Measurement of GPV Released by Activated Platelets Using a Sensitive Immunocapture ELISA – Its Use to Follow Platelet Storage in Transfusion

Summary

Thrombin, the most potent platelet agonist, plays a central role in haemostasis and in the occurrence of thrombotic events. This agonist activates platelets by cleaving the PAR G-protein coupled receptors and by binding to glycoprotein (GP) Ib and also cleaves GPV at the platelet surface to liberate the soluble 69 kDa fragment GPVf1. Monoclonal antibodies (MoAbs) to GPV were developed as tools to study the mechanism of platelet GPV cleavage and measure release of GPV in pathological situations. Specificity of the MoAbs for GPV was confirmed by flow cytometry and immunoprecipitation of proteins from human platelets and Dami megakaryocytic cells. A sensitive immunocapture sandwich ELISA for soluble GPV was developed using two MoAbs recognizing different epitopes of GPV and purified platelet or recombinant GPV as reference protein. This ELISA was employed to determine the mean plasma concentration of GPV in 100 normal individuals (17.3 ng/ml), to demonstrate the dose-dependent release of GPVf1 from washed platelets stimulated with thrombin and to follow the progressive release of GPVf1 during storage of therapeutic platelet concentrates. The present report describes a sensitive GPV ELISA of direct application to survey the processing and storage of platelet concentrates for transfusion and of potential value to monitor platelet activation in thrombotic states.

 

