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In this article, our goal is to offer an introduction and overview of the diagnostic approach to inherited platelet function defects (iPFDs) for clinicians and laboratory personnel who are beginning to engage in the field. We describe the most commonly used laboratory methods and propose a diagnostic four-step approach, wherein each stage requires a higher level of expertise and more specialized methods. It should be noted that our proposed approach differs from the ISTH Guidance on this topic in some points. The first step in the diagnostic approach of iPFD should be a thorough medical history and clinical examination. We strongly advocate for the use of a validated bleeding score like the ISTH-BAT (International Society on Thrombosis and Haemostasis Bleeding Assessment Tool). External factors like diet and medication have to be considered. The second step should rule out plasmatic bleeding disorders and von Willebrand disease. Once this has been accomplished, the third step consists of a thorough platelet investigation of platelet phenotype and function. Established methods consist of blood smear analysis by light microscopy, light transmission aggregometry, and flow cytometry. Additional techniques such as lumiaggregometry, immune fluorescence microscopy, and platelet-dependent thrombin generation help confirm and specify the diagnosis of iPFD. In the fourth and last step, genetic testing can confirm a diagnosis, reveal novel mutations, and allow to compare unclear genetics with lab results. If diagnosis cannot be established through this process, experimental methods such as electron microscopy can give insight into the underlying disease., Competing Interests: MH: travel support: Sobi; Acommodation/Funded education events: Bayer, NovoNordisk., (Thieme. All rights reserved.)

Congenital platelet disorders are rare and targeted treatment is usually not possible. Inherited platelet function disorders (iPFDs) can affect surface receptors and multiple platelet responses such as defects of platelet granules, signal transduction, and procoagulant activity. If iPFDs are also associated with a reduced platelet count (thrombocytopenia), it is not uncommon to be misdiagnosed as immune thrombocytopenia. Because the bleeding tendency of the different platelet disorders is variable, a correct diagnosis of the platelet defect based on phenotyping, function analysis, and genotyping is essential, especially in the perioperative setting. In the case of a platelet receptor deficiency, such as Bernard-Soulier syndrome or Glanzmann thrombasthenia, not only the bleeding tendency but also the risk of isoimmunization after platelet transfusions or pregnancy has to be considered. Platelet granule disorders are commonly associated with either intrinsically quantitative or qualitative granule defects due to impaired granulopoiesis, or granule release defects, which can also affect additional signaling pathways. Functional platelet defects require expertise in the clinical bleeding tendency in terms of the disorder when using antiplatelet agents or other medications that affect platelet function. Platelet defects associated with hematological-oncological diseases require comprehensive information about the patient including the clinical implication of the genetic testing. This review focuses on genetics, clinical presentation, and laboratory platelet function analysis of iPFDs with or without reduced platelet number. As platelet defects affecting the cytoskeleton usually show thrombocytopenia, but less impaired or normal platelet functional responses, they are not specifically addressed., Competing Interests: KA: Speaker's honoraria: Meet the experts, Sobi, CLS Behring; Travel support: Sobi, Biomarin, NovoNordisc; Leadership or fiduciary role in other board, society, committee or advocacy group, paid or unpaid: ISTH Platelet immunology (Co-Chair): no payment., (Thieme. All rights reserved.)

