Emerging evidence implicates platelets as key mediators of venous thromboembolism (VTE). Nevertheless, the pathways by which platelets and circulating procoagulant proteins synergistically orchestrate VTE remain incompletely understood. We prospectively determined whether activated platelets and systemic procoagulant factors were associated with VTE in 32 older orthopedic surgery patients. Circulating platelet-monocyte aggregates (PMAs), p-selectin expression (P-SEL), and integrin αIIbβ3 activation (PAC-1 binding) were assessed pre-operatively and 24 hours post-operatively. The proinflammatory and procoagulant molecule C-reactive protein (CRP), which induces PMA formation in vitro, along with plasma d-dimer and fibrinogen levels were also measured. The primary outcome was VTE occurring within 30 days post-operatively. Overall, 40.6% of patients developed VTE. Patients with VTE had a significant increase in circulating PMAs and CRP post-operatively, compared to those without VTE. Changes in PMA and CRP in VTE patients were significantly correlated (r2 = 0.536, p = 0.004). In contrast, P-SEL expression and PAC-1 binding, fibrinogen levels, and d-dimers were not associated with VTE. This is the first study to identify that increased circulating PMAs and CRP levels are early markers associated with post-surgical VTE. Our findings also provide new clinical evidence supporting the interplay between PMAs and CRP in patients with VTE.

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Dysregulated inflammation is a central component of wound healing following surgery. We prospectively enrolled older patients (n ¼ 25, age 65 + 7 years) undergoing elective total knee arthroplasty or total hip arthroplasty secondary to advanced osteoarthritis (OA) and healthy controls (n ¼ 48). Inflammatory, proangiogenic (vascular endothelial growth factor [VEGF], monocyte chemoattractant protein-1 [MCP-1], and interleukin-8 [IL-8]), and antiangiogenic (interferon g [IFN-g] and IL-4) factors were measured using a high-sensitivity biochip. Patients with OA had significantly higher baseline VEGF (10.5 + 1.2 pg/mL vs 4.8 + 0.2 pg/mL, P < .001), MCP-1 (130.6 + 7.7 pg/mL vs 88.6 + 3.9 pg/mL, P < .0001), and IL-8 (4.0 + 0.5 pg/mL vs 2.6 + 0.1 pg/mL, P < .05). Postoperatively, the levels of VEGF (10.5 + 1.2 pg/mL vs 18.8 + 1.5 pg/mL, P < .001) and MCP-1 (130.6 + 7.7 pg/mL vs 153.1 + 11.5 pg/mL, P < .05) increased significantly. Baseline and postoperative MCP-1 levels correlated positively and significantly with age. The levels of IFN-g and IL-4 (which has anti-inflammatory properties) did not significantly differ at baseline in patients with OA compared to controls and did not significantly rise postoperatively. We conclude that systemic levels of proinflammatory and angiogenic proteins are increased in patients with OA and rise further postoperatively, while proteins that restrain inflammation and angiogenesis do not coordinately rise. These findings implicate imbalance in inflammatory pathways in OA that may contribute to its pathobiology.

Aging and inflammation are independent risk factors for thrombosis and for the development of cardiovascular disease (CVD). Both conditions are characterized by varying degrees of chronic inflammation, mostly due to elevated levels of proinflammatory cytokines such as TNF-α, IL-1ß and IL-6. Furthermore, a significant proportion of thrombotic events associated with these conditions contribute to the elevated morbidity and mortality in this age group. With an expected significant rise in the proportion of older adults over 65 years of age within the next 30 years, it is important to identify the mechanisms by which aging-associated inflammation promotes thrombosis and worsens CVD. Our work has focused on the identification of key aging-associated factors that impact platelet development and function and that result in platelet hyperreactivity. We hypothesize that aging-associated inflammation promotes megakaryocyte reprogramming and results in the development of platelet hyperreactivity increasing the thrombotic risk during aging. Functional characterization of platelets by flow cytometry and microfluidic assay from young (mean age 35± 5 years of age) and aged frail adults (mean age 78±8) as well as from young (2 months) and old (>18 months) mice showed that platelets from old mice and humans are more reactive, demonstrated by increased aIIbb3 activation and phosphatidylserine exposure, and form larger thrombi under flow conditions when compared to platelets from young mice and human. Moreover, this platelet hyperreactive phenotype can be promoted in young mice after daily injections of TNF-α. In addition, TNF-α receptors I and II deficient mice (p55/p65 KO) do not develop platelet hyperreactivity after being injected with TNF-α, suggesting that sustained inflammation by TNF-α is a key promoter of platelet hyperreactivity in acute and chronic TNF-driven inflammation. Similarly, age-associated platelet hyperreactivity in old mice can be abrogated by the use of anti-TNF-α antibodies in vivo. Single-cell RNA-seq analysis of native megakaryocytes from young and old mice revealed significant differences in metabolic and mitochondrial gene pathways. Top differentially regulated pathways between young and old megakaryocytes are: a) protein ubiquitination; b) mitochondrial dysfunction and c) oxidative phosphorylation. Further investigations showed that platelets from old mice exhibit significant mitochondrial dysfunction characterized by elevated mitochondrial mass and oxygen consumption during activation. Finally, we show that platelets from patients with myeloproliferative neoplasms (MPN), characterized by somatic mutations that favor clonal hematopoiesis, elevated TNF-α levels, increased incidence of thrombo-hemorrhagic events and often, thrombocytosis, exhibit a similar mitochondrial phenotype as the one observed in aged murine platelets, suggesting that the high TNF-α levels associated with MPNs may play a significant role in the previously described platelet hyperreactivity of MPN. Disclosures No relevant conflicts of interest to declare.