Christel Poujol

A prototypic platelet septin and its participation in secretion

Studies are presented characterizing platelet CDCrel-1, a protein expressed to high levels by megakaryocytes and belonging to a family of conserved proteins, termed septin. Septin filaments originally were identified in yeast as essential for budding but have become increasingly associated with processes in higher eukaryotic cells involving active membrane movement such as cytokinesis and vesicle trafficking. Direct proof of an in vivo function for septins in higher eukaryotes is limited to the characterization of the Drosophila septin, termed PNUT. We present studies identifying platelet CDCrel-1 as a protein kinase substrate in the presence of known platelet agonists. The immunopurification of CDCrel-1 revealed it to be part of a macromolecular complex containing a protein involved in platelet secretion, syntaxin 4. Moreover, CDCrel-1 was localized in situ to areas surrounding platelet-storage granules. The relevance of CDCrel-1 to normal platelet function was established with the characterization of platelets from a CDCrel-1Null mouse. As compared with platelets from wild-type littermates, CDCrel-1Null platelets aggregate and release stored [14C]serotonin in the presence of subthreshold levels of collagen. These results provide new insights into the mechanisms regulating platelet secretion and identify platelet septins as a protein family contributing to membrane trafficking within the megakaryocyte and platelet.

Absence of GPIbα is responsible for aberrant membrane development during megakaryocyte maturation: Ultrastructural study using a transgenic model

OBJECTIVE The glycoprotein Ib/IX/V complex (GPIb-IX-V) mediates platelet attachment to von Willebrand factor in exposed subendothelium. Molecular defects in the genes for GPIbalpha, GPIbbeta, and GPIX give rise to the Bernard-Soulier syndrome, in which thrombocytopenia and giant platelets suggest that this receptor also is involved in platelet production. To study how giant platelets are produced in vivo, we used a model of GPIbalpha-deficient mice (GPIbalpha(null)) and mice rescued with the human GPIbalpha transgene (GPIbalpha(null;hTg)). MATERIALS AND METHODS Using electron microscopy and immunogold labeling, we examined megakaryocytopoiesis in the bone marrow of these mice and developed a method to quantify the membranes of megakaryocytes (MK) and proplatelets by computer analysis. RESULTS Abnormal membrane development in the perinuclear zone was found in immature MK of GPIbalpha(null) mice. This led to a poorly developed demarcation membrane system and other ultrastructural changes. As a result, well-organized platelet territories were rarely seen within the cytoplasm of mature MK. Membrane quantification confirmed that MK of GPIbalpha(null) mice had a reduced internal membrane pool. Whereas these MK normally crossed the endothelial barrier, their migration was accompanied by the production of unusually large MK fragments or proplatelets in the vascular sinus with an approximately 50% decrease in internal membrane content compared to wild-type. In the rescued GPIbalpha(null;hTg) model, GPIbalpha was normally localized in MK, and there was a total correction of the ultrastructural defects. CONCLUSIONS GPIbalpha is essential for membrane development and distribution in maturing MK. Its absence leads to abnormal partitioning of the membrane systems and abnormal proplatelet production.

Platelet Glycoprotein IIb/IIIa Inhibitors Basic and Clinical Aspects

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 …

Platelet ultrastructural abnormalities in three patients with type 2B von Willebrand disease.

Several reports have described the presence of giant platelets in patients with type 2B von Willebrand disease (VWD). We have now characterized the ultrastructural changes in platelets from three unrelated patients with type 2B VWD and different mutations within exon 28 of the von Willebrand factor (VWF) gene. Electron microscopy showed that each of these subjects had an increased proportion of large platelets when compared with those of a patient with type 2A VWD or control subjects. Immunogold labelling for VWF was performed. Large masses detected by anti‐VWF antibody were seen not only on the platelet surface, but also inside the platelet surface‐connected canalicular system (SCCS) when ultrathin sections were labelled. This suggested translocation of the abnormally bound VWF from the platelet surface. Labelling of the α‐granules was eccentric as for normal platelets. Labelling for glycoprotein (GP) Ib was seen on the surface and within the SCCS, suggesting co‐localization with the bound VWF. However, there was no evidence for VWF in endosomes or other endocytic vesicles. The presence of platelet‐bound VWF was not accompanied by high levels of platelet activation, as detected by electron microscopy, or by using monoclonal antibodies against P‐selectin or activation‐dependent determinants on GP IIb‐IIIa in flow cytometry. Intriguingly, platelet ultrastructure often resembled that seen in patients with congenital thrombocytopathies characteristic of giant platelet syndromes.

