Comparative Analysis of Microfluidics Thrombus Formation in Multiple Genetically Modified Mice: Link to Thrombosis and Hemostasis

Genetically modified mice are indispensable for establishing the roles of platelets in arterial thrombosis and hemostasis. Microfluidics assays using anticoagulated whole blood are commonly used as integrative proxy tests for platelet function in mice. In the present study, we quantified the changes in collagen-dependent thrombus formation for 38 different strains of (genetically) modified mice, all measured with the same microfluidics chamber. The mice included were deficient in platelet receptors, protein kinases or phosphatases, small GTPases or other signaling or scaffold proteins. By standardized re-analysis of high-resolution microscopic images, detailed information was obtained on altered platelet adhesion, aggregation and/or activation. For a subset of 11 mouse strains, these platelet functions were further evaluated in rhodocytin- and laminin-dependent thrombus formation, thus allowing a comparison of glycoprotein VI (GPVI), C-type lectin-like receptor 2 (CLEC2) and integrin α6β1 pathways. High homogeneity was found between wild-type mice datasets concerning adhesion and aggregation parameters. Quantitative comparison for the 38 modified mouse strains resulted in a matrix visualizing the impact of the respective (genetic) deficiency on thrombus formation with detailed insight into the type and extent of altered thrombus signatures. Network analysis revealed strong clusters of genes involved in GPVI signaling and Ca2+ homeostasis. The majority of mice demonstrating an antithrombotic phenotype in vivo displayed with a larger or smaller reduction in multi-parameter analysis of collagen-dependent thrombus formation in vitro. Remarkably, in only approximately half of the mouse strains that displayed reduced arterial thrombosis in vivo, this was accompanied by impaired hemostasis. This was also reflected by comparing in vitro thrombus formation (by microfluidics) with alterations in in vivo bleeding time. In conclusion, the presently developed multi-parameter analysis of thrombus formation using microfluidics can be used to: (i) determine the severity of platelet abnormalities; (ii) distinguish between altered platelet adhesion, aggregation and activation; and (iii) elucidate both collagen and non-collagen dependent alterations of thrombus formation. This approach may thereby aid in the better understanding and better assessment of genetic variation that affect in vivo arterial thrombosis and hemostasis.

