Author response:
The following is the authors’ response to the original reviews.
We thank the editor and the reviewers for their time and efforts to evaluate our manuscript. We have taken into account all the comments and revised the manuscript accordingly which has considerably strengthened the message.
Several parts of the manuscript, have been extensively rewritten to add explanations and clarify our hypothesis and claims, this also led us to add four new references.
In addition, we have added the following new figures:
- New part of figure 2. Figure 2E shows a platelet in a constrained clot after 4h of retraction with the fibrin cage around the platelet center still present. The actin staining of the platelet shows that radial actin fibers are present in each bulb extending to the platelet center. This observation supports our hypothesis that in each bulb an individual cytoskeletal swirling could take place resulting in the accumulation of fibrin fibers at the base of each bulb.
- Modification of figure 3, to include the criteria used to define four categories of platelets and associated fibers in the 2D fiber retraction assay (new Fig. 3C).
- New figure 13, illustrating the quantification of fibrin fibre compaction mediated by platelets in the 2D fiber retraction assay and the rotational movement of a fiber mass (video 9).
- New supplementary figure 1, showing the result of a new model simulation in the absence of cytoskeleton swirling. Under this condition the fibrin fiber does not loop around the platelet bulb.
Public Reviews:
Reviewer #1 (Public review):
This paper reports a previously unrecognized mechanism by which platelets compact fibrin fibers during clot retraction. Rather than simply pulling on fibers, the authors propose that platelets generate swirling motions that wind and loop fibrin into dense structures.
While the results are intriguing, the underlying physical mechanism remains unexplained. In particular, it is unclear how platelets generate swirling motion capable of inducing fibrin coiling, especially when suspended in 3d fibrin mesh. This raises concerns about the conclusions.
The reviewer is right, it is difficult to imagine how platelets in a 3D fibrin mesh can accumulate fibers at the base of their extensions to form a cage-like fiber organisation around the center of the platelets. We therefore developed the 2D fibre-retraction assay, which we believe provides important insight for the coiled fiber accumulations above spread platelets in the 2D situation but also provides a framework for interpreting similar processes that may occur within a 3D clot. In response, we have placed greater emphasis on clarifying and strengthening the comparison between the potential mechanistic aspects in the 2D and 3D assays, in order to better support our proposed model (see Results, section: "Platelets, spread on a 2D surface, organize fibers above them", last paragraph). In addition, the Ideas and Speculations section of the discussion has been extensively rewritten to provide more detailed explanations about the potential mechanism leading to fibrin fiber accumulations around platelet bulbs in a 3D fibrin mesh.
Also, does fibrin have inherent chirality or structural asymmetry that could promote coiling independently of platelet activity?
Yes, double-stranded fibrin protofibrils have a helical twist [1]. Furthermore, a clot formed in the absence of platelets and other cellular components shows intrinsic tensile forces [2]. However, we show that inhibition of actomyosin actions prevents fibrin fiber accumulation in the 2D fibre-retraction assay providing evidence that platelet actions are necessary to observe the coiled fibers above spread platelets. This has been accentuated in the revised version and three references have been added.
Furthermore, platelet retraction typically involves platelet aggregation rather than isolated cells, and it is unclear how fibrin coiling would proceed in clustered platelets.
Under the in vitro fiber retraction conditions used in our study (constrained or unconstrained clots or even in the 2D assay) individual platelets are homogenously distributed within the forming clot or on the coverslip. Therefore, there are no big platelet aggregates or clusters of platelets under our experimental conditions and the results can only demonstrate how individual platelets act on fibrin fibers. This point has been emphasized in the revised version (Discussion, third paragraph).
Reviewer #2 (Public review):
Summary:
Grichine et al. investigate platelet-mediated fibrin compaction using human donor platelets and propose a novel mechanistic model in which platelets generate contractile forces and wind fibrin fibers into compact coiled structures. Using a combination of 2D spread assays, 3D clot imaging via expansion microscopy, live-cell imaging, and computational modelling, the authors present evidence of cage-like fibrin architectures, coiled-fibre morphologies, and platelet centred "rosette" structures present during fibre compaction. They further suggest that actomyosin-driven cytoskeletal dynamics, potentially involving rotational or swirling motion, underlie this proposed winding mechanism, analogous to DNA looping and compaction. The study addresses an important and longstanding question in thrombosis and hemostasis and offers a conceptually novel perspective on clot compaction.
