DOWNLOAD PDF THE FORMATION OF FIBRIN CLOTS RUSSELL F. DOOLITTLE

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1 Chapter 1 : Russell Doolittle Fibrin clots are well known to be permeable to a wide variety of agents. The clot itself, however, is a tangle of fibers, and the permeability of the individual fibers is more problematic. Depending on environmental conditions, fibers may range from a few to hundreds of molecules in diameter. Abstract A blood clot is a meshwork of fibrin fibers built up by the systematic assembly of fibrinogen molecules proteolyzed by thrombin. Here, we describe a model of how the assembly process occurs. The last two of these interactions are responsible for protofibril association and are predicated on intermolecular packing arrangements observed in crystal structures of fibrin double-d fragments cocrystallized with synthetic peptides corresponding to the knobs exposed by the release of the fibrinopeptides A and B. It is generally accepted that the thrombin-catalyzed removal of the fibrinopeptides A leads to the formation of intermediate protofibrils composed of staggered, half-molecule overlaps, the end results of which are two-molecule thick, end-to-end, noncovalent oligomers 6. It is also part of current dogma that the subsequent release of fibrinopeptide B from the units in these oligomers encourages the lateral association that leads to the thick fibers that constitute natural clots 7. These conclusions were reached well before there was any structural information available about the relative locations of the sites involved. Extension of the process leads to the formation of the intermediate protofibrils. And, although synthetic A-knob peptides GPR derivatives are effective inhibitors of fibrin polymerization, synthetic B-knobs peptides GHR derivatives are not, even though they bind to fibrinogen and fragment D 9. Remarkably, GHR peptides actually accelerate fibrin formation 9. Moreover, fibrin can be formed by the removal of fibrinopeptide A only, as can be demonstrated with various snake venom enzymes Nonetheless, such fibrin is not of the same quality as fibrin generated by the thrombin-catalyzed removal of both fibrinopeptides A and B, and the physiological role of removing the latter must be significant. Here, we describe a detailed model for each of the steps in fibrin formation, including especially the lateral association of protofibrils, the least understood step in the process. Our proposal for how this step occurs is mainly based on packing interactions observed in crystal structures of the core fragments D from fibrinogen and DD from crosslinked fibrin complexed with the two different synthetic peptides that correspond to the natural A and B knobs 3 â 5. These considerations have been used in conjunction with a recently reported low-resolution structure of native chicken fibrinogen 13 as the framework model for the units being bundled together. Methods Extensive use was made of the o modeling program 14 ; superposition of double-d structures and that of native chicken fibrinogen was accomplished with the least squares function lsq in that package. The linear growth of model structures was managed with the pdbset routine contained in the CCP4 package 15 ; symmetry operations were based on the chicken fibrinogen structure Estimates of the strengths of interaction at each step of the polymerization process were obtained from measures of lost solvent accessibility 16 ; in particular, the method of Lee and Richards 17 as provided in the CCP4 package 15 was used with a probe radius 1. The program xtalview 21 was used to determine the orientation of objects in the illustrations. Basis for the Model. The model is based on a consideration of packing arrangements in a series of previously reported crystal structures. Our working hypothesis is that interactions that occur in the various crystals are good candidates for those that occur in fiber formation. We know this to be true in the case of the principal end-to-end association between fibrin units 5. These end-to-end interactions are observed in all four double-d structures Fig. Table 1 Unit cells and space groups for four different structures of double-d Structure. Page 1

