Thrombin generation on vascular cells in the presence of factor VIII and/or emicizumab.

BACKGROUND: The effect of factor VIII (FVIII) or emicizumab on thrombin generation is usually assessed in assays using synthetic phospholipids. Here, we assessed thrombin generation at the surface of human arterial cells (aortic endothelial cells [hAECs] and aortic vascular smooth muscle cells [hVSMCs]). OBJECTIVES: To explore the capacity of hAECs (resting or stimulated) and hVSMCs to support thrombin generation by FVIII or emicizumab. METHODS: Primary hVSMCs and hAECs were analyzed for tissue factor (TF)-activity and antigen, phosphatidylserine (PS)-exposure, tissue factor pathway inhibitor (TFPI)-content and thrombomodulin expression. Cells were incubated with FVIII-deficient plasma spiked with FVIII, emicizumab, activated prothrombin complex concentrate (APCC) or combinations thereof. RESULTS: TF activity and PS-exposure were present on both hVSMCs and hAECs. In contrast, thrombomodulin and TFPI were expressed on hAECs, while virtually lacking on hVSMCs, confirming the procoagulant nature of hVSMCs. Tumor necrosis factor α-mediated stimulation of hAECs increased not only TF antigen, TF activity, and PS-exposure but also TFPI and thrombomodulin expression. As expected, FVIII and emicizumab promoted thrombin generation on nonstimulated hAECs and hVSMCs, with more thrombin being generated on hVSMCs. Unexpectedly, FVIII and emicizumab increased thrombin generation to a lesser extent on stimulated hAECs compared with nonstimulated hAECs. Finally, adding emicizumab to FVIII did not further increase thrombin generation, whereas the addition of emicizumab to APCC resulted in exaggerated thrombin generation. CONCLUSION: Tumor necrosis factor stimulation of hAECs increases both pro- and anticoagulant activity. Unexpectedly, the increased anticoagulant activity is sufficient to limit both FVIII- and emicizumab-induced thrombin generation. This protective effect disappears when emicizumab is combined with APCC.

The VWF/LRP4/αVß3-axis represents a novel pathway regulating proliferation of human vascular smooth muscle cells.

AIMS: Von Willebrand factor (VWF) is a plasma glycoprotein involved in primary haemostasis, while also having additional roles beyond haemostasis namely in cancer, inflammation, angiogenesis, and potentially in vascular smooth muscle cell (VSMC) proliferation. Here, we addressed how VWF modulates VSMC proliferation and investigated the underlying molecular pathways and the in vivo pathophysiological relevance. METHODS AND RESULTS: VWF induced proliferation of human aortic VSMCs and also promoted VSMC migration. Treatment of cells with a siRNA against αv integrin or the RGT-peptide blocking αvß3 signalling abolished proliferation. However, VWF did not bind to αvß3 on VSMCs through its RGD-motif. Rather, we identified the VWF A2 domain as the region mediating binding to the cells. We hypothesized the involvement of a member of the LDL-related receptor protein (LRP) family due to their known ability to act as co-receptors. Using the universal LRP-inhibitor receptor-associated protein, we confirmed LRP-mediated VSMC proliferation. siRNA experiments and confocal fluorescence microscopy identified LRP4 as the VWF-counterreceptor on VSMCs. Also co-localization between αvß3 and LRP4 was observed via proximity ligation analysis and immuno-precipitation experiments. The pathophysiological relevance of our data was supported by VWF-deficient mice having significantly reduced hyperplasia in carotid artery ligation and artery femoral denudation models. In wild-type mice, infiltration of VWF in intimal regions enriched in proliferating VSMCs was found. Interestingly, also analysis of human atherosclerotic lesions showed abundant VWF accumulation in VSMC-proliferating rich intimal areas. CONCLUSION: VWF mediates VSMC proliferation through a mechanism involving A2 domain binding to the LRP4 receptor and integrin αvß3 signalling. Our findings provide new insights into the mechanisms that drive physiological repair and pathological hyperplasia of the arterial vessel wall. In addition, the VWF/LRP4-axis may represent a novel therapeutic target to modulate VSMC proliferation.

