To test the hypothesis that skeletal muscle myosins can directly influence blood coagulation and thrombosis, ex vivo studies of the effects of myosin on thrombogenesis in fresh human blood were conducted. Addition of myosin to blood augmented the thrombotic responses of human blood flowing over collagen-coated surfaces (300 s shear rate). Perfusion of human blood over myosin-coated surfaces also caused fibrin and platelet deposition, evidencing myosin's thrombogenicity. Myosin markedly enhanced thrombin generation in both platelet-rich plasma and platelet-poor plasma, indicating that myosin promoted thrombin generation in plasma primarily independent of platelets. In purified reaction mixtures composed only of factor Xa, factor Va, prothrombin, and calcium ions, myosin greatly enhanced prothrombinase activity. The Gla domain of factor Xa was not required for myosin's prothrombinase enhancement. When binding of purified clotting factors to immobilized myosin was monitored using biolayer interferometry, factors Xa and Va each showed favorable binding interactions. Factor Va reduced by 100-fold the apparent of myosin for factor Xa ( ∼0.48 nM), primarily by reducing k, indicating formation of a stable ternary complex of myosin:Xa:Va. In studies to assess possible clinical relevance for this discovery, we found that antimyosin antibodies inhibited thrombin generation in acute trauma patient plasmas more than in control plasmas ( = .0004), implying myosin might contribute to acute trauma coagulopathy. We posit that myosin enhancement of thrombin generation could contribute either to promote hemostasis or to augment thrombosis risk with consequent implications for myosin's possible contributions to pathophysiology in the setting of acute injuries.

Receptor-induced NF-κB activation is controlled by NEMO, the NF-κB essential modulator. Hypomorphic NEMO mutations result in X-linked ectodermal dysplasia with anhidrosis and immunodeficiency, also referred to as NEMO syndrome. Here we describe a distinct group of patients with NEMO C-terminal deletion (ΔCT-NEMO) mutations. Individuals harboring these mutations develop inflammatory skin and intestinal disease in addition to ectodermal dysplasia with anhidrosis and immunodeficiency. Both primary cells from these patients, as well as reconstituted cell lines with this deletion, exhibited increased IκB kinase (IKK) activity and production of proinflammatory cytokines. Unlike previously described loss-of-function mutations, ΔCT-NEMO mutants promoted increased NF-κB activation in response to TNF and Toll-like receptor stimulation. Investigation of the underlying mechanisms revealed impaired interactions with A20, a negative regulator of NF-κB activation, leading to prolonged accumulation of K63-ubiquitinated RIP within the TNFR1 signaling complex. Recruitment of A20 to the C-terminal domain of NEMO represents a novel mechanism limiting NF-κB activation by NEMO, and its absence results in autoinflammatory disease.ctivation of the NF-κB family of transcription factors is required for normal development, innate and adaptive immunity, and the inflammatory response (1, 2). NF-κB-induced transcription of proinflammatory cytokines and chemokines amplifies immune-response programs and stimulates recruitment of inflammatory cells. Transcriptional activity of the classic NF-κB p65/p50 complex is regulated by the inhibitor of NF-κB kinase (IKK), consisting of the α-and β-catalytic subunits and the NEMO (NF-κB essential modulator, IKKγ) regulatory subunit. IKK activity leads to phosphorylation and K48-linked polyubiquitination of IκBα, the inhibitor of NF-κB. Ubiquitinated IκBα is then rapidly degraded, allowing nuclear translocation of NF-κB subunits and gene transactivation. NEMO functions as a scaffold within the IKK complex that is required for canonical IKK enzymatic activity and NF-κB activation (3).Activation of TNF, IL-1R, and Toll-like receptor (TLR) family receptors leads to K63 and linear (also termed M1) ubiquitination of cytosolic adapter proteins, such as receptor interacting protein 1 (RIP1). This in turn promotes recruitment of the IKK complex to the receptor signaling complex (4-8). A well-known negative regulator of NF-κB activation is A20 (encoded by the gene TNFAIP3). In the TNF-R1 complex, A20 removes K63-linked ubiquitin modifications on RIP1 through its deubiquitinase activity and converts these to K48-linked polyubiquitin chains through its E3 ligase activity (9). Editing of polyubiquitin linkages at the receptor complex in this manner results in rapid degradation of RIP1 and other signaling proteins, such as TRAF6, TRAF2, cIAP1, and cIAP2 (9, 10), promoting the termination of receptor-induced NF-κB activation. In addition, A20 directly inhibits IKK activity independently of its deubiquitinase activity in...

Platelets engage cues of pending vascular injury through coordinated adhesion, secretion and aggregation responses. These rapid, progressive changes in platelet form and function are orchestrated downstream of specific receptors on the platelet cell surface, and through intracellular signaling mechanisms that remain systematically undefined. This study brings together cell physiological and phosphoproteomics methods incorporating peptide tandem mass tag (TMT) labeling, sample multiplexing, synchronous precursor selection (SPS) and triple stage tandem mass spectrometry (MS3) to profile signaling mechanisms downstream of the immunotyrosine activation motif (ITAM) platelet collagen receptor GPVI. Altogether, >3,000 significant (FDR<0.05) phosphorylation events on >1,300 proteins were detected in initial and progressing conditions of GPVI-mediated platelet activation. With literature-guided causal inference tools, >300 site-specific signaling relations were mapped from phosphoproteomics data among key and emerging GPVI effectors (i.e., FcRg, Syk, PLCg2, PKCd, DAPP1). Through signaling validation studies and functional screening, other less-characterized targets were also considered within the context of GPVI/ITAM pathways, including Ras/MAPK axis proteins (i.e., KSR1, SOS1, STAT1, Hsp27). Networks highly regulated in GPVI/ITAM signaling out of context of curated knowledge were also illuminated, including a system of >40 Rab GTPases and associated regulatory proteins - where TAK1-mediated Rab7 S72 phosphorylation associated with endolysosomal maturation and GPVI-mediated platelet function. In addition to serving as a model for generating and testing hypotheses from omics datasets, this study puts forth a means to identify hemostatic effectors, biomarkers and therapeutic targets relevant to thrombosis, vascular inflammation and other platelet-associated disease states.

Cancer-associated thrombosis is a common first presenting sign of malignancy and is currently the second leading cause of death in cancer patients after their malignancy. However, the molecular mechanisms underlying cancer-associated thrombosis remain undefined. In this study, we aimed to develop a better understanding of how cancer cells affect the coagulation cascade and platelet activation to induce a prothrombotic phenotype. Our results show that colon cancer cells trigger platelet activation in a manner dependent on cancer cell tissue factor (TF) expression, thrombin generation, activation of the protease-activated receptor 4 (PAR4) on platelets and consequent release of ADP and thromboxane A2. Platelet-colon cancer cell interactions potentiated the release of platelet-derived extracellular vesicles (EVs) rather than cancer cell-derived EVs. Our data show that single colon cancer cells were capable of recruiting and activating platelets and generating fibrin in plasma under shear flow. Finally, in a retrospective analysis of colon cancer patients, we found that the number of venous thromboembolism events was 4.5 times higher in colon cancer patients than in a control population. In conclusion, our data suggest that platelet-cancer cell interactions and perhaps platelet procoagulant EVs may contribute to the prothrombotic phenotype of colon cancer patients. Our work may provide rationale for targeting platelet-cancer cell interactions with PAR4 antagonists together with aspirin and/or ADP receptor antagonists as a potential intervention to limit cancer-associated thrombosis, balancing safety with efficacy.