Surface-connected Canalicular System Research Articles

Platelet Ultrastructure in Familial Platelet Disorder with Associated Myeloid Malignancy (FPDMM)

The familial platelet disorder with associated myeloid malignancy (FPDMM) is an autosomal dominant platelet disorder, caused by germline RUNX1 mutations, with predisposition to develop hematologic malignancies, especially acute myeloid leukemia. In many of the FPDMM families reported, the platelet defect was a delta-storage-pool disease (d-SPD) which can also be found without leukemia propensity. However, it has not been studied whether the two types of d-SPD have a common nature. Platelet ultrastructure, previously very little studied, may be one of the aspects to be analyzed to solve this question. We analyzed the ultrastructural characteristics of platelets in 5 members of a family with FPDMM. The family included three generations and all affected members had a RUNX1 deletion: chr21:36349450-36572837 (Rio-Machin et al. Nat Commun 2020;11:1044). None of the patients studied had developed leukemia at the time of the platelet study. We compared the results with those of 24 patients with d-SPD non-associated with leukemia and with those of 15 healthy individuals. Platelets were processed by transmission electron microscopy by standard methods. On the electron micrographs, morphometric analysis of the following structures was performed: 1) Platelets: size and shape, 2) Intraplatelet corpuscular structures (dense granules, alpha granules, mitochondria, lipid droplets): size and number (per platelet and per square micrometer of platelet area), 3) Surface-connected canalicular system (SCS): mean area of individual channel sections and mean percentage of the total SCS area with respect to the platelet area, 4) Glycogen masses: total area with respect to the platelet area. The morphological traits of the platelets and organelles measured above were also evaluated and the dense granules were classified into 4 different types depending on the appearance of their solid core (Weiss et al. Br J Haematol 1993;83:282). The dense tubular system and other ultrastructural characteristics were evaluated by morphology only. The main features of the platelet ultrastructure in patients with FPDMM were (Fig 1, Table 1): 1) slight increase in platelet size while preserving the discoidal shape, 2) moderate reduction in the number of dense granules, which showed a reduced proportion of type 1 granules (with the solid core occupying more than 50 % of the granule) and an increased proportion of type 2 and type 3 granules, with a solid core reduced or fragmented respectively, 3) marked increase and dilatation of SCS with some elements filled by a substance of unknown origin, 4) moderate increase in dense tubular system with occasional complex formation. The platelet ultrastructure was similar to that described in the non-associated d-SPD group (Pujol-Moix et al. Haematologica 2000;85:619) although there were some differences (Table 1): in FPDMM platelets the dense granules were less reduced but more dysmorphic, and the SCS, equally dilated, contained a substance that was not observed in the d-SPD platelets. Given that all the findings described belong to the same family, it would be necessary to evaluate the platelet ultrastructure in additional families and extend the study to other characteristics of d-SPD. Only in this way, it would be possible to know to what extent the platelet defect of FPDMM and that of non-associated d-SPD shared a pathogenic mechanism. Disclosures No relevant conflicts of interest to declare.

Analysis on the laboratory examination characteristics in 22 patients with acute megakaryoblastic leukemia

目的分析急性巨核细胞白血病（AMKL）患者实验室检查特点。方法采集22例AMKL患者骨髓，分离有核细胞，一部分用于制备普通透射电镜标本，观察AMKL患者巨核细胞的形态结构；另一部分用于制备血小板过氧化物酶（PPO）细胞组化电镜标本，分析AMKL患者巨核细胞PPO阳性反应。同时结合分析患者骨髓细胞形态学、细胞化学和染色体核型等检查结果。结果22例患者中，17例患者骨髓巨核细胞以Ⅰ期巨核细胞为主，核圆，胞质含少量微管、膜性囊腔及微小致密颗粒，无分界膜和表面连接管道系统等特征性结构，致密颗粒和α颗粒少；5例患者骨髓巨核细胞大小不等，除Ⅰ期巨核细胞外，同时含有大量Ⅱ、Ⅲ期巨核细胞。22例患者巨核细胞PPO阳性率0~80%。22例患者骨髓涂片中均可见原始和幼稚巨核细胞，CD41免疫组化染色呈阳性反应。复杂染色体核型异常多见。结论大部分AMKL患者骨髓巨核细胞以Ⅰ期巨核细胞为主，少数患者除Ⅰ期巨核细胞外还伴有大量Ⅱ、Ⅲ期巨核细胞；PPO阳性反应差别明显；巨核细胞CD41免疫组化染色具有特异性；染色体核型复杂。

Phenotypic heterogeneity in the gray platelet syndrome extends to the expression of TREM family member, TLT-1

