Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

Yatin Patel; Salman Rahman; Ashia Siddiqua; Michael J. Wilkinson; Vijay V. Kakkar; Kalwant S. Authi

doi:10.1055/s-0037-1614247

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1998; 79(01): 177-185
DOI: 10.1055/s-0037-1614247

Review Article

Schattauer GmbH

Functional Characterization of PM6/13, a β₃-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

Authors

Yatin Patel^*

¹Present address: Dr. Y. Patel, Institute of Cancer Research, The Haddow Laboratories, 15 Cotswold Road, Sutton, Surrey SN2 5NG, U.K.

¹From the Platelet Section, Thrombosis Research Institute , London, U.K.
Salman Rahman^*

²Present address: Dr. S. Rahman, Coagulation Research Laboratory, Haemophilia Centre, St. Thomas’ Hospital, Lambeth Palace Road, London SE1 7EH, U.K.

¹From the Platelet Section, Thrombosis Research Institute , London, U.K.
Ashia Siddiqua

¹Present address: Dr. Y. Patel, Institute of Cancer Research, The Haddow Laboratories, 15 Cotswold Road, Sutton, Surrey SN2 5NG, U.K.

¹From the Platelet Section, Thrombosis Research Institute , London, U.K.
Michael J. Wilkinson

³Dr. J. M. Wilkinson, Royal College of Surgeons of England, Lincolns Inn Fields, London WC2A 3PN, present address: The Welcome Trust, 183 Euston Road, London NW1 2BE, U.K.

¹From the Platelet Section, Thrombosis Research Institute , London, U.K.
Vijay V. Kakkar

¹Present address: Dr. Y. Patel, Institute of Cancer Research, The Haddow Laboratories, 15 Cotswold Road, Sutton, Surrey SN2 5NG, U.K.

¹From the Platelet Section, Thrombosis Research Institute , London, U.K.
Kalwant S. Authi

¹From the Platelet Section, Thrombosis Research Institute , London, U.K.

Abstract

Summary

We report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC₅₀ values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC₅₀ = 4, 8 and 4 [H9262]g/ml respectively, without affecting [¹⁴C]5-hydroxytryptamine secretion or [³H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca²⁺ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of ¹²⁵I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of ¹²⁵I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of ¹²⁵I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

