R-tPA Resistance Is Specific for Platelet-Rich Stroke Thrombi and Can Be Overcome by Targeting Nonfibrin Components.

BACKGROUND: Resistance to r-tPA (recombinant tissue-type plasminogen activator) is a well-known but poorly understood phenomenon that hampers successful recanalization in patients with acute ischemic stroke. Using clinically relevant thrombi from patients with acute ischemic stroke, we investigated if and how thrombus composition impacts r-tPA-mediated lysis. In addition, we explored strategies to overcome r-tPA resistance. METHODS: Thrombi were split into 2 parts, 1 of which was used for thrombolysis and the other for detailed histological analysis. Thrombolysis was performed in normal human plasma using r-tPA alone, using r-tPA in combination with DNase-1 or using r-tPA in combination with N,N'-diacetyl-l-cystine. Thrombus lysis was calculated as the percentage of residual thrombus weight compared with its initial weight and the degree of lysis was linked to thrombus composition determined via histology. RESULTS: Interestingly, we found that the efficacy of r-tPA-mediated thrombolysis was strongly correlated with the composition of the thrombi. Thrombi containing high amounts of red blood cells and low amounts of DNA and von Willebrand Factor were efficiently degraded by r-tPA, whereas thrombi containing low amounts of red blood cells and higher amounts of DNA and von Willebrand Factor were resistant to r-tPA. Importantly, combination of r-tPA with DNase-1 or N,N'-diacetyl-l-cystine significantly and specifically improved the lysis of these r-tPA-resistant thrombi. CONCLUSIONS: Using patient thrombus material, our results for the first time show that the composition of stroke thrombi largely determines their susceptibility to r-tPA-mediated thrombolysis. Red blood cell-poor thrombi have a specific resistance to r-tPA, which can be overcome by targeting nonfibrin components using DNase-1 or N,N'-diacetyl-l-cystine.

Spatiotemporal profile of neutrophil extracellular trap formation in a mouse model of ischemic stroke.

Background: Thromboinflammatory processes modulate the complex pathophysiology of cerebral ischemia-reperfusion (I/R) injury in ischemic stroke, but the exact underlying mechanisms remain poorly understood. Emerging evidence indicates that neutrophil extracellular traps (NETs) might play an important role in the thromboinflammatory cascade. In addition, the link between von Willebrand factor (VWF) and neutrophil recruitment in the ischemic brain might promote thromboinflammation, possibly by the formation of NETs. Objectives: To study NET formation in a murine model of cerebral I/R injury in ischemic stroke. Methods: The filament-induced transient middle cerebral artery occlusion model was used to induce 60 minutes of focal cerebral ischemia after which reperfusion was allowed. At different time points postischemia, NETs were identified in the ischemic mouse brain using quantitative immunofluorescence microscopy. Results: NETs could be identified in the ipsilateral brain hemisphere. Interestingly, NETs could already be detected at 6 hours poststroke. Their presence increased at 12 hours, was highest at 24 hours, and decreased again 48 hours postischemia. Remarkably, NETs were predominantly localized within the brain vasculature postischemia, suggesting that NETs play a role in secondary microthrombosis. Strikingly, NET formation was significantly decreased in VWF-deficient mice compared to littermate wild-type mice 24 hours postischemia, indicating a possible role for VWF in promoting NETosis in the ischemic brain. Conclusion: This study identified the spatiotemporal profile of NET formation in a mouse model of cerebral I/R injury in ischemic stroke. NETs, potentially in combination with VWF, might be attractive targets for the development of novel therapeutic strategies in ischemic stroke treatment.

Unusual Histopathological Findings in Mechanically Removed Stroke Thrombi - A Multicenter Experience.