International consensus on the prevention of venous and arterial thrombotic events in patients with inflammatory bowel disease.

Patients with inflammatory bowel disease (IBD) are at increased risk of thrombotic events. Therapies for IBD have the potential to modulate this risk. The aims of this Evidence-Based Guideline were to summarize available evidence and to provide practical recommendations regarding epidemiological aspects, prevention and drug-related risks of venous and arterial thrombotic events in patients with IBD. A virtual meeting took place in May 2020 involving 14 international IBD experts and 3 thrombosis experts from 12 countries. Proposed statements were voted upon in an anonymous manner. Agreement was defined as at least 75% of participants voting as 'fully agree' or 'mostly agree' with each statement. For each statement, the level of evidence was graded according to the Scottish Intercollegiate Guidelines Network (SIGN) grading system. Consensus was reached for 19 statements. Patients with IBD harbour an increased risk of venous and arterial thrombotic events. Thromboprophylaxis is indicated during hospitalization of any cause in patients with IBD. Disease activity is a modifiable risk factor in patients with IBD, and physicians should aim to achieve deep remission to reduce the risk. Exposure to steroids should be limited. Antitumour necrosis factor agents might be associated with a reduced risk of thrombotic events.

Mitigation of monocyte driven thrombosis on cobalt chrome surfaces in contact with whole blood by thin film polar/hydrophobic/ionic polyurethane coatings.

Monocytes are active at the crossroads between inflammation and coagulation processes since they can secrete pro-inflammatory cytokines and express tissue factor (TF), a major initiator of coagulation. Cobalt-chrome (CoCr), a metal alloy, used as a biomaterial for vascular stents, has been shown to be potentially pro-thrombotic and pro-inflammatory. Research work with a polymer from a family of degradable-polar hydrophobic ionic polyurethanes (D-PHI), called HHHI, has been shown to exhibit anti-inflammatory responses from human monocytes. We have generated multifunctional polyurethane thin films (MPTF) based on the HHHI chemistry, as a thin coating for CoCr and have evaluated the reactivity of blood with MPTF-coated CoCr. The results showed that the coating of CoCr with MPTF derived from HHHI prevents thrombin generation, reduces coagulation activation, and suppresses fibrin formation in whole blood. Activation of monocytes was also suppressed at the surface of MPTF-coated CoCr and specifically the decrease in thrombin generation was accompanied by a significant decrease in TF and pro-inflammatory cytokine levels. Mass spectroscopy of the adsorbed proteins showed lower levels of fibrinogen, fibronectin and complement C3, C4, and C8 when compared to CoCr. We can conclude that MPTFs reduce the pro-thrombotic and pro-inflammatory phenotype of monocytes and macrophages on CoCr, and prevent clotting in whole blood.

Complex formation with pentraxin-2 regulates factor X plasma levels and macrophage interactions.

Recently, we have identified scavenger receptor class A member I (SR-AI) as a receptor for coagulation factor X (FX), mediating the formation of an FX reservoir at the macrophage surface. Here, we demonstrate that the FX/SR-AI-complex comprises a third protein, pentraxin-2 (PTX2). The presence of PTX2 is essential to prevent internalization of FX by SR-AI, and the presence of FX is needed to interfere with internalization of PTX2. Binding studies showed that FX, SR-AI, and PTX2 independently bind to each other (KD,app: 0.2-0.7 µM). Surprisingly, immunoprecipitation experiments revealed that FX and PTX2 circulate as a complex in plasma, and complex formation involves the FX activation peptide. No binding of PTX2 to other vitamin K-dependent proteins was observed. Short hairpin RNA-mediated inhibition of PTX2 levels in mice resulted not only in reduced levels of PTX2, but also in similarly reduced FX levels. Moreover, PTX2 and FX levels were correspondingly reduced in SR-AI-deficient mice. Analysis of 71 human plasma samples uncovered a strong correlation between FX and PTX2 plasma levels. Furthermore, plasma samples of patients with reduced FX levels (congenital/acquired FX deficiency or after anti-vitamin K treatment) were characterized by concomitantly decreased PTX2 levels. In conclusion, we identified PTX2 as a novel partner for FX, and both proteins cooperate to prevent their SR-AI-mediated uptake by macrophages. Interestingly, their respective plasma levels are interdependent. These findings seem of relevance in perspective of ongoing clinical trials, in which plasma depletion of PTX2 is used as a therapeutical approach in the management of systemic amyloidosis.

