Megakaryocyte and platelet thrombospondin-1 regulates matrix remodeling by stabilizing basement membrane COL6A1 in lung injury

Domain: biology
Published: 2026-03-12
Editorial outcome: major_revision

Abstract
An early event after lung injury is extracellular matrix (ECM) remodeling and the formation of a provisional
matrix. While megakaryocytes and platelets (Mgk/plt) play important roles in hemostasis, their impact on
the matrix in lung injury is not fully understood. Using lung intravital microscopy, 3D large area scanning
with multiphoton confocal hybrid imaging, and label-free quantitative proteomics, the matricellular protein
thrombospondin-1 (TSP1) arising from Mgk/plt protects the lung from alveolar injury. Mgk/plt cell-specific
Thbs1 knockout (cKO) mice show increased alveolar barrier disruption with exaggerated neutrophil-
mediated injury. The cKO mice exhibit striking extracellular matrix re-organization with reduction in
basement membrane protein COL6A1 after injury. Moreover, cKEO mice increase Mgk numbers to regions
of fibrillar collagen deposition and alveolar leak. Our findings indicate Mgk/plt-derived TSP1 represents
a key mechanism of matrix stabilization by protectinNg the basement membrane from neutrophil-mediated
proteolytic damage, regulating injury severity and Mgk numbers at the alveolar interface.

Introduction
Acute lung injury, clinically known as the acute respiratory distress syndrome (ARDS), is a syndrome of
acute hypoxemic respiratory failure caused by alveolar epithelial-capillary membrane barrier disruption1,2.
A prominent feature of ARDS is an exaggerated inflammatory response often dominated by a neutrophil-
rich exudate in the airspaces3. Infection-related insults such as severe pneumonia cause direct lung
injury whereas sepsis causes an indirect form of lung injury. These two risk factors collectively account
for the majority of ARDS worldwide4, although non-infectious injury from aspiration, trauma, and
inhalational toxins also results in ARDS5. An intense inflammatory response in the airspaces of ARDS
patients occurs during the early exudative phase and is characEterized by proinflammatory cytokines and
chemokines, release of neutrophil proteases such as neutrophil elastase (NE) and metalloproteinase-9
(MMP-9), as well as their inhibitors. In addition, thNe early phase of injury is characterized by elevated
procollagen peptide III levels, a marker of collagen synthesis and dysregulated repair6,78.

Original reports in ARDS demonstrated that ECM remodeling is an early event that shifts the balance
towards fibrillar collagen I synthesis and deposition9. This early ECM remodeling event leads to altered
lung tissue mechanics after injury in an experimental rat model10. Persistent elevation in markers of
collagen synthesis in the airspace compartment is associated with ineffective lung repair and increased
risk of death in ARDS11,12. While inflammatory injury is a hallmark of clinical ARDS and thought to
contribute to matrix remodeling, whether components of the early ECM regulate the inflammatory
response and shape the outcome of alveolar injury and repair are less known.

Thrombospondin-1 (TSP1) is a matricellular protein involved in cell-cell and cell-matrix interactions and
a component of the lung ECM13. Multiple cell types produce TSP1 including Mgks and platelets,
endothelial cells13, fibroblasts14, smooth muscle cells15, type II pneumocytes16, macrophages17, and
dendritic cells18. However, platelets19 are the major source of TSP1 in circulation and store large
quantities of TSP1 in α-granules20,21. Upon platelet activation, TSP1 is liberated onto the platelet
membrane and the extracellular space22,24,25. Although direct evidence for TSP1 involvement in the
stabilization of the lung ECM is lacking, TSP1 binds collagen I23, fibrinogen/fibrin, fibronectin24, heparan
sulfate proteoglycans25, laminin26, as well as integrins such as α \beta 24 which is highly expressed by
3 1
alveolar epithelial cells27. In acute lung injury, alveolar barrier disruption leads to exposure of the
subendothelial matrix to the vascular compartment and extravasation of serum proteins, platelet proteins,
and clotting factors. Platelets and their cellular constituents are critical for the formation of the initial
provisional matrix during wound healing comprised of a loose ECM network that permits cell movement,
migration, and dynamic remodeling28,29, although platelet constituentSs that are involved in lung injury are
poorly understood. Given that Mgks and platelets are a major source of TSP1, and that TSP1 is
incorporated into the provisional matrix30,31, we sought to determine whether Mgk/plt TSP1 represents a
key mechanism of matrix stabilization, regulating neut rophil response and Mgk numbers at the alveolar
interface.

Results
Intravital microscopy reveal increased alveolar barrier disruption in global Thbs1-/- reporter mice
following alveolar injury.

Our previous work demonstrated that TSP1 deletion in mice increases lung microvascular permeability
after acute pulmonary infection with Pseudomonas aeruginosa (PA)19,32. Here, we used live intravital
imaging to precisely localize and quantify airspace leakage following alveolar injury.

For this purpose, reporter ROSAmT/mG mice that constitutively express cell membrane-localized td-
Tomato in cells and tissues were crossed to Thbs1-/- global KO mice. To induce experimental lung injury,
mice underwent i.t. instillation of Pseudomonas aeruginosa cell-free supernatant (PA SN). At day 1 post-
injury and just prior to imaging, mice were i.v. injected with FITC-Dextran (70 kDa) to visualize dextran
extravasation into the airspaces by live intravital microscopy (Fig. 1A). Image processing and analysis
were conducted according to the schematic pipeline illustrated in Figure 1B-F, utilizing a custom-
developed NIS-Elements General Analysis (GA3) recipe to ensure precise and reproducible
quantification. Representative raw images of lungs from vehicle treated and PA SN injured mice are
shown with alveolar parenchymal structure outlined in red and intravenously injected FITC-dextran
visualized in green (Fig. 1B), where the injured lung shows greater dispersal of FITC-dextran beyond the
alveolar boundaries. For quantification of alveolar leak, lung structure was segmented and overlaid with
a corresponding binary mask defined by the presence or absence of Td-tomato expressing cell
membrane (Fig. 1C). The resulting binary mask was inverted to identify the airspaces (Fig. 1D). The
emissions from the FITC-dextran were segmented based on the emiSssion intensity of the FITC channel
(Fig. 1E). We quantified airspace leak using area overlap analysis with a Boolean “And” statement where
the area of airspace leak (µm2) was defined as the pixels within the defined airspace mask that were also
positive (“And”) for dextran (Fig. 1F). Quantitative a nalysis confirmed that ROSAmT/mGThbs1-/- mice
exhibited significantly greater leakage of dextranI into the airspaces following alveolar injury compared to
wild-type controls (Fig. 1G).

Megakaryocyte/platelet-specifTic Thbs1 KO mice show normal platelet function

To address the cell-intrinsic role of TSP1, we generated a Thbs1 floxed allele mouse (Thbs1flox) using
CRISPR/Cas9 technology. Targeted insertion of LoxP sites in intron 3 and intron 7 led to the generation
of the floxed allele (Fig. S1A). As platelets and their precursors Mgks are a major source of TSP1,
Thbs1flox mice were subsequently bred with Cre-expressing mice under the control of the platelet factor
4 gene (PF4Cre) to obtain Mgk/platelet-specific Thbs1 knock-out (PF4CreThbs1flox KO) mice. Thrombin-
stimulated platelet releasates from the PF4CreThbs1flox KO mice showed loss of TSP1, while WT littermate
controls (PF4WTThbs1flox) displayed levels comparable to those of C57BL/6 wild-type mice (Fig. 2A).
Moreover, cell-specific deletion of TSP1 from Mgk/platelets (Mgk/plt) did not alter circulating platelet
counts (Fig. 2B) nor did it affect the surface levels of activated αIIbβ3 integrin (Fig. 2C) or P-selectin (Fig.
2D) following stimulation with various concentrations of thrombin or collagen-related peptides (CRP) (Fig.
S1B-C). Platelet aggregation responses to thrombin (Fig. S1D) and CRP (Fig. S1E) were similar between
WT and PF4CreThbs1flox KO mice. Platelet activation and aggregation were also similar between WT
littermates and PF4CreThbs1flox KO mice in response to increasing concentrations of U46619 (a
thromboxane TXA2 analog) or ADP (Fig. 2E-H). These results suggest that Mgk/plt TSP1 does not
regulate platelet activation and adhesive function.

MgK/plt TSP1 regulates neutrophil activity and matrix remodeling-related proteins to limit
alveolar barrier disruption during live bacterial infection.

In a live lower respiratory tract infection model with Pseudomonas aeruginosa (Fig. 3A), PF4CreThbs1flox
KO mice exhibited higher lung bacterial burden at day 1 post-infectionS compared to WT littermate controls
(Fig. 3B). Although global Thbs1 KO mice show significant defects in host defense during acute PA
pneumonia when compared to WT mice19,32, a more modest phenotype was observed in PF4CreThbs1flox
KO mice, suggesting that additional cellular sources o f TSP1 contribute to pulmonary host defense. To
assess the role of MgK/plt-derived TSP-1 in alvIeolar barrier integrity, we measured total BALF protein
levels. Following PA instillation, PF4CreThbs1flox KO mice exhibited significantly elevated BALF protein
levels compared to WT littermates (Fig. 3C). Notably, airspace neutrophil recruitment (Fig. 3D) were
comparable between WT and PTF4CreThbs1flox KO mice. Despite similar numbers of neutrophils in the
airspaces, PF4CreThbs1flox KO mice showed significantly higher BALF neutrophil elastase (NE) activity
(Fig. 3E) and MMP-9 levels (Fig. 3G). Moreover, airspace neutrophils from PF4CreThbs1flox KO mice
demonstrated higher levels of CD63 surface expression, a marker of degranulation (Fig. 3G-H). In the
live infection model, a gating strategy to delineate immune cell subsets in the BALF and lung showed no
differences in other cell types besides neutrophils including alveolar macrophages, eosinophils, and
monocytes between WT and PF4CreThbs1flox KO mice (Fig. S2A-D). Despite similar immune cell
numbers, PF4CreThbs1flox KO mice showed elevated levels of myeloperoxidase (MPO) and NE-DNA
complexes in the BALF, indicative of enhanced neutrophil granule release and neutrophil extracellular
trap (NET) formation (Fig. S2E-F). Levels of cytokines and chemokines involved in neutrophil responses
were similar between WT and PF4CreThbs1flox KO mice (Fig. S2G–K). Moreover, neutrophils obtained
from PF4CreThbs1flox KO mice were not developmentally altered and showed no differences in CD62L
and, \alphaM\beta2 surface levels ex vivo either at baseline or with TNF\alpha, fMLP, or A23187 stimulation (Fig. S2L-
M). The ability to form platelet-neutrophil aggregates was similar between the two groups (Fig. S2N).
Other cell types produce TSP1, in particular myeloid-derived cells, that could influence the course of
inflammation and injury in the live infection model. However, myeloid cell-specific Thbs1 KO mice
(LysMCreThbs1flox) showed no differences in lung CFU following live PA infection, BALF total protein,
BALF NE activity or composition of immune cells in BALF and lung tissue homogenates (Fig. S2O-T).
Collectively, these data strongly suggest that Mgk/plt TSP1 limits neutrophil activation within the lung
tissue microenvironment.

Given the essential role of Mgk/plts in forming the provisional matrix29,30, we explored matrix-related
proteins within the airspace compartment. We utilized mass spectrometry label-free quantitative
proteomics to compare the proteomic profile of BALF in WT and PF4CreThbs1flox KO mice. Neutrophil
degranulation was identified as the top enriched pIathway in BALF following PA infection when comparing
the differential proteomic signatures of WT and PF4CreThbs1flox KO mice (Fig. 3I). To assess the alveolar
matrix niche, we performed laser-capture microdissection (LCM) of injured alveolar tissue from WT and
PF4CreThbs1flox KO mice after live PA infection and coupled with label-free quantitative mass
spectrometry. Strikingly, differential analysis of the alveolar tissue proteomics between WT and
PF4CreThbs1flox KO mice revealed shared pathway enrichment signatures including neutrophil
degranulation, platelet activation, and ECM organization also identified in BALF (Fig. 3J). Differentially
up and down regulated proteins comparing WT and PF4CreThbs1flox KO mice in BALF and alveolar tissue
within these shared pathways feature key proteins involved in early lung injury (Fig. S3A and S3B). Within
alveolar tissue, we identify increases in neutrophil proteases ELANE (or, NE), MMP9, CTSG (or,
Cathepsin G) and markers of the provisional matrix29,33,34 fibronectin (FN1), fibrin or fibrinogen proteins
(FGA, FGB, FGG), F13A1 (which catalyze the conversion of fibrinogen to fibrin), and integrins in
PF4CreThbs1flox KO mice when compared to WT after injury (Fig. S3B). The increase in provisional matrix
proteins observed in PF4CreThbs1flox KO alveolar tissue is likely a consequence of the increased injury
and leak observed in these mice.

Loss of MgK/plt TSP1 but not endothelial TSP1 leads to increased alveolar leak with impaired
lung function following injury.

To exclude the possibility that the injury is from uncontrolled bacterial replication, we induced sterile lung
injury in WT and cKO mice using cell-free PA supernatant (PA SN)35 and evaluated microvascular
permeability by intravital imaging to visually confirm and quantify the degree of airspace leak following
injury. For this purpose, we generated PF4CreROSAmT/mG (WT) and PF4CreROSAmT/mG Thbs1flox (TSP1PF4
KO) reporter mice that constitutively express tdTomato in all cell membranes except in Mgks and
platelets which express green fluorescence protein (GFP) undeSr the control of PF4-driven Cre
recombinase. We utilized segmentation and overlay analysis of WT and TSP1PF4 KO reporter mice lungs
to quantify alveolar leak (Fig. S4A-F). At day 1 post PA SN instillation, mice were i.v. injected with CF680-
Dextran (70 kDa, cyan) and dextran extravasation into the airspaces was visualized by intravital
microscopy (Fig. 4A-B). When WT and TSP1PFI4 KO reporter mice were i.t. instilled with sterile PBS
(vehicle), dextran was visualized within the vessels and, to a limited extent, within the interstitium (Fig.
4B, left upper and lower panels). How C ever, when mice were instilled with PA SN, both WT and TSP1PF4
KO reporter mice showed wideniTng of the interstitial space and dextran extravasation into the airspaces,
whereas TSP1PF4 KO reporter mice showed significantly elevated levels of CF680-Dextran leak into the
alveolar space as measured by airspace leak area (Fig. 4C) and airspace leak intensity (Fig. 4D).
Intravital microscopy details a limited window to the lung, therefore we confirmed these findings by
quantifying the lung injury score in H&E sections (Fig. 4E-L), Evans blue (EB) dye assay to assess lung
permeability (Fig. 4M), and lung mechanics (Fig. 4N-R). PF4CreThbs1flox KO mice showed increase in
hemorrhagic injury, as evidenced by higher lung injury score after PA SN instillation when compared to
WT littermates (Fig. 4H). We did not observe differences between WT and PF4CreThbs1flox KO mice in
the ratio of neutrophils localized to either the alveolar spaces or interstitium (Fig. 4I-J). This finding is
consistent with similar neutrophil numbers found in WT and PF4CreThbs1flox KO mice after live PA
infection (Fig. 3D). The increase in lung injury score observed in the PF4CreThbs1flox KO mice after sterile
injury is driven by the alveolar edema tissue score and the alveolar septal thickness score rather than
the ratio of alveolar or interstitial PMN scores (Fig. 4K-L). This finding is consistent with the airspace
barrier leak observed by lung intravital microscopy (Fig. 4B-D). Moreover, PF4CreThbs1flox KO mice
exhibited increased lung EB extravasation following PA SN challenge when compared with WT
littermates (Fig. 4M). We next tested lung function at baseline (without vehicle) in WT and PF4CreThbs1flox
KO mice. As expected, injury reduced static compliance in both groups of mice (Fig. 4N). Forced
oscillation tests further reduced the dynamic compliance observed in PF4CreThbs1flox KO mice but not in
WT mice group, underscoring the altered lung function in the PF4CreThbs1flox KO mice after injury (Fig.
4O). At day 1 post-injury, PF4CreThbs1flox KO mice had significaSntly increased respiratory system
resistance (Fig. 4P), Newtonian resistance (Fig. 4Q), and tissue elastance (Fig. 4R). These
biomechanical changes are features of lung injury and underscore the importance of MgK/platelet-
derived TSP1 in maintaining lung function and integrity .

