The Deglycosylated Form of 1E12, a Monoclonal Anti-PF4 IgG, Strongly Inhibits Antibody-Triggered Cellular Activation in Vaccine-Induced Thrombotic Thrombocytopenia, and Is a Potential New Treatment for Vιττ

Abstract Introduction Vaccine-induced thrombotic thrombocytopenia (VITT) is a severe complication of recombinant adenoviral vector vaccines used to prevent COVID-19, likely due to anti-platelet factor 4 (PF4) IgG antibodies. The specificity and platelet-activating activity of VITT antibodies strikingly resemble that of antibodies detected in "autoimmune" heparin-induced thrombocytopenia (HIT), but their features remain poorly characterized. In particular, a better knowledge of these antibodies should help to understand the mechanisms leading to hypercoagulability and the particular thrombotic events observed in VITT, but rarely in typical HIT. We have recently developed a chimeric IgG1 anti-PF4 antibody, 1E12, which strongly mimics "autoimmune" HIT antibodies in terms of specificity and cellular effects. Therefore, we assessed whether 1E12 could mimic VITT antibodies. We then evaluated the capability of DG-1E12, a deglycosylated form of 1E12 unable to bind FcγR, to inhibit cellular activation induced by VITT antibodies. Methods and Results Using a PF4-sensitized serotonin release assay (PF4-SRA) (Vayne C, New Engl J Med, 2021), we demonstrated that 1E12 (5 and 10 μg/mL) strongly activated platelets, with a pattern similar to that obtained with human VITT samples (n=7), i.e. in a PF4-dependent manner and without heparin. This platelet activation was inhibited by low heparin concentration (0.5 IU/mL), an effect also observed with VITT samples. Serotonin release induced by 1E12 was also fully inhibited by IV-3, a monoclonal antibody blocking FcγRIIa, or by IdeS, a bacterial protease that cleaves IgG and strongly inhibits the binding of IgG antibodies to FcγRIIa. This inhibitory effect of IV-3 and IdeS strongly supports that interactions between pathogenic anti-PF4 IgG and FcγRIIa play a central role in VITT. Incubation of 1E12 or VITT samples with isolated neutrophils (PMN) and platelets with PF4 (10 µg/mL) strongly induced DNA release and NETosis, supporting that PMN are involved in the processes leading to thrombosis in VITT. Furthermore, when whole blood from healthy donors incubated with 1E12 or VITT plasma was perfused in capillaries coated with von Willebrand Factor, numerous large platelet/leukocyte aggregates containing fibrin(ogen) were formed. To investigate whether 1E12 and VITT antibodies recognize overlapping epitopes on PF4, we then performed competitive assays with a deglycosylated form of 1E12 (DG-1E12), still able to bind PF4 but not to interact with Fcγ receptors. In PF4-SRA, pre-incubation of DG-1E12 (50 µg/mL) dramatically reduced platelet activation induced by VITT antibodies, which was fully abrogated for 9 of the 14 VITT samples tested. Additional experiments using a whole blood PF4-enhanced flow cytometry assay recently designed for VITT diagnosis (Handtke et al, Blood 2021), confirmed that DG-1E12 fully prevented platelet activation induced by VITT antibodies. Moreover, when platelets and neutrophils were pre-incubated with DG-1E12 (100 µg/mL), NETosis and thus DNA release, nuclear rounding, and DNA decondensation induced by VITT antibodies were completely inhibited. Finally, DG-1E12 (100 µg/mL) also fully abolished VITT antibody-mediated thrombus formation in whole blood in vitro under vein flow conditions. Comparatively, DG-1E12 did not inhibit ALB6, a murine monoclonal anti-CD9 antibody, which also strongly activates platelets in a FcγRIIa-dependent manner. Conclusions Our results show that 1E12 exhibits features similar to those of human VITT antibodies in terms of specificity, affinity and cellular effects, and could therefore be used as a model antibody to study the pathophysiology of VITT. Our data also demonstrate that DG-1E12 prevents blood cell activation and thrombus formation induced by VITT antibodies, likely due to the competitive effect of its Fab fragment on antibody binding to PF4. DG-1E12 may allow the development of a new drug neutralizing the pathogenic effect of autoimmune anti-PF4 antibodies, such as those associated with VITT. Disclosures Thiele: Bristol Myers Squibb: Honoraria, Other; Pfizer: Honoraria, Other; Bayer: Honoraria; Chugai Pharma: Honoraria, Other; Novo Nordisk: Other; Novartis: Honoraria; Daichii Sankyo: Other. Pouplard: Stago: Research Funding. Greinacher: Macopharma: Honoraria; Biomarin/Prosensa: Other, Research Funding; Sagent: Other, Research Funding; Rovi: Other, Research Funding; Gore inc.: Other, Research Funding; Bayer Healthcare: Other, Research Funding; Paringenix: Other, Research Funding; BMS: Honoraria, Other, Research Funding; MSD: Honoraria, Other, Research Funding; Boehringer Ingelheim: Honoraria, Other, Research Funding; Aspen: Honoraria, Other, Research Funding; Portola: Other; Ergomed: Other; Instrument Laboratory: Honoraria; Chromatec: Honoraria. Gruel: Stago: Other: symposium fees, Research Funding. Rollin: Stago: Research Funding.