'Click' glycosylation of peptides through cysteine propargylation and CuAAC.

'Click' glycosylation of cysteine-containing peptides were carried out in good yield by Copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC). For that peptides were functionalized though direct propargylation of the cysteine residue allowing their use in CuAAC with suitable free or protected azido sugars of gluco, manno and galacto configuration. Among these free and protected glycopeptides a series of 'glycoRGD' peptides were obtained and submitted to in vitro platelet aggregation tests, showing that the pseudoglycosylation of the adhesion sequence lowers the IC50 value and thus could improve the in vivo pharmacokinetic properties.

Is thrombosis a contributor to heart failure pathophysiology? Possible mechanisms, therapeutic opportunities, and clinical investigation challenges.

Thrombotic events (coronary thrombosis, venous thromboembolism, intraventricular thrombosis, intracranial and systemic thromboembolism) occur frequently in patients with heart failure. These events may be precipitated by several mechanisms including hypercoagulability through enhancement of procoagulant reactions, impairment of the protein C pathway, protease activated receptor (PAR) activation, adenosine-mediated thrombosis, or neurohormonal activation; stasis secondary to low cardiac output; and endothelial dysfunction from neurohormonal activation or systemic inflammation. Pathophysiologic evidence and analyses of retrospective data support the hypothesis that antithrombotic agents may improve outcomes in patients with heart failure. Warfarin has not been shown to reduce clinical events in patients with heart failure, although several of the completed randomized trials were underpowered, and the most recent was not placebo-controlled. Many unanswered questions remain that justify continued research in this area. This paper examines the conceptual framework, opportunities, and challenges of clinical investigative approaches with the newer anti-thrombotic agents in patients with heart failure. Critical questions are raised with regard to clinical trial designs that warrant consideration as the field progresses.

Platelet-dependent thrombography gives a distinct pattern of in vitro thrombin generation after surgery with cardio-pulmonary bypass: potential implications.

BACKGROUND: Bleeding remains a potentially lethal complication of cardio-pulmonary bypass (CPB) surgery. The purpose of this study was to obtain a better insight into in vitro thrombin generation in the context of CPB. METHODS: We used Calibrated Automated Thrombography to assess blood coagulation of 10 low-risk patients operated for valve replacement with CPB, under 2 experimental conditions, one implicating platelets as platelet dysfunction has been described to occur during CPB. RESULTS: Our main finding was that CPB-induced coagulopathy was differently appreciated depending on the presence or absence of platelets: the decrease in thrombin generation was much less pronounced in their presence (mean endogenous thrombin potential change values before and after CPB were -3.9% in the presence of platelets and -39.6% in their absence). CONCLUSION: Our results show that experimental conditions have a profound effect in the study of in vitro thrombin generation in the context of CPB.

Increased microparticle production and impaired microvascular endothelial function in aldosterone-salt-treated rats: protective effects of polyphenols.

