Background-Numerous in vitro studies suggest that sphingosine 1-phosphate (S1P), a bioactive lysosphingolipid associated with high-density lipoproteins, accounts at least partly for the potent antiinflammatory properties of high-density lipoprotein and, thereby, contributes to the antiatherogenic potential attributed to high-density lipoproteins. The present study was undertaken to investigate whether modulation of S1P signaling would affect atherosclerosis in a murine model of disease. Methods and Results-Low-density lipoprotein receptor-deficient mice on a cholesterol-rich diet were given FTY720, a synthetic S1P analogue, at low (0.04 mg/kg per day) or high (0.4 mg/kg per day) doses for 16 weeks. FTY720 dose-dependently reduced atherosclerotic lesion formation, both in the aortic root and brachiocephalic artery, and almost completely blunted necrotic core formation. Plasma lipids remained unchanged during the course of FTY720 treatment. However, FTY720 lowered blood lymphocyte count (at a high dose) and significantly interfered with lymphocyte function, as evidenced by reduced splenocyte proliferation and interferon-␥ levels in plasma. Plasma concentrations of proinflammatory cytokines such as tumor necrosis factor-␣, interleukin (IL)-6, IL-12, and regulated on activation normal T cell expressed and secreted were reduced by FTY720 administration. Moreover, lipopolysaccharide-elicited generation of nitrite/nitrate and IL-6 -two markers of classical (M1) macrophage activation-was inhibited, whereas IL-4 -induced production of IL-1-receptor antagonist, a marker of alternative (M2) macrophage activation, was augmented in peritoneal macrophages from FTY720-treated low-density lipoprotein receptor-deficient mice. Conclusions-The present results demonstrate that an S1P analogue inhibits atherosclerosis by modulating lymphocyte and macrophage function, and these results are consistent with the notion that S1P contributes to the antiatherogenic potential of high-density lipoprotein. (Circulation. 2007;115:501-508.)

Acute thrombotic arterial occlusion is the leading cause of morbidity and mortality in the Western world. Von Willebrand factor is thought to be the only indispensable adhesive substrate to promote thrombus formation in high shear environments. We found that thrombospondin-1, a glycoprotein enriched in arteriosclerotic plaques, might function as an alternative substrate for thrombus formation. Platelets adhered to thrombospondin-1 in a shear dependent manner with an optimum shear as found in stenosed arteries. Adhesion is extremely firm, with no detachment of platelets up to a shear rate of 4000 s(-1). Experiments using platelets from a patient completely lacking von Willebrand factor showed that von Willebrand factor is not involved in platelet binding to thrombospondin-1. Platelet adhesion to thrombospondin-1 is not mediated via beta3-integrins or GPIa. CD36 partially mediates the adhesion of pre-activated platelets. We identified GPIb as high shear adhesion-receptor for thrombospondin-1. Soluble GPIb, as well as antibodies against the GPIb, blocked platelet adhesion almost completely. The new discovered thrombospondin-1-GPIb adhesion axis under arterial shear conditions might be important, not only during thrombus formation but also for pathological processes where other cells bind to the endothelium or subendothelium, including arteriosclerosis, inflammation and tumor metastasis, and a promising therapeutic target.

To cite this article: Jurk K, Lahav J, Van Aken H, Brodde MF, Nofer JR, Kehrel BE. Extracellular protein disulfide isomerase regulates feedback activation of platelet thrombin generation via modulation of coagulation factor binding. J Thromb Haemost 2011; 9: 2278-90.Summary. 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 thrombinmediated 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.

1. Prospective and interventional studies demonstrate an inverse relationship between plasma high-density lipoprotein (HDL)-cholesterol and the incidence of coronary artery disease. Although the atheroprotective effects of HDL are usually attributed to the reverse cholesterol transport, in which HDL shuttles cholesterol from cells in the arterial wall to the liver, other mechanisms are also under investigation. 2. Platelets are involved in both the initiation and progression of atherosclerotic lesions. In addition, the formation of thrombi over ruptured atherosclerotic plaques results in the narrowing or complete occlusion of coronary arteries. Current experimental evidence suggests that HDL may exert antiplatelet effects and thereby counteract the development of atherothrombotic vascular disease. 3. In vitro studies show that HDL inhibits agonist-stimulated platelet aggregation, fibrinogen binding, granule secretion and liberation of thromboxane A(2). Inhibitory effects of HDL are mediated, in part, by scavenger receptor type B1 and/or the apolipoprotein E receptor apoER2/LRP8 and are linked to the induction of intracellular signalling cascades encompassing stimulation of protein kinase C, cytoplasmatic alkalization and generation of nitric oxide. 4. Populational studies demonstrate that there is an inverse association between plasma HDL levels and recurrent venous thromboembolism. In addition, HDL-cholesterol has been identified as an independent predictor of acute platelet thrombus formation. The administration of reconstituted HDL particles in humans attenuates ex vivo platelet activation. 5. The present review summarizes recent advances in understanding HDL-platelet interactions and discusses the potential use of HDL-like particles in the therapy of thrombosis.

