Microaggressions in veterinary communication: what are they? How are they harmful? What can veterinary professionals and educators do?

Trusting relationships between veterinary professionals and clients are important for the well-being of people and the ultimate health of their animals. Yet, microaggressions pose a threat to these relationships. Defined as slights or indignities wielded against people with marginalized identities, microaggressions inflict a unique form of harm that reaffirms negative stereotypes enmeshed in systems of racism, sexism, classism, and beyond. In this article, we explore how microaggressions work and how they are applicable in veterinary settings. We also offer initial suggestions for veterinary professionals and educators to better understand and counteract their damage in the profession.

Kynurenine 3-Monooxygenase Interacts with Huntingtin at the Outer Mitochondrial Membrane.

The flavoprotein kynurenine 3-monooxygenase (KMO) is localised to the outer mitochondrial membrane and catalyses the synthesis of 3-hydroxykynurenine from L-kynurenine, a key step in the kynurenine pathway (KP) of tryptophan degradation. Perturbation of KP metabolism due to inflammation has long been associated with the pathogenesis of several neurodegenerative disorders, including Huntington's disease (HD)-which is caused by the expansion of a polyglutamine stretch in the huntingtin (HTT) protein. While HTT is primarily localised to the cytoplasm, it also associates with mitochondria, where it may physically interact with KMO. In order to test this hypothesis, we employed bimolecular fluorescence complementation (BiFC) and found that KMO physically interacts with soluble HTT exon 1 protein fragment in living cells. Notably, expansion of the disease-causing polyglutamine tract in HTT leads to the formation of proteinaceous intracellular inclusions that disrupt this interaction with KMO, markedly decreasing BiFC efficiency. Using confocal microscopy and ultrastructural analysis, we determined KMO and HTT localisation within the cell and found that the KMO-HTT interaction is localized to the outer mitochondrial membrane. These data suggest that KMO may interact with a pool of HTT at the mitochondrial membrane, highlighting a possible physiological role for mitochondrial HTT. The KMO-HTT interaction is abrogated upon polyglutamine expansion, which may indicate a heretofore unrecognized relevance in the pathogenesis of this disorder.

Does hsa-miR-223-3p from platelet-derived extracellular vesicles regulate tissue factor expression in monocytic cells?

Extracellular vesicles (EVs) released from activated platelets contain microRNAs, the most abundant of which is hsa-miR-223-3p. Endogenous hsa-miR-223-3p suppresses the expression of tissue factor (TF), the initiator of the extrinsic coagulation pathway, in endothelial cells. Monocytes can be induced to express TF to enhance coagulation, but the role of hsa-miR-223-3p in regulating monocyte TF remains unknown. This study examined whether hsa-miR-223-3p from platelet-derived EVs (pdEVs) affects TF expression in monocytes. THP-1 cells, differentiated into a monocyte-like phenotype with 1α,25-dihydroxyvitaminD3, were transfected with hsa-miR-223-3p mimic or control microRNA. Alternatively, THP-1 cells were incubated with pdEVs from PAR1-agonist peptide activated-platelets, as platelet releasate, or pdEVs isolated by ultracentrifugation. Transfection with hsa-miR-223-3p mimic resulted in significant reductions in TF protein, determined by western blotting and flow cytometry and reduced procoagulant activity, measured by a TF-specific factor Xa generation assay, compared to cells transfected with control microRNA. This reduction was reversed by co-transfection with hsa-miR-223-3p inhibitor, AntagomiR-223. Incubation of THP-1 cells with pdEVs also decreased TF expression; however, this was not reversed by AntagomiR-223. Taken together, monocyte TF expression is downregulated by hsa-miR-223-3p, but when transferred via pdEVs the effect was not reversed with Antagomir-223, suggesting other pdEV components may contribute to TF regulation.Abbreviations: Tissue factor (TF), Factor VII (FVII), activated Factor VII (FVIIa), Factor X (FX), activated Factor X (FXa), extracellular vesicles (EVs), microvesicles (MVs), platelet-derived extracellular vesicles (pdEVs), protease-activated receptor 1 agonist peptide (PAR1-AP), lipopolysaccharide (LPS), P-selectin glycoprotein ligand-1 (PSGL-1), Tris-Buffered Saline Tween (TBST), room temperature (RT)[Figure: see text].

