Adhering to adhesion: assessing integrin conformation to monitor T cells.

Monitoring T cells is of major importance for the development of immunotherapies. Recent sophisticated assays can address particular aspects of the anti-tumor T-cell repertoire or support very large-scale immune screening for biomarker discovery. Robust methods for the routine assessment of the quantity and quality of antigen-specific T cells remain, however, essential. This review discusses selected methods that are commonly used for T-cell monitoring and summarizes the advantages and limitations of these assays. We also present a new functional assay, which specifically detects activated ß2 integrins within a very short time following CD8+ T-cell stimulation. Because of its unique and favorable characteristics, this assay could be useful for implementation into our T-cell monitoring toolbox.

ITGA2 as a potential nanotherapeutic target for glioblastoma.

High grade gliomas, including glioblastoma (GBM), are the most common and deadly brain cancers in adults. Here, we performed a quantitative and unbiased screening of 70 cancer-related antigens using comparative flow cytometry and, for the first time, identified integrin alpha-2 (ITGA2) as a novel molecular target for GBM. In comparison to epidermal growth factor receptor (EGFR), a well-established GBM target, ITGA2 is significantly more expressed on human GBM cells and significantly less expressed on normal human glial cells. We also found that ITGA2 antibody blockade significantly impedes GBM cell migration but not GBM cell proliferation. To investigate the utility of ITGA2 as a therapeutic target in GBM, we designed and engineered an ITGA2 antibody-directed liposome that can selectively deliver doxorubicin, a standard-of-care chemotherapeutic agent, to GBM cells. This novel approach significantly improved antitumor efficacy. We also demonstrated that these ITGA2 antibody-directed liposomes can effectively breach the blood-brain tumor barrier (BBTB) in vitro via GBM-induced angiogenesis effects. These findings support further research into the use of ITGA2 as a novel nanotherapeutic target for GBM.

VLA-2 blockade in vivo by vatelizumab induces CD4+FoxP3+ regulatory T cells.

Integrin α2ß1, also known as very late antigen (VLA)-2, is a collagen-binding molecule expressed constitutively on platelets. Vatelizumab, a monoclonal antibody targeting the α2 subunit (CD49b) of VLA-2, was recently investigated for its safety and efficacy during a Phase 2 clinical study in multiple sclerosis patients, as integrin-mediated collagen binding at the site of inflammation is central to a number of downstream pro-inflammatory events. In the course of this study, we could show that VLA-2 is expressed ex vivo on platelets, platelet-T-cell aggregates, as well as a small population of highly activated memory T cells. Even though the clinical trial did not meet its primary clinical end-point (reduction in the cumulative number of new contrast-enhancing lesions on magnetic resonance imaging (MRI)), we observed enhanced frequencies of regulatory T cells (TREG) following vatelizumab treatment. Elevated TREG frequencies might be explained by the inhibition of p38 mitogen-activated protein kinase (MAPK) signaling, which is critically involved in the polarization of T helper 17 (TH17) cells and is activated by the α2 integrin cytoplasmic domain. Our findings suggest that blockade of VLA-2 might be a way to safely shift the TH17/TREG balance by inducing TREGin vivo.

Enhanced Cell Division Is Required for the Generation of Memory CD4 T Cells to Migrate Into Their Proper Location.

CD4 T cell memory is fundamental for long-lasting immunity and effective secondary responses following infection or vaccination. We have previously found that memory CD4 T cells specific for systemic antigens preferentially reside in the bone marrow (BM) and arise from splenic CD49b+T-bet+ CD4 T cells. However, how BM-homing memory precursors are generated during an immune reaction is unknown. We show here that BM memory precursors are generated via augmented rates of cell division throughout a primary immune response. Treatment with the cytostatic drug cyclophosphamide or blockade of the CD28/B7 co-stimulatory pathway at the beginning of the contraction phase abrogates the generation of BM memory precursors. We determine that, following a critical number of cell divisions, memory precursors downregulate CCR7 and upregulate IL-2Rß, indicating that loss of CCR7 and gain of IL-2 signal are required for the migration of memory precursors toward the BM.

A unique phenotype of acquired Glanzmann thrombasthenia due to non-function-blocking anti-αIIbß3 autoantibodies.

