Incorporation of albumin fusion proteins into fibrin clots in vitro and in vivo: comparison of different fusion motifs recognized by factor XIIIa

<p>Abstract</p> <p>Background</p> <p>The transglutaminase activated factor XIII (FXIIIa) acts to strengthen pathological fibrin clots and to slow their dissolution, in part by crosslinking active α₂-antiplasmin (α₂AP) to fibrin. We previously reported that a yeast-derived recombinant fusion protein comprising α₂AP residues 13-42 linked to human serum albumin (HSA) weakened <it>in vitro </it>clots but failed to become specifically incorporated into <it>in vivo </it>clots. In this study, our aims were to improve both the stability and clot localization of the HSA fusion protein by replacing α₂AP residues 13-42 with shorter sequences recognized more effectively by FXIIIa.</p> <p>Results</p> <p>Expression plasmids were prepared encoding recombinant HSA with the following N-terminal 23 residue extensions: H₆NQEQVSPLTLLAG₄Y (designated XL1); H₆DQMMLPWAVTLG₄Y (XL2); H₆WQHKIDLPYNGAG₄Y (XL3); and their 17 residue non-His-tagged equivalents (XL4, XL5, and XL6). The HSA moiety of XL4- to XL6-HSA proteins was C-terminally His-tagged. All chimerae were efficiently secreted from transformed <it>Pichia pastoris </it>yeast except XL3-HSA, and following nickel chelate affinity purification were found to be intact by amino acid sequencing, as was an N-terminally His-tagged version of α₂AP(13-42)-HSA. Of the proteins tested, XL5-HSA was cross-linked to biotin pentylamine (BPA) most rapidly by FXIIIa, and was the most effective competitor of α₂AP crosslinking not only to BPA but also to plasma fibrin clots. In the mouse ferric chloride <it>vena cava </it>thrombosis model, radiolabeled XL5-HSA was retained in the clot to a greater extent than recombinant HSA. In the rabbit jugular vein stasis thrombosis model, XL5-HSA was also retained in the clot, in a urea-insensitive manner indicative of crosslinking to fibrin, to a greater extent than recombinant HSA.</p> <p>Conclusions</p> <p>Fusion protein XL5-HSA (DQMMLPWAVTLG₄Y-HSAH₆) was found to be more active as a substrate for FXIIIa-mediated transamidation than seven other candidate fusion proteins <it>in vitro</it>. The improved stability and reactivity of this chimeric protein was further evidenced by its incorporation into <it>in vivo </it>clots formed in thrombosis models in both mice and rabbits.</p

In Vivo Near-Infrared Imaging of Fibrin Deposition in Thromboembolic Stroke in Mice

imaging of activated factor XIII (FXIIIa), an important mediator of thrombosis or fibrinolytic resistance. The present study was to investigate the fibrin deposition in a thromboembolic stroke mice model by FXIIIa–targeted near-infrared fluorescence (NIRF) imaging., which were correlated with histology after animal euthanasia. NIRF images and lesion volume.Non-invasive detection of fibrin deposition in ischemic mouse brain using NIRF imaging is feasible and this technique may provide an in vivo experimental tool in studying the role of fibrin in stroke

Detection of thrombus size and protein content by <it>ex vivo</it> magnetization transfer and diffusion weighted MRI

<p>Abstract</p> <p>Background</p> <p>To utilize a rabbit model of plaque disruption to assess the accuracy of different magnetic resonance sequences [T1-weighted (T1W), T2-weighted (T2W), magnetization transfer (MT) and diffusion weighting (DW)] at 11.7 T for the <it>ex vivo</it> detection of size and composition of thrombus associated with disrupted plaques.</p> <p>Methods</p> <p>Atherosclerosis was induced in the aorta of male New Zealand White rabbits (n = 17) by endothelial denudation and high-cholesterol diet. Subsequently, plaque disruption was induced by pharmacological triggering. Segments of infra-renal aorta were excised fixed in formalin and examined by <it>ex vivo</it> magnetic resonance imaging (MRI) at 11.7 T and histology.</p> <p>Results</p> <p>MRI at 11.7 T showed that: <it>(i)</it> magnetization transfer contrast (MTC) and diffusion weighted images (DWI) detected thrombus with higher sensitivity compared to T1W and T2W images [sensitivity: MTC = 88.2%, DWI = 76.5%, T1W = 66.6% and T2W = 43.7%, <it>P</it> < 0.001]. Similarly, the contrast-to-noise (CNR) between the thrombus and the underlying plaque was superior on the MTC and DWI images [CNR: MTC = 8.5 ± 1.1, DWI = 6.0 ± 0.8, T1W = 1.8 ± 0.5, T2W = 3.0 ± 1.0, <it>P</it> < 0.001]; <it>(ii)</it> MTC and DWI provided a more accurate detection of thrombus area with histology as the gold-standard [underestimation of 6% (MTC) and 17.6% (DWI) compared to an overestimation of thrombus area of 53.7% and 46.4% on T1W and T2W images, respectively]; <it>(iii)</it> the percent magnetization transfer rate (MTR) correlated with the fibrin (<it>r</it> = 0.73, <it>P</it> = 0.003) and collagen (<it>r</it> = 0.9, <it>P</it> = 0.004) content of the thrombus.</p> <p>Conclusions</p> <p>The conspicuity of the thrombus was increased on MTC and DW compared to T1W and T2W images. Changes in the %MTR and apparent diffusion coefficient can be used to identify the organization stage of the thrombus.</p