Full Text

References

References
1 Plow EF, Pierschbacher MD, Ruoslathi E, Marguerie GA, Ginsberg MH. The effect of Arg-Gly-Asp-containing peptides on fibrinogen and von Willebrand factor binding to platelets.. Proc Natl Acad Sci USA 1985; 82: 8057-61.
2 Gardner JM, Hynes RO. Interaction of fibronectin with its receptor on platelets.. Cell 1985; 42: 438-49.
3 Pytela RM, Pierschbacher MD, Rouslahti E. A 125/115 kDa cell surface receptor specific for vitronectin interacts with the Arg-Gly-Asp adhesion sequence derived from fibronectin.. Proc Natl Acad Sci USA 1985; 82: 5766-70.
4 Ruggeri ZM, De Marlo L, Gath L, Bader R, Montgomery RR. Platelets have more than one binding site for von Willebrand factor.. J Clin Invest 1983; 72: 1-12.
5 Karczewski JK, Knudsen A, Smith L, Murphy A, Rothman VL, Tuszinski GP. The interaction of thrombospondin with platelet glycoprotein IIb/IIIa.. J Biol Chem 1989; 264: 21322-6.
6 Gartner TK, Bennett JS. The tetrapeptide analogue of the cell attachment site of fibronectin inhibits platelet aggregation and fibrinogen binding to activated platelets.. J Biol Chem 1985; 260: 11891-4.
7 Ginsberg MH, Pierschbacher MD, Rouslathi E, Margerie GA, Plow EF. Inhibition of fibronectin binding to platelets by proteolytic fragments and synthetic peptides which support fibroblast adhesion.. J Biol Chem 1985; 260: 3931-6.
8 Kloczewiak M, Timmons S, Hawiger J. Localization of a site interacting with human platelet receptor on carboxy-terminal segment of human fibrinogen gamma chain.. Biochem Biophys Res Commun 1982; 107: 181-7.
9 Kloczewiak M, Timmons S, Lukas TJ, Hawiger J. Platelet receptor recognition site on human fibrinogen. Synthesis and structure-function relationship of peptides corresponding to the carboxy-terminal segment of the gamma chain.. Biochemistry 1984; 23: 1767-74.
10 Farrel DH, Thiagarajan P, Chung DW, Davie EW. Role of fibrinogen alpha and gamma chain sites in platelet aggregation.. Proc Natl Acad Sci USA 1992; 89: 10729-32.
11 Hettasch JM, Bolyard MG, Lord ST. The residues AGDV of recombinant gamma chains of human fibrinogen must be carboxy-terminal to support human platelet aggregation.. Thromb Haemost 1992; 68: 701-6.
12 Bennett JS, Hoxie J, Leitman S, Vilaire G, Cines DB. Inhibition of fibrinogen binding to stimulated human platelets by a monoclonal antibody.. Proc Natl Acad Sci 1983; 80: 2417-21.
13 Coller BS, Peerschke E, Scudder LE, Sullivan CA. A murine monoclonal antibody that competitively blocks the binding of fibrinogen to platelets produces a thrombasthenic-like state in normal platelets and binds to Glycoprotein IIb and/or IIIa.. J Clin Invest 1982; 72: 325-38.
14 Kouns WC, Newman PJ, Puckett KJ, Miller AA, Wall CD, Fox CF, Seyer JM, Jennings LK. Further characterization of the loop structure of platelet glycoprotein IIIa: partial mapping of functionally significant glycoprotein IIIa epitopes.. Blood 1991; 78: 3215-23.
15 Calvete JJ, Arias J, Alvarez MV, Lopez MM, Henchen A, Gonzalez-Rodriguez J. Further studies on the topography of human platelet Glycoprotein IIb. Localization of monoclonal antibody epitopes and the putative glycoprotein IIb and fibrinogen binding regions.. Biochem J 1991; 273: 767-75.
16 Marguerie GA, Plow EF, Eddington TS. Human platelets possess an inducible and saturable receptor specific for fibrinogen.. J Biol Chem 1979; 254: 5357-60.
17 Kinlough-Rathbone RL, Packham MA, Reimers HJ, Cazenave JP, Mustard JF. Mechanisms of platelet shape change, aggregation and release induced by collagen, thrombin or A23187.. J Lab Clin Med 1977; 90: 707-19.
18 Fox JEB, Taylor RG, Tafferel M, Boyles JK, Goll DE. Evidence that activation of platelet calpain is induced as a consequence of binding of adhesive ligand to the integrin, glycoprotein IIb-IIIa.. J Cell Biol 1993; 120: 1501-7.
19 Banga HS, Simmons ER, Brass LF, Rittenhouse SE. Activation of phospholipases A and C in human platelets exposed to epinephrine: role of Glycoprotein IIb/IIIa and dual role of epinephrine.. Proc Natl Acad Sci USA 1986; 83: 9197-201.
20 Ferrel Jr JE, Martin GS. Thrombin stimulates the activities of multiple previously unidentified protein kinases in platelets.. J Biol Chem 1989; 264: 20723-9.
21 Powling MJ, Hardisty RM. Glycoprotein IIbIIIa complex and Ca²⁺ influx into stimulated platelets.. Blood 1985; 66: 731-4.
22 Sinigaglia F, Bisio M, Balduni CJ, Bertolino G, Balduni C. Effect of GPIIb-IIIa complex ligands on calcium ion movement and cytoskeleton organization in activated platelets.. Biochem Biophys Res Commun 1988; 154: 258-64.
23 Hornby EJ, Brown JM, Wilkinson MJ, Mattock C, Authi KS. Activation of human platelets by exposure to a monoclonal antibody, PM6/248, to Glycoprotein IIb-IIIa.. Brit J Haem 1991; 79: 277-85.
24 Silverstein RL. Platelet antigens: section report.. In: Leucocyte Typing V, Schlossman SF et al. (eds). 1995: p1195-1204 Oxford University Press; Oxford, UK.:
