Introduction
Intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH) have limited treatment
options and a high mortality. We have previously shown that both conditions are associated
with early hypercoagulation.[1]
[2]
Histones have been demonstrated to promote thrombin generation.[3] In sepsis and trauma patients, studies have revealed an association between histone-DNA
complexes and free histone levels and mortality or morbidity.[4]
[5]
[6] Theoretically, the effects on coagulation of histones can be investigated in vitro
by attempting to block histones with specific antibodies.
The aim of this study was to quantify histone-DNA complex levels in patients with
ICH or SAH at the time of admission to hospital compared with 24 hours after symptom
onset and to investigate the possible in vitro effect of anti-histone antibodies on
thrombin generation.
Materials and Methods
The study was a prospective cohort study. Inclusion criteria were radiologically verified
spontaneous ICH or SAH. Patients were excluded if they received medications affecting
coagulation prior to inclusion. Further details on the cohort and exclusion criteria
have been previously published.[1]
[2] Blood samples were drawn from a peripheral vein or arterial line at admission to
hospital and 24 ± 2 hours after symptom onset. Samples were batch analyzed shortly
after patient inclusion ended.
Histone-DNA complexes were detected by using the Cell-Death Detection ELISAPLUS kit (Roche, Mannheim, Germany) and reported in absorbance units (AU). A cohort of
20 healthy men and 20 healthy women established the control group for histone-DNA
complexes.[7]
Thrombin generation was measured in platelet-poor plasma with the addition of tissue
factor (5 pm), phospholipids (4 µM), FluCa, hepes, calcium, murine IgG2a kappa isotype control
antibody (StemCell Technologies, Vancouver, Canada) and anti-histone antibodies MHIS
AB1952 (specific for histone H4) and MHIS AB1992 (specific for histones H1 and H3)
kindly donated by Charles T. Esmon, Oklahoma Medical Research Foundation, Oklahoma
City, United States. An antibody concentration equivalent to 0.426 mg/mL was chosen
for thrombin generation analyses after titration experiments based on available murine
studies.[8] Four analyses were performed per sample: one with addition of NaCl, one with addition
of control antibody and two with the addition of AB1952 and AB1992, respectively.
Thrombin generation was quantified with calibrated automated thrombogram (CT, thrombinoscope
BV, Maastricht, Holland). A cohort of 45 healthy men and 45 healthy women established
a control group for thrombin generation results.[9]
Furthermore, international normalized ratio (INR), activated partial thrombin time
(aPTT), antithrombin (functional), thrombin time, fibrinogen (functional, Clauss method),
fibrin D-dimer, hemoglobin, platelet and leucocyte count, C-reactive protein (CRP),
alanine transaminase, albumin, creatinine, and S100 Calcium Binding Protein B (S100B)
were determined.
Descriptive statistics are presented as median with interquartile range (IQR). Paired
data were analyzed with a Wilcoxon's signed rank test (not following Gaussian distribution)
or with a Student's t-test (following Gaussian distribution). Unpaired data were analyzed with a Mann–Whitney
test. Correlation tests were performed by using a Spearman's test. Sample size was
defined by the parent study. The primary endpoint was the change in histone-DNA complex
levels from admission to 24 hours after symptom onset, for which the power was 100%
(mean value 17 AU at admission [standard deviation 16 AU] and 6 AU at 24 hours) and
the significance level, 2α, was 0.05).
The study was conducted in accordance with the Declaration of Helsinki, and the Central
Denmark Region Committees on Health Research Ethics approved the study before initiation
(case no. 1–10–72–95–14).
Results and Discussion
Eighty-seven patients with ICH or SAH were included. The median age was 61 years,
and 67% were females. Of the 46 patients with an SAH, 17 underwent surgical coiling
and thus received unfractionated heparin. Overall 30-day mortality was 26%, with 33%
mortality for SAH patients, and 20% mortality for ICH patients.
[Table 1] shows the standard biochemical profiles at admission and 24 hours after symptom
onset. Seven patients had a prolonged thrombin time 24 hours after symptom onset due
to administration of unfractionated heparin during the coil procedure, close to sample
collection. Exclusion of these patients had no significant impact on the results described
below.
