Plasmodium falciparum-infected erythrocytes: a mutational analysis of cytoadherence via murine thrombomodulin.

The pathophysiologic events leading to organ damage in Plasmodium falciparum malaria infections involve adhesion and sequestration of parasite-infected erythrocytes (PRBC) to the vascular endothelium and syncytiotrophoblast. Several potential receptors to which the PRBCs may bind have recently been identified, one of which is thrombomodulin (TM). TM has been implicated particularly in mediating sequestration of P. falciparum-infected erythrocytes in the placenta and brain, two sites of disease associated with high morbidity. In order to establish that binding of parasite-infected red blood cells to TM is dependent on its containing chondroitin-4-sulfate (CSA), we have mutated the CSA-attachment site of murine TM, and expressed this mutant form (TMsergly) in COS-7 cells. In cytoadhesion assays, we demonstrate that, in contrast to wild-type TM which contains CSA and supports the adhesion of 1466 PRBCs/mm2, TMser-gly does not contain CSA and adhesion of PRBCs to those cells expressing TMser-gly is entirely abrogated (200 PRBCs/mm2). These studies further confirm that the CSA of TM may play a role in the pathophysiology of malaria by providing a binding site for PRBCs.

Thrombomodulin lacking the cytoplasmic domain efficiently internalizes thrombin via nonclathrin-coated, pit-mediated endocytosis.

Thrombomodulin (TM) is a transmembrane vascular endothelial cell receptor that is a cofactor in a major physiologically relevant natural anticoagulant system. We recently developed a cell model to examine one mechanism of regulation of TM cell surface expression and visually demonstrated that the receptor undergoes internalization predominantly via noncoated pits (Conway et al., 1992, J. Cell. Phys., 151:604-612). We have extended these studies to examine the role of the cytoplasmic domain of TM by deleting this region and expressing the truncated version of the molecule in COS cells (COS.Cyto.Del cells). Electron microscopy demonstrated internalization of gold-labeled anti-TM antibody or thrombin in a time- and temperature-dependent manner, similar to that seen with the wild-type transfected cells (COS.TM-CR). Endocytosis was characterized by initial surface clustering of gold particles, followed by aggregation into noncoated pits, early endosome formation, and, finally, entry into multivesicular bodies and lysosomes. There was a notable absence of gold particles in clathrin-coated pits and vesicles. The kinetics of binding and internalization of 125I-labeled ligand in COS.Cyto.Del cells was compared with that of COS.TM-CR cells and was not significantly different. These studies provide ultrastructural and quantitative data to indicate that TM efficiently undergoes endocytosis via nonclathrin-coated pits when the receptor is lacking the cytoplasmic domain. This finding suggests that there may be alternative regions of the molecule that mediate those signals necessary for internalization.

Human neutrophils synthesize thrombomodulin that does not promote thrombin-dependent protein C activation.

Thrombomodulin (TM) is a surface glycoprotein that forms a 1:1 complex with thrombin, thereby interacting to form the basis of a major physiologically relevant natural anticoagulant mechanism. Although initially described as a vascular endothelial cell receptor, TM has been reported to be present in several other cells, including megakaryocytes, platelets, monocytes, and several cultured cells. Other investigators have reported that neutrophils (PMN) may play a role in the hemostatic mechanism by supporting transformation of prothrombin to thrombin. To determine whether PMN might contribute further to the regulation of the coagulation system, we have evaluated these cells for the expression of TM. Large numbers of human leukocytes were isolated by standard techniques, and the PMN fraction was extracted and shown to be free of platelets and monocytes. Membrane preparations were affinity purified on an anti-TM-Affigel-10 matrix and the eluted material was examined by Western blotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and silver staining. The purified material was identical in apparent molecular weight to TM from human placenta and human umbilical vein endothelial cells (HUVEC). Using a sensitive and specific immunoassay, we estimated that there are a minimum of 5,220 +/- 1,658 molecules of TM per PMN, as compared with more than 50,000 in HUVEC. Northern analysis of RNA from PMN indicates that specific messenger RNA for TM, as identified by a single 3.8-kb band, is identical to that from HUVEC, and thereby confirms that PMN can also synthesize the receptor. Localization of TM in PMN was attempted by immunofluorescence, and the receptor was visualized only in permeabilized PMN, but was not seen on the surface of nonpermeabilized cells. Flow cytometry was also used, and could detect TM in 10% to 15% of nonpermeabilized PMN, whereas the antigen was present in greater than 80% of permeabilized cells. Biologic function of the PMN-derived TM, as tested by thrombin-dependent activation of protein C, was absent. Our results suggest that TM is synthesized by PMN, but under nonstimulated conditions, the protein is largely excluded from the membrane surface, and lacks the ability to promote activation of protein C by thrombin. TM from PMN may provide a further link between inflammation and thrombosis and may also be a significant source of plasma TM.

An ultrastructural study of thrombomodulin endocytosis: internalization occurs via clathrin-coated and non-coated pits.

