Search PubMed⌕ Search

Biomedical subjects

E M Cramer

Publications and source records attributed to E M Cramer.

At least 55 records · Page 3Linked to original sources
PubMed1993

Reversible translocation of glycoprotein Ib in plasmin-treated platelets: consequences for platelet function.

Understanding the effect of fibrinolysis on platelet function is of clinical importance. Plasmin is recognized to affect platelet adhesive function by reducing the interaction of platelet glycoprotein (GP) Ib with von Willebrand factor (vWF) bound to the subendothelium. This platelet function is commonly explored in vitro by the ristocetin-induced agglutination test. Our previous study demonstrated a plasmin-induced redistribution of GP Ib molecules from the platelet surface to the linings of the surface-connected canalicular system (SCCS), a critical mechanism for understanding plasmin-induced GP Ib dysfunction. Here, we demonstrate that neutralization of plasmin by its inhibitors, aprotinin or tripeptide Val-Phe-Lys-CH2Cl, permits a time dependent recovery (within 30 min) of ristocetin-induced agglutination in the platelets which were stimulated by plasmin at < 1 CU ml-1. This functional recovery was accompanied with a restoration of a normal amount of GP Ib on the platelet surface, as measured by the binding of both monoclonal anti-GP Ib antibody SZ 2 and 125I-labelled vWF to the platelets. Cytochalasin D did not inhibit this recovery, suggesting that this process may be due to passive actin depolymerization. These findings were further confirmed by immunoelectron microscopic study. Utilizing the platelets pre-labelled with anti-GP Ib antibody prior to plasmin stimulation, it was demonstrated that the observed recovery is due to a reverse translocation from the SCCS to the plasma membrane of the same GP Ib molecules which were present initially at the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Agglutination↗
PubMed1993

Circulating megakaryocytes: delivery of large numbers of intact, mature megakaryocytes to the lungs.

To determine the locus of platelet production, we sought to determine if sufficient megakaryocytes reach the lungs in a state that could produce platelets. Elutriation was used to isolate megakaryocytes from blood reaching and leaving the lungs of 20 patients undergoing routine cardiac catheterizations. A mean of 5.0 intact megakaryocytes/ml were found in pulmonary artery blood, compared to only 0.5 megakaryocytes/ml, with partial cytoplasmic content, in aortic samples. The megakaryocytes in central venous and aortic samples were all mature. The identity of these cells as megakaryocytes, their maturity and normal morphology were confirmed by standard and immunoelectron microscopy. Cardiac outputs were obtained for each patient at the time of blood sampling, allowing an extrapolation that 40 x 10(6) intact, mature megakaryocytes were being delivered to the lungs every day in the average patient, compared to only 4.0 x 10(6) partially spent megakaryocytes exiting the lungs daily. About 98% of megakaryocyte cytoplasm reaching the lungs did not exit as recognizable megakaryocytes or fragments. The number and state of the megakaryocytes apparently filtered in the lungs is consistent with the hypothesis that megakaryocytes may shed platelets within the pulmonary microvasculature, which may be the primary site of platelet production.

Adult↗
PubMed1993

Gray platelet syndrome. Dissociation between abnormal sorting in megakaryocyte alpha-granules and normal sorting in Weibel-Palade bodies of endothelial cells.

The gray platelet syndrome (GPS) is a rare congenital bleeding disorder in which megakaryocytes and platelets are deficient in alpha-granule secretory proteins. Since the Weibel-Palade bodies (WPB) of endothelial cells as well as the alpha-granules contain the von Willebrand Factor (vWF) and P-selectin, we examined by transmission electron microscopy the dermis capillary network of two patients with GPS. Endothelial cells showed the presence of normal WPB with typical internal tubules. Using single and double immunogold labeling for vWF and P-selectin, we detected vWF within WPB, where it was codistributed with the tubules, whereas P-selectin delineated the outline of WPB. Therefore, the fundamental targeting defect in GPS is specific to the megakaryocytic cell line.

Biopsy↗
PubMed1993

[Expression of cyclin B in megakaryocytes and cells of other hematopoietic lines].

