Inhibition of ADAM9 promotes the selective degradation of KRAS and sensitizes pancreatic cancers to chemotherapy.

Kirsten rat sarcoma virus (KRAS) signaling drives pancreatic ductal adenocarcinoma (PDAC) malignancy, which is an unmet clinical need. Here, we identify a disintegrin and metalloproteinase domain (ADAM)9 as a modulator of PDAC progression via stabilization of wild-type and mutant KRAS proteins. Mechanistically, ADAM9 loss increases the interaction of KRAS with plasminogen activator inhibitor 1 (PAI-1), which functions as a selective autophagy receptor in conjunction with light chain 3 (LC3), triggering lysosomal degradation of KRAS. Suppression of ADAM9 by a small-molecule inhibitor restricts disease progression in spontaneous models, and combination with gemcitabine elicits dramatic regression of patient-derived tumors. Our findings provide a promising strategy to target the KRAS signaling cascade and demonstrate a potential modality to enhance sensitivity to chemotherapy in PDAC.

The protease ADAM17 at the crossroads of disease: revisiting its significance in inflammation, cancer, and beyond.

The protease A Disintegrin And Metalloproteinase 17 (ADAM17) plays a central role in the pathophysiology of several diseases. ADAM17 is involved in the cleavage and shedding of at least 80 known membrane-tethered proteins, which subsequently modulate several intracellular signaling pathways, and therefore alter cell behavior. Dysregulated expression and/or activation of ADAM17 has been linked to a wide range of autoimmune and inflammatory diseases, cancer, and cardiovascular disease. In this review, we provide an overview of the current state of knowledge from preclinical models and clinical data on the diverse pathophysiological roles of ADAM17, and discuss the mechanisms underlying ADAM17-mediated protein shedding and the potential therapeutic implications of targeting ADAM17 in these diseases.

Durable remission of thrombotic thrombocytopenic purpura in the setting of pembrolizumab therapy.

BACKGROUND: There is a small but growing number of thrombotic thrombocytopenic purpura (TTP) cases attributed to immune checkpoint inhibitor therapy, with nivolumab and ipilimumab therapy being the most frequently described in the literature. STUDY DESIGN AND METHODS: This report evaluates the course of a patient with a history of metastatic adenocarcinoma of the lung who developed TTP following treatment with the PD-1 inhibitor Pembrolizumab. The patient was treated with six sessions of therapeutic plasma exchange and appeared to be in remission. Exacerbation occurred 4 days later, and seven more sessions of plasma exchange were performed along with four total doses of Rituximab, and a steroid taper with monitoring of platelet counts and ADAMTS13 activity. RESULTS: His platelet count recovered to a peak of 318,000 UL with an ADAMTS13 activity of 77% at the time of discharge. The patient has been following up regularly for outpatient testing with no TTP relapse as of the completion of this report. DISCUSSION: This is one of a few cases of Pembrolizumab-associated TTP reported in the literature with successful complete remission following treatment. Plasma exchange in this setting may be an especially beneficial therapeutic intervention because of the removal of both the anti-ADAMTS13 antibody as well as the immune system upregulating anti-PDL1 monoclonal antibody with replacement of ADAMTS13 from donor plasma. Longer duration of plasma exchange and monitoring for normalization of ADAMTS13 levels in addition to platelet count before cessation of treatment may improve durable remission rates in this entity.

Unfavorable immunotherapy plus tyrosine kinase inhibition outcome of metastatic renal cell carcinoma after radical nephrectomy with increased ADAM9 expression.

