Generation of Anti-Murine ADAMTS13 Antibodies and Their Application in a Mouse Model for Acquired Thrombotic Thrombocytopenic Purpura

<div><p>Thrombotic thrombocytopenic purpura (TTP) is a life-threatening thrombotic microangiopathy linked to a deficiency in the metalloprotease ADAMTS13. In the current study, a novel mouse model for acquired TTP was generated to facilitate development and validation of new therapies for this disease. Therefore, a large panel (n = 19) of novel anti-mouse ADAMTS13 (mADAMTS13) monoclonal antibodies (mAbs) of mouse origin was generated. Inhibitory anti-mADAMTS13 mAbs were identified using the FRETS-VWF73 assay. Four mAbs strongly inhibited mADAMTS13 activity <i>in vitro</i> (∼68–90% inhibition). Injecting a combination of 2 inhibitory mAbs (13B4 and 14H7, 1.25 mg/kg each) in <i>Adamts13</i>^<i>+/+</i> mice resulted in full inhibition of plasma ADAMTS13 activity (96 ± 4% inhibition, day 1 post injection), leading to the appearance of ultra-large von Willebrand factor (UL-VWF) multimers. Interestingly, the inhibitory anti-mADAMTS13 mAbs 13B4 and 14H7 were ideally suited to induce long-term ADAMTS13 deficiency in <i>Adamts13</i>^<i>+/+</i> mice. A single bolus injection resulted in full <i>ex vivo</i> inhibition for more than 7 days. As expected, the mice with the acquired ADAMTS13 deficiency did not spontaneously develop TTP, despite the accumulation of UL-VWF multimers. In line with the <i>Adamts13</i>^-/- mice, TTP-like symptoms could only be induced when an additional trigger (rVWF) was administered. On the other hand, the availability of our panel of anti-mADAMTS13 mAbs allowed us to further develop a sensitive ELISA to detect ADAMTS13 in mouse plasma. In conclusion, a novel acquired TTP mouse model was generated through the development of inhibitory anti-mADAMTS13 mAbs. Consequently, this model provides new opportunities for the development and validation of novel treatments for patients with TTP. In addition, these newly developed inhibitory anti-mADAMTS13 mAbs are of great value to specifically study the role of ADAMTS13 in mouse models of thrombo-inflammatory disease.</p></div

The role of platelet and endothelial GARP in thrombosis and hemostasis

<div><p>Background</p><p>Glycoprotein-A Repetitions Predominant protein (GARP or LRRC32) is present on among others human platelets and endothelial cells. Evidence for its involvement in thrombus formation was suggested by full knockout of GARP in zebrafish.</p><p>Objectives</p><p>To evaluate the role of GARP in platelet physiology and in thrombus formation using platelet and endothelial conditional GARP knock out mice.</p><p>Methods</p><p>Platelet and endothelial specific GARP knockout mice were generated using the Cre-loxP recombination system. The function of platelets without GARP was measured by flow cytometry, spreading analysis and aggregometry using PAR4-activating peptide and collagen related peptide. Additionally, clot retraction and collagen-induced platelet adhesion and aggregation under flow were analyzed. Finally, in vivo tail bleeding time, occlusion time of the mesenteric and carotid artery after FeCl3-induced thrombosis were determined in platelet and endothelial specific GARP knock out mice.</p><p>Results</p><p>Platelet specific GARP knockout mice had normal surface GPIb, GPVI and integrin αIIb glycoprotein expression. Although GARP expression was increased upon platelet activation, platelets without GARP displayed normal agonist induced activation, spreading on fibrinogen and aggregation responses. Furthermore, absence of GARP on platelets did not influence clot retraction and had no impact on thrombus formation on collagen-coated surfaces under flow. In line with this, neither the tail bleeding time nor the occlusion time in the carotid- and mesenteric artery after FeCl3-induced thrombus formation in platelet or endothelial specific GARP knock out mice were affected.</p><p>Conclusions</p><p>Evidence is provided that platelet and endothelial GARP are not important in hemostasis and thrombosis in mice.</p></div

Unaltered bleeding time and thrombus formation in <i>Pf4</i> specific GARP cKO mice.

<p>(<b>A</b>) 2 mm of the tail tip was dissected and tail bleeding times were measured (n = 10). (<b>B</b>) Carotid artery was injured using topical application of 12% FeCl₃ and blood flow was monitored using a flow probe until flow stopped due to the formation of an occlusive thrombus (n = 10). (<b>C</b>) Mesenteric arteries were injured using 10% FeCl₃ and thrombus formation was followed using intravital microscopy until full occlusion was reached (n = 10). Representative pictures after 5, 8 and 10 min of mesenteric thrombus formation are shown on the right. Scale bars represent 50 μm. <b>(D)</b> The right middle cerebral artery of littermate mice (n = 14) and platelet specific GARP knockout mice (n = 12) was occluded during 60 min and reperfusion was allowed during 23 hours.</p

Epitope mapping and epitope overview of the developed anti-mADAMTS13 mAbs.

<p>The epitope of each anti-mADAMTS13 mAb was mapped against both mMDTCS (A) and mT2-CUB2 (B). Individual anti-mADAMTS13 mAbs were coated, recombinant mMDTCS (A) or mT2-CUB2 (B) were added and binding of the respective mADAMTS13 variant was detected using the polyclonal anti-mADAMTS13 rabbit IgG and GAR-HRP. Black and white bars represent respectively anti-mMDTCS and anti-mT2-CUB2 mAbs. The previously reported mAb 20A10 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160388#pone.0160388.ref031" target="_blank">31</a>] was used as a positive (A) and negative (B) control. (C) Epitope overview of the developed anti-mADAMTS13 mAbs. The previously developed mAb 20A10 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160388#pone.0160388.ref031" target="_blank">31</a>] is marked by a dark frame.</p

Schematic representation of the mADAMTS13 variants used in this study.

