Journal: Journal of Thrombosis and Haemostasis

Article Title: A gain‐of‐function filamin A mutation in mouse platelets induces thrombus instability

doi: 10.1111/jth.15864

Figure Lengend Snippet: Schematic representation of the mutant humanized FlnA cloning strategy and FlnA expression in platelets of male mice. AThe region of the mouse Flna gene targeted for homologous recombination is shown. Flna WT includes exons from #38 to 45 (hashed boxes). Exon 45 shows the relative position of the stop codon and the non‐coding 3′ sequence of the exon is drawn as a white box. Flna loxP shows the FLNA gene after recombination of the exon38‐‐45 region with a construct including mouse exon 45 with an upstream lox P site (blue arrowhead), and followed by the neomycin (“neo”) negative selection gene and a second exon 45 harboring the downstream loxP site (blue arrowhead), the human stop mutation (red * ) followed by the 3′ 300 bp human sequence (black box), which includes an in‐frame second stop codon, shown on the drawing. Flna loxP PF4‐Cre + shows the same exon 38–45 region after expression of the Cre recombinase in the platelet–megakaryocyte lineage: deletion of the loxP–loxP fragment (shown by the red lines and the “Cre recombination” mention), leads to swapping of the wild‐type exon 45 for the mutated one. B, PF4‐Cre + males were crossed with Flna LoxP females. PF4‐Cre genomic insertion in Flna loxP male offsprings was investigated by genomic polymerase chain reaction and agarose gel electrophoresis. C, Percentage of male over female mice during breeding. D, In a population of 200 mice, distribution of males and females and of Flna loxP PF4‐Cre + versus Flna loxP PF4‐Cre − males. E, F, FlnA expression in Flna loxP PF4‐Cre + and Flna loxP PF4‐Cre − platelets as assessed by western blotting. The graph represents the means ± standard error of the mean of four independent Flna loxP PF4‐Cre + male mice and four independent Flna loxP PF4‐Cre − male mice, ** p < .01; *** p < .001 (unpaired Student t ‐test)

Article Snippet: A rabbit polyclonal antibody was raised against the extended C‐terminal FLNa sequence of a patient by GenScript, from a mix of two synthetic FLNa peptides (APPSSPQPRPRPPWC and DGGPVHNHPLVLFSSQEEC) coupled to the carrier protein keyhole limpet hemocyanin, followed by affinity purification of the IgG fraction.

Techniques: Mutagenesis, Cloning, Expressing, Homologous Recombination, Sequencing, Construct, Selection, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot

Journal: Journal of Thrombosis and Haemostasis

Article Title: A gain‐of‐function filamin A mutation in mouse platelets induces thrombus instability

doi: 10.1111/jth.15864

Figure Lengend Snippet: Hematological parameters

Article Snippet: A rabbit polyclonal antibody was raised against the extended C‐terminal FLNa sequence of a patient by GenScript, from a mix of two synthetic FLNa peptides (APPSSPQPRPRPPWC and DGGPVHNHPLVLFSSQEEC) coupled to the carrier protein keyhole limpet hemocyanin, followed by affinity purification of the IgG fraction.

Techniques:

Journal: Journal of Thrombosis and Haemostasis

Article Title: A gain‐of‐function filamin A mutation in mouse platelets induces thrombus instability

doi: 10.1111/jth.15864

Figure Lengend Snippet: Mouse tail bleeding time. A, Tail bleeding was assessed in Flna loxP PF4‐Cre + (mutated FlnA) and Flna loxP PF4‐Cre − mice (wild‐type FlnA). B, Quantification of rebleedings is expressed as mean ± standard error of the mean of 11 Flna loxP PF4‐Cre − male mice and 13 Flna loxP PF4‐Cre + male mice

Article Snippet: A rabbit polyclonal antibody was raised against the extended C‐terminal FLNa sequence of a patient by GenScript, from a mix of two synthetic FLNa peptides (APPSSPQPRPRPPWC and DGGPVHNHPLVLFSSQEEC) coupled to the carrier protein keyhole limpet hemocyanin, followed by affinity purification of the IgG fraction.

