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 alpha-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 megakaryocytes in GPV knockout mice.

Lesions in the genes for GPIb alpha, GPIb beta or GPIX result in a bleeding diathesis, the Bernard-Soulier syndrome (BSS), which associates a platelet adhesion defect with thrombocytopenia, giant platelets and abnormal megakaryocytes (MK). The role of GPV, also absent in BSS, was recently addressed by gene targeting in mice. While a negative modulator function for GPV on thrombin-induced platelet responses was found in one model, the absence of GP V had no effect on GPIb-IX expression or platelet adhesion. Our study extends previous results and reports that electron microscopy of bone marrow from the GPV knockout mice revealed a normal MK ultrastructure and development of the demarcation membrane system (DMS). There was a usual presence of MK fragments in the bone marrow vascular sinus. Immunogold labelling of MK from the knockout mice showed a normal distribution of GPIb-IX in the DMS and on the cell surface. The distribution of fibrinogen, vWF and P-selectin was unchanged with, interestingly, P-selectin also localised within the DMS in both situations. Thus GPV is not crucial to MK development and platelet production, consistent with the fact that no mutation in the GPV gene has as yet been described in BSS.

Thrombasthenic mice generated by replacement of the integrin alpha(IIb) gene: demonstration that transcriptional activation of this megakaryocytic locus precedes lineage commitment.

To analyze the transcriptional activity of the gene encoding the alpha subunit of the platelet integrin alpha(IIb)beta(3) during the hematopoietic differentiation, mice were produced in which the herpes virus thymidine kinase (tk) was introduced in this megakaryocytic specific locus using homologous recombination technology. This provided a convenient manner in which to induce the eradication of particular hematopoietic cells expressing the targeted gene. Results of progenitor cell cultures and long-term bone marrow (BM) assays showed that the growth of a subset of stem cells was reduced in the presence of the antiherpetic drug ganciclovir, demonstrating that the activation of the toxic gene occurs before the commitment to the megakaryocytic lineage. Furthermore the knock-in of the tk gene into the alpha(IIb) locus resulted in the knock-out of the alpha(IIb )gene in homozygous mice. Cultures of BM cells of these animals, combined with ultrastructural analysis, established that the alpha(IIb) glycoprotein is dispensable for lineage commitment and megakaryocytic maturation. Platelets collected from alpha(IIb)-deficient mice failed to bind fibrinogen, to aggregate, and to retract a fibrin clot. Moreover, platelet alpha-granules did not contain fibrinogen. Consistent with these characteristics, the mice displayed bleeding disorders similar to those in humans with Glanzmann thrombasthenia. (Blood. 2000;96:1399-1408)

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 (alphaIIbbeta3) 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 alphavbeta3 in 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 alpha-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 alphavbeta3 in vivo. The study revealed no signs that abciximab had accumulated in the marrow.

Expression of markers of platelet activation and the interpatient variation in response to abciximab.

Our study concerns the biological effects of abciximab (c7E3 Fab, ReoPro), a powerful new antiplatelet drug that blocks glycoprotein (GP) IIb-IIIa complexes. Samples were examined from 6 patients with coronary artery disease who received a bolus of abciximab followed by a 10- microg/min infusion for at least 18 hours before percutaneous transluminal coronary angioplasty. Inhibition of ADP-induced PA was maximal for 4 patients but partial (79% and 53%) for 2 others during the infusion. Flow cytometry performed with monoclonal antibodies (PAC-1, AP-6, and F26) specific for the "activated" GP IIb-IIIa complex revealed large decreases in the expression of activation markers on platelets during therapy, but these decreases were less marked when inhibition of ADP-induced PA was incomplete. Residual aggregation was seen for all patients during the infusion when TRAP 14-mer peptide or thrombin was the stimulus. Unblocked GP IIb-IIIa complexes were detected on thrombin-stimulated platelets from the patients by immunoelectron microscopy performed using the monoclonal antibody AP-2. Unblocked GP IIb-IIIa complexes were also detected by flow cytometry when platelets preincubated for 1 hour in vitro with abciximab under saturating conditions were (1) incubated with TRAP 14-mer or (2) permeabilized with Triton X-100. In confirming interpatient variation in the platelet response to a standard dose of abciximab, our results also show that an uninhibited internal pool of GP IIb-IIIa complexes may mediate a residual response to strong agonists.