Strengths:
The integration of multiple imaging modalities is a notable strength of this paper. In particular, the 2D fiber-retraction assay provides a useful model for understanding the spatio-temporal dynamics of platelet-mediated fibrin compaction, which can be applied to other systems and may yield detailed mechanistic insights into biological processes. The live-imaging approaches are particularly well executed and offer valuable dynamic insight.
Weaknesses:
The primary weakness of this paper lies in its descriptive nature and its reliance on correlative rather than causal evidence. Several interpretations are not uniquely supported by the data presented. For example, the categorisation of fibrin accumulation in 2D assays as "fiber winding" and "fibre compaction" remains descriptive without establishing winding as a mechanism.
When introducing the 2D fiber-retraction assay (figure 3) in the revised version, we now only mention the terms fiber accumulation and compaction to better align with the level of evidence, since wound-up fibers cannot be distinguished in this figure. The criteria to establish the four categories of platelets and associated fibers in the 2D fiber retraction assay have now been included in figure 3C.
Nevertheless, coiled fibers above spread platelets are clearly visible in figure 4 and 8 and dynamic fiber rotations or winding-up are observed in figure 12 and video 9. These observations have been presented more cautiously, as indicative rather than definitive evidence of a winding mechanism.
Alternative mechanisms, such as circular bundling, stacked fibers under tension, or fibrin crosslinking-induced aggregation, are neither excluded nor investigated.
For fibrin fiber bundling, staggered or crosslinked protofilaments no platelet actions are necessary as described previously [2,3]. Since we observed a clear difference between +/- blebbistatin conditions in the 2D fiber-retraction assay, the fiber compaction we observe depends on platelet actions. Consequently, we consider these alternative mechanisms unlikely based on our data. This has been stated explicitly in the results section and discussion and three references have been added.
Although the authors present compelling live imaging, establishing winding as a dynamic phenotype would require quantitative analyses, such as measuring angular velocities and coiling rates.
We have incorporated quantitative measurements (new figure 13) about platelet mediated fibrin fiber compaction and angular rotation velocities to complement the observations obtained from live imaging. It is important to note, however, that angular velocities and coiling rates are likely influenced by the number of fiber–fiber contacts present at the time coiling occurs. Specifically, an increased number of contacts is expected to elevate tension within the network, thereby modulating the forces generated by platelets and, consequently, affecting both velocity and coiling dynamics.
The use of a second fluorophore-labelled fibrin population could further strengthen evidence for rotational dynamics.
These live videos are quite difficult to acquire because of the following reasons:
- Small platelet size
- Heterogeneity of platelets within the population (10 d half-life, old platelets may not be able to compact fibers efficiently).
- The speed of the process and the time needed to adjust parameters for image acquisition, necessitates an arbitrary choice of the acquisition window and only one acquisition (90 min) per sample preparation is possible.
- Furthermore, the laser-induced illumination can perturb the observed processes. We therefore use high-spatial-resolution 3D confocal time-lapse imaging, performed in photon-counting mode with very low laser excitation.
For these reasons, the use of additional markers would be technically challenging and could perturb the delicate equilibrium and dynamics of the process under investigation.
Similarly, the inference of rotational contractility or actomyosin "swirling", based on chiral actin organisation and blebbistatin treatment, is not sufficiently supported to conclude that platelets actively wind or loop fibrin fibers.
Importantly, in the 2D fiber-retraction assay, we do not propose that the rotational actomyosin activity leads to a contractility of the platelets which would allow fiber retraction. Rather, we suggest that cytoskeletal actomyosin swirling (as demonstrated for nucleated cells by Bershadsky's team) can induce rotational dragging of extracellular bound fibrin fibers around the pseudonucleus of spread platelets thereby promoting accumulation of fibrin fibers (shown in figure 12C, video 9, third panel). Consistent with this interpretation, inhibition of myosin by blebbistatin prevents the accumulation of fibrin fibers above spread platelets in the 2D fibre retraction assay (Fig. 3).
