Alan T. Nurden

Aminophospholipid exposure, microvesiculation and abnormal protein tyrosine phosphorylation in the platelets of a patient with Scott syndrome: a study using physiologic agonists and local anaesthetics.

The Scott syndrome is a rare inherited haemorrhagic disorder characterized by the inability of blood cells to expose aminophospholipids and to shed microparticles. We have had the opportunity to study a recently reported French patient with this syndrome and have confirmed by means of a fluorescence assay for transbilayer lipid movement a reduced aminophospholipid exposure when platelets were stimulated with the calcium‐ionophore ionomycin, in spite of a normal elevation of intracellular Ca2+. Secretion and calpain activation were also shown to be normal. Significantly, the level of phosphotyrosine‐labelled proteins in platelets treated with thrombin or a thrombinu2003+u2003collagen mixture and in particular the phosphorylation of a 40u2003kD band were severely reduced. Furthermore, inhibition of thiol‐containing enzymes, including tyrosine‐phosphatases, by N‐ethyl maleimide did not lead to aminophospholipid exposure in the patients platelets, in spite of increased tyrosine protein phosphorylation. In contrast, amphiphilic membrane drugs such as tetracaine and propranolol induced both surface aminophospholipid exposure in Scott platelets and the shedding of microparticles, thereby showing that membrane perturbation can lead to loss of phospholipid asymmetry in this syndrome. Our results provide the first insight that the lack of expression of procoagulant phospholipids and microparticle formation in Scott syndrome platelets is associated with a defect of intracellular signalling.

Human CalDAG-GEFI gene (RASGRP2) mutation affects platelet function and causes severe bleeding.

First case of a human RASGRP2 mutation affecting Rap1 activation in platelets and causing severe bleeding.

Identification of platelet membrane thrombospondin binding molecules using an anti-thrombospondin antibody

A rat monoclonal IgG2a antibody, 5G11, was raised against native human platelet thrombospondin (TSP). Western blot analysis revealed that 5G11 bound (i) to TSP before and after disulfide reduction, and (ii) to a 15-kDa fragment released after prolonged trypsin digestion. Crossed immunoelectrophoresis confirmed that the binding epitope was expressed in the presence of Ca2+ and after treatment of TSP with EDTA. Since 5G11 had no effect on platelet aggregation, the antibody was used to immunoprecipitate Ca2+-dependent and Ca2+-independent TSP-binding molecules on the surface of thrombin-activated surface-labeled 125I-platelets. The experimental basis was that ligand-receptor interactions are of high affinity and that anti-ligand antibodies should precipitate the ligand-receptor complex. With platelets activated in the presence of EDTA, 5G11 predominantly precipitated a 125I-labeled band of Mr 88,000, identified as glycoprotein (GP) IV. In contrast, in the presence of 2 mM Ca2+ and 1 mM Mg2+, 5G11 precipitated a complex of five radiolabeled proteins, among which GPIIb, GPIIIa and GPIV were the most prominent.

The transport of high amounts of vascular endothelial growth factor by blood platelets underlines their potential contribution in systemic sclerosis angiogenesis

OBJECTIVESnAltered angiogenesis is a characteristic feature in SSc and remains ill-understood. VEGF is believed to play a central role. Serum VEGF is elevated in SSc patients but questions remain concerning the source of circulating VEGF. Here we investigated platelet activation and the role of platelets as a source of VEGF and other angiogenic mediators in this disease.nnnMETHODSnA cohort of 40 patients with SSc was included. Age- and sex-matched healthy subjects and subjects presenting a primary RP were included as controls. Platelets were isolated, activated with thrombin and the secretion of VEGF, platelet derived growth factor, homodimeric form BB (PDGF-BB), TGF-beta1 and angiopoietins-1 and -2 measured. Plasma concentrations of these mediators and the functionality of platelet-derived VEGF were also studied. Platelet activation was assayed by measuring plasma beta-thromboglobulin and expression of P-selectin on platelets. The effect of iloprost on VEGF secretion by platelets was studied.nnnRESULTSnPlatelets from SSc patients, in contrast to controls, secreted large amounts of VEGF when activated, but not PDGF-BB, TGF-beta1 or angiopoietins. Increased expression of membrane P-selectin confirmed platelet activation in the patients. Iloprost inhibited VEGF secretion by platelets both in vivo and in vitro, through inhibition of platelet activation.nnnCONCLUSIONSnPlatelets transport high levels of VEGF in SSc. They may contribute to circulating VEGF because of ongoing activation in the course of the disease. If activated at the contact of injured endothelium, platelets may be important in the altered angiogenesis associated with the disease through the secretion of high levels of VEGF.

