Description:DIRECT ACTING FIBRINOLYTIC ENZYMES AND BLOOD CLOT DISSOLUTION
Pharmacologic dissolution of an established thrombus has become an accepted therapeutic approach for many patients who develop thrombotic occlusive disease. The major hypothesis of our work is that direct-acting fibrinolytic enzymes offer a unique alternative approach for thrombolytic therapy with theoretical, as well as practical, advantages over presently available plasminogen activator (PA)-based thrombolytic agents, such as tissue plasminogen activator. Fibrolase is a 23 kDa, direct-acting, fibrinolytic metalloproteinase, found in southern copperhead snake venom, that cleaves fibrin independently of plasmin(ogen). It is not inactivated by serine proteinase inhibitors in the blood. Fibrolase lyses fibrin by selective cleavage of peptide bonds in the alpha and beta chains. The thrombolytic actions of fibrolase have been demonstrated in animal models of arterial and venous thrombosis (in the canine and the rabbit). Fibrolase does not activate platelets or coagulation factors,nor does it lyse red blood cells. We have formed a fibrolase-chimera with thrombus specificity and antiplatelet activity. This novel agent may offer a unique mechanistic approach for thrombolytic therapy. The major goal of the construction and evaluation in vitro of the chimeric hybrid of fibrolase has been accomplished. The platelet-avid hybrid, formed by chemical linkage of fibrolase with a fibrinogen receptor antagonist peptide, has been prepared in large quantity and is being tested in vivo to determine clot selectivity and thrombolytic efficacy. We will also test the tolerability and pharmacokinetic behavior of the fibrolase hybrid in vivo.
We are also involved in studies to directly modify fibrolase (PEGylation) to improve circulatory half-life. A number of different fibrinolytic enzymes are also being investigated from the venom of different snake species. In another project we are investigating the crystal structure of fibrolase in collaboration with an x-ray crystallographer on the Main Campus at USC.
DISINTEGRINS: EFFECTIVE INHIBITORS OF CANCER GROWTH AND ANGIOGENESIS
Spread of cancer to remote sites, e.g. bone, lungs, liver, brain, is a characteristic of malignancy and often leads to inoperable disease. Control of metastasis offers an important avenue for cancer treatment. The first step in metastasis involves adhesion of the cancer cells to tissue around the primary tumor site. In the second step, cancer cells secrete digestive enzymes that degrade the surrounding tissues allowing the tumor cells to invade into these tissues. Eventually, the tumor cells enter the blood or lymphatic system where they repeat the adhesion and invasion steps at a distant (metastatic) site. Agents that block any of the above steps should act to inhibit metastasis. In order for a tumor mass to grow beyond a size of 1-2 mm3 the development of a vascular network is required. This process is called angiogenesis or neovascularization. Tumor cells at either a primary site or at metastatic sites induce the formation of new blood vessels, and these vessels supply nutrients and growth factors to the tumor and, importantly, serve as a route for tumor dissemination (metastasis). Since cancer-induced angiogenesis is essential for progressive growth of cancer, therapies that block new blood vessel growth into the tumor will also inhibit tumor growth and are of considerable interest to the clinical community since they may provide a unique and practical way for long term control of cancer. Anti-angiogenic therapy promotes long-term dormancy of the tumor and is non-cytotoxic thereby avoiding side effects, such as gastrointestinal problems, loss of hair and bone marrow suppression, that acccompany chemotherapy. Further, successfully blocking development of the vascular network may provide a useful alternative to chemotherapy that acts directly on tumor cells; attacking vascular cells would avoid the problem of acquired resistance to chemotherapy that results from genetic instability of tumor cells. We have been studying a protein from southern copperhead snake venom that possesses potent anti-tumor activity. A four-step chromatographic procedure was developed to purify the protein of interest, which we call contortrostatin, from the complex mixture of proteins in southern copperhead venom. Contortrostatin is a member of a family of peptides called disintegrins that are found in snake venoms. Members of this family are distinguished by the presence of an amino acid sequence, arginine-glycine-aspartic acid (RGD), that enables them to bind to cell surface receptors called integrins. Originally contortrostatin was characterized as an inhibitor of platelet aggregation. It binds to a specific integrin on the surface of blood platelets, thereby