Genetically modified mice are indispensable for establishing the roles of platelets in arterial thrombosis and hemostasis. Microfluidics assays using anticoagulated whole blood are commonly used as integrative proxy tests for platelet function in mice. In the present study, we quantified the changes in collagen-dependent thrombus formation for 38 different strains of (genetically) modified mice, all measured with the same microfluidics chamber. The mice included were deficient in platelet receptors, protein kinases or phosphatases, small GTPases or other signaling or scaffold proteins. By standardized re-analysis of high-resolution microscopic images, detailed information was obtained on altered platelet adhesion, aggregation and/or activation. For a subset of 11 mouse strains, these platelet functions were further evaluated in rhodocytin-and laminin-dependent thrombus formation, thus allowing a comparison of glycoprotein VI (GPVI), C-type lectin-like receptor 2 (CLEC2) and integrin α 6 β 1 pathways. High homogeneity was found between wild-type mice datasets concerning adhesion and aggregation parameters. Quantitative comparison for the 38 modified mouse strains resulted in a matrix visualizing the impact of the respective (genetic) deficiency on thrombus formation with detailed insight into the type and extent of altered thrombus signatures. Network analysis revealed strong clusters of genes involved in GPVI signaling and Ca 2+ homeostasis. The majority of mice demonstrating an antithrombotic phenotype in vivo displayed with a larger or smaller reduction in multi-parameter analysis of collagen-dependent thrombus formation in vitro. Remarkably, in only approximately half of the mouse strains that displayed reduced arterial thrombosis in vivo, this was accompanied by impaired hemostasis. This was also reflected by comparing in vitro INTRODUCTION Current concepts of platelet activation pathways in thrombosis and hemostasis rely to a large extent on the summation of single observations. Frequently, the role of a particular protein or signaling pathway is deduced from the consequences of a genetic knockout in mouse on platelet responses, such as in comparison to changes in experimental arterial thrombosis and tail bleeding. A large set of such studies has resulted in the concept of collagen-induced arterial thrombus formation (1)(2)(3)(4). Herein, it is stipulated that the exposure of subendothelial collagen to flowing blood is a key trigger to start shear-dependent thrombus formation. Collagen causes platelet adhesion and furthermore binds von Willebrand factor (VWF), which can decelerate flowing platelets at high shear rate. Firm VWF/collagenmediated adhesion and subsequent platelet activation requires synergy between the VWF receptor, glycoprotein (GP)Ib-V-IX, and the collagen receptors, GPVI and integrin α 2 β 1 (5)(6)(7)(8).
Although all these signaling components are known to play a certain role in collagen-dependent thrombus formation, there is still limited insight into the relative contribution of individual proteins. In addition, it is unclear to which extent C-type lectin-like receptor-2 (CLEC2), another receptor that signals via tyrosine kinases (20,21), is capable to regulate the process of thrombus formation. The same holds for integrin α 6 β 1 , an adhesive receptor, which mediates flow-dependent adhesion of platelets to the matrix protein laminin (22).
In a recent synthesis approach, a quantitative evaluation was made of the contribution of 431 mouse genes to experimental arterial thrombosis and hemostasis in vivo, thereby revealing several genes with a role in thrombosis without affecting bleeding (23). For the total cohort of studies and mouse genes, it appeared that microfluidics assays where thrombus formation is measured in vitro-by whole blood perfusion over a collagen surfacepredict the consequences of a gene knockout on thrombosis models in vivo (23). However, the standard microfluidic tests only report on changes in platelet adhesion (surface area coverage, SAC%), which is a limitation given that the recorded microscopic images also contain information on platelet aggregate formation (24,25). In comparison, for human blood samples from a large cohort of healthy subjects, it could be shown that a multiparameter image analysis can provide detailed information on the sub-processes of platelet adhesion, aggregation and activation at the same time (26).
To better understand the alterations in thrombus phenotypes using microfluidics, we applied a similar multi-parameter approach to quantitatively compare the effects of deficiency of 37 signaling proteins on collagen-induced pathways. We therefore re-analyzed earlier recorded microscopic images, in all cases from thrombi generated using the same microfluidic flow chamber setup.

Mice
Mice were included from 38 strains, in each case with a monogenetic or antibody-induced deficiency, as well as 22 sets of corresponding wild-type or control mice, as described in the original publications (see Table 1). As a selection criterion for inclusion, microscopic images needed to be available from n ≥ 3 animals per modified group and n ≥ 4 for wild-types. Scaled effects on in vivo arterial thrombosis and tail bleeding are for the majority of strains described previously (23).
In all cases, mouse blood was collected into anticoagulant medium, consisting of PPACK (40 µM), unfractionated heparin (5 U/ml) and low molecular weight heparin (fragmin, 50 U/ml). Samples were immediately processed. For original data, experiments were approved by the district government of Lower Franconia (Bezirksregierung Unterfranken) and by the Animal Experimental Committee in Hinxton/Cambridge. For previously published data, experiments were approved by the local Animal Experimental Committees as indicated in the original publications (see references in Table 1).
For image recording, different fluorescence microscopic systems were used (see references in Table 1), but always containing a 60/63x oil objective and a sensitive CCD camera for capturing enhanced-contrast, brightfield images.

