fibrin and batimastat

Snakebite envenomation causes over 140,000 deaths every year, predominantly in developing countries. As a result, it is one of the most lethal neglected tropical diseases. It is associated with incredibly complex pathophysiology due to the vast number of unique toxins/proteins present in the venoms of diverse snake species found worldwide. Here, we report the purification and functional characteristics of a Group I (PI) metalloprotease (CAMP-2) from the venom of the western diamondback rattlesnake,

Topics: Animals; Antineoplastic Agents; Antivenins; Binding Sites; Blood Platelets; Catalytic Domain; Collagen; Crotalid Venoms; Crotalus; Drug Repositioning; Erythrocytes; Fibrin; Fibrinolysis; Hemolysis; Humans; Hydroxamic Acids; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Molecular Docking Simulation; Phenylalanine; Protein Binding; Protein Conformation; Structure-Activity Relationship; Substrate Specificity; Thiophenes

Adult angiogenesis, associated with pathologic conditions, is often accompanied by the formation of a fibrinous exudate. This temporary matrix consists mainly of fibrin but is intermingled with plasma proteins and collagen fibers. The formation of capillary structures in a fibrinous matrix in vivo was mimicked by an in vitro model, in which human microvascular endothelial cells (hMVECs) seeded on top of a fibrin-10% collagen matrix form capillarylike tubular structures after stimulation with basic fibroblast growth factor/tumor necrosis factor alpha (bFGF/TNF-alpha) or vascular endothelial growth factor (VEGF)/TNF-alpha. In the fibrin-collagen matrix the metalloproteinase inhibitor BB94 inhibited tubule formation by 70% to 80%. Simultaneous inhibition of plasmin and metalloproteinases by aprotinin and BB94 caused a nearly complete inhibition of tubule formation. Adenoviral transduction of tissue inhibitor of metalloproteinases 1 (TIMP-1) and TIMP-3 into endothelial cells revealed that TIMP-3 markedly inhibited angiogenesis, whereas TIMP-1 had only a minor effect. Immunohistochemical analysis showed the presence of matrix metalloproteinase 1 (MMP-1), MMP-2, and membrane-type 1 (MT1)-MMP, whereas MMP-9 was absent. The endothelial production of these MMPs was confirmed by antigen assays and real-time polymerase chain reaction (PCR). MT1-MMP mRNA was markedly increased in endothelial cells under conditions that induced tubular structures. The presence of MMP-1, MMP-2, and MT1-MMP was also demonstrated in vivo in the newly formed vessels of a recanalized arterial mural thrombus. These data suggest that MMPs, in particular MT-MMPs, play a pivotal role in the formation of capillarylike tubular structures in a collagen-containing fibrin matrix in vitro and may be involved in angiogenesis in a fibrinous exudate in vivo.

Topics: Adenoviridae; Aprotinin; Cells, Cultured; Collagen; Culture Media; Defective Viruses; Drug Synergism; Endothelial Growth Factors; Endothelium, Vascular; Fibrin; Fibroblast Growth Factor 2; Genetic Vectors; Heart Diseases; Humans; Intercellular Signaling Peptides and Proteins; Lymphokines; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Matrix Metalloproteinases, Membrane-Associated; Metalloendopeptidases; Neovascularization, Pathologic; Neovascularization, Physiologic; Phenylalanine; Protease Inhibitors; Thiophenes; Thrombosis; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-3; Transduction, Genetic; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Pericellular proteolysis plays an important role in cell migration and the formation of new capillary structures. The plasminogen activator/plasmin and matrix degrading metalloproteinase (MMP) cascades act together in the remodeling of matrix and cell-matrix contacts. Previously we have shown that the formation of capillary structures by human foreskin microvascular endothelial cells (hMVECs) in a 3-dimensional fibrin matrix requires a functional urokinase-type plasminogen activator receptor (u-PAR). Here we report on the unexpected finding that inhibition of hMVEC-derived MMP activity by BB94 (batimastat) increased the outgrowth of capillary structures in a fibrin matrix. BB94 prevented the release of the u-PA binding domain D1 of u-PAR and thereby increased the number of functional u-PARs on hMVECs without affecting the u-PAR messenger RNA levels. Comparison of various types of protease inhibitors pointed to the prime involvement of MMP activity. Using recombinant MMPs it was shown that MMP-12 activity was able to release the D1 domain of cellularly expressed u-PAR. In addition, the expression of MMP-12 in control and basic fibroblast growth factor/tumor necrosis factor-alpha-stimulated hMVECs was shown by reverse transcriptase-polymerase chain reaction, suggesting that endothelial cell-derived MMP-12 may be involved in angiogenesis-related u-PAR shedding. This new mechanism of u-PAR cleavage provides new insights into the mutual interactions between the MMP and u-PA/plasmin systems. Moreover, it may be helpful in the interpretation of recent data on the use of specific MMP inhibitors in the treatment of several types of cancer.

Topics: Binding Sites; Blotting, Western; Cells, Cultured; Endothelium, Vascular; Fibrin; Fibroblast Growth Factor 2; Gene Expression; Humans; Metalloendopeptidases; Microcirculation; Neovascularization, Physiologic; Phenylalanine; Protease Inhibitors; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiophenes; Transfection; Tumor Necrosis Factor-alpha; Urokinase-Type Plasminogen Activator