Atherosclerosis and unstable angina in Bernard-Soulier syndrome.

Platelets and von Willebrand factor play pathogenetic roles in atherosclerosis and acute coronary artery ischemic syndromes. Patients with Bernard-Soulier Syndrome are deficient in several platelet membrane glycoproteins, including glycoprotein Ib (GpIb). Glycoprotein Ib is the primary platelet receptor for von Willebrand factor and plays a critical role in the initiation of thrombus formation. Glycoprotein Ib, but also GpIIb/IIIa, mediates the adhesion of platelets to damaged endothelium, particularly at the high shear stresses found in small or diseased arteries. A patient with Bernard-Soulier syndrome is described who developed coronary artery atherosclerosis and unstable angina requiring coronary artery bypass grafting. The implications of this experiment in nature on the contribution of platelets and platelet GpIb and GpIIb/IIIa receptors to the development of atherosclerosis and unstable angina are discussed.     

Pertussis toxin activates platelets through an interaction with platelet glycoprotein Ib.

Platelets present a unique model to study the B-oligomer effects of pertussis toxin because they become activated in response to the B oligomer but are not susceptible to ADP-ribosylation by the holotoxin. In these studies, the B oligomer of pertussis toxin caused concentration-dependent platelet activation, as determined by increases in intracellular calcium concentration, dense granule secretion, and platelet aggregation. Stirring was required for pertussis toxin to increase intracellular calcium. A monoclonal antibody against platelet glycoprotein Ib abolished increases in intracellular calcium concentration and increased the latency and reduced the slope of the aggregation response elicited by the B oligomer. Pertussis toxin also evoked [14C]serotonin release from platelets, and this effect was inhibited, though not eliminated, by an antibody against platelet glycoprotein Ib. Binding of pertussis toxin to glycoprotein Ib was observed after nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that the B oligomer of pertussis toxin induces platelet activation mediated, at least in part, by an interaction with platelet glycoprotein Ib.     

Quantitation of cell membrane glycoproteins in pathological conditions using a lectin-bound enzyme-linked immunosorbent assay (ELISA): Application to human platelets in the Bernard-Soulier syndrome

Several techniques are available to study cell membrane glycoprotein in pathological conditions but these either lack sensitivity or require radiolabelled material or expensive apparatus. We have, therefore, developed a sandwich-type enzyme-linked immunosorbent assay (ELISA) to study patients with the Bernard-Soulier syndrome (BSS), a hereditary platelet disorder characterized primarily, at the molecular level, by glycoprotein Ib (GpIb) deficiency. We have used the lectin wheat germ agglutinin to capture GpIb in the microtiter wells. Following incubation with monoclonal antibody AN51 which recognizes an epitope on GpIb, the immune complex was detected using the streptavidin-peroxidase-biotin complex. Platelet samples from 24 normal controls gave a mean value of 106% whereas in the six BSS patients the mean value was 14% and in the eight obligatory heterozygotes it was 78%. The technique is simple, inexpensive and sensitive and does not require the use of radioactive material. The assay method could be applied to quantitate other cellular glycoproteins where specific lectins and monoclonal antibodies are available.     

Participation of IIb-IIIa glycoprotein in spontaneous platelet aggregation

In some patients with stable and unstable angina pectoris and in some donors without clinical manifestations of cardiovascular diseases and other pathologies, spontaneous platelet aggregation was completely suppressed by glycoprotein IIb-IIIa antagonists blocking the interaction of this glycoprotein with fibrinogen. Antibodies inhibiting binding of glycoprotein Ib with von Willebrand factor had no effect on the level and rate of spontaneous platelet aggregation. In the donor group, the level of spontaneous aggregation was almost 1.5-fold higher in persons with a certain genetic polymorphism (Leu-->Pro substitution in position 33 of glycoprotein IIIa). The level of spontaneous aggregation correlated with the amount of glycoprotein IIb-IIIa on the platelet surface (r = 0.41).     

