Lyophilized Phospholipid Vesicles

Overview

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.

References

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.