Inflammation control and improvement of cognitive function in COVID-19 infections: is there a role for kynurenine 3-monooxygenase inhibition?

The novel respiratory virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), emerged during late 2019 and spread rapidly across the world. It is now recognised that the nervous system can be affected in COVID-19, with several studies reporting long-term cognitive problems in patients. The metabolic pathway of tryptophan degradation, known as the kynurenine pathway (KP), is significantly activated in patients with COVID-19. KP metabolites have roles in regulating both inflammatory/immune responses and neurological functions. In this review, we speculate on the effects of KP activation in patients with COVID-19, and how modulation of this pathway might impact inflammation and reduce neurological symptoms.

Advantages of brain penetrating inhibitors of kynurenine-3-monooxygenase for treatment of neurodegenerative diseases.

Kynurenine-3-monooxygenase (KMO) is an important therapeutic target for several brain disorders that has been extensively studied in recent years. Potent inhibitors towards KMO have been developed and tested within different disease models, showing great therapeutic potential, especially in models of neurodegenerative disease. The inhibition of KMO reduces the production of downstream toxic kynurenine pathway metabolites and shifts the flux to the formation of the neuroprotectant kynurenic acid. However, the efficacy of KMO inhibitors in neurodegenerative disease has been limited by their poor brain permeability. Combined with virtual screening and prodrug strategies, a novel brain penetrating KMO inhibitor has been developed which dramatically decreases neurotoxic metabolites. This review highlights the importance of KMO as a drug target in neurological disease and the benefits of brain permeable inhibitors in modulating kynurenine pathway metabolites in the central nervous system.

The Ratio of Factor VIIa:Tissue Factor Content within Microvesicles Determines the Differential Influence on Endothelial Cells.

Tissue factor (TF)-positive microvesicles from various sources can promote cellular proliferation or alternatively induce apoptosis, but the determining factors are unknown. In this study the hypothesis that the ratio of fVIIa:TF within microvesicles determines this outcome was examined. Microvesicles were isolated from HepG2, BxPC-3, 786-O, MDA-MB-231, and MCF-7 cell lines and microvesicle-associated fVIIa and TF antigen and activity levels were measured. Human coronary artery endothelial cells (HCAECs) were incubated with these purified microvesicles, or with combinations of fVIIa-recombinant TF, and cell proliferation/apoptosis was measured. Additionally, by expressing mCherry-PAR2 on HCAEC surface, PAR2 activation was quantified. Finally, the activation of PAR2 on HCAEC or the activities of TF and fVIIa in microvesicles were blocked prior to addition of microvesicles to cells. The purified microvesicles exhibited a range of fVIIa:TF ratios with HepG2 and 786-O cells having the highest (54:1) and lowest (10:1) ratios, respectively. The reversal from proapoptotic to proliferative was estimated to occur at a fVIIa:TF molar ratio of 15:1, but HCAEC could not be rescued at higher TF concentrations. The purified microvesicles induced HCAEC proliferation or apoptosis according to this ruling. Blocking PAR2 activation on HCAEC, or inhibiting fVIIa or TF-procoagulant function on microvesicles prevented the influence on HCAEC. Finally, incubation of HCAEC with recombinant TF resulted in increased surface exposure of fVII. The induction of cell proliferation or apoptosis by TF-positive microvesicles is dependent on the ratio of fVIIa:TF and involves the activation of PAR2. At lower TF concentrations, fVIIa can counteract the proapoptotic stimulus and induce proliferation.

Alteration in endothelial permeability occurs in response to the activation of PAR2 by factor Xa but not directly by the TF-factor VIIa complex.