Effect of recombinant tissue-type plasminogen activator on ten platelet membrane glycoproteins

Recombinant tissue-type plasminogen activator (rt-PA) did not modify densities of nine platelet membrane glycoproteins: collagen receptor subunit GP Ia; fibrinogen receptor GP IIb IIIa; thrombospondin receptor GP IV; von Willebrand factor receptor GP Ib and associated GP IX; thrombospondin; activation glycoprotein GMP 140; vitronectin receptor subunit VNR α and GP Ha. rt-PA induced slight decrease of GP 24 (CD9) density on platelet membrane. Antigen densities were determined after incubation of platelet-rich plasma with therapeutic doses of rt-PA (from 0.5-4 μg/ml) by a quantitative cytofluorometric method. Addition of fibrin did not modify the effect of rt-PA. These results suggest that incubation of platelet-rich plasma with therapeutic doses of rt-PA neither modify glycoproteins involved in platelet adhesion and aggregation nor markedly activate platelets.     

Infusible platelet membranes retain partial functionality of the platelet GPIb/IX/V receptor complex

Infusible platelet membranes (IPMs) prepared from fresh or outdated human platelets have been shown to correct prolonged bleeding times in thrombocytopenic rabbits. In previous trials, IPMs did not seem to be immunogenic and lacked dose-limiting toxicity. The present study was undertaken to explore whether the platelet glycoprotein (GP) Ib/IX/V complex might retain functionality in the IPM preparation. IPMs did not spontaneously bind von Willebrand factor (vWF), but saturable binding could be induced by ristocetin, with a dissociation constant (Kd) of 0.31 +/- 0.03 microgram/mL at 1.0 mg/mL of ristocetin. Of 4 anti-GPIb-alpha monoclonal antibodies tested, AN-51 inhibited vWF binding 67.8% +/- 5.8%, whereas AS-2, AS-7, and SZ-2 were ineffective. Maximal vWF binding induced by botrocetin was only 10% to 15% of that observed with ristocetin. Retention of partial functionality of the GPIb/IX/V receptor allowing vWF binding in a modulated manner seems to represent a critical mechanism by which IPMs may provide hemostatic efficacy.     

血管性血友病因子和血小板表面受体突变的数据库构建及数据分析

背景：血管性血友病因子和血小板表面受体之间的相互作用在血小板黏附、传播、聚集等血栓形成过程中起到关键作用。目前关于血管性血友病因子突变信息的数据库并不完善且血小板表面受体相关的数据库仍未构建。目的：致力于构建血管性血友病因子及血小板糖蛋白Ibα相关突变的数据库,以利于相关领域的研究人员快速查找到该分子对的重要突变信息。方法：以数据库Uniprot、VWFdb及文献报道的突变信息为数据源,采用MySQL和Apache为后台数据库和服务器,运用PHP语言开发血管性血友病因子及血小板糖蛋白Ibα突变数据库。结果与结论：收集了341条血管性血友病因子,13条血小板糖蛋白Ibα的野生或者突变序列数据,并人工构建A1结构域的虚拟突变3 920条,建立了包括背景介绍、相关病理图册和资料下载等登录页面,初步实现了数据检索、突变位点分析以及虚拟突变位点信息等相关功能。该数据库较全面搜集及分析血管性血友病因子及血小板糖蛋白Ibα的数据信息,有利于研究人员对血管性血友病因子和血小板糖蛋白Ibα相关信息的查询,有助于新型抗血栓药物的研发,进一步提高临床诊断和治疗水平。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Detection method for platelet activation markers

The assessment of platelet activation levels may be useful for identifying patients who would benefit from antiplatelet therapy and prediction of ischemic events. Laboratory markers of platelet activation include activation-dependent changes in glycoprotein (GP) IIb/IIIa complex, exposure of granule membrane proteins, binding of secreted platelet proteins, and development of procoagulant surfaces. Whole blood flow cytometry is a popular and useful method for the detection of these markers of platelet activation. Monoclonal antibodies against platelet surface glycoproteins are used to identify activated platelets.     

