Tropomyosin 1 Has Separate Roles in Murine Platelet Function and Developmental Hematopoiesis
Authors: Tsao Victor , Fonar Gennadiy , Peshkova Alina D. D. , Muzykantov Vladimir R. , Thom Christopher S. 
Published: December 2024

Background

Genome-wide association studies (GWAS) have linked thousands of sites with platelet trait variation, but it can be difficult to define causal genes and mechanisms. Genetic polymorphisms that decrease Tropomyosin 1 (Tpm1) expression increase both platelet count and size. Tpm1 is an actin binding protein that impacts many cell types. Our prior studies linked Tpm1 deficiency with increased hematopoietic progenitor cell formation in vitro and in vivo, but it was unclear if these mechanisms extended to postnatal platelet biology. The objectives of this study were to define 1) if Tpm1 deficiency impacted platelet functions and 2) if Tpm1 effects on developmental hematopoiesis were linked to postnatal platelet variation.

Methods

As constitutive Tpm1 knockout (KO) was embryonic lethal, we generated a murine Tpm1 construct with loxP sites flanking Tpm1 exon 3. This exon is retained in all Tpm1 isoforms. Deletion causes a frameshift mutation and premature termination that disrupts function. We analyzed effects of conditional Tpm1 deletion in blood cells using established assays.

Results

Mammalian blood cells originate from embryonic Cdh5+ hemogenic endothelial cells in the aorta-gonad-mesonephros region. Constitutive Tpm1 haploinsufficiency doubles hemogenic endothelial cell formation (Wilken et al, Stem Cell Rep 2024). We confirmed these effects in Cdh5Cre/+ Tpm1fl/fl mice, finding increased Runx1+ hemogenic endothelial cells vs littermate controls by whole mount staining at E9.5 (100±13 vs 55±26 HE cells/mm, mean±SD, p=0.02).

However, these embryonic changes did not alter postnatal hematopoiesis in Cdh5Cre/+ Tpm1fl/fl bone marrow. While adult Cdh5Cre/+ Tpm1fl/fl peripheral blood showed efficient Tpm1 recombination, these mice had normal percentages of bone marrow hematopoietic stem and progenitor cells and normal blood counts at ~2 months of age vs littermate controls. We found similar results in VavCre/+ Tpm1fl/fl mice, which conditionally deleted Tpm1 in all hematopoietic progenitor cells and progeny.

These findings suggested that Tpm1KO had separate impacts on mature blood cells. Genetic colocalization analysis confirmed separate GWAS signals linking the TPM1 gene locus to increased platelet count vs red blood cell trait variation. Rather than effects on hematopoietic stem cells transmitted to progeny, these findings suggested lineage-specific Tpm1KO effects on mature platelets.

We hypothesized that Tpm1KO might impact platelet focal adhesion and hemostatic functions, given focal adhesion defects in heterologous Tpm1KO cells (Kumari et al, Nat Comm 2024). With established in vivo blood clot contraction assays (Tutwiler et al, Blood 2016), we identified delayed lag time (i.e., time from thrombin addition) in Tpm1KO whole blood (380±140 vs 158±26 sec, p=0.02) and overall contraction potential in Tpm1KO whole blood (364±21 vs 253±79 a.u., p=0.03). To confirm platelet impairment, we assessed contraction in platelet-rich plasma. Tpm1KO samples had prolonged lag time (158±51 vs 71±31 sec, p=0.03). However, Tpm1KO did not perturb average velocity or the final extent of plasma clot contraction. Delayed lag time can result from impaired platelet activation or fibrinogen binding. While our previous results confirmed normal activation in TPM1-deficient megakaryocytes (Thom et al, BMC Biol 2020), ex vivo focal adhesion assays demonstrated defective Tpm1KO platelet binding to fibrinogen-coated cover slips (447±149 vs 166±44 platelets per 20x field, p=0.02 by paired t test) and fibronectin (153±37 vs 44±15, p=0.04).

Conclusions

Our findings show a novel role for Tpm1 in platelet focal adhesion and clot contraction that is separate from Tpm1 functions during embryonic hematopoiesis. Tpm1 effects on platelet focal adhesion may impact platelet clearance and account for GWAS-identified changes in human platelet counts. Tpm1 modulation may also represent novel strategies to control platelet adhesion and activation in vivo.