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Abstract 342: Coagulation Factor XII Promotes Platelet Consumption in the Presence of Microbial Polyphosphate Under Shear Flow

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.342 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Jevgenia Zilberman-Rudenko ◽

Stephanie E Reitsma ◽

Chantal P Wiesenekker ◽

Cristina Puy ◽

Stephanie A Smith ◽

...

Keyword(s):

Shear Flow ◽

Platelet Activation ◽

Whole Blood ◽

Thrombin Generation ◽

Thrombus Formation ◽

Coagulation Factor ◽

Dependent Manner ◽

Fibrin Formation ◽

Contact Pathway ◽

Long Chain

Background: Terminal complications of bacterial sepsis include development of consumptive coagulopathy referred to as disseminated intravascular coagulation. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway is capable of promoting thrombin generation and platelet activation and consumption in whole blood distal to thrombus formation under shear ex vivo and in vivo . Aim: Test the hypothesis that the presence of long-chain polyP in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Methods and Results: Presence of long-chain polyP in whole blood promoted platelet aggregation on immobilized collagen surfaces under shear flow. Long-chain polyP enhanced fibrin formation and shortened clotting times of plasma and whole blood. The addition of long-chain polyP promoted platelet P-selectin expression, microaggregate formation and platelet consumption in the bloodstream under shear in a FXII-dependent manner. Moreover, long-chain polyP accelerated thrombus formation on immobilized collagen surfaces under shear flow. Distal to the sites of thrombus formation, platelet consumption was dramatically enhanced in the presence of long-chain polyP in the bloodstream. Inhibiting contact activation of the coagulation pathway reduced fibrin formation on collagen as well as platelet consumption in the bloodstream distal to the site of thrombus formation. Conclusions: This study demonstrates that bacterial-type long-chain polyP promotes FXII-mediated thrombin generation and platelet activation in the flowing blood and could exaggerate sepsis-associated thrombotic processes, consumptive coagulopathy and thrombocytopenia.

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Abstract 56: Coagulation Factor XI Promotes Distal Platelet Activation and Single Platelet Consumption in the Bloodstream Under Shear Flow

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.56 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Jenya Zilberman-Rudenko ◽

Chantal Wiesenekker ◽

Asako Itakura ◽

Owen J McCarty

Keyword(s):

Shear Flow ◽

Platelet Activation ◽

Platelet Adhesion ◽

Ex Vivo ◽

Thrombus Formation ◽

Coagulation Factor ◽

Thrombin Inhibitor ◽

Dependent Manner ◽

Factor Xi ◽

Primate Model

Objective: Coagulation factor XI (FXI) has been shown to contribute to thrombus formation on collagen or tissue factor (TF)-coated surfaces in vitro and in vivo by enhancing thrombin generation. Whether the role of the intrinsic pathway of coagulation is restricted to the local site of thrombus formation is unknown. This study was designed to determine whether FXI could promote both proximal and distal platelet activation and aggregate formation in the bloodstream. Approach and Results: Pharmacological blockade of FXI activation or thrombin activity in blood did not affect local platelet adhesion, yet reduced local platelet aggregation, thrombin localization and fibrin formation on immobilized collagen and TF under shear flow, ex vivo . Downstream of the thrombus formed on immobilized collagen or collagen and 10 pM TF, platelet CD62P expression and microaggregate formation and progressive platelet consumption were significantly reduced in the presence of FXI-function blocking antibodies or a thrombin inhibitor in a shear rate- and time-dependent manner. In a non-human primate model of thrombus formation, we found that inhibition of FXI reduced single platelet consumption in the bloodstream distal to a site of thrombus formation. Conclusions: This study demonstrates that the FXI-thrombin axis contributes to distal platelet activation and procoagulant microaggregate formation in the blood flow downstream of the site of thrombus formation. Our data highlights FXI as a novel therapeutic target for inhibiting distal platelet activation without affecting proximal platelet adhesion.