Microscopic Image Analysis
For all mouse strains, the recorded 16-bit or 8-bit brightfield and fluorescence images were re-analyzed using the same newly developed scripts (one per image type), written in Fiji (56). Scripts always opened a series of images one-by-one with a loop. In each loop run, background illumination was corrected using a fast Fourier transform bandpass filter, followed by manual setting of a threshold and measurement of the surface area coverage. For brightfield images, a series of Gray morphology conversions was applied to reduce striping and to improve the detection quality. Image conversion steps were as follows: a diamond large sized close, followed by a medium sized circle close and a small circle shaped dilate. Note that the first step increased the pixels that were stronger in regions with many neighboring pixels, the second step then rounded the shapes and additionally reduced straight lines, while the final step (allowing alteration by userinterface) served to obtain the best match with the original image. The Fourier transform filter served to flatten the background areas sufficiently for good analysis, with a minimal impact on the structures. For brightfield images, large structures were filtered down to 60 pixels, for the fluorescence images of annexin A5, integrin and P-selectin images large structures were filtered down to 65 pixels. Small structures were not filtered down, as these contained structures of interest within the adhered platelets (Supplementary Figure 1).
Regardless of the image type, parameter values from three images per experiment were averaged, thus resulting in a single value per parameter and experiment. The image analysis and scoring parameters were verified by different observers, who were blinded to the experimental condition. Parameters of experiments from the same mouse strain were combined as proxy measures of platelet adhesion (P1), thrombus signature (P2-5), and platelet activation (P6-8), as described before for human platelets (26).

Network Analysis
A network of protein-protein interactions was built based on 37 investigated genes, using the STRING database (57), taking into consideration the following settings: 1st shell interactors: <20 interactors, 2nd shell interactors: <60 interactors, confidence level: medium to high. Networks were visualized in Cytoscape version 3.7.0 (58).
Network clustering analysis was performed with the Cytoscape app, MCODE to identify highly interacting nodes using the following settings: degree cutoff: 2, node score cutoff: 0.2, K-core: 2 and maximum depth: 100 (59, 60).

Data Processing and Statistical Analysis
For each genetically modified strain and corresponding wildtypes, image data were averaged to obtain one parameter per surface, of which mean and SD values were calculated. For heatmap representation, mean values were univariate scaled from 0 to 10 per parameter (24). Gene effect heatmaps were constructed by subtracting scaled average values of the wild-type (control) strain from those of the modified strain. For statistical evaluation, a filter was applied, considering changes outside the range of composite mean ± SD as a relevant difference between modification and wild-type (26). Heatmap data were visualized by (unsupervised hierarchical) cluster analysis using the program R (61). For comparison of raw parameter values, a Kendall's tau-b correlation analysis was performed using SPSS (IBM SPSS version 24, Armonk, NY, USA).

