Daniel A. Walz

An evolutionary tree for invertebrate globin sequences

SummaryA phylogenetic tree was constructed from 245 globin amino acid sequences. Of the six plant globins, five represented the Leguminosae and one the Ulmaceae. Among the invertebrate sequences, 7 represented the phylum Annelida, 13 represented Insecta and Crustacea of the phylum Arthropoda, and 6 represented the phylum Mollusca. Of the vertebrate globins, 4 represented the Agnatha and 209 represented the Gnathostomata. A common alignment was achieved for the 245 sequences using the parsimony principle, and a matrix of minimum mutational distances was constructed. The most parsimonious phylogenetic tree, i.e., the one having the lowest number of nucleotide substitutions that cause amino acid replacements, was obtained employing clustering and branch-swapping algorithms. Based on the available fossil record, the earliest split in the ancestral metazoan lineage was placed at 680 million years before present (Myr BP), the origin of vertebrates was placed at 510 Myr BP, and the separation of the Chondrichthyes and the Osteichthyes was placed at 425 Myr BP. Local “molecular clock” calculations were used to date the branch points on the descending branches of the various lineages within the plant and invertebrate portions of the tree. The tree divided the 245 sequences into five distinct clades that corresponded exactly to the five groups plants, annelids, arthropods, molluscs, and vertebrates. Furthermore, the maximum parsimony tree, in contrast to the unweighted pair group and distance Wagner trees, was consistent with the available fossil record and supported the hypotheses that the primitive hemoglobin of metazoans was monomeric and that the multisubunit extracellular hemoglobins found among the Annelida and the Arthropoda represent independently derived states.

Thrombin-Elicited Contractile Responses of Aortic Smooth Muscle:

Abstract Human α-thrombin at physiologically relevant concentrations of 0.75 to 225 nM (0.01 to 22 clotting units/ml) caused rabbit thoracic aorta to slowly contract in isolated organ baths. Near maximum contractile tension (10% below the irreversible contraction caused by 22 units/ml) was slowly generated by 75 nM α-thrombin (8 units/ml) over 18 min and was 50% that of the norepinephrine (NE) control. The initial tonus could only be regained by repeated washing and 60 min equilibration. Tissues were refractory to a second 75 nM α-thrombin challenge but responded fully to 1.0 nM NE. Conversion of human α-thrombin to nonclotting but estero/amidolytically active 7-thrombin (<0.1% clotting activity) or nitration of the enzyme (1% clotting activity) did not interfere with the contractile activity, whereas chemical conjugates of the parent enzyme at the catalytic site were inactive. Neither atropine, phentolamine, nor indomethacin blocked the thrombin-induced contractions, whereas D-600 was markedly inhibitory. Preincubation of the tissue with inactive forms of thrombin did not prevent the α-thrombin-induced response. Removal of the vascular endothelium did not prevent contraction. Aorta preparations with intact endothelium relaxed in the presence of very low concentrations (0.75 to 750 pM) of α-thrombin prior to contracting in response to higher concentrations during cumulative dose-response experiments. Our data suggest that catalytically active thrombin forms (α- or nonclotting β- and 7-thrombins) may have hemostatic functions at the vascular levels in hemostasis.

Thrombin enhances tumor cell adhesive and metastatic properties via increased αIIbβ3 expression on the cell surface

Abstract The association between blood coagulation and cancer growth and metastatic dissemination is not yet completely understood. In this study we demonstrate that thrombin is capable of enhancing tumor cell adhesive properties and thereby increases tumor cell metastatic potential. Following exposure to α-thrombin, Walker 256 carcinosarcoma cells and B16 amelanotic melanoma cells became more adherent to both endothelial cell monolayers and the subendothelial matrix component, fibronectin. Preincubation of W256 and B16a cells with doses of α-thrombin from 0.01 to 10.0 U/ml produced a bell shape dose-response curve with the maximal effect (a 2–5-fold increase in adhesion) observed at 0.1 U/ml (corresponding to 0.8 nM). Complexes of α-thrombin with its inhibitors, hirudin and antithrombin III-heparin, diminished its effect on tumor cell adhesion. The effect of thrombin on tumor cell adhesion may be mediated by the αIIbβ3 integrin as thrombin increased cell surface expression of the αIIbβ3 complex. The significance of the in vitro observations was further substantiated by results of in vivo studies. Pretreatment of B16a cells with α-thrombin resulted in a 2-fold increase in the number of metastatic lung colonies in an experimental metastasis model. The data indicate a new role for thrombin in the metastatic spread of cancer.

Inherited platelet-storage pool deficiency associated with a high incidence of acute myeloid leukaemia

Summary A family with an inherited bleeding disorder extending over four generations, and multiple cases of myeloblastic and myelomonoblastic leukaemia was studied. Ten members of the family had, by history, a haemorrhagic diathesis. There were three documented cases of myeloblastic leukaemia, two documented cases of myelomonoblastic leukaemia and two more cases of leukaemia by history. In four of the cases the bleeding diathesis clearly antedated the leukaemia, in two by many years. The bleeding disorder is characterized by a long bleeding time, abnormal platelet aggregation, low platelet ADP and decreased numbers of platelet dense bodies consistent with a dense granule storage pool deficiency. The number of dense granules was decreased by immunofluorescence employing quinacrine or using an antibody to the dense granule membrane protein, granulophysin, confirming an absolute decrease in dense granule numbers rather than the presence of empty granule sacs. This congenital storage pool deficiency is associated with a high incidence of acute myeloid leukaemia in this family.