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FUNDC2 regulates platelet activation through AKT/GSK-3β/cGMP axis

Cardiovascular Research ◽

10.1093/cvr/cvy311 ◽

2018 ◽

Vol 115 (11) ◽

pp. 1672-1679 ◽

Cited By ~ 1

Author(s):

Qi Ma ◽

Weilin Zhang ◽

Chongzhuo Zhu ◽

Junling Liu ◽

Quan Chen

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Rich Plasma ◽

Thrombus Formation ◽

Mitochondrial Protein ◽

Dependent Manner ◽

Clot Retraction ◽

Akt Phosphorylation ◽

Gsk 3Β

Abstract Aims AKT kinase is vital for regulating signal transduction in platelet aggregation. We previously found that mitochondrial protein FUNDC2 mediates phosphoinositide 3-kinase (PI3K)/phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent AKT phosphorylation and regulates platelet apoptosis. The aim of this study was to evaluate the role of FUNDC2 in platelet activation and aggregation. Methods and results We demonstrated that FUNDC2 deficiency diminished platelet aggregation in response to a variety of agonists, including adenosine 5′-diphosphate (ADP), collagen, ristocetin/VWF, and thrombin. Consistently, in vivo assays of tail bleeding and thrombus formation showed that FUNDC2-knockout mice displayed deficiency in haemostasis and thrombosis. Mechanistically, FUNDC2 deficiency impairs the phosphorylation of AKT and downstream GSK-3β in a PI3K-dependent manner. Moreover, cGMP also plays an important role in FUNDC2/AKT-mediated platelet activation. This FUNDC2/AKT/GSK-3β/cGMP axis also regulates clot retraction of platelet-rich plasma. Conclusion FUNDC2 positively regulates platelet functions via AKT/GSK-3β/cGMP signalling pathways, which provides new insight for platelet-related diseases.

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Pathophysiology of Heparin-Induced Thrombocytopenia

Archives of Pathology & Laboratory Medicine ◽

10.5858/2000-124-1657-pohit ◽

2000 ◽

Vol 124 (11) ◽

pp. 1657-1666 ◽

Cited By ~ 4

Author(s):

Fabrizio Fabris ◽

Sarfraz Ahmad ◽

Giuseppe Cella ◽

Walter P. Jeske ◽

Jeanine M. Walenga ◽

...

Keyword(s):

Platelet Activation ◽

Serotonin Release ◽

Platelet Factor 4 ◽

Thrombin Inhibitors ◽

Platelet Factor ◽

Cellular Activation ◽

Heparin Induced Thrombocytopenia ◽

New Information ◽

Study Selection ◽

Release Assay

Abstract Objective.—This review of heparin-induced thrombocytopenia (HIT), the most frequent and dangerous side effect of heparin exposure, covers the epidemiology, pathophysiology, clinical presentation, diagnosis, and treatment of this disease syndrome. Data Sources and Study Selection.—Current consensus of opinion is given based on literature reports, as well as new information where available. A comprehensive analysis of the reasons for discrepancies in incidence numbers is given. The currently known mechanism is that HIT is mediated by an antibody to the complex of heparin–platelet factor 4, which binds to the Fc receptor on platelets. New evidence suggests a functional heterogeneity in the anti-heparin-platelet factor 4 antibodies generated to heparin, and a “superactive” heparin-platelet factor 4 antibody that does not require the presence of heparin to promote platelet activation or aggregation has been identified. Up-regulation of cell adhesion molecules and inflammatory markers, as well as preactivation of platelets/endothelial cells/leukocytes, are also considered to be related to the pathophysiology of HIT. Issues related to the specificity of currently available and new laboratory assays that support a clinical diagnosis are addressed in relation to the serotonin-release assay. Past experience with various anticoagulant treatments is reviewed with a focus on the recent successes of thrombin inhibitors and platelet GPIIb/IIIa inhibitors to combat the platelet activation and severe thrombotic episodes associated with HIT. Conclusions.—The pathophysiology of HIT is multifactorial. However, the primary factor in the mediation of the cellular activation is due to the generation of an antibody to the heparin-platelet factor 4 complex. This review is written as a reference for HIT research.