2 Chapter 2 : Coagulation - Wikipedia A model of fibrin formation based on crystal structures of fibrinogen and fibrin fragments complexed with synthetic peptides Zhe Yang, Igor Mochalkin and Russell F. Doolittle PNAS December 19, Paradise Lost Ib 1 Confusion worse confounded Paradise Lost Ib 1 When the manuscript for the first part of this book was proposed, it was This book is simply wonderful. Although I already knew much of the science, it was explained in a way that helped me to put this work into the context of the larger narrative of molecular evolution. From lamprey to man, Dr. Doolittle writes about the evolution of the blood clotting system with a historical perspective. This was a very enjoyable read. His research interests have centred on the structure and evolution of proteins in general and, more particularly, on blood clotting proteins, an interest he developed while a graduate student at Harvard. He is a member of the U. Laying the Groundwork Chapter 1: Blood Clotting in Humans Chapter 2: Clotting Inhibitors and Fibrinolysis Chapter 3: Localizing Clots Chapter 4: Proteins and Domains Chapter 5: Fibrinogen and Fibrin Chapter 6: Animals, Their Proteins and Phylogenetics Chapter 7: Gene Duplications Chapter 8: Searching Through Genomes Chapter 9: Genomes of Fish with Jaws Chapter Lamprey Clotting Genes Chapter Contact Factors and Other Embellishments Chapter Protochordate Genomes Chapter Page 2

3 Chapter 3 : The Evolution of Vertebrate Blood Clotting: Russell F Doolittle NHBS Book Shop A blood clot is a meshwork of fibrin fibers built up by the systematic assembly of fibrinogen molecules proteolyzed by thrombin. Here, we describe a model of how the assembly process occurs. Publications Research Our group is mainly concerned with the structure and evolution of proteins. In this regard, we have two quite distinct projects under way. One is very general and has to do with reconstructing the evolutionary histories of a wide variety of proteins. This is a computer-based study that draws upon published sequence data for its raw material. We employ a number of sequence searching and alignment programs, many of which we have written ourselves. Among the questions we are trying to answer are: When did prokaryotic organisms diverge from eukaryotes? How many rudimentary families of proteins are there? How are "new proteins" invented? All of these questions can be answered, given enough sequences to compare Our second major research interest is laboratory-based and deals with the invention and evolution of vertebrate blood plasma proteins, and expecially the clotting proteins. In the past, we have cloned and sequenced a number of these proteins from the most primitive of vertebrates, the lamprey. Comparison with the corresponding mammalian proteins has afforded us some important clues as to how these proteins function. We also succeeded in identifying equivalent gene products in even more distantly related creatures, including protochordates and invertebrates. Of all of these, we have focused most on the fibrinogen molecule. Our goal here is to understand how fibrin formation clotting occurs and was invented. We have also solved the structure of various fibrinogen fragments with bound ligands involved in fibrin formation, and also factor XIII-generated crosslinked versions. The crystallography project should shed light not only on how clotting works but also on where some of the components came from. Fibrinogen is a multidomained mosaic protein, a key part of which is found in numerous other animal proteins, including various cytotactins e. Crystal structure of fragment D from human fibrinogen Publications Doolittle RF Coagulation in vertebrates with a focus on evolution and inflammation. Doolittle RF The protochordate Ciona intestinalis has a protein like full-length vertebrate fibrinogen. Correlating structure and function during the evolution of fibrinogen-related domains. TRFII and the evolution of the bilateria. Genes and Development Doolittle RF Clotting of mammalian fibrinogens by papain: Doolittle RF Bioinformatic characterization of Genes and proteins involved in blood clotting in lampreys. Doolittle RF Some important milestones in the field of blood clotting. Doolittle RF The conversion of fibrinogen to fibrin: Matrix Biology epub ahead of print Biography Russell Doolittle received his Ph. Professor Doolittle is a co-recipient of the Paul Ehrlich Prize. Page 3

4 Chapter 4 : Structural Studies on Fibrinogen and Fibrin - Russell Doolittle Russell F. Doolittle* Fibrin clots are well known to contain the seeds of their in the presence of t-pa and plasminogen and following clot formation and lysis. References Abstract Fibrinogen is a soluble plasma protein that is converted to polymeric fibrin in response to damage to the vascular system. The clotting process is initiated when platelets aggregate at the wound site. Their disruption releases biologically active amines and a proteolytic cascade follows which culminates in the conversion of fibrinogen to fibrin. The fibrin polymer forms the matrix of the tangle of cellular and molecular substances called the blood clot. Of necessity, fibrin clots need to be dismantled when they are no longer needed, an operation largely accomplished by the proteolytic enzyme plasmin. Various regulatory phenomena are involved in maintaining the balance between intravascular fluidity and clots that prevent blood loss. A variety of hereditary conditions, including mutant fibrinogens, can predispose individuals to either