Background: Several studies have investigated the histopathology of mechanically retrieved thrombi from stroke patients. Thrombi with unusual components constitute about 1-2% of all stroke thrombi in clinical practice. Knowledge about these rare components is limited. Objectives: To characterize the histopathology of unusual stroke thrombi from a real-world setting with relation to clinical presentation, patient characteristics and procedural aspects of mechanical thrombectomy. Methods: One-thousand and eight thrombi retrieved from stroke patients with mechanical thrombectomy at three different hospitals were retrospectively reviewed for unusual histological components. Fifteen thrombi were included in the study for further histopathological analysis. Clinical data and data on procedural aspects were collected. Results: We identified six cases with large amounts of extracellular DNA, of which three were calcified. All six cases except one received anticoagulant therapy. We describe two types of calcifications that differ with respect to general calcification morphology, von Kossa staining pattern, macrophage immunophenotype and presence of multinucleated giant cells. Cholesterol-rich (n = 3), adipocyte-like pattern-rich (n = 2), collagen-rich (n = 2) and myxomatous (n = 1) thrombi were also identified and are discussed with regard to pathogenesis and clinical and intervention characteristics. Finally, a thrombus with parts of a vascular wall is described. Suggestions for future studies are made and clinical and technical aspects of the management for these rare but important patients are discussed. Conclusion: In our retrospective multicenter study, we characterized stroke thrombi histopathologically and found subgroups of thrombi defined by presence of rarely seen components. These defined subgroups showed relation to underlying cardiovascular disease, patient characteristics, and mechanical thrombectomy technique. Knowledge about these components may increase our understanding of stroke pathophysiology and influence interventional procedures.

Thrombus formation during ECMO: Insights from a detailed histological analysis of thrombus composition.

OBJECTIVES: Intra-device thrombosis remains one of the most common complications during extracorporeal membrane oxygenation (ECMO). Despite anticoagulation, approximately 35% of patients develop thrombi in the membrane oxygenator, pump heads, or tubing. The aim of this study was to describe the molecular and cellular features of ECMO thrombi and to study the main drivers of thrombus formation at different sites in the ECMO circuits. APPROACH AND RESULTS: Thrombi (n = 85) were collected immediately after veno-arterial-(VA)-ECMO circuit removal from 25 patients: 23 thrombi from the pump, 25 from the oxygenator, and 37 from the tubing. Quantitative histological analysis was performed for the amount of red blood cells (RBCs), platelets, fibrin, von Willebrand factor (VWF), leukocytes, and citrullinated histone H3 (H3Cit). ECMO thrombi consist of a heterogenous composition with fibrin and VWF being the major thrombus components. A clustering analysis of the four major histological parameters identified two typical thrombus types: RBC-rich and RBC-poor/fibrin-rich thrombi with no significant differences in VWF and platelet content. Thrombus composition was not associated with the thrombus location, except for higher amounts of H3Cit that were found in pump and oxygenator thrombi compared to tubing samples. We observed higher blood leukocyte count and lactate dehydrogenase levels in patients with fibrin-rich thrombi. CONCLUSION: We found that thrombus composition is heterogenous, independent of their location, consisting of two types: RBC-rich and a fibrin-rich types. We also found that NETs play a minor role. These findings are important to improve current anticoagulation strategies in ECMO.

Detailed histological analysis of a thrombectomy-resistant ischemic stroke thrombus: a case report.

BACKGROUND: Mechanical removal of a thrombus by thrombectomy can be quite challenging. For reasons that are not fully understood, some thrombi require multiple passes to achieve successful recanalization, whereas other thrombi are efficiently removed in a single pass. Since first pass success is associated with better clinical outcome, it is important to better understand the nature of thrombectomy resistant thrombi. The aim of this study was therefore to characterize the cellular and molecular composition of a thrombus that was very hard to retrieve via mechanical thrombectomy. CASE PRESENTATION: In a patient that was admitted with a right middle cerebral artery M1-occlusion, 11 attempts using various thrombectomy devices and techniques were required for removal of the thrombus. This peculiar case provided a rare opportunity to perform an in-depth histopathological study of a difficult to retrieve thrombus. Thrombus material was histologically analyzed using hematoxylin and eosin, Martius Scarlet Blue stain (red blood cells and fibrin), Feulgen stain (DNA), von Kossa stain (calcifications) and immunohistochemical analysis of von Willebrand factor, platelets, leukocytes and neutrophil extracellular traps. Histological analysis revealed abnormally high amounts of extracellular DNA, leukocytes, von Willebrand factor and calcifications. Extracellular DNA stained positive for markers of leukocytes and NETs, suggesting that a significant portion of DNA is derived from neutrophil extracellular traps. CONCLUSION: In this unique case of a nearly thrombectomy-resistant stroke thrombus, our study showed an atypical composition compared to the common structural features found in ischemic stroke thrombi. The core of the retrieved thrombus consisted of extracellular DNA that colocalized with von Willebrand factor and microcalcifications. These results support the hypothesis that von Willebrand factor, neutrophil extracellular traps and microcalcifications contribute to mechanical thrombectomy resistance. Such information is important to identify novel targets in order to optimize technical treatment protocols and techniques to increase first pass success rates.

von Willebrand factor increases in experimental cerebral malaria but is not essential for late-stage pathogenesis in mice.