Immunotherapy plus tyrosine kinase inhibitor (IO-TKI) has become the standard first-line therapy for advanced renal cell carcinoma (RCC). However, the modest response rate of IO-TKI therapy and the absence of biomarkers limited the selection of treatment strategies for RCC patients. There were three cohorts enrolled: two from our facility (ZS-MRCC and ZS-HRRCC) and one from a clinical study (JAVELIN-101). By RNA sequencing, the expression of ADAM9 in each sample was measured. By flow cytometry and immunohistochemistry, immune infiltration and T cell function were examined. Primary outcomes were established as treatment response and progression-free survival (PFS). Patients with low-ADAM9 expression had a higher objective response rate (56.5% vs 13.6%, P = 0.01) and longer PFS in both cohorts. In the ZS-HRRCC cohort, the expression of ADAM9 was associated with increased tumor-infiltrating T cells, which was proved by immunohistochemistry (P < 0.05) and flow cytometry (Spearman's ρ = 0.42, P < 0.001). In the high-ADAM9 group, CD8+ and CD4+ T cells revealed an exhausted phenotype with decreased GZMB (Spearman's ρ = - 0.31, P = 0.05, and Spearman's ρ = - 0.49, P < 0.001, respectively), and fewer Macrophages were identified. A predictive RFscore was further constructed by random forest approach, involving ADAM9 and immunologic genes. Only in the subgroup with the lower RFscore did IO-TKI outperform TKI monotherapy. High-ADAM9 expression was associated with immunosuppression and IO-TKI resistance. Expression of ADAM9 was also associated with the exhaustion and dysfunction of T cells. ADAM9-based RFscore has the potential to be used as a biomarker to distinguish the optimal patient treatment methods between IO-TKI and TKI monotherapy.

Dulaglutide as a demethylating agent to improve the outcome of breast cancer.

Background: Dulaglutide emerged as a promising therapeutic option for diabetes mellitus Type 2 (DM2). Aims: Owing to epigenetic similarities between the pathophysiology of DM2 and breast cancer (BC), we investigated the antitumor effect of dulaglutide. Materials & methods: To investigate the effect of dulaglutide, we analyzed the expression of methylated gene promoter regions in BC (ESR1, CDH1 and ADAM33). Results: Dulaglutide increased the expression of ESR1, CDH1 and ADAM33 up to fourfold in the MDA-MB-231 lineage by demethylating the gene promoter regions. This effect was translated to in vivo antitumoral activity and revealed significant tumor inhibition by combining the half-dose of methotrexate with dulaglutide. Conclusion: This therapy may mitigate the severe side effects commonly associated with chemotherapy.

[Clinical characteristics and outcome of 69 patients with thrombotic thrombocytopenic purpura].

To analyze the clinical characteristics, diagnosis, treatment and outcome of patients with thrombotic thrombocytopenic purpura (TTP). The clinical data of 69 adult patients with TTP were retrospectively analyzed. There were 19 males and 50 females with a median age of 42 (18-79) years. PLASMIC score 6-7 was recognized in 82.8% (53/64) patients. The activity of von Willebrand factor-cleaving protease (ADAMTS13), which was detected in 21 patients before treatment, was less than 5% in 17 patients and 5%-10% in 3 patients. All 69 patients were treated with plasma exchange (PEX) and/or fresh frozen plasma infusion (PI), 43 of whom were also given glucocorticoid. In addition to PEX/PI and glucocorticoid, rituximab and/or immunosuppressants were administrated in 20 patients. The median follow-up time was 12 (1-57) months. The remission rate was 69.6%, while the relapse rate was 11.6%. The 2-year overall survival (OS) rate was 69.6%±5.5%. The univariate and multivariate analysis showed that relapsed/refractory disease was an independent risk factor for OS. The 2-year OS rate of relapsed/refractory patients was significantly lower than that of the rest patients (41.5%±9.8% vs. 83.7%±5.6%, P<0.001). Regarding the unfavorable prognosis in relapsed/refractory patients, rituximab and/or immunosuppressants are strongly recommended for sake of improving the overall survival.

Neutralization of inhibitory antibodies and restoration of therapeutic ADAMTS-13 activity levels in inhibitor-treated rats by the use of defined doses of recombinant ADAMTS-13.

