Production of tissue factor by pancreatic islet cells as a trigger of detrimental thrombotic reactions in clinical islet transplantation

MECHANISMS OF DISEASE

Mechanisms of disease

Production of tissue factor by pancreatic islet cells as a trigger of detrimental thrombotic reactions in clinical islet transplantation L Moberg, H Johansson, A Lukinius, C Berne, A Foss, R Källen, Ø Østraat, K Salmela, A Tibell, G Tufveson, G Elgue, K Nilsson Ekdahl, O Korsgren, B Nilsson

Summary Background Intraportal transplantation of pancreatic islets offers improved glycaemic control and insulin independence in type 1 diabetes mellitus, but intraportal thrombosis remains a possible complication. The thrombotic reaction may explain why graft loss occurs and islets from more than one donor are needed, since contact between human islets and ABOcompatible blood in vitro triggers a thrombotic reaction that damages the islets. We investigated the possible mechanism and treatment of such thrombotic reactions. Methods Coagulation activation and islet damage were monitored in four patients undergoing clinical islet transplantation according to a modified Edmonton protocol. Expression of tissue factor (TF) in the islet preparations was investigated by immunohistochemistry, immunoprecipitation, electron microscopy, and RT-PCR. To assess TF activity in purified islets, human islets were mixed with nonanticoagulated ABO-compatible blood in tubing loops coated with heparin. Findings Coagulation activation and subsequent release of insulin were found consistently after clinical islet transplantation, even in the absence of signs of intraportal thrombosis. The endocrine, but not the exocrine, cells of the pancreas were found to synthesise and secrete active TF. The clotting reaction triggered by pancreatic islets in vitro could be abrogated by blocking the active site of TF with specific antibodies or site-inactivated factor VIIa, a candidate drug for inhibition of TF activity in vivo. Interpretation Blockade of TF represents a new therapeutic approach that might increase the success of islet transplantation in patients with type 1 diabetes, in terms of both the risk of intraportal thrombosis and the need for islets from more than one donor. Lancet 2002; 360: 2039–45 See Commentary page 1999 Department of Radiology, Oncology, and Clinical Immunology, Division of Clinical Immunology, Rudbeck Laboratory (L Moberg MSc, H Johansson MSc, G Elgue PhD, Prof K Nilsson Ekdahl PhD, Prof O Korsgren MD, B Nilsson MD), Department of Genetics and Pathology, Division of Pathology, Rudbeck Laboratory (A Lukinius PhD), Department of Medical Sciences, Division of Medicine (Prof C Berne MD), and Department of Surgical Sciences, Division of Transplantation Surgery (Prof G Tufveson MD), University Hospital, Uppsala, Sweden; Department of Transplantation Surgery, Oslo, Norway (A Foss MD); Department of Nephrology and Transplantation, University Hospital, Malmö, Sweden (R Källen MD); Department of Urology, Skejby Hospital, Arhus, Denmark (Ø Østraat MD); Division of Transplantation, Surgical Hospital, Helsinki University, Helsinki, Finland (K Salmela MD); Department of Transplantation Surgery, Stockholm, Sweden (A Tibell MD); and Department of Chemistry and Biomedical Science, University of Kalmar, Kalmar, Sweden (K Nilsson Ekdahl) Correspondence to: Dr Bo Nilsson, Department of Radiology, Oncology, and Clinical Immunology, Division of Clinical Immunology, Rudbeck Laboratory, University Hospital, S-75185 Uppsala, Sweden (e-mail: [email protected])

