Stanley Zucker

Thrombin Induces the Activation of Progelatinase A in Vascular Endothelial Cells PHYSIOLOGIC REGULATION OF ANGIOGENESIS

Angiogenesis requires degradation of vascular basement membrane prior to migration and proliferation of endothelial cells; proteinases are essential ingredients in this process. Because of thrombins multiple effects on endothelium, we have examined its role in matrix metalloproteinase activation using human umbilical vein endothelial cells. Gelatin zymography of endothelial conditioned media revealed a prominent 72-kDa progelatinase A band. Addition of α-thrombin to endothelial cells resulted in the generation of 64 and 62 kDa gelatinolytic bands which is consistent with the activation of progelatinase A; thrombin had no effect in the absence of cells. This effect requires the proteolytic site of thrombin since progelatinase A activation was abolished by specific inhibitors of thrombin. Matrix metalloproteinase inhibitors diminished thrombin-induced activation of progelatinase A. Pretreatment of endothelial cells with excess tissue inhibitor of metalloproteinase-2 or a COOH-terminal fragment of progelatinase A abrogated thrombin-mediated activation of progelatinase A presumably by competing with the COOH terminus of native progelatinase A for interaction with an activator site on endothelial plasma membranes. Although membrane-type matrix metalloproteinase was demonstrated in endothelial cells by Northern and Western blotting, the receptor function of this molecule in thrombin-induced activation of progelatinase A needs to be clarified. Progelatinase A activation did not require intracellular signal transduction events mediated by the thrombin receptor. These data demonstrate that 1) endothelial cells express a novel activation mechanism for progelatinase A, 2) proteolytically active thrombin regulates this activation mechanism, and 3) activation occurs independently of the functional thrombin receptor.

Membrane type matrix metalloproteinase 1 activates pro-gelatinase A without furin cleavage of the N-terminal domain.

Membrane type matrix metalloproteinase 1 (MT-MMP1), a novel 63-kDa member of the matrix metalloproteinase family, is a membrane-anchored enzyme and an activator for gelatinase A. In addition to its C-terminal hydrophobic transmembrane domain, MT-MMP1 has an insertion of 11 amino acids between its propeptide and catalytic domain encrypted with a RRKR recognition motif for the paired basic amino acid cleaving enzyme, furin. In this report, we investigated whether the cleavage of the RRKR motif of MT-MMP1 by Golgi-associated furin is analogous to a similar enzyme activation mechanism observed with stromelysin-3. Mutant forms of MT-MMP1 were cotransfected into COS-1 cells with cDNAs for pro-gelatinase A and/or furin. Immunoprecipitation and immunoblotting using specific antibodies were employed to characterize cell proteins. Whereas furin readily cleaved soluble MT-MMP1 lacking the transmembrane domain (ΔMT-MMP1), a soluble stromelysin-1/ΔMT-MMP1 chimera without the RRKR basic motif was resistant to furin-induced cleavage. COS-1 cells cotransfected with wild type MT-MMP1 cDNA and furin cDNA demonstrated a 63-kDa protein (latent enzyme) on SDS-polyacrylamide gel electrophoresis rather than the anticipated lower molecular weight activated enzyme. Inhibition of furin activity with α1-protease inhibitorPittsburgh (a furin inhibitor) did not affect the pro-gelatinase A activation mechanism in COS-1 cells cotransfected with MT-MMP1 and pro-gelatinase A cDNAs. Furthermore, substitution of the RRKR motif of MT-MMP1 with alanine residues by site-directed mutagenesis resulted in the same 63-kDa protein without loss of pro-gelatinase A activation function. These data indicate that furin-induced activation of MT-MMP1 is not a prerequisite for pro-gelatinase A activation. The mechanism of activation of cell-bound MT-MMP1 remains to be elucidated.

Localization of tissue inhibitor of matrix metalloproteinases in Alzheimer's disease and normal brain.

Based upon the hypothesis that metalloproteinases and their inhibitors might be involved in the pathogenesis of Alzheimers disease, we studied brain samples of eight cases of Alzheimers disease, six other pathological entities and three elderly controls for tissue inhibitor of metalloproteinase (TIMP) immunorcactivity. Specificity was supported by a loss of immunoreactivity following antigen preabsorption of antisera. Areas studied included ependyma, choroid plexus, frontoparietal, hippocampal and cerebellar cortex, n. basalis of Meynert, basal ganglia, midbrain, pons, and medulla. TIMP positivity was localized to neuritic senile plaques, neurofibrillary tangles and Purkinjc cells. The pattern of TIMP plaque staining was similar to that observed with anti tau and SP18 antibodies. It differed from that observed with anti SP40, HAM 56 and GFAP antibodies. The selective localization of TIMP to the neuritic lesions of Alzheimers disease in a codistribution with the amyloid precursor protein and abnormally phosphorylated and truncated tau supports a possible role for metalloproteinases and their inhibitors in the evolution of these lesions.

Characterization of metalloproteinases and tissue inhibitors of metalloproteinases in human plasma.