BACKGROUND: Acquired thrombotic thrombocytopenic purpura (TTP) is caused by an autoantibody-mediated deficiency of the von Willebrand factor-cleaving protease ADAMTS-13. Acute episodes of the disease are treated with a combination of immunosuppression and repeated cycles of plasma exchange to remove anti-ADAMTS-13 autoantibodies and, at the same time, replenish functional ADAMTS-13. Although this is often effective, the mortality rate has remained between 10% and 20%, highlighting the need for safer treatment options. OBJECTIVES: We previously showed that, in vitro, human recombinant ADAMTS-13 (rADAMTS-13) is able to override neutralizing antibodies and restore ADAMTS-13 activity in plasma from patients with acquired TTP. In the present study, we assessed the in vivo feasibility of this strategy by using a rat model. METHODS: Wild-type rats were adjusted to an ADAMTS-13 inhibitor (inhibitor) titer of ~ 10 BU mL(-1) with goat anti-ADAMTS-13 IgG, and treated with increasing doses of rADAMTS-13. Blood samples were drawn and analyzed for ADAMTS-13-specific parameters, including FRETS-VWF73 activity, inhibitor, and ADAMTS-13-specific immune complexes (ICs). The pharmacokinetics of ADAMTS-13 activity and inhibitors were evaluated. RESULTS: Administration of inhibitor titer-adjusted doses of rADAMTS-13 to inhibitor-treated rats predictably restored activity. Inhibitors were readily neutralized through formation of ADAMTS-13-specific ICs, which were cleared at a higher rate than the free inhibitor. Surplus protease was enzymatically active in plasma, and showed similar pharmacokinetics to ADAMTS-13 in not inhibitor-treated rats. CONCLUSIONS: Defined doses of rADAMTS-13 neutralized circulating anti-ADAMTS-13 antibodies and enabled reconstitution of ADAMTS-13 activity in plasma in our model, indicating that the protease may be a promising candidate for further exploration in treating acute episodes of acquired TTP.

Delayed treatment with ADAMTS13 ameliorates cerebral ischemic injury without hemorrhagic complication.

Tissue plasminogen activator (tPA) is the only approved therapy for acute ischemic stroke. However, delayed tPA treatment increases the risk of cerebral hemorrhage and can result in exacerbation of nerve injury. ADAMTS13, a von Willebrand factor (VWF) cleaving protease, has a protective effect against ischemic brain injury and may reduce bleeding risk by cleaving VWF. We examined whether ADAMTS13 has a longer therapeutic time window in ischemic stroke than tPA in mice subjected to middle cerebral artery occlusion (MCAO). ADAMTS13 (0.1mg/kg) or tPA (10mg/kg) was administered i.v., immediately after reperfusion of after 2-h or 4-h MCAO for comparison of the therapeutic time windows in ischemic stroke. Infarct volume, hemorrhagic volume, plasma high-mobility group box1 (HMGB1) levels and cerebral blood flow were measured 24h after MCAO. Both ADAMTS13 and tPA improved the infarct volume without hemorrhagic complications in 2-h MCAO mice. On the other hand, ADAMTS13 reduced the infarct volume and plasma HMGB1 levels, and improved cerebral blood flow without hemorrhagic complications in 4-h MCAO mice, but tPA was not effective and these animals showed massive intracerebral hemorrhage. These results indicated that ADAMTS13 has a longer therapeutic time window in ischemic stroke than tPA, and ADAMTS13 may be useful as a new therapeutic agent for ischemic stroke.

Antibody-based exosite inhibitors of ADAMTS-5 (aggrecanase-2).

Adamalysin-like metalloproteinases with thrombospondin (TS) motifs (ADAMTS)-5 is the multi-domain metalloproteinase that most potently degrades aggrecan proteoglycan in the cartilage and its activity is implicated in the development of osteoarthritis (OA). To generate specific exosite inhibitors for it, we screened a phage display antibody library in the presence of the zinc-chelating active site-directed inhibitor GM6001 (Ilomastat) and isolated four highly selective inhibitory antibodies. Two antibodies were mapped to react with exosites in the catalytic/disintegrin domains (Cat/Dis) of the enzyme, one in the TS domain and one in the spacer domain (Sp). The antibody reacting with the Sp blocked the enzyme action only when aggrecan or the Escherichia coli-expressed aggrecan core protein were substrates, but not against a peptide substrate. The study with this antibody revealed the importance of the Sp for effective aggrecanolytic activity of ADAMTS-5 and that this domain does not interact with sulfated glycosaminoglycans (GAGs) but with the protein moiety of the proteoglycan. An antibody directed against the Cat/Dis of ADAMTS-5 was effective in a cell-based model of aggrecan degradation; however, the anti-Sp antibody was ineffective. Western blot analysis of endogenous ADAMTS-5 expressed by human chondrocytes showed the presence largely of truncated forms of ADAMTS-5, thus explaining the lack of efficacy of the anti-Sp antibody. The possibility of ADAMTS-5 truncation must then be taken into account when considering developing anti-ancillary domain antibodies for therapeutic purposes.