thrombosis or bleeding. The underlying fabric of blood clots is a protein polymer called fibrin. Fibrin clots are formed in response to injuries to any part of the vascular system. The conversion of soluble fibrinogen molecules to insoluble fibrin depends on thrombin generated from prothrombin. Schematic structure of fibrinogen showing its dimeric nature with three different polypeptide chains proceeding away from a central dyad. Reproduced from Yang et al.. Reprinted with permission from American Chemical Society. Ribbon representation of parts of three fibrinogen molecules showing how the A and B knobs from the central region of one molecule bottom bind to the terminal regions of two other fibrinogens to form fibrin. Adapted from Yang et al.. Ferry JD The mechanism of polymerization of fibrin. Molecular Biology and Medicine 4: Fu Y and Grieninger G Fib Journal of Biophysics, Biochemistry and Cytology 5: Hanss M and Biot F A data base for human fibrinogen variants. Annals of the New York Academy of Science Laudano AP and Doolittle RF Studies on synthetic peptides that bind to fibrinogen and prevent fibrin polymerization. Structural requirements, number of binding sites, and species differences. Thrombosis and Haemostasis Journal of Thrombosis and Haemostasis 1: Genes and Development 9: Weisel JW, Veklich Y and Gorkun O The sequence of cleavage of fibrinopeptides from fibrinogen is important for protofibril formation and enhancement of lateral aggregation. Journal of Molecular Biology New England Journal of Medicine Yang Z, Mochalkin I and Doolittle RF A model of fibrin formation based on crystal structures of fibrinogen and fibrin fragments complexed with synthetic peptides. Page 4

5 Chapter 5 : Fibrinogen and Fibrin Fibrinogen is a soluble plasma protein that is converted to polymeric fibrin in response to damage to the vascular system. The clotting process is initiated when platelets aggregate at the wound site. Plasmin [ edit ] Plasmin is generated by proteolytic cleavage of plasminogen, a plasma protein synthesized in the liver. This cleavage is catalyzed by tissue plasminogen activator t-pa, which is synthesized and secreted by endothelium. Plasmin proteolytically cleaves fibrin into fibrin degradation products that inhibit excessive fibrin formation. Prostacyclin [ edit ] Prostacyclin PGI2 is released by endothelium and activates platelet Gs protein-linked receptors. This, in turn, activates adenylyl cyclase, which synthesizes camp. Fibrinolysis Eventually, blood clots are reorganised and resorbed by a process termed fibrinolysis. The main enzyme responsible for this process plasmin is regulated by various activators and inhibitors. Coagulation can physically trap invading microbes in blood clots. Also, some products of the coagulation system can contribute to the innate immune system by their ability to increase vascular permeability and act as chemotactic agents for phagocytic cells. In addition, some of the products of the coagulation system are directly antimicrobial. For example, beta-lysine, an amino acid produced by platelets during coagulation, can cause lysis of many Gram-positive bacteria by acting as a cationic detergent. In addition, pathogenic bacteria may secrete agents that alter the coagulation system, e. Assessment[ edit ] Numerous tests are used to assess the function of the coagulation system: The contact activation intrinsic pathway is initiated by activation of the "contact factors" of plasma, and can be measured by the activated partial thromboplastin time aptt test. The tissue factor extrinsic pathway is initiated by release of tissue factor a specific cellular lipoprotein, and can be measured by the prothrombin time PT test. PT results are often reported as ratio INR value to monitor dosing of oral anticoagulants such as warfarin. The quantitative and qualitative screening of fibrinogen is measured by the thrombin clotting time TCT. Measurement of the exact amount of fibrinogen present in the blood is generally done using the Clauss method for fibrinogen testing. Many analysers are capable of measuring a "derived fibrinogen" level from the graph of the Prothrombin time clot. If a coagulation factor is part of the contact activation or tissue factor pathway, a deficiency of that factor will affect only one of the tests: If an abnormal PT or aptt is present, additional testing will occur to determine which if any factor is present as aberrant concentrations. Deficiencies of fibrinogen quantitative or qualitative will affect all screening tests. Role in disease[ edit ] Coagulation defects may cause hemorrhage or thrombosis, and occasionally both, depending on the nature of the defect. This protein receptor complex is found on the surface of platelets, and in conjunction with GPV allows for platelets to adhere to the site of injury. Mutations in the genes associated with the glycoprotein