Alterations in the endothelial permeability occur in response to the activation of coagulation mechanisms in order to control clot formation. The activation of the protease activated receptors (PAR) can induce signals that regulate such cellular responses. PAR2 is a target for the coagulation factor Xa (fXa) and tissue factor-factor VIIa (TF-fVIIa) complex. By measuring the permeability of dextran blue across endothelial monolayer, we examined the mechanisms linking coagulation and endothelial permeability. Activation of PAR2 using the agonist peptide (PAR2-AP) resulted in increased permeability across the monolayer and was comparable to that obtained with VEGF at 60 min. Incubation of cells with activated factor Xa (fXa) resulted in an initial decrease in permeability by 30 min, but then significantly increased at 60 min. These responses required fXa activity, and were abrogated by incubation of the cells with a PAR2-blocking antibody (SAM11). Activation of PAR2 alone, or inhibition of PAR1, abrogated the initial reduction in permeability. Additionally, inclusion of Rivaroxaban (0.6 µg/ml) significantly inhibited the response to fXa. Finally, incubation of the endothelial monolayers up to 2 h with TF-containing microvesicles derived from MDA-MB-231 cells, in the presence or absence of fVIIa, did not influence the permeability across the monolayers. In conclusion, fXa but not TF-fVIIa is a noteworthy mediator of endothelial permeability. The rapid initial decrease in permeability requires PAR2 and PAR1 which may act to constrain bleeding. The longer-term response is mediated by PAR2 with increased permeability, presumably to enhance clot formation at the site of damage.

Peptidyl-prolyl isomerase 1 (Pin1) preserves the phosphorylation state of tissue factor and prolongs its release within microvesicles.

The exposure and release of TF is regulated by post-translational modifications of its cytoplasmic domain. Here, the potential of Pin1 to interact with the cytoplasmic domain of TF, and the outcome on TF function was examined. MDA-MB-231 and transfected-primary endothelial cells were incubated with either Pin1 deactivator Juglone, or its control Plumbagin, as well as transfected with Pin1-specific or control siRNA. TF release into microvesicles following activation, and also phosphorylation and ubiquitination states of cellular-TF were then assessed. Furthermore, the ability of Pin1 to bind wild-type and mutant forms of overexpressed TF-tGFP was investigated by co-immunoprecipitation. Additionally, the ability of recombinant or cellular Pin1 to bind to peptides of the C-terminus of TF, synthesised in different phosphorylation states was examined by binding assays and spectroscopically. Finally, the influence of recombinant Pin1 on the ubiquitination and dephosphorylation of the TF-peptides was examined. Pre-incubation of Pin1 with Juglone but not Plumbagin, reduced TF release as microvesicles and was also achievable following transfection with Pin1-siRNA. This was concurrent with early ubiquitination and dephosphorylation of cellular TF at Ser253. Pin1 co-immunoprecipitated with overexpressed wild-type TF-tGFP but not Ser258→Ala or Pro259→Ala substituted mutants. Pin1 did interact with Ser258-phosphorylated and double-phosphorylated TF-peptides, with the former having higher affinity. Finally, recombinant Pin1 was capable of interfering with the ubiquitination and dephosphorylation of TF-derived peptides. In conclusion, Pin1 is a fast-acting enzyme which may be utilised by cells to protect the phosphorylation state of TF in activated cells prolonging TF activity and release, and therefore ensuring adequate haemostasis.

Investigation of the Filamin A-Dependent Mechanisms of Tissue Factor Incorporation into Microvesicles.

