Platelets become labelled by fluorescent fibrin fibers during clot retraction.

A) Time course of unconstrained clot retraction by platelets (4×107 in 400 μl of 12% plasma in PBS) for a total of 60 minutes at room temperature. Experiments have been performed 6x using blood from different donors and typical examples of blood from two different donors are shown (experiments III and IV are repetitions of I and II the day after; although a longer lag-phase is observed, the final retraction volume is similar for the four experiments). B) Unconstrained clot retraction (1×108 platelets per ml in 50% plasma/50% PBS) in presence of fibrinogen-Alexa 488. Image acquisition was started immediately after thrombin addition at a focal plane 100 μm above the bottom of the well and image stacks were collected (61 focal planes, step size 0.5 μm) for a time period of 20:27 min (100 frames with a time interval of 12.5 sec). See also associated Video 1. The experiment has been performed twice using platelets of the same donor. Shown are the first, intermediate and last time points of a depth color-coded time-lapse video (left panel; scale bar 10 μm) and the maximal intensity projections (MIPs) of the same time points (right panel; scale bar 10 μm). C) Constrained clot retraction (1×108 platelets per ml in 50% plasma/50% PBS, fibrinogen-Alexa 488 final concentration 12.5 μg/ml) between two holders. Clots were induced by addition of thrombin (2.5U/ml final), fixed at the indicated retraction times, embedded in gelatin, flash frozen and cryosections (14 μm) were stained for the integrin subunit αIIb (magenta; scale bar 5 μm). The time course was performed twice using PRP from two different donors (retraction assays were repeated, although not for all time points, more than eight times using blood from different donors, with consistent results). Image acquisition was performed using a wide-field epi fluorescence microscope (BX41; Olympus) equipped with a Plan 100x/1.25 NA oil objective, a camera (DP70; Olympus), and the acquisition software analySIS (Olympus).

Fibrin fibers are organized in a “cage-like” fashion around platelets within a constrained clot.

A-D: Two typical examples (out of 39 acquisitions of four experiments using blood from different donors) of platelets in a constrained clot with attached fibrin fibers are shown (1×107 platelets per ml PBS/50% plasma and fibrinogen-Alexa 488). Clot retraction was allowed to take place in an inoculation loop (see methods) for 15 min before fixation and immunofluorescence staining of the integrin subunit αIIb (magenta). Samples were then processed for expansion (scale bars 10 μm = 2.5 μm after correction for expansion; indicated z-levels are not corrected for expansion; see also associated animation, Video 2). A) Four focal planes from a stack of images (z-level as indicated) showing a platelet in a clot (fibrin fibers in green, plasma membrane staining using an antibody against the integrin subunit αIIb in magenta and the merge). B) 3D reconstruction of the image stack used in A (46 planes, step size 0.5 μm), a bulb is indicated by an arrow. C) Four focal planes show another typical platelet in a clot (z-level as indicated). D) 3D image reconstruction of the image stack used in C (28 planes, step size 1 μm), a bulb is indicated by an arrow.

Spread platelets accumulate fibrin fibers above them in a myosin dependent way.

A) Shown is a 2D fiber-retraction assay in the presence of DMSO (control) or 50 μM blebbistatin for myosin inhibition. After a 30 min preincubation of platelets (4 μl plasma per ml PBS, see methods section) with or without blebbistatin, thrombin is added and the assay is continued as described in the methods section. Scale bar 10 μm, arrowheads indicate platelets without fiber contacts, thin arrows with fiber initiation or contact, larger arrows indicate platelets starting to wind-up fibers and even larger arrows point to platelets with compacted fibers around them (see legend below bar graph in B). B) Quantification of the percentage of platelets in the different categories using image acquisitions of four experiments using blood from different donors (for control conditions a total of 636 platelets were counted and for blebbistatin conditions a total of 524). Shown are means + sem, evaluation of significance using the Student’s t-test.

Coiled-up fibrin fibers above spread platelets.