We aimed to characterize circulating microparticles in association with arterial stiffness, inflammation and endothelial dysfunction in aldosterone-salt-induced hypertension in rats and to investigate the preventive effects of red wine polyphenols. Uninephrectomized male Sprague-Dawley rats were treated with aldosterone-salt (1 µg.h(-1)), with or without administration of either red wine polyphenols, Provinols™ (20 mg.kg(-1).day(-1)), or spironolactone (30 mg.kg(-1).day(-1)) for 4 weeks. Microparticles, arterial stiffness, nitric oxide (NO) spin trapping, and mesenteric arterial function were measured. Aldosterone-salt rats showed increased microparticle levels, including those originating from platelets, endothelium and erythrocytes. Hypertension resulted in enhanced aortic stiffness accompanied by increased circulating and aortic NO levels and an upregulation of aortic inducible NO-synthase, NFκB, superoxide anions and nitrotyrosine. Flow-induced dilatation was reduced in mesenteric arteries. These effects were prevented by spironolactone. Provinols™ did not reduce arterial stiffness or systolic hypertension but had effects similar to those of spironolactone on endothelial function assessed by flow-mediated vasodilatation, microparticle generation, aortic NO levels and oxidative stress and apoptosis in the vessel wall. Neither the contractile response nor endothelium-dependent relaxation in mesenteric arteries differed between groups. The in vivo effects of Provinols™ were not mediated by mineralocorticoid receptors or changes in shear stress. In conclusion, vascular remodelling and endothelial dysfunction in aldosterone-salt-mediated hypertension are associated with increased circulating microparticles. Polyphenols prevent the enhanced release of microparticles, macrovascular inflammation and oxidative stress, and microvascular endothelial dysfunction independently of blood pressure, shear stress and mineralocorticoid receptor activation in a model of hyperaldosteronism.

C-reactive protein induces pro- and anti-inflammatory effects, including activation of the liver X receptor alpha, on human monocytes.

Non-specific markers of inflammation such as C-reactive protein (CRP) are associated statistically with an increased risk of atherosclerosis through mechanisms that have not yet been fully elucidated. We investigated the effects of CRP on several aspects of human monocyte biology, a cell type involved in the initiation and progression of atherosclerosis. Blood monocytes isolated from healthy men and premenopausal women (n = 9/group) were exposed to purified CRP (25 microg/ml) for 12 hours. Changes in gene expression were analyzed using a custom-made array containing oligonucleotide sequences of 250 genes expressed by activated monocytes and confirmed by quantitative PCR. CRP increased significantly the expression of the cytokines interleukin (IL)-1alpha, IL-1beta and IL-6, and the chemokines GRO-alpha, GRO-beta and IL-8. CRP also displayed anti-inflammatory effects through upregulation of liver X receptor (LXR) alpha and activin receptor expression, and down-regulation of alpha 2-macroglobulin expression. Increased LXRalpha mRNA expression in both monocytes and the monocytic cell lineTHP-1 was associated with increased LXRalpha protein expression and nuclear translocation, as well as increased ABCA1 mRNA expression, a target gene of LXRalpha. Western blot analysis revealed CRP-induced nuclear translocation of NF-kappaB and activation of p42/44, MAP and Akt kinases. CRP-induced LXRalpha mRNA expression was inhibited by anti-CD64 (FcgammaRI) antibodies and by p42/44 and PI3 kinase inhibitors. This hypothesis-generating study demonstrates that CRP modulates the expression of genes that contribute to both pro- and anti-inflammatory responses in human monocytes. Among these novel anti-inflammatory effects, we show clearly that CRP activates the LXRalpha pathway.

Chronic oxidative stress induces a tissue-specific reduction in telomere length in CAST/Ei mice.

We examine whether increased oxidative stress in vivo promotes telomere shortening in CAST/Ei mice. We explored the effects of L-buthionine sulfoximine treatment (BSO) on telomere length. BSO shortened telomere length in white fat, brown fat, skin, tail, and testis in concert with diminished tissue glutathione content, increased tissue carbonyl content, and increased plasma advanced oxidized protein products. Telomerase activity was mainly detected in testis but no reduction of telomerase activity was observed in response to BSO. In conclusion, BSO-mediated increase in systemic oxidative stress shortens telomeres in several tissues of the mouse. The variable effect of BSO treatment on telomere length in different tissue may result from their different adaptive antioxidative capacity.

Heparin-induced thrombocytopenia associated with interleukin-8-dependent platelet activation in a patient with antiphospholipid syndrome.

