Quantitative and Qualitative Analysis of Platelet GPIb and von Willebrand Factor in Liver Cirrhosis

Summary

Numerous abnormalities of plasmatic coagulation and platelet function may contribute to the bleeding in liver cirrhosis with a defective platelet-von Willebrand factor interaction being a potential mechanism. To analyze GPIb and von Willebrand factor in cirrhosis, we quantified the number of GPIb molecules on the platelet surface by flow cytometry, assessed the total (and indirectly the internal) pool of GPIb by ELISA and measured the circulating amount of glycocalicin in plasma as a measure of proteolytic activity and platelet turnover. Von Willebrand factor was characterized by ELISA, by its ristocetin-cofactor activity and by multimer analysis. Botrocetin-induced agglutination was used for functional analysis.

The data from 8 well-characterized cirrhosis patients indicate that total GPIb is insignificantly increased to 46,000 ± 5,000 molecules/P (normal: 39,500 ± 2,000 [SEM]), surface-GPIb is normal with some variability and that the glycocalicin levels are 2-3 times higher than would be expected from the platelet count (= 100 ±5 × 10⁹/1). Von Willebrand factor antigen levels and -activity were 400-500 % of normal with a 22% reduction of the high molecular weight multimers. A significant hyperagglutination response to botrocetin was observed with platelets from both patients and controls using patient plasma as a source of von Willebrand factor. In conclusion, a hyperresponsiveness rather than a defective platelet-von Willebrand factor interaction can be observed in cirrhosis which may compensate for other hemostatic problems and appears to be mediated primarily by increased levels of von Willebrand factor.

