Background: Protein disulfide isomerase (PDI) controls platelet integrin function, tissue‐factor (TF) activation, and concentrates at fibrin and thrombus formation sites of vascular injury. ...Objective: To investigate the involvement of surface thiol isomerases and especially PDI, in thrombin‐mediated thrombin amplification on human platelets. Methods/results: Using a newly developed thrombin‐dependent platelet thrombin generation assay, we observed that the feedback activation of thrombin generation on the platelet surface does not depend on TF, as anti‐TF antibodies inhibiting TF‐induced thrombin formation in platelet‐depleted plasma had no effect compared with vehicle‐treated controls. Feedback activation of thrombin generation in the presence of platelets was significantly diminished by membrane impermeant thiol blockers or by the thiol isomerase‐inhibitors bacitracin and anti‐PDI antibody RL90, respectively. Platelet thrombin formation depends on binding of coagulation factors to the platelet surface. Therefore, involvement of thiol isomerases in this binding was investigated. As shown by confocal microscopy and flow cytometry, thrombin‐stimulated platelets exhibited increased surface‐associated PDI as well as extracellular disulfide reductase activity compared with unstimulated platelets. Flow cytometric analysis revealed that membrane impermeant thiol blockers or PDI inhibitors, which had been added after platelet stimulation and after phosphatidylserine exposure to exclude their influence on primary platelet activation, significantly inhibited binding of all coagulation factors to thrombin‐stimulated platelets. Conclusions: Thus, surface‐associated PDI is an important regulator of coagulation factor ligation to thrombin‐stimulated platelets and of subsequent feedback activation of platelet thrombin generation. Cell surface thiol isomerases might be therefore powerful targets to control hemostasis and thrombosis.
Essentials
Cofactor‐independent antiphospholipid antibodies (CI‐aPL) are generally considered non‐pathogenic.
We analyzed the effects of human monoclonal CI‐aPL in a mouse model of venous thrombosis.
...As shown in vitro, CI‐aPL induce a procoagulant state in vivo by activation of endosomal NADPH‐oxidase.
Contrary to common belief, CI‐aPL induce venous thrombosis in vivo.
Summary
Background
There is general consensus that the antiphospholipid syndrome (APS) is caused by antiphospholipid antibodies (aPL) with antibodies against β2‐glycoprotein‐I being the most relevant. aPL that bind phospholipids in the absence of protein cofactors are generally considered pathogenetically irrelevant. We showed that cofactor‐independent human monoclonal aPL isolated from APS patients induce proinflammatory and procoagulant cellular responses by activating endosomal NADPH‐oxidase 2 (NOX2). Similar aPL were detected in all IgG fractions from APS patients analyzed.
Objectives
We aimed to clarify if cofactor‐independent aPL can be thrombogenic in vivo and, if so, whether these effects are mediated via activation of NOX2.
Methods
Two cofactor‐independent human monoclonal aPL, HL5B and RR7F, were tested in a mouse model of venous thrombosis. Genetically modified mice and in vitro assays were used to delineate the mechanisms underlying thrombus induction.
Results
HL5B and RR7F dramatically accelerate thrombus formation in this mouse model. Thrombus formation depends on tissue factor activation. It cannot be induced in NOX2‐deficient mice. Bone marrow chimeras of C57BL/6J mice reconstituted with NOX2‐deficient bone marrow showed that leukocyte activation plays a major role in thrombus formation. Neither TLR4 signaling nor platelet activation by our aPL is required for venous thrombus formation.
Conclusions
Cofactor‐independent aPL can induce thrombosis in vivo. This effect is mainly mediated by leukocyte activation, which depends on the previously described signal transduction via endosomal NOX2. Because most APS patients have been shown to harbor aPL with similar activity, our data are of general relevance for the APS.
A significant proportion of patients with peripheral artery disease (PAD) displays a poor response to aspirin and/or the platelet P2Y12 receptor antagonist clopidogrel. This phenomenon is reflected ...by high on-treatment platelet reactivity (HTPR) in platelet function assays in vitro and is associated with an increased risk of adverse cardiovascular events.
This study aimed to elucidate specific plasma protein signatures associated with HTPR to aspirin and clopidogrel in PAD patients.
