A modified clot-based assay to measure negatively charged procoagulant phospholipids.

Growing evidence supports a role for extracellular vesicles (EVs) in haemostasis and thrombosis due to exposure of negatively charged procoagulant phospholipids (PPL). Current commercial PPL-dependent clotting assays use chemically phospholipid depleted plasma to measure PPL activity. The purpose of our study was to modify the PPL assay by substituting the chemically phospholipid depleted plasma with PPL depleted plasma obtained by ultracentrifugation This in order to get readily access to a sensitive and reliable assay to measure PPL activity in human plasma and cell supernatants. The performance of the assay was tested, including the influence of individual coagulation factors and postprandial lipoproteins and compared to a commercial PPL assay (STA-Procoag-PPL). The two PPL assays displayed similar sensitivity to exogenously added standardized phospholipids. The PPL activity measured by the modified assay strongly correlates with the results from the commercial assay. The intraday- and between-days coefficients of variation ranged from 2-4% depending on the PPL activity in the sample. The modified PPL assay was insensitive to postprandial lipoprotein levels in plasma, as well as to tissue factor (TF) positive EVs from stimulated whole blood. Our findings showed that the modified assay performed equal to the comparator, and was insensitive to postprandial lipoproteins and TF+ EVs.

A novel role of bone morphogenetic protein-7 in the regulation of adhesion and migration of human monocytic cells.

BACKGROUND: Bone morphogenetic protein (BMP) 7 is abundant in atherosclerotic plaques and increases monocyte pro-coagulant activity by enhancing tissue factor (TF) expression. While several members of the BMP superfamily are able to serve as chemotactic agents for monocytes, the role of BMP-7 in regulation of monocyte motility is not known. AIMS: To assess the effect of BMP-7 on adhesive and migratory properties of human monocytes. METHODS: Chemokinesis, adhesion, and transendothelial migration of BMP-7-treated THP-1 cells and human monocytes were analysed using live-cell imaging, orbital shear, and Boyden chamber assays. Surface presentation of ß2 integrins and phosphorylation status of Akt & focal adhesion kinase (FAK) were studied by flow cytometry and Western blot. RESULTS: High levels of BMP-7 protein were detectable in intimal regions of atherosclerotic plaques; BMP-7 significantly enhanced THP-1 and monocyte chemokinetic properties in vitro (1.21+0.01 and 1.76+0.21 fold increase in crawling distance, respectively). Under orbital shear, adhesion of monocytic cells to microvascular endothelial cell (MVEC) monolayers was also significantly increased by BMP-7 (3.89+1.56 and 2.57+0.97 fold over vehicle). Moreover, BMP-7 accelerated transendothelial migration of THP-1 cells and monocytes towards MCP-1 (5.91+0.88 and 2.96±0.65 fold increase, respectively). BMP-7 enhanced cell surface presentation of ß2 integrins in the active conformation. Observed effects were determined to be Akt and FAK dependent, as shown by pharmacological inhibition. CONCLUSION: BMP-7 directly upregulates adhesion and migration of human monocytic cells via activation of ß2 integrins, Akt, and FAK. Our findings suggest that BMP-7 may serve as a novel contributor to atherogenesis.

BMP-7 induces TF expression in human monocytes by increasing F3 transcriptional activity.

BACKGROUND: Bone morphogenetic protein (BMP)-7, a major regulator of bone metabolism, inhibits ectopic calcification in atherosclerotic plaques. We have recently reported that BMP-7 is also a potent inducer of tissue factor (TF) in human mononuclear cells (MNCs). While nuclear factor kappa beta (NF-kB) and activation protein-1 (AP-1) are the transcription factors essential for inducible expression of human TF gene (F3), the mechanisms responsible for TF induction by BMP-7 are not known. OBJECTIVE: To elucidate the molecular mechanisms governing BMP-7-triggered TF expression in human MNCs. METHODS: Human blood monocytes were stimulated with BMP-7 and western blotting, qRT-PCR, and flow cytometry studies were carried out to assess F3 expression; promoter studies were also performed using a panel of reporter constructs. Procoagulant TF activity was measured using a validated FXa generation assay. The significance of NF-kB transcriptional activity was verified via pharmacological inhibition. RESULTS: BMP-7 increased TF protein levels, procoagulant activity, surface presentation, and TF mRNA expression. This increase was accompanied by activation of NF-kB as evidenced by reduced IkB-α levels and elevated transcriptional activity of an NF-kB-sensitive reporter in transfected MNCs. Although treatment with BMP-7 also led to a strong phosphorylation of c-Jun, activation of AP-1 alone was not sufficient to induce TF expression: JSH-23, a potent and specific NF-kB inhibitor, completely blocked BMP-7-induced TF expression. CONCLUSIONS: We report that BMP-7-dependent activation of TF in human MNCs is mediated via increased activity of NF-kB, leading to enhanced F3 transcription in human MNCs.

