Glucocorticoids and glucolipotoxicity alter the DNA methylome and function of human EndoC-ßH1 cells.

AIMS: Synthetic glucocorticoids, including dexamethasone (DEX), are clinically prescribed due to their immunoregulatory properties. In excess they can perturb glucose homeostasis, with individuals predisposed to glucose intolerance more sensitive to these negative effects. While DEX is known to negatively impact ß-cell function, it is unclear how. Hence, our aim was to investigate the effect of DEX on ß-cell function, both alone and in combination with a diabetogenic milieu in the form of elevated glucose and palmitate. MAIN METHODS: Human pancreatic EndoC-ßH1 cells were cultured in the presence of high glucose and palmitate (glucolipotoxicity) and/or a pharmacological concentration of DEX, before functional and molecular analyses. KEY FINDINGS: Either treatment alone resulted in reduced insulin content and secretion, while the combination of DEX and glucolipotoxicity promoted a strong synergistic effect. These effects were associated with reduced insulin biosynthesis, likely due to downregulation of PDX1, MAFA, and the proinsulin converting enzymes, as well as reduced ATP response upon glucose stimulation. Genome-wide DNA methylation analysis found changes on PDE4D, MBNL1 and TMEM178B, all implicated in ß-cell function, after all three treatments. DEX alone caused very strong demethylation of the glucocorticoid-regulated gene ZBTB16, also known to influence the ß-cell, while the combined treatment caused altered methylation of many known ß-cell regulators and diabetes candidate genes. SIGNIFICANCE: DEX treatment and glucolipotoxic conditions separately alter the ß-cell epigenome and function. The combination of both treatments exacerbates these changes, showing that caution is needed when prescribing potent glucocorticoids in patients with dysregulated metabolism.

Insulin receptor activation by proinsulin preserves synapses and vision in retinitis pigmentosa.

Synaptic loss, neuronal death, and circuit remodeling are common features of central nervous system neurodegenerative disorders. Retinitis pigmentosa (RP), the leading cause of inherited blindness, is a group of retinal dystrophies characterized by photoreceptor dysfunction and death. The insulin receptor, a key controller of metabolism, also regulates neuronal survival and synaptic formation, maintenance, and activity. Indeed, deficient insulin receptor signaling has been implicated in several brain neurodegenerative pathologies. We present evidence linking impaired insulin receptor signaling with RP. We describe a selective decrease in the levels of the insulin receptor and its downstream effector phospho-S6 in retinal horizontal cell terminals in the rd10 mouse model of RP, as well as aberrant synapses between rod photoreceptors and the postsynaptic terminals of horizontal and bipolar cells. A gene therapy strategy to induce sustained proinsulin, the insulin precursor, production restored retinal insulin receptor signaling, by increasing S6 phosphorylation, without peripheral metabolic consequences. Moreover, proinsulin preserved photoreceptor synaptic connectivity and prolonged visual function in electroretinogram and optomotor tests. These findings point to a disease-modifying role of insulin receptor and support the therapeutic potential of proinsulin in retinitis pigmentosa.

Effect of oral nitrite administration on gene expression of SNARE proteins involved in insulin secretion from pancreatic islets of male type 2 diabetic rats.

BACKGROUND: Nitrite stimulates insulin secretion from pancreatic ß-cells; however, the underlying mechanisms have not been completely addressed. The aim of this study is to determine effect of nitrite on gene expression of SNARE proteins involved in insulin secretion from isolated pancreatic islets in Type 2 diabetic Wistar rats. METHODS: Three groups of rats were studied (n = 10/group): Control, diabetes, and diabetes + nitrite, which treated with sodium nitrite (50 mg/L) for 8 weeks. Type 2 diabetes was induced using a low-dose of streptozotocin (25 mg/kg) combined with high-fat diet. At the end of the study, pancreatic islets were isolated and mRNA expressions of interested genes were measured; in addition, protein expression of proinsulin and C-peptide in pancreatic tissue was assessed using immunofluorescence staining. RESULTS: Compared with controls, in the isolated pancreatic islets of Type 2 diabetic rats, mRNA expression of glucokinase (59%), syntaxin1A (49%), SNAP25 (70%), Munc18b (48%), insulin1 (56%), and insulin2 (52%) as well as protein expression of proinsulin and C-peptide were lower. In diabetic rats, nitrite administration significantly increased gene expression of glucokinase, synaptotagmin III, syntaxin1A, SNAP25, Munc18b, and insulin genes as well as increased protein expression of proinsulin and C-peptide. CONCLUSION: Stimulatory effect of nitrite on insulin secretion in Type 2 diabetic rats is at least in part due to increased gene expression of molecules involved in glucose sensing (glucokinase), calcium sensing (synaptotagmin III), and exocytosis of insulin vesicles (syntaxin1A, SNAP25, and Munc18b) as well as increased expression of insulin genes.