BACKGROUND: Cerebral malaria (CM) is the most severe complication of malaria. Endothelial activation, cytokine release, and vascular obstruction are essential hallmarks of CM. Clinical studies have suggested a link between von Willebrand factor (VWF) and malaria pathology. OBJECTIVES: To investigate the contribution of VWF in the pathogenesis of experimental cerebral malaria (ECM). METHODS: Both Vwf+/+ and Vwf-/- mice were infected with Plasmodium berghei ANKA (PbANKA) to induce ECM. Alterations of plasma VWF and ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), platelet count, neurological features, and accumulation of platelets and leukocytes in the brain were examined following infection. RESULTS: Plasma VWF levels significantly increased upon PbANKA infection in Vwf+/+ animals. While ADAMTS13 activity was not affected, high molecular weight VWF multimers disappeared at the end-stage ECM, possibly due to an ongoing hypercoagulability. Although the number of reticulocytes, a preferential target for the parasites, was increased in Vwf-/- mice compared to Vwf+/+ mice early after infection, parasitemia levels did not markedly differ between the two groups. Interestingly, Vwf-/- mice manifested overall clinical ECM features similar to those observed in Vwf+/+ animals. At day 8.5 post-infection, however, clinical ECM features in Vwf-/- mice were slightly more beneficial than in Vwf+/+ animals. Despite these minor differences, overall survival was not different between Vwf-/- and Vwf+/+ mice. Similarly, PbANKA-induced thrombocytopenia, leukocyte, and platelet accumulations in the brain were not altered by the absence of VWF. CONCLUSIONS: Our study suggests that increased VWF concentration is a hallmark of ECM. However, VWF does not have a major influence in modulating late-stage ECM pathogenesis.

Structural analysis of ischemic stroke thrombi: histological indications for therapy resistance.

Ischemic stroke is caused by a thromboembolic occlusion of cerebral arteries. Treatment is focused on fast and efficient removal of the occluding thrombus, either via intravenous thrombolysis or via endovascular thrombectomy. Recanalization, however, is not always successful and factors contributing to failure are not completely understood. Although the occluding thrombus is the primary target of acute treatment, little is known about its internal organization and composition. The aim of this study, therefore, was to better understand the internal organization of ischemic stroke thrombi on a molecular and cellular level. A total of 188 thrombi were collected from endovascularly treated ischemic stroke patients and analyzed histologically for fibrin, red blood cells (RBC), von Willebrand factor (vWF), platelets, leukocytes and DNA, using bright field and fluorescence microscopy. Our results show that stroke thrombi are composed of two main types of areas: RBC-rich areas and platelet-rich areas. RBC-rich areas have limited complexity as they consist of RBC that are entangled in a meshwork of thin fibrin. In contrast, platelet-rich areas are characterized by dense fibrin structures aligned with vWF and abundant amounts of leukocytes and DNA that accumulate around and in these platelet-rich areas. These findings are important to better understand why platelet-rich thrombi are resistant to thrombolysis and difficult to retrieve via thrombectomy, and can guide further improvements of acute ischemic stroke therapy.

Coculture Method to Obtain Endothelial Networks Within Human Tissue-Engineered Skeletal Muscle.

Skeletal muscle tissue engineering aims at creating functional skeletal muscle in vitro. Human muscle organoids can be used for potential applications in regenerative medicine, but also as an in vitro model for myogenesis or myopathology. However, the thickness of constructs is limited due to passive diffusion of nutrients and oxygen. Introduction of a vascular network in vitro may solve this limitation. Here, we describe tissue engineering of in vitro skeletal muscle consisting of human aligned myofibers with interspersed endothelial networks. To create bio-artificial muscle (BAM), human muscle progenitor cells are cocultured with human umbilical vein endothelial cells (HUVECs) in a fibrin hydrogel. The cell-gel mix is cast into silicone molds with end attachment sites and cultured in endothelial growth medium (EGM-2) for 1 week. The passive forces generated in the contracted hydrogel align the myogenic cells parallel to the long axis of the contracted gel such that they fuse into aligned multinucleated myofibers. This results in the formation of a 2 cm long and ~1.5 mm tick human BAM construct with endothelial networks.

von Willebrand factor deficiency does not influence angiotensin II-induced abdominal aortic aneurysm formation in mice.