The mathematical model, while complementary and well-constructed, relies on multiple assumptions and lacks predictive validation.
We thank the reviewer for this insightful comment and acknowledge that the proposed model relies on several important assumptions. In our view, the most significant assumption is that integrin molecules undergo rotational downstream motion as a consequence of their coupling to the swirling cytoskeleton. To assess the necessity and impact of this assumption, we provide an additional simulation performed in absence of the cytoskeletal swirling. Under this condition the fibrin fibers are not looped around the platelet bulb (this result has been added as supplementary figure 1). This analyses also provides further validation of the proposed model and underlying mechanism. At the same time, it is important to emphasize that the primary purpose of the model was to examine whether the hypothetical swirling dynamics of the cytoskeleton, together with the associated receptors, could in principle reproduce the experimentally observed fibrin organization.
Appraisal:
While the authors successfully document intriguing fibrin architectures and provide a compelling descriptive framework, they do not fully demonstrate a mechanistic model of active fibrin winding by platelets. The conclusions regarding platelet-driven winding and rotational dynamics are not sufficiently supported by direct or quantitative evidence. To substantiate these claims, the study would benefit from experiments that directly link platelet dynamics to fibrin organisation, including coordinated measurements of platelet motion and fibre rearrangement. As it stands, the results are suggestive but do not definitively support the proposed mechanism.
Discussion and Impact:
Despite these limitations, the study addresses an important question in thrombosis and hemostasis and introduces a potentially impactful conceptual framework for understanding clot compaction. The imaging approaches and datasets presented will be valuable to the community, particularly for researchers interested in platelet mechanics and fibrin organisation. However, the overall impact will depend on whether the proposed mechanism can be more rigorously validated. In its current form, the study presents an interesting and thought-provoking model, but would benefit from either stronger experimental support for the proposed mechanisms or a more cautious interpretation of the findings.
We agree that the proposed mechanism requires further validation. In the revised version we have added a new result (figure 2E) showing that radial actin filaments are present in each bulb of a platelet in a constrained clot, supporting the possibility that rotational cytoskeletal movements could take place in individual bulbs. In a new figure 13, we have also quantified fiber compaction and the angular velocity of a rotating fibrin mass observed in video 9. Furthermore, in the revised manuscript, we present a more cautious and explicitly hypothesis-driven interpretation of the mechanism. We hope that the publication of our observations will be of interest to researchers in the field of thrombosis and clot mechanics who possess the specialized tools and expertise necessary to rigorously evaluate and either substantiate or refute the proposed mechanistic model.
Reviewer #3 (Public review):
Summary:
This work aims to understand the mechanisms that platelets use to interact with and compact fibrin fibers during clot formation. This is an important process during wound healing, and recent work has demonstrated that platelets play a critical role in generating the force required to drive the accumulation of fibrin. The authors argue that current models are insufficient to account for the observed reduction in clot volume and propose that platelets actively 'wind up' these fibers by undergoing myosin-dependent rotation. While interesting, the experiments performed by the authors do not directly test this mechanism, and further evidence is required to support their claims.
We do not "propose that platelets actively 'wind up' these fibers by undergoing myosin-independent rotation" of the whole platelet, but rather of the cytoskeleton winding-up extracellular fibrin fibers attached to integrin receptors.
Weaknesses:
(1) The motivation to switch from the system used in Figures 1 and 2 to the '2D fiber-retraction assay' is not clear. While the authors state that this system has 'reduced complexity', the differences between these assays appear to disrupt the 'cage-like' organization of fibrin around platelets shown in Figures 1 and 2 (compare images in Figure 2 with those in Figure 4). An indepth comparison of two methods is needed to support the conclusions from the 2D system.