Table 1
Biochemical values at time of admission to hospital and 24 hours after symptom onset
in 87 patients with intracerebral or subarachnoid hemorrhage
|
Parameter
|
Reference values[a]
|
At admission
|
After 24 hours
|
p-Values
|
|
Hemoglobin (mmol/L) (n
paired = 73)
|
7.3–9.5
|
8.4 (7.7–8.9)
|
7.4 (6.9–8.3)
|
<0.0001
|
|
Platelet count (× 109/L) (n
paired = 72)
|
165–400
|
229 (179–268)
|
212 (170–251)
|
0.003
|
|
INR (n
paired = 71)
|
<1.2
|
1.0 (1.0–1.1)
|
1.1 (1.1–1.2)
|
<0.0001
|
|
aPTT (s) Before April 5, 2016 (n
paired = 68)
|
25–38
|
28 (26–31)
|
31 (28–34)
|
<0.0001
|
|
aPTT (s) After April 5, 2016 (n
paired = 9)
|
20–29
|
27 (25–29)
|
25 (24–31)
|
0.88
|
|
Antithrombin (× 103 IU/L) (n
paired = 37)
|
0.80–1.20
|
0.92 (0.86–1.04)
|
0.92 (0.85–1.01)
|
0.84
|
|
Thrombin time (s) (n
paired = 57)
|
<21
|
16 (16–17)
|
16 (15–17)
|
0.64
|
|
Fibrinogen (μmol/L) (n
paired = 71)
|
5.0–12.0
|
9.2 (7.8–11.1)
|
9.8 (8.3–10.8)
|
0.004
|
|
Fibrin d-dimer (mg/L FEU) (n
paired = 71)
|
<0.5
|
1.3 (0.66–2.20)
|
1.2 (0.6–3.5)
|
0.29
|
|
S100B (μg/L) (n
paired = 71)
|
0.02–0.13
|
0.13 (0.07–0.41)
|
0.13 (0.06–0.24)
|
0.33
|
|
CRP (mg/L) (n
paired = 73)
|
< 8.0
|
2.2 (0.9–4.5)
|
11.9 (3.9–37.8)
|
<0.0001
|
|
Leucocytes (× 109/L) (n
values = 78)
|
3.5–10.0
|
10.9 (7.7–13.4)
|
|
|
|
Alanine Transaminase U/L (n
values = 84)
|
10–70
|
20 (16–30)
|
|
|
|
Albumin (g/L) (n
values = 86)
|
36–45
|
38 (35–40)
|
|
|
|
Creatinine (μmol/L) (n
values = 86)
|
45–105
|
60 (53–75)
|
|
|
Abbreviations: aPTT, activated partial thrombin time. CRP, C-reactive protein; INR,
international normalized ratio; S100B, serum s100 calcium binding protein B.
Note: Values reported as median (interquartile range). As there are missing values
(due to failed blood sampling, patient dying before blood sampling etc.), we have
reported n
paired = number of paired values, n values = number of values. The reference interval for aPTT changed during the inclusion
period, and aPTT data are split in two groups accordingly.
a Reference values for adults, combined for men and women, established at the local
laboratory.
ICH patients and SAH patients did not differ in histone-DNA complex levels measured
at admission (p = 0.34) or 24 hours after symptom onset (p = 0.13). For the total cohort, histone-DNA complex levels decreased from a median = 11.6
AU (IQR = 6.5–24.5) at admission to median = 3.5 AU (1.5–8.2) 24 hours after symptom
onset (p < 0.0001). Patients had significantly higher levels of histone-DNA complexes at admission
compared with the healthy cohort mean = 5.4 AU (2.0–11.4; p = 0.0001).
No difference was demonstrated in histone-DNA complex levels at admission for 30-day
survivors (median = 11.3 AU (IQR = 6.4–23.2) compared with nonsurvivors (11.4 AU [5.1–25.9],
p = 1.00). That histone-DNA complex levels were not found to correlate with mortality
is in accordance with a study performed on trauma patients.[10] Thus, we could not confirm the association between levels of histone-DNA complexes
and morbidity and mortality found in other patient populations.[4]
[5]
[6] These differences between studies could be due to the size of our study or to the
only moderate levels of histone-DNA complexes found in the present study.
[Fig. 1] shows thrombin generation performed on healthy controls as well as patient admission
samples with and without added control and anti-histone antibodies. The patient admission
samples had significantly decreased lagtime and time to peak and increased peak and
endogenous thrombin potential (ETP; all p ≤ 0.003) compared with the healthy controls, signifying an increased thrombin generation.
Fig. 1 Thrombin generation with antibody addition in 87 patients with intracerebral or subarachnoid
hemorrhage at admission. Thrombin generation in healthy controls and in patients with
an intracerebral or subarachnoid hemorrhage, described by lagtime, time to peak, peak
and endogenous thrombin potential, with and without added control antibody and anti-histone
AB1952 and AB1992 antibodies. Boxes and whiskers indicate median, interquartile range,
and range. Number of measurements per group is 81 due to missing values in six patients.
The anti-histone antibodies reduced time to peak when compared with the control antibody
(p < 0.0001 for AB1952 and p = 0.05 for AB1992). However, the addition of anti-histone antibodies did not suppress
thrombin generation more than the control antibody for ETP, peak, and lagtime values.
Thus, the suppression may be due to a physical effect of adding the antibody molecules,
regardless of their specificity. Furthermore, the antibodies only block histones and
not the entire histone-DNA complex.
The main finding of the present study was increased levels of histone-DNA complexes
in the acute phase right after an ICH or SAH when compared with 24 hours after symptom
onset. A higher thrombin generation was demonstrated in patients at admission than
in healthy controls. No convincing effect of adding anti-histone antibodies to thrombin
generation could be discerned. Hence, the present study did not support the hypothesis
that histones have a significant effect on coagulation as expressed by thrombin generation.
