Objective: platelets possess not only haemostatic but also inflammatory properties, which combined are thought to play a detrimental role in thromboinflammatory diseases such as acute coronary syndromes and stroke. Phosphodiesterase (PDE) 3 and -5 inhibitors have demonstrated efficacy in secondary prevention of arterial thrombosis, partially mediated by their antiplatelet action. Yet it is unclear whether such inhibitors also affect platelets’ inflammatory functions. Here, we aimed to examine the effect of the PDE3A inhibitor cilostazol and the PDE5 inhibitor tadalafil on platelet function in various aspects of thromboinflammation. Approach and results: cilostazol, but not tadalafil, delayed ex vivo platelet-dependent fibrin formation under whole blood flow over type I collagen at 1000 s⁻¹. Similar results were obtained with blood from Pde3a deficient mice, indicating that cilostazol effects are mediated via PDE3A. Interestingly, cilostazol specifically reduced the release of phosphatidylserine-positive extracellular vesicles (EVs) from human platelets while not affecting total EV release. Both cilostazol and tadalafil reduced the interaction of human platelets with inflamed endothelium under arterial flow and the release of the chemokines CCL5 and CXCL4 from platelets. Moreover, cilostazol, but not tadalafil, reduced monocyte recruitment and platelet-monocyte interaction in vitro. Conclusions: this study demonstrated yet unrecognised roles for platelet PDE3A and platelet PDE5 in platelet procoagulant and proinflammatory responses.

The critical plasma anticoagulant protein S (PS) circulates in 2 functionally distinct pools: free (anticoagulant) or bound to complement component 4b-binding protein (C4BP; anti-inflammatory). Acquired free PS deficiency is detected in several viral infections, but its cause is unclear. Here, we used biochemical approaches and human patient plasma samples to identify an interaction between PS and von Willebrand factor (VWF), which causes free PS deficiency and reduced PS anticoagulant activity. We first identified a shear-dependent interaction between PS and VWF by mass spectrometry. Consistently, PS and VWF could be crosslinked together in plasma, and plasma PS and VWF comigrated in gel electrophoresis. The PS/VWF interaction was blocked by and tissue factor pathway inhibitor but not activated protein C, suggesting an interaction with the sex hormone binding globulin region of PS. Microfluidic systems demonstrated that PS stably binds VWF as VWF unfolds under turbulent flow. PS/VWF complexes also localized to platelet thrombi under laminar arterial flow. In thrombin generation-based assays, shearing plasma decreased PS activity, an effect not seen in the absence of VWF. Finally, free PS deficiency in patients with COVID-19 correlated with changes in VWF, but not C4BP, and with thrombin generation. Our data indicate that PS binds to a shear-exposed site on VWF, thus sequestering free PS and decreasing its anticoagulant activity, which would account for the increased thrombin generation potential. Because many viral infections present with free PS deficiency, elevated circulating VWF, and increased vascular shear, we propose that the PS/VWF interaction reported here is a likely contributor to virus-associated thrombotic risk.

The high prevalence of chronic wounds, i.e., 2.5-3% of the US population, causes a large social and financial burden. Physiological wound healing is a multi-step process that involves different cell types and growth factors. Platelet-rich plasma or platelet-derived factors have been used to accelerate wound repair, but their use has been controversial with mixed results. Thus, a detailed functional understanding of platelet functions in wound healing beyond hemostasis is needed. This study investigated the importance of platelet α-granule cargo packaging and endocytosis in a dorsal full-thickness excisional skin wound model using mice with defects in α-granule cargo packaging (Nbeal2 ^-/- mice) and endocytosis (platelet-specific Arf6 ^-/- and VAMP2/3 ^Δ mice). We found that proper kinetic and morphological healing of dorsal skin wounds in mice requires both de novo as well as endocytosed platelet α-granule cargo. Histological and morphometric analyses of cross-sectional wound sections illustrated that mice with defects in α-granule cargo packaging or platelet endocytosis had delayed (epi)dermal regeneration in both earlier and advanced healing. This was reflected by reductions in wound collagen and muscle/keratin content, delayed scab formation and/or resolution, re-epithelialization, and cell migration and proliferation. Molecular profiling analysis of wound extracts showed that the impact of platelet function extends beyond hemostasis to the inflammation, proliferation, and tissue remodeling phases via altered expression of several bioactive molecules, including IL-1β, VEGF, MMP-9, and TIMP-1. These findings provide a basis for advances in clinical wound care through a better understanding of key mechanistic processes and cellular interactions in (patho)physiological wound healing. Key points: De novo and endocytosed platelet α-granule cargo support physiological skin wound healing Platelet function in wound healing extends to the inflammation, proliferation, and tissue remodeling phases