Megakaryocytes are normal bone marrow cells which have the unique ability to become polyploid. This phenomenon is termed endomitosis and its mechanism remains poorly understood at present. It is known that the cell cycle of eukaryotes, particularly at G2/M transition, is regulated by a complex with histone H1 kinase activity, the maturation- or M-phase promoting factor (MPF). We have therefore studied the expression of subunits of MPF, the p34cdc2 and cyclin B in normal bone marrow culture megakaryocytic cells, blood leukocytes and platelets as well as in human megakaryoblastic cell lines Dami, Meg-01, HEL and the promyelocytic cell line HL60. Using immunohistochemistry and electron microscopy we have observed that cyclin B was virtually undetectable in megakaryocytes and platelets, but was abundant in granulocytes, monocytes/macrophages and HL60. Studies by RT-PCR showed the presence in large quantities of mRNA of cyclin B in all cell types studied, even in megakaryocytic-like cells. These observations suggest some important implications in the understanding of the mechanisms of megakaryocyte polyploidization and related endomitosis.

Blood Platelets↗
PubMed1993

Effect of thrombin on maturing human megakaryocytes.

Thrombin causes platelet activation and secretion. In some nucleated cells, it is mitogenic. In this study, we have investigated how human megakaryocytes (MKs) respond to this agonist and whether the response depends on the maturation stage. MKs were cultured from bone marrow precursors in liquid culture in the presence of normal plasma. To determine whether thrombin can activate MKs, 14-day MK cultures were incubated with thrombin for 5 minutes, and cells were studied by electron microscopy, either by standard techniques or after embedding in glycol-methacrylate for immunoelectron microscopy. Ultrastructural examination of thrombin-treated MKs revealed dramatic morphological changes reminiscent of those found in platelets, including shape change and organelle centralization that involved immature as well as mature cells. MKs were also able to secrete alpha-granule proteins in the dilated cisternae of the demarcation membrane system, as shown by immunogold staining for thrombospondin and glycoprotein Ib. These changes were rapid (less than 5 minutes) but despite them, MKs remained viable for more than 24 hours. To determine whether thrombin has a mitogenic activity, it was added to the culture of MKs from day 3 to day 10 of culture at concentrations varying from 0.1 to 10 U/ml. Cells were subsequently studied by a double staining technique using flow cytometry to determine MK number and ploidy. No changes were observed in these two parameters, showing that thrombin is not mitogenic for MKs at the concentrations used. In conclusion, this study confirms for human MKs previous observations made about guinea pig MKs (Fedorko et al, Lab Invest 1977, 36:32). In addition, it demonstrates that immature MKs are able to respond to thrombin and that more mature cells can secrete alpha-granule proteins into the demarcation membrane system, which is in continuity with the extracellular space. This phenomenon may have implications for pathological states such as myelofibrosis formation and for megakaryopoiesis autocrine regulation.

Cell Count↗
PubMed1993

von Willebrand factor storage requires intact prosequence cleavage site.

Large multimers of the adhesive glycoprotein von Willebrand factor (vWf) are stored in endothelial cells in rod-shaped granules called Weibel-Palade bodies, while small multimers are secreted constitutively. Expression of pro-vWf in other cells with a regulated pathway of secretion, results in formation of vWf-containing storage granules that have a morphology similar to Weibel-Palade bodies. vWf expressed without its prosequence is not stored. To evaluate the importance of prosequence cleavage in vWf storage, the Arg at position -1, known to be necessary for cleavage, was mutated to Gly. Transfection of this cleavage mutant into two cell lines with a regulated pathway of secretion (RIN 5F and AtT-20 cells) led to the formation of large multimers. However, treatment of the cell lysates by the enzyme endoglycosidase H (Endo-H) did not reveal significant amounts of intracellular Endo-H-resistant vWf, which indicates the absence of a pool of stored processed vWf. In addition, no Weibel-Palade body-like structure was detected in these cells by immunofluorescence labeling with anti-vWf antiserum. Electron microscopy and immunocytochemistry of RIN 5F cells expressing the pro-vWf mutant confirmed the absence of Weibel-Palade body-like structures. In addition, anti-vWf-linked gold particles were found in the ER, occasionally in rounded granules and particularly in lysosomal structures which were abundant. We conclude that the formation of large aggregates is not sufficient to induce efficient vWf storage, and that the lack of cleavage of the prosequence may direct the mutant pro-vWf molecule to a degradative pathway. Therefore, the prosequence cleavage is a requirement for vWf storage.