The Gray platelet syndrome (GPS) is a rare inherited disorder linked to undefined molecular abnormalities that prevent the formation and maturation of alpha-granules. Here, we report studies on two patients from unrelated families that confirm phenotypic heterogeneity in the disease. First we used immunoelectron microscopy (I-EM) to confirm that TREM-like transcript-1 (TLT-1) is mostly localized to alpha-granule membranes of normal platelets. Then we performed Western blotting (WB) and flow cytometry with permeabilized platelets to show that TLT-1 is selectively reduced in the platelets of patient 1, previously noted to be deficient in glycoprotein (GP)VI (Nurden et al., Blood 2004; 104: 107-114). Yet both TLT-1 and GPVI were normally expressed in platelets of patient 2. Usual levels of JAM-C and claudin-5, also members of the immunoglobulin receptor family, were detected in platelets of both patients. In contrast, P-selectin was markedly decreased for patient 1 but not patient 2. Two metalloproteases, MMP-2 and MMP-9 were normally present. As predicted, platelets of patient 1 showed little labelling for TLT-1 in I-EM, whereas residual Fg was seen in small vesicular structures and P-selectin lining vacuoles or channels of what may be elements of the surface-connected canalicular system. Our results identify TLT-1 as a glycoprotein potentially targeted in platelets of GPS patients, while decreases in at least three membrane glycoproteins suggest that an unidentified proteolytic activity may contribute to the phenotype in some patients with this rare disease.

Abnormal Large Platelets Are Associated Not Only with von Willebrand Disease (VWD) Type 2B (Including Type 2B/1 New York) but Also with Severe Type 3: A Potential Role for von Willebrand Factor (VWF) in Megakaryocytopoiesis.

Background: VWD type 2B results from mutations in exon 28 of the VWF gene. Gain of function of this adhesive protein results in an increased affinity for the platelet glycoprotein (GP) Ib-IX-V complex. Recently we reported that impaired megakaryocytopoiesis results from an abnormal interaction between GPIb with newly synthesized VWF in megakaryocytes of a family with the R1308P mutation (Blood 2006; 108:2587–95).Aim of the Study: to further examine the potential consequences of VWF abnormalities on platelet production we have studied a series of patients with different types of VWD.Patients, Methods: 13 VWD patients were enrolled in the study after informed consent. Diagnoses of VWD were performed according to the criteria of the ISTH-SSC-SC. Included were 8 VWD 2B patients from 6 families with the following mutations: R1306W (n=1), R1308C (n=1), I1309V (n=1), V1326M (n=2), R1341Q (n=2) and P1266L (n=1, 2B/1NY). Also studied were 5 additional VWD cases characterized by low/absent VWF in their platelets: VWD 2M (n=1, D1277-E78delInsL), VWD 3 without inhibitors against VWF (n=2, 276delT/257delA and 6182delT/6182delT) and VWD with isoantibodies against VWF (n=2, large deletions of the VWF gene). The platelet count was decreased at the time of examination for 6/8 VWD 2B patients and normal for 2/8 (n=1 V1316M and n=1 P1266L). Platelet counts were normal in the remaining 5 patients with VWD types 3, and 2M. Electron microscopy (EM) of platelets and immunolocalization of VWF were performed.Results: EM showed the presence of an increased population of giant platelets (15 to 40% versus <10% for controls) in all VWD 2B. Characteristics of these platelets were the presence of large vacuoles often filled with material and the presence of numerous membrane complexes. Additional abnormalities were observed in the patient with 2B/1NY; alpha-granule morphology was different with a population of enlarged granules, sometimes giant. There was also a heterogeneneous distribution with some platelets almost devoid of alpha-granules. Immunogold staining for all type 2B patients showed that VWF was present not only inside the granules but also in the surface-connected canalicular system. For 3/8 patients with VWD 2B, cleaved caspase was present in the platelets indicating abnormal caspase activity at least for R1341Q and V1316M. In VWD 2M (mutation D1277-E78delInsL) characterized by low platelet VWF content as well as in the VWD 3 (n=2) with a premature stop codon, no significant modification of platelet morphology was found. Some residual VWF was also seen in the alpha-granules of these 2 VWD 3 patients. In contrast, a significant number of enlarged platelets with numerous vacuoles were found in the 2 VWD 3 with large deletions and isoantibodies directed against VWF. Immunogold labelling for platelet VWF was completely negative for these two patients.Conclusions: Patients with VWD types 2B and 3 (undetectable VWF) show platelet production defects of varying severity, suggesting a major role of VWF in the fine regulation of megakaryocytopoiesis. Up-regulation or loss of the interaction between VWF and GPIb may lead to a variable proportion of giant platelets with or without thrombocytopenia.