MEMBRANE GLYCOPROTEINS AND HUMAN PLATELET FUNCTION

The bulk of the proteins present at the surface of mammalian cells contain varying amouiits of carbohydrate and are therefore glycoproteins (Stoddart & Cook, 1973 ; Hughes, 1976). Considerable evidence suggests that surface bound carbohydrate groupings mediate many cell aggregation, adhesion and surface contact interactions (Roseman, 1970 ; Nicolson, 1976). The blood platelet contributes to the maintenance of haemostasis primarily as a result of its capacity to adhere to exposed subcndothelial tissue and its ability to respond to released stimuli, such as adenosine diphosphatc (ADP) , with the rapid formation of platelet aggregates or throni bi.

CHAPTER 57 – Inherited Disorders of Platelet Function

Genetic defects of platelets give rise to bleeding syndromes of varying severity. Deficiencies of glycoprotein (GP) mediators of adhesion and aggregation, especially the Bernard- Soulier syndrome (BSS, a disorder of the GP Ib-IX-V complex) and Glanzmann thrombasthenia (GT, a disorder of the integrin α IIb β 3 ) are discussed in detail in this chapter. Recent studies have highlighted defects of primary receptors for stimuli, including a pathology of the P2Y12 adenosine diphosphate (ADP) receptor. These lead to agonist-specific deficiencies in the platelet aggregation response. Likewise, abnormalities of signaling pathways that send messages to targets within the platelet give rise to functional defects only when that pathway is used. Defects of secretion from dense bodies and α-granules, of adenosine triphosphate (ATP) production, and of the generation of procoagulant activity are also encountered. Some disorders are exclusive to megakaryocytes and platelets; in others (e.g., the Chediak- Higashi, Hermansky-Pudlak, and Wiskott-Aldrich syndromes), the molecular lesion extends to other cell types. Molecular defects responsible for familial thrombocytopenias (Chapter 54) are also sometimes associated with reduced platelet function.

Quantitation of platelet fibrinogen and thrombospondin in Glanzmann's thrombasthenia by electroimmunoassay

Fibrinogen and thrombospondin are major constituents of human platelet alpha-granules and contribute to cell-cell interactions following their release. Glanzmanns thrombasthenia is characterized by the absence of platelet aggregation and reduced levels of GP IIb-IIIa complexes and platelet fibrinogen. The level of thrombospondin is thought to be normal but has not so far been quantified. Using an electroimmunoassay method adapted from Laurell, we have measured fibrinogen and thrombospondin in platelet extracts of four patients with classical Glanzmanns thrombasthenia and two variants with abnormal platelet aggregation associated with subnormal levels of GP IIb-IIIa complexes. Triton X-100 lysates were prepared in the presence of leupeptin or EDTA to avoid endogenous calcium-dependent protease activation during the solubilization procedure. Platelet fibrinogen was not detected in one patient with type I Glanzmanns thrombasthenia; it was reduced to 5-10% of normal values in two other type I patients and to 65% of normal values in one type II patient. It was normal in patient R.P., a variant of Glanzmanns thrombasthenia with 60% of GP IIb-IIIa complexes but decreased in patient A.P. a newly described variant with 35% of GP IIb-IIIa complexes. These findings support a role for GP IIb-IIIa complexes in the packaging of fibrinogen into alpha-granules. Normal or subnormal amounts of thrombospondin were measured in thrombasthenic platelets. Patient A.P., who was investigated on two different occasions, demonstrated variable levels of thrombospondin. This underlines the need for quantifying this protein when evaluating its expression in this disorder.