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Introduction Homoeostasis is essential for survival. Any disturbance in the haemostatic balance, such as damage to a vessel wall, leads to immediate activation of the coagulation system. In vivo, the coagulation system is triggered mainly by the 47 kDa transmembrane glycoprotein TISSUE FACTOR (TF), which acts both as a receptor and as a cofactor for the cleavage of factor VII to VIIa and for the activity of factor VIIa in the TF (extrinsic) pathway of coagulation. TF is of the CYTOKINE RECEPTOR SUPERFAMILY type 1; when complexed with factor VIIa it triggers intracellular signal transduction involved in angiogenesis, diapedesis, and inflammation.1 TF is constitutively expressed by cells in the adventitia of the blood vessels and is found in richly vascularised tissues such as the cerebral cortex, renal glomeruli, and lungs.2,3 Normally, cells exposed to blood (such as endothelial cells and monocytes) do not express TF, but certain inflammatory stimuli such as lipopolysaccharide, immune complexes, and cytokines can induce TF expression in these cells. TF is strictly regulated by TF pathway inhibitor (TFPI) in the blood.4 For many years, clinical islet transplantation had a success rate, as defined by insulin independence after 1 year, of about 10%. In 2000, Shapiro and colleagues5 showed that insulin independence could be obtained if the patient was treated with repeated transplants from more than one donor. In a follow-up study, however, the same researchers showed that the patients who underwent transplantation had ␤-cell function of only 20% of that in healthy individuals, even though they had received islets from more than one donor; 6 this low activity was thought to be the result of loss of endocrine tissue. A feared complication of islet-cell transplantation is portal thrombosis, and fatal cases were reported in the 1990s.7,8 Although, nowadays, clinical islet transplantation offers patients with type 1 diabetes mellitus improved glycaemic control and insulin independence, these findings emphasise that the treatment protocol is far from optimum and could be improved. We have previously described a thrombotic reaction that occurs in vitro when purified human islets are incubated in ABO-compatible blood, termed the INSTANT BLOODMEDIATED INFLAMMATORY REACTION (IBMIR). The effects of this reaction together cause a disruption of islet morphology within a thrombus entrapping the islets.9,10 Therefore, IBMIR is a likely cause of both the loss of transplanted tissue and the intraportal thrombosis associated with clinical islet transplantation. We describe here the presence of IBMIR in clinical islet transplantation, elucidate the mechanisms underlying this reaction, and suggest a future treatment protocol to combat it.

Methods Islet isolation Islets were isolated from human cadaver donors (under a protocol approved by the local ethics committee)11–13 by 2039

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MECHANISMS OF DISEASE

GLOSSARY CYTOKINE RECEPTOR SUPERFAMILY

All cytokine receptors are characterised by one or more transmembrane proteins in which the extracellular portion contains the cytokine binding sites and the cytoplasmic portions induce intracellular signalling. IMMUNOPRECIPITATION

A technique to extract a specific molecule from a solution by use of an antibody. The antigen-antibody complex is rendered insoluble either by precipitation with a second antibody or by coupling the first antibody to an insoluble particle or bead. INSTANT BLOOD-MEDIATED INFLAMMATORY REACTION (IBMIR)

Characterised by initial activation of the coagulation and complement systems, rapid binding and activation of platelets, and recruitment and infiltration of the islets by leucocytes. TISSUE FACTOR (TF)

A transmembrane protein of 47 kDa that normally is expressed by extravascular cells only. Binding of factor VIIa present in blood to TF activates the coagulation cascade system.

means of Liberase (Roche, Roche Diagnostica, Indianapolis, USA) perfusion followed by continuousdensity Ficoll gradient purification in a refrigerated centrifuge (COBE 2991; COBE Blood Component Technology, Lakewood, CO, USA). Islet preparations were maintained in culture medium (CMRL 1066; ICN Biomedicals, Costa Mesa, CA, USA) at 37⬚C (5% carbon dioxide) for 1–7 days. The volume and purity were assessed by microscopic sizing after staining with diphenylthiocarbazone. Viability was assessed in terms of insulin secretion in response to a glucose challenge in a dynamic perfusion system (in 1·67 mmol/L, 16·7 mmol/L, and then 1·67 mmol/L glucose).

consecutive patients (one female, three male; aged 37–51 years) received intraportal transplants of human islets from a total of nine donors (ie, nine donors for four patients). The grafts were given one at a time with a minimum interval of a few weeks. The mean number of islets in each graft was 270 000 islet equivalent (range 180 000–400 000), and the purity was 70% or more. Patients were already receiving immunosuppressive treatment because they had each undergone kidney transplantation previously. At the time of islet transplantation, the immunosuppressive regimen was switched to the steroid-free protocol applied by Shapiro and colleagues, which includes dacluzimab, sirolimus, and tacrolimus.5 Blood samples were taken either from a central venous catheter or from a peripheral vein. The blood glucose concentration immediately (about 2 h) before each transplantation procedure was below 6 mmol/L. Laboratory procedures Antibodies to human TF (monoclonal antibodies 4509 and 4503 and polyclonal antibody 4502) were purchased from American Diagnostica (Greenwich, CT, USA). Monoclonal antibody 4509 inhibits TF activity, and 4503 recognises a non-functional epitope of TF. Site-inactivated factor VIIa was obtained by treatment of factor VIIa A

Islet transplantation The major inclusion criteria for the study were longstanding type 1 diabetes mellitus, frequent uncontrollable hypoglycaemic attacks and lack of awareness of these problems, and previous transplantation with a cadaver kidney graft to treat end-stage renal disease. Four C-peptide Thrombin-antithrombin

500

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Min Figure 1: Mean (and SE) concentrations of thrombinantithrombin complex and C-peptide after islet transplantation Before=samples obtained within 1 month before transplantation. *p<0·05 for comparison with 0 min values. Error bars represent SE.