Previous studies identified a protective role for endothelial-derived TSP1 in limiting alveolar epithelial
injury13. To determine whether endo C thelial TSP1 contributes to maintaining alveolar barrier integrity
following PA SN challenge, we gTenerated endothelial-specific Thbs1 cKO (Cdh5CreThbs1flox) mice. Loss
of endothelial TSP1 did not significantly alter lung EB accumulation following PA SN challenge (Fig. S5A-
B) compared to WT littermates (Cdh5WTThbs1flox). Unlike MgK/platelet-derived TSP1, these findings
suggest that endothelial TSP1 does not play a major role in limiting alveolar leak or injury-associated
barrier dysfunction.

Mice deficient in MgK/platelet TSP1 show increased MgK recruitment to sites of collagen
exposure and alveolar barrier disruption.

Prior reports indicate that Mgks are localized to the lungs of human and mice36,37. Utilizing publicly
available gene expression data38, we show that the relative Thbs1 gene expression in adult lung MgK is
higher than in adult bone marrow MgK (Fig. S6A). In addition, fetal lung MgKs demonstrate higher Thbs1
gene expression than fetal liver MgKs (Fig. S6A). By lung intravital imaging, we noted striking
appearance of lung MgKs that were > 30 microns in diameter (Fig. 5A, Supplementary Movie 1). To
distinguish MgKs from platelets or platelet aggregates, we confirmed MgK by nuclear staining with DAPI
and high resolution (60X, 1.4NA) large scanning of static lung tissue by confocal microscopy (Nikon A1R)
(Fig. S6B). Advanced 3D image analysis using surface rendering tools in Imaris (Bitplane) confirmed
MgKs with nuclei (Fig. S6C). Quantitative analysis using the NIS-Elements software revealed that, under
vehicle instillation conditions, MgK events were similar in WT and TSP1PF4 KO reporter mice (Fig. 5B).
However, after PA SN-induced lung injury, TSP1PF4 KO reporter mice exhibited a significant increase in
MgK events (defined as > 30 \mu m in size) compared to WT reporter controls (Fig. 5B). The dynamic MgK
accumulation in TSP1PF4 KO reporter mice suggests dysregulated mSobilization or retention in the lung
microenvironment following injury. Lung platelet events were not different between the groups by imaging
analysis or flow cytometric analysis (Fig. S6D-I), while peripheral platelet counts dropped with injury
(Fig. S6J).

Collagen levels play a critical role in Mgk function, mediated by interactions with collagen fibers via
receptors such as GPVI and \alpha2\beta1 integrin39,40. Our BALF proteomic analysis demonstrated enrichment
of key proteins involved in both ECM degradation and collagen formation (Fig. 3I, 5C), suggesting altered
collagen or ECM componeAnt levels. Consistent with these findings, Second Harmonic Generation (SHG)
microscopy of collagen in our lung injury model revealed that fibrillar collagen was markedly increased
in TSP1PF4 KO reporter mice (Fig. 5D-F). Both the total collagen area and collagen deposition near
regions of airspace leak were higher in TSP1PF4 KO reporter mice compared to WT mice (Fig. 5G-H).
Moreover, collagen accumulation in proximity to Mgks and where Mgks localized to regions of airspace
leak were significantly elevated in TSP1PF4 KO reporter mice (Fig. 5I-J). Interestingly, Mgks were
identified in both the extravascular (Fig. 5A, and Supplementary Movie 1) and intravascular spaces (Fig.
5D), and were prominently located at the junctions of alveolar structures marked by Td-Tomato
expression (Fig. 5D). These observations were further validated by 3D large-area multiphoton/confocal
hybrid imaging (Nikon AXR MP/Confocal), which confirmed increased collagen deposition within
adventitial interstitium where Mgks accumulate in TSP1PF4 KO reporter mice lungs (Fig. 5K-L).
Collectively, these results indicate that loss of Mgk/platelet-derived TSP1 leads to increased collagen
deposition and Mgk accumulation, likely reflecting greater degree of injury and leak.

Neutrophil depletion ameliorates lung injury and increases basement membrane protein levels in
mice deficient in Mgk/platelet TSP1.

We next evaluated whether the exaggerated neutrophilic response observed in the lungs of Mgk/plt-
deficient TSP1 mice is a main driver of increased alveolar permeability following lung injury. Pre-
treatment with anti-Ly6G antibody to deplete neutrophils resulted inS a robust decrease in BALF PMN
counts and MPO release (Fig. 6A-C) and reduction of alveolar permeability similar to levels observed in
WT controls (Fig. 6D). These data demonstrate neutrophils as a major contributor to the intense barrier
disruption observed in PF4CreThbs1flox KO mice and su ggest that MgK/platelet TSP1 dampens excessive
neutrophil activation encountered at the barrier inIterface.

In parallel experiments, flow cytometric analysis was performed following anti-Ly6G antibody treatment
(Fig. 6E). While the lung neuTtrophil counts were comparable between isotype-treated WT and
PF4CreThbs1flox KO mice after PA SN injury (Fig. 6F), NE release in BALF were significantly higher in
PF4CreThbs1flox KO mice (Fig. 6G). Depleting neutrophils with anti-Ly6G antibody treatment reduced lung
neutrophil counts and BALF NE levels in PF4CreThbs1flox KO mice (Fig. 6F-G). BALF IgM levels, a
surrogate marker of lung permeability, were increased in PF4CreThbs1flox KO compared to WT mice after
injury (Fig. 6H). Analysis of live CD45+CD41+ Mgk revealed that isotype control antibody-treated
PF4CreThbs1flox KO mice exhibited higher Mgk accumulation than WT littermate controls (Fig. 6I-J),
consistent with lung intravital imaging results (Fig. 5B). Mgks were identified as large cells (FSChigh) that
were CD45+CD41+ and distinct from neutrophils (Fig. S7 and S7A2, respectively). Utilizing the same
gating strategy (Fig. S8A1) or adding lineage markers B220, GR1, and F4/80 in the “dump channel” to
separate lineage committed cells from Mgks (Fig. S8A2), both approaches revealed that the relative
numbers of CD45+CD41+ Mgk numbers increase in cKO lungs and distinct from neutrophils (Fig. S8A3)
when compared to WT after injury (Fig. S8B, S8C).

Importantly, neutrophil depletion diminished Mgk counts and BALF TSP1 levels in PF4CreThbs1flox KO
mice after injury (Fig. 6J-K). These findings suggest that Mgk counts and soluble TSP1 levels are markers
of injury severity. As Mgk can synthesize collagen that contributes to matrix regeneration in the
marrow39,40, we measured pro-COL1A1 and found that levels were increased in PF4CreThbs1flox KO after
injury but were not significantly different between PF4CreThbs1flox KO and WT mice after neutrophil
depletion (Fig. 6L). Neither depleting neutrophils nor reducing MgK numbers in PF4CreThbs1flox KO mice
limits COL1A1 synthesis and point to other cell types (i.e., fibroblastS) as major producers of collagen in
the lung. We also measured components of BALF COL4 and COL6 as these matrix proteins comprise
the alveolar basement membrane41,42 and were significantly reduced in PF4CreThbs1flox KO by BALF
proteomics (Figure S3A, ECM organization heatmap) . While PF4CreThbs1flox KO and WT mice showed
similar levels of BALF COL4A1 with injury, COL6A1 levels were significantly reduced in the
PF4CreThbs1flox KO mice (Fig. 6M-N). Moreover, neutrophil depletion increased COL4A1 and COL6A1
levels in lung injury (Fig. 6M-N). These findings strongly support the concept that Mgk/plt TSP1 prevents
degradation of the alveolar baTsement membrane, specifically COL6A1, from neutrophil-mediated
proteolytic injury (Fig. 6O).

Methods
Sex as a biological variable

Both male and female mice aged-matched (7–22 week olds) were Sused for studies involving experimental
lung injury. We did not observe sex differences in the outcomes measured. Additional information is
provided in the Supplementary Source Data file.

Mice

C57BL/6J (WT, stock #000664), CC57BL/6-Tg(Pf4-icre)Q3Rsko/J (PF4Cre, stock #008535), and
B6.129P2-Lyz2tm1(cre)Ifo/J (LyTsMCre, stock #004781) mice were originally obtained from the Jackson
Laboratories (Bar Harbor, ME) and maintained in the animal facility of University of Pittsburgh and
Washington University in St. Louis. TSP-1 floxed (Thbs1flox) and full body TSP-1 deficient mice (Thbs1-/-)
were generated at the University of Pittsburgh Transgenic and Gene Targeting and Innovative
Technologies Development Cores using CRISPR/Cas9 technology. Briefly, single stranded
oligodeoxynucleotide (ssODN) templates were used to introduce LoxP and BamHI restriction enzyme
sites between exons 3 and 4, and exons 7 and 8 of the Thbs1 gene by homologous directed repair at
the position of the double strand break (DSB) induced by Cas9 nuclease. Genotyping using PCR flanking
the LoxP sites and BamHI restriction digest identified founder mice carrying the Thbs1 floxed allele that
was confirmed by genomic sequencing. The founder mice were backcrossed to the C57Bl/6J mice for 6
generations and homozygous founder colonies of Thbs1fl/fl mice were subsequently established. Thbs1fl/fl
mice were bred with PF4Cre mice to obtain a megakaryocyte and platelet specific TSP-1 knockout mouse
line (PF4CreThbs1flox) and their WT littermate controls (PF4WTThbs1flox).

For intravital imaging, PF4CreThbs1flox were bred with the ROSAmT/mG mice to obtain
PF4CreROSAmT/mGThbs1flox (TSP1PF4 reporter) and PF4CreROSAmT/mGThbs1flox (WT reporter) reporter
mouse line. Additionally, ROSAmT/mG mice were bred with Thbs1-/- mice to obtain a full body reporter
mouse line deficient for TSP-1 (Thbs1-/-ROSAmT/mG). ROSAmT/mG mice were used as reporter WT controls.
To evaluate the role of endothelial TSP-1 in tissue resiliency, Thbs1fl/fl mice were bred with constitutive
Cdh5Cre mice (B6.FVB-Tg(Cdh5-cre)7Mlia/J, JAX stock #006137), kindly provided by Dr. Kyunghee Choi
(Washington University) to obtain endothelial specific TSP-1 knockouSt mouse line (Cdh5CreThbs1flox) and
their WT littermate controls (Cdh5WTThbs1flox). Each mouse line used in this study was co-housed in the
same vivarium and fed the same chow for at least 4 weeks prior to in vivo experiments. All experimental
protocols were reviewed and approved by the IACUC at the University of Pittsburgh and Washington
University in St. Louis.

Pseudomonas aeruginosa live bacterial lung infection and sterile injury models.

Pseudomonas aeruginosa PA14 strain was grown overnight at 37ºC, 2,500 x g in 3 mL of Luria-Bertani
(LB) broth (Sigma-Aldrich L3522) to reach an optical density (OD) = 0.5-0.6 which yielded ~1 x 109 – 5 x
109 CFU/mL. Next, 100 µL of the preparation was resuspended in 10 mL of sterile PBS to reach a stock
inoculum concentration of 1 x 107 – 5 x 107 CFU/mL. Mice were anesthetized with isoflurane in an
anesthesia chamber and intratracheal (i.t.) inoculation with 100 µL of the stock for a final inoculum of ~1-
5 x 106 CFU of PA1419,32,35. In indicated experiments, PA14 strain was grown overnight (16-18 hours) at
37ºC, 2,500 x g in 3 mL of LB broth. The overnight culture was then filtered through a 0.22 \mu m sterile
filter to obtain PA14 cell free SN that was delivered i.t. to induce sterile lung injury35. Mice were i.t.
inoculated with 30% cell free PA SN at 100 \mu l volume. Depending upon the experiment, sex- and age-
matched mice were delivered either live bacteria or PA SN by i.t. delivery. Mice were euthanized after 1
day for tissue processing.

Lung tissue and bronchoalveolar lavage fluid (BALF) collection.

The trachea was cannulated using an 18 G catheter, and the left lung was ligated at the hilum with 2-0
silk suture. BALF from the right lung was subsequently obtained from mice by instilling a first lavage of
600 µL of sterile cold 1x PBS/EDTA (0.6 mM), followed by 3 more lavages of 500 µL each (final volume
of 2.1 mL). BALF cells were pelleted by centrifugation at 300 x g for 15 min for flow cytometry and the
cell-free supernatant was collected and stored in -80oC until further use19,32. BALF measurements
included total BALF protein content (Pierce™ BCA Protein Assay Kit, Thermofisher), free NE activity,
myeloperoxidase (MPO) levels, free DNA NE-DNA complex and untargeted proteomics (LC/MS).

BALF and lung tissue flow cytometry.

BALF recovered from mice was centrifuged at 300 x g for 15 min. After BALF collection, the pellet was
immediatly incubated with 1x Ammonium-EChloride-Potassium (ACK) buffer (150 mM NH Cl (Sigma-
4
Aldrich), 10 mM KHCO (Sigma-AldriCch), 0.1 mM Na EDTA (Sigma-Aldrich), pH 7.4) for 5 min at room
3 2
temperature and cells were wasThed twice with 1x PBS. Cells were subsequently stained for viability
(LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit, Thermofisher) for 30 min at room temperature in the
dark, washed twice with 1x PBS and resuspended in the antibody cocktail in PBS/Newborn calf serum
(NCS) 2%. The following antibodies were included in the staining cocktail CD45-AF700 (clone 30-F11,
cat. 560510 BD), CD11b-PE (clone M1/70, cat. 553311 BD), CD11c-PE-cy7 (clone HL3, cat. 558079
BD), CD64-BV650 (clone X54-5/7.1, cat. 740622 BD), Ly6G-APC (clone 1A8, cat. 560599 BD), SiglecF-
APC-cy7 (clone E50-2440, cat. 565527 BD). Samples were analyzed using a BD LSR FORTESSA. Cells
were counted as (cell/mL) by the addition of countbright absolute counting beads (ThemoFisher, C36950)
immediately before samples were analyzed. Gating analyses were done using Flowjo v10.6.2 MAC (BD
biosciences).

