Plasma protein signatures for high on-treatment platelet reactivity to aspirin and clopidogrel in peripheral artery disease.

BACKGROUND: 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. OBJECTIVE: This study aimed to elucidate specific plasma protein signatures associated with HTPR to aspirin and clopidogrel in PAD patients. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Cofactor-independent human antiphospholipid antibodies induce venous thrombosis in mice.

UNLABELLED: 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 ß2-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.

Analysis of platelet function and dysfunction.

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.

Human neutrophil alpha-defensins induce formation of fibrinogen and thrombospondin-1 amyloid-like structures and activate platelets via glycoprotein IIb/IIIa.

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 α(2)-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.

Extracellular protein disulfide isomerase regulates feedback activation of platelet thrombin generation via modulation of coagulation factor binding.

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.

[Pathophysiology and biochemistry of platelets]. / Pathophysiologie und Biochemie der Thrombozyten.

Although their central role is to control bleeding and to induce thrombosis, platelets are important inflammatory and immune cells as well as modulators of angiogenesis. This review focuses on the different roles of platelets in hemostasis, thrombosis, inflammation, arteriosclerosis, angiogenesis, antimicrobial host defense and hematogenous tumor metastasis. Platelets are the central regulators of hemostasis. On their surface the important thrombin burst takes place. Platelets cause atherothrombotic vascular occlusions. However, they are probably involved in early stages of arteriosclerosis, e.g. extravasation of leukocytes at sites of vascular injury, formation of foam cells and proliferation of smooth muscle cells. These processes are triggered by secretion of proinflammatory substances and growth factors as well as by platelet-cell interactions via specific adhesive axes. During infections platelets kill pathogens through secretion of antimicrobial substances and extracellular traps or nets. Platelets facilitate the revascularisation of ischemic tissue and therefore even promote tumor growth.

[The role of platelets in haemostasis, thrombosis, immune defense and inflammation]. / Die Rolle von Thrombozyten bei Hämostase, Thrombose, Immunabwehr und Entzündung.

The views on the role of platelets in physiology and in pathogenesis have considerably changed in the recent past. While platelets had previously been seen only as contributors in primary haemostasis and as donors of negatively charged phospholipids to support thrombin formation, this view has had to be revised, at least since the discovery of specific receptors for coagulation factors on the platelet surface. Platelets are part of the body's immune defence system. They can interact with bacteria, pathogenic fungi and protozoa. The interaction of platelets with endothelial cells and leukocytes is crucial in innate and adaptive immunity. Platelets participate in the pathogenesis of the initial lesions and in the progression of atherosclerosis by inducing chronic inflammatory processes at the vascular wall, which result in the development of atherosclerotic lesions and atherothrombosis.

[Platelets and the new comprehension of haemostasis]. / Die zentrale Rolle der Thrombozyten im neuen Verständnis der Hämostase.

Platelets are cells with key function in primary haemostasis. They localise coagulation to the haemostatic thrombus. After injury of the vessel wall blood contacts subendothelial matrix proteins as well as cells constitutively exposing tissue factor (TF). Platelets adhere to the subendothelial matrix, become activated, spread and secrete the contents of their granules. On the surface of the TF exposing cells minute amounts of thrombin are formed. These amounts of thrombin are inadequate to yield in a stable fibrin clot, but activate platelets and factors XI, VIII, V. In that way the consolidation pathway is triggered. Activated platelets aggregate and bind leukocytes. On the surface of the activated platelets coagulation (co)factor complexes are formed and protected in an optimal way. Thus large amounts of prothrombin are converted to thrombin, creating a so-called thrombin burst. This leads to the formation of a stable platelet-fibrin-clot. Platelets are not always prothrombotic. They have their own mechanisms to stop activation processes and thrombus growth. Besides, its key role in haemostasis platelets are involved in inflammation and innative immune defence.

P-Selectin glycoprotein ligand 1 (PSGL-1) is expressed on platelets and can mediate platelet-endothelial interactions in vivo.

The platelet plays a pivotal role in maintaining vascular integrity. In a manner similar to leukocytes, platelets interact with selectins expressed on activated endothelium. P-selectin glycoprotein ligand 1 (PSGL-1) is the main P-selectin ligand expressed on leukocytes. Searching for platelet ligand(s), we used a P-selectin-immunoglobulin G (IgG) chimera to affinity purify surface-biotinylated proteins from platelet lysates. P-selectin-bound ligands were eluted with ethylenediaminetetraacetic acid. An approximately 210-kD biotinylated protein was isolated from both human neutrophil and platelet preparations. A band of the same size was also immunopurified from human platelets using a monoclonal anti-human PSGL-1 antibody and could be blotted with P-selectin-IgG. Under reducing conditions, both the predicted PSGL-1 approximately 210-kD dimer and the approximately 120-kD monomer were isolated from platelets. Comparative immunoelectron microscopy and Western blotting experiments suggested that platelet PSGL-1 expression is 25-100-fold lower than that of leukocytes. However, patients with chronic idiopathic thrombocytopenic purpura who harbor predominantly young platelets displayed greater expression, indicating that PSGL-1 expression may be decreased during platelet aging. By flow cytometry, thrombin-activated platelets from normal individuals exhibited greater expression than those unstimulated. An inhibitory anti-PSGL-1 antibody significantly reduced platelet rolling in mesenteric venules, as observed by intravital microscopy. Our results indicate that functional PSGL-1 is expressed on platelets, and suggest an additional mechanism by which selectins and their ligands participate in inflammatory and/or hemostatic responses.

Projectin-thin filament interactions and modulation of the sensitivity of the actomyosin ATPase to calcium by projectin kinase.

The insect muscle protein projectin (900 kDa) belongs to a novel family of cytoskeleton-associated protein kinases (titin, twitchin, and projectin) that are members of the immunoglobulin superfamily. The functions of these kinases are still unknown although recent data suggest a role in modulating muscle activity and generating passive elasticity. An important question is what are the in vivo substrates for these enzymes. We found a thin filament-associated 30 kDa protein that acts as an in vitro substrate for projectin kinase from Locusta migratoria. However, we did not find activators for projectin kinase. Neither calcium, calcium with calmodulin, nor cAMP activated the in vitro activity of projectin kinase. Binding studies revealed a strong interaction between projectin and thin filaments comparable with that of the projectin-myosin interaction. That an interaction might be possible in vivo is suggested by immunological studies showing that projectin is attached to the surface of myosin filaments. Since the molecular weights indicate that the 30 kDa protein might be troponin I, which is known to play a central role in modulating cardiac contractile activity, we studied whether phosphorylation of this protein by projectin changes the calcium sensitivity of the actomyosin ATPase. We found a significant increase in the calcium sensitivity. Thus, our results indicate the existence of a novel mechanism of regulation of muscle activity by a cytoskeleton-associated kinase.