Objectives: The implantation of flow diverters, or stents in general, necessitates the use of dual anti-platelet treatment with typical regimes including aspirin and a P2Y12 inhibitor. This carries an inherent risk of haemorrhage. We sought to compare the thrombogenicity of the anti-thrombogenic p48 hydrophilic polymer coating compared to the standard uncoated p48 flow diverter using an in vitro thrombogenicity assay., Methods: To evaluate the thrombin generation influenced by the different stent types the stents were placed in wells of a 24-well plate with the addition of plasma from healthy volunteers the thrombin calibrator respectively the PPP-reagent was added. Subsequently, the thrombin substrate was added and the thrombin generation was analysed every 60 s using a thrombinoscope. The assay is calibrated using samples containing a known amount of active thrombin in PPP. Thrombin activity is proportional to the change in fluorescence., Results: The p48 hydrophilic polymer coating shows a significantly lower peak thrombin concentration (1.13 ± 0.21 vs. 1.41 ± 0.22) and longer time to peak thrombin concentration (0.96 ± 0.04 vs. 0.74 ± 0.07) compared to the uncoated p48 device ( p  < 0.01). The responses of the p48 hydrophilic polymer coating were similar to that of the negative control., Conclusion: The hydrophilic polymer coating surface modification significantly reduces the thrombogenicity of the p48 flow diverter. These results corroborate the findings from previous in vitro studies.

We examined the structural and functional consequences of oxidative modification of C-reactive protein (CRP) by hypochlorous acid (HOCl), which can be generated in vivo via the myeloperoxidase/H 2 O 2 /Cl - system. HOCl exposure resulted in the oxidation and chlorination of CRP amino acid residues, leading to protein unfolding, greater surface hydrophobicity and the formation of aggregates. After treatment of isolated platelets with 50μg/ml HOCl-CRP, the modified CRP significantly stimulated platelet activation (over 10-fold increase in the fraction of CD62-positive platelets compared to controls, P<0.008), enhanced deposition of platelets onto immobilized fibrinogen (two-fold rise in platelet adhesion compared to controls, P<0.0001), and induced platelet aggregation by up to 79.5%. The ability of HOCl-CRP to interact with several platelet receptors (TLR-4, GPIIbIIIa) and plasma proteins (C1q, IgG) strongly indicates that HOCl-modification leads to structural changes of CRP resulting in the formation of new ligand binding sites, which is characteristic of the monomeric form of CRP exerting pro-inflammatory effects on a variety of cells. Overall, the oxidation of native CRP by HOCl seems to represent an alternative mechanism of CRP modification, by which CRP reveals its pro-inflammatory and pro-thrombotic properties, and as such, it might be of causal relevance in the pathogenesis of atherosclerosis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aberrant immune responses represent the underlying cause of central nervous system (CNS) autoimmunity, including multiple sclerosis (MS). Recent evidence implicated the crosstalk between coagulation and immunity in CNS autoimmunity. Here we identify coagulation factor XII (FXII), the initiator of the intrinsic coagulation cascade and the kallikrein-kinin system, as a specific immune cell modulator. High levels of FXII activity are present in the plasma of MS patients during relapse. Deficiency or pharmacologic blockade of FXII renders mice less susceptible to experimental autoimmune encephalomyelitis (a model of MS) and is accompanied by reduced numbers of interleukin-17A-producing T cells. Immune activation by FXII is mediated by dendritic cells in a CD87-dependent manner and involves alterations in intracellular cyclic AMP formation. Our study demonstrates that a member of the plasmatic coagulation cascade is a key mediator of autoimmunity. FXII inhibition may provide a strategy to combat MS and other immune-related disorders.

Background: We previously identified protein impurities in plasma-derived factor VIII (pdFVIII) products. The goal of the current experiments was to determine whether these impurities might have clinical relevance, by comparing the effects of pdFVIII and recombinant FVIII (rFVIII) products on cellular stress induction., Methods: The in vitro outcomes on cell stress sensors of 2 pdFVIII products and 1 rFVIII product were evaluated. Microparticle formation was assessed in cells treated with the 3 products. Effects on the mitochondrial membrane potential were measured in cells treated with clinically relevant concentrations of each product., Results: Microparticle formation was induced in platelets by 1 pdFVIII product and in monocytes and granulocytes by both pdFVIII products; the rFVIII product did not affect microparticle formation. Both pdFVIII products, but not the rFVIII product, significantly depolarized the mitochondrial membrane potential., Conclusion: The 2 pdFVIII products tested induced cellular stress in in vitro experiments. No such results were seen with the rFVIII product. Chronic activation of the cell stress defense system and chronic cell irritation may have clinical consequences for patients with hemophilia A.