For lung tissue analysis, right after lung collection, they were minced and digested in 5 mL of 0.7 mg/mL
collagenase (in DMEM with 10% FBS and 1% antibiotics) at 37°C for 30 minutes. Digestion was stopped
with FBS-supplemented DMEM, and single-cell suspensions were prepared by passing the tissue
through a 70 μm cell strainer. Red blood cells were lysed using RBC lysis buffer (4 min, room
temperature), and cells were washed and resuspended in FACS staining medium (FSM). Cells were
transferred to 96-well plates and stained with LIVE/DEAD™ Aqua (1:1000 in PBS, Invitrogen) for 15
minutes at 4°C in the dark. Surface marker staining was performed using CD45-BV786 (clone 30-F11),
CD41-PE (MWReg30), CD11b-AF700 (M1/70), and Ly6G-PE-Cy7 (1A8) for 30 minutes at 4°C. After
staining, cells were washed, fixed in 1% paraformaldehyde (10 min), and resuspended in FSM. Samples
were acquired on a Cytek Aurora and analyzed with FlowJo v10.

Neutrophil activation in vivo.

Briefly, 50 µL of BALF samples were incubated in a clear 96-well plate with the specific NE substrate N-
methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (15 mM, Sigma-Aldrich M4765) in the presence of Tris 1
M and NaCl 5 M for 24h at 37ºC. AbsorbLance was read at 410 nm at 0h and 24h after incubation. NE
activity was calculated by: (OD410 – OD410 ). Purified NE (Sigma-Aldrich M4765) was used as the
2I4h 0h
positive control. We also measured the myeloperoxidase (MPO, R&D DY3667), neutrophil elastase (NE,
R&D DY4517) and matrix metalloprotease (MMP)-9 (R&D DY6718) levels in the BALF by ELISA using
R&D DuoSet ELISA kits according to the manufacturer’s instructions. Surface expression of CD63 in
BALF-recruited neutrophils was measured by flow cytometry using the following antibodies: CD11b –
APC (clone M1/70, cat. 561690, BD Biosciences), Ly6G – FITC (clone A18, cat. 551460, BD
Biosciences), and CD63 – AF700 (clone NVG-2, cat. 143924, Biolegend)19,32.

A custom ELISA based on NE associated with DNA was used to quantify soluble NETs in mouse BALF
using a modified protocol for NET quantification in biological fluids50,51. For the capture antibody, 5 μg/ml
anti-NE mAb (M-18 (sc-9521) or G2 (sc-55549), Santa Cruz Biotechnology) was coated onto 96-well
plates (dilution 1:500 in 50 μl) overnight at 4°C. After washing 3 times (300 μl each), 20 μl of samples
was added to the wells with 80 μl incubation buffer containing a peroxidase-labeled anti-DNA mAb (Cell
Death ELISAPLUS, Roche; dilution 1:25). The plate was incubated for 2 hours, shaking at 300 rpm at room
temperature. After 3 washes (300 μl each), 100 μl peroxidase substrate (ABTS) was added. Absorbance
at 405-nm wavelength was measured after 20 minutes incubation at room temperature in the dark.
Values for soluble NET formation are expressed as OD405.

Protein and cytokine quantification.

The left lung was collected, homogenized, resuspended in cytokine buffer (0.5% Triton X-100, 150 mM
NaCl, 15 mM Tris, 1 mM CaCl , and 1 mM MgCl [pH 7.40]) anSd incubated for 30 min at 4°C. Then,
2 2
samples were centrifuged at 10,000 × g for 20 min at room temperature, and supernatants were stored
at −80°C until cytokines were measured. IL-17A (R&D DY421), CXCL-1 (R&D DY453), IL-6 (R&D
DY406), G-CSF (R&D DY414) and IL-1β (R&D DY401) (pg/ml) were measured by ELISA using R&D
DuoSet ELISA kits. Mouse IgM (Invitrogen 88-50470-22, Pro-COL1A1(Abcam ab210579), NE (R&D
DY4517), TSP1 (Abcam ab317551), COL4A1 (Novus Biologicals NBP2-75871), and COL6A1 (Novus
Biologicals NBP3-42425) levels weIre quantified in BALF of mice using ELISA kits according to the
manufacturer’s instructions.

Peripheral platelet counts.

Circulating platelets of mice were counted using the Hemavet 950 (Drew Scientific). 100 µL of blood from
the tail was collected in a 1 mL EDTA-coated vial tube. The blood sample was thoroughly mixed on a
vortex mixer, kept at room temperature and processed on the Hemavet 950 within 1 hour of collection.
Assessment of platelet counts was performed in accordance with the manufacturer’s operating
instructions.

Platelet aggregation assay.

Platelets from PF4CreThbs1flox mice and WT littermates were washed in HEPES-Tyrode buffer and
stimulated with thrombin (0.01-0.02 U/mL, ChronoLog, Cat# 386), collagen-related peptides (0.025-0.05
\mu g/mL, Cambcol, Cat#: CRP-XL), U46119 (0.1-1 \mu M, Sigma D8174), and ADP (5-20 \mu M, Sigma A2754).
Platelet aggregation was measured by a lumi-aggregometer (Chronolog Corp, Havertown, PA) at 37°C
with stirring (1,000 rpm).

Platelet identification by flow cytometry.

For assessment of platelet activation, mouse platelets were washed in HEPES-Tyrode buffer and treated
with or without thrombin (0.01-0.02 U/mL, ChronoLog, Cat# 386), collagen-related peptides (0.025-0.05
\mu g/mL, Cambcol, Cat#: CRP-XL), U46119 (0.1-1 \mu M, SigPma D8174), and ADP (5-20 \mu M, Sigma A2754)
followed by incubation for 20 minutes with fluorescNently labeled antibodies against P-selectin (Wug.E9,
FITC, Emfret analytics, Würzburg, Germany) o r activated \alphaIIb\beta3 integrin (JON/A, PE, Emfret analytics,
Würzburg, Germany). Platelets were anaLlyzed by flow cytometry (CytoFlex, Beckman Coulter). Platelets
in lung tissue were identified as FSClo SSClo CD45- CD41+ events.

Quantitative proteomics.

BALF from PA infected WT, Thbs1-/-, PF4CreThbs1flox and PF4WTThbs1flox mice were collected at day 1
post-infection and centrifuged at 300 x g for 15 min to separate the pellet from the supernatant. BALF
supernatants were processed by the mass spectrometry and proteomics facility at the Ohio State
University for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis.

Protein Digestion via Strap. S-trap was used for the digestion. Briefly, 20% SDS in 200 mM TEAB
(Triethylamonium bicarbonate) was spiked into the sample to make the final concentration of 5% SDS in
50mM TEAB. 5 µL of 50 mM ABC containing 5 µg/µL DTT was added, and the sample was incubated at
65 ºC for 15 min followed by addition of 5 µL of 50 mM ABC containing 15 µg/µL iodoacetamide
(incubated at RT for 15 min in the dark). Samples were then acidified by adding 12% phosphoric acid
(1:10 v/v acid to sample). For every 25 µL of samples, 165 µL of TEAB (Triethylammonium bicarbonate,
1M)/MeOH (10:90 v/v) was added and then loaded to S-trap for further washes. Samples was centrifuged
at 4000 x g for 3 min (4C) to remove supernatant. 150 µL of TEAB (Triethylamonium bicarbonate,
1M)/MeOH (10:90 v/v) was added to the trap as wash solution and the trap was washed 3 times. After
the final wash, sequencing grade trypsin dissolved in 50 mM TEAB was added and digested O/N at 37
ºC. The following day, peptides were eluted from the trap by adding 40 µL of 50mM TEAB, 0.1% FA and
0.1% FA in Acetonitrile (50:50), sequentially. The sample was pooled together and dried in a vacufuge
and resuspended in 20 µL of 50 mM acetic acid. Peptide concentrSation was determined by nanodrop
(A280 nm).

Orbitrap Fusion. Nano-liquid chromatography-nanospr ay tandem mass spectrometry (Nano-LC/MS/MS)
of protein identification was performed on a TIhermo Scientific orbitrap Fusion mass spectrometer
equipped with an nanospray FAIMS Pro™ Sources operated in positive ion mode. Samples (2 µg) were
separated on an easy spray nano column (EASY-Spray™ HPLC Columns, C18 3 µ 100A, 75 µm X 250
mm Thermo Scientific) using a 2D RSLC HPLC system from Thermo Scientific. Each sample was
injected into the trap coluAmn (Acclaim™ PepMap™ C18, 3µ 100A, 75 µm X 20 mm Thermo Scientific)
and desalted with 0.1% Formic Acid in water for 5 minutes. The injector port was then switched to “inject”
and the peptides were eluted off of the trap onto the column. Mobile phase A was 0.1% Formic Acid in
water and acetonitrile (with 0.1% formic acid) was used as mobile phase B. Flow rate was set at 300
nL/min. Mobile phase B was increased from 2% to 16% in 105 min and then increased from 16-25% in
10 min and again from 25-85% in 1 min and then kept at 95% for another 4 min before being brought
back quickly to 2% in 1 min. The column was equilibrated at 2% of mobile phase B (or 98% A) for 15 min
before the next sample injection.

MS/MS data was acquired with a spray voltage of 1.95 KV and a capillary temperature of 305 °C was
used. The scan sequence of the mass spectrometer was based on the preview mode data dependent
TopSpeed™ method: the analysis was programmed for a full scan recorded between m/z 375-1500 and
a MS/MS scan to generate product ion spectra to determine amino acid sequence in consecutive scans
starting from the most abundant peaks in the spectrum in the next 3 seconds. To achieve high mass
accuracy MS determination, the full scan was performed at FT mode and the resolution was set at
120,000 with internal mass calibration. Three compensation voltage (cv=-50, -65 and -80v) were used
for samples acquisition. The AGC Target ion number for FT full scan was set at 4 x 105 ions, maximum
ion injection time was set at 50 ms and micro scan number was set at 1. MSn was performed using HCD
in ion trap mode to ensure the highest signal intensity of MSn spectraS. The HCD collision energy was set
at 32%. The AGC Target ion number for ion trap MSn scan was set at 3.0E4 ions, maximum ion injection
time was set at 35 ms and micro scan number was set at 1. Dynamic exclusion was enabled with a
repeat count of 1 within 60s and a low mass width and high mass width of 10 ppm.

Database searching. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK;
version 2.7.0). Mascot was set up to search the Common_Contamination_Proteins-
cRAP_LZ_20190813.fasta; UnTiprot_Mouse_Reviewed_08272021_20210827 database (unknown
version, 17195 entries) assuming the digestion enzyme strict trypsin. Mascot was searched with a
fragment ion mass tolerance of 0.50 Da and a parent ion tolerance of 10.0 PPM. Carbamidomethyl of
cysteine was specified in Mascot as a fixed modification. Deamidated of asparagine and glutamine and
oxidation of methionine were specified in Mascot as variable modifications.

Criteria for protein identification. Scaffold (version Scaffold_5.0.1, Proteome Software Inc., Portland, OR)
was used to validate MS/MS based peptide and protein identifications. Peptide identifications were
accepted if they could be established at greater than 95.0% probability to achieve an FDR less than 1.0%
by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established
at greater than 98.0% probability to achieve an FDR less than 1.0% and contained at least 2 identified
peptides. Protein probabilities were assigned by the Protein Prophet algorithm. Proteins that contained
similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy
the principles of parsimony.

Relative quantitation using label free spectral counting. Protein abundance was quantified using protein
spectral counts in the BALF of C57Bl/6J WT (n=2), Thbs1-/- (n=2), PF4CreThbs1flox (n=4)
and PF4WTThbs1flox (n=4) mice one day 1 post-infection with Pseudomonas aeruginosa. Fold change
abundance and statistical testing were performed using a two sample Welch t-test after log2 plus one
transformation of the protein spectral counts to compare expressioSn between PF4CreThbs1flox and WT
mice. Protein accessions were mapped to gene identifiers using STRING52. Reactome pathway
enrichment was performed using over-representation analysis with hypergeometric testing of statistically
significant proteins (p < 0.05) between WT and PF4Cre Thbs1flox mice.

For alveolar tissue proteomics, experiments were conducted at the WashU Mass Spec Core, Mass
Spectrometry Technology Access Center at the McDonnell Genome Institute, Washington University in
St. Louis. Briefly, laser capture Tmicrodissection was performed on histopathological sections targeting
regions of alveolar injury within the lung day 1 post i.t. inoculation of PA in PF4creThbs1flox and WT mice.
After laser capture, regions of injury were digested and processed for LC-MS/MS. Mass spectrometry
samples were quantified using label free quantification (LFQ) via data-independent acquisition (DIA) and
mapped against the mouse database in Spectronaut for DIA-LFQ analysis. The precursor filter algorithm
in Spectronaut was utilized to retain proteins with valid label free quantification values and the t-test was
performed for comparisons between WT and PF4creThbs1flox mice. Multiple test correction was performed
using the Benjamini-Hochberg correction. Protein accessions were mapped to gene identifiers using
STRING. Reactome pathway enrichment was performed using over-representation analysis with
hypergeometric testing of statistically significant proteins between WT and PF4creThbs1flox mice.

Intravital lung imaging and quantitative image analysis.

ROSAmT/mG and ROSAmT/mG Thbs1-/- mice were intratracheally (i.t.) instilled with vehicle (PBS) or 30%
Pseudomonas aeruginosa supernatant (PA SN). At 24 h, mice were intraperitoneally (i.p.) anesthetized
using a cocktail of Ketamine (100 mg/kg body weight) and Xylazine (10 mg/kg body weight), and the left
lung was exposed and immobilized. In initial experiments utilizing RosamT/mG and RosamT/mGThbs1-/- mice
to confirm alveolar barrier permeability and quantify dextran extravasation from the circulation to the
airspaces, 70 kDa FITC-dextran (5 mg/ml, ThermoFisher, D1822) was administrated intravenously just
prior to visualization of the lung by multiphoton excitation (MPE) microscopy (Nikon A1R MP). FITC and
TdTomato were excited with the two-photon excitation wavelength 8S90 nm and emissions collected at
480 nm and 560 nm, respectively. Images were analyzed using NIS Elements (Nikon Inc.). The airspace
was identified by segmenting the cell membranes (TdTomato+) and inverting the binary mask to demark
the alveoli. FITC extravasation into the airspace was e stimated based on FITC+ area and the intensity of
the FITC emissions within the alveoli. Septal thicKness was estimated by overlaying a regular series of
horizontal and vertical test lines across each image and measuring the median distance between alveoli.

We determined the impact oTf platelet/megakaryocyte (Mgk)-specific TSP-1 on alveolar barrier
permeability as well as platelet events, Mgks, and collagen fibers surrounding areas of airspace leak by
multi-photon imaging. To evaluate the contribution of platelet/MgK TSP-1 on alveolar barrier integrity,
PF4CreROSAmT/mGThbs1flox and PF4CreROSAmT/mG mice were challenged with vehicle (PBS) or 30% PA
SN by i.t. administration. At 24 h post-injury, mice were anesthetized i.p. with a cocktail of Ketamine (100
mg/kg body weight) and Xylazine (10 mg/kg body weight), intubated, ventilated, and the left lung was
exposed and immobilized. To visualize the pulmonary vasculature and spatially localize airspace leak,
mice were intravenously injected with 70 kDa cyanine-based dye CF680-Dextran (5mg/mL; biotium) just
prior to imaging by Nikon A1R multi-photon microscope equipped with a 20X (1.0NA) water immersion
optic. GFP+ platelets and Mgks were visualized on the background of tdTomato+ alveolar membrane.
Fibrillar collagen was imaged using second harmonic generation (SHG) microscopy. All analysis was
performed using NIS Elements (Nikon Inc.). The airspace was identified by segmenting the cell
membranes based on emission intensity (TdTomato+) and inverting the binary mask to demark the
alveoli. Segmentation of platelet and MgK events was based on the intensity of the GFP emissions and
filtering of discrete objects based on size. Platelet events were defined as GFP+ structures < 4-6 microns
in size. MgKs were defined as GFP+ structures > 30 microns in size with visible nucleus. For each
parameter, 4-5 images from each mouse were analyzed and averaged to obtain a composite value for
each individual mice.