In this study, we have identified and characterized metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs) in human plasma. Treatment of plasma with trypsin or aminophenylmercuric acetate resulted in activation of latent gelatinolytic activity. Fractionation of plasma by gelatin Sepharose chromatography resulted in the isolation of 72 kDa and 92 kDa gelatinases/type IV collagenases. The 72 kDa gelatinase was purified by gel filtration chromatography. Stromelysin-1 was isolated from plasma by Matrex green A affinity chromatography. Immunoblotting of plasma fractions with antibodies to unique peptide regions of human gelatinases differentiated the 72 kDa gelatinase from the 92 kDa gelatinase. Antibodies to the amino terminal peptides of each enzyme were used to determine that plasma gelatinases circulate as latent proenzymes. Immunoblotting with antibodies directed against human stromelysin identified a 57 kDa stromelysin. TIMP-1 (28 kDa) and TIMP-2 (21 kDa) were also identified by immunoblotting of gelatin Sepharose bound plasma proteins using non-crossreacting antibodies to each protein.

The anaemia of chronic disorders: studies of iron reutilization in the anaemia of experimental malignancy and chronic inflammation.

The purpose of this study was to evaluate the contribution of defective reutilization of iron in the pathogenesis of the anaemia of chronic disease (ACD). Normal rats with or without splenectomy and rats with Walker 256 carcinosarcoma with or without splenectomy were studied. The reutilization of 59Fe labelled heat‐damaged red cells (59Fe DRBC), the sequestration of 59Fe DRBC in major organs, red cell mass, 51Cr red cell survival, serum iron, plasma erythropoietin and routine haematological parameters were measured.

IgM Inhibitors of the Contact Activation Phase of Coagulation in Chlorpromazine‐ Treated Patients

Summary. In this report we have described three patients with chronic schizophrenia on long‐term chlorpromazine therapy who developed asymptomatic IgM inhibitors of the intrinsic phase of blood coagulation. The anticoagulant resulted in decreased measurements of all of the plasma clotting factors in the intrinsic pathway (factors VIII, IX, XI, XII, Fletcher factor and Fitzgerald factor). Using crude coagulation reagents, the serum of these patients interfered with the clot promoting activity of contact product. To determine the relationship between drug therapy and these IgM inhibitors, we have studied nine additional schizophrenic patients on long‐term chlorpromazine therapy. All nine chlorpromazine‐treated patients had significantly increased levels of serum IgM and asymptomatic inhibitors of coagulation. We conclude that long‐term high‐dose chlorpromazine treatment of schizophrenic patients results in an increased concentration of IgM which has inhibitory activity in the contact phase of blood coagulation.

Analysis of the Cell Membrane Proteolytic Enzymes of the B16, Fl, F10, and BL6 Melanoma and Their Role in Target Cell Destruction

The tumor-induced red blood cell (RBC) cytolysis assay has been used to demonstrate that three B16 melanoma sublines, the F1, F10, and BL6, cause the cytolysis of normal red blood cells in vitro. RBC cytolysis was inhibited for all three sublines by metalloprotease inhibitors. Cell membrane preparations have been prepared for all three sublines and tumor cell membrane-induced RBC cytolysis was also shown to be inhibited by metalloprotease inhibitors. The F10 and BL6 sublines were shown to have cell membrane-bound proteases. The BL6 subline has a cell membrane enriched in an enzyme with a trypsin-like arginine specificity. The trypsin-like protease may have a metal dependence. The BL6 subline has a collagenolytic cell membrane enzymes and a chymotrypsin-like cell membrane enzyme. B16 cell membrane enzymes may be responsible for RBC cytolysis in vitro in a process requiring divalent cations.

LIPOPROTEIN INHIBITOR OF BQNE MARROW CELLS IN TUMOR‐BEARING RATS

The role of a plasma inhibitor of erythropoiesis is evaluated in rats with Walker‐256 carcinoma (W‐256). Plasma from tumor‐bearing rats was treated by gel filtration chromatography (Sephadex G‐150) and fractions were combined into four pools on the basis of mol. wt. Inhibitory activity was assayed by adding an aliquot of the plasma fractions to normal rat marrow cells which were cultured for 24 hr with and without erythropoietin. 59Fe‐heme synthesis, [3H]thymidine DNA synthesis, and 14C‐leucine protein synthesis were studied. The results indicated that cultures containing the high mol. wt. pool (>400,000 daltons) had significantly decreased heme, DNA and protein synthesis. This inhibitor also diminished the response to erythropoietin in polycythemic mice. The lower mol. wt. pool stimulated heme synthesis in vitro. To identify the inhibitor further, plasma lipo‐protein classes were isolated by density gradient ultracentrifugation. The very low density lipoprotein (VLDL) and chylomicron fractions markedly inhibited DNA, protein and heme synthesis. Low density and high density lipoprotein fractions were inactive. A lipoprotein inhibitor of erythropoiesis was also identified in cancerous ascitic fluid, and to a lesser degree, in normal rat plasma. We suggest that this VLDL inhibitor of marrow erythropoiesis is a contributing factor in the anaemia of cancer.