<p>The proximal (MDTCS) domains are represented in black and include a metalloprotease (M), disintegrin-like (D), thrombospondin type-1 repeat (T1), cysteine-rich (C) and spacer (S) domain. Distal (T2-CUB2) domains are represented in white and consist of seven thrombospondin type-1 repeats (T2 up to T8) and two CUB (Complement component C1r/C1s, Urinary epidermal growth factor (Uegf) and Bone morphogenic protein-1) domains.</p

GARP deficiency in platelets does not affect platelet aggregation.

<p>Aggregation was induced in PRP of GARP^+/+ and GARP cKO mice using 75, 125 or 250 μM PAR4-AP (<b>A</b>) and 0.75, 2.5 or 5 μg/mL CRP (<b>C</b>). Maximal light transmission was calculated and graphed of at least 3 individual experiments. Representative curves for 125 μM PAR4-AP (<b>B</b>) and 2.5 μg/mL CRP (<b>D</b>) induced aggregations in GARP^+/+ (grey) and GARP cKO (black) are shown. (<b>E</b>) Clot retraction of PRP supplemented with erythrocytes was induced using 5 U/mL thrombin and 20 mM CaCl₂ in GARP^+/+ littermates and GARP cKO. Pictures were made every two minutes and clot area was analyzed using ImageJ. Representative pictures are shown on the right. Measured clot area is presented on the right; n = 3. (<b>F</b>) Whole blood of GARP cKO mice and GARP^+/+ littermates was perfused over a Horm collagen (100 μg/mL) coated coverslip at a shear rate of 1600 s^-1. Thrombus formation was visualized during 5 min and surface coverage was quantified using ImageJ; n = 3. Representative pictures of thrombus formation under flow are shown on the right; scale bars represent 50 μm.</p

A subset of anti-mMDTCS mAbs inhibit plasma mADAMTS13 activity.

<p>The effect of anti-mADAMTS13 mAbs on the proteolytic activity of plasma mADAMTS13 was tested <i>in vitro</i> using the FRETS-VWF73 assay. (A) All anti-mADAMTS13 mAbs, both anti-mMDTCS and anti-mT2-CUB2 mAbs, were tested. For each condition, the activity of plasma mADAMTS13 was calculated using a calibration curve of 2.5%, 5%, 10% and 15% NMP. (B) Time profile of the cleavage of the FRETS-VWF73 substrate by plasma mADAMTS13, in the absence or presence of mAbs 13B4 and/or 14H7. Error bars represent the SD of at least three independently performed experiments.</p

GARP expression is elevated upon platelet activation but does not affect outside-in and inside-out signaling.

<p>MFI of P-selectin (anti-CD62P-PE) (<b>A-B</b>), JON/A (anti-GPIIbIIIa-PE) (<b>C-D</b>) and fibrinogen binding (<b>E-F</b>) to unactivated or 200 μM PAR4-AP (<b>A-C-E</b>) or 2.5 μg/mL CRP (<b>B-D-F</b>) activated platelets of littermates and cKO mice are measured using flowcytometry. Platelets were gated based on CD41 (anti-CD41-FITC) expression. Graphs show mean ± SD, n = 3. Washed platelets of littermates and cKO mice were allowed to spread on 100 μg/mL fibrinogen while activated with 200 μM PAR4-AP (<b>G</b>) or 2.5 μg/mL CRP (<b>H</b>) during 5, 10 or 30 min. Platelets are divided in different phases of activation (1) round, (2) only filopodia, (3) filopodia and lamellipodia, (4) fully spread. (<b>I</b>) Representative images of platelet spreading; scale bars represent 5 μm.</p

Generation of platelet specific GARP knockout mice.

<p>GARP and P-selectin expression on 700 μM PAR4-AP (<b>A</b>) or 20 μg/mL CRP (<b>B</b>) activated murine platelets. Mean fluorescence intensity (MFI) of anti-GARP-APC and anti-P-selectin CD62P-PE are shown. <b>(C)</b> Genotypic analysis of genomic DNA from control C57BL/6, littermates and platelet specific cKO mice. <i>Loxp</i> is 181 bp (fl/fl); wt allele is 111 bp, <i>Cre</i> 250 bp. (<b>D</b>) Phenotypic characterization of littermates and cKO mice using flow cytometry. Platelets were labeled with anti-CD41-FITC and anti-GARP-APC and a histogram was made based on APC intensity of CD41+ cells: isotype control (black line), littermates (grey line) and cKO (black).</p

Generation of endothelial specific GARP knockout mice.

<p>(<b>A</b>) Phenotypic characterization of littermates and cKO mice using flow cytometry. Single cells were labeled with anti-CD45-PeCy7, anti-CD31-PE, anti-CD41-FITC and anti-GARP-APC and a histogram was made based on APC intensity of CD45-CD31+CD41- population. Isotype control (black line), littermates (grey line) and cKO (black). (<b>B</b>) Genotypic analysis of genomic DNA from control C57BL/6, littermates and endothelial specific cKO mice. <i>Loxp</i> is 181 bp (fl/fl); wt allele is 111 bp, <i>Cre</i> 450 bp.</p