Journal: Blood

Article Title: High shear-dependent loss of membrane integrity and defective platelet adhesion following disruption of the GPIb?-filamin interaction

doi: 10.1182/blood-2010-07-296194

Figure Lengend Snippet: Caspase-dependent cleavage of cytoskeletal proteins induces a shear-specific defect in platelet membrane tether stability. (A) DiIC12-labeled washed human platelets in the presence of 1 mg/mL ristocetin were perfused over human VWF-coated microchannels and allowed to adhere for 10 minutes in the absence of shear. Shear was applied (at a rate of 600 or 1800 s−1), and platelet adhesive behavior was captured using Metamorph software. Single frame fluorescence images (after subjecting to a low pass filter of 3) show the formation of long membrane tethers from ABT-737–treated (1μM) platelets, which is prevented by pretreatment with Q-VD-OPh (50μM). Scale bar represents 10 μm. (B) Quantitation of mean tether length from 3 independent experiments demonstrates a significant increase in membrane tether length in ABT-737–treated platelets compared with control and QV-D-OPh–treated platelets. ***P < .001. (C) DIC images of platelets demonstrating membrane tethers and the loss of discoid platelet morphology after treatment with ABT-737 and prevention by Q-VD-OPh are shown under the same shear conditions as in panel A. Scale bar represents 10 μm.

Article Snippet: Samples were washed and fixed as initially described in this paragraph, and DIC and fluorescent images were captured and overlayed using Metamorph software.

Techniques: Shear, Membrane, Labeling, Adhesive, Software, Fluorescence, Quantitation Assay, Control

Journal: Blood

Article Title: High shear-dependent loss of membrane integrity and defective platelet adhesion following disruption of the GPIb?-filamin interaction

doi: 10.1182/blood-2010-07-296194

Figure Lengend Snippet: Adhesion and spreading of transgenic mouse platelets to immobilized VWF under static conditions is unaffected by loss of filamin A interaction. (A) hGPIbαWT and hGPIbαFW platelets were allowed to adhere to human VWF (5 μg/mL) for 60 minutes in the presence of ristocetin (1.5 mg/mL), botrocetin (5 μg/mL), or botrocetin + Xia.B2 murine GPIbα blocking antibody (20 μg/mL). Results are mean (± SEM) from at least 4 independent experiments and demonstrate no significant difference in the total number of adherent platelets under all conditions tested. Ristocetin-dependent adhesion and botrocetin-dependent adhesion in the presence of Xia.B2 are identical and represent adhesion to human VWF mediated specifically by hGPIbα. (B) Platelet spreading on VWF (+ 1.5 mg/mL ristocetin) was analyzed from DIC images (at least 3 separate fields from 3 independent experiments) and demonstrated no significant difference between hGPIbαWT and hGPIbαFW platelets. Platelets treated with inhibitors of ADP and thromboxane (1 U/mL apyrase, a combination of 10μM MeSAMP and 100μM MRS2179, or MeSAMP/MRS2179 and 10μM indomethacin) demonstrated that integrin αIIbβ3 activation occurred directly downstream from GPIbα without a contribution from these secondary mediators. (C) Oregon Green fibrinogen binding to platelets adherent to human VWF (in the presence of 1.5 mg/mL ristocetin) was no different between hGPIbαWT and hGPIbαFW mice, indicating that integrin αIIbβ3 activation is unaffected by the loss of filamin A binding to GPIbα. DIC and fluorescent images were overlayed using Metamorph and are representative of 4 independent experiments. Scale bar represents 10 μm.

Article Snippet: Samples were washed and fixed as initially described in this paragraph, and DIC and fluorescent images were captured and overlayed using Metamorph software.

Techniques: Transgenic Assay, Blocking Assay, Activation Assay, Binding Assay