• References

• 1 Kelly DA, Tuddenham EG D. Haemostatic problems in liver disease. Gut 1986; 27: 339-349
• 2 Aoki Y, Hirai K, Tanikawa K. Mechanism of thrombocytopenia in liver cirrhosis: Kinetics of indium-111 tropolone labelled platelets. Eur J Nucl Med 1993; 20: 123-129
• 3 Stein SF, Harker LA. Kinetic and functional studies of platelets, fibrinogen, and plasminogen in patients with hepatic cirrhosis. J Lab Clin Med 1982; 99: 217-230
• 4 Thomas DP, Ream VJ, Stuart RK. Platelet aggregation in patients with Laennec’s cirrhosis of the liver. N Engl J Med 1967; 276: 1344-1348
• 5 Ballard HS, Marcus AJ. Platelet aggregation in portal cirrhosis. Arch Intern Med 1976; 136: 316-319
• 6 Laffi G, Cominelli F, Ruggiero M, Fedi S, Chiarugi VP, La Villa G, Pinzani M, Gentilini P. Altered platelet function in cirrhosis of the liver. Impairment of inositol lipid and arachidonic acid metabolism in response to agonists Hepatology 1988; 8: 1620-1626
• 7 Laffi G, Marra F, Failli P, Ruggiero M, Cecchi E, Carloni V, Giotti A, Gentilini P. Defective signal transduction in platelets from cirrhotics is associated with increased cyclic nucleotides. Gastroenterology 1993; 105: 148-156
• 8 Laffi G, Marra F, Gresele P, Romagnoli P, Palermo A, Bartolini O, Simoni A, Orlandi L, Selli ML, Nenci GG, Gentilini P. Evidence for a storage pool defect in platelets from cirrhotic patients with defective aggregation. Gastroenterology 1992; 103: 641-646
• 9 Cioni G, Cristani A, Mussini C, Grandi S, Pentore R, Zeneroli ML, Tizzanini W, Zagni G, Ventura E. Incidence and clinical significance of elevated fibrin(ogen) degradation products and/or D-dimer levels in liver cirrhosis patients. Ital J Gastroenterol 1990; 22: 70-74
• 10 Desai K, Bagget C, Bellamy MF, Mistry P, Burroughs AK, Owen JS. Inhibition of platelet aggregation by abnormal high density lipoprotein particles in plasma from patients with hepatic cirrhosis. Lancet 1989; 1: 693-695
• 11 Ordinas A, Maragall S, Castillo R, Nurden AT. A glycoprotein I defect in the platelets of three patients with severe cirrhosis of the liver. Thromb Res 1978; 13: 297-302
• 12 Sanchez-Roig MJ, Rivera J, Moraleda JM, Vicente Garcia V. Quantitative defect of glycoprotein Ib in severe cirrhotic patients. Am J Hematol 1994; 45: 10-15
• 13 Jaschonek K, Faul C. Platelets from patients with liver cirrhosis exhibit a defect in the von Willebrand factor-binding domain. Z Gastroenterol 1993; 31: 8-10
• 14 Castillo R, Maragall S, Rodés J, Clemente C, Profitós J, Ordinas A. Increased factor VIII complex and defective ristocetin-induced platelet aggregation in liver disease. Thromb Res 1977; 11: 899-906
• 15 Lu H, Menashi S, Garcia I, Cramer EM, Li H, Tenza D, De Romeuf C, Soria J, Soria C. Reversibility of thrombin-induced decrease in platelet glycoprotein Ib function. Br J Haematol 1993; 85: 116-123
• 16 Kirby EP, Mills DC B. The interaction of bovine factor VIII with human platelets. J Clin Invest 1975; 56: 491-502
• 17 Tomai I, Declerck PJ, Smets L, Arnout J, Deckmyn H, Caekebeke-Peer-linck KM J, Vermylen J. Measurement of von Willebrand factor antigen in plasma and platelets with an enzyme-linked immunosorbent assay based on two murine monoclonal antibodies. Haemostasis 1991; 21: 125-134
• 18 Strieker RB, Wong D, Shin DT, Reyes PT, Shuman MA. Activation of plasminogen by tissue plasminogen activator on normal and thrombasthenie platelets: Effect on surface proteins and platelet aggregation. Blood 1986; 68: 275-280
• 19 Colibretti ML, Berkowitz SD, Coppola R, Lombardi R, Castaman G, Mannucci PM, Federici AB. Von Willebrand factor degradation in 20 patients with acquired clinical conditions characterized by systemic proteolysis. Thromb Res 1991; Suppl (Suppl. 13) 110
• 20 Mannucci PM, Vicente V, Vianello L, Catteneo M, Alberca I, Coccato MP, Faioni E, Mari D. Controlled trial of desmopressin in liver cirrhosis and other conditions associated with a prolonged bleeding time. Blood 1986; 67: 1148-153
• 21 Pugh R NH, Murray-Lyon IM, Dawson JL, Pietroni MC, Williams R. Transection of the oesophagus for bleeding oesophageal varices. Br J Surg 1973; 60: 646-649
• 22 Beer JH, Büchi L, Steiner B. Glycocalicin: a new assay - the normal plasma levels and its potential usefulness in selected diseases. Blood 1994; 83: 691-702
• 23 Fox JE B, Aggerbeck LP, Bemdt MC. Structure of the glycoprotein Ib/IX complex from platelet membranes. J Biol Chem 1988; 263: 4882-4890
• 24 Fox JE B. Linkage of a membrane skeleton to integral membrane glycoproteins in human platelets. Identification of one of the glycoprotein as glycoprotein Ib J Clin Invest 1985; 76: 1673-1683
• 25 Coller BS, Peerschke El, Scudder LE, Sullivan CA. Studies with a murine monoclonal antibody that abolishes ristocetin-induced binding of von Willebrand factor to platelets: Additional evidence in support of GPIb as a platelet receptor for von Willebrand factor. Blood 1983; 61: 99-110
• 26 Luzzatto G, De Franchis G, Fabris F, Gerunda GE, Girolami A. Increased proportion of giant platelets and platelet distribution with are better indicators of altered platelet haemostasis than mean platelet volume in liver cirrhosis. Folia Hematol 1988; 115: 719-726
• 27 Eaton LA, Read MS, Brinkhous KM. Glycoprotein Ib Bioassays. Activity levels in Bemard-Soulier Syndrome and in stored blood bank platelets Arch Pathol Lab Med 1991; 115: 488-493