25 Authi KS, Bokkala S, Patel Y, Kakkar VV, Monkonge F. Ca²⁺ release from platelet intracellular stores by thapsigargin and 2,5 di-(t-butyl) 1,4-benzohydroquinone. Relationship to Ca²⁺ pools and relevance to platelet activation.. Biochem J 1993; 294: 119-26.
26 Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescent properties.. J Biol Chem 1985; 260: 3440-50.
27 Cullen PJ, Patel Y, Kakkar VV, Irvine RF, Authi KS. Specific binding sites for inositol 1,3,4,5-tetrakisphosphate are located predominantly in the plasma membrane of human platelets.. Biochem J 1994; 298: 739-42.
28 Lu X, Williams JA, Deadman JJ, Salmon GS, Kakkar VV, Wlkinson JM, Baruch D, Authi KS, Rahman S. Preferential antagonism of the interactions of the integrin [H9251]IIbβ3 with immobilized glycoprotein ligands by snake venom RGD (Arg-Gly-Asp) proteins. Evidence supporting a functional role for the amino acids residues flanking the tripeptide RGD in determining the inhibitory properties of snake venom RGD proteins.. Biochem J 1994; 304: 929-36.
29 Plow EF, McEver RP, Coller BS, Woods Jr VL, Margerie GA, Ginsberg MH. Related binding mechanims for fibrinogen, fibronectin, von Willi-brand Factor and thrombospondin on thrombin stimulated human platelets.. Blood 1985; 66: 724-7.
30 Honda S, Honda Y, Bauer B, Ruan C, Kunicki TJ. The impact of the three-dimensional structure on the expression of PI^A alloantigens on human integrin β3.. Blood 1995; 86: 234-42.
31 Calvete JJ. Clues for understanding the structure and function of a prototypic human integrin. The platelet glycoprotein IIb-IIIa complex.. Thromb Haemost 1994; 72: 1-15.
32 Ryback ME, Renzulli LA. Ligand inhibition of the platelet glycoprotein IIb-IIIa complex function as a calcium channel in liposomes.. J Biol Chem 1989; 264: 14614-20.
33 Fujimoto T, Fujimura K, Kuramoto A. Functional Ca²⁺ channel produced by purified platelet glycoprotein IIb-IIIa complex incorporated into planar phospholipid bilayer.. Thromb Haemost 1991; 66: 598-603.
34 Fujimoto T, Fujimura K, Kuramoto A. Electrophysiological evidence that glycoprotein IIb/IIIa complex is involved in calcium channel activation on human platelet plasma membranes.. J Biol Chem 1991; 266: 16370-5.
35 Yamaguichi A, Yamamoto N, Kitagawa H, Tanoue K, Yamazaki H. Ca²⁺ influx mediated through the GPIIb/IIIa complex during platelet activation.. FEBS Lett 1987; 225: 228-32.
36 Brown E, Hooper L, Ho T, Gresham H. Integrin associated protein: A 50kDa plasma membrane antigen physically and functionally associated with integrins.. J Cell Biol 1990; 111: 2785-94.
37 Schwartz MA, Brown EJ, Fazel B. A 50 kDa integrin associated protein is required for integrin regulated Ca²⁺ entry in endothelial cells.. J Biol Chem 1993; 268: 19931-4.
38 Ugarova TP, Bubzyrski AZ, Shattil SJ, Ruggeri ZM, Ginsberg MH, Plow EF. Conformational changes in fibrinogen elicited by its interaction with platelet membrane GPIIb-IIIa.. J Biol Chem 1993; 268: 21080-7.
39 Plow EF, Ginsberg MH. Specific and saturable binding of plasma fibronectin to thrombin stimulated human platelets.. J Biol Chem 1981; 256: 9477-82.
40 Bowditch RD, Hallowan CE, Aota S, Obura M, Plow EF, Yamada KM, Ginsberg MH. Integrin [H9251]IIbβ3 (platelet glycoprotein GPIIb-IIIa) recognizes multiple sites in fibronectin.. J Biol Chem 1991; 266: 23323-8.
41 Tanabe J, Fujita H, Iwamatsu A, Ohkubo T. Fibronectin inhibits platelet aggregation independently of RGD sequence.. J Biol Chem 1993; 268: 27143-7.
42 Mohri H, Tanabe J, Katoh K, Okubo T. Identification of a novel binding site to the integrin [H9251]IIbβ3 located in the C-terminal heparin binding domain of human plasma fibronectin.. J Biol Chem 1996; 271: 15724-8.
43 Piotrowicz RS, Orchekowski RP, Nugent DJ, Yamada KY, Kunicki TJ. Glycoprotein Ic-IIa functions as an activation-independent fibronectin receptor on human platelets.. J Cell Biol 1988; 106: 1359-64.
44 Honda S, Felding-Haberman B, Loftus J, Annis D, Kunicki TJ. Report of the CD41,CD61 subsection of the platelet section. Localization of epitopes in integrins [H9251]IIbβ3 (CD41/CD61) and [H9251]vβ3 (CD51/CD61).. In: Leucocyte typing V. Schlossman SF et al. (eds). 1994: p1293-1298. Oxford Univ. Press; Oxford, U.K.:
45 Calvete J, Mann K, Alvarez MV, Lopez MM, Gonzalez-Rodriguez J. Proteolytic dissection of the isolated platelet fibrinogen receptor, integrin GPIIb/IIIa. Localization of GPIIb and GPIIIa sequences putatively involved in the subunit interface and in intrasubunit and intrachain contacts.. Biochem J 1992; 282: 523-32.
46 Calvete J, Mann K, Schafer W, Fernandez-Lafuente R, Guisan JM. Proteolytic degradation of the RGD-binding and non-RGD-binding conformers of human platelet integrin glycoprotein IIb/IIIa: Clues for identification of regions involved in the receptors activation.. Biochem J 1994; 298: 1-7.
47 Loftus JC, Smith JW, Ginsberg MH. Integrin-mediated cell adhesion: the extracellular face.. J Biol Chem 1994; 26: 25235-8.