Essentials Acquired Glanzmann thrombasthenia (aGT) is generally caused by function-blocking antibodies (Abs). We demonstrated a unique aGT case due to marked reduction of αIIbß3 with anti-αIIbß3 Abs. The anti-αIIbß3 Abs of the patient did not inhibit platelet function but reduced surface αIIbß3. Internalization of αIIbß3 induced by the Abs binding may be responsible for the phenotype. SUMMARY: Background Acquired Glanzmann thrombasthenia (aGT) is a bleeding disorder generally caused by function-blocking anti-αIIbß3 autoantibodies. Aim We characterize an unusual case of aGT caused by marked reduction of surface αIIbß3 with non-function-blocking anti-αIIbß3 antibodies (Abs). Methods A 72-year-old male suffering from immune thrombocytopenia since his 50s showed exacerbation of bleeding symptom despite mild thrombocytopenia. Platelet aggregation was absent with all agonists but ristocetin. Analysis of αIIbß3 expression and genetic analysis were performed. We also analyzed effects of anti-αIIbß3 Abs of the patient on platelet function and αIIbß3 expression. Results Surface αIIbß3 expression was markedly reduced to around 5% of normal, whereas his platelets contained αIIbß3 to the amount of 40-50% of normal. A substantial amount of fibrinogen was also detected in his platelets. There were no abnormalities in ITGA2B and ITGB3 cDNA. These results indicated that reduced surface αIIbß3 expression caused a GT phenotype, and active internalization of αIIbß3 was suggested. Anti-αIIbß3 IgG Abs were detected in platelet eluate and plasma. These Abs did not inhibit PAC-1 binding, indicating that the Abs were non-function-blocking. Surface αIIbß3 expression of a megakaryocytic cell line and cultured megakaryocytes tended to be impaired by incubation with the patient's Abs. After 2 years of aGT diagnosis, his bleeding symptom improved and surface αIIbß3 expression was recovered to 20% of normal with reduction of anti-αIIbß3 Abs. Conclusion We demonstrated a unique aGT phenotype due to marked reduction of surface αIIbß3. Internalization induced by anti-αIIbß3 Abs may be responsible in part for the phenotype.

The study of engraftment after hematopoietic stem cell transplantation: From the presence of mixed chimerism to the development of immunological tolerance.

Over the last three decades, allogeneic hematopoietic stem cell transplantation (HSCT) has become an important therapeutic tool that can cure life-threatening diseases affecting children and adults, including a variety of neoplastic and inborn genetic disorders of the hematopoietic system. Engraftment of donor-derived cells represents a crucial event in order to obtain a successful transplant; therefore, many techniques have been developed to monitor engraftment and eventually determine the presence of mixed chimerism (MC) after HSCT. PCR based on the amplification of short tandem repeats is currently the most common technique used to monitor chimerism, although the degree of achievable quantification can be increased by performing quantitative PCR. In hemoglobinopathies, different studies have showed that complete donor hematopoiesis is not essential for sustained engraftment and that the simultaneous presence of hematopoietic cells of both donor and recipient origin is not a rare event after HSCT. In the present study our data confirmed that the presence of MC in thalassemic patients negatively influence the outcome of HSCT early after HSCT, but not if it becomes persistent in the long follow-up. Different studies have shown that T regulatory type 1 (Tr1) cells, characterized by the coexpression of CD49b and LAG-3 and by their ability to secrete IL-10, have been associated with the presence and maintenance of persistent MC in beta-thalassemic patients after HSCT. In the present study we summarize the incidence of MC after HSCT in a single transplant center cohort of thalassemic patients, showing the role of regulatory T cells in promoting and maintaining immune tolerance in some of them.

CD49b defines functionally mature Treg cells that survey skin and vascular tissues.

Regulatory T (Treg) cells prevent autoimmunity by limiting immune responses and inflammation in the secondary lymphoid organs and nonlymphoid tissues. While unique subsets of Treg cells have been described in some nonlymphoid tissues, their relationship to Treg cells in secondary lymphoid organs and circulation remains unclear. Furthermore, it is possible that Treg cells from similar tissue types share largely similar properties. We have identified a short-lived effector Treg cell subset that expresses the α2 integrin, CD49b, and exhibits a unique tissue distribution, being abundant in peripheral blood, vasculature, skin, and skin-draining lymph nodes, but uncommon in the intestines and in viscera-draining lymph nodes. CD49b+ Treg cells, which display superior functionality revealed by in vitro and in vivo assays, appear to develop after multiple rounds of cell division and TCR-dependent activation. Accordingly, single-cell RNA-seq analysis placed these cells at the apex of the Treg developmental trajectory. These results shed light on the identity and development of a functionally potent subset of mature effector Treg cells that recirculate through and survey peripheral tissues.