Abdominal aortic aneurysm (AAA) refers to a localized dilation of the abdominal aorta that exceeds the normal diameter by 50%. AAA pathophysiology is characterized by progressive inflammation, vessel wall destabilization and thrombus formation. Our aim was to investigate the potential involvement of von Willebrand factor (VWF), a thrombo-inflammatory plasma protein, in AAA pathophysiology using a dissection-based and angiotensin II infusion-induced AAA mouse model. AAA formation was induced in both wild-type and VWF-deficient mice by subcutaneous implantation of an osmotic pump, continuously releasing 1000 ng/kg/min angiotensin II. Survival was monitored, but no significant difference was observed between both groups. After 28 days, the suprarenal aortic segment of the surviving mice was harvested. Both AAA incidence and severity were similar in wild-type and VWF-deficient mice, indicating that AAA formation was not significantly influenced by the absence of VWF. Although VWF plasma levels increased after the infusion period, these increases were not correlated with AAA progression. Also detailed histological analyses of important AAA hallmarks, including elastic degradation, intramural thrombus formation and leukocyte infiltration, did not reveal differences between both groups. These data suggest that, at least in the angiotensin II infusion-induced AAA mouse model, the role of VWF in AAA pathophysiology is limited.

Anti-ADAMTS13 Autoantibodies against Cryptic Epitopes in Immune-Mediated Thrombotic Thrombocytopenic Purpura.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is characterized by severe ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13) deficiency, the presence of anti-ADAMTS13 autoantibodies and an open ADAMTS13 conformation with a cryptic epitope in the spacer domain exposed. A detailed knowledge of anti-ADAMTS13 autoantibodies will help identifying pathogenic antibodies and elucidating the cause of ADAMTS13 deficiency. We aimed at cloning anti-ADAMTS13 autoantibodies from iTTP patients to study their epitopes and inhibitory characteristics. We sorted anti-ADAMTS13 autoantibody expressing B cells from peripheral blood mononuclear cells of 13 iTTP patients to isolate anti-ADAMTS13 autoantibody sequences. Ninety-six B cell clones producing anti-ADAMTS13 autoantibodies were identified from which 30 immunoglobulin M (IgM) and 5 IgG sequences were obtained. For this study, we only cloned, expressed and purified the five IgG antibodies. In vitro characterization revealed that three of the five cloned IgG antibodies, TTP73-1, ELH2-1 and TR8C11, indeed recognize ADAMTS13. Epitope mapping showed that antibodies TTP73-1 and TR8C11 bind to the cysteine-spacer domains, while the antibody ELH2-1 recognizes the T2-T3 domains in ADAMTS13. None of the antibodies inhibited ADAMTS13 activity. Given the recent findings regarding the open ADAMTS13 conformation during acute iTTP, we studied if the cloned antibodies could recognize cryptic epitopes in ADAMTS13. Interestingly, all three antibodies recognize cryptic epitopes. In conclusion, we cloned three anti-ADAMTS13 autoantibodies from iTTP patients that recognize cryptic epitopes. Hence, these data nicely fit our recent finding that the conformation of ADAMTS13 is open during acute iTTP.

Neutrophil extracellular traps in ischemic stroke thrombi.

OBJECTIVE: Neutrophil extracellular traps (NETs) have been shown to promote thrombus formation. Little is known about the exact composition of thrombi that cause ischemic stroke. In particular, no information is yet available on the presence of NETs in cerebral occlusions. Such information is, however, essential to improve current thrombolytic therapy with tissue plasminogen activator (t-PA). This study aimed at investigating the presence of neutrophils and more specifically NETs in ischemic stroke thrombi. METHODS: Sixty-eight thrombi retrieved from ischemic stroke patients undergoing endovascular treatment were characterized by immunostaining using neutrophil markers (CD66b and neutrophil elastase) and NET markers (citrullinated histone H3 [H3Cit] and extracellular DNA). Neutrophils and NETs were quantified. In addition, extracellular DNA was targeted by performing ex vivo lysis of retrieved thrombi with DNase 1 and t-PA. RESULTS: Neutrophils were detected extensively throughout all thrombi. H3Cit, a hallmark of NETs, was observed in almost all thrombi. H3Cit-positive area varied up to 13.45% of total thrombus area. Colocalization of H3Cit with extracellular DNA released from neutrophils confirmed the specific presence of NETs. H3Cit was more abundant in thrombi of cardioembolic origin compared to other etiologies. Older thrombi contained significantly more neutrophils and H3Cit compared to fresh thrombi. Interestingly, ex vivo lysis of patient thrombi was more successful when adding DNase 1 to standard t-PA. INTERPRETATION: Neutrophils and NETs form important constituents of cerebral thrombi. Targeting of NETs with DNase 1 might have prothrombolytic potential in treatment of acute ischemic stroke. Ann Neurol 2017;82:223-232.