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.

Diagnostic point of care testing based on thrombelastometry with the aim to fully understand the overall changes in global haemostasis during the perioperative phase is today performed in several operating theatres. Therapeutic measures are based on these laboratory results. Within the scope of a pro and contra outline of the journal this paper comment, why point of care diagnostic solely based on laboratory testing using the ROTEM is not recommendable. On the other hand the author values the thrombelastometry as a supplementary supportive method used directly by haemostasis specialists or in a tight cooperation with such skilled specialists., (© Georg Thieme Verlag Stuttgart · New York.)

Growing evidence supports substantial pathophysiological impact of platelets and their interactions on the development of septic lung failure. We developed a rat model of endotoxemia for direct in situ visualization of pulmonary microcirculation by in vivo fluorescence videomicroscopy. Male Sprague-Dawley rats were assigned to control, endotoxemia (Escherichia coli LPS, 15 mg/kg, i.v.), and fluid management for treatment of LPS-induced hypovolemia (Ringer lactate, hydroxyethyl starch [HES] 6%) groups (n = 7 each). Leukocytes were labeled in vivo by rhodamine, and 5 x 10(6) Calcein-AM-labeled nonactivated platelets were injected. Microcirculatory parameters (vessel diameter, ventilation-perfusion ratio) and adhesive characteristics of platelets and leukocytes (velocity, rolling, sticking) within the pulmonary microcirculation were quantified after endotoxin application under various regimens of fluid substitution for 60 min. A reduction of cell velocity and enhanced cell adhesion was seen in leukocytes and platelets (P < 0.05) after LPS injection. Fluid treatment with HES 6% resulted in a significant increase of platelet's velocity compared with the LPS group (442.86 +/- 20.60 vs. 343.93 +/- 11.17; P < 0.05), whereas Ringer lactate showed no beneficial effects. Similarly, HES 6% normalized LPS-induced platelet rolling and sticking as well as alterations in ventilation-perfusion ratio. Using direct visualization of the pulmonary microcirculation, we observed that platelet and leukocyte interactions are enhanced in the lung during LPS endotoxemia. Fluid therapy with HES 6% seems to have restorative effects on these cellular functions within the pulmonary microcirculation.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that mediates the adaptive response to hypoxia. Increasing evidence suggests a crucial role for HIF-1 in immune reactions. Here we investigated the effect of the Th2 type cytokines IL-4 and IL-10 on HIF-1alpha mediated response in normoxia (21% O2) and hypoxia (1% O2). Incubation of human transformed intestinal cells (HT-29) with IL-4 significantly increased HIF-1alpha protein levels during hypoxia but not during normoxia. Mechanisms involved are IL-4 induced up-regulation of HIF-1alpha gene transcription and the PI3K signaling pathway. The increase in hypoxia-induced accumulation of HIF-1alpha protein after IL-4 treatment did not result in up-regulation of HIF-1 DNA-binding activity or HIF-1 dependent gene expression. IL-10 did not affect HIF-1alpha protein levels.

Myeloid-related protein 8 (MRP8) and MRP14, S100 proteins secreted by activated phagocytes, bind specifically to endothelial cells. The endothelial response to MRP8/MRP14, however, is unknown. Using oligonucleotide microarray analysis, we show for the first time that MRP8/MRP14 induce a thrombogenic, inflammatory response in human microvascular endothelial cells by increasing the transcription of proinflammatory chemokines and adhesion molecules and by decreasing the expression of cell junction proteins and molecules involved in monolayer integrity. All changes on the gene expression level could be confirmed using biochemical and functional assays. We demonstrated that the expression of MRP8/MRP14 closely correlated with the inflammatory activity in systemic vasculitis, confirming the important role of these proteins for distinct inflammatory reactions in endothelia. MRP8/MRP14 may represent novel targets for anti-inflammatory strategies.

