Comparison of the Thrombogenicity of a Bare and Antithrombogenic Coated Flow Diverter in an In Vitro Flow Model.

BACKGROUND: Dual antiplatelet therapy is a pre-requisite for flow diverter (FD) implantation. The purpose of this study was to assess the thrombogenicity of the p48 FD, coated with the newly developed phenox Hydrophilic Polymer Coating (p48_HPC, phenox GmbH, Germany) in comparison with uncoated p48 FDs in an in vitro flow model (Chandler Loop). METHODS: p48 and p48_HPC FDs were implanted into silicon tubes filled with whole human blood and incubated at 37 °C under pulsating flow. After 120 min, platelet count was determined in the blood. Platelet activation markers (PAR1) and formation of microparticles were analyzed in a flow cytometer. Fluorescence microscopy of CD42a positive cells and scanning electron microscopy was used to detect adherent platelets on the wire surface. RESULTS: Platelets in contact with the uncoated p48 FDs are significantly more activated than those incubated with p48_HPC (73 ± 9% vs. 65 ± 6%, p < 0.05) and release more microparticles (1.8 ± 0.5 vs. 1.4 ± 0.4, p < 0.05). The platelet count after 120-min circulation in the Chandler Loop was significantly lower for the uncoated p48 compared to the p48_HPC indicating significantly greater adherence of the platelets to the p48 (71 ± 8% vs. 87 ± 5%, p < 0.05). SEM and fluorescent antibody imaging revealed minimal platelet adherence to the surface of the p48_HPC compared to the uncoated p48. CONCLUSION: The pHPC coating significantly reduces thrombogenicity of the p48 FD. This may help to reduce the risk of thromboembolic complications when using these devices. A reduction in antiplatelet therapy may be possible.

Reversibility of platelet P2Y12 inhibition by platelet supplementation: ex vivo and in vitro comparisons of prasugrel, clopidogrel and ticagrelor.

Essentials Successful outcome of platelet transfusion depends on specific antiplatelet therapy in use. We assessed if ticagrelor, clopidogrel or prasugrel impacts on donor platelet activity ex vivo. Ticagrelor and/or its active metabolite in plasma or bound to platelets can inhibit donor platelets. This might compromise the effectiveness of platelet transfusion therapy. SUMMARY: Background Platelet transfusion is the conventional approach to restore platelet function during acute bleeds or surgery, but successful outcome depends on the specific antiplatelet therapy. Notably ticagrelor is associated with inadequate recovery of platelet function after platelet transfusion. We examined whether plasma and/or platelets from ticagrelor-treated patients influence donor platelet function, in comparison with clopidogrel and prasugrel. Methods Platelet transfusion was mimicked ex vivo by mixing naïve donor platelet-rich plasma (PRP) or gel-filtered platelets (GFP) in defined proportions with PRP, plasma or GFP from cardiovascular patients receiving standard care including medication with prasugrel, clopidogrel or ticagrelor (n = 20 each). Blood was taken 4 h after the previous dose. HLA2/HLA28 haplotyping let us distinguish net (all platelet) and individual patient/donor platelet reactivity in mixtures of patient/donor platelets, measured by flow cytometry analysis of ADP-induced fibrinogen binding and CD62P expression. Results ADP responsiveness of donor platelets was dramatically reduced by even low (10%) concentrations of PRP or plasma from ticagrelor-treated patients. Clopidogrel and prasugrel were associated with more modest donor platelet inhibition. GFP from ticagrelor-treated patients but not patients receiving clopidogrel or prasugrel also suppressed donor GFP function upon mixing, suggesting the transfer of ticagrelor from patient platelets to donor platelets. This transfer did not lead to recovery of ADP responsiveness of patient's platelets. Conclusion Collectively, these observations support the concept that ticagrelor and/or its active metabolite in plasma or bound to platelets can inhibit donor platelets, which might compromise the effectiveness of platelet transfusion therapy.

[Diagnosis of inherited diseases of platelet function. Interdisciplinary S2K guideline of the Permanent Paediatric Committee of the Society of Thrombosis and Haemostasis Research (GTH e. V.)]. / Diagnose angeborener Störungen der Thrombozytenfunktion. Interdisziplinäre S2K-Leitlinie der Ständigen Kommission Pädiatrie der Gesellschaft für Thrombose- und Hämostaseforschung e. V.