Enhanced insulin receptor interaction by a bifunctional insulin-transferrin fusion protein: an approach to overcome insulin resistance.

Bifunctional fusion protein design has been widely utilized as a strategy to increase the efficacy of protein therapeutics. Previously, we proposed a novel application of the bifunctional fusion protein design through the introduction of proinsulin-transferrin (ProINS-Tf) fusion protein as a liver-specific protein prodrug to achieve a glucose-lowering effect in type 1 diabetic mice. In this report, we studied the binding characteristics of this activated fusion protein to the insulin receptor to elucidate its mechanism in eliciting insulin receptor-mediated signaling. We found that, with the assistance of the transferrin moiety binding to the transferrin receptor, the activated ProINS-Tf exhibited significantly higher binding affinity to the insulin receptor compared with the native insulin, resulting in a prolonged and stronger Akt phosphorylation. This enhanced induction by activated ProINS-Tf overcame insulin resistance in palmitate-treated HepG2 cells. ProINS-Tf also demonstrated a better glucose-lowering effect than native insulin, even with a much lower dose and less frequent injections, in non-obese diabetic mice with insulin resistance symptoms. The activated ProINS-Tf, serving as a bivalent protein molecule, could be a new insulin analog to overcome insulin resistance, which is associated with several diseases, including type 2 diabetes and non-alcoholic fatty liver disease.

Maternal Type 1 Diabetes Reduces Autoantigen-Responsive CD4+ T Cells in Offspring.

Autoimmunity against pancreatic ß-cell autoantigens is a characteristic of childhood type 1 diabetes (T1D). Autoimmunity usually appears in genetically susceptible children with the development of autoantibodies against (pro)insulin in early childhood. The offspring of mothers with T1D are protected from this process. The aim of this study was to determine whether the protection conferred by maternal T1D is associated with improved neonatal tolerance against (pro)insulin. Consistent with improved neonatal tolerance, the offspring of mothers with T1D had reduced cord blood CD4+ T-cell responses to proinsulin and insulin, a reduction in the inflammatory profile of their proinsulin-responsive CD4+ T cells, and improved regulation of CD4+ T cell responses to proinsulin at 9 months of age, as compared with offspring with a father or sibling with T1D. Maternal T1D was also associated with a modest reduction in CpG methylation of the INS gene in cord blood mononuclear cells from offspring with a susceptible INS genotype. Our findings support the concept that a maternal T1D environment improves neonatal immune tolerance against the autoantigen (pro)insulin.

Intravitreal Injection of Proinsulin-Loaded Microspheres Delays Photoreceptor Cell Death and Vision Loss in the rd10 Mouse Model of Retinitis Pigmentosa.

PURPOSE: The induction of proinsulin expression by transgenesis or intramuscular gene therapy has been shown previously to retard retinal degeneration in mouse and rat models of retinitis pigmentosa (RP), a group of inherited conditions that result in visual impairment. We investigated whether intraocular treatment with biodegradable poly (lactic-co-glycolic) acid microspheres (PLGA-MS) loaded with proinsulin has cellular and functional neuroprotective effects in the retina. METHODS: Experiments were performed using the Pde6brd10 mouse model of RP. Methionylated human recombinant proinsulin (hPI) was formulated in PLGA-MS, which were administered by intravitreal injection on postnatal days (P) 14 to 15. Retinal neuroprotection was assessed at P25 by electroretinography, and by evaluating outer nuclear layer (ONL) cellular preservation. The attenuation of photoreceptor cell death by hPI was determined by TUNEL assay in cultured P22 retinas, as well as Akt phosphorylation by immunoblotting. RESULTS: We successfully formulated hPI PLGA-MS to deliver the active molecule for several weeks in vitro. The amplitude of b-cone and mixed b-waves in electroretinographic recording was significantly higher in eyes injected with hPI-PLGA-MS compared to control eyes. Treatment with hPI-PLGA-MS attenuated photoreceptor cell loss, as revealed by comparing ONL thickness and the number of cell rows in this layer in treated versus untreated retinas. Finally, hPI prevented photoreceptor cell death and increased AktThr308 phosphorylation in organotypic cultured retinas. CONCLUSIONS: Retinal degeneration in the rd10 mouse was slowed by a single intravitreal injection of hPI-PLGA-MS. Human recombinant proinsulin elicited a rapid and effective neuroprotective effect when administered in biodegradable microspheres, which may constitute a future potentially feasible delivery method for proinsulin-based treatment of RP.