Acquired thrombotic thrombocytopenic purpura: new therapeutic options and their optimal use.

Advances in our understanding of the pathophysiology of both congenital and acquired thrombotic thrombocytopenic purpura (TTP) have led to both an increased understanding of the disease and novel approaches to therapy. The efficacy of rituximab in acquired TTP has led to consideration of rituximab as a prophylactic therapy to prevent relapse of TTP. Novel therapies that target the A1 domain of von Willebrand factor (VWF) to block the formation of microthrombotic disease have also entered clinical study and have demonstrated promise as potential therapeutic options. Additionally, a recombinant ADAMTS13 protease has been developed which may be an important therapeutic option for both congenital and acquired TTP. The development of these new therapeutic options for patients diagnosed with TTP has increased the importance of conducting prospective, randomized studies with these agents to both confirm their efficacy and more importantly understand their most appropriate role in the treatment of patients with TTP.

Antiangiogenic Metargidin Peptide (AMEP) Gene Therapy in Disseminated Melanoma.

Gene delivery by electroporation is an efficient method for transfecting genes into various tissues including tumors. Here we present the treatment protocol used in a phase 1 study on gene electrotransfer of plasmid DNA encoding an antiangiogenic peptide into cutaneous melanoma.

VWF excess and ADAMTS13 deficiency: a unifying pathomechanism linking inflammation to thrombosis in DIC, malaria, and TTP.

Absent or severely diminished activity of ADAMTS13 (A Disintegrin And Metalloprotease with a ThromboSpondin type 1 motif, member 13) resulting in the intravascular persistence and accumulation of highly thrombogenic ultra large von Willebrand factor (UL-VWF) multimers is the pathophysiological mechanism underlying thrombotic thrombocytopenic purpura. Reduced VWF-cleaving protease levels, however, are not uniquely restricted to primary thrombotic microangiopathy (TMA), e. g. thrombotic thrombocytopenic purpura, but also occur in other life-threatening thrombocytopenic conditions: severely decreased ADAMTS13 activity is seen in severe sepsis, disseminated intravascular coagulation (DIC) and complicated malarial infection. The clinical relevance of these secondary thrombotic microangiopathies is increasingly recognised, but its therapeutic implications have not yet been determined. The presence of a secondary TMA in certain diseases may define patient groups which possibly could benefit from ADAMTS13 replacement or a VWF-targeting therapy. This short-review focuses on the role of UL-VWF multimers in secondary TMA and discusses the potential of investigational therapies as candidates for the treatment of TTP. In conclusion, prospective clinical trials on the effectiveness of protease replacementin vivo seem reasonable. Carefully selected patients with secondary TMA may benefit from therapies primarily intended for the use in patients with TTP.

Effect of ADAMTS-13 on cerebrovascular microthrombosis and neuronal injury after experimental subarachnoid hemorrhage.