Summary.  Background:  Human neutrophil α‐defensins (HNPs) are important constituents of the innate immune system. Beyond their antimicrobial properties, HNPs also have pro‐inflammatory features. While HNPs in plasma from healthy individuals are barely detectable, their level is strongly elevated in septic plasma and plasma from patients with acute coronary syndromes. Objectives:  As thrombosis and inflammation are intertwined processes and activation of human polymorphonuclear leukocytes (PMNL) and subsequent degranulation is associated with full activation of surrounding platelets, we studied the effect of HNPs on platelet function. Methods:  The effect of HNPs on platelet activation parameters and apoptosis was investigated via aggregometry, flow cytometry, confocal microscopy and the ELISA technique. Results:  It was found that HNPs activate platelets in pathophysiologically relevant doses, inducing fibrinogen and thrombospondin‐1 binding, aggregation, granule secretion, sCD40L shedding, and procoagulant activity. HNPs bound directly to the platelet membrane, induced membrane pore formation, microparticle formation, mitochondrial membrane depolarization and caspase‐3‐activity. Confocal microscopy revealed the HNP‐induced formation of polymeric fibrinogen and thrombospondin‐1 amyloid‐like structures, which bound microorganisms. Platelets adhered to these structures and formed aggregates. Blocking of glycoprotein IIb/IIIa (GPIIb/IIIa) markedly inhibited HNP‐induced platelet activation. In addition, heparin, heparinoid, serpins and α2‐macroglobulin, which all bind to HNPs, blocked HNP‐1‐induced platelet activation in contrast to direct thrombin inhibitors such as hirudin. Conclusions:  HNPs activate platelets and induce platelet apoptosis by formation of amyloid‐like proteins. As these structures entrapped bacteria and fungi, they might reflect an additional function of HNPs in host defense. The described mechanism links again thrombosis and infection.

ATP binding cassette transporter A1 (ABCA1) is involved in regulation of intracellular lipid trafficking and export of cholesterol from cells to high density lipoproteins. ABCA1 defects cause Tangier disease, a disorder characterized by absence of high density lipoprotein and thrombocytopenia. In the present study we have demonstrated that ABCA1 is expressed in human platelets and that fibrinogen binding and CD62 surface expression in response to collagen and low concentrations of thrombin, but not to ADP, are defective in platelets from Tangier patients and ABCA1-deficient animals. The expression of platelet membrane receptors such as GPVI, ␣ 2 ␤ 1 integrin, and GPIIb/IIIa, the collagen-induced changes in phosphatidylserine and cholesterol distribution, and the collagen-induced signal transduction examined by phosphorylation of LAT and p72 syk and by intracellular Ca 2؉ mobilization were unaltered in Tangier platelets. The electron microscopy of Tangier platelets revealed reduced numbers of dense bodies and the presence of giant granules typically encountered in platelets from Chediak-Higashi syndrome. Further studies demonstrated impaired release of dense body content in platelets from Tangier patients and ABCA1-deficient animals. In addition, Tangier platelets were characterized by defective surface exposure of dense body and lysosomal markers (CD63, LAMP-1, LAMP-2, CD68) during collagen-and thrombin-induced stimulation and by abnormally high lysosomal pH. We conclude that intact ABCA1 function is necessary for proper maturation of dense bodies in platelets. The impaired release of the content of dense bodies may explain the defective activation of Tangier platelets by collagen and low concentrations of thrombin, but not by ADP.

Platelets perform many functions in hemostasis but also in other areas of physiology and pathology. Therefore, it is obvious that many different function tests have been developed, each one conceived and standardized for a special purpose. This review will summarize the different fields in which platelet function testing is currently in use; diagnostics of patients with bleeding disorders, monitoring patients' response to anti-platelet therapy, monitoring in transfusion medicine (blood donors, platelet concentrates, and after transfusion), and monitoring in perioperative medicine to predict bleeding tendency. The second part of the review outlines different methods for platelet function testing, spanning bleeding time, and platelet counting as well as determining platelet adhesion, platelet secretion, platelet aggregation, platelet morphology, platelet signal transduction, platelet procoagulant activity, platelet apoptosis, platelet proteomics, and molecular biology.

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