Animals↗
PubMed1992

Localization of platelet osteonectin at the internal face of the alpha-granule membranes in platelets and megakaryocytes.

Osteonectin is a 32-Kd phosphoglycoprotein originally described in bone but also found in platelets. Platelet and bone osteonectin are different both structurally and immunologically. We have previously shown that platelet osteonectin, by binding to thrombospondin, is involved in the secretion-dependent phase of the platelet aggregation process. In this study, we used antiosteonectin antibodies in combination with immunogold labeling to investigate by electron microscopy the fine localization of osteonectin within normal and gray platelets. Using both a polyclonal and monoclonal antibody ON3, osteonectin was specifically located at the internal face of alpha-granule membranes within normal platelets. Osteonectin was not distributed within all alpha-granules, probably because of its low platelet content. In addition, using immunofluorescence, osteonectin could also be detected in immature and mature megakaryocytes with a granular pattern of staining, suggesting that osteonectin is synthesized by megakaryocytes. Using platelets from two patients with gray platelet syndrome, osteonectin was absent within all abnormal small alpha-granules, but was detected in some rare normal-sized alpha-granules. In separate double-label studies, thrombospondin and von Willebrand factor did not colocalize with osteonectin in resting platelets. However, osteonectin was located at the inner face of the alpha-granules, as it is for alpha-granule membrane protein GMP-140 and glycoprotein IIb-IIIa. These results, taken together with the fact that monoclonal antibodies to osteonectin bind only to the surface of activated platelets, suggest that platelet osteonectin is redistributed to the cell surface during fusion of alpha-granule membranes with the plasma membrane.

Blood Platelets↗
PubMed1992

P-selectin, a granule membrane protein of platelets and endothelial cells, follows the regulated secretory pathway in AtT-20 cells.

P-selectin (PADGEM, GMP-140, CD62) is a transmembrane protein specific to alpha granules of platelets and Weibel-Palade bodies of endotheial cells. Upon stimulation of these cells, P-selectin is translocated to the plasma membrane where it functions as a receptor for monocytes and neutrophils. To investigate whether the mechanism of targeting of P-selectin to granules is specific for megakaryocytes and endothelial cells and/or dependent on von Willebrand factor, a soluble adhesive protein that is stored in the same granules, we have expressed the cDNA for P-selectin in AtT-20 cells. AtT-20 cells are a mouse pituitary cell line that can store proteins in a regulated fashion. By double-label immunofluorescence, P-selectin was visible as a punctate pattern at the tips of cell processes. This pattern closely resembled the localization of ACTH, the endogenous hormone produced and stored by the AtT-20 cells. Fractionation of the transfected cells resulted in the codistribution of P-selectin and ACTH in cellular compartments of the same density. Immunoelectron microscopy using a polyclonal anti-P-selectin antibody demonstrated immunogold localization in dense granules, morphologically indistinguishable from the ACTH granules. Binding experiments with radiolabeled monoclonal antibody to P-selectin indicated that there was also surface expression of P-selectin on the AtT-20 cells. After stimulation with the secretagogue 8-Bromo-cAMP the surface expression increased twofold, concomitant with the release of ACTH. In contrast, the surface expression of P-selectin transfected into CHO cells, which do not have a regulated pathway of secretion, did not change with 8-Br-cAMP treatment. In conclusion, we provide evidence for the regulated secretion of a transmembrane protein (P-selectin) in a heterologous cell line, which indicates that P-selectin contains an independent sorting signal directing it to storage granules.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

Temperature dependence of plasmin-induced activation or inhibition of human platelets.