Combination of worm antigen and proinsulin prevents type 1 diabetes in NOD mice after the onset of insulitis.

Animal studies demonstrated that administration of helminth products can protect from autoimmune diseases. However, the success of such administrations is limited in the case of type 1 diabetes, as protection is only provided if the administration is started before the development of insulitis. In this study we investigated whether inclusion of helminth antigen administrations to an antigen-specific treatment with proinsulin improves the protective effect by triggering non-specific regulatory immune responses. Using a combination therapy of intraperitoneal Litomosoides sigmodontis antigen and intranasal pro-insulin, onset of diabetes was prevented in NOD mice after insulitis started, while either administration alone failed to protect. This protection was associated with an increased frequency of regulatory T cells within the pancreatic lymph nodes and a reduced inflammation of the pancreatic islets. This suggests that inclusion of helminth antigens improve the protective effect provided by antigen-specific therapies and represent a new potential therapeutic approach against autoimmune diseases.

Proinsulin multi-peptide immunotherapy induces antigen-specific regulatory T cells and limits autoimmunity in a humanized model.

Peptide immunotherapy (PIT) is a targeted therapeutic approach, involving administration of disease-associated peptides, with the aim of restoring antigen-specific immunological tolerance without generalized immunosuppression. In type 1 diabetes, proinsulin is a primary antigen targeted by the autoimmune response, and is therefore a strong candidate for exploitation via PIT in this setting. To elucidate the optimal conditions for proinsulin-based PIT and explore mechanisms of action, we developed a preclinical model of proinsulin autoimmunity in a humanized HLA-DRB1*0401 transgenic HLA-DR4 Tg mouse. Once proinsulin-specific tolerance is broken, HLA-DR4 Tg mice develop autoinflammatory responses, including proinsulin-specific T cell proliferation, interferon (IFN)-γ and autoantibody production. These are preventable and quenchable by pre- and post-induction treatment, respectively, using intradermal proinsulin-PIT injections. Intradermal proinsulin-PIT enhances proliferation of regulatory [forkhead box protein 3 (FoxP3(+))CD25(high) ] CD4 T cells, including those capable of proinsulin-specific regulation, suggesting this as its main mode of action. In contrast, peptide delivered intradermally on the surface of vitamin D3-modulated (tolerogenic) dendritic cells, controls autoimmunity in association with proinsulin-specific IL-10 production, but no change in regulatory CD4 T cells. These studies define a humanized, translational model for in vivo optimization of PIT to control autoimmunity in type 1 diabetes and indicate that dominant mechanisms of action differ according to mode of peptide delivery.

Proinsulin-transferrin fusion protein as a novel long-acting insulin analog for the inhibition of hepatic glucose production.

Proinsulin-transferrin (ProINS-Tf) fusion protein was evaluated for its in vivo pharmacokinetics, efficacy, and mechanism. Our previous studies have shown that ProINS-Tf was converted to active insulin-transferrin (INS-Tf) via the transferrin (Tf)-receptor-mediated pathway in hepatoma cells. We hypothesized that this fusion protein can be administered as a prodrug and be converted to a biologically active protein with specificity for the liver versus other insulin (INS)-sensitive tissues (muscle and adipose). Administration as an inactive prodrug with liver-specific action compared with other INS-sensitive tissues conceivably reduces negative side effects seen with other INS analogs. In this report, the data show that ProINS-Tf exhibited a slow, but sustained, in vivo hypoglycemic efficacy and long plasma half-life. The fusion protein showed activity in the liver, as evidenced by decreased expression of two key hepatic glucose production (HGP) enzymes, PEPCK and glucose-6-phosphatase, and increased glycogen levels under feeding conditions. Furthermore, the INS receptor (IR) phosphorylation (activation) in liver and muscle tissues was compared with postinjection of INS or ProINS-Tf. While INS activated IR in both the liver and muscle, ProINS-Tf only showed activation in the liver. Thus, ProINS-Tf fusion protein can potentially be administered as a prodrug with sustained Tf-mediated activation and selectivity in inhibiting HGP.

