Publications

Essential thrombocythemia is a myeloproliferative disorder characterized by frequent bleeding and thrombotic complications. On a molecular level, two abnormalities of platelet thrombospondin have been identified: abnormal glycosylation of the intact 185,000-dalton chain has been detected and a shortened form of the thrombospondin chain is present. We have used two monoclonal antibodies and Lens culinaris lectin to probe the structure of thrombospondin in the platelets from three patients with essential thrombocythemia; one patient with polycythemia vera and two patients with secondary thrombocytosis. The presence of abnormal thrombospondin fragments with molecular weights of 160,000 and 30,000 was detected in the intact platelets and in the supernatant from thrombin-treated platelets, in all of the individuals except one of the secondary thrombocytosis patients. Monoclonal antibody binding studies indicate that both fragments are produced by proteolysis at a single site, which results in the removal of a 30,000-dalton fragment from the NH2-terminal. Lens culinaris lectin-binding studies revealed that some of the carbohydrate moieties of thrombospondin are near this cleavage site. The results are consistent with the hypothesis that the abnormal thrombospondin fragments observed under conditions of increased platelet production are due to increased susceptibility to proteolysis which, in turn, may be due to defective glycosylation.

Thrombospondin is one of a class of adhesive glycoproteins that mediate cell-to-cell and cell-to-matrix interactions. We have used two monoclonal antibodies to isolate cDNA clones of thrombospondin from a human endothelial cell cDNA library and have determined the complete nucleotide sequence of the coding region. Three regions of known amino acid sequence of human platelet thrombospondin confirm that the clones are authentic. Three types of repeating amino acid sequence are present in thrombospondin. The first is 57 amino acids long and shows homology with circumsporozoite protein from Plasmodium falciparum. The second is 50-60 amino acids long and shows homology with epidermal growth factor precursor. The third occurs as a continuous eightfold repeat of a 38-residue sequence; structural homology with parvalbumin and calmodulin indicates that these repeats constitute the multiple calcium-binding sites of thrombospondin. The amino acid sequence arg-gly-asp-ala is included in the last type 3 repeat. This sequence is probably the site for the association of thrombospondin with cells. In addition, localized homologies with procollagen, fibronectin, and von Willebrand factor are present in one region of the thrombospondin molecule.

Thrombospondin is a 450-kDa glycoprotein secreted by a variety of cells including endothelial cells, fibroblasts and platelets. The aim of this study was to compare the structural and immunological properties of human endothelial, fibroblast and platelet thrombospondins. All three thrombospondins were purified, digested with thermolysin, and the subsequent thermolysin-generated fragments isolated on a Superose 12 gel-permeation column using non-denaturating conditions. Each isolated proteolytic fragment of thrombospondins was then detected using either a radioimmunoassay with a polyclonal antibody or an enzyme-linked immunosorbent assay with three monoclonal antibodies (P10, MA-I, MA-II) directed against different epitopes of whole platelet thrombospondin. The fragmentation pattern of human endothelial thrombospondin consists of six major thermolysin-generated fragments (135-110, 98-82, 54-47, 25-20, 18-15 and 10 kDa) having molecular masses very similar to those observed with human fibroblast thrombospondin (115-100, 92-80, 54-49, 27-21, 17-13 and 12-10 kDa). Treatment of platelet thrombospondin with thermolysin only generated four proteolytic fragments having molecular masses of 110, 50, 25 and 12/10 kDa respectively. All these proteolytic fragments of endothelial, fibroblast and platelet thrombospondins were recognized by a polyclonal antibody. Monoclonal antibodies MA-I and P10 essentially recognized two proteolytic fragments (135-110, 98-82 kDa) of endothelial and fibroblast (115-100, 92-80 kDa) thrombospondins, and the 110-kDa fragment of platelet thrombospondin. Monoclonal antibody MA-II recognized three proteolytic fragments (54-47, 25-20, 18-15 kDa) of endothelial and fibroblast (54-49, 27-21, 17-13 kDa) thrombospondins, and two fragments (50, 25 kDa) of platelet thrombospondin, different from those detected by P10 an MA-I. The results clearly demonstrate that, under non-denaturating conditions, endothelial and fibroblast thrombospondins are structurally different from platelet thrombospondin since two fragments of endothelial thrombospondin (98-82, 18-15 kDa), equivalent to those of fibroblast thrombospondin (92-80, 17-13 kDa), are not released from platelet thrombospondin after thermolysin treatment. These three forms of thrombospondin are, however, immunologically indistinguishable. To investigate further the structural differences observed between platelet and the two other forms of thrombospondin, their degree of polymerization was compared. Prior to thermolysin treatment, the three forms of thrombospondin were separated into several oligomers ranging from 450 kDa to 3300 kDa when injected onto a Superose 6 gel-permeation column.(ABSTRACT TRUNCATED AT 400 WORDS)