Isolation and some properties of thrombin-E and other prothrombin derivatives

Abstract Multiple forms of thrombin zymogen occur as prothombin complex, prothrombin, abnormal prothrombin, prethrombin, and prethrombin-E. Prothrombin less PR fragment represents prethrombin and is the stage beyond which degradation of prothrombin does not readily go with thrombin. By removing O fragment from prethrombin, esterase activity develops, due to a structure called prethrombin-E. This enzyme is a single chain structure with molecular weight, amino acid composition and carbohydrate content the same as thrombin. By breaking an Arg-Ile bond in prethrombin-E with autoprothrombin C, thrombin forms and consists of the A chain held to the B chain by a disulfide bond. It has esterase and proteolytic activity, and basically classical thrombin occurs only in this one form. Commonly, thrombin preparations consist of thrombin and thrombin degraded to thrombin-E and B1 chain. During autolysis at 4° C and pH 8.0, proteolytic activity of thrombin is lost while the specific esterase activity increases. The B1 portion of B chain splits off at an Arg-Lys bond. It precipitates in pure form leaving in solution thrombin-E which has B2 chain attached to A chain by a disulfide bond, and has only esterase activity. Breaking the disulfide bond of thrombin-E enables the isolation of A and B2 chains. Either thrombin or autoprothrombin C can remove PR fragment from prothrombin. R fragment was also isolated and probably contains all the carbohydrate of prothrombin which is not associated with B1 chain of thrombin. Thrombin-E is free of carbohydrate. O fragment retarded fibrin formation by thrombin. It enhanced the esterase activity of thrombin. An unidentified procoagulant, probably a form of autoprothrombin C, was closely associated with O fragment, but was removed. The prothrombin activation sequence described by Seegers and Landaburu in 1957 has been confirmed; namely, esterase activity → esterase + proteolytic activity → esterase activity. The respective associated structures are prethrombin-E → thrombin → thrombin-E; and in these, the condition of B1 chain is as follows: bound, free at NH 2 -terminal end, and absent. In addition, an antecedent in the form of prethrombin is easily obtained as a degradation product of prothrombin. The fragments align in prothrombin as follows: P + R + O + A chain + B1 chain + B2 chain. Digestion of prothrombin with thrombin stops with PR removal which is the prethrombin stage, but continues if prothrombin is first denatured. Likewise, autolysis of thrombin stops at the thrombin-E stage (A chain + B2 chain), but if thrombin-E is denatured, thrombin can degrade it further.

Antibodies against a 23Kd heparin binding fragment of thrombospondin inhibit platelet aggregation

Antiserum against a 23Kd heparin binding fragment of thrombospondin inhibits the aggregation of platelets in response to ADP, collagen or thrombin. The antiserum inhibits the secretion-dependent second phase, but not the primary phase of aggregation of platelets responding to ADP. Although immune serum added during the second phase of ADP-induced aggregation causes some inhibition of secretion, it also causes reversal of aggregation to the level produced during primary aggregation. Since thrombospondin is the endogenous lectin of human platelets, these results support the conclusion that the endogenous lectin mediates, at least in part, the secretion-dependent aggregation of platelets. Our data suggest that the region of thrombospondin which contains the heparin binding domain(s) present in the 23Kd fragment play(s) a critical role in secretion-dependent aggregation of platelets.

Quaternary structure of the giant extracellular hemoglobin of the leech Macrobdella decora