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Figure 2: Expression of TF in human pancreatic islets shown by staining with monoclonal antibody 4509 to TF A: section of human pancreas, showing distinct staining of a pancreatic islet (arrow). B: section of an isolated human islet; TF is present in most of the cells in the islet.

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MECHANISMS OF DISEASE

A

kDa

Heavy chain Tissue factor

50 47 37

Light chain

25 1

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0·3 kb 0·2 kb

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Figure 3: Expression of TF of islet lysates A: lanes 1–6 represent three pairs of islets precipitated with monoclonal antibody 4503 (lanes 1, 3, 5) or 4509 (lanes 2, 4, 6); lane 7, monoclonal antibody 4509 without islets. B: RT-PCR of isolated human islets from six individuals (lanes 2–7) yields a 0·3 kb product (284 bp). Lane 1, molecular-weight standards; lane 8, positive control (human placenta); and lane 9, negative control (no DNA template).

and TF was measured with a commercial ELISA kit (Imubind Tissue Factor, American Diagnostica). For ultrastructural analysis, samples of normal pancreatic tissue from two male patients (who had undergone pancreatic resection due to pancreatic tumour) and isolated islets from two pancreas donors were sampled. None of the patients or donors had had any metabolic disease, and all pancreases were macroscopically and microscopically normal and showed no amyloid deposition. To preserve antigenicity, specimens were processed by the lowtemperature method.16 Ultra-thin sections placed on nickel grids were immunolabelled by the immunogold technique.17 Anti-TF monoclonal antibodies 4509 and 4503 and polyclonal antibody 4502 were used at a dilution of 1 in 25, and 10 nm or 15 nm colloidal gold particles were used as electron-dense markers. Polyclonal goat antibodies to mouse and rabbit immunoglobulins labelled with 10 nm and 15 nm gold (GAM-G10/15 and GAR-G 10/15) were purchased from Amersham International (Amersham, UK). Sections were treated with uranyl acetate and lead citrate before being examined in a Philips 201 electron microscope. Clotting time in plasma was measured in a four-channel free oscillating rheometer (ReoRox 4, Global Haemostasis Institute AB, Linköping, Sweden). To measure the IBMIR we used a modification of a tubing loops model previously described.9,18,19 Loops made of polyvinylchloride tubing (inner diameter 6·3 mm, length 390 mm) were treated with a Corline heparin surface (Corline, Uppsala, Sweden) according to the manufacturer’s recommendation.18 4 ␮L (about 4000 islet equivalent) islets (washed twice in CMRL 1066 [Connaught Medical Research Labs])20 were preincubated at room temperature for 10 min with either 15 ␮L monoclonal antibody 4509, control monoclonal antibody 4503, or phosphate-buffered saline. After three washing steps, the islets were resuspended in 150 ␮L CMRL 1066 and placed in the loops, and fresh ABO-compatible human blood (5 mL) was added. We also included a control loop containing blood supplemented with 150 ␮L CMRL 1066 but no islets. Also, 5 ␮L (about 5000 islet equivalent) islets were pretreated with site-inactivated factor VIIa (40 nmol/L) or phosphate-buffered saline before they were added to the tubing loops with 7 mL fresh human blood. To generate a blood flow of about 45 mL/min, we placed the loop devices on a rocker inside a 37ºC incubator for 30 min (monoclonal antibodies) or 60 min (inactivated factor VIIa). The mean glucose concentration in the blood was 5·3 mmol/L (SE 0·04). Blood samples were collected into EDTA (4·1 mmol/L final concentration) before