Two molecular defects involving the spectrin heterodimer (SpD) contact site of the alpha chain (the alpha I domain) were previously identified using limited tryptic digestion followed by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both are characterized by atypical peptide maps which reveal a marked decrease of the 80,000-dalton alpha I domain and a formation of new major peptides of either 74,000 (Sp alpha I/74) or 46,000 (Sp alpha I/46) daltons. We now report a third variant of the spectrin alpha chain, designated Sp alpha I/65, in three unrelated black families. In all three probands, the percentage of SpD in the low ionic strength (O degrees C) membrane extracts was increased to 19% to 32%. One- and two-dimensional electrophoretic separations of limited tryptic digests of spectrin from all three probands revealed a decrease of the alpha I domain of spectrin and the concomitant appearance of peptides at 65,000 daltons and isoelectric points ranging from 5.2 to 5.3. The abnormal 65,000-dalton peptides could be stained with an antiserum which had been raised against the alpha I domain, indicating that it was derived from the alpha I domain.

Two distinct murine monoclonal antibodies, designated MA-I and MA-II, and limited proteolysis with thrombin and trypsin have been used to probe the structure of human platelet thrombospondin. The results indicate that each of the constituent chains of thrombospondin comprise four distinct polypeptide segments. The production of these segments is influenced by the presence of calcium, the enzyme employed, the temperature of digestion, and the enzyme-to-substrate ratio. Thrombin digestion in the presence of calcium results in the release of a 30,000-dalton fragment, designated segment I, which contains the epitope for MA-II and the heparin-binding site. Prior EDTA treatment results in the concomitant cleavage of a 25,000-dalton fragment, designated segment IV, from the other terminus. Limited tryptic digestion in the absence of calcium produces a 47,000-dalton fragment (segment III) which is adjacent to segment IV. Segment III contains the epitope for MA-I. Segment II is an 85,000-dalton fragment which contains the interchain disulfide bonds. Calcium inhibits proteolysis at cleavage sites between segments II and III and between segments III and IV. In the presence of calcium, an 85,000-dalton fragment is produced, which is derived from portions of segments II, III, and possibly IV. Electron microscopy of platinum replicas produced by low angle rotary shadowing reveals that thrombospondin is composed of four well-defined globular regions connected by thin flexible regions. Three of the globular regions, designated globular region C, appear to be at the ends of the three thin connecting regions. The fourth globular region, designated globular region N, appears to be close to the site where the chains are cross-linked. Globular region N can be resolved into three separate smaller globular structures which are 70 +/- 7.1 A in diameter. This region is selectively removed by thrombin digestion in the presence of calcium and binds a monoclonal antibody directed against the heparin-binding peptides. These data indicate that globular region N comprises the three NH2-terminal portions (segment I) from each of the three chains of thrombospondin. Globular region C is located at the ends of each of the three thin connecting regions which are each approximately 291 +/- 46 A long. The removal of calcium results in a decrease in the size of globular region C from 118 +/- 18.6 A to 80 +/- 7.4 A and an increase in the length of the adjacent thin connecting region to 383 +/- 30 A.(ABSTRACT TRUNCATED AT 400 WORDS)