T-lymphocyte recognition of beta cells in type 1 diabetes: clinical perspectives.

Type 1 diabetes (T1D) is an autoimmune disease characterized by the activation of lymphocytes against pancreatic ß cells. Landmarks in the history of T1D were the description of insulitis and of islet cell autoantibodies, and report an association between T1D and a limited number of HLA alleles. Another step was the study of T-lymphocytes, now known to be central to the disease process of T1D whether in mice or men. In humans, T-lymphocytes, and especially CD8⁺ T-cells, are predominant in insulitis. The characterization of antigenic fragments--peptides--recognized by T-cells paves the way towards new assays for predicting T1D and its prevention using antigen- or peptide-specific immunotherapy, while avoiding side effects that may counteract the limited efficacy of immunosuppression and immunomodulation in preserving ß-cells from autoimmune destruction in recent-onset T1D patients. The current need for new preclinical models for testing strategies of antigen-specific immune tolerance is also highlighted.

Proinsulin slows retinal degeneration and vision loss in the P23H rat model of retinitis pigmentosa.

Proinsulin has been characterized as a neuroprotective molecule. In this work we assess the therapeutic potential of proinsulin on photoreceptor degeneration, synaptic connectivity, and functional activity of the retina in the transgenic P23H rat, an animal model of autosomal dominant retinitis pigmentosa (RP). P23H homozygous rats received an intramuscular injection of an adeno-associated viral vector serotype 1 (AAV1) expressing human proinsulin (hPi+) or AAV1-null vector (hPi-) at P20. Levels of hPi in serum were determined by enzyme-linked immunosorbent assay (ELISA), and visual function was evaluated by electroretinographic (ERG) recording at P30, P60, P90, and P120. Preservation of retinal structure was assessed by immunohistochemistry at P120. Human proinsulin was detected in serum from rats injected with hPi+ at all times tested, with average hPi levels ranging from 1.1 nM (P30) to 1.4 nM (P120). ERG recordings showed an amelioration of vision loss in hPi+ animals. The scotopic b-waves were significantly higher in hPi+ animals than in control rats at P90 and P120. This attenuation of visual deterioration correlated with a delay in photoreceptor degeneration and the preservation of retinal cytoarchitecture. hPi+ animals had 48.7% more photoreceptors than control animals. Presynaptic and postsynaptic elements, as well as the synaptic contacts between photoreceptors and bipolar or horizontal cells, were preserved in hPi+ P23H rats. Furthermore, in hPi+ rat retinas the number of rod bipolar cell bodies was greater than in control rats. Our data demonstrate that hPi expression preserves cone and rod structure and function, together with their contacts with postsynaptic neurons, in the P23H rat. These data strongly support the further development of proinsulin-based therapy to counteract retinitis pigmentosa.

Proinsulin binds with high affinity the insulin receptor isoform A and predominantly activates the mitogenic pathway.

Proinsulin is generally regarded as an inactive prohormone because of its low metabolic activity. However, proinsulin appears to regulate embryo development in animal models. In this study, we evaluated whether proinsulin may differentially bind to and activate the two insulin receptor (IR) isoforms (IR-A and IR-B), because IR-A is a relatively low-specificity receptor that is prevalent in fetal and cancer cells and is able to mediate the growth effects of IGF-II. Mouse R(-) fibroblasts devoid of IGF-I receptor (IGF-IR) and stably transfected with cDNA encoding either human IR-A or IR-B (R(-) /IR-A and R(-) /IR-B cells) were used. Three human cancer cell lines were also studied. We found that proinsulin stimulated phosphorylation of IR-A with an EC(50) of 4.5 ± 0.6 nm and displaced [(125)I]insulin from IR-A with a similar EC(50). In contrast, proinsulin EC(50) values for stimulation of IR-B phosphorylation and for [(125)I]insulin displacement from IR-B were approximately 7-fold higher. Proinsulin did not bind or activate IGF-IR or IR/IGF-IR hybrids. Via IR-A, proinsulin activated the ERK/p70S6K pathway to a similar degree as insulin but elicited a weaker Akt response. Despite its low metabolic activity, proinsulin was almost equipotent as insulin in inducing cell proliferation and migration in cells expressing various IR-A levels. In conclusion, proinsulin is a selective IR-A ligand and may induce biological effects through this IR isoform.

