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Biomedical subjects

S V Pizzo

Publications and source records attributed to S V Pizzo.

At least 253 records · Page 14Linked to original sources
PubMed1982

Fibrinolysis and fibrinogenolysis by Val442-plasmin.

Elastase cleavage of Lys77-plasmin results in the formation of Val442-plasmin. This result suggests that small, active plasmin fragments can be produced even under conditions of high plasminogen activator levels such as occur in vivo. We examined the effect of the generation of such fragments by studying the degradation of fibrinogen and fibrin by Val442-plasmin. Val442-plasmin lysis of fibrinogen yielded the same products as obtained with Lys77-plasmin, but at a slightly lower rate. Lysine inhibited fibrinogenolysis by both Lys77-plasmin and Val442-plasmin. The marked inhibition observed at concentrations higher than 10 mM lysine occurred to the same extent for both proteases. In addition, the products and rate of fibrinolysis were the same for both proteases. These results indicate that the lysine binding regions present in Lys77-plasmin but absent in Val442-plasmin do not determine the rate, reaction products, or lysine inhibition of fibrinolysis and fibrinogenolysis by plasmin.

Fibrinogen↗
PubMed1982

Receptors for maleylated proteins regulate secretion of neutral proteases by murine macrophages.

Receptors for maleylated or acetylated proteins as well as for alpha-2-macroglobulin-protease complexes on macrophages serve as scavengers by mediating the uptake of macromolecules from the extracellular compartment. Described in this report is a novel function of these receptors on macrophages: regulation of neutral protease secretion. The binding of maleylated bovine serum albumin to macrophages triggered secretion of three neutral proteases: neutral caseinases, plasminogen activator, and cytolytic proteinase. Release of acid phosphatase, however, was not induced. An important biological consequence of protease secretion by macrophages, tumor-cytolysis, was also triggered by engagement of the receptor for maleylated bovine serum albumin. By contrast, the binding of alpha-2-macroglobulin-protease complexes to the macrophages suppressed secretion of all three proteases. Thus two receptors heretofore believed to serve principally as scavengers also regulate secretory functions of macrophages.

Animals↗
PubMed1982

A unique pathway for the plasma elimination of alpha 2-antiplasmin-protease complexes in mice.

Radiolabeled alpha 2-antiplasmin cleared slowly from the circulation of mice. Complex formation with either plasmin or trypsin resulted in a significant increase in the plasma elimination rate of the protease inhibitor. Approximately 20 min and 14 min were required for 50% of the injected alpha 2-antiplasmin-plasmin and alpha 2-antiplasmin-trypsin to clear from the circulation, respectively. Significant competition was observed when radiolabeled alpha 2-antiplasmin-plasmin was cleared in the presence of a large molar excess of unlabeled alpha 2-antiplasmin-plasmin. alpha 1-Antitrypsin-trypsin failed to complete with radiolabeled alpha 2-antiplasmin-plasmin even when present at 2000 fold molar excess. Organ distribution studies localized the major site of alpha 2-antiplasmin-plasmin clearance in the liver. Microscopic autoradiography data suggested that the cell responsible for the clearance pathway was the hepatocyte.

Animals↗
PubMed1982

The clearance of human fibrinogen fragments D1, D2, D3 and fibrin fragment D1 dimer in mice.

The clearance of human fibrinogen fragments D1, D2, D3 and fibrin fragment D1 dimer were studied in the mouse model. Clearance of these fragments is a complex process involving clearance from blood into three other compartments. The overall clearance of fragment D1 and its dimer were essentially identical. Fragments D2 and D3 cleared at a progressively slower rate. Competition studies were performed between 125I-labeled fragment D1 and large molar excesses of unlabeled human fragments D1, D2, D3, D1 dimer, fragment E, fibrinogen, macroalbumin, mannan and asialoorosomucoid. Of these ligands only the fragment D variants competed for the clearance of 125I-labeled fragment D1. Cross-competition was observed when 125I-labeled fragment D1 dimer was cleared in the presence of a large molar excesses of fragment D1. Autopsies demonstrated that injected fragments D1, D2, D3 and D1 dimer cleared primarily in liver and kidneys. In some clearance studies, livers were perfused with tissue culture fluid, subjected to light microscopic autoradiography, and silver grain counts performed to localize cleared fragment D1. These experiments indicated that 80% of the liver uptake was in hepatocytes. However, when silver grain counts were normalized for the number of parenchymal and nonparenchymal cells, the distribution of silver grains was essentially identical (1.8 and 1.6 grains per cells, respectively). It is concluded that fragments D1, D2, D3 and D1 dimer are recognized by a similar clearance pathway. Since neither fibrinogen nor fragment E competed for the clearance of fragment D1, it is suggested that determinants present in the fragment D domain become exposed after plasmin attack on fibrinogen and are responsible for clearance.