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Omega-6 DPA and its 12-lipoxygenase–oxidized lipids regulate platelet reactivity in a nongenomic PPARα-dependent manner

Blood Advances ◽

10.1182/bloodadvances.2020002493 ◽

2020 ◽

Vol 4 (18) ◽

pp. 4522-4537

Author(s):

Jennifer Yeung ◽

Reheman Adili ◽

Adriana Yamaguchi ◽

Cody J. Freedman ◽

Angela Chen ◽

...

Keyword(s):

Fatty Acid ◽

Platelet Activation ◽

Platelet Reactivity ◽

Thrombus Formation ◽

Docosapentaenoic Acid ◽

Peroxisome Proliferator ◽

Dependent Manner ◽

Cardiovascular Protection ◽

Omega 6 ◽

12 Lipoxygenase

Abstract Arterial thrombosis is the underlying cause for a number of cardiovascular-related events. Although dietary supplementation that includes polyunsaturated fatty acids (PUFAs) has been proposed to elicit cardiovascular protection, a mechanism for antithrombotic protection has not been well established. The current study sought to investigate whether an omega-6 essential fatty acid, docosapentaenoic acid (DPAn-6), and its oxidized lipid metabolites (oxylipins) provide direct cardiovascular protection through inhibition of platelet reactivity. Human and mouse blood and isolated platelets were treated with DPAn-6 and its 12-lipoxygenase (12-LOX)–derived oxylipins, 11-hydroxy-docosapentaenoic acid and 14-hydroxy-docosapentaenoic acid, to assess their ability to inhibit platelet activation. Pharmacological and genetic approaches were used to elucidate a role for DPA and its oxylipins in preventing platelet activation. DPAn-6 was found to be significantly increased in platelets following fatty acid supplementation, and it potently inhibited platelet activation through its 12-LOX–derived oxylipins. The inhibitory effects were selectively reversed through inhibition of the nuclear receptor peroxisome proliferator activator receptor-α (PPARα). PPARα binding was confirmed using a PPARα transcription reporter assay, as well as PPARα−/− mice. These approaches confirmed that selectivity of platelet inhibition was due to effects of DPA oxylipins acting through PPARα. Mice administered DPAn-6 or its oxylipins exhibited reduced thrombus formation following vessel injury, which was prevented in PPARα−/− mice. Hence, the current study demonstrates that DPAn-6 and its oxylipins potently and effectively inhibit platelet activation and thrombosis following a vascular injury. Platelet function is regulated, in part, through an oxylipin-induced PPARα-dependent manner, suggesting that targeting PPARα may represent an alternative strategy to treat thrombotic-related diseases.

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DETECTION OF ACTIVATED PLATELETS IN WHOLE BLOOD BY FLOW CYTOMETRY

10.1055/s-0038-1643830 ◽

1987 ◽

Cited By ~ 4

Author(s):

S J Shattil ◽

J A Hoxie ◽

M Cunningham ◽

C S Abrahms ◽

J O’Brien ◽

...

Keyword(s):