Immunoenzyme assay of glycocalicin, platelet glycoprotein Ib fragment. Evaluation of platelet turnover in circulation and differential diagnosis of thrombocytopenia

We propose a method for measurement of plasma glycocalicin, a fragment of platelet membrane glycoprotein Ib. The concentration of glycocalicin is elevated in thrombocythemia and reduced in thrombocytopenia caused by insufficient platelet production, but not in immune thrombocytopenia due to enhanced platelet degradation. Thus, plasma content of glycocalicin is an indicator of platelet turnover. The proposed assay can be used for differential diagnostics of thrombocytopenia. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 10, pp. 476–479, October, 1999     

Alloantibodies to human platelet glycoprotein antigens (HPA) and HLA class 1 in a cross section of Nigerian antenatal women

The prevalence of antibodies to human platelet antigens (HPA) and human leukocyte antigens (HLA) class 1 antigens among Nigerian pregnant women has not been reported in our country. This study was therefore aimed at screening the obstetric population for evidence of alloimmunization due to human platelet and HLA class 1 antigens. One hundred and forty four (144) pregnant women attending the obstetric clinic of Military Hospital, Port Harcourt, participated in the study. Their sera were tested for antibodies to HPA and HLA class 1 antigens using GTI PakPlus solid phase ELISA Kit. The total prevalence rate of antibody production was 60.5% (87 out of 144). Among the positive samples, 60 had platelet glycoprotein specific antibodies (41.7%) and 27 had HLA class 1 antibodies (18.8%). In 39.6% of the pregnant women, both platelet specific antibodies and HLA class 1 antibodies appeared. The prevalence of platelet specific glycoprotein antibodies were obtained as follows: GP 11b/111a 12 (8.3%), GP 1a/11a 35 (20.8%), GP Ib/IX 18 (12.5%) and GP IV 9 (6.3%). The prevalence of each platelet antibody subgroup was obtained as follows: anti-HPA-1a,-3a,-4a (4.2%), anti-HPA-1b,-3b,-4a (4.2%), anti-HPA-30 5a and anti-GP Ib/IX (12.5% each), anti-HPA-5b (8.3%) and anti-GP IV (6.3%). A high prevalence rate of human platelet arid cytotoxic antibodies has been observed in our obstetric population. There is need to establish platelet serology laboratory for the proper antenatal and postnatal management of pregnant mothers in this region.     

Dynamic redistribution of major platelet surface receptors after contact-induced platelet activation and spreading. An immunoelectron microscopy study.

The authors used an immunogold labeling procedure to investigate the redistribution of platelet receptors and their ligands on the surface of contact-activated adherent platelets before and after thrombin stimulation. During the initial stage of platelet adhesion, a typical segregation of receptors occurred. Gold particles identifying glycoprotein (GP) Ib (CD42b) and GPIIb-IIIa (CD41a) remained distributed over the entire platelet surface, whereas gold particles identifying GPIa-IIa (CDw 49b) and GPIV (CD36) were found essentially overlying the granulomere; p24 (CD9) was present at the peripheral platelet rim and over the cell body. An increased labeling of GPIIb-IIIa, GPIV and p24 was also observed on pseudopods, with GPIIb-IIIa and GPIV concentrated at the enlarged extremities and at sites of contact between two platelets, whereas GPIb was absent from pseudopods. After thrombin stimulation of adherent platelets, GPIb underwent a relocation to the cell center, in contrast to GPIIb-IIIa which still remained randomly distributed over the cell body. To investigate whether ligand distribution paralleled this receptor segregation, platelet released von Willebrand factor (vWF), fibrinogen (Fg) and thrombospondin (TSP) were visualized. During the early stages of platelet activation, surface labeling for all three adhesive proteins was minimal and almost undetectable. Occasionally, intragranular Fg and vWF was accessible to gold-coupled antibodies, with vWF exhibiting the typical eccentric alpha-granular localization. At later stages of activation and especially after thrombin stimulation, no surface labeling for vWF was observed, whereas immunogold particles identifying vWF were still present inside enlarged clear vacuoles. In contrast, labeling of Fg and TSP was increased over the granulomere and extended to the cell periphery and the pseudopods, but was absent from the hyalomere, despite the presence of GPIIb-IIIa molecules. Double labeling experiments showed colocalization of Fg and TSP, GPIV and TSP, as well as Fg and GPIIb-IIIa, although no typical coclustering of GPIIb-IIIa and GPIV or GPIIb-IIIa and p24 was apparent. Our results further suggest that 1) on surface activated adherent platelets, not all GPIIb-IIIa molecules become competent to bind Fg, 2) GPIa-IIa is not anchored to the platelet membrane skeleton, and 3) during the early stage of platelet activation, a communication exists between the alpha granules and the platelet surface.     