Albumins↗
PubMed1982

Physical properties of human alpha 2-macroglobulin following reaction with methylamine and trypsin.

Circular dichroism spectroscopy, sedimentation velocity and ultraviolet difference spectroscopy were used to compare alpha 2-macroglobulin, alpha 2-macroglobulin-trypsin complex and alpha 2-macroglobulin-methylamine complex. The circular dichroic spectrum of native alpha 2-macroglobulin is significantly changed in shape and magnitude following reaction with either trypsin or methylamine. The spectra of alpha 2-macroglobulin-trypsin and alpha 2-macroglobulin-methylamine are, however, indistinguishable. The ultraviolet difference spectrum between alpha 2-macroglobulin-methylamine and native alpha 2-macroglobulin displays a tyrosine blue shift consistent with the exposure of several tyrosine residues to solvent. The conformational change which occurs in alpha 2-macroglobulin during reaction with methylamine follows pseudo-first-order kinetics. T 1/2 was 10.5 min for the reaction with 200 mM methylamine at pH 8.0 and 45 min for the reaction with 50 mM methylamine, also at pH 8.0. Reaction of methylamine with alpha 2-macroglobulin results in loss of trypsin-binding activity which appears to be a direct consequence of the conformational change induced by methylamine. A sedimentation coefficient (S0(20),W) of 20.5 was determined for alpha 2-macroglobulin-methylamine compared to a value of 18.5 for unreacted alpha 2-macroglobulin. This increase in sedimentation velocity is attributed to a 10% decrease in alpha 2-macroglobulin Stokes radius. alpha 2-Macroglobulin-trypsin complex prepared by reaction of the protease at a 2-fold molar excess with the inhibitor was a S0(20),W of 20.3. Although this sedimentation coefficient does reflect compacting of the alpha 2-macroglobulin structure compared to native alpha 2-macroglobulin, it is not large enough to rule out significant protrusion of the proteases from pockets in the alpha 2-macroglobulin structure.

Circular Dichroism↗
PubMed1982

Purification of an almond emulsin fucosidase on Cibacron blue-sepharose and demonstration of its activity toward fucose-containing glycoproteins.

The almond emulsin fucosidase that specifically hydrolyzes fucose in alpha (1-3) linkage to N-acetylglucosamine has been purified 1250-fold. The purification procedure includes ion exchange chromatography on sulfopropyl-Sephadex C-25, gel filtration on Sephacryl S-200, and affinity chromatography on Cibacron blue-Sepharose 4B-CL. The molecular weight of the fucosidase was estimated by gel filtration as approximately 73,000. Enzyme activity was maximal at pH 5.3 in acetate buffer and was dependent on ionic strength; at least 0.1 M NaCl was necessary for optimal activity. The purified enzyme was free of beta-galactosidase activity toward the glycoprotein substrate [3H]galactosyl-asialotransferrin and did not release fucose from substrates containing fucose in alpha (1-6) linkage, (bovine IgG glycopeptides) or in alpha (1-2) linkage, (2'-fucosyllactose). The fucosidase displayed activity toward two glycoprotein substrates known to contain fucose in alpha (1-3) linkage. Extensive incubations resulted in the release of 83% and 43% of the total fucose of asialoorosomucoid and lactoferrin, respectively. The fucosidase did not release fucose from either the "slow" or the "fast" form of alpha 2-macroglobulin, suggesting the absence of fucosyl alpha (1-3) linkages on that glycoprotein.

