Supplementary Figure 1. Microchannel controls & in vivo flow deceleration model.

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1 Mean flow velocity ( m.s ) a c Platelet velocity ( m.s ) [1 m from channel ottom] Localized crush injury & thromus growth acceleration Shear gradient geometry Straight channel (1,8 s laminar flow) deceleration Relative Platelet Path Length ( m) Downstream compression 1 m Micro-needle d 5, 4, 3, 2, 2, s Flow deceleration 1, Arteriole Relative vessel wall compression (%) Supplementary Figure 1. Microchannel controls & in vivo flow deceleration model. (a) CFD simulation (Velocity v displacement plot) showing the velocity change for a platelet (particle) travelling 1 m (½ discoid platelet diameter) from the surface of the micro-channel wall geometry in (Fig. 1c). In the case of a straight microchannel segment (1,8 s laminar flow), the platelet travels at constant velocity throughout its path length. In comparison, note the rapid acceleration phase coupled to the rapid deceleration phase that a platelet undergoes travelling through the shear micro-gradient geometry. () Representative DIC image frame (t = 1 min) showing whole lood flow at a shear rate of 2, s through a straight PDMS microchannel in the asence of a shear micro-gradient geometry. Note that in the asence of the geometry no platelet aggregation was oserved (n = 3). Scale ar = 2 m. (c) Schematic illustrating the in vivo flow deceleration model utilised to examine the impact of ulk flow deceleration on thromus growth in murine mesenteric arterioles. Note that vessel wall compression (flow deceleration) occurred post crush injury, with compression occurring ~1 m downstream of the site of initial crush injury. (d) Intravital micro-piv analysis of lood flow rates (at a focal plane 13 ± 3.5 m from the vessel wall) within a mouse mesenteric arteriole as a function of vessel wall compression. Vessel wall compression is expressed relative to the micromanipulator (micro-needle) z-travel that resulted in complete flow cessation. Note that a relative vessel wall compression of thromosis experiments (Figs. 2c g). 82% was utilized for all Nature Medicine: doi:1.138/nm.1955

2 a Side-view Flow Core Inner discoid layer Peripheral layer Cohesion lifetime (s) Supplementary Figure 2. The inner core of developing thromi consists of a tightly packed amorphous platelet aggregate. (a & ) Representative DIC image of a consolidating thromus typified y the development of an optically amorphous core (dotted marquee in schema) covered y an intermediate layer of stailized discoid platelet aggregates (red, open discs in schema) and an outer sheath of transiently tethering discoid platelets (cyan disks). Scale ar = 1 m. Y ( m) s Zone X ( m) 3 X,Y (s ) Y ( m) s Zone X ( m) X,Y ( s ) Y ( m) s Zone X ( m) 3 X,Y (s ) Supplementary Figure 3. In vitro micro-piv analysis Expanded views of in vitro micro-piv analysis of planar ( x,y ) shear rates around a thromus, within 2 m of the flow channel floor, as a function of applied ulk shear rate (15, 6 & 1,8 s ). Note the overall shear rate acceleration through zone 2 and the formation of a low shear region within zone 3 at all shear rates tested (representative of n = 3 experiments). Nature Medicine: doi:1.138/nm.1955

3 a Planar shear stress (Pa) m 15 m Time (ms) Planar shear stress (Pa) m 15 m Relative platelet path length ( m) Supplementary Figure 4. Effect of ead diameter on dynamic changes in shear (a & ) CFD analysis of individual platelet trajectories at a distance of 1 m (½ platelet diameter) from the lateral surface of 2 and 15 µm eads. Note that platelets will experience varying magnitudes and rate of change in shear ( x,y ) dependent on their position relative to the ead surface. Particle path lines at the ead equators experience a shear increase approaching 1 Pa (15 m eads) that susequently decreases to less than 3.4 Pa in zone 3. Significantly, the rate of change ( x,y v time), spatial distriution ( x,y v path length) and peak shear are significantly reduced for the smaller ead (2 µm), however this shear gradient is still capale of inducing a dynamic discoid platelet aggregation response. 3 s * * * * 1 s 2 s 14 s 23 s * * * * Supplementary Figure s 36 s 39 s 5 s platelet adhesion to a pre-adherent platelet monolayer Memrane tether restructuring during discoid Discoid platelet tethering (yellow arrow: 1 s) to the surface of spread platelets (red asterisk) resulted in the formation of thin memrane tethers from the disc edge (1 14 s) (tether length m; tip to ase, n = 37 platelets analyzed). The platelet cell ody remained discoid during perfusion and moved slowly downstream from the tether attachment point, without forming stale adhesive interactions with the underlying platelet monolayer (Supplementry video 5 Part 2). At a variale stage in the tethering process ( 14 s) the filamentous tether restructured (yellow arrow: 23 s), generating a ulous tether structure proximal to the discoid cell ody (yellow arrow: 29 s). Ongoing tether restructuring resulted in shortening of the platelet tether and translation of the discoid ody against the direction of flow. Scale ar = 2 m. Nature Medicine: doi:1.138/nm.1955

