Thromboinflammation has become a topic of key interest in cardiovascular disease and the prevention of diabetes complications because of the interplay between thrombosis and inflammation in diabetes. Specifically, the significant risk of vascular thrombotic disease in diabetes highlights the need for new and better therapeutic targets to help manage and prevent vascular thrombo‐occlusive disease in this condition. Similarly, the prominent role of inflammation in diabetes has sparked interest in anti‐inflammatory agents to better prevent and control vascular disease. Investigations on the effects of anticoagulation and antiplatelet interventions in patients with diabetes and cardiovascular disease show a potential role for these agents in decreasing morbidity and mortality. Neutrophils and platelets are key players in inflammation and wound‐healing response, respectively. The interaction between neutrophils and platelets is thought to be an important driver of thromboinflammation. Therefore, this review describes the mechanisms involved in platelet–neutrophil interactions that contribute to the development or exacerbation of thromboinflammation in the context of diabetes and its associated comorbidities. The effects observed by the antithrombotic/antidiabetic treatments and physical activity/dietary interventions on attenuating thromboinflammation are discussed. These data suggest that mechanisms involved in platelet–neutrophil interaction, platelet activation/aggregation, and the recruitment of neutrophils have a promising potential to become therapeutic targets to decrease thromboinflammation in patients with diabetes.

Objective: GPVI (glycoprotein VI) is a key molecular player in collagen-induced platelet signaling and aggregation. Recent evidence indicates that it also plays important role in platelet aggregation and thrombus growth through interaction with fibrin(ogen). However, there are discrepancies in the literature regarding whether the monomeric or dimeric form of GPVI binds to fibrinogen at high affinity. The mechanisms of interaction are also not clear, including which region of fibrinogen is responsible for GPVI binding. We aimed to gain further understanding of the mechanisms of interaction at molecular level and to identify the regions on fibrinogen important for GPVI binding. Approach and Results: Using multiple surface- and solution-based protein-protein interaction methods, we observe that dimeric GPVI binds to fibrinogen with much higher affinity and has a slower dissociation rate constant than the monomer due to avidity effects. Moreover, our data show that the highest affinity interaction of GPVI is with the αC-region of fibrinogen. We further show that GPVI interacts with immobilized fibrinogen and fibrin variants at a similar level, including a nonpolymerizing fibrin variant, suggesting that GPVI binding is independent of fibrin polymerization. Conclusions: Based on the above findings, we conclude that the higher affinity of dimeric GPVI over the monomer for fibrinogen interaction is achieved by avidity. The αC-region of fibrinogen appears essential for GPVI binding. We propose that fibrin polymerization into fibers during coagulation will cluster GPVI through its αC-region, leading to downstream signaling, further activation of platelets, and potentially stimulating clot growth. Graphic A graphic abstract is available for this article.

Background Abnormal clot structure has been identified in patients with thrombotic disorders. Anticoagulant therapy offers clear benefits for thrombosis prevention and treatment by reducing blood clot formation and size; nevertheless, there are limited data on the effects of different anticoagulants, where clotting is initiated with different triggers, on clot structure. Objectives Our aim was to investigate the effects of vitamin K antagonists and factor Xa inhibitors on clot structure. Methods Clots from pooled plasma spiked with rivaroxaban, apixaban, or enoxaparin, as well as plasma from patients on warfarin, were compared to plasma without anticoagulation. The kinetic profile of polymerizing clots was obtained by turbidity, fiber density was determined by confocal microscopy, clot pore size was investigated by permeation, and fiber size was analyzed using scanning electron microscopy. Clotting agonist was either tissue factor or thrombin. Results Following clotting with tissue factor, all anticoagulated clots had a significantly increased lag time, with the exception of enoxaparin. Rivaroxaban additionally led to significantly less dense and more permeable clots, with thicker fibers. In contrast, turbidity analysis following initiation with thrombin showed few effects of anticoagulation, with only enoxaparin leading to a prolonged lag time. Enoxaparin clots made with thrombin were less dense and more permeable. Conclusion Our results show that anticoagulants modulate clot structure particularly when induced by tissue factor, most likely due to reduction of thrombin generation. We propose that the effects of different anticoagulants could be assessed with a global clot structure measurement such as permeation or turbidity, providing information on clot phenotype.