Lung Tissue processing and injury score

For left lung inflation, the right lung hilum was identified and secured with silk suture. Mouse trachea was
cannulated by gently passing a 20 G needle into the trachea Eand secured with 2-0 silk suture. The left
lung was inflated using 1% low melting agar at 20 cm H O pressure with a 10 mL syringe attached to a
2
digital manometer. The inflated lung was carefully Nremoved from the thorax and placed in ice cold PBS
to solidify the agar and then immersed in 4% paraformaldehyde overnight for fixation. Samples were
processed, embedded in paraffin, and secLtioned at 4 μm. Slides were stained by a standard H&E staining
protocol (Histowiz, Long Island City, NY). Whole-slide scanning (40×) was performed on an Aperio AT2
(Leica Biosystems). Lung section images were used to score the degree of lung injury using a
modification of a histological grading scheme53. Briefly, whole lung sections were scored randomly in 10
magnified fields (40 X) to count neutrophils in both the alveolar and interstitial compartments to yield the
alveolar neutrophil: total neutrophil ratio and the interstitial neutrophil: total neutrophil ratio. Alveolar
neutrophil and interstitial neutrophil score per field were graded as none = 0, 1-5 =1, >5 =2. Further,
alveolar protein debris and septal thickness were graded as component C (none = 0, 1 = 1, >1 = 2) and
component D (<2x = 0, 2-4x = 1, >4x = 2), respectively. The composite lung inury score was calculated
for each mouse lung section: [(20 x A) + (14 x B) + (7 x C) + (2 x D) /10], where A = airspace neutrophils,
B = interstitial neutrophils, C = proteinaceous debris filling airspaces, D = alveolar septal thickness.

Neutrophil depletion studies.

PF4CreThbs1flox and PF4WTThbs1flox mice were i.p. injected with 200 µg anti-Ly6G (clone 1A8 rat IgG2a,
k, BioLegend) or isotype control (RTK2758, Rat IgG2a, κ, BioLegend) at -24 h and at 0 h when mice
were i.t. inoculated with PA SN (30%). At 24 h post-instillation with PA SN, BALF was collected and
neutrophil depletion was assessed by cell differential counting and MPO production by ELISA (R&D
DY3667). Lung permeability was assessed by quantifying the content of total protein in the BALF
(Pierce™ BCA Protein Assay Kit, Thermofisher).

Respiratory mechanics studies.

Mice were anesthetized with a ketamine/xylazine/acepromazine cocktail (ketamine 90 mg/kg, xylazine
10 mg/kg, acepromazine 2 mg/kg, IP). Mice was trachePotomized with an 18-gauge blunt metal cannula
(typical resistance of 0.18 cmH2O s ml−1) which was secured with a nylon suture. The mouse was then
connected to the flexiVent computer-controlled piston ventilator (SCIREQ) via the cannula, which was
attached to the FX adaptor Y-tubing. Mechanical ventilation was initiated, and a dose of paralytic
vecuronium bromide (2 mg/kg) wIas administered to mice to prevent breathing efforts against the
ventilator and during measuRrements. Mice were ventilated using default settings, and respiratory
mechanics were assessed using the forced oscillation technique using the flexiVent operating software
(flexiWare version 8.2). In brief, the Static compliance (Cst), dynamic compliance (Crs) respiratory
system resistance (Rrs), Newtonian resistance (Rn), and tissue elastance (H) were measured using
forced oscillation maneuvers, at baseline and with increasing concentrations of methacholine (Mch; 0 =
PBS; 2, 4, 8, 16, 25, and 50 mg/ml) aerosol generated with an in-line nebulizer. Data was exported from
the flexiVent operating software (flexiWare version 8.2)54,55. The results were presented at baseline and
with increasing concentration of methacholine.

Megakaryocyte Thbs1 tissue specific expression analysis.

For Megakaryocyte single cell and tissue specific gene expression, tissue specific differentially
expressed gene lists in adult and fetal megakaryocytes were downloaded from the supplemental data of
Yeung, et. al. under the Gene Expression Omnibus accession number GSE15257438. Thbs1 expression
was assessed for differential expression between adult megakaryocyte populations residing in the lung
in comparison to those residing in the bone marrow as well as in fetal megakaryocyte populations
residing in the lung in comparison to those residing in the liver.

