Lyophilized Phospholipid Vesicles

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For the enzymatic reactions of the coagulation cascade to occur at physiologically relevant rates, the protein components must be localized to an appropriate membrane surface comprised of negatively-charged phospholipids (1). Activated platelets (2,3)  and to a lesser extent mononuclear cells (4) provide this membrane surface in vivo, and synthetic phospholipid vesicles comprised of 75% phosphatidylcholine (PC) and 25% phosphatidylserine (PS) (PCPS) can support these reactions in vitro in various assays (5-8). PCPS vesicles can also support membrane-dependent anticoagulant reactions in purified (9) and plasma-based hemostasis assays (10).

Unilamellar PCPS vesicles consisting of 75% PC and 25% PS are prepared from L-α-phosphatidylcholine (chicken egg) and L-α-phosphatidylserine (porcine brain) (11) and quantified by phosphate assay (12).  They are provided as a lyophilizate and should be stored at 4°C prior to and following reconstitution.

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  1. Mann K, Nesheim M, Church W, Haley P, Krishnaswamy S. Surface dependent reactions of the vitamin K dependent enzyme complexes. Blood. 1990;76:1–16.
  2. Chandler A. An overview of thrombosis and platelet involvement in the development of the human atherosclerotic plaque. In: Glagov S, Newman W, Schaffer S, eds. Pathobiology of the Human Atherosclerotic Plaque. New York, NY: Springer-Verlag; 1990:359–378.
  3. White J. Anatomy and structural organization of the platelet. In: Coman R, Hirsh J, Marder V, Salzman E, eds. Hemostasis and Thrombosis: Basic Principles and Clinical Practice Philadelphia, Pa: JB Lippincott Co; 1994:397–413.
  4. Tracy P, Allen D, Worfolk L, Robinson-Lawler R. Monocyte/macrophage regulation of coagulation events. Hemostasis. 1996;26(suppl 1):6–11.
  5. Nesheim M, Taswell J, Mann K. The contribution of bovine factor V and factor Va to the activity of prothrombinase. J Biol Chem. 1979;254:10952–10962.
  6. Krishnaswamy K. Prothrombinase complex assembly. Contributions of protein-protein and protein-membrane interactions toward complex formation. J Biol Chem. 1990;265(7):3708-3718.
  7. Duffy EJ, Parker ET, Mutucumarana VP, Johnson AE, Lollar P. Binding of factor VIIIa and factor VIII to factor IXa on phospholipid vesicles. J Biol Chem. 1992;267(24):17006-17011.
  8. Sorensen BB, Rao LV. Interaction of activated factor VII and active site-inhibited activated factor VII with tissue factor. Blood Coagul Fibrinolysis. 1998; 9 Suppl 1:S67-71.
  9. Kalafatis M, Rand MD, Mann KG. The mechanism of inactivation of human factor V and human factor Va by activated protein C. J Biol Chem. 1994;269(50):31869-31880.
  10. Bajzar L, Nesheim M. The effect of activated protein C on fibrinolysis in cell-free plasma can be attributed specifically to attenuation of prothrombin activation. J Biol Chem. 1993; 268(12):8608-8616.
  11. Barenholz Y, Gibbs D, Litmann B, Goll J, Thompson E, Carlson F. A simple method for the preparation of homogeneous phospholipid vesicles. Biochemistry. 1977;16:2806–2810.
  12. Gomori G. A modification of the colorimetric phosphorous determination for use with a photoelectric colorimeter. J Lab Clin Med. 1942;27:955–960.
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