The inverse correlation between serum levels of high density lipoprotein (HDL) cholesterol and the risk of coronary heart disease, the protection of susceptible animals from atherosclerosis by transgenic manipulation of HDL metabolism, and several potentially anti-atherogenic in vitro-properties have made HDL metabolism an interesting target for pharmacological intervention in atheroslcerosis. We have previously reviewed the concept of reverse cholesterol transport, which describes both the metabolism and the classic anti-atherogenic function of HDL (Arterioscler. Thromb. Vasc. Biol. 20 2001 13). We here summarize the current understanding of additional biological, potentially anti-atherogenic properties of HDL. HDL inhibits the chemotaxis of monocytes, the adhesion of leukocytes to the endothelium, endothelial dysfunction and apoptosis, LDL oxidation, complement activation, platelet activation and factor X activation but also stimulates the proliferation of endothelial cells and smooth muscle cells, the synthesis of prostacyclin and natriuretic peptide C in endothelial cells, and the activation of proteins C and S. These anti-inflammatory, anti-oxidative, anti-aggregatory, anti-coagulant, and pro-fibrinolytic activities are exerted by different components of HDL, namley apolipoproteins, enzymes, and even specific phospholipids. This complexity further emphasizes that changes in the functionality of HDL rather than changes of plasma HDL-cholesterol levels determine the anti-atherogenicity of therapeutic alterations of HDL metabolism.

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.

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.

The vasodilator-stimulated phosphoprotein (VASP) is associated with actin filaments and focal adhesions, which form the interface between the cytoskeleton and the extracellular matrix. VASP is phosphorylated by both the cAMP- and cGMP-dependent protein kinases in a variety of cells, including platelets and smooth muscle cells. Since both the cAMP and cGMP signalling cascades relax smooth muscle and inhibit platelet activation, it was speculated that VASP mediates these effects by modulating actin filament dynamics and integrin activation. To study the physiological relevance of VASP in these processes, we inactivated the VASP gene in mice. Adult VASP-deficient mice had normal agonist-induced contraction, and normal cAMP- and cGMP-dependent relaxation of intestinal and vascular smooth muscle. In contrast, cAMP- and cGMP-mediated inhibition of platelet aggregation was significantly reduced in the absence of VASP. Other cAMP- and cGMP-dependent effects in platelets, such as inhibition of agonist-induced increases in cytosolic calcium concentrations and granule secretion, were not dependent on the presence of VASP. Our data show that two different cyclic, nucleotide-dependent mechanisms are operating during platelet activation: a VASP-independent mechanism for inhibition of calcium mobilization and granule release and a VASP-dependent mechanism for inhibition of platelet aggregation which may involve regulation of integrin function.

We demonstrate that physiological concentrations of HDL3 inhibit the thrombin-induced platelet fibrinogen binding and aggregation in a time- and concentration-dependent fashion. The underlying mechanism includes HDL3-mediated inhibition of phosphatidylinositol 4,5-bis-phosphate turnover, 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate formation, and intracellular calcium mobilization. The inhibitory effects of HDL3 on inositol 1,4,5-tris-phosphate formation and intracellular calcium mobilization were abolished after covalent modification of HDL3 with dimethylsuberimidate. Furthermore, they could be blocked by calphostin C and bis-indolylmaleimide, 2 highly selective and structurally unrelated protein kinase C inhibitors. However, the inhibitory effects of HDL3 were not blocked by H89, a protein kinase A inhibitor. In addition, HDL3 failed to induce cAMP formation but stimulated the phosphorylation of the protein kinase C 40- to 47-kD major protein substrate. We observed a close temporal relationship between the HDL3-mediated inhibition of thrombin-induced inositol 1,4,5-tris-phosphate formation, intracellular calcium mobilization, and fibrinogen binding and the phosphorylation of the protein kinase C 40- to 47-kD major protein substrate. Taken together, these findings indicate that the HDL3-mediated inhibition of thrombin-induced fibrinogen binding and aggregation occurs via inhibition of phosphatidylinositol 4,5-bis-phosphate turnover and formation of 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate. Protein kinase C may be involved in this process.