Microfluidics Analysis of Thrombus Formation on Collagen of Multiple Wild-Type Mouse Datasets
Microscopic brightfield and (annexin A5) fluorescence images were collected from earlier performed whole blood perfusion experiments with blood from 38 different strains of modified mice and 22 corresponding wild-types ( Table 1). Included were experiments with strains (in majority published), that were judged to be of sufficiently high quality and power to allow re-analysis by newly developed image analysis scripts (Supplementary Figure 1). From each experiment, five parameters of thrombus formation were obtained ( Table 2). Platelet adhesion was quantified by the conventional analysis of platelet SAC% (P1). Thrombus signature (26) was composed of four parameters related to the thrombus buildup (ΣP2-5), i.e., platelet aggregate SAC% (P2), thrombus morphology score (P3), thrombus multilayer score (P4), and thrombus contraction score (P5). As far as available, platelet activation was assessed from fluorescence images of PS exposure (P6) (see Supplementary Figure 1).
To establish the overall consistency of the combined databases of the whole blood experiments, we compared the mean values plus variation for each of the image analysis parameters P1-5 for the 22 wild-type control datasets. The calculated coefficients of variation of means across the wild-type datasets ranged from <12% (P3-5) to 23-25% (P1,2) (Supplementary Table 1). For heatmap presentation, mean values for all wild-types (and corresponding transgenic animals) were univariate scaled from 0-10 ( Figure 1A). The obtained heatmap illustrated an overall high cohesion of the data, yet also suggesting that wild-type strains with mixed C57Bl/6 x 129SV background (databases 04, 05, 08, 16, 20) had a tendency for smaller P3-5 values. Overall, these findings point to a high degree of comparability between the various wild-type datasets.
For assessment of the effects of (genetic) deficiency, scaled values per parameter were calculated for each of the 38 modified mouse strains and compared to those of the corresponding wild-types (Supplementary Figure 2). A subtraction heatmap was generated to pinpoint the effects of genetic modification, in which differences outside the range of (composite) means ± SD were considered as being relevant. The heatmap data could be ranked based on alterations in thrombus signature (Figure 2A) or on differences in platelet adhesion ( Figure 2B). With either way of ranking, profound quantitative differences were observed in the majority of the thrombus formation parameters, when comparing the 38 modified mouse strains. Thrombus signatures were suppressed, in a decreasing order (Figure 2A), by deficiencies in Syk, GPVI/CLEC2, GPVI, Fcer1g, Gnaq, Rac1, Prkd2, Prkca, Stim1, Itga2, Orai1, CLEC2, Mpig6b, Pik3cg, Csk, and Ptprj. In contrast, this thrombus marker was elevated, in an increasing order, by deficiencies in Prkcq, Capn1, Plcg2 (gain-of-function mutation), Apoe, Prkcd, and Fpr2.
The ranking based on altered platelet adhesion revealed several similarities, but also marked differences. Defects in Syk, GPVI, Rac1, Stim1, and Itga2 resulted in a strong suppression of both platelet adhesion and thrombus signature. Relative larger effects on thrombus signature-in comparison to platelet adhesion-were apparent for mice with defects in Fcer1g, Prkd2, and Prkca (reduced thrombus formation), as well as for mice with defective members of the PKC family, Prkcd and Prkcq (increased thrombus formation). Relative increases in platelet adhesion were only seen for mice with deficiencies in Kcnn4, Ano1, and Capn1. In general, these heatmaps demonstrated that the ranking based on changes in thrombus signature (Figure 2A) can provide additional insight into the 'thrombogenic" consequences of a gene defect, in comparison to a ranking based on altered platelet adhesion ( Figure 2B).

Microfluidics Analysis of Thrombus Formation on Collagen and Other Surfaces
For a subset of 11 genetically modified mice strains, the same microfluidic device was used to assess whole blood thrombus formation on collagen-I (M1) plus two additional microspots, i.e., rhodocytin/laminin (M2) and laminin (M3), both co-coated with VWF-BP to induce shear-dependent platelet adhesion ( Table 2). FIGURE 2 | Multi-parameter comparison of collagen-dependent thrombus formation for 38 (genetically) deficient mice. Whole blood from mice with indicated genetic or antibody-induced defects as well as from corresponding wild-type mice was perfused over collagen-I. Parameters of thrombus formation were as indicated in Figure 1. For detailed information on mouse strains, see Table 1. Mean parameters per strain were scaled (0-10) across all wild-types (n = 22) and (genetically) modified mice (n = 38), as for Figure 1. Subtraction heatmaps showing differences between indicated genetic (or antibody-mediated) deficiency in comparison to wild-type, after filtering for differences outside the range of (composite) mean ± SD, to select relevant changes. As clarified before, immobilized rhodocytin triggers CLEC2induced platelet activation (26,54), whereas laminin surfaces allow platelet adhesion via integrin α 6 β 1 (22). For the same subset of mice, the formed thrombi were post-stained in three colors to quantify PS exposure (P6), P-selectin expression (P7) and integrin α IIb β 3 activation (P8), using procedures previously established for human platelet thrombi (26). Representative images from each of the microspots using wild-type blood are depicted in Figure 3. In comparison to collagen-I (M1), rhodocytin/laminin (M2) was less thrombogenic, with only moderately activated platelets that formed small aggregates. The laminin microspot (M3) only triggered adhesion of a monolayer of spreading platelets.
For these 11 genetically modified mouse strains plus corresponding wild-type mice, we again listed the scaled parameters (P1-8) for each microspot (M1-3) (Supplementary Figure 3). The ensuing subtraction heatmap revealed major reductions in platelet adhesion and thrombus signature at surface M1 for the animals with deficiencies in Csk, Ptprj, Mpig6b (tyrosine protein kinase, phosphatases and ITIM receptor, respectively); and, to a clearly lesser extent, for deficiencies in Ifnar1, Vps13a and Anxa1 (Figure 4). For the kinase and phosphatase knockouts, this extended to a reduction in platelet activation parameters (P6-8) at M1, and furthermore to lower P1-2 values at the other microspots M2-3.
Markedly, for several of these mice, also gain-of-plateletfunctions could be detected. In this case, increased parameters P4-6 were distributed over M1 (deficiencies in Apoe and Fpr2) and M2 (deficiency in Grm8). Another remarkable finding was that, for the majority of mice, parameters at the laminin surface (M3) were unchanged. This suggested that lamininplatelet interactions are relatively insensitive to these genetic modifications. Next to the ranking based on changes in platelet adhesion (Figure 4B), the ranking of genes according to changes in overall thrombus signature ( Figure 4A) appeared to be a valuable addition in the description of the alterations in platelet properties.