Two representative examples (out of 18 acquisitions from three independent experiments using blood from different donors) of fibrin fiber accumulations above spread platelets in the 2D fiber-retraction assay (4 μl plasma per ml PBS, see methods section) are shown (see also animation, Video 3). Samples were stained for the αIIb integrin subunit and expanded (scale bars 10 μm = 2.5 μm after correction for expansion, indicated z-levels are not corrected for expansion). A) Three different focal planes (z-levels as indicated) from an image stack showing two spread platelets and attached fibrin fibers. B) 3D reconstruction of the image stack used in A (51 planes, step size 0.25 μm), shown are three different view angles. C) Three different focal planes from an image stack illustrating a spread platelet and attached, coiled-up fibrin fibers. D) 3D reconstruction of the image stack used in C (50 planes, step size 0.25 μm), shown are three different view angles. The fibrin fibers are additionally displayed as depth-color coded (Fire LUT) reconstructions to highlight the straight fiber parts along the z-axis. E) Same fiber image stacks as in C and D. Left panels are MIPs of the green fibrin fibers along the z, x and y axes. Right panels are the same projections color coded in the HSB space (Hue, Saturation, Brightness) according to the orientation of individual fiber segments. The fibers are well aligned along the z-axis, while they crisscross horizontally.

Strongly compacted fibrin fibers around the center of platelets in the 2D fiber-retraction assay.

Two representative examples of platelets are shown with bulbs encircled by fibrin fibers in the 2D fiber-retraction assay (4 μl plasma/ml, see methods section) behaving similar to platelets in a clot (see also animation, Video 4). Samples were stained for the αIIb integrin subunit (A, B) or the myosin light chain (MLC; C,D) and processed for expansion (scale bars 10 μm = 2.5 μm after correction for expansion, indicated z-levels are not corrected for expansion). Experiment was repeated 4x with blood from different donors with a total of 18 image stacks acquired for fibrin/integrin and 62 for fibrin/myosin staining. A) Three different focal planes from an image stack showing two platelets, one spread and one with bulbs and attached, compacted fibrin fibers. B) 3D image reconstruction of the image stack used in A (34 planes, step size 0.25 μm), shown are three different view angles. C) Three different focal planes from an image stack illustrating another platelet with bulbs and attached, rolled-up, compacted fibrin fibers. D) 3D image reconstruction of the image stack used in C (55 planes, step size 0.25 μm), shown are three different view angles.

Fibrin fibers are twisted in the proximity of platelets.

Two representative examples of platelets with compacted fibrin fibers are shown in the 2D fiber-retraction assay (4 μl plasma/ml, see also animation, Video 5). Samples were stained for myosin II (A, B) or phosphorylated myosin light chain (p-MLC; C-E) and processed for expansion (scale bars 10 μm = 2.5 μm after correction for expansion, indicated z-levels are not corrected for expansion). Please note also the strong fiber accumulation around the platelets similar to platelets shown in figure 5. A) 3D image reconstruction of an image stack (72 planes, step size 0.25 μm) illustrating a platelet with bulbs and wound-up compacted fibrin fibers. B) Three different focal planes (z-level as indicated) from the image stack used in A, depth color-coded. Rectangles indicate twisted fibrin fibers and a corresponding zoom is shown below. C) Three different focal planes (z-level as indicated) from an image stack illustrating two platelets with bulbs and attached, wound-up, compacted fibrin fibers. Twisted thick fiber bundles are observed between the two platelets. D) 3D image reconstruction of the image stack (91 planes, step size 0.25 μm) used in C, shown are three different view angles. E) The same focal planes as in C and the top view of the 3D image reconstruction shown in D of the fibrin fibers, depth color-coded to better distinguish the twist of the thick fiber bundles between the two platelets.

A fibrin rosette is located close to a ring-like actin organization in spread platelets.

Four representative examples (out of 20 acquisitions, experiment repeated 4x using blood from different donors) of a fibrin rosette associated with spread platelets in the 2D fiber-retraction assay (7 μl plasma per ml PBS, see methods section) are shown (see also animation, Video 6). Samples were stained for the αIIb integrin subunit as well as for actin and processed for expansion (scale bars 10 μm = 2.5 μm after correction for expansion). A) 3D image reconstruction of an image stack (30 planes, step size 0.33 μm) of fibrin fibers (green) and integrin or actin staining (upper and lower panels respectively, magenta). B) Similar example as in A (17 planes, step size 0.33 μm). C) Another example of a fibrin rosette with intercalated actin nodules (34 planes, step size 0.33 μm). D) An example showing a long fibrin fiber associated with the fibrin rosette (35 planes, step size 0.38 μm).