Glycoprotein VI (GPVI) plays a key role in collagen-induced platelet aggregation. Affimers are engineered binding protein alternatives to antibodies. We screened and characterized GPVI-binding Affimers as novel tools to probe GPVI function. Among the positive clones, M17, D22, and D18 bound GPVI with the highest affinities (dissociation constant (KD) in the nanomolar range). These Affimers inhibited GPVI-collagen-related peptide (CRP)-XL/collagen interactions, CRP-XL/collagen-induced platelet aggregation, and D22 also inhibited in vitro thrombus formation on a collagen surface under flow. D18 bound GPVI dimer but not monomer. GPVI binding was increased for D18 but not M17/D22 upon platelet activation by CRP-XL and adenosine 5′-diphosphate. D22 but not M17/D18 displaced nanobody 2 (Nb2) binding to GPVI, indicating similar epitopes for D22 with Nb2 but not for M17/D18. Mapping of binding sites revealed that D22 binds a site that overlaps with Nb2 on the D1 domain, whereas M17 targets a site on the D2 domain, overlapping in part with the glenzocimab binding site, a humanized GPVI antibody fragment antigen-binding fragment. D18 targets a new region on the D2 domain. We found that D18 is a stable noncovalent dimer and forms a stable complex with dimeric GPVI with 1:1 stoichiometry. Taken together, our data demonstrate that Affimers modulate GPVI-ligand interactions and bind different sites on GPVI D1/D2 domains. D18 is dimer-specific and could be used as a tool to detect GPVI dimerization or clustering in platelets. A dimeric epitope regulating ligand binding was identified on the GPVI D2 domain, which could be used for the development of novel bivalent antithrombotic agents selectively targeting GPVI dimer on platelets.

Background: Polyphenols have been shown to decrease oxidative stress and modulate glycemic response. Nevertheless, their effect on platelet bioenergetics and clot structure in diabetes and hyperglycemia is unknown. Objectives: To investigate the effect of polyphenols on human platelet bioenergetics and its subsequent effect on clot structure in normoglycemia vs acute hyperglycemia in vitro. Methods: Four polyphenols (resveratrol, hesperetin, epigallocatechin gallate [EGCG], and quercetin) were selected for initial analysis. Healthy volunteers' isolated platelets/ platelet-rich plasma were treated with 5 or 25 mM glucose to represent normoglycemia and acute hyperglycemia, respectively. Platelet-derived reactive oxygen species (ROS), citrate synthase activity (mitochondrial density), mitochondrial calcium flux, and mitochondrial respiration were performed following exposure to polyphenols (20 mM, 1 hour) to determine their effects on platelet bioenergetics. Procoagulant platelets (annexin V) and fibrin fiber density (Alexa Fluor-488 fibrinogen; Invitrogen) were analyzed by laser scanning confocal microscopy, while clot porosity was determined using platelet-rich plasma following exposure to polyphenols (20 mM, 20 minutes). Results: Acute hyperglycemia increased ROS, mitochondrial calcium flux, maximal respiration, and procoagulant platelet number. Resveratrol, quercetin, and EGCG reduced platelet ROS in normoglycemic and acute hyperglycemic conditions. Mitochondrial density was decreased by quercetin and EGCG in normoglycemia. Resveratrol and EGCG reduced mitochondrial calcium flux in acute hyperglycemia. Resveratrol also decreased procoagulant platelet number and attenuated oxygen consumption rate in normoglycemia and acute hyperglycemia. No effect of hyperglycemia or polyphenols was observed on fibrin fiber density or clot pore size. Conclusion: Our results suggest polyphenols attenuate increased platelet activity stemming from hyperglycemia and may benefit thrombosis-preventative strategies in patients with diabetes.