Electronic Supplementary Material
INCREASED MORTALITY IN SEPTIC SHOCK WITH THE
4G/4G GENOTYPE OF PLASMINOGEN ACTIVATOR
INHIBITOR 1 IN PATIENTS OF CAUCASIAN DESCENT
Gloria García-Segarra1, M.D., Gerard Espinosa1, M.D., Dolors Tassies2, M.D.,
Josep Oriola3, Ph.D., Jesús Aibar1, M.D., Albert Bové1, M.D., Pedro Castro1,
M.D., Joan-Carles Reverter2, M.D., and Josep-Maria Nicolás1, M.D.
Medical Intensive Care Unit1, Department of Hemotherapy and Hemostasis2,
and Biochemistry Laboratory3. Hospital Clínic, IDIBAPS – University of
Barcelona. Spain.
PATIENTS AND METHODS
Patients
Along a 12-month period (from January 2003 to January 2004), 301 patients
were admitted to the Medical ICU of the Hospital Clínic of Barcelona. This is an
eight-bed intensive care facility in which patients are mainly admitted because
of infectious medical problems. Of these patients, 27 non-septic subjects with
an ICU stay less than 48 hours (mostly uncomplicated cardiac postoperative
patients), 40 patients with short-term irreversible diseases (21 cases with
hematological tumors mainly admitted due to infectious complications after
transplantation, 10 patients with stroke that ended as cadaveric organ donors, 6
subjects with advanced solid neoplasia mainly admitted due to infectious
complications, and three AIDS cases admitted with opportunistic diseases), six
subjects with unresolved surgical problems, and four patients in whom futile
care (stroke patients) was applied during ICU stay, were not considered for the
study in order to avoid the confounding effect of the primary disease in the
outcome (Figure 1). The remaining patients were included and followed until
hospital discharge. Furthermore, 80 healthy blood donors recruited
consecutively were used as the control population. All the subjects were of
Caucasian descent, living in or around Barcelona (Catalonia, Spain). Informed
consent was obtained from the patients or their relatives within 24 hours after
admission and none refused to participate. The protocol was approved by the
Institutional Review Board of the Hospital Clínic of Barcelona.
Epidemiological data, the primary site of infection and infection-related
organisms were recorded. Clinical infections were defined according to the
Centers for Diseases Control and Prevention criteria (25). Severe sepsis and
septic shock were defined according to SCCM/ESICM/ACCP/ATS/SIS
consensus conference (26). Severe sepsis was considered when oliguria
(diuresis < 0.5 ml/Kg/h for at least 1 hour), metabolic acidosis (pH < 7.3 or base
excess > 5.0 mmol/L or lactate > 2 mmol/L), or mental confusion (GCS < 14 in
absence of sedation) was present. Septic shock was diagnosed in patients with
sepsis who were hypotensive (systolic blood pressure < 90 mmHg or reduction
> 40 mmHg from usual in hypertensive patients) for at least one hour despite
adequate fluid resuscitation or vasopressor therapy (dopamine > 5 µg/Kg/min,
any dose of noradrenaline or adrenaline for at least one hour) to maintain a
systolic blood pressure > 90 mmHg within 48 hours from ICU admission. Septic
shock was considered nosocomial (late) when observed after 48 hours after
ICU entry.
Severity indexes including APACHE II (acute physiology and chronic health
evaluation score), SAPS II (simplified acute physiologic score) and SOFA
(sequential-organ failure assessment) were calculated at ICU admission, and
thereafter on a daily basis. Multiorgan failure was considered in case of acute
progressive dysfunction of two or more organs systems, with a minimum failure
score of 3 points for each organ. Chest X-ray examination and oxygenation
parameters (PaO2, PaO2/FIO2 ratio) were recorded. The presence of acute
respiratory distress syndrome (ARDS) was considered according to the
European Consensus score (27), with patients showing a Murray’s score ≥ 2.5
(28). Blood samples and the information used in the study were coded and
patient confidentiality was preserved. Biochemical studies and genotyping were
performed in the patients after completion of follow-up.