Ultrastructural analysis of the distribution of the vitronectin receptor (αvβ3) in human platelets and megakaryocytes reveals an intracellular pool and labelling of the α‐granule membrane

The vitronectin receptor (VnR or αvβ3) belongs to the cytoadhesin subclass of the integrin family. This subclass consists of two receptors which have the β3 subunit in common: GP IIb–IIIa complexes (or αIIbβ3) and VnR. We report the subcellular distribution of VnR within human platelets as determined by immunogold staining of ultrathin frozen sections and transmission electron microscopy. Monoclonal antibodies directed against: (i) the αv subunit (LM142, AMF7, CLB‐706), or (ii) an epitope specific to the complex (LM609) were used. Although VnR is present on platelets, it is a minor component. We therefore first compared several different staining procedures to detect this integrin. Optimal localization of VnR was obtained using a multistep procedure in which biotinylated anti‐mouse IgG and a monoclonal anti‐biotin antibody provided staining enhancement. Results showed that although present on the surface, αvβ3 was mostly detected in internal membrane systems including those of α‐granules. Occasionally, platelet sections showed special vesicular structures covered by gold particles. These were often localized at the edge or immediately under the plasma membrane and their origin remains unclear. An internal pool of αvβ3 was confirmed by flow cytometry and by using platelets from a patient with type I Glanzmanns thrombasthenia arising from a GP IIb gene defect. We also investigated the presence of VnR in megakaryocytes (MK) obtained from normal human bone marrow. A fluorescence study showed VnR in small MK with unilobulated nuclei, suggesting that synthesis of this integrin occurs early during megakaryocytopoiesis. In mature cells, VnR expression had decreased relative to GP IIb–IIIa, although intracellular staining was present in EM and α‐granules were again labelled.

1 The evolution of megakaryocytes to platelets

Megakaryocytes (MKs) arise from pluripotent stem cells by a process of cell division, endoreplication and maturation. Progressively, the MK cytoplasm is invaded by the demarcation membrane system speculated to delimit pre-formed platelets. One theory is that the passage of entire MKs (or fragments) into the blood stream is followed by their physical break-up into platelets in the pulmonary circulation. A second theory is that MKs produce beaded processes (proplatelets) which then separate into platelets. Functionally vital platelet receptors such as GPIIb-IIIa and GPIb-IX complexes are specific markers of the MK lineage. CD34 and CD4 are present in progenitors but progressively disappear as MKs mature. Stroma cells secrete cytokines, produce extracellular matrix proteins and mediate cellular contact interactions that regulate MK development. Studies on thrombopoietin and the use of transgenic mouse models are helping to clarify MK biology.

Ultrastructural analysis of the distribution of von Willebrand factor and fibrinogen in platelet aggregates formed in the PFA-100

The PFA-100 is a new apparatus used to detect platelet dysfunction in vitro . Anticoagulated blood flows under constant pressure through a capillary, and across an aperture that pierces a membrane coated with collagen and either epinephrine or ADP. Through their ability to adhere and aggregate, platelets occlude the orifice and the closure time is a test of platelet function. Using electron microscopy and immunogold staining, we have analyzed the ultrastructure of platelet aggregates formed within the aperture and that are responsible for the occlusion. Standard electron microscopy showed that the aggregates formed on both collagen-epinephrine and collagen-ADP cartridges presented the same morphological features. The aggregates were exclusively composed of platelets, some of which were degranulated. Degranulation was particularly intense at the periphery of the aggregate where platelets were often totally devoid of secretory organelles. Immunogold staining on ultrathin frozen sections with polyclonal antibodies, allowed us to evaluate the distribution of adhesive proteins such as fibrinogen and von Willebrand factor (vWF) within the aggregate. The latter was found to be abundant in the intercellular spaces between adjoining platelets. Although fibrinogen was also present, its labeling was less intense suggesting that vWF is the major protein implicated in the platelet-platelet interactions in the aggregates formed in the PFA-100 system. This may be because of the high shear rate that occurs across the aperture which suggests that the PFA-100 is particularly sensitive for detecting abnormalities of vWF-platelet interactions.