It is known that at 37 degrees C plasmin may have two opposite effects on platelets: at high concentrations (greater than 1.5 caseinolytic units [CU]/mL), plasmin activates platelets; at lower concentrations (0.1 to 1.0 CU/mL) it inhibits platelet activation induced by thrombin, collagen, or calcium ionophore A23187. In this study, we report that when lowering the incubation temperature to 22 degrees C, plasmin at low concentrations (0.1 to 0.5 CU/mL) fully activated platelets. When platelets were treated with 0.2 CU/mL of plasmin, lowering the incubation temperature from 37 degrees C to 22 degrees C resulted in an increase in the expression of fibrinogen receptors, in platelet release and aggregation. Thromboxane A2 was not generated by plasmin treatment at either temperature. Ultrastructural studies showed that platelets responded to low-dose plasmin at 37 degrees C by forming pseudopods, centralizing granules without fibrinogen release, whereas at 22 degrees C the same dose of plasmin caused platelet degranulation with the appearance of alpha-granule fibrinogen within the lumen of the surface connected canalicular system. In addition, at 22 degrees C plasmin at doses insufficient to induce platelet aggregation potentiated platelet response to thrombin. Thus, we suggest that plasmin may initiate both activating and inhibitory processes within platelets and that the change of temperature could influence this balance. These results may be of clinical relevance, because the fibrinolytic system was found activated during cardiopulmonary bypass in which the temperature of patient's blood circulation was reduced. This temperature-dependent behavior is also an interesting model for a further study on platelet response to serine proteinases.

Antibodies, Monoclonal↗
PubMed1991

Differential redistribution of platelet glycoproteins Ib and IIb-IIIa after plasmin stimulation.

The subcellular localization of the platelet membrane receptors glycoproteins (GP) Ib and IIb/IIIa [corrected] has been studied within resting platelets by a combination of biochemical and cytochemical techniques. While both GPIb and GPIIb/IIIa are localized within the plasma membrane and surface-connected canalicular system (SCCS) membranes, only GPIIb/IIIa is present within the internal face of alpha-granular membranes. Previous studies demonstrated that plasmin can induce platelet stimulation and also decrease ristocetin-induced platelet aggregation; it was suggested that this was because of GPIb degradation by plasmin. In this study, the respective localizations of both GPIb and GPIIb/IIIa were visualized during in vitro plasmin stimulation of platelets. Generally, plasmin induced shape change, pseudopod formation, organelle centralization either with or without alpha-granule release depending on the conditions of stimulation. Plasmin treatment of platelets at 37 degrees C resulted in the disappearance of GPIb from the cell surface and its subsequent redistribution into the channels and vesicles of the SCCS with no significant modification of GPIIb/IIIa remaining on the plasma membrane. Within degranulated platelets, GPIIb/IIIa was expressed on the plasma membrane and within membranes of large vacuoles containing the alpha-granule proteins. GPIb was virtually absent from these structures and mainly restricted to the SCCS. Addition of cytochalasin D inhibited the migration of GPIb to the SCCS. Biochemical measurements confirmed that no important hydrolysis of GPIb had occurred because only very little amounts of glycocalicin were generated during the reaction. In conclusion, in plasmin-treated platelets GPIIb/IIIa is externalized to the plasma membrane while GPIb is internalized into the SCCS. Although previous studies have suggested that plasmin degrades GPIb, the reduction in ristocetin-induced aggregation may be explained by its apparent redistribution within the membranes of the SCCS.

Blood Platelets↗
PubMed1991

Induction of specific storage organelles by von Willebrand factor propolypeptide.

Endothelial cells store the multimeric adhesive glycoprotein von Willebrand factor (vWf), which promotes the formation of a platelet plug at the site of vessel injury. To investigate the packaging of vWf into the granules called Weibel-Palade bodies, we expressed pro-vWf cDNA and cDNA lacking the prosequence in a variety of cell lines. Storage granules formed only in cells that contain a regulated pathway of secretion. Furthermore, packaging required the prosequence. Pro-vWf, lacking the C-terminal region involved in interchain disulfide bonding, formed granules. We conclude that the signal for storage is universal in that an adhesive glycoprotein can be stored by a hormone-secreting cell; the storage of vWf is independent of its covalent multimeric structure; the unusual rod shape of Weibel-Palade bodies is due to vWf; and the vWf propolypeptide is necessary for the formation of vWf storage granules.

