Average chest tube output within first 24 h (mL)
|
115
|
294
|
|
Blood products
|
|
|
|
WB prime
|
11
|
6
|
|
PRBC:FFP prime
|
14
|
10
|
|
WB:FPRBC:FP prime
|
3
|
|
|
Non-prime FFP (mL)
|
45 ± 100
|
138 ± 219
|
p = 0.002
|
Non-prime PRBC (mL)
|
56 ± 134
|
133 ± 216
|
p = 0.0009
|
Non-prime platelets (mL)
|
41 ± 52
|
47 ± 44
|
p = 0.3235
|
Non-prime cryoprecipitate (mL)
|
10 ± 16
|
12 ± 17
|
p = 0.2975
|
Abbreviations: AP, aortopulmonary; aPTT, activated partial thromboplastin time; ASD,
atrial septal defect; ASO, arterial switch operation; CPB, cardiopulmonary bypass;
DORV, double outlet right ventricle; FFP, fresh frozen plasma; FP, frozen plasma;
FPRBC, frozen packed red blood cell; IAA, interrupted aortic arch; PRBC, packed red
blood cell; PT, pro-thrombin time; RACHS-1, Risk Adjusted Classification for Congenital
Heart Surgery-1; TAPVR, total anomalous pulmonary venous return; TOF, Tetralogy of
Fallot; VSD, ventricular septal defect; WB, whole blood.
Change in Surgical Total Plasma Protein
Blood products collected from adults and/or teenagers were used to prime the bypass
pump in amounts exceeding the total blood volume of the neonate. This increased the
total plasma protein concentration across the surgical procedure ([Fig. 1]). Baseline plasma protein averaged 60% than that of adults, increased to 89% on-CPB,
further increased to 98% post-CPB and then decreased to 83% post-op. Despite differences
in amounts of blood products transfused, there was no difference in plasma total protein
between the bleeder and non-bleeder groups at any time point. Since the change in
the concentration of each individual protein is associated with the change in total
plasma protein concentration, the individual protein concentrations were modelled
adjusting for the amount of total plasma protein in statistical analyses. However,
the results of these analyses indicated that total plasma protein was not a confounding
variable for changes observed in the concentration of individual plasma proteins.
Fig. 1 Total plasma protein of the neonate increases upon transfusion of blood products
at the beginning of surgery. Box and whisker plots for total plasma protein at the
four surgical time points as per cent adult plasma are presented. The central line
represents median, the boxes range from 25th to 75th percentiles, the whiskers extend
from the 5th to 95th percentile and the dots depict outliers. There were no differences
between the non-bleeder (black bars) and bleeder groups (grey bars) at any time point.
Change in Surgical Fibrinogen and D-Dimer
Fibrinogen consumption and elevation of D-dimer suggest activation of coagulation
and fibrinolytic cascades and have been related to the propensity for bleeding during
paediatric CPB.[26] Average plasma fibrinogen was above 200 mg/dL at baseline and remained near or above
this level throughout surgery in both the non-bleeder and bleeder groups as per the
surgical transfusion protocol ([Fig. 2A]). There were no differences in fibrinogen between the two groups. Plasma D-dimer
was elevated at baseline, consistent with previous reports of neonates with congenital
heart defects.[27] It decreased from baseline at the post-CPB and post-op time points (p < 0.0001), perhaps reflecting dilution following transfusion or inhibition of fibrinolysis
by tranexamic acid ([Fig. 2B]). D-dimer was not measured at on-CPB due to interference from the high amounts of
heparin present in the samples. There were no differences in D-dimer between the non-bleeder
and bleeder groups at the three time points measured.
Fig. 2 Change in fibrinogen and D-dimer across surgery. Box and whisker plots as described
in [Fig. 1] for fibrinogen and D-dimer concentration at the four surgical time points in the
non-bleeder (black bars) and bleeder (grey bars) groups are presented. (A) Fibrinogen decreased from baseline to on-cardiopulmonary bypass (CPB), p = 0.0005 for non-bleeder; p = 0.0041 for bleeder. At post-CPB and post-op, fibrinogen increased compared with
on-CPB, p < 0.0001 for non-bleeder at both time points; p < 0.0001 and p = 0.0009 for bleeder at post-CPB and post-op, respectively. (B) D-dimer decreased at post-CPB and post-op compared with baseline, p < 0.0001 for both groups at both time points. D-dimer was not measured at on-CPB
due to heparin interference with the assay. There were no differences in fibrinogen
or D-dimer between the non-bleeder and bleeder groups at any time point, p = 0.1735 and p = 0.3809, respectively.