4 Displacement ( m) a 2+ [Ca ] c (nm) Transient tethering Sustained tethering Core platelet Time (s) Time (s) c d e Supplementary Figure 6. In vivo Ca 2+ flux & P-selectin staining (a & ) Intravital calcium flux ( [Ca 2+ ] c ) profiles and concomitant translocation velocity graphs for representative platelets (n = 3 experiments) adhering to a developing thromus in vivo: Platelets displaying transient tethering interactions display minimal [Ca 2+ ] c (dashed cyan lines). Platelets displaying sustained surface interactions show elevated [Ca 2+ ] c that initiates periods of sustained tethering interactions (solid red lines). Platelets found within the core of the thromus display sustained oscillatory [Ca 2+ ] c and concomitant stationary adhesion (dotted grey lines). (c e) DIC and epi-fluorescence acquisition during thromus formation in mouse mesenteric venules. P- selectin antiody RMP Alexa488 was administered systemically (1.3 mg/kg; (c) Panel showing P- selectin expression during initial discoid platelet recruitment to sites of vascular injury. Localized vascular injury was induced y the microinjection of 6% FeCl 3 into the tissue adjacent to the arteriolar wall; (d) Panel showing discoid aggregate growth downstream from the site of vascular injury. Note the asence of P-selectin expression; (e) Panel showing a representative thromus formed ~1 minutes after vascular injury. Note that P-selectin positive platelets are only detected within the thromus core. The outer stailized layers of discoid platelets remain P-selectin negative. Scale ar = 2 m. Nature Medicine: doi:1.138/nm.1955

5 SUPPLEMENTARY METHODS Micro-particle image velocimetry (micro-piv) In order to perform micro-piv experiments at the surface of preformed thromi, hirudin anti-coagulated whole lood was perfused over immoilized type I collagen (1 µg/ml) for 9 seconds and susequently washed out with Tyrode s uffer. Thromi were fixed with 3% paraformaldehyde prior to PIV flow experiments. Physiological saline was seeded with 1% w/v red fluorescent particles (612 nm em ) having a mean diameter of.9 µm and 1% washed red lood cells. The particles were illuminated using light from a continuous laser source of (532nm ex ) and maximum energy output of 2 mw. Image pairs were captured using a high speed Motion-Pro X3 CMOS camera with a maximum resolution of pixels. Each image pair was processed using in-house PIV software 33. This software uses a doule-frame, crosscorrelation multi-window algorithm, with temporal correlation averaging, to extract a grid of velocity vectors from the PIV images. PIV was performed using a cross-correlation type analysis, with the dynamic range enhanced using an iterative approach to select the Sampling Window Size (SWS) y starting at pixels to a final window size of pixels with an overlap of 5%. By performing the analysis in this fashion the largest displacement vectors were determined y using a large SWS. The accuracy and spatial resolution was increased y then reducing the SWS and offsetting successive pairs of sample windows y the displacement calculated from the previous iteration. Erroneous vectors were rejected y comparing them to a local fit of the data (in an asolute sense) and any vector which deviated from that fit (y more than 2 pixels in this case) were rejected and replaced y the local fit. Eventually the two-dimensional velocity vectors otained from the PIV processing were converted to shear rates 34. A twenty one-point two-dimensional local fit to the data was used. The ias and random error introduced y this approximation into the shear value have een investigated y Fouras & Soria (1998) 34 and have een shown to e smaller in relative magnitude compared to the velocity data. Analysis of platelet calcium flux Isolated platelets (3 x 1 8 /ml) suspended in PWB (4.3 mm K 2 HPO 4, 4.3 mm Na 2 HPO 4, 24.3 mm NaH 2 PO 4, 113 mm NaCl, 5.5 mm D-glucose, and 1 mm theophylline, ph 6.5) were incuated for 3 minutes at 37 o C with Oregon Green BAPTA, AM (2.5 µm) and Fura Red, AM (5 µm). Unincorporated dye was removed y washing once with PWB. Platelets were finally resuspended at a concentration of 3 x 1 8 /ml in modified Tyrode's uffer (1 mm Hepes, 12 mm NaHCO 3, 137 mm NaCl, 2.7 mm KCl, and 5 mm glucose, ph 7.3) containing calcium (1 mm), magnesium (1 mm) and apyrase Nature Medicine: doi:1.138/nm.1955

6 (.2 units/ml) (ADPase activity). Real-time platelet calcium flux was monitored via Epi Fluorescence microscopy as the ratio of Oregon Green BAPTA fluorescence over Fura Red fluorescence (with emission wavelengths of 51nm and 66nm, respectively). Real time stacks were acquired and analyzed using MM6. Intravital calcium measurements were performed y dye loading murine platelets from a donor animal (as aove). Laeled platelets were infused via the inferior vena cava into a second experimental animal and intravital microscopy performed (as aove) using a Leica DMIRBE laser scanning confocal microscope. Bead Collision Assay Rectangular glass microcapillary tues (Vitrotues; Vitro Com, mountain Lakes, NJ) were derivatized with 3-aminopropylsilazane (3APS) for 15 minutes at room temperature and washed with 1% ethanol and dried under nitrogen. Microslides were flushed with 2.5% Glutaraldehyde and incuated at room temperature for 15 minutes efore washing with unsupplemented Tyrode s uffer. Polystyrene microspheres (.5% w/v) [Bangs Laoratories] of 2, 5, 9 and 15 µm diameter were incuated overnight at 4 o C with 2 µg/ml purified human VWF and susequently washed twice in un supplemented Tyrode s uffer at.5% w/v. VWF eads were perfused into derivatized microcapillaries via capillary action and incuated overnight at 4 o C and finally flushed with un supplemented Tyrode s uffer. Whole lood (hirudin anticoagulated) perfusion experiments were carried out at 1, s and monitored via DIC video microscopy. Aggregate surface area was determined y manual thresholding in Metamorph 6. (MM6) on a frame y frame asis over 2 mins perfusion. Nature Medicine: doi:1.138/nm.1955