Animals↗
PubMed1991

Phenotypic modulations of human umbilical vein endothelial cells and human dermal fibroblasts using two angiogenic assays.

Different angiogenic assays in vitro have helped to define various events underlying angiogenesis. In this report we have compared the phenotypic modifications of human umbilical vein endothelial cells (HUVE cells) and human dermal fibroblasts using Matrigel and collagen gels. Both HUVE cells and human dermal fibroblasts form a network of anastomosing cords that apparently resemble blood capillaries when grown on Matrigel. The whole network was formed by several cellular aggregates joined to each other by cellular cords. Lumen formation was not observed in this angiogenic system. In opposite, considerable differences between HUVE cells and human dermal fibroblasts were observed in the three-dimensional angiogenic assay on collagen gels described by Montesano et al [14]. These results indicate that data obtained with angiogenic systems using Matrigel must be interpreted with caution and that the assay described by Montesano et al [14], is more reliable to describe angiogenesis.

Cells, Cultured↗
PubMed1991

Platelets of the Wistar Furth rat have reduced levels of alpha-granule proteins. An animal model resembling gray platelet syndrome.

Rats of the Wistar Furth (WF) strain have hereditary macrothrombocytopenia (large mean platelet volume [MPV] with increased platelet size heterogeneity and reduced platelet count). Ultrastructural studies suggest that this anomaly results from erratic subdivision of megakaryocyte cytoplasm into platelets. In this study, we have examined protein profiles of platelets of WF rats for biochemical abnormalities associated with this anomaly. Marked decreases in protein bands with an Mr of 185, 57, 53, 16, 13, and 8 kd were observed in one-dimensional reduced SDS-PAGE gels in WF platelets compared with platelets of Wistar, Long Evans, and Sprague-Dawley rats. These proteins were released into the supernatant when washed platelets were treated with thrombin suggesting that they were alpha-granule proteins. These abnormalities were not present in offspring of crosses between Wistar Furth and Wistar rats; however, they were present in platelets of offspring with large MPV derived from backcrosses of (WF X Wistar) F1 males to WF females, but not in backcross offspring with normal platelet size. Immunoblotting confirmed decreased levels of thrombospondin, fibrinogen, and platelet factor 4 in WF platelets. Electron microscopic examination revealed that platelet alpha granules were usually smaller in Wistar Furth than in Wistar rats. In addition, immunogold electron microscopy demonstrated that the surface connected canalicular system of the large Wistar Furth platelets, contained dense material composed of alpha-granule proteins, not present in Wistar platelets. From these results, we conclude that the Wistar Furth rat platelet phenotype of large mean platelet volume and decreased levels of alpha-granule proteins represents an animal model resembling gray platelet syndrome. The autosomal recessive pattern of inheritance of the large MPV phenotype and platelet alpha-granule protein deficiencies suggests that a component common to both formation of platelet alpha granules, and subdivision of megakaryocyte cytoplasm into platelets, is quantitatively or qualitatively abnormal in Wistar Furth rat megakaryocytes and platelets.

Afibrinogenemia↗
PubMed1991

Ultrastructural localization of the CD68 macrophage-associated antigen in human blood neutrophils and monocytes.

The ultrastructural localization of the CD68 antigen, a 110-kd intracellular glycoprotein associated with myeloid cells and with monocytes/macrophages, was investigated in human neutrophil granulocytes by postembedding immunogold staining, using monoclonal antibody KP1. The antigen was found in the primary granules of neutrophils, although not all primary granules were labeled. It was absent from the plasma membrane. In monocytes, it was also detected within cytoplasmic granules, colocalized with lysozyme and myeloperoxidase. This observation confirms and completes results obtained by immunofluorescence and other light-microscopic methods. Moreover this study shows that the CD68 epitope recognized by antibody KP1 is able to resist fixation and embedment and therefore emphasizes the value of using KP1 as a marker for this macrophage-associated molecule.

Antibodies, Monoclonal↗
PubMed1991

[Gray platelet syndrome, an example of myelofibrosis of megakaryocytic origin].