Change in Surgical Plasma Anti-Thrombin Concentration and Conformation
Anti-thrombin inhibits thrombin and FXa, as well as other proteases of the coagulation
cascade in the presence of heparin.[28] It was approximately 45 to 50% of the adult plasma concentration at baseline and
fluctuated slightly higher during surgery ([Fig. 3A]). The overall pattern of change mirrored that of total protein, but the anti-thrombin
increase lagged total protein, suggesting consumption, particularly in on-CPB samples.
There were no differences between the non-bleeder and the bleeder groups at any time
point. Anti-thrombin TUG gel conformational analysis revealed primarily a fully active,
virgin form at baseline ([Fig. 4]). At later time points, a rapidly migrating band that did not unfold, representing
proteolytically inactivated anti-thrombin, as well as multiple higher molecular weight
bands that did not unfold, representing polymerized and protease complexed anti-thrombin
emerged ([Fig. 4]). These changes, most prevalent in on-CPB samples, were also present post-CPB and
post-op. There were no identifiable patterns in anti-thrombin TUG gels consistently
associated with non-bleeders or bleeders.
Fig. 3 Change in anti-thrombin, antitrypsin, anti-plasmin and C1-inhibitor across surgery.
Box and whisker plots as described in [Fig. 1] for plasma serpin concentrations at the four surgical time points as per cent adult
plasma in the non-bleeder (black bars) and bleeder groups (grey bars) are presented.
(A) Anti-thrombin was increased at post-cardiopulmonary bypass (CPB) compared with baseline
and on-CPB, p < 0.0001 for non-bleeders; p = 0.0009 and p = 0.0386 for bleeders. (B) Antitrypsin was decreased at all three surgical time points compared with baseline,
p < 0.0001 for the non-bleeder and bleeder groups. (C) Anti-plasmin decreased between baseline and on-CPB, p = 0.0006 for non-bleeders; p = 0.0285 for bleeders. (D) C1-inhibitor increased at on-CPB and post-CPB compared with baseline, p < 0.0001 for the non-bleeder group at on-CPB and post-CPB; p < 0.0001 for the bleeder group at on-CPB and p = 0.0002 at post-CPB, but was not different from baseline at post-op, p = 0.0864 for non-bleeders and p = 0.2236 for bleeders. There were no differences between the non-bleeder and bleeder
groups at any time point for any serpin, p > 0.1203 for all.
Fig. 4 Transverse urea gradient (TUG) gel electrophoresis with Western blot to assess conformational
stability of serpins across neonatal cardiopulmonary bypass (CPB). Each panel represents
a single gel with migration from top to bottom and 0–8 M urea gradient from left to
right. There were minor differences for different serpins at the various time points
observed in some patients, but overall results were consistent. The most representative
gels from individual neonates are presented. All serpins exhibited the virgin conformation
at baseline. There are minor forms of anti-plasmin and C1-inhibitor that unfold differently
(arrowheads) than the primary form. Other than anti-thrombin and C1-inhibitor, the
serpins remained in virgin conformation across surgery. New conformations of anti-thrombin
appeared in samples collected during surgery. These include reactive site cleaved
anti-thrombin present as a rapidly migrating band that did not unfold in 8 M urea
(arrowhead), as well as multiple slower migrating bands, that represent anti-thrombin-protease
complexes, or polymerized protein. A new form of slowly migrating C1-inhibitor, likely
representing a protease-complexed form, was observed at post-CPB and post-op (arrows).
Change in Surgical Plasma Antitrypsin Concentration and Conformation
Antitrypsin plasma concentration is over 1.0 g/L, higher than other plasma protease
inhibitor. As a rapid neutrophil elastase inhibitor, it protects tissues from enzymatic
degradation during inflammation.[29] The neonatal plasma concentration was approximately 85 to 95% of adults at baseline
and decreased by 20 to 40% over time during surgery (p < 0.0001; [Fig. 3B]), suggesting consumption. No differences in antitrypsin levels between the non-bleeder
and bleeder groups were found. Antitrypsin TUG gel conformational analysis revealed
only the fully active, virgin form across surgery ([Fig. 4]). Interestingly, in addition to the classical signature ([Fig. 5A]),[19] three unique signatures were seen at baseline; a slightly less stable form that
unfolded at low urea concentration ([Fig. 5B]), a form that migrated slightly faster following unfolding at high urea concentration
([Fig. 5C]) and a double heterozygous form combining these two less common forms ([Fig. 5D]). An association between the antitrypsin TUG gel baseline forms and non-bleeder
versus bleeder group status was not identified.