Statistics

Parametric t tests or one-way analysis of variance (ANOVA) was performed for data that were normally
distributed. For data that were not normally distributed, a non-parametric analysis using a two-tailed
Mann-Whitney U test for single comparisons and KruskaEll-Wallis for multiple comparisons was
performed. For cytokine production, the data was log transformed and a two tailed t test analysis was
used. A two-way ANOVA test with multiple compaNrison was performed to evaluate platelet aggregation
across time. Repeated measures (RM) one-way or two-way ANOVA was applied for paired samples
under different conditions. Statistical sLignificance was assigned for P <0.05. Comparisons were
performed using the GraphPad Prism software, version 10.6.1.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Summary\nThis is an exciting paper from an excellent group. The primary novel finding is that platelet and megakaryocyte TSP1 plays a\nmajor role in lung injury. They show data on barrier function, structural changes, and inflammation. The paper is clearly\nwritten and the figures are nice. The biggest issue with the manuscript is that the main message does not really follow the\ndata. What I mean is, the authors focus here on permeability and barrier dysfunction but the data supporting this are not very\nconvincing. They also have some very cool data on neutrophil function in cKO mice and these findings are underdeveloped.\nI have a high level of enthusiasm for the main message but the details of the story don’t entirely hold together. I have some\nspecific suggestions to improve the manuscript below.\nComments\n1. With the focus on lung barrier function, it would be helpful to include more traditional measures of lung edema such as\nBAL IgM and/or lung wet to dry weight ratio. In a prior JCI Insight publications, the authors do show BAL protein in these\ntransgenic mice which does help. This is important because there are dramatic structural abnormalities in the alveolar wall\nthat make assessment of edema formation challenging.\n2. Along these same lines, the intravital microscopy images are beautiful but it’s not clear that they provide additional\ninformation beyond more standard measures of barrier dysfunction and edema formation. In other words, the authors don’t\nreally explore the spatial aspect of edema formation that is provided by intravital microscopy.\n3. The term “conditional knockout” is usually used for constructs that can be turned on with exposure to doxycycline,\ntamoxifen, etc. The transgenic mouse is better described as a constitutive cell specific knockout.\n4. In figure 5, the cKO vehicle treated lungs look quite different from the WT lungs. This raises concern for potential\ndevelopmental or structural effects of platelet TSP1. Could the authors show flexivent data at baseline from the cKO mice?\nThe flexivent data shown is fold change and does not allow for assessment of baseline structural differences between WT\nand cKO. The data in figure 6 showing differences in baseline collagen heighten this concern.\n5. In figure 7, the only data supporting a role for plt TSP1 in lung structure/barrier function is panel D. All other panels simply\nshow the effects of knocking out neutrophils on the function of neutrophils. G and H actually suggest that there is no\nrelationship between neutrophils and structural changes in TSP1 cKO.", "reviewer_2": "Overall this is an interesting study that adds knowledge to our understanding of lung injury and the role that\nMks and platelets have in the injury pathogenesis. There is interesting and meaningful data. Most of my critique revolves around\ngaining a better understanding of the Mk/platelet and PMN interface.\n1. Fig 1 and 2 are basically establishing the model for the reader. This is really important to show, but could be reduced to a\nsingle figure with the rest of the data in supplemental files.\n2. The platelet activation data is not sufficient to conclude the cKO mice have no defect in response. Higher and lower doses\nof the agonists chosen as well as adding thromboxane and ADP as stimuli should be included for completeness.\n3. The BALF data in Figure 3 is intriguing and leads to more questions about direct effects of platelets. WT and cKO mice\nhave similar PMN numbers in the BALF, yet the immune/injury markers are different. Do the authors have data regarding\nPMN functions? Can they rule out that developmentally or in the circulation that PMNs in the cKO mice are not altered?\nThere is abundant literature that platelets affect immune cell functions and differentiation so this should be ruled in or out as\na mechanism.\n4. Without validation not sure what Fig 4 is adding to the manuscript. Is just a list at this point.\n5. The authors in Fig 7G indicate that the lung Mks are CD45/CD41+. These could be immune cells with adherent platelets.\nThis should be clarified experimentally.\n6. The increase in lung Mks following injury is intriguing - are these intra or extra vascular? Is there any change in post-injury\nplatelet counts?\n7. Do lung Mks express more thrombospondin compared to bone marrow Mks?", "reviewer_3": "The authors present a very nice story describing acute alveolar injury model where by megakaryocyte and platelet\nproduction of thrombospondin-1 appears to play a key role in limiting neutrophil activation, megakaryocyte accumulation and\nlung injury. Interestingly, animals that have Tsp-1 deficient megakaryocyte/platelets have an altered ECM in the lungs. I think\nthis represents an exciting area of research that capitalises on a growing interest in immune-ECM cross-regulation in health\nand disease and hence will be of interest to many fields. Data analysis is well described and presented in detail and I think\nthe authors have done a good job here.\nThere are a few points that I think need better clarifying or describing, in particular centred around the term provisional\nmatrix. Also I think it would be important to show or least mention whether the immune phenotype of these cKO mice are\nnormal in the lung injury model – particularly focusing on cells like macrophages which might produce Tsp1 and therefore\ninfluence the findings especially if changed in cell number in the lungs.\nComments\n• Typically, the provisional matrix is described as a matrix largely devoid of collagens, yet in this study you are using\ncollagen as main indicator of a provisional matrix. I just wonder whether you need to provide further background to the\nprovisional matrix and perhaps also talk about how other molecules (e.g. Fn1, Vtn etc) are upregulated in cKO mice versus\nWT. Why was the focus on collagen and collagen-1? In line with this, whilst the data is compelling, I’m not convinced it\npoints specifically to Tsp-1 “stablising” the provisional matrix – I feel this needs a bit more explanation or perhaps the\nauthors should pair back the wording a little?\n• In relation to point above, for proteomic analysis looking at ECM molecules in BAL, it was interesting to see less Col4, Col6\netc, things that are expressed in the alveoli basement membrane in cKO v WT; also correlates with higher neutrophil\nelastase levels. This isn’t specifically discussed, but is Tsp1 from platelets/megakaryocytes preventing degradation of the\nbasement membrane and hence preventing lung injury which may explain the change in collagen 1? Maybe a discussion\nand/or schematic model (graphical abstract) of mechanism would be useful?\n• For the cKO mice, the authors show levels of Tsp-1 from platelets. What are total TSP1 levels in the lungs/BAL of these\nmice, ie. how much of the Tsp1 is from platelets/megakaryocytes. Are other cells compensating for a loss of Tsp1, are there\ndefects in other immune cells (e.g macrophages/monocytes) in cKO v WT that might influence the findings?\n• Just wondering whether the authors examined neutrophil localisation? Neutrophil numbers in the BAL didn’t change but\nmarkers of activation increased in cKO vs WT mice. Are these neutrophils more concentrated around the damaged alveoli?\n• Size of the alveoli in vehicle WT v cKO mice look very different in Fig 5. During the steady state, are the function/structure\nof the alveoli compromised in these mice even before they are challenged? Also looks to be more collagen in the steady\nstate in cKO mice – which may fit with these observations?\nMinor considerations\n• Approach to data analysis in Figure 1 is great. It might also be worth showing a representative overlay of segmented areas\nin WT v cKO mice to really highlight the changes visually\n• I would consider putting colour labels on the images in the figures so people don’t have to read the figure legends\neverytime to see what colour represents what cell or protein", "reviewer_4": "General Comments\nIn the manuscript NCOMMS-25-26357 “Megakaryocyte/platelet-derived thrombospondin-1 stabilizes the early provisional\nmatrix in lung injury by exercising spatioltemporal control of neutrophil function and matrix remodeling”, the authors\ndemonstrate protein thrombospondin-1 (TSP1) protects the lung from acute alveolar injury using MgK/plt Thbs1 conditional\nknockout mice.\nMy comments are specific to the proteomic portion of the manuscript.\nSpecific Comments\n1. Overall, I thought the method section was lacking. Speaking to the proteomic section only (including the supplemental\nmethods), there is inadequate information on how the proteomic samples were prepared, analyzed by mass spec, which\nmass spectrometer was used, and how the data was analyzed and by what program.\n2. Shotgun proteomics terminology has been updated to label-free quantitation proteomics to reflect more accurately the\nmethod. It would be advised to update the terminology throughout the manuscript.\n3. The biological replicates for BALF of WT, and Thbs1+/- was n=2. That a significant deficiency of the proteomics data as\ntrue statistical power requires a minimum of n=3. This diminishes the strength of the results and conclusions as a n=2 is\ngenerally not considered statistically significant for a robust conclusion for this section of the manuscript (Fig S4 and\ncorresponding results)."}, "author_response": "Response to reviewers.\nWe thank the reviewers for their time in evaluating our manuscript and their constructive comments. We have\ncarefully reviewed each comment and performed additional experiments to comprehensively address the\nconcerns raised. To provide a concise and informative response, we did not embed the experimental figures\nbelow the text of each reply given the substantial number of additional experiments and revisions undertaken.\nRather, we provide the location of the 16 additional experiments and 1 graphical figure in the Figures section.\n1. Platelet agonist stimuli experiments with U46619 and ADP to evaluate aIIbb3, P-selectin, % platelet\naggregation (Fig. 2E-H).\n2. Laser capture microdissection of alveolar parenchymal tissue after lung infection, label-free quantitation\nproteomics to assess the provisional matrix (Fig. 3J, 3L, S3B).\n3. Flow cytometry analysis of immune cells in BAL and lung tissue in Mgk/plt Thbs1 KO vs WT after live\ninfection (Fig. S2C-D).\n4. Neutrophil agonist stimuli experiments with TNFa, fMLP, A23187, and platelet-neutrophil aggregate\nformation ex vivo (Fig. S2L-N).\n5. Phenotyping of LysMcre Thbs1 KO vs WT mice after live infection: Flow cytometry analysis of immune\ncells in BAL and lung tissue, lung CFU, total BALF protein, BALF NE activity after live infection (Fig.\nS2O-T).\n6. Lung histology scoring of Mgk/plt Thbs1 KO vs WT mice at baseline and after PA SN injury (Fig. 4F-L).\n7. Lung function studies at baseline and after PA SN injury in the presence or absence of airway\nprovocation to assess static compliance, dynamic compliance, resistance, Newtonian resistance,\nTissue elastance (Fig. 4N-R).\n8. Localization of Mgks in relation to the underlying lung vasculature (Fig. 5A, suppl video 1).\n9. New segmentation analysis figure comparing Mgk/plt Thbs1 KO and WT after PA SN injury (Fig. S4A-\nF).\n10. Comparison of Mgk Thbs1 gene expression in adult lung versus adult bone marrow, fetal lung versus\nfetal liver using publicly available gene expression data (Fig. S6A).\n11. Lung platelets counts after PA SN injury (platelet gating strategy, lung platelet count) (Fig. S6H-I).\n12. Peripheral platelet counts before and after PA SN injury (Fig. S6J).\n13. Measurement of BALF IgM (Fig. 6H).\n14. Measurement of BALF TSP1 levels (Fig. 6K).\n15. Measurement of BALF COL4A1 and COL6A1 levels (Fig. 6M and 6N).\n16. Mgk gating strategy comparison with and without Lin- markers in the dump channel and enumeration\n(Fig. S8).\n17. Graphical figure to highlight mechanism (Fig. 6O).\nIn addition, the mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium\nvia the PRIDE [1] partner repository with the dataset identifier PXD071231 with the following submission\ndetails:\nReviewer access details\nLog in to the PRIDE website using the following details:\nProject accession: PXD071231\nToken: SNJkrjGWL18y\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nSummary\nThis is an exciting paper from an excellent group. The primary novel finding is that platelet and megakaryocyte\nTSP1 plays a major role in lung injury. They show data on barrier function, structural changes, and\ninflammation. The paper is clearly written and the figures are nice. The biggest issue with the manuscript is\nthat the main message does not really follow the data. What I mean is, the authors focus here on permeability\nand barrier dysfunction but the data supporting this are not very convincing. They also have some very cool\ndata on neutrophil function in cKO mice and these findings are underdeveloped. I have a high level of\nenthusiasm for the main message but the details of the story don’t entirely hold together. I have some specific\nsuggestions to improve the manuscript below.\nResponse: We appreciate the positive comment of reviewer 1. We performed additional experiments and\nhave clarified the details of our work to ensure that the main message is supported by the presented data.\nPlease find a response for each of the comments below.\nComments\n1. With the focus on lung barrier function, it would be helpful to include more traditional measures of lung\nedema such as BAL IgM and/or lung wet to dry weight ratio. In a prior JCI Insight publications, the authors do\nshow BAL protein in these transgenic mice which does help. This is important because there are dramatic\nstructural abnormalities in the alveolar wall that make assessment of edema formation challenging.\nResponse: Thank you for the comment. In addition to our novel method of visualizing and quantifying\nairspace barrier leak with lung intravital microscopy, we would like to point out that we measured BALF protein\nin the cKO mice in the live PA infection model (Figure 3C), and Evan’s blue dye lung permeability study in the\nPA SN sterile injury model (old Figure 5F, new Figure 4M). Both the BALF total protein and Evan’s blue dye\nlung permeability assay are traditional measures of lung edema as described in a recent Update on Features\nand Measurements of Experimental Acute Lung injury: an Official American Thoracic Society Workshop\nReport1.\nWe provide new lung histology data from WT and cKO mice and show increase in lung injury score in cKO\nmice after PA SN when compared to WT littermates (new Figure 4F-L). We provide lung histology data at\nbaseline (absence of injury stimuli, without vehicle PBS instillation) and following injury in WT and cKO lung\ntissue sections (new Figure 4F, G). The increase in lung injury score observed in the cKO after sterile injury is\ndriven by the alveolar edema tissue score and the alveolar septal thickness score rather than the ratio of\nalveolar or interstitial PMN: total PMN scores. This is consistent with the airspace barrier leak observed by lung\nintravital microscopy, BAL total protein, and Evan’s blue dye lung permeability assay. Moreover, we measured\nBALF total protein in the PA SN sterile injury model (old Figure 7D, new Figure 6D), where cKO shows higher\nBALF total protein when compared with WT littermates, and neutrophil depletion restores barrier integrity. In\nresponse to reviewer’s request, we also measured BALF IgM and show increase in cKO after injury. There was\na trend towards decrease in IgM with neutrophil depletion (p=0.05) (new Figure 6H).\nResults section: \"Intravital microscopy details a limited window to the lung, therefore we confirmed these\nfindings by quantifying the lung injury score in H&E sections (Fig. 4E-L), Evans blue (EB) dye assay to assess\nlung permeability (Fig. 4M), and lung mechanics (Fig. 4N-R). PF4CreThbs1flox KO mice showed increase in lung\ninjury score after PA SN sterile injury when compared to WT littermates (Fig. 4H). We did not observe\ndifferences between WT and PF4CreThbs1flox KO mice in the ratio of neutrophils localized to either the alveolar\nspaces or interstitium (Fig. 4I-J). This finding is consistent with similar neutrophil numbers found in WT and\nPF4CreThbs1flox KO mice after live PA infection (Fig. 3D). The increase in lung injury score observed in the\nPF4CreThbs1flox KO mice after sterile injury is driven by the alveolar edema tissue score and the alveolar septal\nthickness score rather than the ratio of alveolar or interstitial PMN scores (Fig. 4K-L). This finding is consistent\nwith the airspace barrier leak observed by lung intravital microscopy (Fig. 4B-D).\n“BALF IgM levels, a surrogate marker of lung permeability, were increased in PF4CreThbs1flox KO compared to\nWT mice after injury (Fig. 6H).”\n2. Along these same lines, the intravital microscopy images are beautiful but it’s not clear that they provide\nadditional information beyond more standard measures of barrier dysfunction and edema formation. In other\nwords, the authors don’t really explore the spatial aspect of edema formation that is provided by intravital\nmicroscopy.\nResponse: Thank you for the comment and opportunity to clarify the additional information and novelty the\nlung intravital microscopy and analysis provide. It may have not been clear from Figure 1 that we are doing\nmore than visualizing and quantifying airspace leak and edema. Not only does this method identify where the\nleak is, this approach allowed us to identify Mgks in the lung, spatially co-localize leak with Mgks, quantify\ncollagen by SHG, overlaying the SHG and lung structures and cells of interest to discover that Mgks localize to\nregions of collagen deposition at sites of airspace leak (old Figure 6A-J, new Figure 5A-B, 5D-J). In response\nto Reviewer 3’s request, we created overlay images comparing WT and cKO to highlight the innovative\nimaging analysis performed (Revised Fig. S4). In response to Reviewer 1 and 2’s queries, we now include a\nshort video to show the dynamic process in real time that identifies the Mgks and their localization in relation to\nthe vasculature (new Figure 5A, and accompanying Suppl video 1).\nResults section:\n“We utilized segmentation and overlay analysis of WT and TSP1PF4 KO reporter mice lungs to quantify alveolar\nleak (Fig. S4A-F).”\n“Interestingly, Mgks were identified in both the extravascular (Fig. 5A, and Suppl Video 1) and intravascular\nspaces (Fig. 5D), and were prominently located at the junctions of alveolar structures marked by Td-Tomato\nexpression (Fig. 5D).”\n3. The term “conditional knockout” is usually used for constructs that can be turned on with exposure to\ndoxycycline, tamoxifen, etc. The transgenic mouse is better described as a constitutive cell specific knockout.