(NovoSeven, Novo Nordic, Denmark) with dansyl-GluGly-Arg chloromethyl ketone.14 For immunohistochemistry, pieces of whole pancreas (from 13 pancreases; not the same pancreases as those used for islet transplantation) and isolated islets (from seven donors) were collected in embedding medium (Tissue-Tek, Miles, Elkhart, IN, USA) and snap-frozen in liquid nitrogen. The samples were sectioned and stained with monoclonal antibody 4509 (1 in 50 dilution), followed by rabbit antibody to mouse immunoglobulin (1 in 25) and mouse peroxidase-anti-peroxidase (DAKO A/S, Glostrup, Denmark). For IMMUNOPRECIPITATION, 2000 islets were washed five times by centrifugation at 9 g at room temperature in phosphate-buffered saline containing 5 mmol/L EDTA (edetic acid), 10 mmol/L benzamidine, 0·1 g/L soybean trypsin inhibitor, and 1 mmol/L phenyl methyl sulfonyl fluoride. The pellet was incubated in 0·5 mL of the same buffer supplemented with 1% Triton X-100 (Sigma) at 37°C for 30 min. Thereafter, the cell debris was removed by centrifugation at 10 000 g for 5 min. 3 ␮g monoclonal antibody 4509 or 4503 was incubated with 250 ␮L of the cell lysate for 30 min at 37°C and precipitated with protein-G sepharose (Pharmacia Upjohn, Stockholm, Sweden). The samples were subjected to polyacrylamide gel electrophoresis in sodium dodecyl sulphate (12% by volume) and westernblot analysis with rabbit polyclonal antibody 4502 (5 mg/L) and horseradish-peroxidase-conjugated antibodies (2·5 mg/L) to rabbit immunoglobulins 100 nm 100 nm 100 nm (Dako A/S).15 100 nm C A B D To measure the islets were harvested on days 0, 2, and 7 and transferred into Figure 4: Electron micrographs showing representative results from immunogold phosphate-buffered saline containing labelling with monoclonal antibody 4509 of sections from isolated islets 5 mmol/L EDTA, 10 mmol/L benzami- Gold particles are 15 nm in diameter. A: TF molecules in smooth endoplasmic reticulum of a ␤ cell (⫻54 000). B: Golgi apparatus in an ␣ cell, with gold particles showing TF in the Golgi stacks dine, 0·1 g/L soybean trypsin inhibitor, (arrowheads), in transitory vesicles budded off from the Golgi trans region (large arrows), and in a and 1 mmol/L phenyl methyl sulfonyl secretory granule (small arrow) (⫻36 000). C: TF (arrows) storage in the core of the ␤-cell granules fluoride. The islets were homogenised, (⫻54 000). D: TF (arrows) storage randomly distributed in the ␣-cell granules (⫻36 000). THE LANCET • Vol 360 • December 21/28, 2002 • www.thelancet.com

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MECHANISMS OF DISEASE

A

PBS-treated islets Islets with antibody to non functional epitope of TF Islets treated with anti-TF to functional epitope Medium without islets

Platelets

60 250

Platelet number (⫻109/L)

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A: representative serial dilution of medium from one islet batch. B: 60 ␮L culture medium (from islets of three different individuals) treated with 15 ␮L phosphate-buffered saline, monoclonal antibody 4503 to a non-functional epitope of TF, or anti-TF monoclonal antibody 4509 (mean and SE). *p=0·005 for comparison with islets in phosphatebuffered saline alone.

perfusion and at 5, 15, 30, and 60 min after the start of perfusion. Platelets and leucocytes were counted with a Coulter AcT diff analyser (Beckman Coulter, FL, USA). Plasma concentrations of prothrombin fragments 1 and 2 and thrombin-antithrombin complex were quantified with commercial EIA kits (Enzygnost F1+2 and TAT, Dade Behring, Marburg, Germany). Plasma factor-XIaantithrombin complexes were quantified according to Sanchez and colleagues.21 ␤ thromboglobulin was analysed by Asserachrom (Diagnostica Stago, Asnières-sur-Seine, France). Complement activation products C3a and sC5b-9 were quantified as previously described.22,23 C-peptide was measured with an EIA (Mercodia, Uppsala, Sweden). Cytoplasmic RNA was isolated from islets.24 Singlestranded cDNAs were prepared by use of oligo (dT)

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FXIa-AT (␮mol/L)

Figure 5: Clotting time

0·75

0·50

0·25 *

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Figure 6: Blockade by anti-TF of IBMIR triggered by human islets PBS=phosphate-buffered saline. IBMIR monitored by platelet count and EIAs for thrombin-antithrombin complex and factor XIa-antithrombin (mean and SE). *p<0·05 for comparison with the loop containing islets alone.