We agree that the cage-like fibrin organization around platelets is disrupted in the 2D fibre-retraction assay when platelets are completely spread on the coverslip before they have encountered fibrin fibers (Fig. 4). This has been explicitly stated in the revised version. However, some platelets in the 2D fiber-retraction assay form the same number of extensions as platelets in a 3D clot (Fig. 9 A, B) and are not completely spread on the glass surface. For these platelets a cage-like fibrin organisation is retained under the 2D conditions (Fig. 5 and 6). Nevertheless, the fiber density at the base of the bulbs is higher in the 2D assay than under the constrained 3D clot retraction conditions (Fig. 1C and Fig. 2), probably because in the 2D condition the fibers are less constrained and readily available for compaction.
Furthermore, the change in plasma volume (Figure 2 vs Figure 7) should also be tested - the authors state that this increases fibrin fiber formation, but this is not quantified or demonstrated in the figures. Notably, this appears to change the morphology of the fibrin fibers shown (comparing Figure 2 and Figure 7).
We thank the reviewer for raising this point. We would like to clarify that Figure 2 and Figure 7 correspond to two distinct experimental setups: the constrained clot retraction assay (Figure 2) and the 2D fiber-retraction assay (Figure 7). As such, they are not directly comparable. We understand, however, that the reviewer is likely referring to the apparent differences between Figures 3–6 (lower plasma volume, higher fiber density) and Figures 7–8 (higher plasma volume, lower apparent fiber density).
The reduced number of visible fibers in the latter condition is not solely a consequence of plasma volume per se, but rather results from the formation of a labile fibrin gel at higher plasma concentrations, which is lost during the fixation and aspiration steps. This effect was initially observed across samples from two donors with differing plasma fibrinogen levels. In one case, an unusually low fibrinogen concentration allowed the addition of higher plasma volumes without inducing gel formation. In contrast, in the other sample, a more typical fibrinogen level resulted in gel formation under the same conditions.
Importantly, we performed all experiments using matched donor plasma and platelets. As a result, the precise fibrinogen concentration could not be determined prior to experimentation. Nonetheless, post hoc measurements confirmed that fibrinogen levels in most donor samples fell within the normal physiological range, which allowed us to always use the same plasma volumes for low and high plasma concentrations (4ul/ml PBS and 7 ul/ml PBS, respectively) except for one donor as mentioned above.
(2) It is unclear how the classification of platelets as 'fiber-winding' versus 'fiber compaction' differs in Figure 2. The criteria used for these classifications should be stated. Further, it seems premature to characterize fibers as wound without having established this earlier in the manuscript.
The reviewer probably refers to figure 3 and he is right; it is premature to mention fiber winding at this stage of the results section (see our response to reviewer #2). In the revised version, we have modified figure 3 to include the criteria used to classify the platelets into four different categories (Fig. 3C).
(3) Is the 'gearwheel' different from the 'cage' of fibrin fibers? They appear similar, but it is difficult to distinguish between them with only qualitative descriptions of these phenotypes.
The "gearwheel" is observed for completely spread platelets in the 2D fiber-retraction assay and a figure illustrating our hypothetical speculations to compare the 2D gearwheel with the 3D clot situation is presented in the discussion under the "Ideas and Speculations" paragraph (now Fig. 14). We have given a more comprehensive explanation of the proposed mechanism in the revised version.
(4) The quantification of platelet extensions in Figure 9 is confusing. While those in 9A are clear, those in 9B are not. For instance, what is the difference between #7 and #8 in the middle panel of 9B? It does not seem like #8 is labeling an extension.
For the platelet shown in the middle panel of Figure 9B, the extensions cannot be clearly distinguished in the MIP (Maximum Intensity Projection) image because extension #8 is positioned above extension #7 and is therefore superimposed in the projection. However, the two extensions can be differentiated when examining the 3D image stack (Video 4, upper panel). As indicated in the figure legend, the number of extensions was determined manually by scrolling through the z-stack image sequence. In the revised version, we will also define the abbreviation “MIP” as Maximum Intensity Projection.
(5) It is unclear what the modeling accomplishes, as there is no comparison between the results of these simulations and their experiments.