Flow Cytometry ◽

Monoclonal Antibodies ◽

Platelet Activation ◽

Whole Blood ◽

Thrombus Formation ◽

Membrane Surface ◽

White Blood Cells ◽

Color Flow ◽

Platelet Surface ◽

Activated Platelets

Platelets may become activated in a number of clinical disorders and participate in thrombus formation. We have developed a direct test for activated platelets in whole blood that utilizes dual-color flow cytometry and requires no washing steps. Platelets were distinguished from erythrocytes and white blood cells in the flow cytometer by labeling the platelets with biotin-AP1, an antibody specific for membrane glycoprotein lb, and analyzing the cells for phycoerythrin-streptavidin fluorescence. Membrane surface changes resulting from platelet activation were detected with three different FITC-labeled monoclonal antibodies: 1) PAC1, an antibody specific for the fibrinogen receptor on activated platelets; 2) 9F9, which binds to the D-domain of fibrinogen and detects platelet-bound fibrinogen; and 3) S12, which binds to an alpha-granule membrane protein that associates with the platelet surface during secretion. Unstimulated platelets demonstrated no PAC1, 9F9, or S12-specific fluorescence, indicating that they did not bind these antibodies. Upon stimulation with agonists, however, the platelets demonstrated a dose-dependent increase in FITC-fluorescence. The binding of 9F9 to activated platelets required fibrinogen. Low concentrations of ADP and epinephrine, which induce fibrinogen receptors but little secretion, stimulated near-maximal PAC1 or 9F9 binding but little S12 binding. On the other hand, a concentration of phorbol myristate acetate that evokes full platelet aggregation and secretion induced maximal binding of all three antibodies. When blood samples containing activated and non-activated platelets were mixed, as few as 0.8% activated platelets could be detected by this technique. There was a direct correlation between ADP-induced FITC-PAC1 binding and binding determined in a conventional 125I-PAC1 binding assay (r = 0.99; p < 0.001). These studies demonstrate that activated platelets can be reliably detected in whole blood using activation-dependent monoclonal antibodies and flow cytometry. This method may be useful to assess the degree of platelet activation and the efficacy platelet inhibitor therapy in thrombotic disorders.

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A New Mechanism of Platelet Activation and Oxidative Death Induced by ADAMTS-18 and Regulating Bleeding Time.

Blood ◽

10.1182/blood.v110.11.133.133 ◽

2007 ◽

Vol 110 (11) ◽

pp. 133-133

Author(s):

Zongdong Li ◽

Michael Nardi ◽

Ruimin Pan ◽

Herman Yee ◽

Simon Karpatkin

Keyword(s):

Amino Acid ◽

Platelet Activation ◽

Bleeding Time ◽

Ex Vivo ◽

Thrombus Formation ◽

Activation Mechanism ◽

Dependent Manner ◽

Cell Secretion ◽

Concentration Dependent Manner ◽

Buffer Control

Abstract Anti-platelet integrin GPIIIa49-66 Ab obtained from HIV-ITP patients (or raised in rabbits) induces complement-independent platelet oxidative fragmentation and death by activating platelet 12-lipoxygenase (generation of 12(S)-HETE) and NADPH oxidase (JCI, 113:973, 2004). Platelet oxidative fragmentation is measured by flow cytometry of generated microparticles as well as intracellular DCFH oxidation. We now report that oxidative fragmentation in human platelets is preceded by Ca++ flux and P-selectin activation, n=6. However, the activation mechanism is different from classic platelet activation in that it is not inhibited by PGE1 or dibutryl cyclic AMP and is operative with Gαq−/− mouse platelets, whereas under these conditions, thrombin-induced platelet activation is completely inhibited, n=5–6. We chose to identify putative physiologic ligands that behave similarly to the GPIIIa49-66 Ab, and are therefore capable of regulating platelet reactive oxygen species (ROS) as well as arterial thrombus formation. The GPIIIa49-66 platelet peptide was used as bait to screen a 7-mer peptide phage display library. A peptide was found with 70% homology at the C-terminal position of ADAMTS-18, an ‘orphan’ disintegrin and metalloproteinase with thrombospondin (TSR)-like motifs, with unknown substrate. We have found it present in HUVEC as well as human pulmonary artery endothelial cells, on fixed sections of pathology specimens employing immunohistochemistry with a specific rabbit Ab raised against a C-terminal 18 mer peptide ADAMTS-18 (no staining with preimmune Ab). Recombinant ADAMTS-18 was produced in HEK 293 T cells and shown to induce ROS and oxidative platelet fragmentation in an identical kinetic fashion as anti-GPIIIa49-66 Ab. HUVEC ADAMTS-18 activity could be inhibited by a human scFv Ab raised against its C-terminal 18 mer peptide, as well as the ADAMTS-18 peptide itself, but not by a rabbit Ab against the N-terminal domain or an irrelevant peptide. Endothelial cell secretion and activation of ADAMTS-18 was optimally induced with 0.5 u/ml thrombin at 2 – 4 hrs, n=3–4. The truncated 385 amino acid C-terminal rADAMTS-18 fragment containing the 4 TSR motifs (produced in E.coli) had full activity at (<0.3 uM) whereas the C-terminal 66 amino acid fragment not containing the 18-mer binding site was inactive at 65 fold higher concentration, n=4. The physiologic significance of ADAMTS-18 was supported by demonstrating its secretion into plasma following iv injection of 4–16 u/ml thrombin into mice. Wild type mice have no detectable ADAMTS-18 in their plasma, with a sensitive ELISA assay (1 ng detectability). Thrombin stimulated mice secrete ADAMTS-18 in a concentration dependent manner. Platelet aggregates produced ex vivo with ADP and fibrinogen were destroyed with ADAMTS-18 as documented by LDH release at 1, 2 and 4 hrs of 83, 241 and 260 fold respectively, of PBS buffer control. In vivo tail vein bleeding time was shortened 4.5 fold with 1 hr prior infusion of 25 ug of a polyclonal rabbit IgG against ADAMTS-18, but not with preimmune IgG, n=10. Thus, a new mechanism is proposed for platelet activation, ROS release, death and platelet thrombus regulation, via platelet membrane oxidative fragmentation induced by thrombin-induced secretion and activation of ADAMTS-18.