Heterogeneity in the Gray Platelet Syndrome: Variable Expression of the TREM Family Member, TLT-1, in Platelets of Patients of Two Unrelated Families.

The Gray platelet syndrome is a rare inherited disorder of platelets linked to undefined molecular abnormalities that prevent the normal formation and maturation of alpha-granules. This leads to the spontaneous release of endogenously synthesized alpha-granule proteins from megakaryocytes into the marrow, a factor favouring the development of fibrosis. The platelet functional response varies greatly in patients in the GPS. Here we examine patients from two unrelated families, patient 1 has platelets that have a much reduced aggregation response to collagen, and patient 2 has a normal response. Both patients possess platelets severely deficient in alpha-granules. Features of their internal stucture include numerous vacuoles, thin channels and a reduced surface-connected canalicular system (SCCS) and small vesicular structures (SVs) some of which are open to the surface and rapidly accessed by abciximab used as a probe for ligand trafficking in immunoelectron microscopy (I-EM). Residual amounts of fibrinogen, and VWF are often associated with the SVs, P-selectin delimits some elements of the SCCS and vacuoles while most internal and surface membranes are labelled for seven transmembrane receptors such as PAR-1 and TPalpha, Immunoblotting showed that unlike platelets of patient 1 where GPVI was severely decreased (Nurden P et al, Blood 2004; 104:107–114), those of patient 2 normally expressed this collagen receptor. TREM-like transcript-1 (TLT-1), a membrane glycoprotein, has recently been predominantly localized to the alpha-granules of human platelets on the basis of its codistribution with P-selectin. Using a rabbit antibody to a cytoplasmic domain sequence we have confirmed this distribution in normal platelets. Significantly, we then further showed by Western blotting (WB) that TLT-1, like GPVI, is severely reduced in the platelets of patient 1 but normally expressed in those of patient 2. Flow cytometry using permeabilized platelets showed that this decrease was selective compared to normal levels of JAM-3 and claudin-5, also members of the immunoglobulin family. I-EM showed residual TLT-1 associated with both internal and external membranes. One possibility is that a pathology-related metalloprotease activity (MMP) leads to spontaneous cleavage of the extracellular domains of GPVI and TLT-1 in the platelets of some patients. We therefore examined the platelets of patient 1 to see if MMP-2 and MMP-9, two proteases expressed in normal platelets, were present. I-EM first showed that while for normal platelets both proteases were in their majority localized to alpha-granules, other pools were present in the cytoplasmic and surface-linked membrane systems. The latter were retained in the platelets of patient 1 but flow cytometry with permeabilized platelets failed to show over-expression. Our results identify TLT-1 as a new glycoprotein potentially cleaved in GPS platelets and further show that so-far undefined protease activity contributes to phenotypic heterogeneity in this rare disease.

Lentivirus degradation and DC-SIGN expression by human platelets and megakaryocytes.

As platelets are able to endocytose human immunodeficiency virus (HIV), we have investigated the fate of lentiviruses when endocytosed by human platelets and megakaryocytes (MK), and have characterized a specific receptor directly involved in this function. Genetically modified (non-replicative) lentiviruses with an HIV envelope (HIV-e) or with a vesicular stomatitis virus protein G envelope (VSV-e) were alternatively used and their interaction with platelets and MK analyzed by electron microscopy (EM) and immunoEM. When incubated with platelets, HIV-e and VSV-e lentiviruses were internalized in specific endocytic vesicles and trafficked to the surface connected canalicular system (SCCS). Double immunolabeling for the viral P24 core protein and alpha-granule markers showed that lentiviruses were degraded in the SCCS after contact with alpha-granule proteins. In culture MK, lentiviruses were found in endocytic vesicles and accumulated in acid phosphatase-containing multivesicular bodies (MVB). The expression of the pathogen receptor dendritic cell-specific ICAM-grabbing non-integrin (DC-SIGN) was then demonstrated in platelets by flow cytometry, immunoEM and Western blot. Anti-DC-SIGN antibodies decreased HIV-e lentivirus internalization by platelets, showing that the receptor is functional. Specific signals for DC-SIGN protein and mRNA were also found in MK. This study indicates that platelets and MK can internalize lentiviruses in a pathway, which either provide a shelter to lentiviral particles or alternatively disrupts viral integrity. The receptor DC-SIGN is involved in this function.

Redistribution of Activation-Dependent Antigens on the Platelet Surface In Vivo and In Vitro.