We have previously shown that phosphorylation of tissue factor (TF) at Ser253 increases the incorporation of TF into microvesicles (MVs) following protease-activated receptor 2 (PAR2) activation through a process involving filamin A, whereas phosphorylation of TF at Ser258 suppresses this process. Here, we examined the contribution of the individual phosphorylation of these serine residues to the interaction between filamin A and TF, and further examined how filamin A regulates the incorporation of TF into MVs. In vitro binding assays using recombinant filamin A C-terminal repeats 22-24 with biotinylated phospho-TF cytoplasmic domain peptides as bait showed that filamin A had the highest binding affinities for phospho-Ser253 and double-phosphorylated TF peptides, while the phospho-Ser258 TF peptide had the lowest affinity. Analysis of MDA-MB-231 cells using an in situ proximity ligation assay revealed increased proximity between the C-terminus of filamin A and TF following PAR2 activation, which was concurrent with Ser253 phosphorylation and TF-positive MV release from these cells. Knock-down of filamin A expression suppressed PAR2-mediated increases in cell surface TF procoagulant activity without reducing cell surface TF antigen expression. Disrupting lipid rafts by pre-incubation with methyl-ß-cyclodextrin prior to PAR2 activation reduced TF-positive MV release and cell surface TF procoagulant activity to the same extent as filamin A knock-down. In conclusion, this study shows that the interaction between TF and filamin A is dependent on the differential phosphorylation of Ser253 and Ser258. Furthermore, the interaction of TF with filamin A may translocate cell surface TF to cholesterol-rich lipid rafts, increasing cell surface TF activity as well as TF incorporation and release into MVs.

Comparison of tissue factor expression and activity in foetal and adult endothelial cells.

: Tissue factor (TF) is not usually expressed by endothelial cells but can be induced in these cells by inflammatory cytokines. Many studies have used human umbilical vein endothelial cells (HUVEC) as a model to examine the regulation of TF expression. However, there is a question as to whether this reflects TF expression in adult endothelial cells. This study compared TF expression and the release of TF-positive microvesicles in HUVEC and adult human dermal blood endothelial cells (HDBEC) in response to tumour necrosis factor α (TNFα) and interleukin-1 ß (IL-1ß). Cells were treated with the inflammatory cytokines and TF mRNA and total protein expression was examined by real-time RT-PCR and TF ELISA. Cell surface TF activity was measured in the calibrated automated thrombogram assay, as were microvesicle concentrations and microvesicle-associated TF activity. The TF antigen content of the microvesicles was determined by TF ELISA. Both HUVEC and HDBEC expressed increased levels of TF mRNA in response to TNFα and IL-1ß within 2 h. TF antigen expression increased in both cell types, reaching a maximum at 6 h, with HUVEC expressing significantly higher levels compared with HDBEC in response to TNFα. However, increases in TF-specific thrombin generation were similar on both HUVEC and HDBEC and both cell types also released comparable levels of TF-positive microvesicles. HUVEC and HDBEC respond similarly to TNFα and IL-1ß in terms of TF expression, and both are suitable models to examine cell surface TF activity and TF-positive microvesicle release in endothelial cells.

Oligoubiquitination of tissue factor on Lys255 promotes Ser253-dephosphorylation and terminates TF release.

Restriction of tissue factor (TF) activity at the cell surface and TF release are critical for prevention of excessive coagulation. This study examined the regulation of TF dephosphorylation and its release through ubiquitination. A plasmid containing the sequence to express the tandem protein TF-tGFP was mutated to include an arginine-substitution at Lys255 within TF. MDA-MB-231 cell line, and HCAEC endothelial cells were transfected and subsequently activated with PAR2-agonist peptide. The wild-type and mutant TF-tGFP were immunoprecipitated from the cell lysates and the ubiquitination and phosphorylation state of TF examined. Analysis of the proteins showed that arginine-substitution of Lys255 within TF prevented its ubiquitination while the wild-type TF-tGFP was oligoubiquitinated. The TF-associated oligoubiquitin chain was estimated to contain up to 4 ubiquitin units, with the linkage formed between Lys63 of one ubiquitin unit, and the C-terminus of the next unit. The Lys255→Arg substitution of TF-tGFP prolonged the phosphorylation of Ser253 within TF, compared to the wild-type TF-tGFP, lengthened the presence of TF-tGFP at the cell surface and extended the duration of TF-tGFP release from cells following PAR2 activation. A biotinylated 19-mer peptide corresponding to the C-terminus of TF (TFc) was used as substrate to show that the ubiquitination of TF was mediated by the Ube2D family of E2-enzymes and involved Mdm2. Moreover, double-phosphorylation of TFc was prerequisite for ubiquitination, with subsequent dephosphorylation of Ser253 by phosphatase PP2A. In conclusion, oligoubiquitination of Lys255 within TF permits PP2A to bind and dephosphorylate Ser253 and occurs to terminate TF release and contain its activity.