Biochemical studies
Venous blood samples were collected the morning after admission with a clean
antecubital venipuncture without venocclusion. Samples for coagulation and
fibrinolysis studies were obtained in tubes containing 3.8% trisodium citrate (1/9
volume/volume; Becton Dickinson, Rutherford, NJ). Plasma was aliquoted,
snap-frozen in a mixture of dry ice/ethanol (1/2 v/v), and stored. For genotype
studies, samples were drawn in trisodium EDTA tubes (Becton Dickinson), and
100 µl of whole blood was immediately transferred into tubes containing lysis
buffer (5M guanidine thiocyanate, 1.3% [weight/volume] Triton X-100, and 50
mM Tris HCl, pH 6.4) and frozen at -80°C. Sera for biochemistry were drawn in
tubes containing no anticoagulants (Becton Dickinson).
General biochemical and basic coagulation determinations were performed
using standard semiautomated methods. Prothrombin and activated partial
thromboplastin times were determined in an automated coagulometer CA-6000
(Dade Behring, Marburg, Germany) using standard reagents (Thromborel and
Actin FS; Dade Behring) and were expressed as ratios (patient time : control
time). Fibrinogen level was measured by the Clauss technique. D-Dimer was
measured using an automated latex-enhanced turbidimetric assay (Dade
Behring) (29) Plasma antigen related to PAI-1 was measured by an ELISA
(Biopool, Umea, Sweden) based on a double antibody principle (30).
Genotype studies
Genomic DNA was extracted from 100 µL of whole blood with a silica gel
column method (QIAamp DNA blood mini kit, Qiagen GmbH, Hilden, Germany),
and stored at –80ºC until further use. Genotyping was validated by direct
sequencing of a group of random samples.
PAI-1 4G/5G polymorphism genotyping. Detection of the PAI-1 4G/5G
polymorphism was made as previously described (31), with minor modifications.
The following primers were used: 5’-CAC AGA GAG AGT CTG GC*C ACG T-
3’, forward primer, position -697/-676, with a C ->A substitution (*) in position 681; and 5’-CCA ACA GAG GAC TCT TGG TCT- 3’, reverse primer, position 598/-619. The mutated forward primer inserts a site for the Bsi YI enzyme in the
product of amplification that enables the identification of the extra G base. PCR
was performed using 30 cycles at 95°C for 1 minute, 60°C for 1 minute, and
72°C for 1 minute, and a final extension cycle at 72°C for 7 minutes. The
expected size of the amplified products was 99bp for the 5G allele and 98bp for
the 4G allele. The PCR product was digested for 150 minutes at 55°C with 5
units of the Bsi YI restriction enzyme (Roche Diagnostics, Basel, Switzerland).
Detection was performed by 4% agarose gel electrophoresis. The digested
fragments were a single 98bp band for the 4G allele and 2 bands (77bp and
22bp) for the 5G allele.
TNF--252G polymorphism genotyping. Analysis of the TNF-B1/B2 intron 1
gene polymorphism was performed as follows. Forward and reverse primers
have been previously described (32) 5’-CCG TGC TTC GTG CTT TGG ACTA3’ and 5’- AGA GGG GTG GAT GCT TGG GTT C-3´ respectively. Briefly, PCR
conditions were: 50 ng of genomic DNA was added to 25 µL of reaction mixture.
The PCR reaction contained 1 µM of each primer and 1U Taq polymerase
(Invitrogen, Life technologies, Carlsbad, CA) and 2% of DMSO. Amplification
was carried out using 35 cycles of denaturation at 94ºC (30 sec), annealing at
58ºC (30 sec) and extension at 72ºC (1 min). PCR amplification was digested
with Nco I restriction enzyme and PCR product yielded fragments of 586bp and
196bp (allele B1) and 782bp (allele B2). The analysis was monitored with
electrophoresis on 2% agarose gels containing ethidium bromide.
IL-1ra A2 polymorphism genotyping. Analysis of the 86bp variable number
tandem repeat present in the intron 2 of the IL-1ra gene was performed as
follows: 50 ng of genomic DNA was amplified by PCR using primers 5’-CTC
AGC AAC ACT CCT AT-3’ and 5’-TCC TGG TCT GCA GGT AA-3’ described
previously (33). The PCR reaction contained 1 µM of each primer and 1U Taq
polymerase (Roche, Switzerland). Amplification was carried out using 35 cycles
of denaturation at 94ºC (10 sec), annealing at 60ºC (10 sec) and extension at
72ºC (30 sec). The final PCR products were analyzed on a 7% polyacrylamide
gel and stained with ethidium bromide. We found three alleles: A1 (410bp), A2
(240bp), and A3 (500bp).
