Platelet C3G: a key player in vesicle exocytosis, spreading and clot retraction.

C3G is a Rap1 GEF that plays a pivotal role in platelet-mediated processes such as angiogenesis, tumor growth, and metastasis by modulating the platelet secretome. Here, we explore the mechanisms through which C3G governs platelet secretion. For this, we utilized animal models featuring either overexpression or deletion of C3G in platelets, as well as PC12 cell clones expressing C3G mutants. We found that C3G specifically regulates α-granule secretion via PKCδ, but it does not affect δ-granules or lysosomes. C3G activated RalA through a GEF-dependent mechanism, facilitating vesicle docking, while interfering with the formation of the trans-SNARE complex, thereby restricting vesicle fusion. Furthermore, C3G promotes the formation of lamellipodia during platelet spreading on specific substrates by enhancing actin polymerization via Src and Rac1-Arp2/3 pathways, but not Rap1. Consequently, C3G deletion in platelets favored kiss-and-run exocytosis. C3G also controlled granule secretion in PC12 cells, including pore formation. Additionally, C3G-deficient platelets exhibited reduced phosphatidylserine exposure, resulting in decreased thrombin generation, which along with defective actin polymerization and spreading, led to impaired clot retraction. In summary, platelet C3G plays a dual role by facilitating platelet spreading and clot retraction through the promotion of outside-in signaling while concurrently downregulating α-granule secretion by restricting granule fusion.

New functions of C3G in platelet biology: Contribution to ischemia-induced angiogenesis, tumor metastasis and TPO clearance.

C3G is a Rap1 guanine nucleotide exchange factor that controls platelet activation, aggregation, and the release of α-granule content. Transgenic expression of C3G in platelets produces a net proangiogenic secretome through the retention of thrombospondin-1. In a physiological context, C3G also promotes megakaryocyte maturation and proplatelet formation, but without affecting mature platelet production. The aim of this work is to investigate whether C3G is involved in pathological megakaryopoiesis, as well as its specific role in platelet mediated angiogenesis and tumor metastasis. Using megakaryocyte-specific C3G knockout and transgenic mouse models, we found that both C3G overexpression and deletion promoted platelet-mediated angiogenesis, induced by tumor cell implantation or hindlimb ischemia, through differential release of proangiogenic and antiangiogenic factors. However, only C3G deletion resulted in a higher recruitment of hemangiocytes from the bone marrow. In addition, C3G null expression enhanced thrombopoietin (TPO)-induced platelet production, associated with reduced TPO plasma levels. Moreover, after 5-fluorouracil-induced platelet depletion and rebound, C3G knockout mice showed a defective return to homeostatic platelet levels, indicating impaired platelet turnover. Mechanistically, C3G promotes c-Mpl ubiquitination by inducing Src-mediated c-Cbl phosphorylation and participates in c-Mpl degradation via the proteasome and lysosome systems, affecting TPO internalization. We also unveiled a positive role of platelet C3G in tumor cell-induced platelet aggregation, which facilitated metastatic cell homing and adhesion. Overall, these findings revealed that C3G plays a crucial role in platelet-mediated angiogenesis and metastasis, as well as in platelet level modulation in response to pathogenic stimuli.

A novel nonsense variant in TPM4 caused dominant macrothrombocytopenia, mild bleeding tendency and disrupted cytoskeleton remodeling.

BACKGROUND: Rare inherited thrombocytopenias are caused by alterations in genes involved in megakaryopoiesis, thrombopoiesis and/or platelet release. Diagnosis is challenging due to poor specificity of platelet laboratory assays, large numbers of culprit genes, and difficult assessment of the pathogenicity of novel variants. OBJECTIVES: To characterize the clinical and laboratory phenotype, and identifying the underlying molecular alteration, in a pedigree with thrombocytopenia of uncertain etiology. PATIENTS/METHODS: Index case was enrolled in our Spanish multicentric project of inherited platelet disorders due to lifelong thrombocytopenia and bleeding. Bleeding score was recorded by ISTH-BAT. Laboratory phenotyping consisted of blood cells count, blood film, platelet aggregation and flow cytometric analysis. Genotyping was made by whole-exome sequencing (WES). Cytoskeleton proteins were analyzed in resting/spreading platelets by immunofluorescence and immunoblotting. RESULTS: Five family members displayed lifelong mild thrombocytopenia with a high number of enlarged platelets in blood film, and mild bleeding tendency. Patient's platelets showed normal aggregation and granule secretion response to several agonists. WES revealed a novel nonsense variant (c.322C>T; p.Gln108*) in TPM4 (NM_003290.3), the gene encoding for tropomyosin-4 (TPM4). This variant led to impairment of platelet spreading capacity after stimulation with TRAP-6 and CRP, delocalization of TPM4 in activated platelets, and significantly reduced TPM4 levels in platelet lysates. Moreover, the index case displayed up-regulation of TPM2 and TPM3 mRNA levels. CONCLUSIONS: This study identifies a novel TPM4 nonsense variant segregating with macrothrombocytopenia and impaired platelet cytoskeletal remodeling and spreading. These findings support the relevant role of TPM4 in thrombopoiesis and further expand our knowledge of TPM4-related thrombocytopenia.

Characterization of the Platelet Phenotype Caused by a Germline RUNX1 Variant in a CRISPR/Cas9-Generated Murine Model.

