Figure 1
Figure 1. Shear induces loss of platelet GPVI and release of sGPVI in PRP. (A) PRP from a single donor was subjected to a shear rate of 7500 seconds−1 for up to 10 minutes in the presence of an inhibitor of αIIbβ3, or treated with 5mM NEM for 15 minutes. Aliquots of plasma and PRP were isolated for measurement of sGPVI by ELISA (light bars) and platelet surface levels of GPVI by flow cytometry (black bars), respectively. Data are expressed as percentage of total intact GPVI on resting platelets, or sGPVI released from platelets treated with 5mM NEM. (B) Citrated PRP from a single donor was left untreated or subjected to different shear rates for 5 minutes or (C) subjected to a shear rate of 10 000 seconds−1 for indicated times, or (D) subjected to 10 000-second−1 shear rate for 5 minutes in the presence of 100μM GM6001 or 2μM GI254023. Platelets were pelleted by centrifugation and assayed for sGPVI by ELISA in triplicate (sGPVI is shown as mean ± SD). Data are representative of at least 3 independent experiments using different single donors. (E) Human washed platelets in Tyrode buffer containing 10μM ADAM-cleavable quenched fluorogenic peptide were left untreated or exposed to 10 000-second−1 shear for 1 minute in the presence or absence of 2μM GI254023 or 10mM EDTA. Fluorescence activity (320/405 nm) at 30 minutes was quantified, and all values were corrected for background fluorescence measured in an untreated platelet sample containing substrate alone. Data are representative of at least 3 independent experiments using different single donors. (F) Human washed platelets (5 × 108/mL) were resuspended in Tyrode buffer and left untreated (NT) or treated with 5mM NEM as a positive control, or with 10 μg/mL collagen in the absence or presence of 2μM GI254023 (GI) for 1 hour at room temperature and then lysed and subjected to SDS-PAGE and Western blotted with anti-GPVI cytoplasmic tail antibody. (G) Human washed platelets (5 × 108/mL) were lysed and subjected to SDS-PAGE and Western blotted with anti-ADAM10 cytoplasmic tail antibody (ADAM10 CT) or anti-Syk (Syk). Vertical lines indicate a repositioned lane.

Shear induces loss of platelet GPVI and release of sGPVI in PRP. (A) PRP from a single donor was subjected to a shear rate of 7500 seconds−1 for up to 10 minutes in the presence of an inhibitor of αIIbβ3, or treated with 5mM NEM for 15 minutes. Aliquots of plasma and PRP were isolated for measurement of sGPVI by ELISA (light bars) and platelet surface levels of GPVI by flow cytometry (black bars), respectively. Data are expressed as percentage of total intact GPVI on resting platelets, or sGPVI released from platelets treated with 5mM NEM. (B) Citrated PRP from a single donor was left untreated or subjected to different shear rates for 5 minutes or (C) subjected to a shear rate of 10 000 seconds−1 for indicated times, or (D) subjected to 10 000-second−1 shear rate for 5 minutes in the presence of 100μM GM6001 or 2μM GI254023. Platelets were pelleted by centrifugation and assayed for sGPVI by ELISA in triplicate (sGPVI is shown as mean ± SD). Data are representative of at least 3 independent experiments using different single donors. (E) Human washed platelets in Tyrode buffer containing 10μM ADAM-cleavable quenched fluorogenic peptide were left untreated or exposed to 10 000-second−1 shear for 1 minute in the presence or absence of 2μM GI254023 or 10mM EDTA. Fluorescence activity (320/405 nm) at 30 minutes was quantified, and all values were corrected for background fluorescence measured in an untreated platelet sample containing substrate alone. Data are representative of at least 3 independent experiments using different single donors. (F) Human washed platelets (5 × 108/mL) were resuspended in Tyrode buffer and left untreated (NT) or treated with 5mM NEM as a positive control, or with 10 μg/mL collagen in the absence or presence of 2μM GI254023 (GI) for 1 hour at room temperature and then lysed and subjected to SDS-PAGE and Western blotted with anti-GPVI cytoplasmic tail antibody. (G) Human washed platelets (5 × 108/mL) were lysed and subjected to SDS-PAGE and Western blotted with anti-ADAM10 cytoplasmic tail antibody (ADAM10 CT) or anti-Syk (Syk). Vertical lines indicate a repositioned lane.

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