Regulation of cathepsin G reduces the activation of proinsulin-reactive T cells from type 1 diabetes patients.

Autoantigenic peptides resulting from self-proteins such as proinsulin are important players in the development of type 1 diabetes mellitus (T1D). Self-proteins can be processed by cathepsins (Cats) within endocytic compartments and loaded to major histocompatibility complex (MHC) class II molecules for CD4(+) T cell inspection. However, the processing and presentation of proinsulin by antigen-presenting cells (APC) in humans is only partially understood. Here we demonstrate that the processing of proinsulin by B cell or myeloid dendritic cell (mDC1)-derived lysosomal cathepsins resulted in several proinsulin-derived intermediates. These intermediates were similar to those obtained using purified CatG and, to a lesser extent, CatD, S, and V in vitro. Some of these intermediates polarized T cell activation in peripheral blood mononuclear cells (PBMC) from T1D patients indicative for naturally processed T cell epitopes. Furthermore, CatG activity was found to be elevated in PBMC from T1D patients and abrogation of CatG activity resulted in functional inhibition of proinsulin-reactive T cells. Our data suggested the notion that CatG plays a critical role in proinsulin processing and is important in the activation process of diabetogenic T cells.

Receptor-mediated activation of a proinsulin-transferrin fusion protein in hepatoma cells.

A proinsulin-transferrin (ProINS-Tf) recombinant fusion protein was designed and characterized for the sustained release of an active form of insulin (INS) by hepatoma cells. During incubation with H4IIE hepatoma cells, a gradual decline of ProINS-Tf concentration, with a concomitant generation of the immuno-reactive insulin-transferrin (irINS-Tf), was detected in the culture medium by using INS- or proinsulin (ProINS)-specific radioimmunoassay (RIA) system. Further studies indicated that the conversion of ProINS-Tf to irINS-Tf was a transferrin receptor (TfR) mediated process that was pH-sensitive, and temperature- and microtubule-dependent. These results suggest that the conversion occurred during the slow recycling route of transferrin (Tf)-TfR pathway, possibly processed by proteases in the slow recycling compartments juxtaposed to the trans-Golgi network (TGN). ProINS-Tf exhibited little activity in the short-term promotion of glucose uptake in adipocytes, indicating that it was in an inactive form similar to ProINS. Stimulation of Akt phosphorylation by ProINS-Tf was detected only after prolonged incubation with H4IIE cells. On the other hand, ProINS-Tf pre-incubated with H4IIE cells for 24h acquired an immediate activity of stimulating Akt phosphorylation. Furthermore, ProINS-Tf elicited a strong activity in the inhibition of glucose production following 24h incubation with H4IIE cells. Based on these findings, we conclude that the Tf-TfR endocytosis and recycling pathway enables the conversion and release of ProINS-Tf in an active form of irINS-Tf. Results from this study suggest that the Tf-TfR pathway can be exploited for the design of prohormone-Tf fusion proteins as protein prodrugs for their sustained and targeted activation.

Suppression of dendritic cell activation by diabetes autoantigens linked to the cholera toxin B subunit.

