Ya-Ping Wu

Role of ADP Receptor P2Y12 in Platelet Adhesion and Thrombus Formation in Flowing Blood

ADP plays a central role in regulating platelet function. It induces platelet aggregation via the activation of 2 major ADP receptors, P2Y1 and P2Y12. We have investigated the role of P2Y12 in platelet adhesion and thrombus formation under physiological flow by using blood from a patient with a defect in the gene encoding P2Y12. Anticoagulated blood from the patient and from healthy volunteers was perfused over collagen-coated coverslips. The patient’s thrombi were smaller and consisted of spread platelets overlying platelets that were not spread, whereas control thrombi were large and densely packed. Identical platelet surface coverage, aggregate size, and morphology were found when a P2Y12 antagonist, N6-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-&bgr;,&ggr;-dichloromethylene ATP (also known as AR-C69931 MX), was added to control blood. The addition of a P2Y1 antagonist (adenosine-3′,5′-diphospate) to control blood resulted in small, but normally structured, thrombi. Thus, the ADP-P2Y12 interaction is essential for normal thrombus buildup on collagen. The patient’s blood also showed reduced platelet adhesion on fibrinogen, which was not due to changes in morphology. Comparable results were found by using control blood with AR-C69931 MX and also with adenosine-3′,5′-diphospate. This suggested that P2Y12 and P2Y1 were both involved in platelet adhesion on immobilized fibrinogen, thereby revealing it as ADP dependent. This was confirmed by complete inhibition on the addition of creatine phosphate/creatine phosphokinase.

Platelet Thrombus Formation on Collagen at High Shear Rates Is Mediated by von Willebrand Factor–Glycoprotein Ib Interaction and Inhibited by von Willebrand Factor–Glycoprotein IIb/IIIa Interaction

We studied the role of von Willebrand Factor (vWF) in platelet thrombus formation in flowing blood by using a perfusion system and mutant forms of vWF lacking either interaction with glycoprotein Ib (GpIb) or with glycoprotein IIb/IIIa (alphaIIb-beta3). These mutants were added to the blood of patients with severe von Willebrands disease (vWD) or to normal blood reconstituted with a human albumin solution instead of plasma. This blood was then perfused over collagen type III spray-coated on a glass surface and preincubated for 2 hours with 20 microg/mL plasma vWF. In this way, the adhesion step was mediated by the preincubated plasma vWF bound to collagen type III, whereas thrombus formation was mediated by mutant vWF added to the perfusate. Thrombus formation was absent at all 3 shear rates studied (300, 800, and 2600 s(-1)) when DeltaA1-vWF, lacking interaction with GpIb, was added to the perfusate, indicating the importance of GpIb-vWF interaction for thrombus formation. The interaction of vWF and GpIb is currently thought to be possible under physiological conditions in which the conformation of vWF has been changed by adsorption to a surface. Our results regarding the role of GpIb-vWF interaction in thrombus formation suggest that a second mechanism may operate by which a change may occur in GpIb on the surface of adhered platelets either by activation of the molecule or as a consequence of shear stress. Increased thrombus formation was observed when the Arg-Gly-Gly-Ser-vWF, which does not interact with alphaIIb-beta3, was added to vWD blood and perfused at 2600 s(-1). This increase was not observed in vWD blood at lower shear rates or after addition of Arg-Gly-Gly-Ser-vWF to reconstituted normal blood. Thrombus formation at a high shear rate was largest when either vWF or fibrinogen was present as a single ligand for alphaIIb-beta3 at a high shear rate. When both were present, thrombus formation was decreased. We postulate that thrombus formation is less efficient because of incomplete bridge formation when vWF and fibrinogen are both present as ligands for alphaIIb-beta3.

