Objectives: Activated thrombin-activatable fibri-nolysis inhibitor
is a coagulation factor in some thrombotic diseases. However, available data on
whether thrombin-activatable fibrinolysis inhibitor is activated in islet
transplant are limited. In this study, changes of plasma-activated
thrombin-activatable fibrinolysis inhibitor levels in instant blood-mediated
inflammatory reaction after islet transplant were assessed.
Materials and Methods: Plasma concentrations of
thrombin-antithrombin complex, D-dimer, C-peptide, and activated
thrombin-activatable fibrinolysis inhibitor were assessed at 0 minutes, 30
minutes, 1 hour, 6 hours, 12 hours, and 24 hours after an intraportal islet
transplant using rats via an enzyme-linked immunosorbent assay, or solid-phase,
2-site chemiluminescent immunometric assay. We recovered the liver at 1 hour
after the transplant for histologic examination.
Results: Thrombin-antithrombin complex, C-peptide, and activated
thrombin-activatable fibrinolysis inhibitor levels increased immediately after
we stopped islet infusion, and their peak levels occurred at 1 hour after islet
infusion. D-dimer levels increased continually after islet infusion was stopped,
and peaked 24 hours after infusion. Histologic examination of the liver 1 hour
after islet infusion revealed frequent portal venous thrombi, with entrapped
islets. The entrapped islets showed a disrupted morphology.
Conclusions: Activated thrombin-activatable fibrinolysis inhibitor
was generated and peaked 1 hour after islet transplant according with activating
coagulation, indicating that thrombin-activatable fibrinolysis inhibitor is
activated and accumulated at levels in instant blood-mediated inflammatory
reaction was sufficient to affect fibrinolysis.
Key words: Diabetes mellitus, Instant blood-mediated inflammatory
reaction, Islet transplant, Thrombin-activatable fibrinolysis inhibitor
Intraportal islet allotransplant has become promising therapy for patients
with type 1 diabetes mellitus.1 However, the procedure’s success is
hindered by the islet loss that occurs immediately after transplant, leaving a
final islet cell survival of around 20% to 40% of a healthy nondiabetic subject.2
A major cause of early islet loss is instant blood-mediated inflammatory
reaction (IBMIR), which occurs rapidly when islets come in direct contact with
blood after infusion of the portal vein. It is characterized by coagulation
activation, inflammatory cell infiltration, and subsequent insulin dumping.
Inflammatory thrombosis is driven by a tissue factor and manifests itself
clinically with formation of thrombi with entrapped islets.3,4
The most recently identified coagulation factor is activated
thrombin-activatable fibrinolysis inhibitor (TAFIa).5,6 Upon
activation by thrombin/thrombomodulin, TAFI becomes the active TAFI (TAFIa), and
modulates fibrinolysis in vivo by cleaving C-terminal lysine residues from
partially degraded fibrin.6 Because these residues serve as
plasminogen binding sites, removal of them by TAFIa reduces local concentrations
of plasmin at the site of the clot.7 One of the properties of plasmin
is anticoagulation. Thrombin-activatable fibrinolysis inhibitor serves to
inhibit plasmin recruitment at the forming thrombus; thus, TAFIa is a potent
inhibitor of clot lysis, protecting fibrin clots against fibrinolytic attack.7
Some studies have shown that increased TAFIa is a risk factor for ischemic
stroke, acute myocardial infarction, and venous thrombosis.8-10
Although not all thrombotic disorders will cause activation of TAFI. Coagulation
activation in a baboon model of low-dose E. coli induces sepsis not accompanied
by activated TAFI, while continuous TAFI activation up to 8 hours, is seen in
coagulation activation seen by high-dose E. coli.11,12 However, the
available data on activation of TAFI in IBMIR are limited. In this study, we
assessed the changes of plasma TAFIa levels after islet transplant.