RUNX1-related disorder (RUNX1-RD) is caused by germline variants affecting the RUNX1 gene. This rare, heterogeneous disorder has no specific clinical or laboratory phenotype, making genetic diagnosis necessary. Although international recommendations have been established to classify the pathogenicity of variants, identifying the causative alteration remains a challenge in RUNX1-RD. Murine models may be useful not only for definitively settling the controversy about the pathogenicity of certain RUNX1 variants, but also for elucidating the mechanisms of molecular pathogenesis. Therefore, we developed a knock-in murine model, using the CRISPR/Cas9 system, carrying the RUNX1 p.Leu43Ser variant (mimicking human p.Leu56Ser) to study its pathogenic potential and mechanisms of platelet dysfunction. A total number of 75 mice were generated; 25 per genotype (RUNX1WT/WT, RUNX1WT/L43S, and RUNX1L43S/L43S). Platelet phenotype was assessed by flow cytometry and confocal microscopy. On average, RUNX1L43S/L43S and RUNX1WT/L43S mice had a significantly longer tail-bleeding time than RUNX1WT/WT mice, indicating the variant's involvement in hemostasis. However, only homozygous mice displayed mild thrombocytopenia. RUNX1L43S/L43S and RUNX1WT/L43S displayed impaired agonist-induced spreading and α-granule release, with no differences in δ-granule secretion. Levels of integrin αIIbß3 activation, fibrinogen binding, and aggregation were significantly lower in platelets from RUNX1L43S/L43S and RUNX1WT/L43S using phorbol 12-myristate 13-acetate (PMA), adenosine diphosphate (ADP), and high thrombin doses. Lower levels of PKC phosphorylation in RUNX1L43S/L43S and RUNX1WT/L43S suggested that the PKC-signaling pathway was impaired. Overall, we demonstrated the deleterious effect of the RUNX1 p.Leu56Ser variant in mice via the impairment of integrin αIIbß3 activation, aggregation, α-granule secretion, and platelet spreading, mimicking the phenotype associated with RUNX1 variants in the clinical setting.

C3G contributes to platelet activation and aggregation by regulating major signaling pathways.

C3G is a GEF (guanine nucleotide exchange factor) for Rap GTPases, among which the isoform Rap1b is an essential protein in platelet biology. Using transgenic mouse models with platelet-specific overexpression of C3G or mutant C3GΔCat, we have unveiled a new function of C3G in regulating the hemostatic function of platelets through its participation in the thrombin-PKC-Rap1b pathway. C3G also plays important roles in angiogenesis, tumor growth, and metastasis through its regulation of the platelet secretome. In addition, C3G contributes to megakaryopoiesis and thrombopoiesis. Here, we used a platelet-specific C3G-KO mouse model to further support the role of C3G in hemostasis. C3G-KO platelets showed a significant delay in platelet activation and aggregation as a consequence of the defective activation of Rap1, which resulted in decreased thrombus formation in vivo. Additionally, we explored the contribution of C3G-Rap1b to platelet signaling pathways triggered by thrombin, PMA or ADP, in the referenced transgenic mouse model, through the use of a battery of specific inhibitors. We found that platelet C3G is phosphorylated at Tyr504 by a mechanism involving PKC-Src. This phosphorylation was shown to be positively regulated by ERKs through their inhibition of the tyrosine phosphatase Shp2. Moreover, C3G participates in the ADP-P2Y12-PI3K-Rap1b pathway and is a mediator of thrombin-TXA2 activities. However, it inhibits the synthesis of TXA2 through cPLA2 regulation. Taken together, our data reveal the critical role of C3G in the main pathways leading to platelet activation and aggregation through the regulation of Rap1b.

C3G, through its GEF activity, induces megakaryocytic differentiation and proplatelet formation.

BACKGROUND: Megakaryopoiesis allows platelet formation, which is necessary for coagulation, also playing an important role in different pathologies. However, this process remains to be fully characterized. C3G, an activator of Rap1 GTPases, is involved in platelet activation and regulates several differentiation processes. METHODS: We evaluated C3G function in megakaryopoiesis using transgenic mouse models where C3G and C3GΔCat (mutant lacking the GEF domain) transgenes are expressed exclusively in megakaryocytes and platelets. In addition, we used different clones of K562, HEL and DAMI cell lines with overexpression or silencing of C3G or GATA-1. RESULTS: We found that C3G participates in the differentiation of immature hematopoietic cells to megakaryocytes. Accordingly, bone marrow cells from transgenic C3G, but not those from transgenic C3GΔCat mice, showed increased expression of the differentiation markers CD41 and CD61, upon thrombopoietin treatment. Furthermore, C3G overexpression increased the number of CD41+ megakaryocytes with high DNA content. These results are supported by data obtained in the different models of megakaryocytic cell lines. In addition, it was uncovered GATA-1 as a positive regulator of C3G expression. Moreover, C3G transgenic megakaryocytes from fresh bone marrow explants showed increased migration from the osteoblastic to the vascular niche and an enhanced ability to form proplatelets. Although the transgenic expression of C3G in platelets did not alter basal platelet counts, it did increase slightly those induced by TPO injection in vivo. Moreover, platelet C3G induced adipogenesis in the bone marrow under pathological conditions. CONCLUSIONS: All these data indicate that C3G plays a significant role in different steps of megakaryopoiesis, acting through a mechanism dependent on its GEF activity.