Delineating the roles of the GPIIb/IIIa and GP-Ib-IX-V platelet receptors in mediating platelet adhesion to adsorbed fibrinogen and albumin

Platelet adhesion to adsorbed plasma proteins, such as fibrinogen (Fg), has been conventionally thought to be mediated by the GPIIb/IIIa receptor binding to Arg-Gly-Asp (RGD)-like motifs in the adsorbed protein. In previous studies, we showed that platelet adhesion response to adsorbed Fg and Alb was strongly influenced by the degree of adsorption-induced protein unfolding and that platelet adhesion was only partially blocked by soluble RGD, with RGD-blocked platelets adhering without activation. Based on these results, we hypothesized that in addition to the RGD-specific GPIIb/IIIa receptor, which mediates both adhesion and activation, a non-RGD-specific receptor set likely also plays a role in platelet adhesion (but not activation) to both Fg and albumin (Alb). To identify and elucidate the role of these receptors, in addition to GPIIb/IIIa, we also examined the GPIb-IX-V receptor complex, which has been shown to mediate platelet adhesion (but not activation) in studies by other groups. The platelet suspension was pretreated with either a GPIIb/IIIa-antagonist drug Aggrastat(?) or monoclonal antibodies 6B4 or 24G10 against GPIb-IX-V prior to adhesion on Fg- and Alb-coated OH- and CH(3)-functionalized alkanethiol self-assembled monolayer surfaces. The results revealed that GPIIb/IIIa is the primary receptor set involved in platelet adhesion to adsorbed Fg and Alb irrespective of their degree of adsorption-induced unfolding, while the GPIb-IX-V receptor complex plays an insignificant role. Overall, these studies provide novel insights into the molecular-level mechanisms mediating platelet interactions with adsorbed plasma proteins, thereby assisting the biomaterials field develop potent strategies for inhibiting platelet-protein interactions in the design of more hemocompatible cardiovascular biomaterials and effective anti-thrombotic therapies.     

Formation of viscoelastic protein layers on polymeric surfaces relevant to platelet adhesion

The hemocompatibility of biomaterials is highly dependent on the adhesion and activation of platelets. Surface-adsorbed fibrinogen has a dominant role in promoting platelet adhesion to artificial surfaces by binding glycoprotein IIb-IIIa (GPIIb-IIIa), the major platelet membrane receptor. Using quartz crystal microbalance with dissipation monitoring (QCM-D), we have investigated the material-dependent binding kinetics of purified GPIIb-IIIa to polymer-adsorbed fibrinogen. The following ranking of polymer-adsorbed mass (fibrinogen and GPIIb-IIIa) to test polymers could be established: poly[desaminotyrosyl-tyrosine ethyl (DTE) carbonate]/poly(lactide-co-glycolide)>poly[DTE co-5% poly(ethylene glycol) carbonate]. The QCM-D fibrinogen adsorption data were confirmed using an immunofluorescence assay. A synthetic RGD-containing peptide, but not a control peptide, inhibited GPIIb-IIIa binding to polymer-adsorbed fibrinogen, demonstrating the specificity of binding. Importantly, the binding efficiency of purified GPIIb-IIIa to polymer-adsorbed fibrinogen correlated with increased platelet adhesion in an in vitro model. Theoretical simulations using a Voight-based model provided quantitative data on the thickness and viscoelastic properties of the polymer-adsorbed protein layers. The precision of the modeling technique was limited with respect to the shear moduli values, leading to large variations. However, the other modeling parameters showed reproducible results. The thickness of both protein layers was polymer-dependent and ranged from 5 to 35 nm and the viscosity from 0.001 to 0.005 kg/ms, whereas the protein layer densities showed little differences between the test polymers. These results suggest that material-dependent changes in the thickness and viscoelastic properties of adsorbed fibrinogen-GPIIb-IIIa layers are crucial factors in the binding behavior of platelets to biomaterials.     