Accelerated uptake of VWF/platelet complexes in macrophages contributes to VWD type 2B-associated thrombocytopenia

Von Willebrand disease (VWD) type 2B is characterized by mutations causing enhanced binding of von Willebrand factor (VWF) to platelets. Bleeding tendency is associated with heterogeneous clinical manifestations, including moderate to severe thrombocytopenia. The underlying mechanism of the thrombocytopenia has remained unclear. Here, a mouse model of VWD type 2B was used to investigate pathways contributing to thrombocytopenia. Immunohistochemical analysis of blood smears revealed that mutant VWF was exclusively detected on platelets of thrombocytopenic VWD type 2B mice, suggesting that thrombocytopenic VWD type 2B mice were elevated two- to threefold upon chemical macrophage depletion. Colocalization of platelets with CD68-positive Kupffer cells and CD168-positive marginal macrophages in liver and spleen, respectively, confirmed the involvement of macrophages in the removal of VWF/platelet complexes. Significantly more platelets were found in liver and spleen of VWD type 2B mice compared with control mice. Finally, platelet survival was significantly shorter in VWD type 2B mice compared with control mice, providing a rationale for lower platelet counts in VWD type 2B mice. In conclusion, our data indicate that VWF type 2B binds to platelets and that this is a signal for clearance by macrophages, which could contribute to the thrombocytopenia in patients with VWD type 2B.

Thrombopoietin increases platelet adhesion under flow and decreases rolling.

Summary. Thrombopoietin (TPO) is known to sensitize platelets to other agonists at 20 ng/ml, and above 100 ng/ml it is an independent activator of aggregation and secretion. In studies with a perfusion chamber, TPO, between 0·01 ng/ml and 1 ng/ml, increased platelet adhesion to surface‐coated fibrinogen, fibronectin and von Willebrand Factor (VWF) but not to a collagen‐coated surface. Increased adhesion was observed at shear rates of 300/s and 800/s in perfusions with whole blood as well as in suspensions of platelets and red blood cells reconstituted in plasma. The by the cyclooxygenase inhibitor, indomethacin, and the thromboxane A2‐receptor blocker, SQ30741, abolished the stimulation by TPO. The effect of TPO was mimicked by a very low concentration (10 nmol/l) of the thromboxane TxA2 analogue, U46619. Real‐time studies of platelet adhesion to a VWF‐coated surface at a shear of 1000/s showed that about 20% of the platelets were in a rolling phase before they became firmly attached. TPO (1 ng/ml) pretreatment reduced this number to < 5%, an effect again abolished by indomethacin. Thus, TPO potentiates the direct and firm attachment of platelets to surface‐coated ligands for αIIbβ3, possibly by increasing the ligand affinity of the integrin.

The proteome of the insoluble Schistosoma mansoni eggshell skeleton

In schistosomiasis, the majority of symptoms of the disease is caused by the eggs that are trapped in the liver. These eggs elicit an immune reaction that leads to the formation of granulomas. The eggshell, which is a rigid insoluble structure built from cross-linked proteins, is the site of direct interaction between the egg and the immune system. However, the exact protein composition of the insoluble eggshell was previously unknown. To identify the proteins of the eggshell of Schistosoma mansoni we performed LC-MS/MS analysis, immunostaining and amino acid analysis on eggshell fragments. For this, eggshell protein skeleton was prepared by thoroughly cleaning eggshells in a four-step stripping procedure of increasing strength including urea and SDS to remove all material that is not covalently linked to the eggshell itself, but is part of the inside of the egg, such as Reynolds layer, von Lichtenbergs envelope and the miracidium. We identified 45 proteins of which the majority are non-structural proteins and non-specific for eggs, but are house-keeping proteins that are present in large quantities in worms and miracidia. Some of these proteins are known to be immunogenic, such as HSP70, GST and enolase. In addition, a number of schistosome-specific proteins with unknown function and no homology to any known annotated protein were found to be incorporated in the eggshell. Schistosome-specific glycoconjugates were also shown to be present on the eggshell protein skeleton. This study also confirmed that the putative eggshell protein p14 contributes largely to the eggshell. Together, these results give new insights into eggshell composition as well as eggshell formation. Those proteins that are present at the site and time of eggshell formation are incorporated in the cross-linked eggshell and this cross-linking does no longer occur when the miracidium starts secreting proteins.

Differential platelet adhesion to distinct life-cycle stages of the parasitic helminth Schistosoma mansoni.