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MECHANISMS OF DISEASE No islets (n=7)

Islets Untreated (n=7)

Blood cell counts (⫻109/L) Platelets Lymphocytes Monocytes Granulocytes Coagulation Thrombin-antithrombin (␮g/L) Prothrombin F1+2 (nmol/L) Factor XIa-antithrombin (mmol/L) ␤ thromboglobulin (IU/mL) Complement C3a (␮g/L)† sC5b-9 (AU/mL)†

Control anti-TF (n=7)

Inhibitory anti-TF (n=7)

160 (15) 1·8 (0·1) 0·3 (0·1) 3·4 (0·2)

12 (6·9) 1·8 (0·1) 0·2 (0·1) 2·0 (0·3)

24 (8·1) 1·7 (0·1) 0·3 (0·1) 2·5 (0·1)

100 (14)* 1·8 (0·1) 0·3 (0) 3·0 (0·2)*

83 (20) 5·8 (1·7) 0·1 (0) 1800 (300)

5600 (2300) 97 (1·7) 0·8 (0·3) 3700 (290)

2100 (430) 100 (31) 0·4 (0·1) 3400 (200)

870 (190)* 38 (5·6)* 0·2 (0) 2500 (240)*

1200 (190) 310 (84)

1200 (170) 330 (80)

770 (160) 220 (61)

600 (140) 140 (32)*

Data are mean (SE) at 30 min. *Significant difference for comparison with loop with untreated islets after 30 min (60 min for C3a and sC5b-9) of perfusion. †Data represent values after 60 min.

Blood cell counts, coagulation, and complement variables before and after human-islet perfusion with fresh ABO-compatible blood

priming (Amersham Pharmacia). PCR primers were combined to generate products spanning two exons25 of the TF transcript to amplify cDNA alone.1 RT-PCR was carried out at 95⬚C for 10 min to activate the polymerase, and amplification was done by 35 cycles of switching between 95⬚C for 15 s and 54⬚C for 60 s with high-fidelity PCR components (Expand, Boehringer-Mannheim, Germany), and the products were analysed on 3% agarose gels with 0·5 mg/L ethidium bromide. Statistical analysis Mean values were compared by Friedman’s ANOVA. Role of the funding source The sponsors had no role in study design, data collection, data analysis, data interpretation, or writing of the report.

Results Two of the patients became insulin independent shortly after a second transplant (one donor was used at each patient’s transplant). These patients are still off exogenous insulin therapy, with stable blood-glucose control. One patient has so far received only one islet transplant and is awaiting a second dose. The fourth patient who received islets from four donors, has stable blood-glucose control and requires about 20 U exogenous insulin per day (less than half the daily dose before transplantation). No clinical signs of portal thrombosis or general discomfort occurred in association with any of the islet transplantations. We monitored coagulation activation (thrombinantithrombin complex) and C-peptide concentrations in plasma before and after the infusion of purified human islets into the portal vein of patients undergoing islet transplantations (figure 1). Thrombin-antithrombin complex peaked after 15 min of infusion and thereafter began to taper off. After 1 day the concentrations were normal. C-peptide concentrations also increased by 15 min but peaked at 60 min or later. Staining of pancreatic sections with monoclonal antibody 4509 showed that TF was present in the islets of Langerhans in the pancreas (figure 2). Most, but not all, of the endocrine cells of the human islets of Langerhans were stained. The staining pattern suggested that TF was located in the granules. TF was also found in the adventitia of larger blood vessels. The endothelial cells were not stained, indicating that TF was not expressed in response to any inflammatory signals. No TF was found in the acinar cells of the exocrine pancreas. Also, in isolated islets from human pancreases, we found a similar distribution of TF, showing that expression was not affected by the isolation procedure. To confirm that TF was present in human islets, we