Analysis of the potential of cancer cell lines to release tissue factor-containing microvesicles: correlation with tissue factor and PAR2 expression.

BACKGROUND: Despite the association of cancer-derived circulating tissue factor (TF)-containing microvesicles and hypercoagulable state, correlations with the incidence of thrombosis remain unclear. METHODS: In this study the upregulation of TF release upon activation of various cancer cell lines, and the correlation with TF and PAR2 expression and/or activity was examined. Microvesicle release was induced by PAR2 activation in seventeen cell lines and released microvesicle density, microvesicle-associated TF activity, and phoshpatidylserine-mediated activity were measured. The time-course for TF release was monitored over 90 min in each cell line. In addition, TF mRNA expression, cellular TF protein and cell-surface TF activities were quantified. Moreover, the relative expression of PAR2 mRNA and cellular protein were analysed. Any correlations between the above parameters were examined by determining the Pearson's correlation coefficients. RESULTS: TF release as microvesicles peaked between 30-60 min post-activation in the majority of cell lines tested. The magnitude of the maximal TF release positively correlated with TF mRNA (c = 0.717; p < 0.001) and PAR2 mRNA (c = 0.770; p < 0.001) expressions while the percentage increase correlated with PAR2 mRNA (c = 0.601; p = 0.011) and protein (c = 0.714; p < 0.001). There was only a weak correlation between resting TF release, and microvesicle release. However, TF release in resting cells did not significantly correlate with any of the parameters examined. Furthermore, TF mRNA expression correlated with PAR2 mRNA expression (c = 0.745; p < 0.001). DISCUSSION AND CONCLUSIONS: In conclusion, our data suggest that TF and PAR2 mRNA, and PAR2 protein are better indicators of the ability of cancer cells to release TF and may constitute more accurate predictors of risk of thrombosis.

Zoledronic Acid-Induced Interface Dermatitis.

Zoledronic acid (ZA) is a bisphosphonate given intravenously, most commonly for the treatment of postmenopausal osteoporosis. Increase in usage of ZA because it was FDA-approved has resulted in increasing reports of side effects. For the most part, these are systemic. Cutaneous side effects associated with ZA are infrequent and limited to 2 reports of dermatomyositis to date. In both, patients presented with clinical and laboratory stigmata of dermatomyositis soon after initiation of therapy. In this report, we describe a 62-year-old woman who presented with diffuse, erythematous scaly plaques over the right thigh after 12 hours of infusion of ZA. Histopathologic examination of a skin biopsy from the right thigh revealed patchy scale crust containing neutrophils and inspissated serum, interface change with scattered individually necrotic keratinocytes, and a mild, superficial perivascular lymphocytic infiltrate with scattered eosinophils and pigment incontinence-findings consistent with an interface dermatitis. Given that the patient had no other systemic manifestations or laboratory abnormalities, to the best of our knowledge, ours is the first report of interface dermatitis secondary to ZA with the caveat that longer follow-up is required to definitively exclude the development of drug-induced connective tissue disease.

Culture Change to Promote Patient Engagement: It Isn't How It's Who.

Patient and family engagement is no longer a "why," "when," or even "how" conversation. So why are many health care organizations still struggling to embrace the patient as a partner? Now is the time to shift the conversation to a personal level.

Extracellular vesicles, tissue factor, cancer and thrombosis - discussion themes of the ISEV 2014 Educational Day.

Although the association between cancer and venous thromboembolism (VTE) has long been known, the mechanisms are poorly understood. Circulating tissue factor-bearing extracellular vesicles have been proposed as a possible explanation for the increased risk of VTE observed in some types of cancer. The International Society for Extracellular Vesicles (ISEV) and International Society on Thrombosis and Haemostasis (ISTH) held a joint Educational Day in April 2014 to discuss the latest developments in this field. This review discusses the themes of that event and the ISEV 2014 meeting that followed.