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Vascular Consequences of Cocaine Exposure.

Blood ◽

10.1182/blood.v110.11.3718.3718 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3718-3718

Author(s):

William E. Hobbs ◽

Chen Jun-Mei ◽

López A. José

Keyword(s):

Endothelial Cells ◽

Platelet Activation ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Vascular Endothelium ◽

Whole Blood ◽

Fold Increase ◽

Dependent Manner ◽

Cocaine Exposure ◽

Cocaine Use

Abstract Cocaine use is associated with sudden cardiac death, cardiac ischemia, and stroke in patients with no additional risk factors and is a frequent cause of these syndromes in patients with sickle cell disease. Pathologic findings include platelet-rich microthrombi and an increased plasma concentration of von Willebrand Factor (VWF). These findings suggest that, in addition to the well-known cocaine effects of vasoconstriction and increased tissue oxygen demand, activation of platelets and/or endothelial cells contributes to cocaine-induced ischemia. However, studies investigating the effect of cocaine on platelet functions have been inconclusive, finding both platelet activation and inhibition depending on the assay used. Further, the ability of cocaine to activate the vascular endothelium has not been examined, in particular, the endothelial secretion of the most adhesive forms of VWF, the ultralarge forms (ULVWF). ULVWF are long VWF multimers that remain tethered to the endothelial surface upon secretion, extend into the blood vessel lumen under laminar flow in long strings measuring up to 0.5 cm in length, and have multiple exposed binding sites for receptors on platelets, erythrocytes, and leukocytes. Elevated levels of ULVWF, due either to enhanced secretion or defective processing, have been implicated in diseases such as thrombotic thrombocytopenic purpura (TTP) and sickle cell anemia. We hypothesized that a major consequence of cocaine exposure is activation of the vascular endothelium to secrete ULVWF, which would provide a platform for blood cell adhesion and subsequent thrombosis or vaso-occlusion. We evaluated the ability of cocaine to stimulate ULVWF from cultured endothelial cells in a parallel-plate flow chamber assay and found that 1 μg/ml cocaine, a level comparable to peak blood levels detectable in cocaine abusers, efficiently induced secretion of ULVWF capable of binding platelets under flow conditions similar to that induced by histamine 6 μg/ml (3.92 ULVWF strings/field with cocaine vs. 4.62 strings/field with histamine). We also assessed the activation of platelets exposed in vitro to cocaine by flow cytometry, using two markers of platelet activation: P-selectin expression (which signals a-granule release) and conformational activation of the platelet integrin aIIbb3, detected with the antibody PAC-1. We found that when platelet-rich plasma was incubated with cocaine at concentrations from 0.1 μg/ml to 10 μg/ml, there was no increase in P- selectin exposure or PAC-1 binding. Furthermore, pretreatment of platelets with cocaine inhibited the ability of platelets to subsequently be activated by ADP in a dose-dependent manner. We did not observe any increase in mean fluorescence above background in ADP stimulated platelets pre-incubated with 1 μg/ml cocaine for P-selectin or PAC-1 binding. However, exposure of platelets in whole blood to 1 μg/ml cocaine resulted in a 3.2-fold increase in P- selectin exposure and a 5.4-fold increase in PAC-1 binding. These results indicate that cocaine directly activates the vascular endothelium to secrete ULVWF, and activates platelets indirectly, involving as yet unknown factors in whole blood, resulting in the formation of microthrombi. These effects of cocaine are likely to have pathogenic roles in cardiovascular syndromes associated with cocaine use, including the triggering of vaso-occlusive crises in sickle cell anemia and may explain the observed association of cocaine use with TTP.

