Platelet Membrane Glycoproteins and the Interaction between Bovine Factor VIII Related Protein and Human Platelets

Summary

The “GP I fraction” seen on polyacrylamide gel electrophoresis of reduced samples of whole normal platelets contains three glycopolypeptides corresponding to the integral membrane protein GP I (GP Ia), the easily solubilized membrane protein GPS (GP Ib, glycocalicin) and a granule-located glycoprotein.

Freezing and thawing of platelets in tris-buffered saline leads to a lysis of platelets and platelet granules with the result that both GPS and the granule glycoprotein is found in the soluble fraction. The two glycoproteins can be separated by SDS polyacrylamide gel electrophoresis both in reduced and unreduced samples when urea and EDTA is incorporated into the gels. This permitted electrophoretic studies of GPS using the granule glycoprotein as a control and marker substance.

A working hypothesis stating that the presence of GPS on the platelet surface is a prerequisite to the agglutination of human platelets with bovine factor VIII related protein, has been investigated. The hypothesis was supported by the observation that storage of platelets in tris-buffered saline at 4° C led to the elution of GPS and loss of agglutination, as was also the case when platelets were frozen and thawed in tris-buffered saline, or preincubated in 3 M KCl and resuspended in either tris-buffered saline or the EDTA-containing medium A. GPS was not, or only slightly, solubilized when platelets were frozen and thawed in the EDTA-containing medium and the resulting platelet ghosts still agglutinated.

Platelets from 1 patient of the Bernard-Soulier type did not agglutinate with the bovine factor VIII-related protein, nor did the platelets contain GPS. An improved technique for the isolation of such platelets is described.

• References

• 1 Bithell T. C, Parekh S. J, Strong R. R. 1972; Platelet function studies in the Bernard-Soulier syndrome. Annals of the New York Academy of Sciences 201: 145.
• 2 Bøyum A. 1974; Separation of blood leucocytes, granulocytes and lymphocytes. Tissue Antigens 4: 269.
• 3 Caen J, Levy-Toledano S, Sultan Y, Bernard J. 1973; La dystrophie thrombocytaire hémorragipare (interaction des plaquettes et du facteur Willebrand). Nouvelle revue française d’hématologie 13: 595.
• 4 Caen J. P, Nurden A. T, Jeanneau C, Michel H, Tobelem G, Levy-Toledano S, Sultan Y, Valensi F, Bernard J. 1976; Bernard-Soulier syndrome: A new platelet glycoprotein abnormality. Its relationship with platelet adhesion to subendothelium and with factor VIII von Willebrand protein. Journal of Laboratory and Clinical Medicine 87: 586.
• 5 Clemetson K. J, Pfueller S. L, Lüscher E. F, Jenkins C. S. P. 1977; Isolation of the membrane glycoproteins of human blood platelets by lectin affinity chromatography. Biochimica et biophysica acta 464: 493.
• 6 Evensen S. A, Solum N. O, Grottum K. A, Hovig T. 1974; Familial bleeding disorder with a moderate thrombocytopenia and giant blood platelets. Scandinavian Journal of Haematology 13: 203.
• 7 French P, Holme R. 1974; A method for blood plate let homogenization using the Aminco-French pressure cell. Thrombosis et Diathesis Haemorrhagica 32: 432.
• 8 George J. N. 1976; Platelet membrane glycoproteins: Alteration during storage of human platelet concentrates. Thrombosis Research 8: 719.
• 9 Grøttum K. A, Solum N. O. 1969; Congenital thrombocytopenia with giant platelets: A defect in the platelet membrane. British Journal of Haematology 16: 277.
• 10 Hagen I. 1975; Effects of thrombin on washed, human platelets: changes in the subcellular fractions. Biochimica et biophysica acta 392: 242.
• 11 Hagen L, Olsen T, Solum N. O. 1976; Studies on subcellular fractions of human platelets by the lactoperoxidase-iodination technique. Biochimica et biophysica acta 455: 214.
• 12 Howard M. A, Hutton R. A, Hardisty R. M. 1973; Hereditary giant platelet syndrome: a disorder of a new aspect of platelet function. British Medical Journal 4: 586.
• 13 Jenkins C. S. P, Phillips D. R, Clemetson K. J, Meyer D, Larrieu M.-J, Lüscher E. F. 1976; Platelet membrane glycoproteins implicated in ristocetin-induced aggregation. Studies of the proteins on platelets from patients with Bernard-Soulier syndrome and von Willebrand’s disease. Journal of Clinical Investigation 57: 112.
• 14 Nachman R. L, Jaffe E. A, Weksler B. B. 1977; Immunoinhibition of ristocetin-induced platelet aggregation. Journal of Clinical Investigation 59: 143.
• 15 Newman J, Johnson A. J, Karpatkin M. H, Puszkin S. 1971; Methods for the production of clinically effective intermediate- and high-purity factor VIII concentrates. British Journal of Haematology 21: 1.
• 16 Nurden A. T. 1976. Glycoprotein components of the human platelet surface. Symposium on the structure and functions of platelet membranes. Barcelona: 1976. Abstract.
• 17 Nurden A. T, Caen J. P. 1975; Specific roles for platelet surface glycoproteins in platelet function. Nature 255: 720.
• 18 Nurden A. T, Caen J. P. 1976; Role of surface glycoproteins in human platelet function. Thrombosis and Haemostasis 35: 139.
• 19 Okumura T, Jamieson G. A. 1976; a Platelet glycocalicin: A single receptor for platelet aggregation induced by thrombin or ristocetin. Thrombosis Research 8: 701.
• 20 Okumura T, Jamieson G. A. 1976; b Platelet glycocalicin. I. Orientation of glycoproteins of the human platelet surface. Journal of Biological Chemistry 251: 5944.
• 21 Okumura T, Lombart C, Jamieson G. A. 1976; Platelet glycocalicin. II. Purification and characterization. Journal of Biological Chemistry 251: 5950.
• 22 Phillips D. 1972; Effect of trypsin on the exposed polypeptides and glycoproteins in the human platelet membrane. Biochemistry 11: 4582.
• 23 Solum N. O. 1968; Aggregation of human platelets by bovine platelet fibrinogen. Scandinavian Journal of Haematology 5: 474.
• 24 Solum N. O, Hagen I, Peterka M. 1977; Human platelet glycoproteins. Further evidence that the “GP I band” from whole platelets contains three different polypeptides one of which may be involved in the interaction between platelets and factor VIII. Thrombosis Research 10: 71.
• 25 Solum N. O, Peterka M. 1977. Interaction of platelets with factor VIII related protein. In: Mills D. C. B, Pareti F. (eds.) Platelets and thrombosis. Academic Press; London: in press.
• 26 Stormorken H, Lund-Riise A, Rorvik T. O. 1965; Platelet adhesiveness to glass beads. Methodological investigations using automatic platelet counting. Scandinavian Journal of Clinical and Laboratory Investigations 17 (Suppl. 84) 183.
• 27 Van Mourik J. A, Mochtar I. A. 1970; Purification of human antihemophilic factor (factor VIII) by gel chromatography. Biochimica et biophysica acta 221: 677.
• 28 Walsh P. N, Mills D. C. B, Pareti F. I, Stewart G. J, MacFarlane D. E, Johnson M. M, Egan J. J. 1975; Hereditary giant platelet syndrome. Absence of collagen-induced coagulant activity and deficiency of factor–XI binding to platelets. British Journal of Haematology 29: 639.