The fibrin rosette and attached fibers are in close proximity to myosin II in spread platelets

Three representative examples (out of 62 acquisitions, experiment repeated 4x using blood from different donors) of fibrin and myosin localization in spread platelets (see also animation, Video 7) in the 2D fiber-retraction assay (4 μl plasma/ml for A, B, E, F or 7 μl plasma/ml C, D; see methods section). Samples were stained for myosin (A, B, C, D) or phosphorylated myosin light chain (p-MLC; E, F) and expanded (scale bars 10 μm = 2.5 μm after correction for expansion, indicated z-levels are not corrected for expansion). A) Three different focal planes (z-levels as indicated) from an image stack showing a spread platelet and an attached fibrin fiber in the center of a faint fibrin rosette. B) 3D image reconstruction of the image stack used in A (36 planes, step size 0.3 μm), shown are three different view angles. C) Three different focal planes from an image stack illustrating a spread platelet with a fibrin rosette and an attached, rolled-up fibrin fiber. D) 3D image reconstruction of the image stack used in C (31 planes, step size 0.33 μm), shown are three different view angles. E) Three different focal planes from an image stack illustrating a spread platelet with attached, rolled-up fibrin fibers. F) 3D image reconstruction of the image stack used in E (54 planes, step size 0.25 μm), shown are three different view angles.

Determination of platelet protrusions and fibrin fiber curvatures in proximity of platelets

A) Three examples of platelets within a clot stained for the aIIb integrin subunit (including platelets shown in figure 2). MIPs are shown, the number of platelet extensions is indicated and has been determined manually by scrolling through the image z-stack. Scale bars 10 μm = 2.5 μm after correction for expansion. B) Three examples of platelets attached to a glass surface (including platelets shown in figure 5) and stained for the MLC (upper panel) or the aIIb integrin subunit (middle and lower panel). MIPs are shown, the number of platelet extensions is indicated and has been determined manually by scrolling through the image z-stack. Scale bars 10 μm = 2.5 μm after correction for expansion. C) The comparison of all data points between platelets in clots or attached to a glass surface does not show a statistically significant difference between protrusions of platelets in a clot or when spread on a glass surface (Mann-Whitney U test). D-F: The degree of fiber curvature in examples shown in D and E is determined as κ=1/r using the radius of a circle fitting the region of strongest curvature of the fibers. D) Three examples of TEM images used to determine the curvature of fibrin fibers close to platelets within a clot (measured examples are indicated by arrows). E) Three examples of fluorescent images of fibers wound around platelets attached to a glass surface (including platelets shown in figure 5). A single focal plane of each image stack is shown (scale bars 10 μm = 2.5 μm after correction for expansion). F) Comparison of all data points obtained using TEM images and fluorescent images (curvature in κ μm-1). For quantification, 31 EM acquisitions of three clots from different donors were used as well as 12 Fluo acquisitions of platelets from 4 different donors.

The model parameters used in the simulation.

CS – current study.

Simulation of platelet mediated winding-up of fibrin fibers.

A) Time evolution of the fibrin fiber configuration around the platelet bulb. Two different angles between the fiber and the x-axis were used ([1]: α = π/4, lime color; [2]: α = π/2.5, dark green color). Left: initial positions, top view and side view. Center: intermediate position, top view and side view. The fibrin fiber began to form a loop around the bulb. Right: final position, top view and side view. A compact fibrin loop was formed around the bottom of the bulb (see also animation, Video 8). B) Time when the first node bound to the bulb and thus the formation of the fibrin loop started. The fibrin winding-up started earlier for a larger angle between fiber and x-axis (and consequently smaller angle between fiber and tangent line at the point of the node 0 binding). The number of nodes that were bound to the bulb at the end of the simulation was slightly higher for larger α. Thus, larger α led to a more compact fibrin fiber organization. C) The distance between nodes and the bottom of bulb along z-axis. The distance was smaller for larger α and the fibrin organization was slightly more compact. D) Comparison of final fiber configurations around the bulb for two different α.

Features predicted by the model/simulation are observed on TEM images.

Interpreted, potential fiber organisations are highlighted in green on an identical image on the right. A) Fibrin fibers appear to encircle a bulb. B) Cross sectioned fibrin fibers at the base of a bulb, which potentially curve around the bulb. C) Several cross sectioned fibers at the base of a bulb, which may surround the bulb. D) Several cross sectioned fibers along a platelet extension potentially forming a spiral. E) Cross sectioned fibrin fibers around a forming bulb. F) Cross sectioned fibers at the base of a bulb and two fibers along a filopodial extension.

Live imaging of spread platelets winding-up fluorescent fibrin fibers.