BACKGROUND: Microthrombosis and reactive inflammation contribute to neuronal injury after subarachnoid hemorrhage (SAH). ADAMTS-13 cleaves von Willebrand factor multimers, and inhibits thrombus formation and, seemingly, inflammatory reactions. OBJECTIVE: To investigate the effect of ADAMTS-13 in experimental SAH. METHODS: A total of 100 male C57/BL6 mice were randomly assigned to four groups: sham (n = 15), SAH (n = 27), vehicle (n = 25), and ADAMTS-13 (n = 23; 100 µL per 10 g of body weight of 100 µg of ADAMTS-13 per 1 mL of 0.9% NaCl; 20 min after SAH). Neurologic performance was assessed on days 1 and 2 after SAH. Animals were killed on day 2. The amounts of subarachnoid blood, microthrombi, apoptosis and degenerative neurons were compared. The degree of neuronal inflammation and vasospasm was also compared. In five mice each (SAH and ADAMTS-13 groups), bleeding time was assessed 2 h after SAH. RESULTS: Systemic administration of ADAMTS-13 achieved significant amelioration of microthrombosis and improvement in neurologic performance. ADAMTS-13 reduced the amount of apoptotic and degenerative neurons. A tendency for decreased neuronal inflammation was observed. ADAMTS-13 did not show any significant effect on vasospasm. The degree of systemic inflammation was not changed by ADAMTS-13 administration. ADAMTS-13 neither increased the amount of subarachnoid blood nor prolonged the bleeding time. CONCLUSIONS: ADAMTS-13 may reduce neuronal injury after SAH by reducing microthrombosis formation and neuronal inflammation, thereby providing a new option for mitigating the severity of neuronal injury after SAH.

Thrombotic thrombocytopenic purpura: basic pathophysiology and therapeutic strategies.

VWF is a multimeric plasma glycoprotein that specifically recruits platelets to sites of vessel injury. VWF multimeric size is central to this function, with larger multimers being more hemostatically active. Regulation of VWF multimeric size is mediated by the plasma metalloprotease ADAMTS13 (A Disintegrin And Metalloproteinase with ThromboSpondin type 1 motifs, member 13). This enzyme can only recognize and cleave VWF when it is unraveled by rheological shear forces of the flowing blood. After the exposure of cryptic exosites, VWF recognition by ADAMTS13 involves multiple interactions that enable the protease to cleave VWF. Loss of VWF multimer size regulation caused by severe ADAMTS13 deficiency (either inherited or acquired) is associated with the microvascular thrombotic disorder thrombotic thrombocytopenic purpura (TTP). The sequelae associated with TTP are widely thought to be linked to hyperreactive circulating VWF that cause unwanted platelet aggregation in the high shear environment of the microvasculature. Diagnosis of TTP is primarily made through a combination of symptoms, analysis of plasma ADAMTS13 activity, and detection of inhibitory anti-ADAMTS13 antibodies. Current frontline treatments for TTP include plasma exchange, which serves to remove inhibitory antibodies (in acquired TTP) and provide a source of functional ADAMTS13, and steroids to treat the autoimmune component of acquired TTP. The use of anti-CD20 therapy has also exhibited encouraging results in the treatment of acquired TTP. Newer therapeutic strategies that are currently being explored or are in development include recombinant ADAMTS13, a hyperreactive ADAMTS13 variant, and anti-VWF therapy. This review discusses the basic biochemistry of VWF and ADAMTS13, their dysfunction in TTP, and therapeutic approaches for the amelioration of TTP.

Development of novel treatment options for patients with haemophilia.

UNLABELLED: Treatment of haemophilia has vastly improved over the last years, but many needs are still unmet. Baxter is continuously pursuing the aim to provide new therapeutic options to patients with haemophilia and to their treating physicians. In fact, there are several opportunities to improve existing therapies, e.g., by new indications for existing products, the introduction of new products, and by novel therapeutic approaches other than factor replacement. Among these, Baxter is working on a number of innovations, such as pharmacokinetics-tailored factor VIII prophylaxis, bypassing agent prophylaxis with FEIBA in inhibitor patients, development of a longer acting pegylated recombinant FVIII, a new recombinant factor IX, a new recombinant factor FVIIa, the first recombinant von Willebrand factor, recombinant ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) as well as gene therapy to cure haemophilia B. CONCLUSION: Baxter is truly committed to the benefit for the patient, and therefore engaged in providing a more and more individualized treatment, in increasing efficiency of current products, in developing new products and new approaches with added value.