Techniques:

Journal: Journal of Thrombosis and Haemostasis

Article Title: A gain‐of‐function filamin A mutation in mouse platelets induces thrombus instability

doi: 10.1111/jth.15864

Figure Lengend Snippet: Aggregation, secretion, and αIIbβ3 activation induced by thrombin. A, B, Aggregation of washed Flna loxP PF4‐Cre − (Ctrl) and Flna loxP PF4‐Cre + platelets was induced by thrombin (30, 40, and 80 mU/ml). C, Dense granule secretion measured after 3 min of platelet aggregation was quantified by measuring adenosine triphosphate release. D, E, Quantification of P‐selectin exposure and αIIbβ3 activation in conditions of unstirred platelets was assessed by flow cytometry using an anti‐P‐selectin antibody and the JON/A monoclonal antibody specific for the activated form of αIIbβ3. The graph represents the means ± standard error of the mean of three independent determinations. * p < .05, *** p < .001 (one‐way analysis of variance with Sidak's post hoc test for multiple comparisons)

Article Snippet: A rabbit polyclonal antibody was raised against the extended C‐terminal FLNa sequence of a patient by GenScript, from a mix of two synthetic FLNa peptides (APPSSPQPRPRPPWC and DGGPVHNHPLVLFSSQEEC) coupled to the carrier protein keyhole limpet hemocyanin, followed by affinity purification of the IgG fraction.

Techniques: Activation Assay, Flow Cytometry

Journal: Journal of Thrombosis and Haemostasis

Article Title: A gain‐of‐function filamin A mutation in mouse platelets induces thrombus instability

doi: 10.1111/jth.15864

Figure Lengend Snippet: Cell signaling induced by convulxin. Western blotting of phosphorylated Syk (A), LAT (B), and PKC substrates (C) of platelets activated for 5 min by various doses of Cvx (100 to 400 pM) in the presence of apyrase (5 U/ml) and indomethacin (5 μM). Phosphorylation levels were normalized in relation to the expression level of total Syk (A), total LAT (B) of 14–3‐3ζ (C), and then compared with each other to the phosphorylation of Flna loxP PF4‐Cre − (Ctrl) at 400 pM Cvx which was set to 100%. The graphs represent the means of phosphorylation ± standard error of the mean of at least three independent determinations. ** p < .01, *** p < .001 (one‐way analysis of variance with Sidak's post hoc test for multiple comparisons)

Article Snippet: A rabbit polyclonal antibody was raised against the extended C‐terminal FLNa sequence of a patient by GenScript, from a mix of two synthetic FLNa peptides (APPSSPQPRPRPPWC and DGGPVHNHPLVLFSSQEEC) coupled to the carrier protein keyhole limpet hemocyanin, followed by affinity purification of the IgG fraction.

Techniques: Western Blot, Phospho-proteomics, Expressing

Journal: Journal of Thrombosis and Haemostasis

Article Title: A gain‐of‐function filamin A mutation in mouse platelets induces thrombus instability

doi: 10.1111/jth.15864

Figure Lengend Snippet: In vivo thrombus instability of Flna loxP PF4‐Cre + mice. The impact of the FlnA mutation on thrombus formation was investigated in vitro . A, A whole‐blood perfusion assay was carried out on collagen matrix (50 μg/ml) at an arterial shear rate (1500 s −1 ). Thrombus formation was evaluated by fluorescence microscopy and the thrombus size was quantified by measurement of the mean fluorescence intensity (MFI) of thrombi. B‐D, In vivo thrombosis was assessed in exposed mesenteric vessels (venules and arterioles) after FeCl 3 ‐induced injury. Thrombus formation of fluorescently labeled platelets was monitored by intravital videomicroscopy. The graphs represent (B) the time point of the formation of the first thrombus of 30 μm, (C) the occlusion time defined as the stopping blood flow for at least 1 min, and (D) the number of emboli shedding from thrombi in venules throughout the procedure. The graph represents the means ± standard error of the mean of eight Flna loxP PF4‐Cre + and seven Flna loxP PF4‐Cre − mice. Statistical difference between Flna loxP PF4‐Cre − mice and Flna loxP PF4‐Cre + mice was evaluated by the unpaired Student t ‐test (** p < .01)

Article Snippet: A rabbit polyclonal antibody was raised against the extended C‐terminal FLNa sequence of a patient by GenScript, from a mix of two synthetic FLNa peptides (APPSSPQPRPRPPWC and DGGPVHNHPLVLFSSQEEC) coupled to the carrier protein keyhole limpet hemocyanin, followed by affinity purification of the IgG fraction.

Techniques: In Vivo, Mutagenesis, In Vitro, Shear, Fluorescence, Microscopy, Labeling