A-C: Shown are three experiments using blood from different donors. A) Washed platelets were adjusted to 5×106 platelets per 2 ml PBS and 5 μl plasma was added as well as fibrinogen-Alexa 488. Thrombin was added to induce fibrin fiber formation and platelet activation. After 10 min, the suspension was transferred into a petri dish (WPI Fluoro), centrifuged and installed in the microscope incubator at 37°C to start image acquisition (see also Video 9, upper panel). The first and last time points of the time-lapse video are shown; acquired using the fluorescence and transmission channels. Projections of four focal fluorescent image planes of fibrin fibers (green) and projections of the transmission planes as well as the merged channels are shown. An arrow indicates a forming kink in a fibrin fiber (scale bar 10 μm). Below are shown Individual time points of the time-lapse video zooming on the rectangle indicated in the panels above. This region shows rotational fiber movements. B) Independent experiment under conditions as described in A except that the petri dish was kept at room temperature to slow down the platelet mediated fiber reorganizations (scale bar 10 μm). An arrow shows platelet mediated fiber compaction. Below are shown Individual time points of the time-lapse video zooming on the rectangle indicated in the panels above. This region shows a fibrin fiber getting curved by the platelet and finally ruptures followed by a rotational movement of the ruptured fiber. C) Platelets of a third donor (not washed, i.e., 5 μl PRP with 4×106 platelets per 2 ml PBS) and fibrinogen-Alexa 488 were added to 2 ml of PBS and the experiment was continued as in B (scale bar 10 μm). Below are shown Individual time points of the time-lapse video zooming on the rectangle indicated in the panels above. This region shows fibrin fibers getting coiled around the pseudo-nucleus of the spread platelet. The last zoomed image shows a temporal color-coded projection of the transmission channel to illustrate the rotational movement of the pseudo-nucleus (scale bar 5 μm). D) Samples shown in B and C were fixed 3h after the start of time-lapse acquisitions and stained for the integrin subunit aIIb. Shown are fibrin fibers (green) integrin staining (magenta) and the merge (samples B and C, upper and lower panels, respectively; scale bar 10 μm). E) Preformed fluorescent fibrin fibers were prepared in the absence of platelets. PRP was adjusted to 2.5×106 platelets per ml with PBS and 1800 μl was transferred into a petri dish (WPI Fluoro). The dish was centrifuged to allow spreading of the platelets and the supernatant was replaced by the preformed fibrin fibers. The dish was installed in the microscope incubator to start image acquisition (see also Video 9, last panel). The experiment was performed twice with blood of the same donor. The last fluorescent image of the time-lapse video is shown (left image, scale bar 10 μm). Fifteen of the fibrin accumulations in the middle of the platelets are rotating in a counterclockwise direction (indicated as “1”), four rotate clockwise (“2”) and for 16 no clearly visible turn is observed (“3”). A projection of all fluorescent time points, temporal color-coded using the LUT spectral, is shown. White corresponds to the sum of all the colors at each time point, meaning no movement during the time-lapse video. (right image, scale bar 10 μm).

Schematic illustration of the hypothesis explaining the potential mechanism by which platelets may wind-up fibrin fibers and compact them.

Represented is a platelet in the 2D fiber-retraction assay (upper part) or in a clot (lower part). At the beginning of platelet fiber interactions, a fibrin rosette is present around the pseudo-nucleus and in each bulb with a dense fiber mass at the internal periphery of the rosette possibly representing an initiation complex. Radial actin fibers extend to the center of the platelet and an actomyosin rotational movement along the radial actin fibers may lead to the winding-up and the downward sliding of the extracellular attached fibers to encircle the pseudo-nucleus or the base of the bulbs at the final state of compaction (3D rendering of the scheme was produced using an open AI).

Illustration of clot retraction assays used in this study.

A) Unconstrained clot retraction B) Constrained clot retraction between two holders C) Constrained clot retraction within an inoculation loop

Model of fibrin fiber binding to the platelet bulb and fibrin winding-up.

A) The model of a fibrin fiber in solution. The fiber was modeled as a sequence of N nodes (black dots) which were connected by elastic springs (blue). Fi,i-1/ Fi,i+1 were vector forces which acted on the node i according to the Hook’s law, the black vectors show the force directions. B) The system configuration at time point T=0. Magenta circle: internal volume of a bulb. Light-magenta: the binding region. If the node i entered the binding region, it bound to the integrin molecule. The black arrow shows the rotation direction of the cytoskeleton and integrin molecules.