ADAMTS13 content in plasma-derived factor VIII/von Willebrand factor concentrates.

Thrombotic thrombocytopenic purpura (TTP) is a microangiopathy syndrome caused by a congenital or acquired deficiency of ADAMTS13, a plasma metalloprotease that cleaves von Willebrand factor (VWF) and thus prevents the formation of platelet-rich thrombi in the microcirculation. TTP can be fatal if not appropriately and timely treated with the infusion of fresh frozen plasma (FFP) or exchange plasmapheresis, that reverse the process of microangiopathy by removing anti-ADAMTS13 autoantibodies and replacing functional ADAMTS13. The treatment of TTP with FFP is not free from risks and must be administered in hospitals or clinics, owing to the substantial amount of plasma volume infused or exchanged and the frequent need of catheter application. Moreover, most FFPs are not subjected to treatments to remove or inactivate blood-borne infectious agents. A number of recent reports indicate that certain plasma-derived VWF-factor VIII (FVIII) concentrates are clinically effective in the treatment of congenital TTP. In this study, we measured ADAMTS13 levels in various plasma-derived VWF-FVIII concentrates, showing that Koate(®) -DVI (Grifols), contained relatively high amounts of ADAMTS13 and that Alphanate(®) (Grifols) was the closest other product in terms of protease content. Koate(®) -DVI contains, on average (five lots tested), 0.091 ± 0.007 Units of ADAMTS13 activity per IU of FVIII. On the basis of this analysis and other reports of VWF-FVIII concentrate utilization in congenital TTP, potential dosing, and future clinical developments are discussed.

Chloroquine promotes the anticancer effect of TACE in a rabbit VX2 liver tumor model.

BACKGROUND: To investigate the efficacy of TACE combined with CQ, an autophagic inhibitor, in a rabbit VX2 liver tumor model. METHODS: Tumor size was measured. And tumor growth rate was calculated to examine the effect of the combined treatment. Apoptosis was detected by TUNEL assay. Meanwhile, autophagic activity was detected by immunohistochemistry and Western blotting to investigate the mechanism underlying. Liver function was also examined to assess feasibility and safety of the combined therapy. RESULTS: Tumors in the control grew more than 4 times bigger after 14 days, while that in the group of TACE alone just showed mild growth. But a slight shrinkage was shown after the treatment of CQ+TACE. Growth ratio of TACE alone was 96.45% ± 28.958% while that of CQ+TACE was -28.73% ± 12.265%. Compared with TACE alone, necrosis in CQ+TACE showed no significant difference, however, the apoptosis was much higher. There were only 14.8±3.11% apoptotic cells in TACE, but 33±4.18% in CQ+TACE, which suggests the increased apoptosis in CQ+TACE contributed to the decrease of tumor volume. In terms of autophagic activity, the result is negative when we immunostained sections of the control with LC3 antibody, but positive in TACE alone and CQ+TACE. And the result of Western blot showed that there was just a low level of LC3Ⅱexpressed in the control and CQ alone, but higher in TACE, and much higher in CQ+TACE because CQ inhibited its degradation in autophagy. Compared with control, p62 decreased in TACE, but the decrease was partially reversed in CQ+TACE. In addition, toxicity of CQ+TACE was assessed not higher than TACE alone, which supports the safety of CQ+TACE. CONCLUSION: CQ+TACE works better than TACE alone in rabbit VX2 liver tumor model because CQ inhibits autophagy induced by TACE. The inhibited autophagy loses its resistance to apoptosis that apoptosis increased, which contributes to the inhibition of tumor growth. This study indicates CQ may be a promising adjuvant to promote the effect of TACE.

Thrombotic thrombocytopenic purpura in children.

