Search

Platelets are critical mediators of hemostasis and thrombosis. Platelets circulate as discs in their resting form but change shape rapidly upon activation by vascular damage and/or soluble agonists such as thrombin. Platelet shape change is driven by a dynamic remodeling of the actin cytoskeleton. Actin filaments interact with the protein myosin, which is phosphorylated on the myosin light chain (MLC) upon platelet activation. Actin-myosin interactions trigger contraction of the actin cytoskeleton, which drives platelet spreading and contractile force generation. Filamin A (FLNA) is an actin cross-linking protein that stabilizes the attachment between subcortical actin filaments and the cell membrane. In addition, FLNA binds multiple proteins and serves as a critical intracellular signaling scaffold. Here, we used platelets from mice with a megakaryocyte/platelet-specific deletion of FLNA to investigate the role of FLNA in regulating platelet shape change. Relative to controls, FLNA-null platelets exhibited defects in stress fiber formation, contractile force generation, and MLC phosphorylation in response to thrombin stimulation. Blockade of Rho kinase (ROCK) and protein kinase C (PKC) with the inhibitors Y27632 and bisindolylmaleimide (BIM), respectively, also attenuated MLC phosphorylation; our data further indicate that ROCK and PKC promote MLC phosphorylation through independent pathways. Notably, the activity of both ROCK and PKC was diminished in the FLNA-deficient platelets. We conclude that FLNA regulates thrombin-induced MLC phosphorylation and platelet contraction, in a ROCK- and PKC-dependent manner. [ABSTRACT FROM AUTHOR]

Objective: To gain insights into how proteases signal to connective tissues cells in the periodontium. Background: The connective tissue degradation observed in periodontitis is largely due to matrix metalloproteinase (MMP) release by gingival fibroblasts. Granzyme B (GzmB) is a serine protease whose role in periodontitis is undefined. Methods: Human gingival crevicular fluid (GCF) samples were obtained from sites with periodontal disease and healthy control sites. GzmB was quantified in the GCF ([GzmB]GCF) by ELISA. Gingival fibroblasts (GF) were cultured in the presence or absence of recombinant GzmB. Culture supernatants were analyzed by ELISA to quantify GzmB‐induced release of interstitial collagenase (MMP‐1). In some experiments, cells were pre‐treated with the inhibitor PD98059 to block MEK/ERK signaling. The protease‐activated receptor‐1 (PAR‐1) was blocked with ATAP‐2 neutralizing antibody prior to GzmB stimulation. Systemic MMP‐1 levels were measured in plasma from wild‐type (WT) and granzyme‐B‐knockout (GzmB−/−) mice. Results: The [GzmB]GCF in human samples was ~4–5 fold higher at sites of periodontal disease (gingivitis/periodontitis) compared to healthy control sites, suggesting an association between GzmB and localized matrix degradation. GzmB induced a ~4–5‐fold increase in MMP‐1 secretion by cultured fibroblasts. GzmB induced phosphorylation of Erk1/2, which was abrogated by PD98059. GzmB‐induced upregulation of MMP‐1 secretion was also reduced by PD98059. Blockade of PAR‐1 function by ATAP‐2 abrogated the increase in MMP‐1 secretion by GF. Circulating MMP‐1 was similar in WT and GzmB−/− mice, suggesting that GzmB's effects on MMP‐1 release are not reflected systemically. Conclusion: These data point to a novel GzmB‐driven signaling pathway in fibroblasts in which MMP‐1 secretion is upregulated in a PAR1‐ and Erk1/2‐dependent manner. [ABSTRACT FROM AUTHOR]

Thrombosis, a key factor in most cardiovascular diseases, is a major contributor to human mortality. Existing antithrombotic agents carry a risk of bleeding. Consequently, there is a keen interest in discovering innovative antithrombotic agents that can prevent thrombosis without negatively impacting hemostasis. Platelets play crucial roles in both hemostasis and thrombosis. We have previously characterized calcium- and integrin-binding protein 1 (CIB1) as a key regulatory molecule that regulates platelet function. CIB1 interacts with several platelet proteins including integrin α IIb β 3 , the major glycoprotein receptor for fibrinogen on platelets. Given that CIB1 regulates platelet function through its interaction with α IIb β 3 , we developed a fluorescence polarization (FP) assay to screen for potential inhibitors. The assay was miniaturized to 1536-well and screened in quantitative high-throughput screening (qHTS) format against a diverse compound library of 14,782 compounds. After validation and selectivity testing using the FP assay, we identified 19 candidate inhibitors and validated them using an in-gel binding assay that monitors the interaction of CIB1 with α IIb cytoplasmic tail peptide, followed by testing of top hits by intrinsic tryptophan fluorescence (ITF) and microscale thermophoresis (MST) to ascertain their interaction with CIB1. Two of the validated hits shared similar chemical structures, suggesting a common mechanism of action. Docking studies further revealed promising interactions within the hydrophobic binding pocket of the target protein, particularly forming key hydrogen bonds with Ser180. The compounds exhibited a potent antiplatelet activity based on their inhibition of thrombin-induced human platelet aggregation, thus indicating that disruptors of the CIB1- α IIb β 3 interaction could carry a translational potential as antithrombotic agents., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)