Linking Microfluidics Outcomes to Thrombosis and Hemostasis in vivo
Recently, we described a systematic procedure to compare the consequences of genetic knockout in mice for experimentally induced (collagen-dependent) arterial thrombosis and hemostasis (23). For the 38 deficiencies, it was thus of interest to compare the results of the current extended microfluidic assay (M1, P1-5) with the previously reported changes in arterial thrombosis tendency and tail bleeding in vivo. Hence, per modified mouse strain, the recorded changes ( Table 1) were listed as being a reduced/unchanged/increased thrombosis phenotype and as a prolonged/unchanged/shortened bleeding time. Figure 5 gives the comparison of these in vivo effects with altered parameters of in vitro thrombus formation using microfluidics.
Strikingly, the majority of mice demonstrating an antithrombotic phenotype in vivo manifested with a larger or smaller reduction in collagen-dependent thrombus formation in vitro (Figure 5). We noted only a few exceptions: (i) mice deficient in Cdc42 with an apparently prothrombotic phenotype, but for unknown reasons no effect in vitro; (ii) mice deficient in Pld1 (where in vitro thrombus formation was only impaired at a higher shear rate of 1700 s −1 ) (14); and (iii) Cd36-deficient mice (requiring a thrombospondin surface for altered in vitro thrombus formation) (29). Also, the impaired arterial thrombosis reported for Capn1 −/− mice (27) did not match with a measured higher platelet adhesion under flow, although it should be noted that the latter mice showed a complex pattern of increased and decreased platelet activation parameters in vitro (28).
Markedly, in the mouse strains with a reduced arterial thrombosis tendency in vivo, only approximately half of these were accompanied with a hemostatic defect (Figure 5). This was also reflected by comparing in vitro thrombus formation (by microfluidics) with alterations in bleeding time. Mice with reduced in vitro thrombus formation, but unchanged or slightly prolonged bleeding times, included animals with deficiencies in the collagen receptors Gp6 and Itga2; the protein kinases Prkd2 and Prkca; and the protein phosphatases Ptprj and Dusp3. This may suggest that the collagen receptors (and hence collagen itself) and the (de)phosphorylating proteins within the downstream signaling pathways are not essential for hemostasis. This may suggest that the collagen receptors (and hence collagen itself) and the (de)phosphorylating proteins are not uniquelyperhaps redundantly-required for platelet functions at lower shear rates such as during hemostasis. The same mouse genes were also previously characterized as having a distinct role in arterial thrombosis and hemostasis (23).