ANNEXIN V RELOCATES TO THE PERIPHERY OF ACTIVATED PLATELETS FOLLOWING THROMBIN ACTIVATION: AN ULTRASTRUCTURAL IMMUNOHISTOCHEMICAL APPROACH

We have previously shown biochemically that the physiological agonist thrombin can cause translocation of endogenous annexin V to a fraction containing all platelet membranes. This paper reports ultrastructural immunohistochemical data revealing that annexin V molecules localize with plasma membranes of blood platelets following thrombin activation. When ultrathin sections of resting platelets were examined by immunogold staining, annexin V was found to be cytosolic, having a generalized distribution throughout the platelet. After thrombin activation, annexin V became peripheral in location and plasmalemma association increased. Morphometric analysis of gold particles shows that annexin V relocates specifically to the plasma membrane and its underlying cytoskeleton following treatment with thrombin. In control platelets 6.1%±0.78 of annexin V is present at the plasma membrane and 15.0%±0.82 in the region corresponding to the membrane cytoskeleton (10–80nm); after stimulation with 0.5unit/ml thrombin for 2min this increased to 16.7%±0.22 and 40.4%±0.53, respectively.

Accessibility of abciximab to megakaryocytes and endothelial cells in the bone marrow compartment: studies on a patient receiving antithrombotic therapy

Abciximab, chimaeric Fab fragments of the monoclonal antibody 7E3 (c7E3 Fab), has achieved widespread use as an anti‐platelet agent for blocking GP IIb–IIIa (αIIbβ3) function and preventing ischaemic complications after coronary artery angioplasty. However, its accessibility to the bone marrow compartment during therapy is unknown, as is its ability to bind αvβ3in vivo. Using electron microscopy and immunogold labelling, we have looked for abciximab in the bone marrow of a patient who became thrombocytopenic during treatment. The presence of abciximab was assessed on ultrathin frozen sections of a marrow aspirate, the drug being revealed by a rabbit antibody to c7E3 Fab. Labelling was maximal on fragmenting megakaryocytes (MK) and proplatelets in the vascular sinus and in direct access to the blood compartment. Not only the plasma membrane but also the demarcation membrane system (DMS) and the membranes of α‐granules were labelled. Abciximab was also revealed on the luminal surface of endothelial cells lining the marrow sinuses, thereby confirming for the first time its ability to bind to αvβ3in vivo. The study revealed no signs that abciximab had accumulated in the marrow.

Effect of infusing rat monoclonal antibodies to the murine GPIb-IX-V complex on platelet and megakaryocyte morphology in mice

In the Bernard-Soulier syndrome, the absence of GPIb-IX-V leads to thrombocytopenia and giant platelets. In autoimmune thrombocytopenia in man, anti-platelet antibodies are associated with changes in megakaryocyte (MK) morphology and platelet size heterogeneity. We have compared the ultrastructural changes in mature MK following the infusion of rat monoclonal antibodies (MoAbs) to different epitopes of the murine GPIb-IX-V complex in mice. Blood and marrow samples were examined during both the acute thrombocytopenic phase and during the recovery phase. A MoAb to GPV induced neither thrombocytopenia nor changes in platelet morphology. During the acute thrombocytopenic phase with anti-GPIb f MoAbs, the size of residual platelets was heterogeneous and included large forms and platelets with few granules. During recovery, platelet size heterogeneity continued, and some platelets showed signs of activation. But only rare platelets were giant forms with ultrastructural defects resembling BSS. Megakaryocytopoiesis during acute thrombocytopenia was already abnormal, with some mature cells often showing vacuoles and an irregular development of the demarcation membrane system which varied in extent. These changes continued into the recovery phase. The anti-GPV MoAb had no effect on MK. Thus, anti-platelet antibodies can induce a different medullary response even when binding to the same receptor.