Characterization of physical properties of tissue factor-containing microvesicles and a comparison of ultracentrifuge-based recovery procedures.

Microvesicles were isolated from the conditioned media of 3 cell lines (MDA-MB-231, AsPC-1 and A375) by ultracentrifugation at a range of relative centrifugal forces, and the tissue factor (TF) protein and activity, microvesicle number, size distribution and relative density compared. Also, by expressing TF-tGFP in cells and isolating the microvesicles, the relative density of TF-containing microvesicles was established. Nanoparticle tracking analysis (NTA) indicated that the larger-diameter microvesicles (>200 nm) were primarily sedimented at 100,000g and possessed TF-dependent thrombin and factor Xa generation potential, while in the absence of factor VII, all microvesicles possessed some thrombin generation capacity. Immuno-precipitation of TF-containing microvesicles followed by NTA also indicated the range of these microvesicles to be 200-400 nm. Analysis of the microvesicles by gradient density centrifugation showed that lower-density (<1.1 g/ml) microvesicles were mainly present in the samples recovered at 100,000g and were associated with TF antigen and activity. Analysis of these fractions by NTA confirmed that these fractions were principally composed of the larger-diameter microvesicles. Similar analysis of microvesicles from healthy or patient plasma supported those obtained from conditioned media indicating that TF activity was mainly associated with lower-density microvesicles. Furthermore, centrifugation of healthy plasma, supplemented with TF-tGFP-containing microvesicles, resulted in 67% retrieval of the fluorescent microvesicles at 100,000g, but only 26% could be recovered at 20,000g. Pre-centrifugation of conditioned media or plasma at 10,000g improved the speed and yield of recovered TF-containing microvesicles by subsequent centrifugation at either 20,000g or 100,000g. In conclusion, TF appears to be associated with low-density (1.03-1.08 g/ml), larger-diameter (200-350 nm) microvesicles.

Filamin-A is required for the incorporation of tissue factor into cell-derived microvesicles.

We previously reported that the incorporation of tissue factor (TF) into cell-derived microvesicles (MVs) is regulated by the phosphorylation of the cytoplasmic domain of TF. Since the cytoskeletal protein filamin-A is known to bind to the cytoplasmic domain of TF in a phosphorylation-dependent manner, the involvement of filamin-A in the incorporation of TF into MVs was examined. Endothelial cells were transfected to express TF, whereas MDA-MB-231 cells were used to examine endogenously expressed TF. MV release was induced by activating protease-activated receptor-2 (PAR2). Partial suppression of filamin-A expression using two different filamin-A siRNA sequences resulted in significant reductions in the incorporation of TF antigen into MVs as determined by TF-ELISA and western blot analysis, and was reflected in reduced thrombin-generation and FXa-generation capacities of these MVs. Deletion of the cytoplasmic domain of TF also resulted in reduced incorporation of TF into MVs, whereas the suppression of filamin-A expression had no additional effect on the incorporation of truncated TF into MVs. Partial suppression of filamin-A expression had no effect on the number and size distribution of the released MVs. However, >90% suppression of filamin-A expression resulted in increased MV release, possibly as a result of increased instability of the plasma membrane and underlying cytoskeleton. In conclusion, the presence of filamin-A appears to be essential for the incorporation of TF into MVs following PAR2 activation, but is not required for the process of MV formation and release following PAR2 activation.

Randomized phase II study of sunitinib versus standard of care for patients with previously treated advanced triple-negative breast cancer.