PURPOSE OF REVIEW: Thrombotic thrombocytopenic purpura (TTP) is a rare life-threatening disease in children, due to a severe deficiency of ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 repeats, member 13), inherited in congenital TTP or secondary to anti-ADAMTS13 antibodies in acquired TTP. Rapid techniques for ADAMTS 13 assays, long-term follow-up of patients, phenotype-genotype analysis, improved therapeutic schedules, and new therapies have emerged. RECENT FINDINGS: Rapid techniques for ADAMTS13 assays now permit rapid confirmation of diagnosis. In congenital TTP, mutations affecting the N-terminal domains of ADAMTS13 are associated with lower residual ADAMTS13 activity and more severe phenotype. Early initiation of plasma infusion treatment and lifelong prophylactic plasma infusion have decreased mortality and sequels and prevent relapses. In acquired TTP, a disease of adolescents but also of children less than 2, adding rituximab to plasma exchange is beneficial. Recombinant ADAMTS13 ought to be soon available for congenital TTP, while acquired TTP children might benefit from its administration, alone or in association with rituximab, to avoid or limit plasma exchange duration. SUMMARY: Progress in the understanding of TTP has boosted physicians' awareness that diagnosis and treatment are medical emergencies. New therapies hopefully will decrease treatment burden and improve prognosis.

Protective anti-inflammatory effect of ADAMTS13 on myocardial ischemia/reperfusion injury in mice.

Coronary heart disease is a major cause of death in the western world. Although essential for successful recovery, reperfusion of ischemic myocardium is inevitably associated with reperfusion injury. To investigate a potential protective role of ADAMTS13, a protease cleaving von Willebrand factor multimers, during myocardial ischemia/reperfusion, we used a mouse model of acute myocardial infarction. We found that Adamts13(-/-) mice developed larger myocardial infarctions than wild-type control mice, whereas treatment of wild-type mice with recombinant human ADAMTS13 (rhADAMTS13) led to smaller infarctions. The protective effect of ADAMTS13 was further confirmed by a significant reduction of cardiac troponin-I release and less myocardial apoptosis in mice that received rhADAMTS13 compared with controls. Platelets adherent to the blood vessel wall were observed in few areas in the heart samples from mice treated with vehicle and were not detected in samples from mice treated with rhADAMTS13. However, we observed a 9-fold reduction in number of neutrophils infiltrating ischemic myocardium in mice that were treated with rhADAMTS13, suggesting a potent anti-inflammatory effect of ADAMTS13 during heart injury. Our data show that ADAMTS13 reduces myocardial ischemia/reperfusion injury in mice and indicate that rhADAMTS13 could be of therapeutic value to limit myocardial ischemia/reperfusion injury.

ADAMTS13 exerts a thrombolytic effect in microcirculation.

Recombinant tissue plasminogen activator (r-tPA) is the drug of choice for thrombolysis, but it is associated with a significant risk of bleeding and is not always successful. By cleaving von Willebrand factor (VWF), the metalloprotease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I repeats-13) down-regulates thrombus formation in injured vessels. We investigated whether recombinant ADAMTS13 (r-ADAMTS13) induces thrombolysis in vivo in mice. Thrombosis was produced by ferric chloride-induced (FeCl(3)) injury in the venules of a dorsal skinfold chamber. Phosphate-buffered saline (PBS, vehicle), r-tPA or r-ADAMTS13, supplemented with hirudin (to stop on-going thrombin generation), was directly applied onto the occluded vessel, and thrombus dissolution was evaluated by intravital microscopy. The incidence of blood flow restoration significantly increased 30 minutes (min) after r-ADAMTS13 vs. PBS treatment (60% vs. 0%, p<0.05) and 60 min after r-tPA treatment (75% vs. 17%, p<0.05). Both r-tPA and r-ADAMTS13 significantly reduced thrombus size 60 min after their superfusion (53.2% and 62.3% of the initial thrombus size, p<0.05 and p<0.01, respectively). Bleeding occurred in all r-tPA-treated chambers, while it was absent in mice treated with r-ADAMTS13 or PBS. We observed that, similar to r-tPA, r-ADAMTS13 can dissolve occlusive thrombi induced by FeCl(3) injury in venules. In contrast to r-tPA, the in vivo thrombolytic effect of ADAMTS13 was not associated with any signs of haemorrhage. ADAMTS13 could represent a new therapeutic option for thrombolysis.