Platelets play a crucial role in thrombosis, platelet activation induces an alteration of platelet membrane glycoproteins between the platelet surface and surface connected canalicular system (SCCS). In an attempt to better understand this process and assess its significance in the study of thrombosis disease, we used flowcytometry for detecting the redistribution of antigens on the platelet surface following activation. The expression of platelet membrane glycoproteins (GP) Ib, IIIa and P-selectin as well as the platelet agglutination induced by ristocetin were measured in 20 normal subjects, whose platelets were stimulated by the peptide SFLLRNP(TRAP, thrombin receptor activated peptide) at different time points (0~30minute). The same antigens were also analyzed in whole blood in 30 patients with cerebral infarction. As expected, a stable increase of P-selectin was obtained from 2 minute upon TRAP activation, while a reversible reduction of GPIbα and agglutinability by ristocetin were showed at the same time, with a maximum decrease at 2 minute, then followed by a return of GP Ibα to platelet surface. In addition, the platelet membrane GPIIIa was not significantly changed in the initial stage after platelet stimulation and increased by degrees 10 minutes later. Compared with those in normal group, there was an apparent increase of P-selectin and a lower expression of GPIb in the patients with cerebral infarction, while GPIIIa was not significantly changed in all the samples. In summary, TRAP can mediate platelet activation, which leads to the redistribution of GPIb and GPIIIa in a time-dependent manner, together with the release of P-selectin. Meanwhile, the change of activation-dependent antigens on the platelet surface could be an important index reflecting the platelet activation in vivo.

Gene Transfer to Macrophages with Nanoparticle-Loaded Platelets.

Hemorrhagic fever, traumatic brain injury, and inflammatory bowel syndrome are among a wide range of pathologies involving inflammation that results in bleeding. The vascular injury that occurs in these conditions is closely associated with macrophage activation. An inherent function of macrophages is to phagocytose platelets that have adhered to sites of vascular injury, and thus platelets are an ideal vehicle for site-specific delivery of anti-inflammatory genes to macrophages at wound sites. Here we report the preparation of reporter gene nanoparticle-platelet conjugates and gene transfer to macrophages. Plasmid-containing nanoparticles were prepared by condensing cDNA for β-galactosidase, green fluorescent protein, or luciferase with polyethyleneimine. Scanning electron microscopic analysis showed that this anion/cation agglomeration reaction yields spherical particles with diameters of 100 to 300 nm. Plasmid-containing nanoparticles were then incubated with platelets that were isolated from human blood by differential centrifugation. Flow cytometric and transmission electron microscopic analysis demonstrated that the nanoparticles associated with the surface-connected canalicular system with most platelets containing one or more particles. The ability of the nanoparticle-loaded platelets to transfer the reporter genes to macrophages was tested in tissue culture using monocyte-derived macrophages incubated with the nanoparticle-loaded platelets. Subsequent fluorescent microscopic, luminescent and histological analysis demonstrated that the macrophages readily phagocytosed the nanoparticle-loaded platelets and expressed the reporter genes. This result provides the basis for studies to deliver anti-inflammatory genes to macrophages in animal model systems.

The 3-Dimensional Architecture of Platelets in the Native State by Electron Cryotomography.

Conventional electron microscopy requires that samples be chemically fixed, embedded in plastic and stained prior to examination. This technique always raises uncertainty as to whether the observations are faithful to the native cellular organization. These limitations are overcome with the technique of electron cryomicroscopy, in which samples are prepared and observed in the frozen, hydrated state. A modification of this technique, electron cryotomography, is a method capable of recording images from the same specimen area at different tilt angles under low electron dose conditions, revealing the spatial relationships of organelles and molecular assemblies within a cell that has not been subjected to chemical fixation or metal staining.Here, we used electron cryotomography to examine the 3-dimensional architecture of platelets in the resting state and in various stages of activation. Platelets in citrated plasma isolated from healthy human subjects were allowed to settle onto a perforated carbon grid and frozen immediately in liquid ethane or were allowed to settle for different time periods on a grid coated with fibrillar type 1 collagen before being frozen. The platelets suspended across holes were embedded in a thin layer of vitreous ice. Successive images of these frozen, hydrated platelets were then acquired as the stage was tilted through a range of ±70° using a JEM2010F electron cryomicroscope. Each image was recorded using a 4kx4k CCD camera. The digital images were computationally aligned and merged to reconstruct a 3-dimensional density map with an 4.0 gigapixel volume. Multiple tilt-series were reconstructed and analyzed using segmentation and visualization tools. The platelet reconstructions were annotated in terms of the sizes, shapes and relative locations of the subcellular components, including mitochondria, α and dense granules, dense clusters, the dense tubular system, microtubules, and the surface-connected canalicular system. We observed great variability in the extent of ordered structure in the α granules, and in the filopodia adhering to the grid surface. The tomograms resolve the fibrous actin bundles driving filopod extension and the individual actin cross-links within the bundles. In some platelets, we observed membrane tethers that attached to the surface but were devoid of actin. This represents the first 3-D visualization of human platelets under native conditions. These observations and the ease of generating such data suggest that electron cryotomography can become a useful tool for dissecting the molecular sequence of cytoskeletal rearrangements occurring during platelet activation and granule secretion and for understanding the structural basis of platelet diseases.