The regulation of thrombomodulin (TM) expression has been reported to occur by several mechanisms. We have examined constitutive internalization of TM using immunofluorescent and electron microscopic (EM) methods. A cell model was developed to study this process by introducing TM DNA into COS-7 cells for expression. The recombinant TM was determined to behave similarly to native TM from human umbilical vein endothelial cells (HUVEC) with respect to M(r) and cell surface functional activity. The transfected cells expressed 8-100-fold more functional TM per cell than HUVEC. Immunofluorescent studies on these cells indicated that anti-TM antibody-TM complex was internalized in a time- and temperature-dependent manner, with internalization detectable within 10 minutes. When the cells were incubated at 4 degrees C with gold-labelled anti-TM antibody, most of the gold particles were surface bound and detected by EM as individual particles or clusters of 2 or 3 particles. Initiation of endocytosis for 10 to 60 minutes resulted in a redistribution of gold particles into small clusters predominantly in non-coated pits and rarely in clathrin-coated pits, subsequently in early endosomes, multivesicular bodies, and lysosomes. Similar studies were performed with gold-conjugated thrombin, demonstrating a similar route of intracellular processing. These studies provide ultrastructural evidence that the process of endocytosis of TM involves the participation of both clathrin-coated and non-coated pits and vesicles, but that the latter process predominates. Further structure/function studies are indicated using our cell model, since defects in the endocytic pathway of this important anticoagulant receptor may contribute to the development of thromboembolic disease.

Heat shock of vascular endothelial cells induces an up-regulatory transcriptional response of the thrombomodulin gene that is delayed in onset and does not attenuate.

Thrombomodulin is a vascular endothelial cell transmembrane protein that forms a 1:1 complex with thrombin, this interaction product forming the basis of a physiologically important natural anticoagulant system. Transcriptional down-regulation of thrombomodulin occurs following exposure of cultured endothelial cells to cytokines, while up-regulation is induced by retinoic acid and dibutyryl cyclic AMP. Thrombomodulin is also regulated developmentally, appearing in the parietal endoderm of 7.5-day-old mouse embryos. We determined that cell surface functional thrombomodulin in cultured human umbilical vein endothelial cells (HUVEC) and A549 cells increased 3.2- and 6.7-fold, respectively, in response to 24 h of continuous 42 degrees C heat shock stress. Northern analyses of thrombomodulin mRNA accumulation also showed a delayed response that was characterized by an augmentation in mRNA levels that started 12-18 h after the initiation of the stress, and continued to rise, without attenuation, during 48 h of continuous heat shock. Nuclear run-on studies confirmed that the predominant mechanism of augmentation was transcriptional. Furthermore, the heat shock-induced up-regulation of thrombomodulin in HUVEC abrogated the suppressive effect of tumor necrosis factor. Analysis of the 5' region of the thrombomodulin gene revealed six highly conserved tandem copies of the five base pair recognition unit that is the consensus sequence for a heat shock element. We hypothesize that the stress-induced augmentation in thrombomodulin gene transcription is mediated via heat shock factors binding to the heat shock element and that the stress response of thrombomodulin may have a biological role to protect the vascular endothelium during a variety of stresses, including inflammation, infection, and/or development.

The amino terminal lectin-like domain of thrombomodulin is required for constitutive endocytosis.

Thrombomodulin (TM) is a multidomain protein that serves as a cofactor in a major natural anticoagulant system. To further characterize the structure-function of TM, we have transfected COS cells with different truncated forms of TM. In the first form, COS cells expressing TM that lacks the putative signal peptide (17 residues); the lectin-like, hydrophobic N-terminal domain (226 residues); and 12 residues of the first epidermal growth factor (EGF)-like repeat (COSdel.238 cells) were found to function normally with respect to TM transport to the cell surface and thrombin-dependent protein C activation. However, in contrast to wild-type TM, as visually studied by immunofluorescence and immunogold electron microscopy, the COSdel.238 cells did not constitutively internalize anti-TM-TM or thrombin-TM complexes. To identify the region responsible for mediating the endocytic process, deletant forms of TM lacking either the lectin-like region (residues 2-155) or the hydrophobic region of the N-terminal domain (residues 161-202) were expressed in COS cells (COSdel.2-155 and COSdel.161-202, respectively). Protein C cofactor activity was maintained in both cells. Although the COSdel.161-202 cells behaved similarly to wild-type TM-transfected cells, visual studies showed a lack of constitutive internalization of thrombin-TM or anti-TM-TM complexes in the COSdel.2-155 cells. We conclude that the lectin-like domain of human TM serves to regulate cell surface expression of TM via the endocytic route and therefore may also play a major physiologic role in controlling intracellular and extracellular accumulation of thrombin in a variety of biologic systems.

Heat shock-sensitive expression of calreticulin. In vitro and in vivo up-regulation.