Fig. 5 Transverse urea gradient (TUG) gel electrophoresis and Western blot reveals unique
unfolding signatures of virgin antitrypsin present in different neonates at baseline.
(A) The classical signature; (B) A slightly less stable form that unfolded at low urea concentration (arrowhead);
(C) A form that migrated slightly faster following unfolding at high urea concentration
(arrowhead); and (D) a neonate heterozygous for the two variant forms (arrowheads).
Change in Surgical Plasma Anti-Plasmin Concentration and Conformation
Anti-plasmin is the primary plasma inhibitor of plasmin, the enzyme that degrades
fibrin.[30] It measured approximately 85 to 90% of the adult plasma concentration at baseline,
with a fluctuating pattern across surgery ([Fig. 3C]). No differences between the non-bleeder and bleeder groups were found. Anti-plasmin
TUG gel conformational analysis revealed only the fully active, virgin form across
surgery ([Fig. 4]). A minor band that migrated slightly faster than the anti-plasmin primary form
was also present in all samples across surgery. This minor band appears to reflect
the heterogeneity of plasma anti-plasmin at both its N- and C-termini.[31]
[32]
[33]
Change in Surgical Plasma Anti-Chymotrypsin Conformation
Anti-chymotrypsin is an inhibitor of neutrophil cathepsin G and limits tissue injury
at sites of inflammation.[29] Measurement of anti-chymotrypsin plasma concentration by ELISA was not performed,
because we were unable to identify an antibody pair that worked consistently. Anti-chymotrypsin
TUG gel conformational analysis revealed only the fully active, virgin form across
surgery ([Fig. 4]) with no differences between the non-bleeder and bleeder groups.
Change in Surgical Plasma C1-Inhibitor Concentration and Conformation
C1-inhibitor inhibits several proteases of the complement cascade.[34] It was approximately 85 to 90% of the adult plasma concentration at baseline, increased
to 45 to 75% in on-CPB and post-CPB samples, before returning towards baseline at
post-op ([Fig. 3D]). There were no differences between the non-bleeder and bleeder groups across surgery.
C1-inhibitor TUG gel conformational analysis revealed primarily the fully active,
virgin form at baseline ([Fig. 4]). There were several minor bands on the C1-inhibitor TUG gels that were most apparent
at post-CPB and post-op ([Fig. 4]). One band unfolded in a similar pattern to virgin C1-inhibitor and may represent
a form that has undergone N- or C-terminal proteolysis or a form with altered glycosylation.[35] There also were high molecular weight bands that did not unfold, a pattern consistent
with circulating C1-inhibitor-protease complex and low level consumption. An association
between the presence or apparent amount of C1-inhibitor-protease complex and bleeding
status was not identified.
Change in Surgical Plasma TFPI Concentration and Degradation
TFPI, a Kunitz-type protease inhibitor, dampens early stages of a pro-coagulant response
by inhibiting tissue factor, FVIIa in a FXa-dependent manner,[36] and early forms of pro-thrombinase, the catalytic complex containing FXa and FVa.[37] Plasma TFPI is present in the full-length form, termed TFPIα, and C-terminally truncated
forms.[38] Plasma total TFPI (TFPIα plus truncated TFPI) and TFPIα concentrations were measured
across surgery. At baseline, total TFPI was approximately 20 to 25% the adult concentration
with TFPIα representing approximately 10% of the total plasma TFPI ([Fig. 6A]). Total TFPI and TFPIα promptly increase two- to fourfold upon heparin infusion
in adults.[39]
[40] Heparin-releasable TFPI was similarly observed in neonates, as total TFPI increased
over twofold between baseline and on-CPB. At post-CPB and post-op, it declined slightly
but remained elevated at twofold over baseline. TFPIα increased fivefold between baseline
and on-CPB ([Fig. 6B]). It then declined to about fourfold over baseline at post-CPB and twofold at post-op.
Despite these large changes in plasma TFPI across surgery, there were no differences
in the plasma concentration of either total TFPI or TFPIα between the non-bleeder
and bleeder groups. TFPI Western blots ([Fig. 6C]) mirror the ELISA data with greatly increased band intensity in samples collected
after baseline. Of note, the presence of a small amount of degraded TFPI apparent
at post-CPB and post-op may partially explain the continued elevation of plasma TFPI
following reversal of heparin with protamine in the post-CPB and post-op samples.