\nResponse: Thank you for the comment. We have clarified the reference of cKO from “conditional KO” to “cell-\nspecific KO”. We now mention Mgk/plt Thbs1 KO mice (PF4cre Thbs1flox) by name throughout the manuscript to\navoid confusion. When referring to the reporter mice (PF4CreROSAmT/mGThbs1flox), we use the nomenclature\nTSP1PF4 KO reporter mice. We have also made use of experimental schematic figures to orient the reader to\nwhich mice are being used.\n4. In figure 5, the cKO vehicle treated lungs look quite different from the WT lungs. This raises concern for\npotential developmental or structural effects of platelet TSP1. Could the authors show flexivent data at baseline\nfrom the cKO mice? The flexivent data shown is fold change and does not allow for assessment of baseline\nstructural differences between WT and cKO. The data in figure 6 showing differences in baseline collagen\nheighten this concern.\nResponse: Thank you for raising these concerns. In response to Reviewer 1’s query, we performed new\nFlexivent experiments at baseline (without vehicle instillation) as well as with forced oscillation technique using\ncKO and WT littermates (Fig. 4N-R). There are no significant differences in baseline lung mechanics between\ncKO and WT mice. Given the complexity of the graphics now shown, we split out static and dynamic lung\ncompliance in order to address the reviewer’s concern. As expected, injury reduced static compliance in both\ngroups of mice (new Fig. 4N). Methacholine challenge further reduced dynamic compliance in cKO mice but\nnot in WT mice group, underscoring the altered lung function in cKO mice after injury (new Fig. 4O). As\nrequested, the Flexivent data is now shown in actual unit measurements. Old Figure 5B showed differences in\nalveolar size of WT reporter versus cKO reporter mouse lungs with vehicle instillation. We now confirm that the\nscalebars for all images depicted in new Figure 4B as 100 microns. For enhanced clarity, we have replaced the\nfigure of the WT vehicle-treated control image (left upper panel of new Figure 4B) using a different field from\nthe same mouse within the same experiment.\nResults section: “We next tested lung function at baseline (without vehicle) in WT and PF4CreThbs1flox KO mice.\nAs expected, injury reduced static compliance in both groups of mice (Fig. 4N). Forced oscillation tests further\nreduced the dynamic compliance observed in PF4CreThbs1flox KO mice but not in WT mice group, underscoring\nthe altered lung function in the PF4CreThbs1flox KO mice after injury (Fig. 4O).”\n5. In figure 7, the only data supporting a role for plt TSP1 in lung structure/barrier function is panel D. All other\npanels simply show the effects of knocking out neutrophils on the function of neutrophils. G and H actually\nsuggest that there is no relationship between neutrophils and structural changes in TSP1 cKO.\nResponse: We thank the reviewer for the comment. To address Reviewer 1 and 3’s comments regarding lung\nstructure/barrier function, we provide new data showing the levels of alveolar basement membrane proteins\nCOL4 and COL6 in cKO mice following neutrophil depletion or with isotype control Ab (new Fig. 6M-N). COL4\nand COL6 are both structural matrix proteins that comprise the lung basement membrane that were identified\nin the BALF proteomics of cKO mice compared to WT littermates after injury (Figure 5C and S3A, ECM\norganization). Notably, COL6 levels are reduced in cKO mice, and neutrophil depletion restores COL6 to levels\nobserved in WT littermates. As COL6 has been previously reported as a substrate of NE, the findings support\nthe concept that Mgk/plt TSP1 shields the basement membrane matrix protein COL6A1 from neutrophil-\nmediated proteolytic damage and thereby protects against barrier disruption. In the response to query 1 by\nreviewer 1, BALF IgM levels were measured, and the data are now provided (new Fig. 6H).\nReviewer #2 (Remarks to the Author):\nOverall this is an interesting study that adds knowledge to our understanding of lung injury and the role that\nMks and platelets have in the injury pathogenesis. There is interesting and meaningful data. Most of my\ncritique revolves around gaining a better understanding of the Mk/platelet and PMN interface.\n1. Fig 1 and 2 are basically establishing the model for the reader. This is really important to show, but could be\nreduced to a single figure with the rest of the data in supplemental files.\nResponse: We thank the reviewer for the positive comments. Based on Reviewer 2’s suggestion, the CRISPR\nstrategy used to generate the floxed mice (old Figure 2A-C) are now shown as Suppl Figure 1A. Based on\nReviewer 3’s positive comments, we have retained Figure 1 to illustrate the segmentation analysis that is\ncritical for understanding the novelty of our methods.\n2. The platelet activation data is not sufficient to conclude the cKO mice have no defect in response. Higher\nand lower doses of the agonists chosen as well as adding thromboxane and ADP as stimuli should be included\nfor completeness.\nResponse: Thank you for the comment. Please note that higher and lower doses of agonists thrombin and\nCRP were previously included in prior Fig. S1A-B, now presented in new Fig. S1B-C. To address Reviewer 2’s\nconcerns, we performed additional experiments that include cKO and WT mouse platelet responses at\nbaseline and with increasing concentrations of thromboxane (U46619, a TXA2 analog) or ADP. We examined\nplatelet aIIbb3 activation, P-selectin surface expression, and platelet aggregation as readouts (new Figure 2E-\nH). Given the importance of this negative data and for completeness, we provide the data in revised Figure 2.\n3. The BALF data in Figure 3 is intriguing and leads to more questions about direct effects of platelets. WT and\ncKO mice have similar PMN numbers in the BALF, yet the immune/injury markers are different. Do the authors\nhave data regarding PMN functions? Can they rule out that developmentally or in the circulation that PMNs in\nthe cKO mice are not altered? There is abundant literature that platelets affect immune cell functions and\ndifferentiation so this should be ruled in or out as a mechanism.\nResponse: To address Reviewer 2’s comments, we performed new experiments to show cKO and WT\nneutrophil responses at baseline and with either TNFa, fMLP, or A23187 stimulation are similar, as is the ability\nto form platelet-neutrophil aggregates (New Fig. S2L-M). We used CD62L shedding, aMb2 surface expression,\nand platelet-neutrophil aggregate formation as readouts. We found no differences in cKO and WT neutrophil\nfunction ex vivo, further supporting the idea the lung tissue microenvironment having a role in regulating\nneutrophil function.\n4. Without validation not sure what Fig 4 is adding to the manuscript. Is just a list at this point.\nResponse: We thank the reviewer for this comment and have removed old Fig. 4. In the revised manuscript,\nwe combined the relevant portions of the BALF proteomics data from old Fig. 4 into new Fig. 3I, 3K and Fig.\nS3. In response to Reviewer 3 comments, we provide data from additional experiments involving label-free\nquantitation proteomics of alveolar parenchymal tissue obtained by laser capture microdissection. The alveolar\ntissue was obtained after live infection. Notably, there is striking similarity in the enrichment pathway analysis\nof the BALF and alveolar tissue proteomics (new Fig. 3I and 3J). We also validated the increase in neutrophil\ndegranulation and BALF MMP-9 shown in the BALF proteomics (new Fig. 3K) by measuring BALF MMP-9\nlevels by ELISA (new Fig.3F). Moreover, the proteomics data showing increased BALF MMP-9 and alveolar\ntissue NE levels corroborate the exaggerated neutrophil activation markers observed in cKO mice (new Fig.\n3E-H).\nResults section: “Moreover, neutrophils obtained from PF4CreThbs1flox KO mice were not developmentally\naltered and showed no differences in CD62L and, aMb2 surface levels ex vivo either at baseline or with TNFa,\nfMLP, or A23187 stimulation (Fig. S2L-M). The ability to form platelet-neutrophil aggregates was similar\nbetween the two groups (Fig. S2N).”\n5. The authors in Fig 7G indicate that the lung Mks are CD45/CD41+. These could be immune cells with\nadherent platelets. This should be clarified experimentally.\nResponse: We thank the reviewer for the comment. Please note that old Figure 7G (now new Fig. 6I-J)\nidentifying lung MgKs as CD45/CD41+ utilized a gating strategy where we gated on large cells and excluded\nsmaller cells such as neutrophils (new Fig. S7). To clarify this approach experimentally, we performed new\nexperiments showing the lung MgKs identified by FSC and CD45+/CD41+ surface markers are separate from\nCD45+CD11c-CD11b+Ly6G+ neutrophils (new Fig. S8(3)). In these new experiments, we used the prior gating\nstrategy where we gate on large cells and identify MgKs as CD45/CD41+ (new Fig. S8(1)) and compare the\nsame findings using a “dump channel” to exclude cells from the CD3, B220, Gr1, F4/80, or CD11b lineage from\nlarge cells that are Lineage negative (Lin-) CD45/CD41+ identified as MgKs (new Fig. S8(2)). Regardless of\nthe approach taken (i.e., whether or not Lin- markers in the dump channel used), the CD45/CD41+ Mgk\nnumbers increase in cKO lungs when compared to WT after injury (new Fig. S8B-C).\nResults section: “Mgks were identified as large cells (FSChigh) that were CD45+CD41+ and distinct from\nneutrophils (Fig. S7A1 and S7A2, respectively). Utilizing the same gating strategy (Fig. S8A1) or adding\nlineage markers B220, GR1, and F4/80 in the “dump channel” to separate lineage committed cells from Mgks\n(Fig. S8A2), both approaches revealed CD45+CD41+ Mgk numbers increase in cKO lungs and distinct from\nneutrophils (Fig. S8A3) when compared to WT after injury (Fig. S8B, S8C).”\n6. The increase in lung Mks following injury is intriguing - are these intra or extra vascular? Is there any change\nin post-injury platelet counts?\nResponse: The Mgks localize to both intravascular and extravascular compartments. We now provide a new\nimage and corresponding video clip to show that MgKs are extravascular (new Fig. 5A, and Suppl video 1).\nThe lung intravital static images (old Figure 6C-F) identify intravascular MgKs in regions of collagen deposition\nand barrier leak (new Fig. 5D-F). The large tissue scanning by confocal clearly identify Mgks within the\ninterstitium (old Fig. 6K-N, now new Fig. 5L indicated by blue arrows). The post-injury platelet counts in the\nperipheral blood are reduced after injury in both WT and cKO mice (new Fig. S6J).\nResults section: “Interestingly, Mgks were identified in both the extravascular (Fig. 5A, and Suppl Video 1)\nand intravascular spaces (Fig. 5D), and were prominently located at the junctions of alveolar structures\nmarked by Td-Tomato expression (Fig. 5D).”\n“The dynamic MgK accumulation in TSP1PF4 KO mice suggests dysregulated mobilization or retention in the\nlung microenvironment following injury. Lung platelet events were not different between the groups by imaging\nanalysis or flow cytometric analysis (Fig. S6D-G), while peripheral platelet counts (defined as Lin-CD45-CD41+\nsmall cells) dropped with injury (Fig. S6H-J).”\n7. Do lung Mks express more thrombospondin compared to bone marrow Mks?\nResponse: This is an interesting question. We attempted to quantify surface as well as intracellular TSP1 in\nlung MgKs and compare to bone marrow MgKs by flow cytometry. We were unable to address the question on\nthe protein level, as we encountered technical limitations due to non-specific labeling using three different\nantibody clones. However, we were able to download publicly available data from the article by Yeung, et al\ncomparing lung Mgks to bone marrow Mgks, as well as fetal lung Mgks to fetal liver Mgks in mice2. We show\nthat the relative Thbs1 gene expression in the lung is higher in both adult and at the fetal stage (new Fig. S6A).\nResults section: “Utilizing publicly available gene expression data, we show that the relative Thbs1 gene\nexpression in adult lung MgK is higher than in adult bone marrow MgK (Fig. S6A). In addition, fetal lung MgKs\ndemonstrate higher Thbs1 gene expression than fetal liver MgKs (Fig. S6A).”\nReviewer #3 (Remarks to the Author):\nThe authors present a very nice story describing acute alveolar injury model where by megakaryocyte and\nplatelet production of thrombospondin-1 appears to play a key role in limiting neutrophil activation,\nmegakaryocyte accumulation and lung injury. Interestingly, animals that have Tsp-1 deficient\nmegakaryocyte/platelets have an altered ECM in the lungs. I think this represents an exciting area of research\nthat capitalises on a growing interest in immune-ECM cross-regulation in health and disease and hence will be\nof interest to many fields. Data analysis is well described and presented in detail and I think the authors have\ndone a good job here.\nThere are a few points that I think need better clarifying or describing, in particular centred around the term\nprovisional matrix. Also I think it would be important to show or least mention whether the immune phenotype\nof these cKO mice are normal in the lung injury model – particularly focusing on cells like macrophages which\nmight produce Tsp1 and therefore influence the findings especially if changed in cell number in the lungs.\nResponse: We appreciate the reviewer comments. The term provisional matrix was originally described from\nthe wound healing literature. However, the provisional matrix in the lung after injury is poorly understood.\nBased on the reviewer’s comments, we present new data from laser capture microdissection of alveolar\nparenchymal tissue and label-free quantitation proteomics from these regions of interest (new Fig. 3J, 3L and\nS3B). We also note the striking similarity in the enrichment pathway analysis between the BALF and alveolar\ntissue proteomics in the live infection model (new Fig. 3I-J). Our response is further detailed under\n“Comments” section below. Regarding the reviewer’s comment about the immune phenotype of the Mgk/plt\nThbs1 cKO mice, the immune profile is not significantly different from WT littermates in the live infection model.\nWe now provide the immune cell subsets in the Mgk/plt Thbs1 cKO versus wildtype mice in the BALF and lung\nafter live PA infection (new Fig. S2C-D). To further address the reviewer’s concern, we also provide new data in\nmyeloid-specific Thbs1 cKO mice (LysMcreThbs1flox) (new Fig. S2O-T). These mice do not show any\ndifferences in CFU following live PA infection, BALF total protein, BALF NE activiy or immune cell subsets in\nBALF and lung tissue homogenates.\nComments\n1. Typically, the provisional matrix is described as a matrix largely devoid of collagens, yet in this study you are\nusing collagen as main indicator of a provisional matrix. I just wonder whether you need to provide further\nbackground to the provisional matrix and perhaps also talk about how other molecules (e.g. Fn1, Vtn etc) are\nupregulated in cKO mice versus WT. Why was the focus on collagen and collagen-1? In line with this, whilst the\ndata is compelling, I’m not convinced it points specifically to Tsp-1 “stablising” the provisional matrix – I feel\nthis needs a bit more explanation or perhaps the authors should pair back the wording a little?\nResponse: We thank the reviewer for these insightful comments. As the reviewer points out, the provisional\nmatrix is largely devoid of collagens and is comprised of fibrin, fibronectin, platelets, integrins, and\ninflammatory cells. To directly assess the lung matrix niche, we performed laser-capture microdissection (LCM)\nof injured alveolar tissue from cKO and WT mice after live PA infection. We now provide a description of the\nprovisional matrix in the Results section.\nResults section: “To assess the alveolar matrix niche, we performed laser-capture microdissection (LCM) of\ninjured alveolar tissue from WT and PF4CreThbs1flox KO mice after live PA infection and coupled with label-free\nquantitative mass spectrometry. Strikingly, differential analysis of the alveolar tissue proteomics between WT\nand PF4CreThbs1flox KO mice revealed shared pathway enrichment signatures including neutrophil\ndegranulation, platelet activation, and ECM organization also identified in BALF (Fig. 3J). Differentially up and\ndown regulated proteins comparing WT and PF4CreThbs1flox KO mice in BALF and alveolar tissue within these\nshared pathways feature key proteins involved in early lung injury (Fig. S3A and S3B). Within alveolar tissue,\nwe identify increases in neutrophil proteases ELANE (or, NE), MMP9, CTSG (or, Cathepsin G) and markers of\nthe provisional matrix29,33,34 fibronectin (FN1), fibrin or fibrinogen proteins (FGA, FGB, FGG), F13A1 (which\ncatalyze the conversion of fibrinogen to fibrin), and integrins in PF4CreThbs1flox KO mice when compared to WT\nafter injury (Fig. S3B). The increase in provisional matrix proteins observed in PF4CreThbs1flox KO alveolar\ntissue is likely a consequence of the increased injury and leak observed in these mice.”\nTo the reviewer’s point regarding collagen, collagen has been shown to be an early marker of matrix\nremodeling in human ARDS. Increased collagen synthesis (in particular, fibrillar collagen such as COL1) in the\nBALF of ARDS patients is associated with increased mortality. Collagen’s prominence in human ARDS and our\ndata identifying increased fibrillar collagen deposition in regions of airspace leak by lung intravital imaging (old\nFig. 6C-F, new Figure 5D-J) and by large area scanning of lung tissue using MPE/confocal hybrid imaging\nprompted us to focus on collagen and COL1. Based on our new LCM proteomics data and the Reviewers’\ncomments, we have revised the manuscript to focus on other collagens in addition to pro-COL1A1. We now\nclarify the role of TSP1 in protecting structural basement membrane protein COL6 from neutrophil-mediated\nproteolytic damage (new Fig. 6N). Please see below response to query\n2. In relation to point above, for proteomic analysis looking at ECM molecules in BAL, it was interesting to see\nless Col4, Col6 etc, things that are expressed in the alveoli basement membrane in cKO v WT; also correlates\nwith higher neutrophil elastase levels. This isn’t specifically discussed, but is Tsp1 from\nplatelets/megakaryocytes preventing degradation of the basement membrane and hence preventing lung injury\nwhich may explain the change in collagen 1? Maybe a discussion and/or schematic model (graphical abstract)\nof mechanism would be useful?\nResponse: To address the reviewer’s comment, we measured BAL COL6 levels during injury as this matrix\nprotein is significantly reduced in cKO by BALF proteomics (new Fig. S3B, ECM organization heatmap). We\nnow validate that BALF COL6A1 levels are indeed reduced in the cKO mice compared to WT mice by ELISA,\nand that neutrophil depletion restores COL6A1 levels in lung injury (new Fig. 6N). These findings strongly\nsupport the concept that Mgk/plt TSP1 prevents degradation of the matrix protein COL6A1, a key constituent of\nthe alveolar basement membrane, from neutrophil-mediated lung injury. We pared back the wording “stabilizes\nthe provisional matrix” to suggest TSP1 ‘shields’ the matrix by preventing proteolytic degradation of the\nbasement membrane matrix protein COL6A1. As a result of this exciting finding, we have revised the title of the\nmanuscript as well as sections of the abstract, results, and discussion to highlight the novel finding. In addition,\nwe now provide a graphical abstract of the mechanism in new Figure 6O.