Dependence of innate lymphoid cell 1 development on NKp46.

NKp46, a natural killer (NK) cell-activating receptor, is involved in NK cell cytotoxicity against virus-infected cells or tumor cells. However, the role of NKp46 in other NKp46+ non-NK innate lymphoid cell (ILC) populations has not yet been characterized. Here, an NKp46 deficiency model of natural cytotoxicity receptor 1 (Ncr1)gfp/gfp and Ncr1gfp/+ mice, i.e., homozygous and heterozygous knockout (KO), was used to explore the role of NKp46 in regulating the development of the NKp46+ ILCs. Surprisingly, our studies demonstrated that homozygous NKp46 deficiency resulted in a nearly complete depletion of the ILC1 subset (ILC1) of group 1 ILCs, and heterozygote KO decreased the number of cells in the ILC1 subset. Moreover, transplantation studies confirmed that ILC1 development depends on NKp46 and that the dependency is cell intrinsic. Interestingly, however, the cell depletion specifically occurred in the ILC1 subset but not in the other ILCs, including ILC2s, ILC3s, and NK cells. Thus, our studies reveal that NKp46 selectively participates in the regulation of ILC1 development.

Engineered T Regulatory Type 1 Cells for Clinical Application.

T regulatory cells, a specialized subset of T cells, are key players in modulating antigen (Ag)-specific immune responses in vivo. Inducible T regulatory type 1 (Tr1) cells are characterized by the co-expression of CD49b and lymphocyte-activation gene 3 (LAG-3) and the ability to secrete IL-10, TGF-ß, and granzyme (Gz) B, in the absence of IL-4 and IL-17. The chief mechanisms by which Tr1 cells control immune responses are secretion of IL-10 and TGF-ß and killing of myeloid cells via GzB. Tr1 cells, first described in peripheral blood of patients who developed tolerance after HLA-mismatched fetal liver hematopoietic stem cell transplantation, have been proven to modulate inflammatory and effector T cell responses in several immune-mediated diseases. The possibility to generate and expand Tr1 cells in vitro in an Ag-specific manner has led to their clinical use as cell therapy in patients. Clinical grade protocols to generate or to enrich and expand Tr1 cell medicinal products have been established. Proof-of-concept clinical trials with Tr1 cell products have demonstrated the safety and the feasibility of this approach and indicated some clinical benefit. In the present review, we provide an overview on protocols established to induce/expand Tr1 cells in vitro for clinical application and on results obtained in Tr1 cell-based clinical trials. Moreover, we will discuss a recently developed protocol to efficient convert human CD4+ T cells into a homogeneous population of Tr1-like cells by lentiviral vector-mediated IL-10 gene transfer.

Dengue Virus and Its Relation to Human Glycoprotein IIb/IIIa Revealed by Fluorescence Microscopy and Flow Cytometry.

Understanding dengue virus (DENV)-induced hemorrhage remains a challenging jigsaw puzzle with many pieces missing to understand the complex interactions between DENV and blood coagulation system. To use flow cytometry studying the interactions between DENV and human platelet aggregation receptor, glycoprotein IIb/IIIa (gpIIb/IIIa), directly conjugated fluorochrome monoclonal antibody (mAb) is essential to facilitate multifluorochrome immunostaining. However, the obstacle was that no directly conjugated fluorochrome-anti-DENV mAb had been commercially available. To overcome, we directly conjugated fluorochrome to a primary anti-DENV mAb using a LYNX rapid conjugation kit. Flow cytometry analysis showed that this conjugated antibody and anti-gpIIb/IIIa mAb were able to detect DENV and CD41a simultaneously. Fluorescence microscopy analysis further demonstrated CD41a superficially and DENV intracellularly. Potentially, this strategy can facilitate virologists for directly conjugating any virus-specific primary antibodies, which are not commercially available with fluorochrome, to study the infectivity in any surface marker-specific hosts through flow cytometry. Together, DENV can interact with both human gpIIb/IIIa- and gpIIb/IIIa+ cells revealed by flow cytometry and fluorescence microscopy for the first time.

Exosomes from eosinophils autoregulate and promote eosinophil functions.