blocking the ability of platelets to adhere to one another (thus it acts as an inhibitor of platelet aggregation). Integrins are a family of adhesive proteins found on the surface of many different types of cells; they mediate interactions between cells and their surroundings. Integrins on cancer cells play important roles in tumor invasion and spread. We postulated that since contortrostatin disrupts integrin interactions on platelets, it should act similarly on cancer cell integrins and may, therefore, have significant anti-angiogenic and anti-metastatic activity. In fact, we have recently shown that contortrostatin acts as an effective inhibitor of breast cancer progression. Importantly, contortrostatin displays impressive inhibitory activity on the growth of new blood vessels into the breast cancer. CN not only inhibits human breast cancer cells from adhering to the surrounding tissue (the first step in tumor spread), but it also blocks the ability of the cells to migrate into the surrounding tissue. The RGD sequence in contortrostatin plays a critical role in binding to and inhibiting growth and spread of breast cancer cells. The mechanism by which the protein inhibits new blood vessel growth involves a direct interaction with adhesive proteins on the surface of the newly growing vascular cells. Recent evidence indicates that contortrostatin has an interesting activity due to its unique dimeric structure. It is able to cross-link and cluster integrins resulting in a temporally and spatially inappropriate activation of phosphorylation of proteins involved in integrin-mediated signal transduction. This results in disruption of the actin cytoskeleton and disturbance of focal adhesions resulting in inhibition of cell motility. Thus, in addition to disruption of adhesive and invasive activity at the surface of the cell, contortrostatin interferes with normal signaling processes to cause an internal effect on cell structure and motility. The net result is an effective disruption of cancer cell motility and dissemination. We hypothesize that similar effects occur in vascular endothelial cells leading to inhibition of angiogenesis. Recent studies on the effect of contortrostatin on gene expression, using DNA array technology, have resulted in some interesting preliminary findings. In ongoing investigations in our laboratory, we are using breast, prostate and ovarian cancer and glioma (a devastating brain tumor) models to demonstrate the anti-tumor and anti-angiogenic activities of contortrostatin. We are also examining the mechanism of its induction of integrin-mediated signal transduction pathways in both tumor and endothelial cells. Additionally, studies are aimed at developing more effective systems to deliver CN to the tumor site and we are also developing an anti-idiotype monoclonal antibody which will serve as a functional surrogate for contortrostatin and eliminate the problems related to its short circulatory half life and its recognition by the immune system. Finally, in collaboration with an investigator in the chemistry department on the Main Campus at USC we are determining the three-dimensional structure of contortrostatin.
For more information about Dr. Markland's research, please visit his PIBBS Research Faculty webpage.
- Cardiovascular Diseases
- Cancer Treatment
- Signal Transduction
- Protein Chemistry/Enzymology
- Gene Therapy
HISTORY & EDUCATION
- Johns Hopkins University, PHD, 1964
- University of California at Los Angeles, California, 1964 – 1966
- Schmitmeier S, Markland FS, Ritter MR, Sawcer DE, Chen TC. Functional effect of contortrostatin, a snake venom disintegrin, on human glioma cell invasion in vitro. Cell Commun Adhes. [ 2003 ] 10(1):1-16. PubMed
- Moiseeva N, Swenson SD, Markland FS Jr, Bau R. Purification, crystallization and preliminary X-ray analysis of the disintegrin contortrostatin from Agkistrodon contortrix contortrix snake venom. Acta Crystallogr D Biol Crystallogr. [ 2002 ] Dec;58(Pt 12):2122-4. PubMed
- Markland FS, Shieh K, Zhou Q, Golubkov V, Sherwin RP, Richters V, Sposto R. A novel snake venom disintegrin that inhibits human ovarian cancer dissemination and angiogenesis in an orthotopic nude mouse model. Haemostasis. [ 2001 ] May-Dec;31(3-6):183-91. PubMed
- Ritter MR, Zhou Q, Markland FS Jr. Contortrostatin, a homodimeric disintegrin, actively disrupts focal adhesion and cytoskeletal structure and inhibits cell motility through a novel mechanism. Cell Commun Adhes. [ 2001 ] 8(2):71-86. PubMed
- Bolger MB, Swenson S, Markland FS Jr. Three-dimensional structure of fibrolase, the fibrinolytic enzyme from southern copperhead venom, modeled from the X-ray structure of adamalysin II and atrolysin C. AAPS PharmSci. [ 2001 ] 3(2):E16. PubMed
Direct link to profile: http://keck.usc.edu/profiles?facid=735