Rubicon (RUN domain Beclin-1–interacting and cysteine-rich–containing protein) is a negative regulator of autophagy in nucleated cells; however, its role in platelets is unstudied. Here, we identify an autophagy-independent role for Rubicon in arterial thrombosis. Mice with a platelet/megakaryocyte-specific deletion of Rubicon (RUBCN−/−) showed normal circulating platelet counts, with a slight increase in platelet size. The basal levels of autophagy-related, vesicle-elongation proteins (ie, ATG5, ATG7, syntaxin 17, LC3, and Rab7) were unaffected, and there was no disruption in canonical platelet signaling pathways upon activation. Platelet secretion from all 3 granule classes remained intact. Aggregation/Agglutination in response to thrombin, convulxin, or botrocetin was unaffected. There was no overt defect in activation-dependent autophagic flux (ie, loss of LC3 or p62). However, RUBCN−/− mice had a significant thrombosis defect in the FeCl3-induced, carotid artery injury model, but no defect in the tail-transection hemostasis model. An intrinsic platelet defect was confirmed using microfluidics, in which RUBCN−/− platelets had reduced collagen binding under flow at high shear flow but not low shear. RUBCN−/− platelets also showed impaired surface exposure of phosphatidylserine (PS) after thrombin and convulxin activation, though there was no effect on total anoctamin 6, which contributes to PS exposure. There was no effect on platelet endocytosis, and only modest reductions in mitochondrial membrane potential with a slight delay in clot contraction, suggesting that Rubicon contributes to late stages of platelet function. Collectively, our findings demonstrate that Rubicon contributes to thrombosis and procoagulant platelet formation in a manner that appears independent of classical autophagy.

Platelet-rich plasma or platelet-derived factors are often used to promote acute and chronic wound healing, an intricate, multistep process involving multiple cell types and factors. However, their use is controversial with mixed results, highlighting the need for detailed functional insights into how platelets affect wound healing beyond initial hemostasis. In this study, we investigated the importance of platelet function in physiological healing using a dorsal full-thickness excisional skin wound model in mice with defects in α-granule cargo packaging (Nbeal2−/− [neurobeachin-like 2 knockout] mice) and endocytic trafficking (platelet-specific Arf6−/− [ADP-ribosylation factor 6–deficient] and VAMP2/3Δ [vesicle-associated membrane protein 2/3–deficient] mice). Wound healing dynamics and skin structure, as established by histology, were significantly disrupted in all 3 mouse strains. Each showed unique kinetic, morphological, and structural healing patterns that differed from wild-type controls. Notably, Nbeal2−/− mice had delayed (epi)dermal regeneration, which was reflected by reductions in scab formation and/or resolution, re-epithelialization, and changes in cell migration and proliferation. In the platelet-specific endocytosis-deficient mice, Arf6−/− and VAMP2/3Δ, slower re-epithelialization was accompanied by defects in structural skin characteristics, including wound collagen and muscle/keratin content. Molecular profiling of bioactive peptides from skin (wound) extracts showed how modulating platelet α-granule cargo or endocytosis differentially affected healing, beyond initial hemostasis. Based on the changing profiles in the presence of several bioactive molecules (eg, interleukin-1β, vascular endothelial growth factor, matrix metalloproteinase 9, and tissue inhibitor of metalloproteinase 1), the inflammation, proliferation, and tissue remodeling phases all appear to be affected by platelet function(s). These findings provide a better understanding of platelets’ role in all wound healing phases, thereby advancing clinical wound care.

Protein S (PS), the critical plasma cofactor for the anticoagulants tissue factor (TF) pathway inhibitor (TFPI) and activated protein C (APC), circulates in two functionally distinct pools: free (anticoagulant) or bound to complement component 4b-binding protein (C4BP) (anti-inflammatory). Acquired free PS deficiency is detected in several viral infections, but its cause is unclear. Here, we identified a shear-dependent interaction between PS and von Willebrand Factor (VWF) by mass spectrometry. Consistently, plasma PS and VWF comigrated in both native and agarose gel electrophoresis. The PS/VWF interaction was blocked by TFPI but not APC, suggesting an interaction with the C-terminal sex hormone binding globulin (SHBG) region of PS. Microfluidic systems, mimicking arterial laminar flow or disrupted turbulent flow, demonstrated that PS stably binds VWF as VWF unfolds under turbulent flow. PS/VWF complexes also localized to platelet thrombi under laminar arterial flow. In thrombin generation-based assays, shearing plasma decreased PS activity, an effect not seen in the absence of VWF. Finally, free PS deficiency in COVID-19 patients, measured using an antibody that binds near the C4BP binding site in SHBG, correlated with changes in VWF, but not C4BP, and with thrombin generation. Our data suggest that PS binds to a shear-exposed site on VWF, thus sequestering free PS and decreasing its anticoagulant activity, which would account for the increased thrombin generation potential. As many viral infections present with free PS deficiency, elevated circulating VWF, and increased vascular shear, we propose that the PS/VWF interaction reported here is a likely contributor to virus-associated thrombotic risk. Key Points: Von Willebrand Factor (VWF) binds Protein S (PS) in a shear-dependent manner, reducing the free PS pool and its anticoagulant activity. The PS/VWF complex forms under turbulent flow conditions, is stable in whole blood, and localizes to growing platelet thrombi.