In this study, the clinical history of two patients with the gray platelet syndrome, a rare congenital disorder associating thrombopathia and myelofibrosis is recalled. Complementary studies on platelets and megakaryocytes were performed, mainly with an immunocytochemical approach. In gray platelets, a general decrease of alpha-granule proteins, including PF4, beta tg and PDGF was observed. The decrease in platelet mitogenic activity (PDGF) was confirmed by biological and radio-immunological measurements. An abnormally high level of these compounds was also found in the plasma. In megakaryocytes cultured from the bone marrow of these patients, alpha-granule proteins were normally expressed in early maturation stages, whereas they were found to be absent in the mature megakaryocytes. An alpha-granule membrane glycoprotein, GMP 140 has been studied in resting and thrombin stimulated gray platelets and was found to be normally expressed at the surface of stimulated platelets. GMP140 was studied in resting platelets by immunoelectron microscopy and found to be present in vacuole probably corresponding to empty granules. This observation allows to conclude that alpha-granule membrane is formed in the gray platelet syndrome, but that there is a storage defect of alpha-granule soluble proteins, possibly due to an abnormal targetting of these proteins to the alpha-granule. Synthesis and subsequent release of these proteins, namely of the mitogenic factors, which can induce myelofibrosis and lung fibrosis by abnormal fibroblast stimulation, is discussed.

Adult↗
PubMed1991

Alpha 1-antitrypsin is present within the primary granules of human polymorphonuclear leukocytes.

Elastase is a potent proteolytic enzyme found within human neutrophil primary granules. Its major inhibitor in the serum is alpha 1-antitrypsin, a protein that is synthesized by hepatocytes but which has recently also been shown to be synthesized by circulating neutrophils. The authors have therefore carried out an immunocytochemical study at the light microscopic and ultrastructural level to determine the intracellular localization of alpha 1-antitrypsin. Double labeling with colloidal gold showed that alpha 1-antitrypsin is localized at the same site as neutrophil elastase, i.e., within primary granules. Secondary granules (detected by labeling for lactoferrin) were unstained for alpha 1-antitrypsin. Elastase and its major inhibitor therefore coexist within the same granule population within human neutrophils. Some difference in their intraorganelle distribution existed at the ultrastructural level (in that elastase tended to be localized at the periphery of the granules whereas alpha 1-antitrypsin was usually diffusely present in the matrix of the granules), but further studies are required to determine whether the two molecules are already complexed with each other within the neutrophil.

Colloids↗
PubMed1990

Alpha-granule pool of glycoprotein IIb-IIIa in normal and pathologic platelets and megakaryocytes.

Using an immunogold staining technique and electron microscopy, we investigated the localization of the alpha-granule pool of glycoprotein (GP) IIb-IIIa in normal platelets and maturing megakaryocytes (MK), in pathologic platelets from a patient with type I Glanzmann's thrombasthenia (GT), and from three patients with the gray platelet syndrome (GPS). In normal resting platelets, GPIIb-IIIa was observed on the plasmatic side of the plasma membrane, the open canicular system (OCS) membranes, and along the internal face of the alpha-granule membrane. This location was found with three monospecific polyclonal antibodies: one anti-GPIIb-IIIa antibody, the second specific for GPIIb, and the third specific for GPIIIa. After thrombin stimulation, the alpha-granule labeling disappeared whereas membrane labeling increased. Platelets from GT did not display labeling on plasma membranes, OCS membranes, or alpha-granule membranes. Platelets from the three patients with GPS displayed intense labeling of the plasma membrane and the OCS membrane, as well as the abnormal small alpha-granules and along the inside of large vacuoles (which contain the granule membrane protein [GMP]-140). In cultured immature MK from normal progenitors, both peptide components of GPIIb-IIIa appeared in the Golgi saccules and vesicles, and in the small precursors of alpha-granules, labeling both their membranes and their matrix. It was then observed only on the membrane of the mature MK alpha-granules, although labeling was less consistent than on the platelet granules. The MK plasma membrane and demarcation membrane system also displayed GPIIb-IIIa labeling. In conclusion, this study demonstrates that GPIIb-IIIa is present on the internal face of the alpha-granule membranes of platelets (where it appears early during MK maturation) as well as in the abnormal alpha-granules of gray platelets; it is absent from GT type I platelets.

Antibody Specificity↗