Glycopeptides↗
PubMed1982

In vivo catabolism of alpha 1-proteinase inhibitor-trypsin, antithrombin III-thrombin and alpha 2-macroglobulin-methylamine.

The clearances of 125I-labeled alpha 1-proteinase inhibitor-trypsin, antithrombin III-thrombin and alpha 2-macroglobulin-methylamine (CH3NH2) were compared in our previously described mouse model. alpha 1-Proteinase inhibitor-trypsin cleared with a t 1/2 of 20 min, antithrombin III-thrombin of 7 min and 125I-labeled alpha 2-macroglobulin-methylamine of 2 min. Competition studies were performed to determine whether one or several pathways clear these three ligands. The clearance of 125I-labeled alpha 1-proteinase inhibitor-trypsin and 125I-labeled antithrombin III-thrombin was blocked by large molar excesses of either ligand, but not by alpha 2-macroglobulin-methylamine. The clearance of 125I-labeled alpha 2-macroglobulin-methylamine can be blocked by a large molar excesses of unlabeled alpha 2-macroglobulin-methylamine but not by alpha 1-proteinase inhibitor-trypsin. These studies demonstrate that the clearance of alpha 1-proteinase inhibitor-trypsin complexes is independent of alpha 2-macroglobulin-methylamine and utilizes the same pathway which is involved in the clearance of antithrombin III-thrombin complexes.

Animals↗
PubMed1982

Localization of factor VIII/von Willebrand factor and glial fibrillary acidic protein in the hemangioblastoma: implications for stromal cell histogenesis.

The histogenesis of hemangioblastoma stromal cells is unresolved. Ultrastructural observations suggest that the stromal cells, endothelial cells, and pericytes that compose this neoplasm are all derived from angiogenic mesenchyme. The expression of factor VIII/von Willebrand factor (FVIII/vWF), a specific marker for endothelial cells, and of glial fibrillary acidic protein (GFAP), a specific marker for glial cells, was examined in 16 hemangioblastomas using the peroxidase-antiperoxidase immunohistochemical method. Endothelial cell staining for FVIII/vWF was intense in 14 tumors, weak in one, and absent in another. There was no stromal cell staining in any of the neoplasms. Process-bearing, GFAP-positive cells were observed near the tumor margin in 13 cases, and deeper in the neoplasm in 8. In two of these tumors there were also occasional GFAP-positive cells that lacked processes and had a vacuolated cytoplasm. Virtually all of the GFAP-positive cells were interpreted as trapped astrocytes rather than stromal cells. The lack of expression of FVIII/vWF by the stromal cells indicates that they are antigenically distinct from endothelial cells. Several alternatives for stromal cell histogenesis remain open. The stromal undergone antigenic loss, or from angiogenic mesenchymal cells that do not express FVIII/vWF. Alternatively, the stromal cells may originate from non-angiogenic mesenchymal cells derived from the mesoderm or neuroectoderm.

Adolescent↗
PubMed1982

Catabolic pathways for streptokinase, plasmin, and streptokinase activator complex in mice. In vivo reaction of plasminogen activator with alpha 2-macroglobulin.