Antigen presenting cells, specifically dendritic cells (DCs) are a focal point in the delicate balance between T cell tolerance and immune responses contributing to the onset of type I diabetes (T1D). Weak adjuvant proteins like the cholera toxin B subunit when linked to autoantigens may sufficiently alter the balance of this initial immune response to suppress the development of autoimmunity. To assess adjuvant enhancement of autoantigen mediated immune suppression of Type 1 diabetes, we examined the cholera toxin B subunit (CTB)-proinsulin fusion protein (CTB-INS) activation of immature dendritic cells (iDC) at the earliest detectable stage of the human immune response. In this study, Incubation of human umbilical cord blood monocyte-derived immature DCs with CTB-INS autoantigen fusion protein increased the surface membrane expression of DC Toll-like receptor (TLR-2) while no significant upregulation in TLR-4 expression was detected. Inoculation of iDCs with CTB stimulated the biosynthesis of both CD86 and CD83 co-stimulatory factors demonstrating an immunostimulatory role for CTB in both DC activation and maturation. In contrast, incubation of iDCs with proinsulin partially suppressed CD86 co-stimulatory factor mediated DC activation, while incubation of iDCs with CTB-INS fusion protein completely suppressed iDC biosynthesis of both CD86 and CD83 costimulatory factors. The incubation of iDCs with increasing amounts of insulin did not increase the level of immune suppression but rather activated DC maturation by stimulating increased biosynthesis of both CD86 and CD83 costimulatory factors. Inoculation of iDCs with CTB-INS fusion protein dramatically increased secretion of the immunosuppressive cytokine IL-10 and suppressed synthesis of the pro-inflammatory cytokine IL12/23 p40 subunit protein suggesting that linkage of CTB to insulin (INS) may play an important role in mediating DC guidance of cognate naïve Th0 cell development into immunosuppressive T lymphocytes. Taken together, the experimental data suggests Toll like receptor 2 (TLR-2) plays a dominant role in CTB mediated INS inhibition of DC induced type 1 diabetes onset in human Type 1 diabetes autoimmunity. Further, fusion of CTB to the autoantigen was found to be essential for enhancement of immune suppression as co-delivery of CTB and insulin did not significantly inhibit DC costimulatory factor biosynthesis. The experimental data presented supports the hypotheses that adjuvant enhancement of autoantigen mediated suppression of islet beta cell inflammation is dependent on CTB stimulation of dendritic cell TLR2 receptor activation and co-processing of both CTB and the autoantigen in the same dendritic cell.

Retuning the immune system: the future of type 1 diabetes therapy?

Strategies that restore immune tolerance are attracting growing interest in their potential to advance the treatment of type 1 diabetes beyond lifelong insulin therapy.

Autoimmunity to both proinsulin and IGRP is required for diabetes in nonobese diabetic 8.3 TCR transgenic mice.

T cells specific for proinsulin and islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) induce diabetes in nonobese diabetic (NOD) mice. TCR transgenic mice with CD8(+) T cells specific for IGRP(206-214) (NOD8.3 mice) develop accelerated diabetes that requires CD4(+) T cell help. We previously showed that immune responses against proinsulin are necessary for IGRP(206-214)-specific CD8(+) T cells to expand. In this study, we show that diabetes development is dramatically reduced in NOD8.3 mice crossed to NOD mice tolerant to proinsulin (NOD-PI mice). This indicates that immunity to proinsulin is even required in the great majority of NOD8.3 mice that have a pre-existing repertoire of IGRP(206-214)-specific cells. However, protection from diabetes could be overcome by inducing islet inflammation either by a single dose of streptozotocin or anti-CD40 agonist Ab treatment. This suggests that islet inflammation can substitute for proinsulin-specific CD4(+) T cell help to activate IGRP(206-214)-specific T cells.

Proinsulin C-peptide and insulin: Limited pattern similarities of interest in inter-peptide interactions but no C-peptide effect on insulin and IGF-1 receptor signaling.

The recently reported influence of proinsulin C-peptide on insulin prompted us to examine structural features of the C-peptide. Four sets of limited pattern similarities towards inter-chain end regions of insulin were noticed, involving secondary structure elements, binding residues and intra- as well as inter-peptide residue similarities. Using surface plasmon resonance, we examined insulin binding to truncated, soluble insulin receptor A and IGF-1 receptor, but C-peptide effects on these bindings were not detectable. Two forms of the insulin receptor, differing in activation of gene transcription with regards to (pre)proinsulin and glucokinase, respectively, were also uninfluenced by C-peptide. We conclude that the pattern similarities, if functional, reflect C-peptide interactions with molecules other than both insulin A and B receptors and IGF-1 receptors. Any such effects are of interest in relation to reported binding interactions between insulin and C-peptide.

Inhibition of platelet aggregation of a mutant proinsulin chimera engineered by introduction of a native Lys-Gly-Asp-containing sequence.

An eight amino acid sequence, CAKGDWNC, from disintegrin barbourin, was introduced into an inactive human proinsulin molecule between the B28 and A2 sites to construct a chimeric, anti-thrombosis recombinant protein. The constructed Lys-Gly-Asp (KGD)-proinsulin gene was expressed in Escherichia coli and then purified. The KGD-proinsulin chimera protein inhibits human platelet aggregation, induced by ADP, with an IC50 value (molar concentration causing 50% inhibition of platelet aggregation) of 830 nM: and demonstrates also specific affinity to glycoprotein IIb/IIIa receptor. Its insulin receptor binding activity remains as low as 0.04% with native insulin as a control.