[...]... of these models of hemostasis, both laboratory and mathematical, have proven to be useful, and show that for all the interactions and complexity of different systems combined with flow and cellular interaction, we do have a considerable understanding of the processes of hemostasis Suggested Reading Bloom, A L., Forbes, C D., Thomas, D P., and Tuddenham, E G D (1994) Haemostasis and Thrombosis, 3rd ed... copies the DNA template, generating a double-stranded DNA molecule For the amplification of RNA sequences, an additional step is required to convert the single-stranded RNA into a single-stranded cDNA, which is then suitable for amplification by PCR From: Methods in Molecular Medicine, Vol 31: Hemostasis and Thrombosis Protocols Edited by: D J Perry and K J Pasi © Humana Press Inc., Totowa, NJ 31 32... Haemostasis 74, 204–209 3 White, J G (1994) Anatomy and structural organisation of the platelet, in Hemostasis and Thrombosis: Basic Principles and Clinical Practice (Coleman, R W., et al., eds.), 3rd ed., J B Lippencott Co., Philadelphia, PA 4 Levy-Toledano, S., Gallet, C., Nadel, F., Bryckaert, M., Macloug, J., and Rosa, J.-P (1997) Phosphorylation and dephosporylation mechanisms in platelet function:... amounts, such that one primer is exhausted and linear amplification then takes place or alternatively the PCR product forms the basis of a second PCR in which only one amplification primer is present From: Methods in Molecular Medicine, Vol 31: Hemostasis and Thrombosis Protocols Edited by: D J Perry and K J Pasi © Humana Press Inc., Totowa, NJ 39 40 Perry Two different protocols are covered in this chapter... Protein C caused by the Arg506 to Gln mutation in factor V as a pathogenic risk factor for venous thrombosis Thromb Haemostasis 74, 139–148 12 Collen, D and Lijen, H R (1991) Basic and clinical aspects of fibrinolysis and thrombolysis Blood 78, 3114–3124 Isolation of DNA and RNA 25 2 Isolation of DNA and RNA David J Perry 1 Introduction Blood samples for most coagulation tests are collected into 3.8%... for several years and generates high-quality RNA suitable for a wide variety of uses A number of commercial kits are now available for rapid RNA isolation, e.g, RNeasy™ kit (Qiagen Ltd, UK) Although often more expensive than “in-house” methods, these kits are capable of isolating highFrom: Methods in Molecular Medicine, Vol 31: Hemostasis and Thrombosis Protocols Edited by: D J Perry and K J Pasi © Humana... the pellet in 400 µL of TE, pH 8.0 10 Check the yield and quality of the RNA by electrophoresing 5–10 µL in a 1.5% agarose gel in 1X Trios-Borate-EDTA (TBE) The 28S and 18S (and sometimes the 5S) ribosomal bands should be clearly visible For long-term storage, the RNA should be precipitated by adding 21⁄2 vol of ethanol and 1/10 vol of 3 M NaOAc and then placed at –70°C 4 Notes 1 For packed cells, i.e.,... Tris-containing solutions cannot be treated in the manner and should be made using DEPC-treated water and then autoclaved 5 To make DEPC-treated water add 1 mL of DEPC to 1 L of distilled water Incubate at 37°C overnight and then autoclave Store at room temperature 6 DEPC is a potential carcinogen and must be handled with care 7 Cells may be lysed in Solution D and then stored at –70°C until convenient We have... that factor VII and tissue factor, released from damaged vessels, directly activate factor X, and coagulation factor lying below factor X in the final common pathway The division into extrinsic and intrinsic systems and the ability to test these two systems in the laboratory (the prothrombin time and activated partial thromboplastin time, respectively) have been valuable in understanding clinical bleeding... process The recent improved understanding has in part been brought about by improved knowledge of the individual components of the different elements of the overall process of hemostasis and cellular repair Although increasing by appreciated to be complex, attempts have been made to model and reproduce this system in vitro to validate research findings and increase understanding of the interactions For . h0" alt="" Hemostasis 3 3 From: Methods in Molecular Medicine, Vol. 31: Hemostasis and Thrombosis Protocols Edited by: D. J. Perry and K. J. Pasi © Humana Press Inc., Totowa, NJ 1 Hemostasis Components. understanding of the interactions. For all its complexity, many of these models of hemostasis, both laboratory and mathematical, have proven to be useful, and show that for all the interactions and complexity. sites and a significantly increased activity through increased binding of fibrin and plasminogen. t-PA has a short half-life (5 min) and is rapidly cleared from the circulation. sct-PA and tct-PA