Fig. 6 Change in tissue factor pathway inhibitor (TFPI) concentration and partial degradation
across surgery. In panels A and B, box and whisker plots as described in [Fig. 1] with black bars representing the non-bleeding group and grey bars the bleeding group
are presented. (A) Total TFPI increased over twofold in on-cardiopulmonary bypass (CPB) samples compared
with baseline and remained twofold elevated across surgery, p < 0.0001. There were no differences in total TFPI or TFPIα between the non-bleeder
and bleeder groups at any time point, p = 0.9353. (B) TFPIα increased fivefold in on-CPB samples compared with baseline and remained over
fourfold elevated across surgery, p < 0.0001. There were no differences in total TFPI or TFPIα between the non-bleeder
and bleeder groups at any time point, p = 0.1486. (C) TFPI Western blot demonstrates a marked increase in the amount of plasma TFPI after
baseline. Degraded forms of TFPI were present in post-CPB and post-op samples (arrow).
Discussion
Physiological responses to the haemostatic stress of CPB were evaluated by examining
changes in concentration and structural conformation of plasma protease inhibitors
regulating the coagulation, inflammatory, fibrinolytic and complement cascades in
neonates undergoing complex cardiac surgical procedures. TUG gel analysis identifies
these different serpin conformations in plasma in a simple, low cost manner that requires
only micro-litres of plasma.[19]
[20] These analyses were used to obtain ‘snapshots’ of serpin conformational changes
occurring across the cardiac surgical procedures. Although a decrease in fibrinogen
concentration was not observed, large effects on the structural conformation of anti-thrombin
were observed, indicating activation of the coagulation cascade. The heparin infusion
occurring between baseline and on-CPB was associated with the accumulation of non-virgin
forms of anti-thrombin in plasma. These remained following protamine infusion at post-CPB
or post-op, suggesting continued activation of coagulation proteases at the end of
surgery. However, no associations were identified between change in plasma concentration
or structure of anti-thrombin and post-operative bleeding. Despite activation of the
blood coagulation cascade, only minimal conformational change of other protease inhibitors
was observed. Thus, based on serpin conformational change, there was no evidence for
substantial CPB-mediated activation or cross-activation by coagulation proteases of
other plasma proteolytic cascades in the neonates. Further, ample amounts of the virgin
form of each serpin examined were present across surgery indicating that widespread
or uncontrolled proteolysis did not occur, even in the patients experiencing bleeding.
The presence of the inactive forms of anti-thrombin in on-CPB, post-CPB and post-op
samples indicates that ELISA measurement of antigen likely over-estimates the active
anti-thrombin in plasma. However, examination of the TUG gels revealed that typically
75% or more of the anti-thrombin remained in the virgin conformation and severe depletion
of active anti-thrombin was never observed. These data suggest that the neonates had
ample anti-thrombin, either endogenous or transfused, and would not have further benefitted
from infusion of anti-thrombin concentrate.
TFPIα is a heparin-releasable plasma protein in adults.[39]
[40] The neonates had a similar response to heparin, as plasma TFPIα increased an average
of fivefold between baseline and on-CPB. The neonates differed from adult CPB patients
in that TFPIα remained elevated over twofold following protamine reversal in the post-CPB
and post-op samples, while it returned to baseline in adults.[41] Similar to adults, C-terminal degradation of a small portion of circulating TFPIα
occurred during surgery.[41] One possible cause for the elevated TFPI remaining in the circulation would be incomplete
protamine reversal of heparin. However, it may also result from a unique interaction
between neonatal TFPI and vasculature that is not present in adults. Further studies
are needed to understand this interaction. TFPI is a Kunitz-type protease inhibitor
with inhibitory domains defined by disulphide bonds; therefore, unfolding transitions
were not examined using TUG gel conformation analysis. While the elevated plasma TFPIα
at the end of surgery is expected to increase plasma anticoagulant activity, it remains
of uncertain physiological significance because it was not associated with post-operative
bleeding in the neonates.
Although previous studies have found activation of complement, and inflammatory cascades
in paediatric CPB,[11] there was remarkably little change in the concentration or structural conformation
of the serpins regulating inflammatory, fibrinolytic and complement proteolytic cascades.
Plasma concentrations of antitrypsin, anti-plasmin and C1-inhibitor each remained
at 60% or more of baseline with only small amounts of non-virgin conformations observed
in TUG gels, providing little evidence for extensive unregulated cross-activation
of non-coagulation proteolytic cascades by coagulation proteases or cascade activation
by exposure to artificial surfaces in the bypass pump. Of the changes observed, perhaps
the most relevant was the plasma antitrypsin concentration. Antitrypsin is an acute
phase reactant and, therefore, may have been expected to increase across surgery.