To determine environmental requirements for erythroid burst formation in primary culture, we added human bone marrow cells to serum-depleted methylcellulose, agar, or fibrin clot cultures. Optimal BFU-E proliferation was present in cultures prepared with Iscove's modified Dulbecco medium, 248 micrograms/ml transferrin, 1.63 micrograms/ml ferric chloride, 117 micrograms/ml bovine serum albumin, and each of seven preparations of erythropoietin. Burst number was comparable to that in serum containing culture. Reducing sodium dodecyl sulfate electrophoresis of commercial albumin preparations showed them to contain abundant lipoproteins. Results of experiments with human plasma albumin found to be greater than 98% pure by one- and two-dimensional gel electrophoresis and delipidated albumin indicate that an albumin source is needed for burst formation to occur. Together with albumin, exogenous phosphatidylcholine but not phosphatidylserine augmented burst number. Bursts routinely appeared in serum-depleted culture without added burst-promoting activity (BPA). However, leukocyte-conditioned medium (LCM) and its high-speed supernatant and pellet fractions enhanced burst formation. Antimembrane IgG capable of neutralizing BPA reduced burst number to a level below that achieved in LCM-depleted culture, suggesting that endogenous BPA was inactivated. We conclude that human marrow BFU-E proliferation requires iron-saturated transferrin, albumin, and erythropoietin. Exogenous BPA and phospholipids enhance but are not essential for burst formation to proceed in primary culture.

Spectrin tetramers and oligomers in normal erythrocytes are cross-linked by actin and protein 4.1 to form a two-dimensional membrane skeletal network. In the present study, we find that hemin, a breakdown product of hemoglobin, progressively (a) alters the conformation of spectrin as revealed by electron microscope studies and by the decreased resistance of spectrin to proteolytic degradation, (b) alters the conformation of protein 4.1 as revealed by the increased mobility of protein 4.1 on nondenaturing gel electrophoresis, (c) weakens spectrin dimer alpha beta-dimer alpha beta, spectrin alpha-spectrin beta, as well as spectrin-protein 4.1 associations as analyzed by nondenaturing gel electrophoresis, and (d) diminishes the structural stability of erythrocyte membrane skeletons (i.e. Triton-insoluble ghost residues) subjected to mechanical shearing. Since hemin may be liberated from oxidized or unstable mutant hemoglobin under pathological conditions, these hemin-induced effects on spectrin, protein 4.1, and membrane skeletal stability may play a role in the membrane lesion of these erythrocytes.

Thrombospondin synthesized and secreted by human endothelial cells in culture binds specifically to fibronectin immobilized on Sepharose beads. It can also bind to immobilized platelet-derived thrombospondin but not to immobilized gelatin or albumin. These interactions are not dependent on the presence of divalent cations or of other secreted materials. Purified platelet thrombospondin binds to fibronectin and fibrinogen immobilized on plastic surfaces with dissociation constants of 1.12 +/- 0.37 X 10(-7) M and 1.27 +/- 0.41 X 10(-7) M respectively, and to thrombospondin immobilized on plastic with dissociation constant of 4.82 +/- 1.01 X 10(-7) M. The affinities of interaction are not significantly affected by removal of divalent cations. Soluble fibrinogen inhibits binding of thrombospondin to fibronectin regardless of which of the latter two is surface-bound. Thrombospondin-fibronectin interaction is also inhibited by soluble thrombospondin. The binding of soluble thrombospondin to surface-bound fibrinogen is inhibited both by soluble fibronectin and soluble fibrinogen. These results suggest that thrombospondin plays a role both in platelet-platelet aggregation and in platelet-substratum adhesion, and that it may also take part in the construction of the extracellular matrix.