BMP-2 inhibits TF expression in human monocytes by shutting down MAPK signaling and AP-1 transcriptional activity.

BACKGROUND: We have recently identified bone morphogenetic protein (BMP)-2 as a novel inhibitor of tissue factor (TF) expression in lipopolysaccharide (LPS)-activated human monocytes, and now sought for intracellular mechanisms. METHODS: Here, we studied activation status of mitogen activated protein kinases (MAPKs) extracellular signal-regulated protein kinase (Erk) 1/2, p38, and c-Jun N-terminal kinase (JNK) as well as transcription factors activator protein (AP)-1 and nuclear factor kappa B (NF-kB), which regulate inducible expression of TF. RESULTS: Human mononuclear cells (MNCs) responded to BMP-2 stimulation with activation of canonic Smad1/5/8 signaling. Pretreatment with BMP-2 prevented LPS-induced increase in surface presentation, intracellular accumulation, and fraction of TF-positive MNCs. Similarly, LPS-induced increase in levels of phosphorylated Erk1/2, p38, and JNK was markedly diminished by BMP-2 pretreatment. BMP-2 pretreatment prior to LPS significantly diminished LPS-induced transcriptional activation of AP-1-dependent reporter. In contrast, BMP-2 given prior to LPS did not dampen the transcriptional activation of NF-kB-sensitive luciferase reporter. CONCLUSIONS: BMP-2 can inhibit LPS-induced TF protein expression and surface presentation in human MNCs by downregulation of Erk1/2, p38, and JNK signaling, as well as reduced transcriptional activity of AP-1, but not NF-kB.

Increased expression of TF in BMP-7-treated human mononuclear cells depends on activation of select MAPK signaling pathways.

Bone morphogenetic protein (BMP)-7 regulates atherosclerotic plaque calcification, and it contributes to increased thrombogenicity of lipid-rich lesions by enhancement of TF expression in monocytes/macrophages by unknown mechanism. Since Erk1/2, JNK and p38 mitogen activated protein kinases (MAPKs) regulate TF expression, we studied involvement of MAPK pathways in BMP-7-induced activation of TF in human mononuclear cells (MNCs). Whole blood from healthy volunteers was treated with BMP-7, MNCs were isolated, and TF expression was assessed by western blot (WB) and In-Cell Western assay. Phosphorylation and nuclear translocation of Smad1/5/8 in response to BMP-7 stimulation of MNCs was evaluated by WB and confocal microscopy. Activation of MAPKs was judged by measuring the levels of phoshorylated Erk1/2, JNK, and p38 in the lysates of MNCs. The impact of Erk1/2 and p38 activation was studied by use of PD98059 and SB203580 inhibitors, respectively. Stimulation of whole blood with BMP-7 increased the levels of TF in the lysates of MNCs by 7-fold as compared to 12-fold after LPS stimulation. It was followed by elevation in TF fuctional activity. It was accompanied by elevated levels of phosphorylated Smad 1/5/8 and nuclear translocation of Smad1/5/8 proteins. Treatment of whole blood with BMP-7 led to a phosphorylation of Erk1/2, JNK and p38 MAPKs. BMP-7-induced TF expression was partially inhibited by Erk1/2 inhibitor, whereas TF expression was completely abolished by p38 inhibitor. BMP-7-dependent activation of TF in human MNCs by BMP-7 is accompanied by activation of canonic Smad1/5/8 signaling pathway and depends on activation of Erk1/2 and p38.