Instead, it decreased 20 to 40% from baseline, suggesting the possibility of consumption.
It is possible that there was rapid clearance of antitrypsin-protease complexes from
the circulation, as only the virgin form was observed on TUG gel conformational analysis,
but antitrypsin-protease complexes are cleared from the circulation by the same receptor
as anti-thrombin-protease complexes,[21] which were observed in the TUG gels. Thus, the mechanism for the decrease in the
plasma antitrypsin concentration remains unclear.
In contrast to antitrypsin, C1-inhibitor, which also is an acute phase reactant, was
increased in on-CPB and post-CPB samples. There was a small amount of protease-complexed
C1-inhibitor in samples collected after baseline, but over 90% remained in the virgin
conformation. The increase in C1-inhibitor observed here contrasts with a previous
study where a significant drop was observed in < 12-month-old patients undergoing
CPB.[42] However, several of these babies experienced capillary leak syndrome, which is associated
with the decreased C1-inhibitor and did not develop in neonates studied here.
In addition to identifying serpin conformational changes occurring across surgery,
the TUG gels performed using baseline samples uncovered natural variants in antitrypsin,
anti-plasmin and C1-inhibitor. Antitrypsin had two altered unfolding patterns, one
at lower and one at higher urea concentrations. Neonates having neither, either or
both patterns at baseline were identified, suggesting normal polymorphic variation
in the protein, although altered glycosylation may also cause these variants. Further
studies are needed to biochemically define these variants and how they alter the structural
stability of antitrypsin. Anti-plasmin and C1-inhibitor had minor bands that unfolded
in a non-classical pattern in TUG gels. These were identically present in all samples
and may represent partial N- or C-terminal variants or degradation or altered glycosylation.[31]
[32]
[33]
[35]
A limitation to this study is that excessive bleeding in some patients with post-operative
bleeding began in the operating room, as evidenced by the increased number of blood
products they received during surgery. To account for this, the analyses comparing
protein concentrations in non-bleeders to bleeders were adjusted for the amount of
total protein in each plasma sample to account for the effects of transfusion, but
this did not alter the changes observed for the individual plasma protein concentrations.
Thus, while transfusion produced a major change in plasma protein concentration at
the beginning of surgery, the transfusions administered in response to patient bleeding
that occurred primarily in the post-CPB period had minimal impact on plasma protein
concentration. However, the intra-operative transfusions may have masked significant
declines in the plasma protease inhibitors among the bleeding patients. Similarly,
the transfusions may have impacted the conformations of the serpins observed on the
TUG gels, particularly the virgin forms, which are in FFP.[43] However, the consistent presence of non-virgin forms of anti-thrombin across the
surgical procedure in all neonates suggests that if other serpins existed in non-virgin
conformations, they also would have been observed on the TUG gels.
None of the protease inhibitor plasma concentrations or conformational changes were
associated with post-operative bleeding in the neonates, reflecting the multiple possible
causes of bleeding in this population with a complex disease undergoing a complex
surgical procedure. Direct comparisons of the findings presented here to those of
previous studies of neonatal or paediatric CPB are difficult due to variations in
study protocols and patient management. These include differences in the age of the
patients enrolled, exclusion criteria, priming solutions and differences in transfusion
of blood products, colloid solutions and recombinant anti-thrombin. Therefore, our
findings and conclusions about cross-activation of plasma proteolytic cascades by
coagulation proteases and the effects of changes in plasma protease inhibitor concentration
and conformation on post-operative bleeding following neonatal CPB are applicable
to patients treated with a transfusion protocol similar to that used here.
What is known about this topic?
-
Exposure of blood to artificial materials during cardiopulmonary bypass can activate
coagulation, complement and inflammatory pathways.
-
Serpin conformation can be readily detected using transverse urea gradient gels and
serve as a plasma biosensor for proteolytic cascade activation.
-
Tissue factor pathway inhibitor is a heparin-releasable protein.
What does this paper add?
-
Blood samples from 44 neonates were obtained at four times points during cardiopulmonary
bypass surgery.
-
Coagulation was activated as evidenced by changes in the conformation of anti-thrombin,
but extensive cross-activation of other cascades was not observed.
-
Plasma tissue factor pathway inhibitor increased upon heparin infusion and remained
elevated following protamine administration. However, it was not associated with bleeding
in this population.