Materials and Methods
Preparation of rat islets
Animals were supplied by the experimental animal center of Tongji University in
Shanghai, China. The investigation conforms to the Guide for the Care and Use of
Laboratory Animals published by the National Institutes of Health (publication
no. 85-23, revised 1985) and is approved by the Ethics Committee of Tongji
University. Pancreatic islets were obtained from adult male Sprague-Dawley rats,
weighing 250 to 300 grams, by collagenase V (Sigma-Aldrich, St. Louis, MO, USA);
digestion and discontinuous Ficoll density gradient centrifugation (MEDIATECH,
INC., Herndon, VA, USA). After washing and handpicking, islets were cultured for
24 hours in RPMI-1640 (Gibco [now Invitrogen Corporation], Carlsbad, CA, USA)
medium supplemented with 10% fetal bovine serum in a humidified 5% CO2
incubator at 37ºC. Islet purity was assessed by dithizone (Sigma) staining after
isolation. Islet viability was assessed via fluorescence staining with acridine
orange and propidium iodide (Sigma). Islet isolations with > 90% viability and >
90% purity were used. Before transplant, islets were washed 3 times and
suspended in phosphate buffered saline (PBS).
Animal experiment design
Seventy-two normal Sprague-Dawley rats received 30 islet grafts. Receptors
were randomly divided into 2 groups (experimental and control). Each group was
further divided into 6 subgroups with 6 rats in each subgroup to obtain blood
before infusion and at 30 minutes, 1 hour, 6 hours, 12 hours, and 24 hours after
infusion. Rats in the experimental group received intraportal injection of
islets at 800 IEQ/rat, and those in the control group received only PBS.
When they were killed, rats were anesthetized by intraperitoneal injection
of pentobarbital (50 mg/kg). Blood was drawn from the inferior vena cava into
tubes containing 3.8% sodium citrate (9 parts blood, 1 part anticoagulant).
Plasma was obtained by centrifugation and stored at -80ºC. Plasma
thrombin-antithrombin complex (TAT) level was determined using a commercial
enzyme- linked immunosorbent assay (ELISA) kit (Behringwerke, Marburg, Germany).
D-dimer level was measured with ELISA kits (American Diagnostica Inc.,
Greenwich, CT, USA). C-peptide level was measured using solid-phase, 2-site
chemiluminescent immunometric assay kits (Mercodia AB, Uppsala, Sweden). Plasma
TAFIa levels were measured with ELISA kits (American Diagnostica Inc.).
Livers were recovered 1 hour after islet infusion. Liver biopsy samples were
fixed in paraformaldehyde. Sections of the liver biopsies then were stained with
hematoxylin and eosin stain.
The data are presented as mean values ± standard deviations. The analysis of
variance (ANOVA) test was used to assess statistical significance at different
times within groups; the paired t test was used to assess statistical
significance between 2 groups at the same time. Values of P < .05
were considered statistically significant.
Expression of TAT, D-dimer, C-peptide, TAFIa during islet transplant
Reflecting the activation of coagulation, TAT levels increased immediately
after islet infusion was stopped, and the peak level in the series was reached
after only 1 hour, and then decreased gradually. After 12 hours, the levels
returned to baseline values (Figure 1A). Reflecting the activation of
fibrinolysis, D-dimer levels increased continually after islet infusion was
stopped and peaked at 24 hours after infusion (Figure 1B). C-peptide was
released immediately after infusion. The levels peaked 1 hour after infusion and
declined thereafter, but never reached baseline levels (Figure 1C). TAFIa levels
increased immediately after islet infusion was stopped, and peaked 1 hour after
infusion, and then decreased rapidly. After 6 hours, levels had returned to
baseline values (Figure 1D). No obvious changes in the levels of TAT, D-dimer,
C-peptide, and TAFIa were observed in the vehicle-treated control rats.
Histologic evaluation of the liver after intraportal islet transplant
Plasma analyses showed that activation of coagulation and release of
C-peptide occurred mainly during the first hour after infusion, so the time
point of histologic examination for the liver was chosen 1 hour after islet
infusion. Results revealed frequent portal vein thrombi with entrapped islets,
and the entrapped islets showed a disrupted morphology (Figure 2).
