Research ArticleTHROMBOSIS

Platelet decoys inhibit thrombosis and prevent metastatic tumor formation in preclinical models

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Science Translational Medicine  13 Feb 2019:
Vol. 11, Issue 479, eaau5898
DOI: 10.1126/scitranslmed.aau5898
  • Fig. 1 The preparation of platelet decoys by detergent extraction procedure leads to smaller, less granular platelets.

    (A) Scheme of the preparation of platelet decoys. PRP, platelet-rich plasma; PPP, platelet-poor plasma; RBCs, red blood cells. (B) Platelet and platelet decoy SEM (top) and TEM (bottom) images presenting the morphology and ultrastructure of platelets. (C) Flow cytometry density plots displaying the size [forward scatter (FSC)] and granularity [side scatter (SSC)] of platelets and platelet decoys. (D) Geometric mean of the size and granularity of platelets and platelet decoys (n = 5 individual donors; **P ≤ 0.01 and ***P ≤ 0.001). a.u., arbitrary units. (E) Platelet decoy yield after detergent treatment of healthy platelets (n = 17).

  • Fig. 2 Platelet decoys retain major platelet receptors but do not activate under stimuli.

    (A) Percentage of remaining GPIIb and GPIb on the surface of platelet decoys (n = 6 individual donors) and representative flow cytometry histograms for each receptors. FITC, fluorescein isothiocyanate; APC, allophycocyanin. (B) GPIIb recruitment from α-granules to the platelet surface upon stimuli with ADP, TRAP, and collagen as detected by flow cytometry (n = 4 to 6 individual donors; *P < 0.05 and ****P < 0.0001). (C) Histogram representation of platelets and platelet decoy activation with supraphysiological concentrations of platelet agonists (TRAP, ADP, and collagen) shown as activation of PAC-1 (n = 3 to 5 individual donors; ****P ≤ 0.0001). ns, not significant.

  • Fig. 3 Platelet decoys do not aggregate under platelet agonists, unlike parent platelets.

    (A) LTA curves and (B) SEM micrographs of platelets and decoys after incubation without (control) or with supraphysiological concentrations of agonists [50 μM ADP, 50 μM TRAP, or collagen (5 μg/ml)] (n = 2 to 3 individual donors).

  • Fig. 4 Platelet decoys decrease platelet aggregation under supraphysiological concentration of agonists.

    (A) Aggregation response in control mixtures of platelets and platelet decoys at various P:D (platelet-to-decoy) ratios without agonist (n = 3). Aggregation responses of platelets versus 20% decoy-supplemented platelets stimulated with 50 μM ADP (B), collagen (5 μg/ml) (C), or 50 μM TRAP (D) (n = 2). Platelets (plts) preincubated with 0.4 mM aspirin (E) or abciximab (1.25 μg/ml) (F) and subsequently stimulated with ADP, TRAP, and collagen.

  • Fig. 5 Platelet decoys do not adhere to collagen under flow using a focal collagen chip model and decrease platelet adhesion to this thrombogenic surface.

    (A) Microfluidic chip device made of four channels containing multiple collagen-coated ROIs along the channel. The collagen chip allows real-time monitoring of platelet adhesion on the collagen strips via fluorescence imaging of tagged platelets. Images of (B) fibrillar collagen on ROIs and (C) absence of collagen between ROIs, (D) positive control showing fluorescent platelet adhesion on a collagen ROI, (E) decoy adhesion to collagen in whole blood perfused in the microchannel compared to positive blood control (P:D, platelet to decoy ratio), (F) adhesion of decoys alone to the collagen patch, (G) negative control showing abciximab-inhibited platelets, and (H) quantification of platelet adhesion on collagen over time whether perfused alone or in combination with decoys or abciximab antiplatelet treatments (****P < 0.0001 and **P = 0.0064; n = 20). The Folt model of in vivo thrombosis was created to study the effect of platelet decoys on thrombus formation. (I) Representative flow reduction measurement during the common carotid artery (CCA) clamping procedure. Arrows in (I) indicate the three clamps of the CCA at the site of the stenosis created bilaterally in the CCA [arrowhead in (J)]. Arrow in (J) represents the ultrasound flow probe placed distally on the CCA. Representative image of (K) thrombus formation (arrow) and (L) thrombus release. (M) Representative flow reduction measurement after flow restoration (arrow). (N) CFRs measured by Doppler ultrasonography after transfusion of saline (n = 6), human platelets (n = 4), or platelet decoys at 20% (n = 6) or 40% (n = 2) of total circulating platelets in the rabbit carotid injury model (*P < 0.04).

  • Fig. 6 Platelet decoys decrease MDA-MB-231 arrest and extravasation within the vasculature and delay metastasis in vivo.

    (A) Graphs on the left show the number of tumor cells that arrest within vessels per device after 2 hours of perfusion with medium and the percentage of perfused tumor cells that extravasated per device at 6 hours. Right: Representative confocal fluorescence microscopic images of MDA-MB-231 breast cancer cells (teal, GFP expression) arrested within the HUVEC-lined microvascular network [magenta, red fluorescent protein, LifeAct] after being perfused through engineered vessels on chip either alone (MDA) or preincubated with platelets (MDA + platelets), decoy (MDA + decoys), or a mixture of platelets and decoys at a ratio of 5:1 (MDA + P:D) (n = 2 to 4; *P < 0.05, **P < 0.003, and ***P ≤ 0.0008). Graphs showing quantification of fluorescence intensity of whole-body images of (B) the dorsal, ventral, or (C) both sides of nude mice over 5 weeks after intracardiac injection of luciferase expressing D3H2LN tumor cells. (D) Representative noninvasive fluorescence dorsal and ventral views of the whole animals are shown at the bottom right. P:D 1 and P:D 2 represent a mixture of platelets and decoys at ratios of 5:1 and 5:2, respectively. The P:D 2 group was statistically significant compared to the D3H2LN control at week 5 (P = 0.0411) and decoy (P = 0.0102) groups.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/479/eaau5898/DC1

    Fig. S1. Characterization of platelet decoy size (FSC) and granularity (SSC) parameters after their transfusion in NOD/SCID mice and blood collection.

    Fig. S2. Effect of platelet decoys on coagulation and assessment of microparticle release as well as histological staining.

    Fig. S3. Adhesion of platelets and platelet decoys on collagen and fibrinogen surfaces under static conditions.

    Fig. S4. Platelet decoys interact with breast cancer cells as much as platelets do.

    Fig. S5. Quantification of the surface area of cancer cell aggregates using ImageJ, NIH.

    Fig. S6. Platelets extracted from whole blood display similar GPIIb characteristics both before and after detergent extraction.

    Table S1. Raw data (Excel file).

  • The PDF file includes:

    • Fig. S1. Characterization of platelet decoy size (FSC) and granularity (SSC) parameters after their transfusion in NOD/SCID mice and blood collection.
    • Fig. S2. Effect of platelet decoys on coagulation and assessment of microparticle release as well as histological staining.
    • Fig. S3. Adhesion of platelets and platelet decoys on collagen and fibrinogen surfaces under static conditions.
    • Fig. S4. Platelet decoys interact with breast cancer cells as much as platelets do.
    • Fig. S5. Quantification of the surface area of cancer cell aggregates using ImageJ, NIH.
    • Fig. S6. Platelets extracted from whole blood display similar GPIIb characteristics both before and after detergent extraction.
    • Legend for Table S1

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    Other Supplementary Material for this manuscript includes the following:

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