Congenital disorders of platelet function are a heterogeneous group of disorders that are often not detected until bleeding occurs. In clinical settings only a few methods have proven to be useful for identification and classification of inherited platelet disorders. For a rational diagnostic approach, a stepwise algorithm is recommended. Patient history and clinical investigation are mandatory. Von Willebrand disease and other coagulation disorders should always be ruled out prior to specific platelet testing. Platelet count, size, volume (MPV) and morphology may guide further investigations. The PFA-100® CT is suited for screening for severe platelet defects. Platelet aggregometry allows assessment of multiple aspects of platelet function. Flow cytometry enables diagnosis of thrombasthenia Glanzmann, Bernard-Soulier syndrome and storage pool defects. Molecular genetics may confirm a putative diagnosis or pave the way for identifying new defects. We present an unabridged version of the interdisciplinary guideline.

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: biochemistry and physiology]. / Blutplättchen: Biochemie und Physiologie.

This article reviews the roles of blood platelets in haemostasis as well as in the pathogenesis of thromboembolic diseases. Besides the basic processes in primary haemostasis, platelet adhesion, platelet secretion, platelet aggregation, clot retraction, the new model of thrombin formation on the platelet surface is presented. The different signal transduction pathways in platelets are a main focus of this review.

[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.

[Blood platelets: biochemistry and physiology]. / Blutplättchen: biochemie und physiologie.

This article reviews the roles of blood platelets in different physiologic and pathologic processes in the human body. Besides the basic processes in primary haemostasis, platelet adhesion, platelet secretion, platelet aggregation, clot retraction, the new model of thrombin formation on the platelet surface is presented. The different signal transduction pathways in platelets are a main focus of this review.

Aggretin, a heterodimeric C-type lectin from Calloselasma rhodostoma (Malayan pit viper), stimulates platelets by binding to α2ß1 integrin and glycoprotein Ib, activating Syk and phospholipase Cγ 2, but does not involve the glycoprotein VI/Fc receptor γ chain collagen receptor.

Aggretin, a potent platelet activator, was isolated from Calloselasma rhodostoma venom, and 30-amino acid N-terminal sequences of both subunits were determined. Aggretin belongs to the heterodimeric snake C-type lectin family and is thought to activate platelets by binding to platelet glycoprotein alpha(2)beta(1). We now show that binding to glycoprotein (GP) Ib is also required. Aggretin-induced platelet activation was inhibited by a monoclonal antibody to GPIb as well as by antibodies to alpha(2)beta(1). Binding of both of these platelet receptors to aggretin was confirmed by affinity chromatography. No binding of other major platelet membrane glycoproteins, in particular GPVI, to aggretin was detected. Aggretin also activates platelets from Fc receptor gamma chain (Fcgamma)-deficient mice to a greater extent than those from normal control mice, showing that it does not use the GPVI/Fcgamma pathway. Platelets from Fcgamma-deficient mice expressed fibrinogen receptors normally in response to collagen, although they did not aggregate, indicating that these platelets may partly compensate via other receptors including alpha(2)beta(1) or GPIb for the lack of the Fcgamma pathway. Signaling by aggretin involves a dose-dependent lag phase followed by rapid tyrosine phosphorylation of a number of proteins. Among these are p72(SYK), p125(FAK), and PLCgamma2, whereas, in comparison with collagen and convulxin, the Fcgamma subunit neither is phosphorylated nor coprecipitates with p72(SYK). This supports an independent, GPIb- and integrin-based pathway for activation of p72(SYK) not involving the Fcgamma receptor.

Alboaggregin A activates platelets by a mechanism involving glycoprotein VI as well as glycoprotein Ib.

The snake venom C-type lectin alboaggregin A (or 50-kd alboaggregin) from Trimeresurus albolabris was previously shown to be a platelet glycoprotein (GP) Ib agonist. However, investigations of the signal transduction induced in platelets showed patterns of tyrosine phosphorylation that were different from those of other GPIb agonists and suggested the presence of an additional receptor. In this study, the binding of biotinylated alboaggregin A to platelet lysates, as well as affinity chromatography evaluations of platelet lysates on an alboaggregin A-coated column, indicated that this other receptor is GPVI. Additional experiments with reagents that inhibit either GPIb or GPVI specifically supported this finding. These experiments also showed that both GPIb and GPVI have a role in the combined signaling and that the overall direction this takes can be influenced by inhibitors of one or the other receptor pathway.

The GPIb thrombin-binding site is essential for thrombin-induced platelet procoagulant activity.

The role of the platelet glycoprotein (GP) Ib-V-IX receptor in thrombin activation of platelets has remained controversial although good evidence suggests that blocking this receptor affects platelet responses to this agonist. The mechanism of expression of procoagulant activity in response to platelet agonists is also still obscure. Here, the binding site for thrombin on GPIb is shown to have a key role in the exposure of negatively charged phospholipids on the platelet surface and thrombin generation, in response to thrombin, which also requires protease-activated receptor-1, GPIIb-IIIa, and platelet-platelet contact. Von Willebrand factor binding to GPIb is not essential to initiate development of platelet procoagulant activity. Inhibition of fibrinogen binding to GPIIb-IIIa also failed to block platelet procoagulant activity. Both heparin and low molecular weight heparin block thrombin-induced platelet procoagulant activity, which may account for part of their clinical efficacy. This study demonstrates a new, critical role for platelet GPIb in hemostasis, showing that platelet activation and coagulation are tightly interwoven, which may have implications for alternative therapies for thrombotic diseases.