\nResults section: “We also measured components of BALF COL4 and COL6 as these matrix proteins comprise\nthe alveolar basement membrane41,42 and were significantly reduced in PF4CreThbs1flox KO by BALF\nproteomics (Figure S3A, ECM organization heatmap). While PF4CreThbs1flox KO and WT mice showed\nsimilar levels of BALF COL4A1 with injury, COL6A1 levels were significantly reduced in the PF4CreThbs1flox KO\nmice (Fig. 6M-N). Moreover, neutrophil depletion increased COL4A1 and COL6A1 levels in lung injury (Fig.\n6M-N). These findings strongly support the concept that Mgk/plt TSP1 prevents degradation of the alveolar\nbasement membrane, specifically COL6A1, from neutrophil-mediated proteolytic injury (Fig. 6O).”\nTitle: “Megakaryocyte/platelet-derived thrombospondin-1 regulates neutrophil function and matrix remodeling\nby stabilizing basement membrane COL6A1 in lung injury”\nAbstract: “…These cKO mice exhibit an early airspace proteomic signature featuring neutrophil degranulation,\nplatelet activation, and extracellular matrix organization pathways with reduction in basement membrane\nprotein COL6A1 after injury. In response to the increased injury, the cKO mice display increased Mgk\nrecruitment to areas of fibrillar collagen deposition in regions of alveolar leak and extending into the adventitial\ninterstitium. Depleting neutrophils restores barrier integrity in cKO mice, reduces lung Mgk numbers, and\nincreases COL6A1 levels. Thus, Mgk/plt-derived TSP1 represents a key mechanism of matrix stabilization by\nprotecting the basement membrane from neutrophil-mediated proteolytic damage, regulating injury severity\nand Mgk numbers at the alveolar interface.”\nDiscussion section:\n“Our findings show Mgk/platelet-derived TSP1 protects the alveolar basement membrane by preventing the\nmatrix protein COL6A1 from neutrophil-mediated proteolytic damage and limiting Mgk recruitment to regions of\nalveolar leak.”\n“In contrast, PF4CreThbs1flox KO mice showed significant reductions in the basement matrix protein COL6 and\nneutrophil depletion increased both COL4 and COL6 levels in lung injury. The degradation of the basement\nmembrane COL6 may be driving new fibrillar collagen synthesis in PF4CreThbs1flox KO mice that are\nsusceptible to lung injury.”\n3. For the cKO mice, the authors show levels of Tsp-1 from platelets. What are total TSP1 levels in the\nlungs/BAL of these mice, ie. how much of the Tsp1 is from platelets/megakaryocytes. Are other cells\ncompensating for a loss of Tsp1, are there defects in other immune cells (e.g macrophages/monocytes) in cKO\nv WT that might influence the findings?\nResponse: The reviewer correctly points out that cKO mice express TSP1, as other cells besides Mgks and\nplatelets are still able to make TSP1 following injury. To address the reviewer’s question, we measured TSP1\nlevels by proteomics. In the BAL fluid, uninjured cKO and WT mice show minimal TSP1 expression based on\nLC-MS/MS data. Because TSP1 levels are not significantly different in BALF by proteomics, they do not show\nup in the heatmap comparisons of cKO:WT. At 24 h post-injury, we also confirmed TSP1 levels in the BAL fluid\nof cKO and WT mice and do not see a difference by ELISA (Fig. 6K). By alveolar parenchymal tissue\nproteomics, TSP1 levels are increased in cKO compared to WT lungs (new Fig. S3B) which may reflect the\ncompensatory increase in TSP1 release by other cell types. Our proteomic findings show that under basal\nconditions, there is minimal TSP1 release in the BALF. We did not compare TSP1 levels from different cell\nsources in vivo during acute injury which is beyond the scope of this manuscript. However, global Thbs1 KO\nmice have a more pronounced phenotype than Mgk/plt Thbs1 KO mice in terms of host defense, cytokine\nproduction, and immune cell recruitment3. This implies other cellular sources of TSP1 exert a compensatory\neffect in host defense but not alveolar barrier integrity which is plt/MgK TSP1 specific. Please note that neither\nthe LysMcreThbs1flox (Fig. S2O-T) nor the Cdh5creThbs1flox (Fig. S5) show impaired alveolar barrier integrity\nduring infectious or sterile lung injury, respectively. We speculate surface bound TSP1 on plts/Mgks are\ninvolved in dampening neutrophil proteolytic activity given their spatial proximity to neutrophils at the site of\nlung injury. We have added this perspective to the Discussion section.\nDiscussion section: “Previous data have shown that TSP1 is surface bound on activated platelets and\nundergoes a conformational change that enables enhanced interactions with integrins4. In addition, activated\nneutrophils have been shown to express surface bound neutrophil proteases such as NE and cathepsin G that\nretain their catalytic activity, allowing for finer control of enzymatic degradation and tissue infiltration5.”\n4. Just wondering whether the authors examined neutrophil localisation? Neutrophil numbers in the BAL didn’t\nchange but markers of activation increased in cKO vs WT mice. Are these neutrophils more concentrated\naround the damaged alveoli?\nResponse: Please see our response to Reviewer 1, comment 1 regarding the lung histology scoring of lungs\nof cKO vs WT mice in the live infection model. As part of the lung histology scoring, we evaluated neutrophils\nlocalized to the alveolar space and interstitium in cKO and WT mice. We did not observe differences in\nneutrophil localization in the alveolar spaces or interstitium in cKO when compared to WT mice (new Fig. 4H-\nL). This finding implies that TSP1 regulates neutrophil activation rather than neutrophil numbers or recruitment.\n6. Size of the alveoli in vehicle WT v cKO mice look very different in Fig 5. During the steady state, are the\nfunction/structure of the alveoli compromised in these mice even before they are challenged? Also looks to be\nmore collagen in the steady state in cKO mice – which may fit with these observations?\nResponse: Please see our response to Reviewer 1, Query 4. Old Figure 5B showed differences in alveolar\nsize of WT reporter versus cKO reporter mouse lungs with vehicle instillation. We have replaced the figure of\nthe WT vehicle-treated control image (left upper panel of new Figure 4B) using a different field from the same\nmouse within the same experiment. We now confirm that the scalebars for all images depicted in new Figure\n4B as 100 microns. To address whether there was any baseline structural differences that could affect lung\nfunction, we performed new Flexivent experiments at baseline (without vehicle) as well as with forced\noscillation technique using cKO and WT littermates (Fig. 4N-R). There are no significant differences in baseline\nlung mechanics between cKO and WT mice. As expected, injury reduced static compliance in both groups of\nmice (new Fig. 4N). Given the complexity of the graphics now shown, we split out static and dynamic lung\ncompliance in order to address the reviewer’s concern. Methacholine challenge further reduced dynamic\ncompliance in cKO mice but not in WT mice group, underscoring the altered lung function in cKO mice after\ninjury (new Fig. 4O). The Flexivent data is now shown in actual unit measurements. Please note that the\nincrease in collagen shown in new Fig. 5G are not in steady state but following vehicle instillation and PA SN.\nMinor considerations\n7. Approach to data analysis in Figure 1 is great. It might also be worth showing a representative overlay of\nsegmented areas in WT v cKO mice to really highlight the changes visually\nResponse: We thank the reviewer for the excellent suggestion. We now provide a segmentation analysis\nshowing a representative overlay of lung images in WT and cKO mice during injury. This is provided in new\nFig. S4.\n8. I would consider putting colour labels on the images in the figures so people don’t have to read the figure\nlegends everytime to see what colour represents what cell or protein\nResponse: We have now added color labels on the images in the figures for added clarity.\nReviewer #4 (Remarks to the Author):\nGeneral Comments\nIn the manuscript NCOMMS-25-26357 “Megakaryocyte/platelet-derived thrombospondin-1 stabilizes the early\nprovisional matrix in lung injury by exercising spatioltemporal control of neutrophil function and matrix\nremodeling”, the authors demonstrate protein thrombospondin-1 (TSP1) protects the lung from acute alveolar\ninjury using MgK/plt Thbs1 conditional knockout mice.\nMy comments are specific to the proteomic portion of the manuscript.\nResponse: We appreciate the constructive comments of the reviewer. As requested, we have revised the\nmanuscript to clarify the queries related to the proteomic assays.\nSpecific Comments\n1. Overall, I thought the method section was lacking. Speaking to the proteomic section only (including the\nsupplemental methods), there is inadequate information on how the proteomic samples were prepared,\nanalyzed by mass spec, which mass spectrometer was used, and how the data was analyzed and by what\nprogram.\nResponse: We have included the full detail of the methods used in our experiments, including sample\npreparation for proteomics, the mass spectrometer used, how data was analyzed and by which software. We\nalso include new experimental data with alveolar parenchymal tissue proteomics. Please refer to the new\nsupplementary methods and Fig. 3 and Fig. S3 legends.\nQuantitative proteomics for BALF in Supplementary methods: “Protein Digestion via Strap. S-trap was used for\nthe digestion. Briefly, 20% SDS in 200mM TEAB (Triethylamonium bicarbonate) was spiked into the sample to\nmake the final concentration of 5% SDS in 50mM TEAB. 5 µL of 50 mM ABC containing 5 µg/µL DTT was\nadded and the sample was incubated at 65 °C for 15 min followed by addition of 5 µL of 50 mM ABC\ncontaining 15 µg/µL iodoacetamide (incubated at RT for 15 min in the dark). Samples were then acidified by\nadding 12% phosphoric acid (1:10 v/v acid to sample). For every 25uL of samples, 165uL of TEAB\n(Triethylammonium bicarbonate, 1M)/MeOH (10:90 v/v) was added and then loaded to S-trap for further\nwashes. Samples was centrifuged at 4000g for 3 min (4C) to remove supernatant. 150uL of TEAB\n(Triethylamonium bicarbonate, 1M)/MeOH (10:90 v/v) was added to the trap as wash solution and the trap was\nwashed 3 times. After the final wash, sequencing grade trypsin dissolved in 50mM TEAB was added and\ndigested O/N at 37 °C. The following day, peptides were eluted from the trap by adding 40uL of 50mM TEAB,\n0.1% FA and 0.1% FA in Acetonitrile (50:50), sequentially. The sample was pooled together and dried in a\nvacufuge and resuspended in 20 µL of 50 mM acetic acid. Peptide concentration was determined by nanodrop\n(A280nm).\nOrbitrap Fusion. Nano-liquid chromatography-nanospray tandem mass spectrometry (Nano-LC/MS/MS) of\nprotein identification was performed on a Thermo Scientific orbitrap Fusion mass spectrometer equipped with\nan nanospray FAIMS Pro™ Sources operated in positive ion mode. Samples (2ug) were separated on an\neasy spray nano column (EASY-Spray™ HPLC Columns, C18 3µ 100A, 75µm X250mm Thermo Scientific)\nusing a 2D RSLC HPLC system from Thermo Scientific. Each sample was injected into the trap column\n(Acclaim™ PepMap™ C18, 3µ 100A, 75µm X20mm Thermo Scientific) and desalted with 0.1% Formic Acid in\nwater for 5 minutes. The injector port was then switched to “inject” and the peptides were eluted off of the trap\nonto the column. Mobile phase A was 0.1% Formic Acid in water and acetonitrile (with 0.1% formic acid) was\nused as mobile phase B. Flow rate was set at 300nL/min. mobile phase B was increased from 2% to 16% in\n105 min and then increased from 16-25% in 10 min and again from 25-85% in 1 min and then kept at 95% for\nanother 4 min before being brought back quickly to 2% in 1 min. The column was equilibrated at 2% of mobile\nphase B (or 98% A) for 15 min before the next sample injection.\nMS/MS data was acquired with a spray voltage of 1.95 KV and a capillary temperature of 305 °C is used. The\nscan sequence of the mass spectrometer was based on the preview mode data dependent TopSpeed™\nmethod: the analysis was programmed for a full scan recorded between m/z 375-1500 and a MS/MS scan to\ngenerate product ion spectra to determine amino acid sequence in consecutive scans starting from the most\nabundant peaks in the spectrum in the next 3 seconds. To achieve high mass accuracy MS determination, the\nfull scan was performed at FT mode and the resolution was set at 120,000 with internal mass calibration.\nThree compensation voltage (cv=-50, -65 and -80v) were used for samples acquisition. The AGC Target ion\nnumber for FT full scan was set at 4 x 105 ions, maximum ion injection time was set at 50 ms and micro scan\nnumber was set at 1. MSn was performed using HCD in ion trap mode to ensure the highest signal intensity of\nMSn spectra. The HCD collision energy was set at 32%. The AGC Target ion number for ion trap MSn scan\nwas set at 3.0E4 ions, maximum ion injection time was set at 35 ms and micro scan number was set at 1.\nDynamic exclusion is enabled with a repeat count of 1 within 60s and a low mass width and high mass width of\n10ppm.\nDatabase searching. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version\n2.7.0). Mascot was set up to search the Common_Contamination_Proteins-cRAP_LZ_20190813.fasta;\nUniprot_Mouse_Reviewed_08272021_20210827 database (unknown version, 17195 entries) assuming the\ndigestion enzyme stricttrypsin. Mascot was searched with a fragment ion mass tolerance of 0.50 Da and a\nparent ion tolerance of 10.0 PPM. Carbamidomethyl of cysteine was specified in Mascot as a fixed\nmodification. Deamidated of asparagine and glutamine and oxidation of methionine were specified in Mascot\nas variable modifications.\nCriteria for protein identification. Scaffold (version Scaffold_5.0.1, Proteome Software Inc., Portland, OR) was\nused to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they\ncould be established at greater than 95.0% probability to achieve an FDR less than 1.0% by the Scaffold Local\nFDR algorithm. Protein identifications were accepted if they could be established at greater than 98.0%\nprobability to achieve an FDR less than 1.0% and contained at least 2 identified peptides. Protein probabilities\nwere assigned by the Protein Prophet algorithm (Nesvizhskii, Al et al Anal. Chem. 2003;75(17):4646-58).\nProteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were\ngrouped to satisfy the principles of parsimony.\nRelative quantitation using label free spectral counting. The spectral count of each protein identified in the\ndataset were used to quantify the abundance of each protein in the BALF of WT (n=2), Thbs1-/- (n=2),\nPF4CreThbs1flox (n=4) and PF4WTThbs1flox (n=4) mice at 1 day post-infection. Data was Log normalized and\n2\nexpressed as the fold change between Thbs1-/-: WT or PF4CreThbs1flox: WT mice. For BAL proteomics data,\nstatistical testing was performed using a two sample Welch t-test after log2 plus one transformation of the\nprotein spectral counts to compare expression between PF4CreThbs1flox and WT mice. Protein accessions were\nmapped to gene identifiers using STRING7. Reactome pathway enrichment was performed using over-\nrepresentation analysis with hypergeometric testing of statistically significant proteins (p < 0.05) between WT\nand PF4CreThbs1flox mice.”\nQuantitative proteomics for alveolar tissue in Supplemental Methods: “For alveolar tissue proteomics, laser\ncapture microdissection was performed on histopathological sections targeting regions of alveolar injury within\nthe lung day 1 post i.t. inoculation of PA in PF4creThbs1flox and WT mice. After laser capture, regions of injury\nwere digested and processed for LC-MS/MS. Mass spectrometry samples were quantified using label free\nquantification (LFQ) via data-independent acquisition (DIA) and mapped against the mouse database in\nSpectronaut for DIA-LFQ analysis. The precursor filter algorithm in Spectronaut was utilized to retain proteins\nwith valid label free quantification values and the t-test was performed for comparisons between WT and\nPF4creThbs1flox mice. Multiple test correction was performed using the Benjamini-Hochberg correction. Protein\naccessions were mapped to gene identifiers using STRING. Reactome pathway enrichment was performed\nusing over-representation analysis with hypergeometric testing of statistically significant proteins between WT\nand PF4creThbs1flox mice.”\nFigure 3 legend: “(I) BALF LC-MS/MS differentially enriched pathways identified in the airspaces of WT and\nPF4CreThbs1flox mice at day 1 post-infection by Reactome pathway analysis. n = 4 mice per group. (J) Alveolar\ntissue LC-MS/MS differentially enriched pathways identified by Reactome pathway analysis. Alveolar\nparenchymal tissues were obtained from WT and PF4CreThbs1flox mice at day 1 post-infection. n = 3 mice per\ngroup comprised of multiple alveolar parenchymal regions obtained via laser capture microdissection per\nmouse post DIA-LFQ quality filtering. (K) BALF LC-MS/MS MMP-9 relative quantification. n = 4-6 mice per\ngroup. (L) Alveolar tissue LC-MS/MS NE relative quantification. n = 3 mice per group comprised of multiple\nalveolar parenchymal regions obtained via laser capture microdissection per mouse post DIA-LFQ quality\nfiltering.”\nFigure S3 legend: “Quantitative proteomics reveals dysregulated neutrophil activation and ECM related\nproteins in the airspaces and alveolar tissue of MgK/platelet-specific Thbs1 KO mice following live PA\ninfection. WT (PF4WTThbs1flox) and PF4CreThbs1flox KO mice were i.t. inoculated with vehicle (sterile PBS) or\nPF4CreThbs1flox KO and WT mice within the significantly enriched Reactome pathways (neutrophil\ndegranulation, platelet activation, ECM organization, BALF n = 4 mice/group, alveolar tissue n = 3 mice/group\nwith 2-4 parenchymal regions obtained per mouse). Data are shown as the mean log -fold difference between\n2. Shotgun proteomics terminology has been updated to label-free quantitation proteomics to reflect more\naccurately the method. It would be advised to update the terminology throughout the manuscript.\nResponse: Thank you for the comment. As requested, we have updated the terminology to label-free\nquantitative proteomics throughout the main manuscript, the main figures and the supplementary material.\n3. The biological replicates for BALF of WT, and Thbs1+/- was n=2. That a significant deficiency of the\nproteomics data as true statistical power requires a minimum of n=3. This diminishes the strength of the results\nand conclusions as a n=2 is generally not considered statistically significant for a robust conclusion for this\nsection of the manuscript (Fig S4 and corresponding results).\nResponse: Thank you for the comment. The initial rationale for including the global KO proteomics data is to\nsupport and reinforce the findings observed in the cKO mice, rather than to draw independent conclusions from\nit. Given the reviewer’s concerns, we have removed the data from the Supplemental figures.