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.

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 (10-80 nm); after stimulation with 0.5 unit/ml thrombin for 2 min this increased to 16.7% +/- 0.22 and 40.4% +/- 0.53, respectively.

Ultrastructural analysis of bone marrow hematopoiesis in mice transgenic for the thymidine kinase gene driven by the alpha IIb promoter.

Transgenic mice have been generated with expression of the herpes virus thymidine kinase gene directed by a 2.7-kb fragment of the alphaIIb murine promoter of the gene encoding the alphaIIb-subunit of the platelet integrin alphaIIbbeta3 (Tropel et al, Blood 90:2995, 1997). Administration of ganciclovir (GCV) to these mice resulted not only in an acute cessation of platelet production due to the depletion of the megakaryocytic lineage, but also a decrease in erythrocyte and leukocyte numbers. Immunogold staining on ultrathin frozen sections and electron microscopy has now shown that the remaining population of immature hematopoietic cells contain a high proportion of Sca-1(+) and CD34(+) cells, with CD45R+ cells of the lymphopoietic lineage being maintained. Stromal cells were also preserved. Blood thrombopoietin levels were high. At 4 days of the recovery phase, Sca-1 and CD34 antigen expression decreased with intense proliferation of cells of the three lineages, with megakaryocyte (MK) progenitors being identified by their positivity for glycoprotein IIb-IIIa. These results suggest that transcriptional activity for the alphaIIb gene promoter was present on pluripotent hematopoietic stem cells. At 6 to 8 days after cessation of GCV, numerous mature MK were observed, some of them with deformed shapes crossing the endothelial barrier through thin apertures. Proplatelet production was visualized in the vascular sinus. After 15 days, circulating platelet levels had increased to approximately 65% of normal. Transgenic alphaIIb-tk mice constitute a valuable model to study in vivo megakaryocytopoiesis.

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.

A 2.7-kb portion of the 5' flanking region of the murine glycoprotein alphaIIb gene is transcriptionally active in primitive hematopoietic progenitor cells.

The continuous generation of mature blood cells from primitive multipotent progenitor cells requires a highly complex series of cellular events that are still largely unknown. To examine the molecular events associated with the commitment of these hematopoietic progenitor cells to the megakaryocytic lineage, the alpha subunit of the platelet integrin alphaIIb beta3 was used as marker. Despite an abundance of information regarding the role of this integrin in platelet adhesion and aggregation, the mechanisms that control the expression of the genes that code for these proteins are poorly understood and the earliest hematopoietic cell capable of expressing them has not been clearly identified. Thus, a strategy was developed to eradicate, using a conditional toxigene, all the hematopoietic cells capable of expressing the alphaIIb gene in mice. This was achieved by targeting the expression of the gene encoding the herpes simplex virus thymidine kinase (tk), specifically to these cell types, using a 2.7-kb fragment of the 5'-flanking region of the murine alphaIIb gene. Three transgenic lines having 1, 3, and 4 copies of the transgene, respectively were produced and analyzed. Administration of ganciclovir (GCV) to these mice induced a severe thrombocytopenia, which was due to the depletion of the entire megakaryocytic lineage, as shown by bone marrow (BM) culture and electron microscopy analysis. The time required to attain a severe thrombocytopenia was dependent on the level of the expression of the transgene and varied from 7 to 11 days. This condition was completely reversed when GCV treatment was discontinued. Progenitor cell assays showed that the alphaIIb promoter was active in primitive hematopoietic progenitor cells possessing myeloid, erythroid, and megakaryocytic potential and that the transcriptional activity of the promoter decreased progressively as differentiation proceeded towards the erythroid and myeloid lineages.

Ultrastructural analysis of the distribution of the vitronectin receptor (alpha v beta 3) in human platelets and megakaryocytes reveals an intracellular pool and labelling of the alpha-granule membrane.