Based on targeted plasma proteomics, 184 proteins from two cardiovascular Olink panels were measured in 105 PAD patients. VerifyNow ASPI- and P2Y12-test values were transformed to a continuous variable representing HTPR as a spectrum instead of cut-off level-defined HTPR. Using the Boruta random forest algorithm, the importance of 3 plasma proteins for HTPR in the aspirin, six in clopidogrel and 10 in the pooled group (clopidogrel or aspirin) was confirmed. Network analysis demonstrated clusters with CD84, SLAMF7, IL1RN and THBD for clopidogrel and with F2R, SELPLG, HAVCR1, THBD, PECAM1, TNFRSF10B, MERTK and ADM for the pooled group. F2R, TNFRSF10B and ADM were higher expressed in Fontaine III patients compared to Fontaine II, suggesting their relation with PAD severity.
A plasma protein signature, including eight targets involved in proatherogenic dysfunction of blood cell-vasculature interaction, coagulation and cell death, is associated with HTPR (aspirin and/or clopidogrel) in PAD. This may serve as important systems-based determinants of poor platelet responsiveness to aspirin and/or clopidogrel in PAD and other cardiovascular diseases and may contribute to identify novel treatment strategies.
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•A spectrum of high on-treatment platelet reactivity (HTPR) was established for PAD.•HTPR-associated plasma protein signatures differ between aspirin and clopidogrel.•Pooled HTPR relates to PSGL1, PECAM1, TM, PAR1, TRAILR2, ADM, KIM1 and MERTK.•Higher plasma levels of PAR1, TRAILR2 and ADM are associated with PAD severity.•Systems-based plasma protein signatures may determine antiplatelet therapy response.
Although platelets act as central players of haemostasis only their cross-talk with other blood cells, plasma factors and the vascular compartment enables the formation of a stable thrombus. Multiple ...activation processes and complex signalling networks are responsible for appropriate platelet function. Thus, a variety of platelet function tests are available for platelet research and diagnosis of platelet dysfunction. However, universal platelet function tests that are sensitive to all platelet function defects do not exist and therefore diagnostic algorithms for suspected platelet function disorders are still recommended in clinical practice. Based on the current knowledge of human platelet activation this review evaluates point-of-care related screening tests in comparison with specific platelet function assays and focuses on their diagnostic utility in relation to severity of platelet dysfunction. Further, systems biology-based platelet function methods that integrate global and specific analysis of platelet vessel wall interaction (advanced flow chamber devices) and post-translational modifications (platelet proteomics) are presented and their diagnostic potential is addressed.
Background: Human neutrophil α‐defensins (HNPs) are important constituents of the innate immune system. Beyond their antimicrobial properties, HNPs also have pro‐inflammatory features. While HNPs in ...plasma from healthy individuals are barely detectable, their level is strongly elevated in septic plasma and plasma from patients with acute coronary syndromes.
Objectives: As thrombosis and inflammation are intertwined processes and activation of human polymorphonuclear leukocytes (PMNL) and subsequent degranulation is associated with full activation of surrounding platelets, we studied the effect of HNPs on platelet function.
Methods: The effect of HNPs on platelet activation parameters and apoptosis was investigated via aggregometry, flow cytometry, confocal microscopy and the ELISA technique.
Results: It was found that HNPs activate platelets in pathophysiologically relevant doses, inducing fibrinogen and thrombospondin‐1 binding, aggregation, granule secretion, sCD40L shedding, and procoagulant activity. HNPs bound directly to the platelet membrane, induced membrane pore formation, microparticle formation, mitochondrial membrane depolarization and caspase‐3‐activity. Confocal microscopy revealed the HNP‐induced formation of polymeric fibrinogen and thrombospondin‐1 amyloid‐like structures, which bound microorganisms. Platelets adhered to these structures and formed aggregates. Blocking of glycoprotein IIb/IIIa (GPIIb/IIIa) markedly inhibited HNP‐induced platelet activation. In addition, heparin, heparinoid, serpins and α2‐macroglobulin, which all bind to HNPs, blocked HNP‐1‐induced platelet activation in contrast to direct thrombin inhibitors such as hirudin.
Conclusions: HNPs activate platelets and induce platelet apoptosis by formation of amyloid‐like proteins. As these structures entrapped bacteria and fungi, they might reflect an additional function of HNPs in host defense. The described mechanism links again thrombosis and infection.