• References

• 1 Ware JA, Coller BS. Platelet morphology, biochemistry, and function. In: Williams Hematology. Fifth edition. Beutler E, Lichtman MA, Coller BS, Kipps TJ. eds. New york, NY: Mc Graw-Hill, Inc.; 1995: 1161-200.
• 2 Chesebro JH, Zoldhelyi P, Badimon L, Fuster V. Role of thrombin in arterial thrombosis. Implication for therapy. Thromb Haemost 1991; 66: 1-5.
• 3 Vu TH, Hung DT, Wheaton I V, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991; 64: 1057-68.
• 4 Rasmussen UB, Vouret-Craviari V, Jallat S, Schlesinger Y, Pages G, Pavirani A, Lecocq J-P, Pouyégur J, Van Obberghen-Schilling E. cDNA cloning and expression of a hamster α-thrombin receptor coupled to Ca²⁺ mobilization. FEBS Lett 1991; 288: 123-8.
• 5 Brass LF, Vassallo Jr RR, Belmonte E, Ahuja M, Cichowski K, Hoxie JA. Structure and function of the human platelet thrombin receptor: studies using monoclonal antibodies against a defined epitope within the receptor N-terminus. J Biol Chem 1992; 267: 13795-8.
• 6 Berndt MC, Phillips DR. Purification and preliminary physicochemical characterization of human platelet membrane glycoprotein V. J Biol Chem 1981; 256: 59-65.
• 7 Modderman PW, Admiraal LG, Sonnenberg A, von dem Borne AEGKr. Glycoproteins V and Ib-IX form a non covalent complex in the platelet membrane. J Biol Chem 1992; 267: 364-9.
• 8 Hickey MJ, Hagen FS, Yagi M, Roth GJ. Human platelet glycoprotein V: characterization of the polypeptide and the related Ib-V-IX receptor system of adhesive, leucine-rich glycoproteins. Proc Natl Acad Sci USA 1993; 90: 8327-31.
• 9 Lanza F, Morales M, de la Salle C, Cazenave J-P, Clemetson KJ, Shimomura T, Phillips DR. Cloning and characterization of the gene encoding the human platelet glycoprotein V: a member of the leucine-rich glycoprotein family cleaved during thrombin-induced platelet activation. J Biol Chem 1993; 270: 20801-7.
• 10 Lopez JA. The platelet glycoprotein Ib-IX complex. Blood Coag Fibrinol 1994; 5: 97-119.
• 11 Connolly AJ, Ishihara H, Kahn ML, Farese RV, Coughlin SR. Role of the thrombin receptor in development and evidence for a second receptor. Nature 1996; 381: 516-9.
• 12 Li CQ, Dong J-F, Lanza F, Sanan DA, Sae-Tung G, Lopez JA. Expression of platelet glycoprotein (GP) V in heterologous cells and evidence for its association with GPIbα in forming a GP Ib-IX-V complex on the cell surface. J Biol Chem 1995; 270: 16302-7.
• 13 Dong J-F, Sae-Tung G, Lopez JA. Role of glycoprotein V in the formation of the platelet high-affinity thrombin-binding site. Blood 1997; 89: 4355-63.
• 14 Wicki AN, Clemetson KJ. Structure and function of platelet membrane glycoprotein Ib and V. Effect of leucocyte elastase and other proteases on platelet response to von Willebrand factor and thrombin. Eur J Biochem 1985; 153: 1-11.
• 15 Kobe B, Deisenhofer J. The leucine-rich repeat: a versatile binding motif. Trends Biochem Sci 1994; 19: 415-21.
• 16 Beer JH, Büchi L, Steiner B. Glycocalicin: a new assay-the normal plasma level and its potential usefulness in selected diseases. Blood 1994; 83: 691-702.
• 17 Lanza F, Beretz A, Stierlé A, Hanau D, Kubina M, Cazenave J-P. Adrenaline activates human platelets but is not an aggregating agent. Amer J Physiol 1988; 225: H1276-88.
• 18 Shimomura T, Fujimura K, Maehama S, Takemoto M, Oda K, Fujimoto T, Oyama R, Suzuki M, Ichihara-Tanaka K, Titani K, Kuramoto A. A rapid purification and characterization of human platelet glycoprotein V: the amino acid sequence contains leucine-rich repetitive modules as in glycoprotein Ib. Blood 1990; 75: 2349-56.
• 19 Hildreth JEK, Derr D, Azorsa DO. Characterization of a novel self-associating Mr 40,000 platelet protein. Blood 1991; 77: 121-32.
• 20 Hildreth JEK, Gotch FM, Hildreth PDK, McMichael AJ. A human lymphocyte-associated antigen involved in cell-mediated lympholysis. Eur J Immunol 1983; 13: 202-8.
• 21 Ravanat C, Morales M, Azorsa DO, Moog S, Schuhler S, Grunert P, Loew D, Van Dorsselaer A, Cazenave J-P, Lanza F. Gene cloning of rat and mouse platelet glycoprotein V (GPV). Identification of megakaryocyte-specific promoters and demonstration of functional thrombin cleavage. Blood 1997; 89: 3253-62.
• 22 Takafuta T, Fujimura K, Kawano H, Noda M, Fujimoto T, Oda K, Shimomura T, Kuramoto A. Expression of platelet membrane glycoprotein V in human megakaryocytes and megakaryocytic cell lines: a study using a novel monoclonal antibody against GPV. Thromb Haemost 1994; 75: 762-9.
• 23 Fox JEB. Shedding of adhesion receptors from the surface of activated platelets. Blood Coag Fibrinol 1994; 5: 291-304.
• 24 Michelson AD, Benoit SE, Furman I M, Barnard MR, Nurden P, Nurden AT. The platelet surface expression of glycoprotein V is regulated by two independent mechanisms: proteolysis and a reversible cytoskeletal-mediated redistribution to the surface-connected canalicular system. Blood 1996; 87: 1396-408.
• 25 Berger G, Maé J-M, Cramer EM. Alpha-granule membrane mirrors the plasma membrane and contains the glycoproteins Ib, IX, and V. Blood 1996; 87: 1385-95.
• 26 Rinder HM, Snyder E. Activation of platelet concentrates during preparation and storage. Blood Cells 1992; 18: 445-56.
• 27 Michelson AD, Benoit SE, Barnard MR, Mac Gregor H, Valeri CR. Blood bank storage of platelet concentrates results in loss of platelet surface gpIb (CD42b) and gpV (CD42d) but not gpIX (CD42a). In: Leucocyte typing V. White cell differentiation antigens, vol 1. Schlossman SF, Boumsell L, Gilks W, Harlam JM, Kishimoto T, Morimoto C, Ritz J, Shaw S, Silverstein R, Springer T, Tedder TF, Todd RF. eds Oxford, UK: Oxford University Press; 1995: 1331-2.
• 28 Bode AP, Miller DT. Generation of fibrinopeptide A in stored platelet concentrates. Vox Sang 1986; 51: 192-6.
• 29 Ravanat C, Freund M, Azorsa DO, Schuhler S, Moog S, Grunert P, Cazenave J-P, Lanza F. GPV is a marker of in vivo thrombin-induced platelet activation in a rat thrombosis model. Thromb Haemost 1997; 77: 632
• 30 Files JC, Malpass TW, Yee EK, Ritchie JL, Harker LA. Studies of human platelet α-granule release in vivo. Blood 1981; 58: 607-18.
• 31 Ramachandran R, Klufas AS, Molino M, Ahuja M, Hoxie JA, Brass LF. Release of the thrombin receptor (PAR-1) N-terminus from the surface of human platelets activated by thrombin. Thromb Haemost 1997; 78: 1119-24.