PURPOSE: This randomized, open-label phase II study compared the efficacy of sunitinib monotherapy with that of single-agent standard-of-care (SOC) chemotherapy in patients with previously treated advanced triple-negative breast cancer (TNBC). METHODS: Patients with advanced TNBC, relapsed after anthracycline- and taxane-based chemotherapy, were randomized to receive either sunitinib (37.5 mg/day) or the investigator's choice of SOC therapy. Progression-free survival was the primary endpoint. RESULTS: Median progression-free survival was 2.0 months with sunitinib and 2.7 months with SOC chemotherapy (one-sided P = 0.888). Median overall survival was not prolonged with sunitinib (9.4 months) compared with SOC chemotherapy (10.5 months; one-sided P = 0.839). The objective response rate was 3% with sunitinib and 7% with SOC chemotherapy (one-sided P = 0.962). CONCLUSIONS: Sunitinib monotherapy did not improve efficacy compared with SOC chemotherapy in patients with previously treated advanced TNBC, for which identification of effective treatments and therapeutic targets remains an urgent need. TRIAL REGISTRATION: NCT00246571.

Enhanced binding of tissue factor-microparticles to collagen-IV and fibronectin leads to increased tissue factor activity in vitro.

The role of tissue factor (TF)-containing microparticles in clot propagation has been established, but the ability of circulating microparticles to initiate coagulation has been disputed. However, TF-bearing microparticles, particularly endothelial-microparticles generated during disease, may interact with extracellular matrices which in turn can localise circulating TF to sites of injury. In order to examine this hypothesis in vitro , microparticles were isolated from human coronary artery endothelial cells transfected to overexpress TF, tumour-necrosis factor (TNF) α-treated cells or non-transfected cells lacking TF. The ability of microparticles to bind collagen-IV, fibronectin and fibrin was examined under static conditions and arterial shear rates (650 s⁻¹), and also in the presence of inhibitory antibodies against ß1-, ß3-, α3- and αv-integrins or an anti-TF antibody. TF-microparticles showed increases of up to 43% and 24% in adherence to collagen-IV and fibronectin, respectively, compared to control microparticles under shear flow. Furthermore, TF-containing microparticles, but not the transfected parent cells had increased levels of ß1-integrin compared to TF-deficient microparticles. Pre-incubation of microparticles with a ß1-integrin-blocking antibody counteracted the additional adhesion of TF-microparticles compared to control microparticles. Finally, adherence of TF microparticles to collagen-IV or fibronectin resulted in increased TF activity by concentrating TF onto the surface. In conclusion, the presence of TF within microparticles enhances the interactions of endothelial cell-derived microparticles with extracellular matrices in an integrin-dependent manner. Accumulation and localisation of these microparticles in turn results in the enhancement of TF activity. This may be an innate mechanism by which TF-bearing microparticles induce coagulation upon vascular injury.

p38α phosphorylates serine 258 within the cytoplasmic domain of tissue factor and prevents its incorporation into cell-derived microparticles.

We previously showed that the phosphorylation of Ser253 within the cytoplasmic domain of human tissue factor (TF) initiates the incorporation and release of this protein into cell-derived microparticles. Furthermore, subsequent phosphorylation of Ser258 terminates this process. However, the identity of the kinase responsible for the phosphorylation of Ser258 and mode of action of this enzyme remain unknown. In this study, p38α was identified as the proline-directed kinase capable of phosphorylating Ser258 specifically, and without any detectable activity towards Ser253. Furthermore, using synthetic peptides, it was shown that the Km for the reaction decreased by approximately 10 fold on substitution of Ser253 with phospho-Ser253. Either inhibition of p38 using SB202190 or knockdown of p38α expression in coronary artery endothelial cells overexpressing wild-type TF, resulted in decreased phosphorylation of Ser258, following activation of cells with PAR2-agonist peptide (PAR2-AP). In agreement with our previous data, inhibition of phosphorylation of this residue maintained the release of TF. Activation of PAR2 in cells transfected to overexpress TF, resulted in two separate peaks of p38 activity at approximately 40 and 120 min post-activation. Furthermore, overexpression of Ala253-substituted TF enhanced the second p38 activation peak. However, the second peak was absent in cells devoid of TF or in cells overexpressing the Asp253-substituted TF. Our data clearly identifies p38α as a kinase capable of phosphorylating Ser258 within the cytoplasmic domain of TF. Moreover, it appears that the presence of TF within the cells regulates the late activation of p38 and consequently the termination of TF release into microparticles.