Calreticulin (CRT) is an ubiquitous, highly conserved, Ca(2+)-binding protein of the sarcoplasmic and endoplasmic reticulum. The precise function(s) of CRT is unknown. However, based on sequence analyses and observations that it may bind to steroid receptors and integrins and store Ca2+ within the cell, it has been postulated to play a "housekeeping" role. To determine whether the level of expression of CRT is affected by stress, we examined the heat shock response of CRT from a variety of cultured cells, including vascular endothelial, lung epithelial, and lung fibroblasts. Following exposure of the cells to 42 degrees C, CRT mRNA transiently accumulated 2.5-4.2-fold at 1-6 h. Nuclear run-on studies and mRNA stability experiments confirmed that the predominant mechanism of augmentation was transcriptional. Chloramphenicol acetyltransferase assays further indicated that the promoter region, containing a putative heat shock element between -172 and -158 of the human CRT gene, is heat shock-sensitive. Finally, we demonstrated the in vivo significance of these findings by exposing rats to hyperthermia. This resulted in accumulation of CRT mRNA and an augmentation of CRT protein in lung tissue. We hypothesize that this stress-induced up-regulation of CRT contributes to the mechanism(s) by which the vascular endothelium and lung tissue, and possibly other organ systems, maintain homeostasis when exposed to a variety of pathophysiological conditions.

Three differentially expressed survivin cDNA variants encode proteins with distinct antiapoptotic functions.

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is believed to play a role in oncogenesis. To elucidate further its physiologic role(s), we have characterized the murine survivin gene and complementary DNA (cDNA). The structural organization of the survivin gene, located on chromosome 11E2, is similar to that of its human counterpart, both containing 4 exons. Surprisingly, 3 full-length murine survivin cDNA clones were isolated, predicting the existence of 3 distinct survivin proteins. The longest open reading frame, derived from all 4 exons, predicts a 140-amino acid residue protein, survivin(140), similar to human survivin, which contains a single IAP repeat and a COOH-terminal coiled-coil domain that links its function to the cell cycle. A second cDNA, which retains intron 3, predicts the existence of a 121-amino acid protein, survivin(121) that lacks the coiled-coil domain. Removal of exon 2-derived sequences by alternative pre-messenger RNA (mRNA) splicing results in a third 40-amino acid residue protein, survivin(40), lacking the IAP repeat and coiled-coil structure. Predictably, only recombinant survivin(140) and survivin(121) inhibited caspase-3 activity. All 3 mRNA species were variably expressed during development from 7.5 days postcoitum. Of the adult tissues surveyed, thymus and testis accumulated high levels of survivin(140) mRNA, whereas survivin(121)-specific transcripts were detected in all tissues, while those representing survivin(40) were absent. Human counterparts to the 3 survivin mRNA transcripts were identified in a study of human cells and tissues. The presence of distinct isoforms of survivin that are expressed differentially suggests that survivin plays a complex role in regulating apoptosis. (Blood. 2000;95:1435-1442)

Structure-function analyses of thrombomodulin by gene-targeting in mice: the cytoplasmic domain is not required for normal fetal development.

Thrombomodulin (TM) is a widely expressed glycoprotein receptor that plays a physiologically important role in maintaining normal hemostatic balance postnatally. Inactivation of the TM gene in mice results in embryonic lethality without thrombosis, suggesting that structures of TM not recognized to be involved in coagulation might be critical for normal fetal development. Therefore, the in vivo role of the cytoplasmic domain of TM was studied by using homologous recombination in ES cells to create mice that lack this region of TM (TMcyt/cyt). Cross-breeding of F1 TMwt/cyt mice (1 wild-type and 1 mutant allele) resulted in more than 300 healthy offspring with a normal Mendelian inheritance pattern of 25.7% TMwt/wt, 46.6% TMwt/cyt, and 27.7% TMcyt/cyt mice, indicating that the tail of TM is not necessary for normal fetal development. Phenotypic analyses showed that the TMcyt/cyt mice responded identically to their wild-type littermates after procoagulant, proinflammatory, and skin wound challenges. Plasma levels of plasminogen, plasminogen activator inhibitor 1 (PAI-1), and alpha2-antiplasmin were unaltered, but plasmin:alpha2-antiplasmin (PAP) levels were significantly lower in TMcyt/cyt mice than in TMwt/wt mice (0.46 +/- 0.2 and 1.99 +/- 0.1 ng/mL, respectively; P <.001). Tissue levels of TM antigen were also unaffected. However, functional levels of plasma TM in the TMcyt/cyt mice, as measured by thrombin-dependent activation of protein C, were significantly increased (P <.001). This supported the hypothesis that suppression in PAP levels may be due to augmented activation of thrombin-activatable fibrinolysis inhibitor (TAFI), with resultant inhibition of plasmin generation. In conclusion, these studies exclude the cytoplasmic domain of TM from playing a role in the early embryonic lethality of TM-null mice and support its function in regulating plasmin generation in plasma.