The general characteristics of IBMIR are activation of coagulation and
disruption of normal islet morphology 1 hour after islet infusion. Reflecting
the magnitude of the IBMIR, activation of coagulation indicated by elevated TAT
peaked 1 hour after islet infusion. The generation of D-dimer further
strengthens the appearance of the coagulation after transplant. In parallel with
coagulation, a rapid liberation of C-peptide and disruption of islet morphology
were observed during the first hour, suggesting that transplanted islets were
damaged severely during the thrombotic reaction. Release of C-peptide as a
result of glucose stimulation is highly unlikely, because the glucose
concentration in nondiabetic rat receptors is normal. Therefore, IBMIR after
islet transplant may be responsible for this rapid release of C-peptide.
This is the first study to report on the kinetics of TAFIa plasma
concentrations during the development of IBMIR after an islet transplant. This
rapid and continuous increasing of TAFIa concentration during the first hour
after infusion is consistent with the increasing level of TAT, which might be
indicative of higher levels of thrombin/thrombomodulin complex generated after
islet transplant, this is thought to be the physiologic TAFI activator.5
Although definite proof of a causal relation is currently unavailable, it is
reasonable to interpret this temporal profile as a rapid increase of TAFIa
during the first hour after transplant, indicating that TAFIa favors islet
clotting because of its antifibrinolytic activity.13-15
To obviate the effects of IBMIR, some methods of regulating early
coagulation, including systemic administration of anticoagulants such as
heparin, melagatran, dextran sulfate, and nacystelyn, have been shown to prevent
islet-induced coagulation and reduce islet loss to a certain extent.3,16-18
However, it is difficult to apply these anticoagulants in the clinical
environment because systemic administration is associated with an increased risk
of severe bleeding.19 Therefore, there is an urgent need to explore
new therapeutic target for treating IBMIR with fewer bleeding complications. The
present study shows that TAFI is activated and accumulated during the first hour
after islet infusion, indicating that TAFIa is sufficient to affect fibrinolysis
Previous studies have shown that endogenous thrombolysis is enhanced, and
thrombosis is reduced, by inhibiting TAFIa in different thrombus models.20-23
Additionally, inhibition of TAFIa represents a potentially subtle adjustment of
the clotting and lysis balance, without affecting the coagulation cascade.
Ideally, this subtlety may reduce the risk of bleeding when using TAFIa
inhibitor, compared with other potential mechanism for treating thrombotic
disease.24 Because administration of TAFIa inhibitor does not produce
an increase in bleeding time in a rat-transection bleeding model,25
TAFIa would be a safe and effective drug for inhibiting thrombosis in IBMIR.
TAFIa peaks 1 hour after infusion then declines to its baseline level,
suggesting that 1 hour after infusion might be the best intervention time for
Taken together, the present study shows that TAFIa is generated and peaks 1
hour after islet transplant in accord with activation of coagulation, indicating
that TAFI is activated and accumulates at levels in which IBMIR is sufficient to
affect fibrinolysis. Taking into account that inhibition of TAFIa may enhance
endogenous thrombolysis, while leaving the coagulation system intact, TAFIa
might be an innovative drug target for eliminating the effects of IBMIR.
- Shapiro AM, Ricordi C, Hering BJ, et al. International trial of the
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- Ryan EA, Lakey JR, Rajotte RV, et al. Clinical outcomes and insulin
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Volume : 12
Issue : 1
Pages : 62-66
From the Department of General Surgery, Shanghai East Hospital, Tongji
University, Shanghai, PR China
Acknowledgements: This study was funded by grants from the Project of
Shanghai Science and Technology Commission of China (No. 10411968400).
Corresponding author: Guangjun Suo, MD, PhD, Department of General
Surgery, Shanghai East Hospital, Tongji University, Shanghai, 200120, PR China
Phone: +86 021 131 2064 5697
Fax: +86 021 5879 8999
Figure 1. Alterations in the Concentration of TAT, D-dimer, C-peptide,
and TAFIa During IBMIR After Islet Transplant
Figure 1. Histopathologic Examination of Liver 1 Hour After an
Intraportal Islet Allotransplant