The catabolic pathways of streptokinase, plasmin, and activator complex prepared with human plasminogen were studied in mice. (125)I-streptokinase clearance occurred in the liver and was 50% complete in 15 min. Incubation with mouse plasma had no effect on the streptokinase clearance rate. Complexes of plasmin and alpha(2)-plasmin inhibitor were eliminated from the plasma by a specific and saturable pathway. Competition experiments demonstrated that this pathway is responsible for the clearance of injected plasmin. Streptokinase-plasminogen activator complex formed with either (125)I-plasminogen or (125)I-streptokinase cleared in the liver at a significantly faster rate than either of the uncomplexed proteins (50% clearance in <3 min). Streptokinase incubated with human plasma also demonstrated this accelerated clearance. p-Nitrophenyl-p'-guanidinobenzoate-HCl or pancreatic trypsin inhibitor-treated complex cleared slowly compared with untreated complex independent of which protein was radiolabeled. Significant competition for clearance was demonstrated between alpha(2)-macroglobulin-trypsin and activator complex only when the plasmin(ogen) was the radiolabeled moiety. Large molar excesses of alpha(2)-plasmin inhibitor-plasmin failed to retard the clearance of activator complex. Hepatic binding of streptokinase-plasmin, in liver perfusion experiments, was dependent upon prior incubation with plasma (8-10% uptake compared to a background of approximately 2.5%). Substitution of human alpha(2)-macroglobulin for plasma also resulted in binding when the incubation was performed for 10 min at 37 degrees C (7.5%). Electrophoresis experiments confirmed the transfer of 0.8 mol plasmin/mol alpha(2)-macroglobulin when activator complex was incubated at 37 degrees C with alpha(2)-macroglobulin for 40 min. Streptokinase transfer from activator complex to alpha(2)-macroglobulin was negligible. The in vivo clearance of activator complex is proposed to involve active attack of the complex on the alpha(2)-macroglobulin "bait region," resulting in facilitated plasmin transfer. Dissociated streptokinase is rapidly bound and cleared by sites in the liver.

Animals↗
PubMed1982

Specificity and sensitivity of immunohistochemical detection of factor VIII/von Willebrand factor antigen in formalin-fixed paraffin-embedded tissue.

The immunohistochemical detection of factor VIII/von Willebrand factor antigen (FVIII/vWF-AG) in formalin-fixed paraffin-embedded tissues was investigated using the peroxidase-antiperoxidase (PAP) method. Highly purified human FVIII/vWF was used to raise rabbit anti-FVIII/vWF-AG serum. In addition to anti-FVIII/vWF-AG activity, the unabsorbed antiserum had anti-IgG, anti-IgM, and anti-alpha2-macroglobulin specificities. Following exhaustive absorption with these proteins, the antiserum reacted monospecifically for FVIII/vWF-AG in immunodiffusion, immunoelectrophoresis, and PAP immunohistochemistry. Sections of normal tissues from six patients and a total of 43 neoplasms were examined. Treatment of the tissue sections with trypsin prior to application of the antiserum markedly increased the sensitivity of FVIII/vWF-AG detection. The positive staining for FVIII/vWF-AG was restricted to endothelial cells in both neoplastic and nonneoplastic tissue. In general, the hyperplastic endothelia in neoplastic and reactive tissues stained more intensely than those in normal tissues. Expression of FVIII/vWF-AG by nonendothelial neoplastic cells was not observed. FVIII/vWF-AG is a reliable marker for endothelial cells.

Blood Coagulation Factors↗
PubMed1982

A colorimetric assay for releasable plasminogen activator.

We describe an equilibrium assay for measuring release of plasminogen activator form blood-vessel walls and report data from 125 individuals free of overt thromboembolic disease. Excess human plasminogen is added to the euglobulin fraction of plasma obtained before and after venous occlusion at mean systolic pressure. To measure plasmin generation in these samples, we used the chromogenic plasmin substrate D-Val-Leu-Lys-p-nitroanilide, which liberates p-nitroaniline upon cleavage. Releasable plasminogen activator in 24 subjects was determined by this colorimetric assay and by the radiocasein assay previously reported by this laboratory (Am. J. Clin. Pathol. 76,403-409, 1981), and the results were compared. The correlation coefficient was 0.97. The colorimetric assay offers several advantages over the radiocasein assay: shorter incubation (6 vs 16 h) and no preparation or quantification of a radioactive substrate and its cleavage products.

Adult↗
PubMed1982

Immunohistochemical detection of factor VIII/von Willebrand factor in hyperplastic endothelial cells in glioblastoma multiforme and mixed glioma-sarcoma.