Y . P . W U,* P . J . L ENT ING ,* A . G . M. T I ELENS , P . G . DE GROOT* and J . J . VAN H ELLEMOND *Department of Haematology, University Medical Centre Utrecht, Utrecht; Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht; and Department of Medical Microbiology and Infectious Diseases, Erasmus MC Medical Centre and Harbour Hospital Institute for Tropical Medicine, Rotterdam, the Netherlands

Plasma Surfactant Protein D Levels and the Relation to Body Mass Index in a Chinese Population

Surfactant protein D (SP‐D) is a member of the collectin family and is an important component of the pulmonary innate host defence. The protein has a widespread distribution in the human body and is present in multiple epithelia, in endothelium and in blood. Various studies have looked at the relationship between serum SP‐D levels and pulmonary inflammatory diseases. The SP‐D distribution has been most thoroughly described in European populations and appears with a broad range of serum values highly influenced by genetic factors. In the present study, we investigated the plasma SP‐D distribution in a Chinese population from the Tai An region comprising 268 individuals. We found that (i) plasma SP‐D in the Chinese population was distributed with a median value of 380.2 ng/ml (324.9; 418.7) and a range from 79.4 to 3965.3 ng/ml, (ii) significantly higher plasma SP‐D in men than in women, and no significant effect of age, and (iii) a significant inverse association between serum SP‐D and body mass index (BMI) (P = 0.012). The data indicate that racial differences in SP‐D expression exist as the median plasma SP‐D in the Chinese population was approximately two times lower than the median serum SP‐D previously measured in a Danish population using the same immuno‐assay. The inverse association between serum SP‐D and BMI found in the Chinese population indicates that serum SP‐D is related to obesity in similar ways in Chinese and Danes.

Role of glycoprotein Ibα mobility in platelet function

Incubation at 0 degrees C is known to expose b- N -acetyl-D-glucosamine residues on glycoprotein (GP) Ibalpha inducing receptor clustering and alpha(M)beta(2)-mediated platelet destruction by macrophages. Here we show that incubation at 0/37 degrees C (4 hours at 0 degrees C, followed by 1 hour at 37 degrees C to mimic cold-storage and post-transfusion conditions) triggers a conformational change in the N -terminal flank (NTF, amino acids, aa 1-35) but not in aa 36-282 of GPIbalpha as detected by antibody binding. Addition of the sugar N -acetyl-D-glucosamine (GN) inhibits responses induced by 0/37 degrees C. Incubation at 0 degrees C shifts GPIbalpha from the membrane skeleton to the cytoskeleton. Different GPIbalpha conformations have little effect on VWF/ristocetin-induced aggregation, but arrest of NTF change by GN interferes with agglutination and spreading on a VWF-coated surface under flow. Strikingly, incubation at 0/37 degrees C initiates thromboxane A(2) formation through a von Willebrand factor (VWF)-independent and GPIbalpha-dependent mechanism, as confirmed in VWF- and GPIbalpha-deficient platelets. We conclude that the NTF change induced by 0/37 degrees C incubation reflects clustering of GPIbalpha supports VWF/ristocetin-induced agglutination and spreading and is sufficient to initiate platelet activation in the absence of VWF.

Elevated plasma surfactant protein D (SP-D) levels and a direct correlation with anti-severe acute respiratory syndrome coronavirus-specific IgG antibody in SARS patients.

Pulmonary SP‐D is a defence lectin promoting clearance of viral infections. SP‐D is recognized to bind the S protein of SARS‐CoV and enhance phagocytosis. Moreover, systemic SP‐D is widely used as a biomarker of alveolar integrity. We investigated the relation between plasma SP‐D, SARS‐type pneumonia and the SARS‐specific IgG response. Sixteen patients with SARS, 19 patients with community‐acquired pneumonia (CAP) (Streptococcus pneumonia) and 16 healthy control subjects were enrolled in the study. Plasma SP‐D and anti‐SARS‐CoV N protein IgG were measured using ELISA. SP‐D was significantly elevated in SARS‐type pneumonia [median (95% CI), 453 (379–963) ng/ml versus controls 218 (160–362) ng/ml, P < 0.05] like in patients with CAP. SP‐D significantly correlated with anti‐SARS‐CoV N protein IgG (r2 = 0.5995, P = 0.02). The possible re‐emergence of SARS or SARS‐like infections suggests a need for minimal traumatic techniques for following the alveolar compartment, e.g. during testing of antivirals. We suggest that monitoring systemic SP‐D may be useful in monitoring the alveolar integrity in SARS‐type pneumonia. The significant correlation between plasma SP‐D and anti‐SARS‐CoV‐specific antibodies support the role for SP‐D in interlinking innate and adaptive immune pathways.