• 28 Beer JH, Rabaglio M, Berchtold P, von Felten A, Clemetson KJ, Tsakiris DA, Kehrel B, Brandenberger S. Autoantibodies against the platelet glycoproteins (GP)IMIIa, Ia/IIa, and IV and partial deficiency in GPIV in a patient with a bleeding disorder and a defective platelet collagen interaction. Blood 1993; 82: 820-829
• 29 Brinkhous KM, Barnes DS, Potter JY, Read MS. Von Willebrand syndrome induced by a bothrops venom factor: Bioassay for venom coagglutinin. Proc Natl Acad Sci (USA) 1981; 78: 3230-3234
• 30 Mazurier C, Parquet-Gemez A, Goudemand M. Dosage de l’antigène lié au facteur VIII par la technique ELISA. Interêt dans l’étude de la maladie de Willebrand Pathol Biol 1977; Suppl (Suppl. 25) 18
• 31 Caduff T, Straub PW. Die Verwendung gefrorener Thrombozyten für die Bestimmung des von Willebrand Faktors mit Ristocetin. Schweiz med Wschr 1979; 109: 399-402
• 32 Weiss HJ. Abnormalities of factor VIII and platelet aggregation. Use of ristocetin in diagnosing the von Willebrand syndrome Blood 1975; 45: 403-412
• 33 Baillod P, Affolter B, Kurt GH, Pflugshaupt R. Multimeric analysis of von Willebrand factor by vertical sodium dodecyl sulphate agarose gel electrophoresis, vacuum blotting technology and sensitive visualization by alkaline phosphatase anti-alkaline phosphatase complex. Thromb Res 1992; 66: 745-755
• 34 Budde U, Scharf RE, Franke P, Hartmann-Budde K, Dent J, Ruggeri ZM. Elevated platelet count as a cause of abnormal von Willebrand factor multimer distribution in plasma. Blood 1993; 82: 1749-1757
• 35 Federici AB, Bader R, Pagani S, Colibretti ML, De Marco L, Mannucci PM. Binding of von Willebrand factor to glycoproteins Ib and IIb/IIIa complex: affinity is related to multimeric size. Br J Haematol 1989; 73: 93-99
• 36 Ruggeri ZM, Zimmerman TS, Russell S, Bader R, De Marco L. (23) Von Willebrand factor binding to platelet glycoprotein Ib complex. Methods Enzymol 1992; 215: 263-275
• 37 Asbert M, Ginès A, Ginès P, Jiménez W, Clária J, Saió J, Arroyo V, Rivera F, Rodés J. Circulating levels of endothelin in cirrhosis. Gastroenterology 1993; 104: 1485-1491
• 38 De Nucci G, Gryglewski RJ, Warner TD, Vane JR. Receptor mediated release of endothelium derived reflexing factor and prostacylin from bovine endothelial cells is coupled. Proc Natl Acad Sci USA 1988; 85: 2334-2338
• 39 Vallance P, Moncada S. Hyperdynamic circulation in cirrhosis: A role for nitric oxide. Lancet 1991; 337: 776-778
• 40 Cruz MA, Handin RI, Wise RJ. The interaction of the von Willebrand factor-A1 domain with platelet glycoprotein Ib/IX. The role of glycosylation and disulfide bonding in a monomeric recombinant A1 domain protein J Biol Chem 1993; 268: 21238-21245
• 41 Cowan DH. Effect of alcoholism on hemostasis. Seminars in Hematol 1980; 17: 137-147
• 42 Spertini O, Hauert J, Bachmann F. Reaction of acetaldehyde with human platelets. Thromb Haemost 1992; 67: 126-130
• 43 Michelson AD, Barnard MR. Plasmin-induced redistribution of platelet glycoprotein lb. Blood 1990; 76: 2005-2010
• 44 Zheng T, Ren ZY. Activities of serum enzymes in patients with viral hepatitis B, posthepatic cirrhosis and hepatocellular carcinoma. Chin Med J 1990; 103: 729-734
• 45 Langley PG, Hughes RD, Rolando N, Williams R. Increased elastase-α-antitrypsin complex in fulminant hepatic failure: Relationship to bacterial infection and activation of coagulation. Clin Chim Acta 1991; 200: 211-219
• 46 Michelson AD, Barnard MR. Thrombin-induced changes in platelet membrane glycoproteins Ib, IX, and IIb-IIIa complex. Blood 1987; 70: 1673-1678
• 47 Cramer EM, Lu H, Caen JP, Soria C, Bemdt MC, Tenza D. Differential redistribution of platelet glycoproteins Ib and IIb/IIIa after plasmin stimulation. Blood 1991; 77: 694-699
• 48 De Noronha R, Taylor BA, Wild G, Triger DR, Greaves M. Inter-relationships between platelet count, platelet IgG, serum IgG, immune complexes and severity of liver disease. Clin Lab Haemat 1991; 13: 127-135
• 49 Michelson AD, Adelman B, Barnard MR, Carroll E, Handin RI. Platelet storage results in a redistribution of glycoprotein Ib molecules. Evidence for a large intraplatelet pool of glycoprotein Ib J Clin Invest 1988; 81: 1734-1740
• 50 Wencel-Drake JD, Plow EF, Kunicki TJ, Woods VL, Keller DM, Ginsberg MH. Localization of internal pools of membrane glycoproteins involved in platelet adhesive responses. Am J Pathol 1986; 124: 324-334
• 51 Bode 34 AP, Knupp CL, Miller DT. Effect of platelet activation inhibitors on the loss of glycoprotein Ib during storage of platelet concentrates. J Lab Clin Med 1990; 115: 669-679
• 52 Coller BS, Beer JH, Scudder LE, Steinberg MH. Collagen-platelet interactions: Evidence for a direct interaction of collagen with platelet GP Ia/IIa and an indirect interaction with platelet GP IIb/IIIa mediated by adhesive proteins. Blood 1989; 74: 182-192
• 53 Read MS, Smith SV, Lamb MA, Brinkhous KM. Role of botrocetin in platelet agglutination: Formation of an activated complex of botrocetin and von Willebrand factor. Blood 1989; 74: 1013-1035
• 54 Adelman B, Michelson AD, Handin RI, Ault KA. Evaluation of platelet glycoprotein Ib by fluorescence flow cytometry. Blood 1985; 66: 423-427
• 55 Beer JH, Springer KT, Coller BS. Immobilized Arg-Gly-Asp (RGD) peptides of varying lengths as structural probes of the platelet glycoprotein IIb/IIIa receptor. Blood 1992; 79: 117-128