Network Modeling to Predict Novel Proteins Implicated in Mouse Platelet Functions
Based on the 37 different mouse genes/proteins that were analyzed for effects on collagen-dependent whole blood thrombus formation, we constructed a network using the biological STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) database, in order to be capable to depict additional protein-protein interactions. Accordingly, a network was established containing in total 117 nodes (37 core and 80 novel nodes) and 1142 edges (interaction score: medium to highest confidence: 0.40-0.99). Reactome pathways that were highly represented in the network were: platelet activation,

DISCUSSION
In this paper, we applied standardized analysis procedures in order to allow detailed and quantitative comparison of the changes in platelet functions in multiple genetically modified mice, using microfluidic methods of collagendependent thrombus formation under shear conditions. For this purpose, we re-analyzed sets of microscopic brightfield and fluorescence images, using defined semi-automated scripts, resulting in quantitative parameters of platelet adhesion, platelet aggregation (defined as thrombus signature) (26), and platelet activation. The underlying rationale was that earlier captured images provide more relevant information than only a platelet surface area coverage, and thus can provide additional insight into the complex process of thrombus formation. For a subset of mice, it was also possible to extend this complex phenotyping of thrombus formation to other, non-collagen surfaces with additional parameters of platelet activation.
Recent work has shown that multiple platelet function assessment by whole blood microfluidic assays provides novel insights, for instance into the changes in human platelets linked to normal genetic variation (26,62) and in mouse FIGURE 5 | Effects of genetic modification on arterial thrombosis and tail bleeding in comparison to collagen-dependent thrombus formation using microfluidics. Effects of genetic or antibody-induced defects of concerning mouse strains on arterial thrombosis and tail bleeding was obtained from the literature (see Table 1). Effects were classified as being unchanged (black), antithrombotic/prolonged bleeding (green) or prothrombotic/shortened bleeding (red), according to procedures described before (23). White color indicates that information is lacking. Heat mapped data were ranked based on summative effects on all thrombus formation parameters (ΣP1-5) from surface M1.
platelets due to co-activation by interacting chemokines (63). The present comparative quantitative analysis of changes in thrombus formation in multiple genetically or antibody-induced modified mice now also allows to evaluate the assay outcomes for changes in specific platelet functions. Here, we could identify a partial distinction between genes/protein affecting platelet adhesion and those altering platelet aggregation properties (collectively termed as thrombus signature). For instance, it seems that the murine Csk, Mpig6b and Ptprj have a relatively large role in flowdependent platelet adhesion. Markedly, for Csk, Mpig6b, and Ptprj this effect is extended to also a reduced adhesion at the non-collagen surface M2. On the other hand, the processes of platelet adhesion and aggregate formation are also related, as shown by an overall consistent correlation between adhesion and aggregation parameters (Kendall's tau-b = 0.42-0.68). A similar conclusion was drawn earlier from the analysis of thrombus formation in blood samples from 94 healthy subjects, also pointing to the existence of a subject-dependent thrombus signature (26).
In comparison to the collagen surface (M1), for the 11 mouse strains analyzed, we observed in general smaller gene effects at the two other surfaces (M2-3), which mediate platelet adhesion via GPIb-V-IX and α 6 β 1 with or without CLEC2 (rhodocytin). Accordingly, it seems that, at least in part, distinct sets of genes/proteins are implicated in the platelet adhesion to non-collagen surfaces than to the collagen surface. Clearly in vivo thrombus formation is not purely mediated by collagen, but rather is the result of platelet interactions with a mixture of different extracellular matrix proteins.
The present data set also includes new findings with unpublished blood samples from mice deficient in Rhoa and Syk. The Rhoa defect did not appear to influence platelet adhesion nor thrombus signature parameters at the shear rate of 1000 s −1 , which is in support of the earlier conclusion that platelet RHO-A becomes relevant for thrombus formation at high (pathological) shear rates (13). In contrast, genetic deficiency in Syk resulted in a strong reduction of all platelet and thrombus parameters on collagen. This highlights the importance of SYK signaling in collagen-dependent thrombus formation.