Eosinophils are able to secrete exosomes that have an undefined role in asthma pathogenesis. We hypothesized that exosomes released by eosinophils autoregulate and promote eosinophil function. Eosinophils of patients with asthma (n = 58) and healthy volunteers (n = 16) were purified from peripheral blood, and exosomes were isolated and quantified from eosinophils of the asthmatic and healthy populations. Apoptosis, adhesion, adhesion molecules expression, and migration assays were performed with eosinophils in the presence or absence of exosomes from healthy and asthmatic individuals. Reactive oxygen species (ROS) were evaluated by flow cytometry with an intracellular fluorescent probe and nitric oxide (NO) and a colorimetric kit. In addition, exosomal proteins were analyzed by mass spectrometry. Eosinophil-derived exosomes induced an increase in NO and ROS production on eosinophils. Moreover, exosomes could act as a chemotactic factor on eosinophils, and they produced an increase in cell adhesion, giving rise to a specific augmentation of adhesion molecules, such as ICAM-1 and integrin α2. Protein content between exosomes from healthy and asthmatic individuals seems to be similar in both groups. In conclusion, we found that exosomes from the eosinophils of patients with asthma could modify several specific eosinophil functions related to asthma pathogenesis and that they could contribute fundamentally to the development and maintenance of asthma.

CD4+Foxp3-IL-10+ Tr1 Cells Promote Relapse of Diffuse Large B Cell Lymphoma by Enhancing the Survival of Malignant B Cells and Suppressing Antitumor T Cell Immunity.

Diffuse large B cell lymphoma (DLBCL) is a common B cell malignancy. Complete remission can be achieved in most patients by conventional treatment with rituximab and chemotherapy. However, a subset of remission individuals will develop a relapsed disease for obscure reasons. CD4+Foxp3-IL-10+ cell (Tr1) is a novel cell subtype with the capacity to suppress pro-inflammatory responses, but has not been extensively studied in most tumors. In this study, we investigated the potential role of Tr1 cells in DLBCL. We found that compared to that in healthy controls, the frequency of Tr1 cells was significantly increased in DLBCL patients, even during complete remission. Further study showed that these Tr1 cells were enriched in the CD25low/-Foxp3-CD49b+LAG-3+ fraction and could be developed in vitro from naive CD45RA+ CD4+ T cells. To examine the effect of Tr1 upregulation, we cocultured the enriched in vitro-induced Tr1 cells (iTr1) with autologous primary DLBCL cells and CD3+ T cells and found that iTr1 cells both enhanced the survival of CD20+ DLBCL tumor cells and suppressed the antitumor response of CD3+ T cells through the production of IL-10. Furthermore, the frequency of CD4+Foxp3-IL-10+ Tr1 cells in DLBCL patients during complete remission is directly associated with the risk of relapse. Together, these results suggested that Tr1 cells contributed to tumor cell maintenance and may serve as a prognostic marker and therapeutic target.

Analysis of Extracellular Vesicles Using Magnetic Nanoparticles in Blood of Patients with Acute Coronary Syndrome.

Extracellular vesicles (EVs) are released from various cell types and play an important role in intercellular interactions. In our study, we investigated abundance of individual EVs in patients with acute forms of ischemic heart disease. Previously, we developed an approach for individual analysis of EVs conjugated with magnetic nanoparticles (MNPs), which was applied in the current study for analyzing phenotypic composition of EVs (by staining for markers CD31, CD41a, and CD63). EVs were isolated using fluorescently labeled MNPs containing anti-CD31, CD41a, or CD63 antibodies and analyzed by combining fluorescently labeled anti-CD41a and CD63, CD31 and CD63, or CD41a and CD31 antibodies, respectively. EVs were analyzed in 30 individuals: 17 healthy volunteers and 13 patients with acute coronary syndrome (ACS). Six and seven ACS patients were with acute myocardial infarction and unstable angina, respectively. It was found that patients with ACS and healthy volunteers contained a dominant subset of EVs expressing surface CD41a antigen, suggesting that they originated from platelets. In addition, the total number of EVs isolated using either of the surface markers examined in our study was higher in patients with ACS compared to healthy volunteers. The subgroup of patients with acute myocardial infarction was found to contain significantly higher number of blood EVs compared to the control group. Moreover, increased number of EVs in patients with ACS is mainly due to the increased number of EVs in the subset of EVs bearing CD41a. By analyzing individual EVs, we found that plasma of patients with ACS, particularly upon developing of myocardial infarction, contained dominant platelet-derived EVs fraction, which may reflect activation of platelets in such patients.

Immunisation against αIIbß3 and αvß3 in a type 1 variant of Glanzmann's thrombasthenia caused by a missense mutation Gly540Asp on ß3.