Initial characterization of human platelet mepacrine-labelled granules isolated using a short metrizamide gradient

Summary. A method for the purification of human platelet mepacrine‐labelled granules is described. Characterization of these isolated granules allowed them to be identified as the serotonin storage organelles or dense bodies. Each step of the purification procedure has been controlled in order to obtain a minimum of leakage of the granule content during initial isolation of the platelets from the blood, the platelet washing procedures, and platelet lysis and the subcellular separation. A key step in the procedure was the centrifugation of the labelled granules across a short, discontinuous metrizamide gradient. The pellet of isolated mepacrine‐fluorescent granules consisted almost entirely of granules with the typical appearance of dense bodies, as shown by electron microscopy, and was relatively free from membranes and other granule populations as evaluated by the presence of the different markers (tritiated lectin, beta‐glucuronidase, monoamine oxidase, platelet factor 4). The method is simple, reproducible and allows the highest enrichment in dense bodies obtained hitherto with human platelets: x 177 in calcium and x 115 in [14C]serotonin after fractionation of [14C]serotonin‐labelled whole platelets. Functional studies performed with the isolated granules showed that they rapidly accumulated [14C]serotonin.

Distribution of glycoprotein IIb-IIIa complexes in the surface membranes of human platelets and megakaryocytes

Summary. The distribution of glycoprotein (GP) IIb–IIIa complexes in the surface membranes of human platelets and megakaryocytes was investigated by transmission electron microscopy of cells that had been incubated with Fab fragments of a human alloantibody (IgG L) specific for the complex. Binding was visualized by a second antibody conjugated to peroxidase or adsorbed onto gold particles. Initial studies showed that the peroxidase reaction product and the gold particles were to be found at the outer surface of unactivated platelets. The occasional small cluster of particles was present. A positive reaction, more apparent with peroxidase labelling, was also seen in the channels of the platelet open canalicular system. Gold particles were abundant on the outer surface of mature megakaryocytes, and their distribution resembled that on unactivated platelets. As with platelets, peroxidaselabelled antibodies penetrated better, and revealed GP IIb–IIIa in the demarcation membrane system. A double immunofluorescence study, involving Fab fragments of IgG L and rhodamine‐conjugated antibodies to factor VIII R:Ag, demonstrated the presence of GP IIb–IIIa in megakaryocyte precursor cells. Our results show that the GPIIb–IIIa complex is present in megakaryocyte membranes and that it appears at the same time as the other platelet antigens.

Membrane glycoprotein IIb is the major endogenous acceptor for human platelet ectosialyltransferase

1. Introduction Platelet surface glycoconjugates have been suggested to play roles in the major biological functions of this cell, namely aggregation and adhesion [ 141. Bosmann [5] first showed that platelet suspension as well as plasma membrane fraction possessed sialyltransferase activity which would play a role in platelet aggregation. In fact, a parallel inhibition of aggregation and sialyl- transferase activity by aspirin [5,6] and an apparent stimulation of sialyltransferase activity with various platelet aggregating agents such as collagen, epinephrin and ADP plus fibrinogen [7] have been noted, while cancer patients with a high incidence of thrombosis have markedly elevated levels of platelet sialyltransfer- ase activity [6]. Despite the possible functional signi- ficance of the above studies, the presence of ectosial- yltransferase activity at the human platelet surface has not been rigorously defined. This is due to possible errors introduced into most assay systems by: (i) Precursor degradation and intracellular utilisation of the free radioactive sialic acid: (ii) The liberation of intracellular enzymes as a result of cell lysis during the assay; (iii) Phagocytosis of added exogenous acceptors [8]. Using exogenous non-phagocytosable acceptors [9] and the methodology developed to prove the presence of ectoglycosyltransferases at the surface of other cell types [ lo,1 11, we demonstrate here that the human blood platelet exhibits ectosialyltransferase activity and that the major endogenous acceptor is the plama membrane glycoprotein GP IIb.