The molecular dimensions of the extracellular hemoglobin of the leech Macrobdella decora, determined by scanning transmission electron microscopy were 29.8 nm x 19.5 nm (diameter x height) for negatively stained specimens. Measurements of molecular mass (Mm) of unstained specimens with the microscope gave Mm = 3560 +/- 160 kDa. Small-angle X-ray scattering measurements gave a diameter of 28.0(+/- 0.5) nm, radius of gyration 10.5(+/- 0.2) nm and volume 7500(+/- 300) nm3. The hemoglobin had no carbohydrate and its iron content was found to be 0.23(+/- 0.02)% (w/w), corresponding to a minimum Mm of 24,000(+/- 1300) kDa. SDS/polyacrylamide gel electrophoresis of the unreduced hemoglobin showed that it consisted of three subunits, which have apparent Mm values of 12 (1), 25 (2) and 29 kDa (3). The reduced hemoglobin consisted of four subunits, I (12 kDa), II (14 kDa), III (26 kDa) and IV (30 kDa). Subunit 1 corresponded to subunit I, subunit 2 to subunits III and IV and subunit 3 to subunit II. Partial N-terminal sequences were obtained for subunit 1, the two chains of subunit 2 and one of the two chains of subunit 3, suggesting that the hemoglobin consists of at least five different polypeptide chains. The percentage fraction of the three unreduced subunits was determined by densitometry of SDS/polyacrylamide gel patterns and quantitative determination of Coomassie R-250 dye bound to the individual bands in reduced and unreduced patterns to be, monomer (subunit I) : non-reducible subunit (subunit 2) : reducible dimer (subunit 3) = 0.35 : 0.29 : 0.35 (S.D. = +/- 0.05). This corresponded to a stoichiometry of 74 +/- 11 : 37 +/- 5 : 38 +/- 6, assuming the molecular masses to be 17 kDa, 30 kDa and 34 kDa, taking into account the anomalously high mobility of annelid globins in SDS-containing gels. The stoichiometry calculated from the amino acid compositions of the hemoglobin and the three subunits was 82 +/- 12 : 29 +/- 4 : 40 +/- 8. Gel filtration of the hemoglobin at pH 9.8, at neutral pH subsequent to dissociation at pH 4 and at neutral pH in the presence of urea and Gu.HCl provided no evidence for the existence of a putative 1/12 of the whole molecule (Mm approx. 300 kDa). Furthermore, the largest subunits obtained had Mm of 60 to 100 kDa and had a much decreased content of subunit 2, suggesting that the hemoglobin was not a simple multimeric protein. Three-dimensional reconstruction from microscope images provided a model of Macrobdella hemoglobin that is very similar to the reconstruction of Lumbricus hemoglobin: the radial mass distribution curves are virtually superimposable.(ABSTRACT TRUNCATED AT 400 WORDS)

Thrombospondin as a mediator of cancer cell adhesion in metastasis

SummaryThrombospondin (TSP) is a 450 kDa adhesive glycoprotein. It is present in high concentrations in the platelet α-granule and can readily be secreted following platelet activation where local concentrations can be increased by 3–4 orders of magnitude. TSP is also synthesized by a variety of other cells and is incorporated into their extracellular matrix. TSP is a homotrimer with a number of functional domains, at least four of which might serve as receptor recognizing regions. The amino-terminal heparin binding domain interacts with heparin, other glycosaminoglycans and glycolipids and likely recognizes specific cell surface proteoglycans. The central disulfide cross-linked region, 210 kDa non-reduced and 70 kDa reduced, contains a peptide motif CSVTCG which is apparently responsible for binding to glycoprotein IV (CD36) with high affinity. Immediately adjacent to the calcium binding region of TSP, which undergoes considerable molecular relaxation in the absence of calcium, is an RGDA sequence. TSP has been demonstrated to bind to integrins of the αvβ3 and αIIbβ3 class. The carboxy-terminal region of TSP also contains at least one binding epitope for a cell receptor. There are 2 well characterized genes for TSP and truncated forms of TSP have been detected which have inhibitory effects on angiogenesis. Finally, TSP can interact with fibrinogen and fibronectin, perhaps on cellular surfaces, which might serve as secondary receptor-like mechanisms for TSP binding and subsequent mediation of cell adhesion.

Proboscidean Origins of Mastodon and Woolly Mammoth Demonstrated Immunologically

Immunologically reactive protein substances were extracted from bone samples of an American mastodon (Mammut americanum), 10,200 yr old by radiocarbon dating, and from musde samples of three woolly mammoths (Mammuthus primigenius), 10,000, 40,000 and 53,000 yr old, respectively. The mas- todon samples contained significant quantities of the amino acids hydroxylysine and hydroxyproline, both of which are usually found in collagens and not in albumins. Using these products and other comparable extracts, as well as sera and purified collagens from modern elephants and other living mammals, as test antigens, immunological comparisons were carried out with the following antisera: rabbit anti-mastodon bone; chicken anti-mammoth musde; chicken anti-elephant muscle; rabbit anti-elephant albumin and rabbit anti-elephant collagen, as well as with rabbit antisera to purified albumins and collagens of other mammals. For the first time, mastodon bone was found to have elephant-like proteins, which elicited antibodies that reacted strongly with collagen and serum proteins of extant elephants. Mammoth muscle strongly reacted with anti-elephant collagen and anti-elephant albumin, but the concentrations of the recoverable mammoth collagen and albumin decreased with increasing chronological age of the mammoth specimens. Nevertheless, in the immunological comparisons, the mammoth was doser to Asian (Elephas maximus) and African (Loxodonta africana) elephants than to the mastodon; in turn, the mastodon was doser to these elephantid species than to mammals outside the order Proboscidea.

Connective tissue activation XXXIII. Biologically active cleavage products of CTAP-III from human platelets

Evidence for three new isoforms of CTAP-III from human platelets is presented; two NH2-terminal cleavage products were identified, CTAP-III (des 1-13) and CTAP-III (des 1-15). CTAP-III (des 1-13) has a pI of 8.6 and is a relatively stable proteolytic cleavage product that retains the capacity to stimulate [14C]GAG synthesis in human synovial cell cultures. CTAP-III (des 1-15) appears to be an elastase or chymotrypsin cleavage product and identical to NAP-2, an entity thought to have neutrophil activating properties.