Treatment of bleeding in patients with Glanzmann's thrombasthenia (GT) can be hampered by iso-antibodies against the αIIbß3 integrin, which cause rapid clearance of transfused donor platelets. Type 1 GT patients with a total absence of αIIbß3 from the platelet surface are known to be susceptible to form such isoantibodies. In this study, we describe a type 1 GT patient with a missense mutation (Gly540Asn) located in the EGF3 domain of the ß3 integrin subunit. Cotransfection analysis in CHO cells demonstrates total absence of αIIbß3 from the surface, based on inappropriate αIIb maturation. The patient's serum was reactive with αIIbß3 and αvß3 integrins in a capture assay, when platelets and endothelial cells were used. Two specificities could be isolated from the patient's serum, anti-αIIbß3 and anti-αvß3 isoantibodies. Both specificities did not interfere with platelet aggregation. In contrast, isoantibodies against αvß3, but not against αIIbß3, were able to disturb endothelial cell adhesion onto vitronectin, triggered endothelial cell apoptosis and interfered with endothelial tube formation. This intriguing finding may explain more recently observed features of fetal/neonatal iso-immune thrombocytopenia in children from type 1 GT mothers with intracranial haemorrhage, which could be related to anti-endothelial activity of the maternal antibodies. In conclusion, we give evidence that two isoantibody entities exist in type 1 GT patients, which are unequivocally different, both in an immunological and functional sense. Further research on the clinical consequences of immunisation against αvß3 is required, predominantly in GT patients of childbearing age.

A pilot study showing associations between frequency of CD4(+) memory cell subsets at diagnosis and duration of partial remission in type 1 diabetes.

In some patients with type 1 diabetes the dose of insulin required to achieve euglycemia is substantially reduced soon after diagnosis. This partial remission is associated with ß-cell function and good glucose control. The purpose of this study was to assess whether frequencies of CD4(+) T cell subsets in children newly diagnosed with type 1 diabetes are associated with length of partial remission. We found that the frequency of CD4(+) memory cells, activated Treg cells and CD25(+) cells that express a high density of the IL-7 receptor, CD127 (CD127(hi)) are strongly associated with length of partial remission. Prediction of length of remission via Cox regression is significantly enhanced when CD25(+) CD127(hi) cell frequency is combined with either Insulin Dependent Adjusted A1c (IDAA1c), or glycosylated hemoglobin (HbA1c), or C-peptide levels at diagnosis. CD25(+) CD127(hi) cells do not express Foxp3, LAG-3 and CD49b, indicating that they are neither Treg nor Tr1 cells.

Tr1 Cells, but Not Foxp3+ Regulatory T Cells, Suppress NLRP3 Inflammasome Activation via an IL-10-Dependent Mechanism.

The two best-characterized types of CD4(+) regulatory T cells (Tregs) are Foxp3(+) Tregs and Foxp3(-) type 1 regulatory (Tr1) cells. The ability of Foxp3(+) Tregs and Tr1 cells to suppress adaptive immune responses is well known, but how these cells regulate innate immunity is less defined. We discovered that CD44(hi)Foxp3(-) T cells from unmanipulated mice are enriched in Tr1 cell precursors, enabling differentiation of cells that express IL-10, as well as Tr1-associated cell surface markers, CD49b and LAG-3, and transcription factors, cMaf, Blimp-1, and AhR. We compared the ability of Tr1 cells versus Foxp3(+) Tregs to suppress IL-1ß production from macrophages following LPS and ATP stimulation. Surprisingly, Tr1 cells, but not Foxp3(+) Tregs, inhibited the transcription of pro-IL-1ß mRNA, inflammasome-mediated activation of caspase-1, and secretion of mature IL-1ß. Consistent with the role for IL-10 in Tr1 cell-mediated suppression, inhibition of inflammasome activation and IL-1ß secretion was abrogated in IL-10R-deficient macrophages. Moreover, IL-1ß production from macrophages derived from Nlrp3(A350V) knockin mice, which carry a mutation found in cryopyrin-associated periodic syndrome patients, was suppressed by Tr1 cells but not Foxp3(+) Tregs. Using an adoptive transfer model, we found a direct correlation between Tr1 cell engraftment and protection from weight loss in mice expressing a gain-of-function NLRP3. Collectively, these data provide the first evidence for a differential role of Tr1 cells and Foxp3(+) Tregs in regulating innate immune responses. Through their capacity to produce high amounts of IL-10, Tr1 cells may have unique therapeutic effects in disease-associated inflammasome activation.

NK cell development in bone marrow and liver: site matters.