RESULTS
Baseline patient characteristics
The 224 patients studied had a mean age of 62.3 ± 15.9 years (range, 18 to
85), male predominance was of 63.8%, with APACHE II, SAPS II and SOFA
scores of 16.8 ± 6.3, 37.4 ± 12.3, and 8.6 ± 3.1 at ICU entry, respectively.
Eighty-eight and 26 patients exhibited septic shock and severe sepsis,
respectively, within 48 hours of ICU entry (Figure 1). Of the remaining
patients, 51 had sepsis and 59 had non-infectious SIRS. Epidemiological and
clinical data of the groups of patients are shown in Table 1. Patients with
septic shock required surgery more frequently than patients with sepsis (P =
0.03). The most frequent primary site of infection was community-acquired
pneumonia (38.4 to 48.8% among septic groups), although abdominal
infection was reported in 19.3% of the patients with septic shock. Coagulation
parameters at ICU entry did not differ among the groups of patients, except
for greater fibrinogen levels in septic subjects than in those with noninfectious SIRS (P < 0.05, all) (Table 1). Plasma glucose, assessed every 6
hours, was maintained in the range of 80 to 140 mg/dL in 82% of the
determinations. For patients mechanically ventilated, a protective strategy
(tidal volume 6-8 mL/Kg, plateau pressure ≤ 30 cmH20) was routinely used.
No patient had received treatment with activated Protein-C because at the
time of the study was not yet introduced in our Hospital.
DISCUSSION
It is already known that virtually all patients with sepsis have coagulation
abnormalities, which range from a small decrease in the platelet count and a
subclinical prolongation of global clotting times, to fulminant disseminated
intravascular coagulation characterized by simultaneous widespread
microvascular fibrin deposition, which contributes to organ dysfunction and
profuse bleeding from various sites (35). Inhibition of the fibrinolytic system is a
key element of the pathogenesis of fibrin deposition during severe inflammation.
In sepsis-induced generalized activation of coagulation, generation of thrombin
also initiates fibrinolysis through the release of tissue plasminogen activator.
However, the activation of the fibrinolytic system is transient due to a strongly
pro-inflammatory cytokine-induced PAI-1 expression and secretion by
endothelial cells (36,37). Thus, the net result is a proinflammatory and
prothrombotic disorder with exhaustion of fibrinolysis and coagulation inhibitors
(38). In agreement with previous findings (39), we found that both plasma PAI-1
and D-Dimer were elevated in patients with septic shock, mainly in those who
died, and correlated with the first to organ failure. Elevated PAI-1 levels may
contribute to a greater development of intravascular micro-thrombi by protecting
formed fibrin from the fibrinolysis mediated by tissue type plasminogen
activator. This effect may be responsible, at least in part, for augmented
accumulation of cross-linked fibrin in patients with high PAI-1 levels. Then, in
these patients a greater total fibrin mass is subsequently presented for
fibrinolysis mediated by plasmin resulting finally in increased D-Dimer formation.
The G4 variant of the PAI-1 gene has also been implicated in many other
diseases. Initial studies showed that the 4G/4G genotype was associated with
a 20% increased risk of myocardial infarction (42), but recent studies do not
agree with these results, giving a 6% increased risk (19) o even none (43). The
association of this genetic variant with stroke has been less consistent
(20,43,44). The presence of 4G/4G PAI-1 genotype may also confer an
additional risk for hemodyalisis access thrombosis (45). These conflicting
results may be related to the fact that plasma levels of PAI-1 are influenced by
other genetic factors involved in the fibrinolysis, rennin-angiotensin and
bradykynin systems, as well as gender of patients (46). Finally, high plasmatic
levels of PAI-1 has been related to incident type 2 diabetes (47), and may also
present a prognostic factor for patients with head and neck squamous cell
carcinoma (48) and in nonsmall-cell lung carcinoma (49).
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