We thank the reviewer for this valuable concern. We chose not to combine the experimental fibrin organization and the modeling results within the same figure panel, as the resulting image would be too complex and difficult to interpret. We have, however, added a supplementary figure 3 showing the results of a new simulation in the absence of cytoskeletal swirling. Under these conditions no winding of the fibrin fiber around the platelet bulb can be observed. It is also important to emphasize that the comparison between the model and the experimental data was intended to be primarily qualitative rather than quantitative.
(6) The data presented in Figure 12 provides the most direct support for their mechanism, but falls short of directly testing their claims. These experiments should be repeated to include blebbistatin to test the contribution of myosin and include quantitative rather than qualitative comparisons of these experiments.
As mentioned already above, these live videos are quite tricky to acquire because of the following reasons: - small platelet size
- Heterogeneity of platelets within the population (10 d half-life, old platelets may not be able to compact fibers efficiently).
- The speed of the process and the time required to optimize imaging parameters, necessitate the selection of an arbitrary acquisition window. Consequently, only a single acquisition of approximately 90 min can be performed per sample preparation, with no guarantee that relevant platelet-fibrin interactions can be acquired in the acquisition window.
- Furthermore, after blood donation, the first sample is usually ready to be acquired around 3 pm, acquisition time 90 min. At least 10 successful acquisitions per condition would be required to ensure statistical robustness, but maximal 4 can be acquired per donor, because platelet samples start to deteriorate within twelve hours after blood donation.
Taken together, the intrinsic heterogeneity of the platelet population, the low likelihood of capturing informative events, and the limited availability of suitable imaging resources at our institute render a robust and quantitative comparison between conditions with and without blebbistatin extremely challenging, if not impractical, within a reasonable timeframe.
In accordance with the reviewer's request, we have added a new figure 13 to the revised version, presenting quantitative data on the platelet-mediated fibre compactions and the speed of angular fibrin rotations observed in video 9.
Recommendations for the authors:
Reviewer #3 (Recommendations for the authors):
Throughout the manuscript, it is difficult to map the data presented in the figures to the text in the results section. Often, many subpanels are referred to collectively (for example, 'Fig 4 AE and animation, Video 3' on line 150), and the reader is left to piece together how this data fits into the statements in the results section. More guidance from the authors would help to understand the connection between these data and their conclusions.
In the revised version, we have provided clearer explanations to make it easier to understand the conclusions drawn from the data. Concerning the indication "Fig 4 A-E and animation, Video 3" just means that platelets shown in panels A-E of figure 4 can also be visualized in the animation video 3. We have also put an effort to clearly indicate which figure part is presented in the associated video.
There are also many figures that contain redundant information. The authors should consider revising these figures and including some of these repeated images as supplemental figures.
As noted by the reviewers, our study provides predominantly qualitative observations essentially because it is not obvious to choose parameters which would be pertinent and could be quantified accurately using expansion microscopy. A quantitative analysis would allow to show the quantification and a representative image to describe the phenotypes of platelet-mediated fibre organisations. Without a quantitative analysis, we consider it more appropriate to provide multiple examples, enabling the reader to assess the consistency as well as the variability across repeated observations.
Additional References
(1) Jansen KA, Zhmurov A, Vos BE, et al. Molecular packing structure of fibrin fibers resolved by X-ray scattering and molecular modeling. Soft Matter. 2020;16(35):8272-8283.
(2) Spiewak R, Gosselin A, Merinov D, et al. Biomechanical origins of inherent tension in fibrin networks. J Mech Behav Biomed Mater. 2022;133:105328.
(3) Ramanujam RK, Lavi Y, Poole LG, Bassani JL, Tutwiler V. Understanding blood clot mechanical stability: the role of factor XIIIa-mediated fibrin crosslinking in rupture resistance. Res Pract Thromb Haemost. 2025;9(4):102871.
(4) Gaertner F, Ahmad Z, Rosenberger G, et al. Migrating Platelets Are Mechano-scavengers that Collect and Bundle Bacteria. Cell. 2017;171(6):1368-1382 e1323.