ADAMTS13-mediated thrombolysis of t-PA-resistant occlusions in ischemic stroke in mice.

Rapid vascular recanalization forms the basis for successful treatment of cerebral ischemia. Currently, tissue plasminogen activator (t-PA) is the only approved thrombolytic drug for ischemic stroke. However, t-PA does not always result in efficient thrombus dissolution and subsequent blood vessel recanalization. To better understand thrombus composition, we analyzed thrombi retrieved from ischemic stroke patients and found a distinct presence of von Willebrand factor (VWF) in various samples. Thrombi contained on average 20.3% ± 10.1% VWF, and this was inversely correlated with thrombus red blood cell content. We hypothesized that ADAMTS13 can exert a thrombolytic effect in VWF-containing thrombi in the setting of stroke. To test this, we generated occlusive VWF-rich thrombi in the middle cerebral artery (MCA) of mice. Infusion of t-PA did not dissolve these MCA occlusions. Interestingly, administration of ADAMTS13 5 minutes after occlusion dose-dependently dissolved these t-PA-resistant thrombi resulting in fast restoration of MCA patency and consequently reduced cerebral infarct sizes (P < .005). Delayed ADAMTS13 administration 60 minutes after occlusion was still effective but to a lesser extent (P < .05). These data show for the first time a potent thrombolytic activity of ADAMTS13 in the setting of stroke, which might become useful in treatment of acute ischemic stroke.

Endothelial Network Formation Within Human Tissue-Engineered Skeletal Muscle.

The size of in vitro engineered skeletal muscle tissue is limited due to the lack of a vascular network in vitro. In this article, we report tissue-engineered skeletal muscle consisting of human aligned myofibers with interspersed endothelial networks. We extend our bioartificial muscle (BAM) model by coculturing human muscle progenitor cells with human umbilical vein endothelial cells (HUVECs) in a fibrin extracellular matrix (ECM). First, the optimal medium conditions for coculturing myoblasts with HUVECs were determined in a fusion assay. Endothelial growth medium proved to be the best compromise for the coculture, without affecting the myoblast fusion index. Second, both cell types were cocultured in a BAM maintained under tension to stimulate myofiber alignment. We then tested different total cell numbers containing 50% HUVECs and found that BAMs with a total cell number of 2 × 10(6) resulted in well-aligned and densely packed myofibers while allowing for improved interspersed endothelial network formation. Third, we compared different myoblast-HUVEC ratios. Including higher numbers of myoblasts improved endothelial network formation at lower total cell density; however, improvement of network characteristics reached a plateau when 1 × 10(6) or more myoblasts were present. Finally, addition of Matrigel to the fibrin ECM did not enhance overall myofiber and endothelial network formation. Therefore, in our BAM model, we suggest the use of a fibrin extracellular matrix containing 2 × 10(6) cells of which 50-70% are muscle cells. Optimizing these coculture conditions allows for a physiologically more relevant muscle model and paves the way toward engineering of larger in vitro muscle constructs.

Comparative analysis of different peptidyl-prolyl isomerases reveals FK506-binding protein 12 as the most potent enhancer of alpha-synuclein aggregation.