Protein A is the von Willebrand factor binding protein on Staphylococcus aureus.

Endovascular infection is a highly critical complication of invasive Staphylococcus aureus disease. For colonization, staphylococci must first adhere to adhesive endovascular foci. Von Willebrand factor (vWF) is a large, multimeric glycoprotein mediating platelet adhesion at sites of endothelial damage. Earlier it was demonstrated that vWF binds to and promotes the surface adhesion of S. aureus, prompting this effort to identify the vWF adhesin. In Western ligand assays of S. aureus lysates, staphylococcal protein A (SPA) was recognized by purified vWF. Surface plasmon resonance demonstrated the binding of soluble vWF to immobilized recombinant protein A with a K(d) of 1.49 x 10(-8) mol/L. Using flow cytometry, the binding of fluorescein isothiocyanate-labeled vWF to S. aureus was found to be saturable and inhibitable by unlabeled vWF, antiprotein-A antibodies, or IgG. Isogenic Deltaspa::Tc(r) mutants were constructed by the insertion of a tetracycline resistance cassette into spa using allelic replacement, and it exhibited decreased binding of soluble vWF and decreased adhesion to vWF-adsorbed surfaces. The interaction was restored on complementation of the mutants with spa-containing plasmid pSPA7235. In conclusion, protein A confers interaction of S. aureus with soluble and immobilized vWF in a newly discovered function characterizing protein A as a novel member of the staphylococcal surface protein adhesin superfamily and suggesting its potential role in the pathogenesis of endovascular staphylococcal disease.

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.

Anticoagulative management of patients requiring left ventricular assist device implantation and suffering from heparin-induced thrombocytopenia type II.

BACKGROUND: Heparin-induced thrombocytopenia type II (HIT II) is a rare but life-threatening side effect of heparin therapy. We describe the perioperative anticoagulative management of patients tested positive for HIT II and requiring implantation of a left ventricular assist device (LVAD). METHODS: We report on 3 patients with a different perioperative anticoagulative management (preoperative, intraoperative, and postoperative anticoagulation with danaparoid-sodium; preoperative anticoagulation with recombinant hirudin, anticoagulation with danaparoid-sodium intraoperatively and postoperatively; preoperative anticoagulation with recombinant hirudin, intraoperative anticoagulation with heparin, and postoperative anticoagulation with danaparoid-sodium) and discuss the difficulties of the treatment. RESULTS: Anticoagulation with alternative drugs such as recombinant hirudin and danaparoid-sodium led to serious and life-threatening bleeding complications as well as to thromboembolic events in the first 2 patients. Therefore the third patient underwent LVAD implantation using heparin for intraoperative anticoagulation to avoid administration of high doses of recombinant hirudin or danaparoid-sodium. Despite very low anti-factor Xa activities, when using danaparoid-sodium postoperatively, the patient suffered from a bleeding complication on the 4th day after LVAD implantation requiring reexploration. CONCLUSIONS: In selected cases (negative heparin-induced platelet aggregation (HIPA) test at the time of LVAD implantation and continuation of postoperative anticoagulation with recombinant hirudin or danaparoid-sodium), heparin may be used for LVAD implantation in HIT II patients to reduce bleeding complications.

New strategies in diagnosis and treatment of thrombotic thrombocytopenic purpura: case report and review.

UNLABELLED: The pentad of thrombocytopenia, haemolytic anaemia, mild renal dysfunction, neurological signs and fever, classically characterizes the syndrome of thrombotic thrombocytopenic purpura (TTP). TTP usually occurs in adults but also children have been described with this condition. The disorder may take a relapsing course, termed chronic relapsing TTP (CRTTP), which although very rare, may also begin in childhood. Deficiency of a recently identified enzyme, the von Willebrand factor (vWF)-cleaving protease, seems to play a major role in the development of TTP. We report on a 3-year-old boy with a dramatic but typical clinical course of CRTTP. At the time of diagnosis, neurological deficits and multiple cerebral infarctions had already occurred. In plasma, vWF-cleaving protease was completely absent, both during acute TTP and in remission. There was no protease inhibitor detected. Regular infusions of fresh frozen plasma were successfully given for replacement on a prophylactic basis. CONCLUSION: Assay of von Willebrand factor-cleaving protease helps to diagnose a form of thrombotic thrombocytopenic purpura which may be managed by prophylactic treatment with fresh frozen plasma.