Many patients with chronic kidney disease (CKD) receive anticoagulation or antiplatelet therapy due to atrial fibrillation, coronary artery disease, thromboembolic disease, or peripheral artery ...disease. The treatment usually includes vitamin K antagonists (VKAs) and/or platelet aggregation inhibitors. The direct oral anticoagulants (DOAC) inhibiting factor Xa or thrombin represent an alternative for VKAs. In patients with acute and chronic kidney disease, caution is warranted, as DOACs can accumulate as they are partly eliminated by the kidneys. Thus, they can potentially increase the bleeding risk in patients with CKD. In patients with an estimated glomerular filtration rate (eGFR) above 60 mL/min, DOACs can be used safely with greater efficacy and safety as compared to VKAs. In patients with CKD 3, DOACs are as effective as VKAs with a lower bleeding rate. The more the renal function declines, the lower is the advantage of DOACs over VKAs. Thus, use of DOACs should be avoided in patients with an eGFR below 30 mL/min, particularly, the compounds with a high renal elimination. Available data suggest that DOACs can also be used safely in older patients. In this review, use of DOACs in comparison with VKAs, heparins, and heparinoids, together with special considerations in patients with impaired renal function will be discussed.
Abstract
Background
Arterial hypertension is one of the most important modifiable risk factors for all-cause morbidity and mortality worldwide. Angiotensin II (Ang II) plays a pathogenic role in the ...development of hypertension, vascular dysfunction, inflammation and tissue damage. In the context of hypertension, heme-oxygenase 1 (HO-1) gene expression is upregulated as an antioxidant defense system in response to AngII through its action on heme catabolism, which generates carbon monoxide (CO), ferritin and biliverdin, which is reduced to bilirubin by biliverdin reductase A (BLVRA). Previous studies have shown the important role of HO-1 in the maturation and migration of immune cells. Myeloid HO-1 modulates macrophage polarization and protects against ischemia-reperfusion damage. However, the role of myeloid cell specific HO-1 in the detrimental effects of AngII induced vascular dysfunction has not yet been explored.
Objectives
To investigate the potential vascular protection of myeloid cell specific overexpression of HO-1 in AngII-induced arterial hypertension.
Methods
Hypertension was induced in 8–13 weeks old male mice with selective over-expression of HO-1 (HO-1indLysMcre) in myelomonocytic cells versus LysMCre/wt by AngII infusion (1mg/kg/d). Blood pressure was recorded by tail-cuff. Bilirubin levels were quantified in plasma by High Performance Liquid Chromatography (HPLC). The quantification of adherent and rolling leukocytes in carotid arteries was detected by intravital video microscopy (IVM). Endogenuos Thrombin potential (ETP) was measured in platelet rich plasma (PRP) and platelets poor plasma (PPP) by Calibrated Automated Thrombogram (CAT) assay. Endothelium-dependent vasodilation was assessed in isolated aortic rings by concentration-relaxation curves in response to acetylcholine (ACh).
Results
AngII-infused HO-1indLysMcre had decreased blood pressure values and improved endothelial function as compared to LysMcre controls. IVM revealed reduced leukocyte rolling and adhesion to the vascular endothelium in AngII infused HO-1indLysMcre mice compared to controls, paralleled by reduced expression of NOX-2 mediated oxidative stress and vascular inflammation in AngII-induced arterial hypertension by decreasing VCAM-1, CCR2 and MCP-1 expression. mRNA analysis revealed an increased expression of BLVRA in liver, spleen, heart and aortic tissues in response to AngII, which was higher in HO-1indLysMcre mice than in controls. By CAT assay we registered a decrease ETP in PPP and PRP in HO-1ind mice infused with AngII compatible with less abundance of inflammatory platelets, potentially regulated by BLVRA activity. This was supported by increased in bilirubin levels in response to AngII, which was higher in HO-1indLysMcre mice than in controls.
Conclusion
Myeloid cell specific overexpression of HO-1 confers anti-inflammatory protection to the vasculature in AngII induced hypertension. This effect is, at least in part, mediated by BLVRA.
Funding Acknowledgement
Type of funding sources: None.