\nReferences\n1. Kulkarni HS, Lee JS, Bastarache JA, Kuebler WM, Downey GP, Albaiceta GM, Altemeier WA, Artigas\nA, Bates JHT, Calfee CS, Dela Cruz CS, Dickson RP, Englert JA, Everitt JI, Fessler MB, Gelman AE,\nGowdy KM, Groshong SD, Herold S, Homer RJ, Horowitz JC, Hsia CCW, Kurahashi K, Laubach VE,\nLooney MR, Lucas R, Mangalmurti NS, Manicone AM, Martin TR, Matalon S, Matthay MA, McAuley DF,\nMcGrath-Morrow SA, Mizgerd JP, Montgomery SA, Moore BB, Noël A, Perlman CE, Reilly JP, Schmidt\nEP, Skerrett SJ, Suber TL, Summers C, Suratt BT, Takata M, Tuder R, Uhlig S, Witzenrath M, Zemans\nRL, Matute-Bello G. Update on the features and measurements of experimental acute lung injury in\nanimals: an official american thoracic society workshop report. Am J Respir Cell Mol Biol. 2022\nFeb;66(2):e1–e14. PMCID: PMC8845128\n2. Yeung AK, Villacorta-Martin C, Hon S, Rock JR, Murphy GJ. Lung megakaryocytes display distinct\ntranscriptional and phenotypic properties. Blood Adv. 2020 Dec 22;4(24):6204–6217. PMCID:\nPMC7757004\n3. Qu Y, Olonisakin T, Bain W, Zupetic J, Brown R, Hulver M, Xiong Z, Tejero J, Shanks RM,\nBomberger JM, Cooper VS, Zegans ME, Ryu H, Han J, Pilewski J, Ray A, Cheng Z, Ray P, Lee JS.\nThrombospondin-1 protects against pathogen-induced lung injury by limiting extracellular matrix\nproteolysis. JCI Insight. 2018 Feb 8;3(3). PMCID: PMC5821195\n4. Rodrigues RG, Guo N, Zhou L, Sipes JM, Williams SB, Templeton NS, Gralnick HR, Roberts DD.\nConformational regulation of the fibronectin binding and alpha 3beta 1 integrin-mediated adhesive\nactivities of thrombospondin-1. J Biol Chem. 2001 Jul 27;276(30):27913–27922. PMID: 11358957\n5. Owen CA, Campbell MA, Sannes PL, Boukedes SS, Campbell EJ. Cell surface-bound elastase and\ncathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and\npreserve catalytic activity of serine proteinases. J Cell Biol. 1995 Nov;131(3):775–789. PMCID:\nPMC2120617\nResponse to reviewers.\nResponse: We thank the reviewers for their time in carefully reviewing the revision and their encouraging\nresponse. There were a few additional clarifications requested. Below are the point-by-point response.\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors did a very nice job responding to my questions and suggestions and the manuscript is much\nimproved as a result. I do have two additional suggestions.\n1. In figure 4G, The PF4.Cre/Thbs1.Flox PA SN lungs appear to have airways filled with blood, Did these mice\nexperience alveolar or airway hemorrhage? Can the authors explain this observation?\nResponse: The reviewer astutely comments on the alveolar and airway hemorrhage shown in Figure 4G. We\npreviously published the live PA infection model where global TSP1 KO mice show increased barrier disruption\nin lung injury compared to WT1,2. In a subsequent manuscript, we show that mice with low platelets (Mpl-/-) exhibit\nsignificant alveolar hemorrhagic injury 2, prompting us to examine the role of Mgk/plt cell specific TSP1 KO mice.\nBased on the findings reported in the current manuscript, we suggest the alveolar hemorrhage [editorial note:\nconfidential information redacted] and airway hemorrhage observed in mice deficient in megakaryocyte and\nplatelet TSP1 are due to barrier disruption from matrix degradation of basement membrane protein COL6. COL6\nis found not only in the alveolar basement membrane but also along perivascular and peribronchiolar regions of\nthe lung3,4. [editorial note: confidential information redacted]. Although beyond the scope of this manuscript,\nwe modified the results section to add the point about increased hemorrhage in the Mgk/plt cKO mice.\n[editorial note: confidential figure redacted]\nOriginal results section, page 10, lines 262-263 from: “PF4CreThbs1flox KO mice showed increase in lung injury\nscore after PA SN sterile injury when compared to WT littermates (Fig. 4H).”\nRevised results section:“PF4CreThbs1flox KO mice showed increase in hemorrhagic injury, as evidenced by higher\nlung injury score after PA SN instillation when compared to WT littermates (Fig. 4H).”\n2. The authors use both male and female mice which is a strength. It would be helpful for the authors to indicate\nmale and female mice separately (ex. squares and triangles, etc). In addition, the authors should comment on\nwhether they saw an sex differences.\nResponse: Please see representative figures from 2H, 3B, 4C, 4D, 4H below which depict major phenotype\nresults that display sex disaggregated data. Given the overlap between male and female data, we provide\nuniform presentation of individual data points in each of the figures to maintain clarity and focus. Please note the\nmales and female sex of individual mice for each experiment are provided in the data source file in the\nSupplementary file section. We have revised the Methods section to state we did not observe sex differences\nand the sex of individual mice for each figure is located in the data source file.\nOriginal methods section: “Sex as a biological variable. We used both male and female mice and age-matched\nthem based on availability for each experiment.”\nRevised methods section: “Sex as biological variable. We used both male and female mice and age-matched\nthem based on availability for each experiment. We did not observe sex differences in the outcomes\nmeasured. Please refer to the Source Data File in the Supplementary Information for details of sex of individual\nmice used.”\nReviewer #2 (Remarks to the Author):\nOverall the authors have done an excellent job addressing reviewers concerns. I just ask for some clarification\nand further lung Mk quantification and characterization analysis.\nThe authors state -\"gating strategy where we gated on large cells and excluded smaller cells such as\nneutrophils\". Lung megakaryocytes are not all large cells, they are largely 2N, so a size based exclusion shown\nin Fig S7 there are likely a large number of Mks that are missing from the analysis. It is strongly suggested that\nadditionally markers such as CD42c be added to the lung flow panel, less size exclusion employed and more\nnegative/dump channel markers used in the analysis. This will greatly improve the overall rigor and make readers\nmore confident in the proper cell identities.\nResponse: We thank the reviewer for the comment and raising this important point. We performed both size\nexclusion and negative/dump channel markers to remove the possibility of platelet-neutrophil aggregates as a\nconfounder in the lung Mgk quantification as this was a concern previously raised by the same reviewer. We\nagree that CD42c is another specific Mgk and platelet marker. However, adding CD42c together with CD41 will\nexclude non-activated Mgk which is not the goal of the current experiment. We utilized size exclusion as a second\nmethod to minimize the counting of platelet-neutrophil aggregates in addition to the use of negative/dump\nchannel markers (i.e., B220, CD3, Gr1, F4/80) to exclude B cell, T cell, myeloid cell, and macrophage\npopulations, as shown in Figure S7. Using the two methods together to exclude platelet-neutrophil or platelet-\nmonocyte aggregates improved rigor, albeit at the expense of identifying higher numbers of potential lung Mgks.\nPlease note that our focus was relative and not absolute quantification of the lung Mgk, as indicated in\nsupplemental Figure S8 legend. Our main question pertains to whether lung Mgk numbers increase after injury\nin the lung which we believe we have effectively addressed. To the reviewer’s point, we have emphasized in the\nresults section that our focus is relative quantification of lung Mgk. As suggested by the reviewer, we will add\nCD42c to our panel for future studies focusing on the role of lung Mgks in injury.\nOriginal results section: “Mgks were identified as large cells (FSChigh) that were CD45+CD41+ and distinct from\nneutrophils (Fig. S7 and S7A2, respectively). Utilizing the same gating strategy (Fig. S8A1) or adding lineage\nmarkers B220, GR1, and F4/80 in the “dump channel” to separate lineage committed cells from Mgks (Fig.\nS8A2), both approaches revealed CD45+CD41+ Mgk numbers increase in cKO lungs and distinct from\nneutrophils (Fig. S8A3) when compared to WT after injury (Fig. S8B, S8C).”\nRevised results section: “Mgks were identified as large cells (FSChigh) that were CD45+CD41+ and distinct from\nneutrophils (Fig. S7 and S7A2, respectively). Utilizing the same gating strategy (Fig. S8A1) or adding lineage\nmarkers B220, GR1, and F4/80 in the “dump channel” to separate lineage committed cells from Mgks (Fig.\nS8A2), both approaches revealed that the relative numbers of CD45+CD41+ Mgk numbers increase in cKO lungs\nand distinct from neutrophils (Fig. S8A3) when compared to WT after injury (Fig. S8B, S8C).”\nReviewer #3 (Remarks to the Author):\nThe authors have performed extensive revision experiments and additional analysis that in my opinion addresses\nall the reviewers comments including mine.\nI think the additional data/reorganisation and text changes really strengthen the message of the manuscript and\nthe authors should be congratulated for the hard work in putting this together. Well done, no additional points\nhave been raised.\nResponse: Thank you.\nReviewer #4 (Remarks to the Author):\nThe authors did an excellent job of addressing the reviewer concerns and thus produced a much stronger\nmanuscript for publication.\nResponse: Thank you.\nReferences:\n1. Qu, Y. et al. Thrombospondin-1 protects against pathogen-induced lung injury by limiting extracellular\nmatrix proteolysis. JCI Insight 3, (2018).\n2. Bain, W. et al. Platelets inhibit apoptotic lung epithelial cell death and protect mice against infection-\ninduced lung injury. Blood Adv. 3, 432–445 (2019).\n3. Mereness, J. A. et al. Collagen VI deficiency results in structural abnormalities in the mouse lung. Am.\nJ. Pathol. 190, 426–441 (2020).\n4. Mereness, J. A. & Mariani, T. J. The critical role of collagen VI in lung development and chronic lung\ndisease. Matrix Biology Plus 10, 100058 (2021)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors did a very nice job responding to my questions and suggestions and the manuscript is much improved as a\nresult. I do have two additional suggestions.\n1. In figure 4G, The PF4.Cre/Thbs1.Flox PA SN lungs appear to have airways filled with blood, Did these mice experience\nalveolar or airway hemorrhage? Can the authors explain this observation?\n2. The authors use both male and female mice which is a strength. It would be helpful for the authors to indicate male and\nfemale mice separately (ex. squares and triangles, etc). In addition, the authors should comment on whether they saw an sex\ndifferences.", "reviewer_2": "Overall the authors have done an excellent job addressing reviewers concerns. I just ask for some clarification and further\nlung Mk quantification and characterization analysis.\nThe authors state -\"gating strategy where we gated on large cells and excluded smaller cells such as neutrophils\". Lung\nmegakaryocytes are not all large cells, they are largely 2N, so a size based exclusion shown in Fig S7 there are likely a\nlarge number of Mks that are missing from the analysis. It is strongly suggested that additionally markers such as CD42c be\nadded to the lung flow panel, less size exclusion employed and more negative/dump channel markers used in the analysis.\nThis will greatly improve the overall rigor and make readers more confident in the proper cell identities.", "reviewer_3": "The authors have performed extensive revision experiments and additional analysis that in my opinion addresses all the\nreviewers comments including mine.\nI think the additional data/reorganisation and text changes really strengthen the message of the manuscript and the authors\nshould be congratulated for the hard work in putting this together. Well done, no additional points have been raised.", "reviewer_4": "The authors did an excellent job of addressing the reviewer concerns and thus produced a much stronger manuscript for\npublication."}, "author_response": "Response to reviewers.\nWe thank the reviewers for their time in carefully reviewing the revision and their encouraging response. There were a few additional clarifications requested. Below are the point-by-point response.\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors did a very nice job responding to my questions and suggestions and the manuscript is much improved as a result. I do have two additional suggestions.\n1. In figure 4G, The PF4.Cre/Thbs1.Flox PA SN lungs appear to have airways filled with blood, Did these mice experience alveolar or airway hemorrhage? Can the authors explain this observation?\nResponse: The reviewer astutely comments on the alveolar and airway hemorrhage shown in Figure 4G. We previously published the live PA infection model where global TSP1 KO mice show increased barrier disruption in lung injury compared to WT1,2. In a subsequent manuscript, we show that mice with low platelets (Mpl-/-) exhibit significant alveolar hemorrhagic injury 2, prompting us to examine the role of Mgk/plt cell specific TSP1 KO mice. Based on the findings reported in the current manuscript, we suggest the alveolar hemorrhage [editorial note: confidential information redacted] and airway hemorrhage observed in mice deficient in megakaryocyte and platelet TSP1 are due to barrier disruption from matrix degradation of basement membrane protein COL6. COL6 is found not only in the alveolar basement membrane but also along perivascular and peribronchiolar regions of the lung3,4. [editorial note: confidential information redacted]. Although beyond the scope of this manuscript, we modified the results section to add the point about increased hemorrhage in the Mgk/plt cKO mice.\n[editorial note: confidential figure redacted]\nOriginal results section, page 10, lines 262-263 from: “PF4CreThbs1flox KO mice showed increase in lung injury score after PA SN sterile injury when compared to WT littermates (Fig. 4H).”\nRevised results section:“PF4CreThbs1flox KO mice showed increase in hemorrhagic injury, as evidenced by higher lung injury score after PA SN instillation when compared to WT littermates (Fig. 4H).”\n2. The authors use both male and female mice which is a strength. It would be helpful for the authors to indicate male and female mice separately (ex. squares and triangles, etc). In addition, the authors should comment on whether they saw an sex differences.\nResponse: Please see representative figures from 2H, 3B, 4C, 4D, 4H below which depict major phenotype results that display sex disaggregated data. Given the overlap between male and female data, we provide uniform presentation of individual data points in each of the figures to maintain clarity and focus. Please note the males and female sex of individual mice for each experiment are provided in the data source file in the Supplementary file section. We have revised the Methods section to state we did not observe sex differences and the sex of individual mice for each figure is located in the data source file.\nOriginal methods section: “Sex as a biological variable. We used both male and female mice and age-matched them based on availability for each experiment.”\nRevised methods section: “Sex as biological variable. We used both male and female mice and age-matched them based on availability for each experiment. We did not observe sex differences in the outcomes measured. Please refer to the Source Data File in the Supplementary Information for details of sex of individual mice used.”\nReviewer #2 (Remarks to the Author):\nOverall the authors have done an excellent job addressing reviewers concerns. I just ask for some clarification and further lung Mk quantification and characterization analysis.\nThe authors state -\"gating strategy where we gated on large cells and excluded smaller cells such as neutrophils\". Lung megakaryocytes are not all large cells, they are largely 2N, so a size based exclusion shown in Fig S7 there are likely a large number of Mks that are missing from the analysis. It is strongly suggested that additionally markers such as CD42c be added to the lung flow panel, less size exclusion employed and more negative/dump channel markers used in the analysis. This will greatly improve the overall rigor and make readers more confident in the proper cell identities.\nResponse: We thank the reviewer for the comment and raising this important point. We performed both size exclusion and negative/dump channel markers to remove the possibility of platelet-neutrophil aggregates as a confounder in the lung Mgk quantification as this was a concern previously raised by the same reviewer. We agree that CD42c is another specific Mgk and platelet marker. However, adding CD42c together with CD41 will exclude non-activated Mgk which is not the goal of the current experiment. We utilized size exclusion as a second method to minimize the counting of platelet-neutrophil aggregates in addition to the use of negative/dump channel markers (i.e., B220, CD3, Gr1, F4/80) to exclude B cell, T cell, myeloid cell, and macrophage populations, as shown in Figure S7. Using the two methods together to exclude platelet-neutrophil or platelet-monocyte aggregates improved rigor, albeit at the expense of identifying higher numbers of potential lung Mgks. Please note that our focus was relative and not absolute quantification of the lung Mgk, as indicated in supplemental Figure S8 legend. Our main question pertains to whether lung Mgk numbers increase after injury in the lung which we believe we have effectively addressed. To the reviewer’s point, we have emphasized in the results section that our focus is relative quantification of lung Mgk. As suggested by the reviewer, we will add CD42c to our panel for future studies focusing on the role of lung Mgks in injury.\nOriginal results section: “Mgks were identified as large cells (FSChigh) that were CD45+CD41+ and distinct from neutrophils (Fig. S7 and S7A2, respectively). Utilizing the same gating strategy (Fig. S8A1) or adding lineage markers B220, GR1, and F4/80 in the “dump channel” to separate lineage committed cells from Mgks (Fig. S8A2), both approaches revealed CD45+CD41+ Mgk numbers increase in cKO lungs and distinct from neutrophils (Fig. S8A3) when compared to WT after injury (Fig. S8B, S8C).”\nRevised results section: “Mgks were identified as large cells (FSChigh) that were CD45+CD41+ and distinct from neutrophils (Fig. S7 and S7A2, respectively). Utilizing the same gating strategy (Fig. S8A1) or adding lineage markers B220, GR1, and F4/80 in the “dump channel” to separate lineage committed cells from Mgks (Fig. S8A2), both approaches revealed that the relative numbers of CD45+CD41+ Mgk numbers increase in cKO lungs and distinct from neutrophils (Fig. S8A3) when compared to WT after injury (Fig. S8B, S8C).”\nReviewer #3 (Remarks to the Author):\nThe authors have performed extensive revision experiments and additional analysis that in my opinion addresses all the reviewers comments including mine.\nI think the additional data/reorganisation and text changes really strengthen the message of the manuscript and the authors should be congratulated for the hard work in putting this together. Well done, no additional points have been raised.\nResponse: Thank you.\nReviewer #4 (Remarks to the Author):\nThe authors did an excellent job of addressing the reviewer concerns and thus produced a much stronger manuscript for publication.\nResponse: Thank you.\nReferences:\n1. Qu, Y. et al. Thrombospondin-1 protects against pathogen-induced lung injury by limiting extracellular matrix proteolysis. JCI Insight 3, (2018).\n2. Bain, W. et al. Platelets inhibit apoptotic lung epithelial cell death and protect mice against infection-induced lung injury. Blood Adv. 3, 432–445 (2019).\n3. Mereness, J. A. et al. Collagen VI deficiency results in structural abnormalities in the mouse lung. Am. J. Pathol. 190, 426–441 (2020).\n4. Mereness, J. A. & Mariani, T. J. The critical role of collagen VI in lung development and chronic lung disease. Matrix Biology Plus 10, 100058 (2021)."}]

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