Ib-IX-V complex are characteristic of Bernard-Soulier syndrome Platelet conditions may be congenital or acquired. Most are rare conditions. Most inborn platelet pathologies predispose to hemorrhage. Von Willebrand disease is due to deficiency or abnormal function of von Willebrand factor, and leads to a similar bleeding pattern; its milder forms are relatively common. Decreased platelet numbers may be due to various causes, including insufficient production e. Most consumptive conditions lead to platelet activation, and some are associated with thrombosis. Disease and clinical significance of thrombosis[ edit ] The best-known coagulation factor disorders are the hemophilias. Hemophilia A and B are X-linked recessive disorders, whereas Hemophilia C is a much more rare autosomal recessive disorder most commonly seen in Ashkenazi Jews. Von Willebrand disease which behaves more like a platelet disorder except in severe cases, is the most common hereditary bleeding disorder and is characterized as being inherited autosomal recessive or dominant. This binding helps mediate the activation of platelets and formation of primary hemostasis. Bernard-Soulier syndrome is a defect or deficiency in GPIb. GPIb, the receptor for vwf, can be defective and lead to lack of primary clot formation primary hemostasis and increased bleeding tendency. This is an autosomal recessive inherited disorder. Thrombasthenia of Glanzmann and Naegeli Glanzmann thrombasthenia is extremely rare. In liver failure acute and chronic forms, there is insufficient production of Page 5

6 coagulation factors by the liver; this may increase bleeding risk. Deficiency of Vitamin K may also contribute to bleeding disorders because clotting factor maturation depends on Vitamin K. Thrombosis is the pathological development of blood clots. These clots may break free and become mobile, forming an embolus or grow to such a size that occludes the vessel in which it developed. An embolism is said to occur when the thrombus blood clot becomes a mobile embolus and migrates to another part of the body, interfering with blood circulation and hence impairing organ function downstream of the occlusion. This causes ischemia and often leads to ischemic necrosis of tissue. Most cases of venous thrombosis are due to acquired states older age, surgery, cancer, immobility or inherited thrombophilias e. Mutations in factor XII have been associated with an asymptomatic prolongation in the clotting time and possibly a tendency toward thrombophlebitis. Other mutations have been linked with a rare form of hereditary angioedema type III essentialism. Procoagulants[ edit ] The use of adsorbent chemicals, such as zeolites, and other hemostatic agents are also used for sealing severe injuries quickly such as in traumatic bleeding secondary to gunshot wounds. Thrombin and fibrin glue are used surgically to treat bleeding and to thrombose aneurysms. Desmopressin is used to improve platelet function by activating arginine vasopressin receptor 1A. Coagulation factor concentrates are used to treat hemophilia, to reverse the effects of anticoagulants, and to treat bleeding in patients with impaired coagulation factor synthesis or increased consumption. Prothrombin complex concentrate, cryoprecipitate and fresh frozen plasma are commonly used coagulation factor products. Recombinant activated human factor VII is increasingly popular in the treatment of major bleeding. Tranexamic acid and aminocaproic acid inhibit fibrinolysis, and lead to a de facto reduced bleeding rate. Before its withdrawal, aprotinin was used in some forms of major surgery to decrease bleeding risk and need for blood products. Rivaroxaban drug bound to the coagulation factor Xa. The drug prevents this protein from activating the coagulation pathway by inhibiting its enzymatic activity. Antiplatelet drug and Anticoagulant Anticoagulants and anti-platelet agents are amongst the most commonly used medications. Of the anticoagulants, warfarin and related coumarins and heparin are the most commonly used. A newer class of drugs, the direct thrombin inhibitors, is under development; some members are already in clinical use such as lepirudin. Also under development are other small molecular compounds that interfere directly with the enzymatic action of particular coagulation factors e. Chapter 6 : The Evolution of Vertebrate Blood Clotting - Russell F Doolittle - Bok () Bokus Russell Doolittle is an Emeritus Professor at the University of California, San Diego, USA, where he has spent most of his long career. He received his PhD in Biochemistry from Harvard University in Chapter 7 : - NLM Catalog Result Yang Z, Mochalkin I and Doolittle RF () A model of fibrin formation based on crystal structures of fibrinogen and fibrin fragments complexed with synthetic peptides. Proceedings of the National Academy of Sciences of the USA Page 6