CD36, also known as GPIIIb or GPIV, is a main protein of the platelet membrane. Accumulating evidence implicates CD36 in the pathogenesis of atherosclerosis. CD36 binds to cholesteryl-hemisuccinate coupled to agarose. This binding may be of physiologic relevance since CD36 has a long extracellular hydrophobic region at its N-terminus and belongs to a family of proteins involved in lipid metabolism. This newly discovered binding property of CD36 was used to develop a rapid, efficient and gentle two-step purification method for CD36 from human platelets.

Simple collagen-related peptides (CRPs) containing a repeat Gly-Pro-Hyp sequence are highly potent platelet agonists. Like collagen, they must exhibit tertiary (triple-helical) and quaternary (polymeric) structure to activate platelets. Platelet signaling events induced by the peptides are the same as most of those induced by collagen. The peptides do not recognize the alpha 2 beta 1 integrin. To identify the signaling receptor involved, we have evaluated the response to the CRP, Gly-Lys-Hyp(Gly-Pro-Hyp)10-Gly-Lys-Hyp-Gly of platelets with defined functional deficiencies. These studies exclude a primary recognition role for CD36, von Willebrand factor (vWF), or glycoprotein (GP) IIb/IIIa. Thus, both CD36 and vWF-deficient platelets exhibited normal aggregation, normal fibrinogen binding, and normal expression of CD62 and CD63, measured by flow cytometry, in response to the peptide, and there was normal expression of CD62 and CD63 on thrombasthenic platelets. In contrast, GPVI-deficient platelets were totally unresponsive to the peptide, indicating that this receptor recognizes the Gly-Pro-Hyp sequence in collagen. GPVI-deficient platelets showed some fibrinogen binding in response to collagen but failed to aggregate and to express CD62 and CD63. Collagen, but not CRP-XL, contains binding sites for alpha 2 beta 1. Therefore, it is possible that collagen still induces some signaling via alpha 2 beta 1, leading to activation of GPIIb/IIIa. Our findings are consistent with a two-site, two-step model of collagen interaction with platelets involving recognition of specific sequences in collagen by an adhesive receptor such as alpha 2 beta 1 to arrest platelets under flow and subsequent recognition of another specific collagen sequence by an activatory receptor, namely GPVI.

Activated platelets provide a procoagulant surface for the assembly and expression of prothrombinase complex. Expression of activity is associated with the binding of the protease factor Xa (FXa) and the co-factor Va (FVa) to the procoagulant surface. A flow cytometric methodology to measure annexin V-FITC as well as FVa and FXa binding to ionophore A 23187 activated platelets is described. Annexin V-FITC was used to determine platelet exposure of phosphatidylserine. The binding was calcium-dependent and excess of unlabelled annexin V (10-fold) prevented the binding of the labelled protein. The binding of FVa and FXa to platelets was measured using specific FITC-labelled monoclonal antibodies. The FITC labelled antibodies were displaced by 10-to 20-fold excess of unlabelled antibodies. Binding was strictly Ca2+-dependent. Fixation of platelets by formaldehyde caused artificial binding of annexin V, FVa and FXa as well, irrespective of the platelet activation status. Using gel-filtered platelets, the binding of FVa increased with alpha -granule secretion but the amount of stored FVa was not sufficient to saturate the available platelet binding sites. Exogenous FVa was needed for maximal FVa binding to occur. No binding of FXa from internal platelet stores was observed. Addition of exogenous FVa and FXa resulted in FXa binding to the platelet surface. The methodology might be of use for the study of platelets from patients with bleeding disorders.