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immunoprecipitated the protein from lysates of pure islets with two different monoclonal antibodies to TF (figure 3). In parallel experiments, we immunoprecipitated TF with polyclonal antibody and identified the molecule with each of the two monoclonal antibodies (not shown). The polypeptide had a molecular mass of 47 kDa, identical to that of TF.4 We then calculated the amount of TF per cultured islet after quantification of TF in the lysates by ELISA. Immediately after islet isolation the TF concentration was 0·0024 pmol/␮g DNA (SE 0·0005), on day 2 0·0071 pmol/␮g DNA (SE 0·003), and on day 7 0·0037 pmol/␮g DNA (SE 0·0008; the differences were not significant, p=0·08). The corresponding insulin concentrations were 47 pmol/␮g DNA (SE 4), 32 (SE 15), and 26 (SE 10). We also confirmed the expression of TF at the mRNA level by use of RT-PCR on pure, hand-picked isolated islets (figure 3). We then examined in-situ islets from two healthy pancreases and two batches of isolated islets for the presence of TF by electron microscopy with the immunogold technique (figure 4). Ultrastructurally, the endocrine cells in all the examined islets were well preserved, although the contrast in the intracellular structures was not optimum because the tissues had not been treated with osmium. In pancreatic islets in situ and isolated islets, gold particles showed the presence of TF in both ␣ and ␤ cells, localised to the smooth endoplasmic reticulum, the Golgi stacks and transitory vesicles budding from the trans-Golgi stacks, and the secretory granules. TF was detected at a moderate concentration in the ␤-cell granules, particularly in the core, but at a higher concentration and randomly distributed throughout the matrix of the ␣-cell granules. We saw no TF immunoreactivity in the ␦ or pancreatic polypeptide cells, and all negative control labelling experiments were negative. We found procoagulant activity in the culture medium of isolated human islets when the medium was mixed with human plasma (figure 5). In the presence of culture medium, plasma clotted within 5 min and TF activity was dose dependent. Clotting activity was blocked by inhibitory monoclonal antibody 4509, but control monoclonal antibody 4503 had no effect. After ultracentrifugation of the medium at 100 000 g, the supernatants showed no clotting activity, but the pellet had twice the activity of the untreated culture medium (data not shown). Thus, TF activity is apparently associated with a high-molecular-weight fraction; since TF is a membrane-bound protein with a transmembrane region,4 the protein is likely to be associated with micro particles. Incubation of human islets of Langerhans with fresh human plasma that had no additives induced gelation (as

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MECHANISMS OF DISEASE

Platelets Islets with inactivated factor VIIa Islets without inactivated factor VIIa Medium without islets

Platelet number (⫻109/L)

300

* 200

*

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0 Thrombin-antithrombin

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TF (monoclonal antibody 4509), gelation was 5·6 times slower (after 53 min), compared with 1·8 times slower for the islets exposed to control monoclonal antibody. To investigate whether TF triggers the IBMIR, we perfused human islets with fresh human ABO-compatible blood in the tubing loop model9 for 30 min (figure 6). In the control samples (blood with islets alone or with the noninhibitory anti-TF (4503), clotting occurred within 15 min; however, with the inhibitory anti-TF (4509), clotting was inhibited over the whole observation period. This difference was reflected in the consumption of platelets (platelet count), in the release of platelet ␣-granule content (␤ thromboglobulin), and in the generation of thrombinantithrombin complex, prothrombin fragment 1+2, and factor XIa-antithrombin complexes, all of which were suppressed by monoclonal antibody 4509 but not by 4503 (figure 6; table). An even more pronounced inhibition of IBMIR was obtained with site-inactivated factor VIIa, an efficient inhibitor of TF activity. Blood containing this inhibitor completely stopped the fall in platelet count and the increases in thrombin-antithrombin complex, factor XIa-antithrombin, and C3a (figure 7). The effect of these two inhibitors strongly indicates that TF is the trigger of the IBMIR.

Discussion TAT (␮g/L)

10 000 8000 6000 4000 2000 *

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Figure 7: Blockade by site-inactivated factor VIIa of IBMIR triggered by human islets *p<0·05 for comparison with the loop without inactivated factor VIIa.

monitored by viscosimetry) after about 9·5 min, whereas the buffer alone induced no clotting after 60 min; thus, the islets were able to induce coagulation activation (n=7; not shown). To link the expression of TF in the islets with the procoagulant activity, we attempted to block the gelation with antibodies to TF. In the presence of an inhibitory anti-