411. Trapping of Adenovirus Vectors in Blood

In an attempt to circumvent the limitations of Adenovirus (Ad) vectors derived from group C serotype Ad5 we recently developed vectors based on group B serotype Ad11. These vectors are benefited by the low prevalence of Ad11-neutralizing antibodies in humans, and the CD46-dependent tropism of Ad11. CD46 is expressed at high levels in cell types including hematopoietic stem cells, dendritic cells and metastatic tumor cells. Following systemic intravenous delivery of an Ad5 vector, a chimeric Ad5 vector possessing the CD46 interacting Ad11 fiber (Ad5/11), and an Ad11 vector to CD46 transgenic mice we discovered that the kinetics of vector clearance from blood are different. Using real time quantitative PCR to detect viral genomes we found that the number of Ad11 vector genomes in mouse serum at 3 minutes post tail vein injection was 2 orders of magnitude lower than for Ad5 and Ad5/11 vectors, but at 15 and 120 minutes post injection the number of vector genomes was comparable between Ad5, Ad5/11 and Ad11. To investigate the differences between the numbers of vector genomes in serum at 3 minutes post injection the association of vector genomes with blood cells was also monitored by real time quantitative PCR at all time points. The number of vector genomes associated with blood cells was more than one order of magnitude higher for Ad11 than Ad5 or Ad5/11 at all time points tested with the highest difference seen at 3 minutes post injection (|[sim]|2 orders of magnitude higher). To study which type of blood cells associate with Ad11 vectors, CD46 transgenic mice were injected in the tail vein with 3H-[thymidine] labeled Ad vector. Individual cell types were separated from blood collected at 3 minutes post injection and the highest levels of tritium labeled Ad11 were associated with the platelet fraction. Subsequent analyses revealed that Ad5, Ad5/11 and Ad11 vectors associate with platelets within minutes after intravenous injection but that Ad11 vectors associate at higher levels. Electron microscopic analysis of platelets isolated 3 minutes after injection of Ad5, Ad5/11 or Ad11 vectors revealed that Ad particles were associated with the platelet surface and structures of the surface-connected canalicular system. We also noticed that Ad11 vectors associate with mononuclear cells more efficiently than Ad5/11 vectors. When taken together, the observations that Ad11 associates with platelets and mononuclear cells more efficiently than Ad5/11 suggests that capsid proteins other than fiber may mediate blood cell interactions. We are currently investigating the mechanism(s) by which Ad capsids and blood cells interact. The roles of the serotype specific (Ad5 or Ad11) capsid proteins hexon, penton and fiber in Ad/blood cell interactions will be presented. Our aim is to develop new Ad vectors that are not trapped in blood upon systemic intravascular administration.

Subcellular localization of thrombopoietin in human blood platelets and its release upon thrombin stimulation.

Thrombopoietin (TPO) is a major regulator of platelet production. The concentration of circulating TPO seems to be determined by its binding and internalization by megakaryocytes and platelets. To elucidate the platelet compartments involved in TPO metabolism, we investigated intraplatelet TPO by post-embedding immunoelectron microscopy, incubated platelets with recombinant human (rh)TPO coupled to colloidal gold and visualized the TPO uptake using electron microscopy. TPO concentrations were measured in 12 platelet concentrates (PC) before and after stimulation with thrombin and after disruption of platelets by freezing-thawing. In resting platelets, immunogold labelling revealed a prevailing cytoplasmic localization of TPO antigen and minor labelling within the surface-connected canalicular system (SCCS); storage granules were devoid of labelling. In tracer experiments, TPO-gold was observed on the plasma and SCCS membranes and within the cytoplasm. Upon thrombin stimulation, endogenous TPO was still detected within the cytoplasm by immunolabelling, and tracer experiments revealed TPO-gold within the cytoplasm and on fibrin fibres. After thrombin stimulation of PC, the plasma TPO levels increased to an average of 535%, and after platelet lysis to an average of 1625% compared with plasma values in unstimulated PC. We conclude that platelets contain releasable immunoreactive TPO within the SCCS and within their cytoplasm, but not within granular compartments. Stored immunoreactive TPO is released upon thrombin stimulation, but only to a minor degree.