Given the earlier established correlation between the outcome of microfluidic tests (in terms of platelet surface area coverage) and experimental arterial thrombosis in mice (23), it was of interest to evaluate for the current mouse strains how a more detailed analysis (considering more thrombus parameters) contributes to this relationship. Such a comparison clearly has limitations, such as the wide variety of methods and test outcomes of the in vivo thrombosis measurements, making a clear differentiation between moderate and strong phenotypes difficult (23); and furthermore, the absence of coagulation and vessel wall components other than collagen in the in vitro approach. Nevertheless, a ranking of the investigated mouse strains according to overall changes in thrombus parameters (including platelet adhesion and aggregation) showed a good reflection with published changes in arterial thrombosis in vivo. On the other hand, for a considerable set of genes, a changed thrombus formation in vitro (and mostly in vivo) was not associated with an altered bleeding time.
In several cases, a discrepancy is well explainable. For instance, defects in platelet Clec1b (as a non-collagen receptor) (64) or in Ano6 (an isolated defect of platelet-dependent coagulation) (18), will not be picked up by flow assays over collagen with anticoagulated blood. A striking example is Gp6. Whereas, GPVI deficiency overall impairs thrombus formation in vitro as well as arterial thrombosis in vivo, mouse tail bleeding times are only moderately prolonged (37,38). In line with that, mild bleeding symptoms have been reported in patients with a defect in the GP6 gene (65,66). Hence, this gene does likely have a restricted role, which is in line with the constructed network indicating multiple GPVI-linked proteins that contribute to both thrombosis and hemostasis. FIGURE 6 | Network of protein-protein interactions in collagen-dependent thrombus formation. Network, constructed from the STRING database and visualized in Cytoscape, of murine protein-protein interactions with as seed the 37 investigated core genes. The network contained 117 nodes (37 core nodes, 80 novel nodes) and 1142 edges. Core nodes of the network were color-and size-coded based on altered thrombus signature (ΣP2-5) at surface M1. Green color intensity (larger size) of nodes points to a stronger reducing effect, and red color intensity (large size) to a stronger stimulating effect in comparison to wild-type. Novel nodes are indicated in gray.
Translation of the current evaluation of genes in murine thrombus formation to human pathophysiology can-taking into account the above-provide better insight into the genetic background of human platelet function abnormalities and how these can relate to thrombotic and bleeding disorders. Indeed, for homologs of several of the genes analyzed in this paper, e.g., human ORAI1 and STIM1, mutations have been identified that link to defective collagen-dependent thrombus formation in vitro (67). By applying an extended image analysis, a detailed description of the formed thrombi can be generated. This can aid in the identification of the defective part (platelet adhesion, aggregation or procoagulant response) of the process of thrombus formation in patients with an unexplained increased risk for either thrombosis or bleeding. Extended analysis of the available mouse data and summation in the form of a network can also contribute to a more targeted approach to select novel candidate genes and proteins, possibly affecting platelet functions in a positive or negative way.
Moreover, by applying new knowledge and techniques, in the present paper, new insights were gained from previously performed mouse experiments. In such a manner, our study may contribute to the 3R approaches by reducing, refining and replacing animal experiments.
Taken together, we conclude that the presently developed multi-parameter analysis of thrombus formation on microspots using microfluidics can be used to: (i) determine the severity of platelet abnormalities; (ii) distinguish between altered platelet adhesion, aggregation and activation; and (iii) elucidate both collagen and non-collagen dependent platelet changes. This approach may thereby aid in the better understanding and better assessment of the changes in platelets that affect arterial thrombosis and hemostasis.

DATA AVAILABILITY
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

ETHICS STATEMENT
For original data, experiments were approved by the district government of Lower Franconia (Bezirksregierung Unterfranken) and by the Animal Experimental Committee in Hinxton/Cambridge. For previously published data, experiments were approved by the local Animal Experimental Committees as indicated in the original publications (see references in Table 1).

AUTHOR CONTRIBUTIONS
MN and JvG analyzed and interpreted the data and wrote the manuscript. SdW and MK analyzed the data. RV created scripts used for image analysis. DA, AB, ME, KK, CO, JP, IP, AP, JM, MG, YS, and CW provided the mice, other tools, and revised the manuscript. DS and BN provided the mice, original published and unpublished images, and revised the manuscript. JH and CB provided expert supervision, analyzed and interpreted data, and wrote the manuscript.