The sarcomatous components of most glioma-sarcomas are thought to arise from the neoplastic transformation of hyperplastic endothelial and adventitial vascular cells in a preexisting glioblastoma multiforme. The expression of factor VIII/von Willebrand factor (FVIII/vWF), a marker for endothelial cells, and of glial fibrillary acidic protein (GFAP), a marker for glial cells, was examined in 10 glioblastomas and seven mixed glioma-sarcomas using the peroxidase-antiperoxidase immunohistochemical technique. Hyperplasia of small blood vessels was observed in all 10 glioblastomas; in five, the vascular proliferation had resulted in the formation of prominent glomeruloid structures. FVIII/vWF was detected in the endothelial cells in these vascular structures, but not in the adventitial cells. In the mixed glioma-sarcomas. FVIII/vWF was detected only in endothelial cells; there was no staining for FVIII/vWF in the neoplastic mesenchymal cells. The gliomatous components of the mixed tumors stained intensely for GFAP. These observations indicate that both endothelial and nonendothelial cell types contribute to the small vessel hyperplasia in glioblastomas, and that the sarcomatous components of mixed glioma-sarcomas are derived from either non-endothelial cells or endothelial cells that have undergone antigenic loss following transformation.

Blood Coagulation Factors↗
PubMed1982

Immunohistochemical studies of cell differentiation in a juxtaglomerular tumor.

A renal tumor removed from a 29-year-old woman, who recently had had onset of primary hypertension, showed the histologic and ultrastructural features of a renin-secretory juxtaglomerular cell tumor. The expression of Factor VIII-related antigen (FVIII-RAG) was evaluated to identify endothelial differentiation of tumor cells in this lesion. Control tissue and vessels within the tumor showed normal expression of FVIII-RAG, but tumor cells as well as normal juxtaglomerular cells showed no localization of this antigen, which suggests the nonendothelial nature of these specialized cells.

Adult↗
PubMed1981

Inactivation of the plasma protease inhibitor alpha 2-macroglobulin by the antitumor drug cis-dichlorodiamineplatinum(II).

The plasma protease inhibitor alpha 2-macroglobulin (alpha 2M) was reacted in vitro with cis-dichlorodiamineplastinum(II) (cis-DDP). Following the reaction, alpha 2M demonstrated a significantly decreased ability to bind trypsin as determined by esterase activity assays in the presence of soybean trypsin inhibitor and studies with radiolabeled trypsin. Inactivation of alpha 2M by cis-DDP was not associated with a conversion to the "fast" electrophoretic form, as determined on nondenaturing gels, in contrast to the inactivation of alpha 2M by proteases and certain amine salts. The extent of reaction increased with the elevation of temperature within the thermal stability range of the protein; however, variation of pH within the range 6.82-8.55 had little effect. Binding of [14C]methylamine to alpha 2M was not affected by cis-DDP. The conformational change, however, which normally accompanies this reaction did not occur. It is concluded that the alpha 2M thiolesters are most likely not reactive sites for cis-DDP. cis-DDP-treated alpha 2M failed to dissociate into quarter subunits under denaturing and reducing conditions, suggesting cross-linking of subunits. This cross-linking may be responsible for locking the alpha 2M quarternary structure into the "slow conformation."

Cisplatin↗
PubMed1981

Altered clearance of human alpha 2-macroglobulin complexes following reaction with cis-dichlorodiamineplatinum(II).

Clearance studies were performed in mice using alpha 2-macroglobulin (alpha 2M), alpha 2M-trypsin complex and alpha 2M-CH3NH2 complex. All three species were incubated with cis-dichlorodiamine platinum(II) (cis-DDPt) at concentrations between 9.0 microM and 1.67 mM for 4 h and then dialyzed. The clearance rate of native alpha 2M was unchanged following incubation with cis-DDPt. alpha 2M-trypsin and alpha 2M-CH3NH2 cleared rapidly from the circulation; however, reaction with cis-DDPt significantly decreased the plasma elimination rate of both complexes. Non-denaturing gel electrophoresis and alpha 2M activity assays demonstrated relative stability following incubations with cis-DDPt which markedly altered clearance. Evidence for cis-DDPt crosslinking of alpha 2M subunits was obtained; however, whether this crosslinking is involved in altered clearance remains undetermined. Iodoacetamide treatment of alpha 2M did not duplicate the effect of cis-DDPt on alpha 2M clearance, nor did it inhibit the effect of cis-DPPt on alpha 2M clearance. Plasma elimination of alpha 2M complex was also unaltered by pretreatment of mice with intravenous free cis-DDPt.

Animals↗