Localization of protein kinase A and vitronectin in resting platelets and their translocation onto fibrin fibers during clot formation

Physiological stimulation of platelets with thrombin brings about the release of protein kinase A (PKA) into the plasma. In human blood, this kinase singles out and phosphorylates vitronectin (Vn), a multifunctional regulatory protein, which was proposed to play an important role in the control of fibrinolysis. Here we present immuno-cytochemical evidence to show: (i) that intact platelets possess on their surface an ecto-PKA which can preferentially phosphorylate Vn; (ii) that in the resting platelet, both the catalytic and the regulatory subunits of PKA are present on the platelet surface, in the surface-connected canalicular system, and within the alpha-granules of the platelets; (iii) that the process initiated upon platelet activation, which leads to the formation of fibrin fibers and consequently forms the fibrin net, is accompanied by a translocation of PKA, of Vn, and of PAI-1 onto the fibrin fibers. We propose that the localization and the translocation of these proteins in the fibrin net, together with our finding that PKA phosphorylation of Vn reduces its grip of PAI-1, can unleash PAI-1 in its free form. The free PAI-1 can then assume its latent (non inhibitory) conformation, allow plasminogen activators to trigger the formation of active plasmin, and to initiate fibrinolysis.

The Cleaved Peptide of PAR1 Results in a Redistribution of the Platelet Surface GPIb-IX-V Complex to the Surface-Connected Canalicular System

The only known function of the 41 amino acid cleaved peptide (TR1-41) of the seven transmembrane domain thrombin receptor (PARI) is to activate platelets (as determined by aggregation, surface P-selectin, and fibrinogen binding to activated GPIIb-IIIa). We now demonstrate that TR1-41 results in a concentration-dependent decrease in the platelet surface expression of each component of the GPIb-IX-V complex, as determined by flow cytometry with a panel of monoclonal antibodies (including 6D1, directed against the von Willebrand factor binding site on GPIbalpha, and TM60, directed against the thrombin binding site on GPIbalpha). TR1-41 also decreased ristocetin-induced platelet agglutination. Immunoblotting after incubation of platelets with TR1-41 revealed neither a loss of platelet GPIb nor increase in supernatant GPIb fragments. As demonstrated by immunoelectron microscopy, TR1-41 resulted in a redistribution of GPIb, GPIX, and GPV from the platelet surface to the surface-connected canalicular system (SCCS). In summary, the cleaved peptide (TR1-41) of PAR1 results in a redistribution of the platelet surface GPIb-IX-V complex to the SCCS, thereby negatively regulating the GPIbalpha binding sites for von Willebrand factor and thrombin.

Platelet Glycoprotein IIb/IIIa Inhibitors

Glycoprotein IIb/IIIa (GPIIb-IIIa) complexes (integrin αIIbβ3) mediate platelet aggregation by binding fibrinogen or von Willebrand factor (vWF), protein cofactors that form bridges between adjacent platelets. The cross-linked adhesive proteins assemble platelets into the aggregate. Agents that block the function of the GPIIb-IIIa complex of platelets constitute a powerful new generation of antithrombotic drugs.1 Among the short- and long-term aims of such drugs are (1) to provide immediate relief in the case of ongoing arterial thrombosis and (2) to eliminate excessive platelet reactivity in diseased vessels so that occlusive thrombi and restenosis do not occur, while allowing sufficient hemostasis to prevent spontaneous bleeding. It should be emphasized that stenosis and partial occlusion are both prothrombotic, with increased shear stress promoting platelet activation. Under these conditions, vWF plays a major role in the mediation of thrombus formation, interacting with GPIIb-IIIa and the adhesion receptor GPIb.2 Otherwise, fibrinogen is the major cofactor of platelet aggregation, essentially binding through a dodecapeptide sequence (aa400 to aa411) present at the carboxy terminus of each γ chain. Binding of vWF and other adhesive proteins, such as fibronectin, to GPIIb-IIIa is mediated by the Arg-Gly-Asp (RGD) sequence, a universal mediator of cellular interactions with the extracellular matrix.1 2 3 Anti–GPIIb-IIIa drugs block this final step of the platelet aggregation process. They also block the “outside-in” signaling that follows the binding of adhesive proteins to activated GPIIb-IIIa and the onset of platelet aggregation.3 This signaling may promote events such as secretion, clot retraction, and the expression of procoagulant activity; therefore, its inhibition extends the influence of anti–GPIIb-IIIa drugs beyond the blocking of platelet-to-platelet cohesion. The present review will mostly be illustrated by results obtained with abciximab (c7E3 Fab, ReoPro), a chimeric antibody fragment that is the most widely used of the new …