Platelets: Physiology and Biochemistry Jurk, Kerstin; Kehrel, Beate E
Seminars in thrombosis and hemostasis,
2005, Letnik:
31, Številka:
4
Journal Article
Recenzirano
Odprti dostop
ABSTRACT
Platelets are specialized blood cells that play central roles in physiologic and pathologic processes of hemostasis, inflammation, tumor metastasis, wound healing, and host defense. ...Activation of platelets is crucial for platelet function that includes a complex interplay of adhesion and signaling molecules. This article gives an overview of the activation processes involved in primary and secondary hemostasis, for example, platelet adhesion, platelet secretion, platelet aggregation, microvesicle formation, and clot retraction/stabilization. In addition, activated platelets are predominantly involved in cross talk to other blood and vascular cells. Stimulated “sticky” platelets enable recruitment of leukocytes at sites of vascular injury under high shear conditions. Platelet-derived microparticles as well as soluble adhesion molecules, sP-selectin and sCD40L, shed from the surface of activated platelets, are capable of activating, in turn, leukocytes and endothelial cells. This article focuses further on the new view of receptor-mediated thrombin generation of human platelets, necessary for the formation of a stable platelet-fibrin clot during secondary hemostasis. Finally, special emphasis is placed on important stimulatory and inhibitory signaling pathways that modulate platelet function.
Multicellular interactions of platelets, leukocytes, and the blood vessel wall support coagulation and precipitate arterial and venous thrombosis. High levels of angiotensin II cause arterial ...hypertension by a complex vascular inflammatory pathway that requires leukocyte recruitment and reactive oxygen species production and is followed by vascular dysfunction. We delineate a previously undescribed, proinflammatory coagulation-vascular circuit that is a major regulator of vascular tone, blood pressure, and endothelial function. In mice with angiotensin II-induced hypertension, tissue factor was up-regulated, as was thrombin-dependent endothelial cell vascular cellular adhesion molecule 1 expression and integrin α
β
- and platelet-dependent leukocyte adhesion to arterial vessels. The resulting vascular inflammation and dysfunction was mediated by activation of thrombin-driven factor XI (FXI) feedback, independent of factor XII. The FXI receptor glycoprotein Ibα on platelets was required for this thrombin feedback activation in angiotensin II-infused mice. Inhibition of FXI synthesis with an antisense oligonucleotide was sufficient to prevent thrombin propagation on platelets, vascular leukocyte infiltration, angiotensin II-induced endothelial dysfunction, and arterial hypertension in mice and rats. Antisense oligonucleotide against FXI also reduced the increased blood pressure and attenuated vascular and kidney dysfunction in rats with established arterial hypertension. Further, platelet-localized thrombin generation was amplified in an FXI-dependent manner in patients with uncontrolled arterial hypertension, suggesting that platelet-localized thrombin generation may serve as an inflammatory marker of high blood pressure. Our results outline a coagulation-inflammation circuit that promotes vascular dysfunction, and highlight the possible utility of FXI-targeted anticoagulants in treating hypertension, beyond their application as antithrombotic agents in cardiovascular disease.
Platelets and neutrophils are the first blood cells accumulating at sites of arterial thrombus formation, and both cell types contribute to the pathology of thrombotic events. We aimed to identify ...key interaction mechanisms between these cells using microfluidic approaches.
Whole-blood perfusion was performed over a collagen surface at arterial shear rate. Platelet and leukocyte (in majority neutrophil) activation were microscopically visualized using fluorescent markers. The contributions of platelet-adhesive receptors (integrin, P-selectin, CD40L) and chemokines were studied by using inhibitors or antibodies and using blood from patients with Glanzmann thrombasthenia lacking platelet-expressed αIIbβ3.
We observed (1) an unknown role of activated platelet integrin αIIbß3 preventing leukocyte adhesion, which was overcome by short-term flow disturbance provoking massive adhesion; (2) that platelet-expressed CD40L controls the crawling pattern and thrombus fidelity of the cells on a thrombus; (3) that continued secretion of platelet substances promotes activation of identified neutrophils, as assessed by (fMLP induced) Ca
rises and antigen expression; (4) and that platelet-released chemokines activate the adhered cells in the order of CXCL7>CCL5>CXCL4. Furthermore, postsilencing of the platelets in a thrombus suppressed the leukocyte activation. However, the leukocytes on thrombi did no more than limitedly form neutrophil extracellular traps, unless stimulated with phorbol ester of lipopolysaccharide.
Together, these findings reveal a multifaceted regulation of adhesion and activation of neutrophils by platelets in a thrombus, with a balanced role of several platelet-adhesive receptors and a promoting role of platelet-released substances. This multivalent nature of neutrophil-thrombus interactions offers novel prospects for pharmacological intervention.
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