Platelet factor 4 heparin enzyme immunoassay, platelet aggregation test, and serotonin release assay are commonly used to diagnose and confirm heparin-induced thrombocytopenia. We describe a case of recurrent thrombocytopenia appearing in a few hours after each heparin administration and who tested negative for the three assays. Further analysis revealed anti-interleukin (IL)-8 antibodies and IL-8-dependent platelet activation facilitated by heparin, which may explain this unusual case of heparin-induced thrombocytopenia.

In vivo transglutaminase type 1 expression in normal lung, preinvasive bronchial lesions, and lung cancer.

Transglutaminase type 1 (TGase 1) is a member of a class of enzymes that catalyze the cross-linking of proteins, a characteristic feature of epidermal differentiation and squamous metaplasia. The role of TGase 1 has been extensively studied in epidermis but not in the lung. Using a polyclonal anti-TGase 1 antibody prepared in our laboratory (TGase-lac), TGase 1 expression in normal bronchial epithelium, bronchial preinvasive lesions, and lung cancer was characterized. The specificity of the antibody was confirmed by the presence in squamous differentiated bronchial cells of specific 106-kD and 92-kD bands by Western blotting. In addition, immunohistochemistry displayed a recognized pattern of labeling in both normal and tumor cells beneath the cytoplasmic membrane and within the cytosol. TGase 1 was shown to be expressed by cells from bronchial epithelium and bronchial preinvasive lesions, strongly expressed in most non-small-cell lung cancer tumor cells and in apoptotic bodies, but weakly expressed in small-cell lung cancer. The distribution of TGase 1 mRNA correlated with the immunohistochemical profile. These observations suggest that TGase 1 expression is a normal feature of bronchial epithelium and is linked to the process of squamous differentiation occurring in preinvasive lesions. Its role in lung cancer remains to be clarified.

Thrombinography shows acquired resistance to activated protein C in patients with lupus anticoagulants.

In patients with lupus anticoagulants (LA), acquired resistance to activated protein C (APC) is difficult to demonstrate with clot-based assays due to the presence of the anticoagulant. Via the conversion of a fluorogenic substrate (thrombinography), we monitored the complete process of thrombin formation and decay and its delimitation by the protein C system in eight consecutive LA-patients without anticoagulant therapy and non-carriers of the V Leiden polymorphism. Thrombin generation was triggered in platelet-poor and platelet-rich plasma by recalcification in the presence of a low concentration of tissue factor. In 7 out of 8 patients we observed a long lag-time before the thrombin burst (LA effect) together with a marked inability of APC to diminish the thrombin activity. The lag-phase was however prolonged to some degree by APC. The effects were more outspoken in the presence of phospholipids from patients' platelets than with added phospholipids. Thrombinography thus demonstrates APC resistance in LA-patients despite the occurrence of long lag-times (clotting times). The amount of thrombin activity generated in the presence of APC could be a better indicator of the thrombotic risk than the moment at which the thrombin burst starts.

Platelet activation induced by human antibodies to interleukin-8.

Some cases of heparin-induced thrombocytopenia (HIT) have been reported to be associated with antibodies against interleukin-8 (IL-8), a chemokine related to platelet factor 4. We found that sera from 5 HIT patients containing immunoglobulin G (IgG) or IgM antibodies to IL-8, as evidenced using surface plasmon resonance spectroscopy, were able to trigger IL-8-dependent activation of washed platelets, leading to procoagulant activity. This activation occurred at IL-8 concentrations achievable in vivo and was facilitated by heparin (0.1 U/mL). Activation was also induced by affinity-purified anti-IL-8 IgG and involved FcgammaRIIa. In the 2 patients who could be followed up, antibodies were no longer detectable 4 months after heparin withdrawal. One additional patient with paraneoplastic recurrent thrombosis without thrombocytopenia was found to have platelet-activating anti-IL-8 IgM, but in this case heparin was inhibitory. This is another example of potentially pathogenic platelet activation by antibodies.