Labeling of the Internal Pool of GP IIb-IIIa in Platelets by c7E3 Fab Fragments (abciximab): Flow and Endocytic Mechanisms Contribute to the Transport

Abciximab is a new antiplatelet therapeutic in ischemic cardiovascular disease. The drug, chimeric Fab fragments of a murine monoclonal antibody (MoAb) (c7E3), blocks GP IIb-IIIa function. However, its capacity to reach all receptor pools in platelets is unknown. Electron microscopy and immunogold labeling were used to localize abciximab in platelets of patients receiving the drug for up to 24 hours. Studies on frozen-thin sections showed that c7E3 Fab, in addition to the surface pool, also labeled the surface-connected canalicular system (SCCS) and alpha-granules. Analysis of gold particle distribution showed that intraplatelet labeling was not accumulative and in equilibrium with the surface pool. After short-term incubations of platelets with c7E3 Fab in vitro, gold particles were often seen in lines within thin elements of the SCCS, some of which appeared in contact with alpha-granules. Little labeling was associated with Glanzmann's thrombasthenia platelets, confirming that the channels contained bound and not free c7E3 Fab. Endocytosis of abciximab in clathrin-containing vesicles was visualized by double staining and constitutes an alternative mechanism of transport. The remaining free pool of GP IIb-IIIa was evaluated with the MoAb AP-2; flow cytometry showed it to be about 9% on the surface of nonstimulated platelets but 33% on thrombin-activated platelets. The ability of drugs to block all pools of GP IIb-IIIa and then to be associated with secretion-dependent residual aggregation must be considered when evaluating their efficiency in a clinical context.

Labeling of the Internal Pool of GP IIb-IIIa in Platelets by c7E3 Fab Fragments (abciximab): Flow and Endocytic Mechanisms Contribute to the Transport

AbstractAbciximab is a new antiplatelet therapeutic in ischemic cardiovascular disease. The drug, chimeric Fab fragments of a murine monoclonal antibody (MoAb) (c7E3), blocks GP IIb-IIIa function. However, its capacity to reach all receptor pools in platelets is unknown. Electron microscopy and immunogold labeling were used to localize abciximab in platelets of patients receiving the drug for up to 24 hours. Studies on frozen-thin sections showed that c7E3 Fab, in addition to the surface pool, also labeled the surface-connected canalicular system (SCCS) and α-granules. Analysis of gold particle distribution showed that intraplatelet labeling was not accumulative and in equilibrium with the surface pool. After short-term incubations of platelets with c7E3 Fab in vitro, gold particles were often seen in lines within thin elements of the SCCS, some of which appeared in contact with α-granules. Little labeling was associated with Glanzmann's thrombasthenia platelets, confirming that the channels contained bound and not free c7E3 Fab. Endocytosis of abciximab in clathrin-containing vesicles was visualized by double staining and constitutes an alternative mechanism of transport. The remaining free pool of GP IIb-IIIa was evaluated with the MoAb AP-2; flow cytometry showed it to be about 9% on the surface of nonstimulated platelets but 33% on thrombin-activated platelets. The ability of drugs to block all pools of GP IIb-IIIa and then to be associated with secretion-dependent residual aggregation must be considered when evaluating their efficiency in a clinical context.

The surface-connected canalicular system in the sinus endothelial cells of rat spleen.

The existence of a surface-connected canalicular system in the splenic sinus endothelial cells of the rat has been demonstrated by transmission electron microscopy with lanthanum nitrate acting as a tracer for the extracellular space. In addition, the three-dimensional arrangement of the canaliculi has been revealed by computer-aided reconstruction. The surface-connected canalicular system of the sinus endothelial cells consists of slender canaliculi that are branched, anastomosed, and that show continuity with the plasma membrane. They twist in and out among the organelles and are often found in close apposition to the spherical invaginations of the plasma membrane and run alongside them. Canaliculi which are not infiltrated by lanthanum nitrate take the form of electron-lucent tubules and are accompanied by numerous spherical invaginations of the plasma membrane. From a computer-aided reconstruction, the canaliculi, which invaginate from various sites of the plasma membrane, have been found to be continuous with each other and to penetrate to the surface of the sinus endothelial cell; they also branch and anastomose to form a complex network in the cytoplasm. Although the surface-connected canalicular system in blood platelets and thrombocytes is believed to function as the main route for the discharge of granules and the uptake of foreign materials and also to take part in the storage and transport of calcium, it is unclear at present whether the network of the surface-connected canalicular system in splenic sinus endothelial cells has any physiological significance.

Platelet internalization in early thrombogenesis.