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Our results show that IBMIR occurs regularly in vivo during the transplantation of pancreatic islets, even without clinical signs of intraportal thrombosis. TF was produced and secreted by the endocrine cells of the islets of Langerhans in the islet preparations. In vitro, IBMIR was inhibited by anti-TF and the potential TF-inhibitory drug site-inactivated factor VIIa; these findings show that IBMIR is triggered by TF. The immunoprecipitation, RT-PCR, and electron-microscopic findings point to expression of TF by both the ␣ and ␤ cells but not by ␦ or pancreatic polypeptide cells. Furthermore, the TF activity found in culture medium showed that the agent is released from the islets bound to micro particles. The localisation to granules in the ␣ and ␤ cells showed that TF is released together with insulin and glucagon. The mechanisms that regulate its synthesis are, however, unknown. The fact that the IBMIR is triggered by TF, taken together with the recent finding that most of the process of IBMIR is driven and amplified by thrombin,10 allows us to propose a hypothesis to explain how this reaction might move forward. After the initial generation of thrombin by islet-expressed TF, thrombin-activated platelets start to bind to the islet surface. The ligand to which the platelets bind on the islet surface is still unidentified, but collagen is one likely candidate that surrounds human islets.26 Via the amplification loop involving factor XI (factor XIaantithrombin) and activated platelets,27 more thrombin is formed, generating a fibrin capsule surrounding the islets. This binding is followed by a rapid loss of platelets from the blood. IBMIR occurs in clinical islet transplantation as shown by an increase in concentrations of thrombin-antithrombin complex immediately after islet infusion. The in-vivo concentrations are much lower than those in the closed tubing loop model but they are of the same order of magnitude as those found in patients undergoing orthopaedic surgery and in patients with sepsis and thromboembolism.28,29 The increased C-peptide concentrations verify liberation of insulin from the transplanted islets and suggest that the islets were affected by the blood contact similarly to our findings in vitro. This idea also accords with our previous studies in an allogeneic porcine islet transplantation model; we found that the islets were

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MECHANISMS OF DISEASE

morphologically damaged by the transplantation procedure. No other signs, such as increased intraportal pressure, were observed in the pigs. However, several groups have reported increased intraportal pressure and even portal thrombosis in association with clinical islet transplantation.7,8 Even without clinical signs, intraportal thrombosis can occur since the thrombus does not originate from the vessel wall but from the transplanted islets and is therefore not occlusive. There is therefore a need to control IBMIR in clinical islet transplantation. Inhibition of islet-bound TF activity before transplantation with monoclonal antibodies or treatment of the patient with site-inactivated factor VIIa might prevent IBMIR. Pretreatment protocols involving agents that can block TF expression or synthesis (eg, antisense reagents30) will be developed in the near future. Pretreatment of the islets before transplantation would have clear clinical benefit, since, by contrast with systemic inhibition, it would have no adverse effect on haemostasis in the recipient. Our findings have implications for several diabetesrelated disorders. In particular, the finding that IBMIR is initiated by TF and consistently occurs in clinical islet transplantation without clear clinical signs of intraportal thrombosis shows that inhibition of the process might increase the success rate of clinical islet transplantation and reduce the number of donors needed for each patient. Contributors L Moberg was responsible for conduct of the study, including compilation of data and statistical analysis, and writing the report. H Johansson did the viscosimetry analyses and the iFVIIa study. A Lukinius was responsible for electron microscopy. K Nilsson Ekdahl carried out immunoprecipitation and western-blot studies. G Elgue undertook EIAs. C Berne, A Foss, R Källen, Ø Østraat, K Salmela, A Tibell, and G Tufveson are members of the Nordic network for islet transplantation and were responsible for procurement and delivery of human pancreases to Uppsala and for the clinical islet transplantations including collection of blood samples. O Korsgren and B Nilsson led the research groups, supervised the study, and wrote the report. All the investigators contributed to critical revision of the final version of the paper.

Conflict of interest statement

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None declared.

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Acknowledgments

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We thank Margareta Engkvist, Selina Parvin, and Ulrika Johansson for technical assistance and Deborah McClellan for editing the text. This study was supported by grants from the Juvenile Diabetes Foundation International, the Swedish Research Council (16P-13568, 16X-12219, 16X-5674, 72X-06817-19), the Åke Wiberg Foundation, the Nordic Insulin Fund, the Novo Nordisk Foundation, the Torsten and Ragnar Söderbergs Foundation, the Ernfors Family Fund, Barn Diabetes Fonden, the Swedish Diabetes Association, the Clas Groschinsky Foundation, and the Knut and Alice Wallenberg Foundation.

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References 1

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