Recombinant TGF-beta 1 is synthesized as a two-component latent complex that shares some structural features with the native platelet latent TGF-beta 1 complex

The entire coding region of the human transforming growth factor beta 1 (TGF-beta 1) precursor cDNA has been stably expressed in a human renal carcinoma cell line. Like platelet TGF-beta 1, the recombinant TGF-beta 1 is secreted in a biologically latent form. Immunoblot analysis and gel-filtration indicate that the recombinant latent TGF-beta 1 is a 100-kDa complex in which active 25-kDa TGF-beta 1 is noncovalently associated with the remaining 75 kDa of the processed precursor. Unlike the platelet latent complex, the recombinant latent complex contains no 135-kDa component. Thus, the processed precursor peptide alone is sufficient to confer latency on active TGF-beta 1, and the 135-kDa platelet component has a different role. The processed precursor is similarly glycosylated in recombinant and platelet complexes, and in both has an exposed heparin binding site that may be involved in targeting of the latent complex. Finally, acid activation of recombinant and platelet complexes is reversible, suggesting that the activation process does not cause major structural modifications in the components of the latent complex.     

Staphylococcus aureus induces platelet aggregation via a fibrinogen-dependent mechanism which is independent of principal platelet glycoprotein IIb/IIIa fibrinogen-binding domains.

Platelet aggregation by bacteria is felt to play an important role in the pathogenesis of infective endocarditis. However, the mechanisms involved in bacterium-induced platelet aggregation are not well-defined. In the present study, we examined the mechanisms by which Staphylococcus aureus causes rabbit platelet aggregation in vitro. In normal plasma, the kinetics of S. aureus-induced platelet aggregation were rapid and biphasic. The onset and magnitude of aggregation phase 1 varied with the bacterium-platelet ratio, with maximal aggregation observed at a ratio of 5:1. The onset of aggregation phase 2 was delayed in the presence of apyrase (an ADP hydrolase), suggesting that this later aggregation phase may be triggered by secreted ADP. The onset of aggregation phase 2 was delayed in the presence of prostaglandin I2-treated platelets, and this phase was absent when paraformaldehyde-fixed platelets were used, implicating platelet activation in this process. Platelet aggregation phase 2 was dependent on S. aureus viability and an intact bacterial cell wall, and it was mitigated by antibody directed against staphylococcal clumping factor (a fibrinogen-binding protein) and by the cyclooxygenase inhibitor indomethacin. Similarly, aggregation phase 2 was either delayed or absent in three distinct transposon-induced S. aureus mutants with reduced capacities to bind fibrinogen in vitro. In addition, a synthetic pentadecapeptide, corresponding to the staphylococcal binding domain in the C terminus of the fibrinogen delta-chain, blocked aggregation phase 2. However, phase 2 of aggregation was not inhibited by two synthetic peptides (alone or in combination) analogous to the two principal fibrinogen-binding domains on the platelet glycoprotein (GP) IIb/IIIa integrin receptor: (i) a recognition site on the IIIa molecule for the Arg-Gly-Asp (RGD) sequence of the fibrinogen alpha-chain and (ii) a recognition site on the IIb molecule for a dodecapeptide sequence of the fibrinogen delta-chain. This differs from ADP-induced platelet aggregation, which relies on an intact platelet GP IIb/IIIa receptor with an accessible RGD sequence and dodecapeptide recognition site for fibrinogen. Furthermore, a monoclonal antibody directed against the RGD recognition site on rabbit platelet GP IIb/IIIa receptors failed to inhibit rabbit platelet aggregation by S. aureus. Collectively, these data suggest that S. aureus-induced platelet aggregation requires bacterial binding to fibrinogen but is not principally dependent upon the two major fibrinogen-binding domains on the platelet GP IIb/IIIa integrin receptor, the RGD and dodecapeptide recognition sites.     

Influence of tuftsin-like synthetic peptides derived from C-reactive protein (CRP) on platelet behaviour.