FK506-binding proteins (FKBPs) are members of the immunophilins, enzymes that assist protein folding with their peptidyl-prolyl isomerase (PPIase) activity. Some non-immunosuppressive inhibitors of these enzymes have neuroregenerative and neuroprotective properties with an unknown mechanism of action. We have previously shown that FKBPs accelerate the aggregation of α-synuclein (α-SYN) in vitro and in a neuronal cell culture model for synucleinopathy. In this study we investigated whether acceleration of α-SYN aggregation is specific for the FKBP or even the PPIase family. Therefore, we studied the effect of several physiologically relevant PPIases, namely FKBP12, FKBP38, FKBP52, FKBP65, Pin1, and cyclophilin A, on α-SYN aggregation in vitro and in neuronal cell culture. Among all PPIases tested in vitro, FKBP12 accelerated α-SYN aggregation the most. Furthermore, only FKBP12 accelerated α-SYN fibril formation at subnanomolar concentrations, pointing toward an enzymatic effect. Although stable overexpression of various FKBPs enhanced the aggregation of α-SYN and cell death in cell culture, they were less potent than FKBP12. When FKBP38, FKBP52, and FKBP65 were overexpressed in a stable FKBP12 knockdown cell line, they could not fully restore the number of α-SYN inclusion-positive cells. Both in vitro and cell culture data provide strong evidence that FKBP12 is the most important PPIase modulating α-SYN aggregation and validate the protein as an interesting drug target for Parkinson disease.

The conformation and the aggregation kinetics of α-synuclein depend on the proline residues in its C-terminal region.

The neuronal protein α-synuclein (α-syn) plays a central role in Parkinson's disease (PD). The pathological features of PD are the loss of dopaminergic neurons in the substantia nigra pars compacta and the presence of Lewy bodies. The C-terminal domain of α-syn is characterized by the presence of 15 acidic amino acids and all five proline residues of the protein (P108, P117, P120, P128, and P138). The aggregation of this natively unfolded protein is accelerated in vitro by FK506 binding proteins (FKBPs) showing peptidyl-prolyl cis-trans isomerase activity. These proteins catalyze the cis-trans conformational change of the X-Pro peptide bond, often a rate-limiting step in protein folding. The acceleration of the folding of α-syn by FKBPs may accelerate disease-associated aggregation. To further elucidate the role of the proline residues in the conformation and aggregation of α-syn, we constructed several mutants of α-syn in which one or more proline residues are mutated to alanine via site-directed mutagenesis. For this purpose, we produced and purified His-WT α-syn, a recombinant α-syn with a polyhistidine tag (six His residues) and a linker, and a number of Pro-to-Ala mutants. The aggregation kinetics of these mutants and His-WT α-syn were studied by turbidity, thioflavin T fluorescence, and CD measurements. We can conclude that mutation of the proline residues to alanine accelerates the aggregation kinetics of α-syn while all proline mutants formed fibrils similar to His-WT α-syn, as visualized via transmission electron microscopy. We also demonstrate that the accelerating effect of hFKBP12 is abolished via removal of the proline residues from the C-terminus. Finally, we show that the mutant of His α-syn with all five proline residues mutated to alanine is more structured (more α-helix) than His-WT α-syn, indicating the role of the Pro residues as potential helix breakers in the inhibitory conformation of the C-terminus.

FK506 binding protein 12 differentially accelerates fibril formation of wild type alpha-synuclein and its clinical mutants A30P or A53T.

Aggregation of alpha-synuclein (alpha-SYN) plays a key role in Parkinson's disease. We have previously shown that aggregation of alpha-SYN in vitro is accelerated by addition of FK506 binding proteins (FKBP) and that this effect can be counteracted by FK506, a specific inhibitor of these enzymes. In this paper, we investigated in detail the effect of FKBP12 on early aggregation and on fibril formation of wild-type, A53T and A30P alpha-SYN. FKBP12 has a much smaller effect on the fibril formation of these two clinical mutants alpha-SYN. Using an inactive enzyme, we were able to discriminate between catalytic and non-catalytic effects that differentially influence the two processes. A model explaining non-linear concentration dependencies is proposed.

Differential interaction of HIV-1 integrase and JPO2 with the C terminus of LEDGF/p75.