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Neutrophil Extracellular Traps Induce Platelet Adhesion and Thrombus Formation.

Blood ◽

10.1182/blood.v114.22.1345.1345 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1345-1345 ◽

Cited By ~ 4

Author(s):

Tobias Fuchs ◽

Alexander Brill ◽

Daniel Dürschmied ◽

Daphne Schatzberg ◽

John H. Hartwig ◽

...

Keyword(s):

Whole Blood ◽

Platelet Adhesion ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Neutrophil Extracellular Traps ◽

Thrombin Inhibitor ◽

Human Neutrophils ◽

Dependent Manner ◽

Extracellular Traps ◽

Deep Vein

Abstract Abstract 1345 Introduction Thrombus stability is provided by very large polymers adhering to platelets and anchoring the thrombus to the vessel wall. The best described polymers are fibrin and von Willebrand Factor (VWF). Activated neutrophils and other leukocytes can form an extracellular fibrous network which is composed of DNA, histones, and granular proteins. These neutrophil extracellular traps (NETs) are present in various inflammatory diseases. In deep vein thrombosis (DVT) inflammation closely cooperates with thrombosis. Here we examine whether NETs provide a new means to support the adhesion and recruitment of platelets and whether NETs are present in DVT. Methods and Results: To study the interaction of platelets with NETs, we isolated human neutrophils, induced NET formation and perfused over the NETs human platelets in plasma or whole blood anticoagulated with the thrombin inhibitor PPACK. Microscopic analysis revealed that under flow platelets adhere avidly to NETs. Perfusion of whole blood at physiological shear resulted in formation of thrombi on NETs in a time dependent manner. Addition of DNase1 degraded NETs and removed all platelets and thrombi demonstrating their adhesion to NETs. Thrombus formation on NETs was absent if blood was supplemented with EDTA indicating the requirement for divalent cations. Perfusion of NETs with heparinized blood dismantled NETs and prevented thrombus formation. Incubation of NETs with heparin alone released histones from NETs, indicating that heparin destroys the chromatin backbone of NETs. Furthermore, immunocytochemistry revealed that NETs were able to bind platelet adhesion molecules VWF and fibronectin from human plasma. Immunohistochemical analysis of a baboon deep vein thrombus showed abundant extracellular chromatin which co-localized with fibronectin and VWF. Conclusions: We show that extracellular traps are able to promote thrombosis in vitro and are abundant in vivo in DVT. We propose that extracellular chromatin provides a new type of scaffold that promotes platelet adhesion, activation, and aggregation and may be important for thrombus initiation or stability. Disclosures No relevant conflicts of interest to declare.

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CLEC-2 is an essential platelet-activating receptor in hemostasis and thrombosis

Blood ◽

10.1182/blood-2009-05-222273 ◽

2009 ◽

Vol 114 (16) ◽

pp. 3464-3472 ◽

Cited By ~ 130

Author(s):

Frauke May ◽

Ina Hagedorn ◽

Irina Pleines ◽

Markus Bender ◽

Timo Vögtle ◽

...

Keyword(s):

Platelet Activation ◽

Thrombus Formation ◽

Antibody Treatment ◽

Cellular Activation ◽

Primary Hemostasis ◽

Arterial Thrombus ◽

Normal Adhesion

Abstract Damage to the integrity of the vessel wall leads to exposure of the subendothelial extracellular matrix (ECM), triggering platelet activation and aggregation. This process is essential for primary hemostasis but it may also lead to arterial thrombosis. Although the mechanisms underlying platelet activation on the ECM are well explored, it is less clear which receptors mediate cellular activation in a growing thrombus. Here we studied the role of the recently identified C-type lectin-like receptor 2 (CLEC-2) in this process. We show that anti–CLEC-2 antibody treatment of mice leads to complete and highly specific loss of CLEC-2 in circulating platelets for several days. CLEC-2–deficient platelets displayed normal adhesion under flow, but subsequent aggregate formation was severely defective in vitro and in vivo. As a consequence, CLEC-2 deficiency was associated with increased bleeding times and profound protection from occlusive arterial thrombus formation. These results reveal an essential function of CLEC-2 in hemostasis and thrombosis.

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