The vitronectin receptor (VnR or alpha v beta 3) belongs to the cytoadhesin subclass of the integrin family. This subclass consists of two receptors which have the beta 3 subunit in common: GP IIb-IIIa complexes (or alpha IIb beta 3) 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 alpha v 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, alpha v beta 3 was mostly detected in internal membrane systems including those of alpha-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 alpha v beta 3 was confirmed by flow cytometry and by using platelets from a patient with type I Glanzmann's 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 alpha-granules were again labelled.

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.

Distribution of ligand-occupied alpha IIb beta 3 in resting and activated human platelets determined by expression of a novel class of ligand-induced binding site recognized by monoclonal antibody AP6.

The sequence beta (3)203-228 is involved, in a yet undetermined manner, in alpha IIb beta 3 function. We now show that murine monoclonal antibody (MoAb) AP6, specific for beta (3)211-221, binds to alpha IIb beta 3 on adenosine diphosphate (ADP)-activated platelets only when the receptor is occupied by intact fibrinogen. The ligand-induced binding-site reported by AP6 is unique in that it is not expressed following occupancy by either RGD peptides or the gamma-chain carboxy-terminal dodecapeptide. Binding of AP6 to platelets coincides temporally with the binding of the MoAb 9F9, specific for a receptor-induced binding site on fibrinogen. Thus, AP6 reports the binding of fibrinogen to the recognition pocket of alpha IIb beta 3. Its binding to thrombin-stimulated washed platelets correlates with secretion as determined using an MoAb to P-selectin. When ultrathin sections of nonactivated platelets were examined by immunogold staining and electron microscopy, AP6 identified a pool of alpha IIb beta 3 colocalizing with P-selectin and suggesting the presence of alpha IIb beta 3-ligand complexes in the alpha-granule membrane. There was little binding of AP6 to surface alpha IIb beta 3 of unstimulated platelets. After ADP-induced activation, AP6 was abundantly distributed over the entire platelet surface, including pseudopods, but only when fibrinogen was present in the medium. ADP had little effect on AP6 reactivity within platelets. This contrasted with washed platelets and thrombin, where extensive AP6 binding was observed within internal membrane pools as early as 10 to 15 seconds after stimulation. Surface labeling with AP6 followed slower kinetics. Flow cytometry on Triton X-100 permeabilized fixed platelets confirmed AP6 binding to alpha IIb beta 3 within the platelet. Thus, our results provide evidence of (1) a pool of alpha-granule alpha IIb beta 3 occupied by ligand in nonactivated platelets; (2) thrombin-induced activation of alpha IIb beta 3 within the platelet, and (3) thrombin-induced mobilization of ligand-bound alpha IIb beta 3 to the surface.

[Effort-induced malignant hyperthermia. Electromyographic anomalies with a myogenic component. 10 cases]. / L'hyperthermie maligne d'effort. Anomalies électromyographiques d'aspect myogène. Dix observations.

Exertion malignant hyperthermia, usually regarded as a form of heat stroke, is mainly observed in young apparently healthy men enlisted in the army and subjected to intensive physical exertion in a warm and damp environment. It is frequently lethal. Ten cases with favourable outcome are reported. In 8 patients, EMG tracings recorded several months after the acute episode showed myogenic abnormalities. Computer-aided analysis of motor units showed a 42% reduction in mean duration of the motor unit collected from the brachial biceps and a 44% reduction of signal energy as compared with controls. Two possible reasons for these abnormalities are discussed: they may result from rhabdomyolysis or from a pre-existing muscular pathology.

[Malignant hyperthermia of effort or "heat stroke" (author's transl)]. / L'hyperthermie maligne d'effort ou "coup de chaleur".

Heat stroke following effort is not confined to hot regions. The authors have seen five cases in the Paris region between 1967 and 1974. It particularly affects young subjects, in pour training or living away from home. Clinically very similar to anaesthetic malignant hyperthermia, it has the same gravity, with a high mortality rate. It may be characterised by the triad: coma, muscular hypertonicity and hyperthermia of over 40 degrees C. Refrigeration, sedation and rehydration are all the more effective when started early. Improved knowledge of malignant hyperthermia of effort, within the more confused context of heat stroke, will ensure that it is recognised more frequently, limit its consequences and lead to better understanding of its underlying cause, the origin of which is undoubtedly muscular.