The transcriptional co-activator lens epithelium-derived growth factor (LEDGF) has been shown to protect cells against environmental stress. The protein has been implicated in auto-immunity and cancer, and is present in cells as the p52 or p75 splice variant. Recently, LEDGF/p75, but not p52, was identified as the prominent interaction partner of human immunodeficiency virus type 1 (HIV-1) integrase. This interaction of HIV-1 integrase with the C-terminal integrase-binding domain of LEDGF/p75 is crucial for HIV-1 replication. To gain insight into the cell biology of LEDGF/p75, we were interested in identifying cellular binding partners of its C-terminal domain. By yeast-two-hybrid screening with a CEMC7 cDNA-library, we were able to identify JPO2 as a binding partner of the C-terminal part of LEDGF/p75. The specific interaction between JPO2 and LEDGF/p75 was verified by pull-down, AlphaScreen, and co-immunoprecipitation. Competition assays using recombinant proteins show a mutually exclusive binding of either JPO2 or HIV-1 integrase to LEDGF/p75. However, differing mechanisms of binding were suggested by continuing interaction of JPO2 with some LEDGF/p75 mutants (I365A, D366A, F406A) that are totally defective for interaction with HIV-1 integrase. This finding is of significance for the development of specific inhibitors targeting only the interaction between LEDGF/p75 and HIV-1 integrase, without disturbing interaction with other cellular factors. Over-expression of JPO2 resulted in a modest but reproducible inhibition of HIV-1 replication, consistent with competition between integrase and JPO2 for binding to LEDGF/p75. Furthermore, JPO2 over-expression activated transcription from the HIV-1 LTR.

Identification of the LEDGF/p75 binding site in HIV-1 integrase.

Lens epithelium-derived growth factor (LEDGF)/p75 is an important cellular co-factor for human immunodeficiency virus (HIV) replication. We originally identified LEDGF/p75 as a binding partner of integrase (IN) in human cells. The interaction has been mapped to the integrase-binding domain (IBD) of LEDGF/p75 located in the C-terminal part. We have subsequently shown that IN carrying the Q168A mutation remains enzymatically active but is impaired for interaction with LEDGF/p75. To map the integrase/LEDGF interface in more detail, we have now identified and characterized two regions within the enzyme involved in the interaction with LEDGF/p75. The first region centers around residues W131 and W132 while the second extends from I161 up to E170. For the different IN mutants the interaction with LEDGF/p75 and the enzymatic activities were determined. IN(W131A), IN(I161A), IN(R166A), IN(Q168A) and IN(E170A) are impaired for interaction with LEDGF/p75, but retain 3' processing and strand transfer activities. Due to impaired integration, an HIV-1 strain containing the W131A mutation in IN displays reduced replication capacity, whereas virus carrying IN(Q168A) is replication defective. Comparison of the wild-type IN-LEDGF/p75 co-crystal structure with that of the modelled structure of the IN(Q168A) and IN(W131A) mutant integrases corroborated our experimental data.

The aggregation of alpha-synuclein is stimulated by FK506 binding proteins as shown by fluorescence correlation spectroscopy.

Aggregation of alpha-synuclein (alpha-SYN) plays a key role in Parkinson's disease (PD). We have used fluorescence correlation spectroscopy (FCS) to study alpha-SYN aggregation in vitro and discovered that this process is clearly accelerated by addition of FK506 binding proteins (FKBPs). This effect was observed both with E. coli SlyD FKBP and with human FKBP12 and was counteracted by FK506, a specific inhibitor of FKBP. The alpha-SYN aggregates formed in the presence of FKBP12 showed fibrillar morphology. The rotamase activity of FKBP apparently accelerates the folding and subsequent aggregation of alpha-SYN. Since FK506 and other non-immunosuppressive FKBP inhibitors are known to display neuroregenerative and neuroprotective properties in disease models, the observed inhibition of rotamase activity and alpha-SYN aggregation, may explain their mode of action. Our results open perspectives for the treatment of PD with immunophilin ligands that inhibit a specific member of the FKBP family.

Biotinylated Stealth magnetoliposomes.

Dimyristoylphosphatidylethanolamine (DC(14:0)PE) and the dioleoyl analogue (DC(18:1cis)PE) were mixed with alpha-biotinylamido-omega-N-succinimidoxycarbonyl-poly(ethylene glycol) (NHS-PEG-biotin) and quantitatively converted to alpha-biotinylamido-omega-(dimyristoylphosphatidylethanolamino-carbonyl)polyethylene glycol (DC(14:0)PE-PEG-biotin) and the dioleoyl analogue DC(18:1cis)PE-PEG-biotin, respectively. As shown by thin-layer chromatography and 1H NMR spectroscopy, PEGylation of both phosphatidylethanolamine types went to completion if the reaction was performed in organic solvent in the presence of triethylamine. The resulting derivatives were successfully incorporated into both classical phospholipid vesicles and a phospholipid bilayer surrounding nanometer-sized magnetite cores. In the latter case, the so-called activated Stealth(1) magnetoliposomes were produced which very efficiently immobilized streptavidinylated alkaline phosphatase.