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Pathologic shear triggers shedding of vascular receptors: a novel mechanism for down-regulation of platelet glycoprotein VI in stenosed coronary vessels

Mohammad Al-Tamimi, Chee Wee Tan, Jianlin Qiao, Gabrielle J. Pennings, Ashkan Javadzadegan, Andy S. C. Yong, Jane F. Arthur, Amanda K. Davis, Jing Jing, Fi-Tjen Mu, Justin R. Hamilton, Shaun P. Jackson, Andreas Ludwig, Michael C. Berndt, Christopher M. Ward, Leonard Kritharides, Robert K. Andrews and Elizabeth E. Gardiner

Article Figures & Data

Figures

  • 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.

  • Figure 2

    Shear induces GPVI shedding in washed platelets. Washed platelets (5 × 108 platelets/mL) were left untreated (NT), or treated with either 0.5 μg/mL convulxin for 15 minutes, subjected to shear (10 000 seconds−1) for various times, or subjected to different shear rates for 5 minutes, with and without 100μM GM6001 (A-E) or up to 2.5μM GI254023 (C). (A) Platelet pellets were lysed and analyzed by SDS-PAGE and immunoblotting using anti-GPVI cytoplasmic tail antibody (top panel) or anti-αIIbβ3 mAb CRC54 (bottom panel). (B) Supernatants were blotted with anti-GPVI mAb 12H1. (C) Platelet pellets were lysed and analyzed by SDS-PAGE and immunoblotting using anti-GPVI cytoplasmic tail antibody. Densitometry measurements (relative intensities) of each GPVI tail fragment in blot C were compared relative to the densitometry of the fragment detected in the 10 000-second−1/5-minute sample. (D-E) Platelet pellets were lysed and analyzed by SDS-PAGE/Western blotting using (D) anti-GPV or (E) anti-GPIbα cytoplasmic tail antibodies.

  • Figure 3

    Shear-induced GPVI shedding does not require platelet aggregation, intracellular signaling, or platelet activation. Citrated PRP was either untreated or preincubated with TS buffer alone or containing 1mM RGD peptide, 1mM control peptide, 10 μg/mL AK2, 10 μg/mL SZ2, 10μM PP1, 10μM PP2, 100 nM wortmannin, or 10μM LY294002 as indicated and then subjected to a shear rate of 10 000 seconds−1 for 5 minutes. Aggregation was measured in samples by obtaining platelet particle count (A-C), and then plasma was generated and assayed for sGPVI by ELISA in triplicate (D-F). Panels A and D, B and E, and C and F contain data from 3 different donors that are representative of 3 independent experiments.

  • Figure 4

    Shear-induced shedding of GPVI occurs in the absence of VWF. Citrated PRP or washed platelets were prepared from blood isolated from a patient with VWD type 3 or a healthy donor. (A) To assess levels of VWF and GPIbα in each sample, equivalent concentrations of washed platelets were lysed in nonreducing sample loading buffer and subjected to SDS-PAGE and Western blot using 1 μg/mL anti-VWF mAb, 6G1 (top panel) or 1 μg/mL anti-glycocalicin antibody (bottom panel). (B) To assess VWF function, citrated PRP from the same patient or a healthy donor was subjected to ristocetin-induced platelet aggregation using light transmission aggregometry. Arrow indicates the time at which 1.5 mg/mL ristocetin was added. Absent ristocetin-induced aggregation in the patient sample was rescued by inclusion of 10 μg/mL purified VWF before addition of ristocetin. (C-D) Citrated PRP from the patient or a healthy donor was subjected to 10 000-second−1 shear for 5 minutes, and samples were then analyzed for (C) platelet aggregation by particle counting or (D) sGPVI levels by ELISA. (E) VWD type 3 washed platelets were either left untreated (NT) or treated for 15 minutes with 5mM NEM, or exposed to 10 000 seconds−1 shear for 5 minutes in the presence of TS buffer alone, or containing 10 μg/mL AK2, 10 μg/mL purified VWF, or 100μM GM6001. Samples were then lysed in nonreducing sample loading buffer and subjected to SDS-PAGE and Western blot using 1 μg/mL anti-GPVI cytoplasmic tail antibody. A vertical line indicates a repositioned lane.

  • Figure 5

    Brief exposure to high shear triggers significant GPVI shedding over time. Washed platelets (5 × 108/mL) from a healthy donor were resuspended in (A) Tyrode buffer alone or (B) containing 50μM DM-BAPTA or 1 U/mL apyrase and then subjected to a shear rate of 10 000 seconds−1 for 1 minute. Samples were then left at room temperature for the indicated time before addition of EDTA followed by centrifugation and lysis of platelet pellets. Platelet lysates were analyzed by SDS-PAGE and Western blot using 1 μg/mL anti-GPVI cytoplasmic tail antibody. Data are representative of 3 independent experiments with different donors. (C) PRP from healthy donors was subjected to the indicated level of shear for 1 minute before addition of EDTA (dark bars) or incubated at room temperature for 30 minutes followed by addition of EDTA (light bars) as shown in the pictogram. All samples were then processed to isolate plasma and analyzed for sGPVI levels by ELISA. Data are representative of 4 independent experiments with different donors. (D) In the presence of an inhibitor of αIIbβ3, washed platelets in Tyrode buffer were exposed to 10 000-second−1 shear for 2 minutes and then mixed with equal amounts of buffer or unsheared platelets for up to 30 minutes (left panel), or PRP was subjected to 7500 seconds−1 for 1 minute and then mixed with equal amounts of buffer or unsheared PRP for up to 30 minutes (right panel). Aliquots of supernatant and PRP were isolated for measurement of sGPVI by ELISA (right panel) and platelet surface levels of GPVI by flow cytometry (left panel), respectively.

  • Figure 6

    sGPVI is elevated in patients with stable single vessel coronary disease. Ten patients with SAP and single vessel coronary disease (mean % diameter stenosis, 57.0% ± 2.9%) confirmed by quantitative coronary angiography and 10 age-matched controls were assessed for plasma sGPVI levels. sGPVI levels were significantly higher in the SAP group than in the control group (62.0 ± 7.5 vs 36.8 ± 5.5 ng/mL, mean ± SEM, P = .014, unpaired t test). *P < .05.

Tables

  • Table 1

    Calculated shear across authentic human coronary stenosis

    Peak shear, s−1Mean shear, s−1QCA diameter stenosis, %
    No. of values666
    Mean19 224293555.00
    SD26 40630448.167
    SE10 78012433.334
    • Intracoronary shear rate was calculated by computational hemodynamics in 6 of the subjects in the SAP group described in Figure 6, and summary data are as shown.

    • s−1 indicates seconds−1; QCA, quantitative coronary angiography; SD, standard deviation; and SE, standard error.