The NKp46 protein is found on resting and activated natural killer (NK) cells and is involved in the recognition of malignant and infected cells. The expression of NKp46 is believed to precede that of DX5 in early NK cell development. We show that this is not the case in the bone marrow (BM). Here, NKp46 is predominantly expressed after DX5, whereas the liver harbors a subpopulation that expresses NKp46 but not DX5. NK cell precursors in the liver show much lower levels of Eomesodermin than NK cell precursors in the BM, although they express higher levels of granzymes and unlike the NK cell precursors in the BM are fully able to degranulate and produce interferon gamma (IFN-γ). The development of NK cells thus differs between the two organs. This needs to be considered when using NKp46 and DX5 as NK cell markers and when performing NK cell-specific gene deletion in Ncr1 transgenic mice.

The effect of immune microenvironment on the progression and prognosis of colorectal cancer.

Cancer cells may escape from host immune responses through active suppression of the immune response, but the detailed mechanisms in colorectal cancer remain to be elucidated. In this study, 108 colorectal tumor samples and their peritumoral tissues were collected for immunohistochemistry of infiltrating lymphocytes. Th1 and Tregs cells were determined by the positive expression of T-bet and FOXP3 proteins, respectively. The Tr1 cells were identified by CD49b and LAG-3 protein expression. IL-17-positive cells were identified by IL-17 expression. Results showed that the percentage of T-bet-positive cells was significantly decreased, while the percentages of IL-17-, FOXP3-, CD49b-, and LAG-3-positive cells were significantly increased in tumor tissues compared to that in peritumoral tissues. The ratio of IL-17-, FOXP3-, CD49b-, and LAG3-positive cells to T-bet-positive cells was significantly higher in tumor tissues than in peritumoral tissues. The percentage of infiltrating IL-17-positive cells in tumor tissues was negatively associated with lymph metastasis, invasion, and TNM stage. The percentage of CD49b- and LAG-3-positive cells was positively associated with differentiation, lymph metastasis, invasion, TNM, and Duke stage of colorectal cancer. The percentage of positive Th17 and Tr1 cells is a marker for poor prognosis in patients with CRC. In conclusion, decreased composition of regulative Th1 cells and increased composition of FOXP(+) Tregs-, CD49b(+)/LAG-3(+) Tr1-, and IL-17-positive cells in tumor tissues may be associated with the progression of colorectal cancer. The high percentage of IL-17- and Tr1-positive cells in tumor tissues is a predictive marker for poor prognosis of colorectal cancer.

Towards effective and safe thrombolysis and thromboprophylaxis: preclinical testing of a novel antibody-targeted recombinant plasminogen activator directed against activated platelets.

RATIONALE: Fibrinolysis is a valuable alternative for the treatment of myocardial infarction when percutaneous coronary intervention is not available in a timely fashion. For acute ischemic stroke, fibrinolysis is the only treatment option with a very narrow therapeutic window. Clinically approved thrombolytics have significant drawbacks, including bleeding complications. Thus their use is highly restricted, leaving many patients untreated. OBJECTIVE: We developed a novel targeted fibrinolytic drug that is directed against activated platelets. METHODS AND RESULTS: We fused single-chain urokinase plasminogen activator (scuPA) to a small recombinant antibody (scFvSCE5), which targets the activated form of the platelet-integrin glycoprotein IIb/IIIa. Antibody binding and scuPA activity of this recombinant fusion protein were on par with the parent molecules. Prophylactic in vivo administration of scFvSCE5-scuPA (75 U/g body weight) prevented carotid artery occlusion after ferric chloride injury in a plasminogen-dependent process compared with saline (P<0.001), and blood flow recovery was similar to high-dose nontargeted urokinase (500 U/g body weight). Tail bleeding time was significantly prolonged with this high dose of nontargeted urokinase, but not with equally effective targeted scFvSCE5-scuPA at 75 U/g body weight. Real-time in vivo molecular ultrasound imaging demonstrates significant therapeutic reduction of thrombus size after administration of 75 U/g body weight scFvSCE5-scuPA as compared with the same dose of a mutated, nontargeting scFv-scuPA or vehicle. The ability of scFvSCE5-scuPA to lyse thrombi was lost in plasminogen-deficient mice, but could be restored by intravenous injection of plasminogen. CONCLUSIONS: Targeting of scuPA to activated glycoprotein IIb/IIIa allows effective thrombolysis and the potential novel use as a fibrinolytic agent for thromboprophylaxis without bleeding complications.