C-reactive protein (CRP) is an acute-phase reactant that modifies platelet function differently, depending upon its physiochemical state. Aggregated and ligand-complexed forms of CRP initiate the activation of platelets, whereas naturally occurring CRP peptides inhibit platelet activation. The present study documents neutral proteases of the polymorphonuclear leucocyte (PMN) to cleave CRP into reaction products with the potential to inhibit platelet activation, and explore the structure-function relationships involved in the regulation of platelet activation by CRP using synthetic CRP peptides. Evidence was obtained that (i) a minimum of two linear functional domains exist within CRP that influence platelet activation; (ii) they reside in the mid-portion and at the C-terminus of the CRP molecule; (iii) the mid-portion domain inhibits platelet activation stimulated by adenosine diphosphate (ADP) or acid-soluble collagen, whereas the C-terminal domain initiates platelet activation; (iv) the functional expression of the C-terminal domain is maximized when the linear peptide is immobilized on latex; and (v) both CRP domains contain a homologue of the immunoregulatory signal peptide, tuftsin. These data suggest that the molecular mechanisms by which platelet processes are modulated by CRP may be related to the presence of tuftsin homologues in CRP.     

Facilitating roles of murine platelet glycoprotein Ib and αIIbβ3 in phosphatidylserine exposure during vWF–collagen-induced thrombus formation

Vessel wall damage exposes collagen fibres, to which platelets adhere directly via the collagen receptors glycoprotein (GP) VI and integrin α₂β₁ and indirectly by collagen-bound von Willebrand factor (vWF) via the GPIb-V-IX and integrin αIIbβ3 receptor complexes. Platelet–collagen interaction under shear stimulates thrombus formation in two ways, by integrin-dependent formation of platelet aggregates and by surface exposure of procoagulant phosphatidylserine (PS). GPVI is involved in both processes, complemented by α2β1. In mouse blood flowing over collagen, we investigated the additional role of platelet–vWF binding via GPIb and αIIbβ3. Inhibition of GPIb as well as blocking of vWF binding to collagen reduced stable platelet adhesion at high shear rate. This was accompanied by delayed platelet Ca²⁺ responses and reduced PS exposure, while microaggregates were still formed. Inhibition of integrin αIIbβ3 with JON/A antibody, which blocks αIIbβ3 binding to both vWF and fibrinogen, reduced PS exposure and aggregate formation. The JON/A effects were not enhanced by combined blocking of GPIb–vWF binding, suggesting a function for αIIbβ3 downstream of GPIb. Typically, with blood from FcR γ-chain +/− mutant mice, expressing 50% of normal platelet GPVI levels, GPIb blockage almost completely abolished platelet adhesion and PS exposure. Together, these data indicate that, under physiological conditions of flow, both adhesive receptors GPIb and αIIbβ3 facilitate GPVI-mediated PS exposure by stabilizing platelet binding to collagen. Hence, these glycoproteins have an assistant procoagulant role in collagen-dependent thrombus formation, which is most prominent at reduced GPVI activity and is independent of the presence of thrombin.     

Changes in platelet membrane glycoproteins and platelet-leukocyte interaction during hemodialysis

Platelet aggregation and interaction with other vascular cells play a key role in hemostatic events during hemodialysis. We studied seven patients with end-stage renal failure on long-term hemodialysis treatment. Flow-cytometric techniques and platelet-specific monoclonal antibodies were used to measure the platelet surface expression of glycoproteins - fibrinogen receptor on glycoprotein IIb-IIIa (GP IIb-IIIa; CD41) and -granule membrane protein (GMP-140; CD62). In addition, adhesion of platelets or platelet microparticles with leukocytes was evaluated by appearance of the platelet-specific antigen (GP IIb-IIIa) on leukocytes. Blood samples were taken before the start of dialysis and 15, 60, and 240 min thereafter. There was a significant increase in fibrinogen receptor activation on circulating platelets after 15 min of dialysis treatment (P < 0.001) and enhanced degranulation of GMP-140 (P < 0.05). In parallel, the interaction of platelets with neutrophils and monocytes also increased with the duration of dialysis and was maximal after 15 min (P < 0.001). We conclude that the platelet fibrinogen receptor on GP IIb-IIIa in circulating platelets is activated during hemodialysis and is associated with increased adhesion of platelets or platelet microparticles with circulating leukocytes. Thus, the phenomenon described here of platelet-leukocyte interaction could be pathophysiologically important for the development of dialysis-associated leukopenia.Abbreviations GP IIb-IIIa glycoprotein IIb-IIIa - GMP140 granule membrane protein 140 - LIBS ligand-induced binding site - FACS flow-activated cell sorting - mAb monoclonal antibody Correspondence to: M. Gawaz     

Localization of internal pools of membrane glycoproteins involved in platelet adhesive responses.