Activated platelets rapidly internalize ligand-alpha(IIb)beta 3 complexes and particulates. The platelets sequester these primarily to the surface-connected canalicular system (SCCS), alpha-granules or evaginated surfaces. This occurs swiftly as activation initiates shape change, internal transformation, and secretion out of the platelet SCCS. The transmembrane signaling mechanism for internalization is through the platelet submembranous, detergent-resistant, cytochalasin B-resistant cytoskeletal filaments, not cytoplasmic actin filaments. This work proposes the model that platelet internalization responses contribute in the retraction of clots and participate in vivo in thrombus development at sites of coronary artery injury. In isometric clot retraction, the retracting platelets show internalization of fibrinogen-gold and fibrin while spreading on the polymerizing fibrin strands. The combination of platelet internalization (retracting) with evagination (spreading) on fibrin strands appear to approximate and organize nearby platelets into the developing thrombi. Ex vivo findings are similar under flowing conditions on denuded rabbit aortic endothelium with fibrinogen-gold markers. Early thrombi developing in vivo at sites of congenital coronary artery lesions in perinatal piglets show fibrin strands internalized into the SCCS of platelets. A thrombogenesis model is proposed that shows that platelet internalization responses anchor fibrin and approximate platelets, thus enhancing thrombus development and retraction.

Visualization of activation-dependent epitopes on glycoprotein IIb-IIIa complexes of platelets stimulated by thrombin: immunogold staining of ultrathin sections.

An immunoglobulin M monoclonal antibody (IgM MAb; AP-6) recognizing the sequence 211-221 of glycoprotein (GP) IIIa enabled a study of the distribution of this epitope on unstimulated or thrombin-activated platelets. Flow cytometry was used to evaluate the expression of this epitope on platelets and immunogold staining on ultrathin sections to analyze its distribution within the cell. There was little or no binding of AP-6 to unstimulated platelets, but immunogold staining showed labeling associated with the membranes of alpha-granules. The binding of AP-6 to thrombin-stimulated platelets was compared with that of anti-RIBS MAbs and antifibrinogen polyclonal antibodies. An increased expression of the AP6 epitope was observed on membranes of the surface-connected canalicular system and at the periphery of the cell as early as 10 to 15 seconds after platelet activation by thrombin. At the same time, binding of anti-RIBS MAbs confirmed that at least part of the endogenous fibrinogen had left the alpha-granules and was bound to platelet membranes. Staining with polyclonal antifibrinogen antibody also revealed fibrinogen in vesicles resulting from granule fusion. Rapidly, the pool of internal membranes with bound fibrinogen became exposed to the outside of the platelet, a process involving the unfolding of membranes and pseudopod formation. Our results provide evidence that activation of GP IIb-IIIa and binding of fibrinogen to platelet membranes can occur before their expression at the platelet surface. They also suggest the presence of an alpha-granule pool of GP IIb-IIIa linked to fibrinogen in unstimulated platelets.

Isolation of InsP4 and InsP6 binding proteins from human platelets: InsP4 promotes Ca2+ efflux from inside-out plasma membrane vesicles containing 104 kDa GAP1IP4BP protein

A low-density membrane fraction from human platelets contained the plasma membrane marker glycoprotein Ib (GpIb) and selective binding sites for InsP4 and InsP6. It was separated from the bulk of InsP3-receptor-containing membranes, but was heterogeneous, probably also containing surface-connected canalicular system and some lighter elements of the internal dense tubule system. After loading with calcium oxalate and re-centrifugation on Percoll gradients, this mixed fraction was subfractionated into light membranes containing all of the GpIb, high-affinity InsP4 binding sites (KD = 18 nM) and phosphate-stimulated Ca2+ transport activity. InsP4 (EC50 0.6 microM), but not InsP3 or InsP6, released up to 35% of the accumulated Ca2+ from these vesicles, which were shown to be inside-out plasma membrane vesicles by a biotinylation labelling technique and selective removal of right-side-out plasma membrane vesicles with streptavidin-agarose. Most of the InsP4, and all of the InsP6, binding was present in the much denser calcium oxalate-loaded subfractions, which were free of GpIb. InsP6 binding activity was chromatographically purified as a 116 kDa protein (KD for InsP6 = 5.9 nM), with an amino acid content and two internal peptide sequences identical to those of 116 kDa vinculin. A 104 kDa InsP4 binding protein (KD for InsP4 = 12 nM), probably identical to GAP1IP4BP described by Cullen, Hsuan, Truong, Letcher, Jackson, Dawson and Irvine [(1995) Nature (London) 376, 527-530], was also isolated. This InsP4 receptor may mediate Ca2+ influx in platelets that occurs subsequent to receptor-stimulated production of InsP3 and unloading of internal Ca2+ stores.