To investigate the existence of intracellular pools of membrane glycoproteins involved in platelet adhesive reactions, the authors have studied the distribution of glycoprotein (GP) Ib and IIb/IIIa by immunofluorescence and immunoelectron microscopy. Studies on whole cells and frozen thick sections revealed a rim pattern of fluorescence for GPIb and GPIIb/IIIa consistent with a surface distribution. In addition, extensive staining occupying the entire cell interior was observed for anti-GPIIb/IIIa, whereas anti-GPIb revealed staining of large intracellular structures that contained no stainable fibrinogen. On the ultrastructural level, the extracellular face of the plasma membrane and the intraluminal face of vacuolar structures were stained with both anti-GPIb and anti-GPIIb/IIIa. Additionally, GPIIb/IIIa antigen was localized to alpha-granule membranes. To determine whether alpha-granule GPIIb/IIIa could be transported to the cell surface, the authors employed a calcium-dependent monoclonal anti-GPIIb/IIIa antibody. Incubation of platelets with EGTA at 37 C abolished staining of plasma membrane and vacuolar but not alpha-granule GPIIb/IIIa. Recalcification of these cells failed to restore the epitope; however, thrombin treatment of recalcified cells reconstituted surface staining with a concurrent loss of internal staining. These data suggest that GPIIb/IIIa is present in alpha-granule membranes and may be transported to the cell surface in response to thrombin treatment. In addition, both GPIb and GPIIb/IIIa antigens are present in intracellular membrane-bounded vacuolar structures which are closed to antibody probes in fixed cells. Redistribution of these internal pools of adhesive protein "receptors" may participate in the regulation of platelet adhesive properties.     

Endothelin attenuates endothelium-dependent platelet inhibition in man

Aim: The vascular endothelium produces several substances, including nitric oxide (NO) and endothelin-1 (ET-1), which participate in the regulation of vascular tone in humans. Both these substances may exert other actions of importance for cardiovascular disease, e.g. effects on vascular smooth muscle cell proliferation and inflammation, and NO inhibits platelet function. Experiments were designed to investigate the effect of ET-1 on endothelium-dependent vasodilatation and attenuation of platelet activation. Methods: In 25 healthy male subjects (25 ± 1 years), forearm blood flow was measured by venous occlusion plethysmography, and platelet activity was assessed by whole blood flow cytometry (platelet fibrinogen binding and P-selectin expression) in unstimulated and adenosine diphosphate (ADP)-stimulated samples during administration of ET-1, the endothelium-dependent vasodilator acetylcholine and the NO synthase inhibitor l -NMMA. Results: Acetylcholine increased forearm blood flow and significantly inhibited platelet activation in both unstimulated and ADP-stimulated samples. In samples stimulated with 0.3 μm ADP, fibrinogen binding decreased from 41 ± 4% to 31 ± 3% (P < 0.01, n = 11) after acetylcholine administration. The vasodilator response to acetylcholine was significantly impaired during infusions of ET-1 and l -NMMA. ET-1 did not affect platelet activity per se, whereas l -NMMA increased platelet P-selectin expression. Both ET-1 and l -NMMA attenuated the acetylcholine-induced inhibition of platelet activity. Conclusions: Our study indicates that, further to inhibiting endothelium-dependent vasodilatation, ET-1 may also attenuate endothelium-dependent inhibition of platelet activation induced by acetylcholine. An enhanced ET-1 activity, as suggested in endothelial dysfunction, may affect endothelium-dependent platelet modulation and thereby have pathophysiological implications.