Species Specific Immunoassays to Measure Blood Platelet and Coagulation Activation in the Rat

Summary

The purpose of this study was to develop specific and sensitive immunoassays to detect early indices of hypercoagulability in the rat. Rat platelet factor 4 (rPF4) and rat fibrinopeptide A (rFPA) assays, tools for the detection of activation of platelets and coagulation respectively, were designed using antibodies raised against purified rPF4 and against synthetic rFPA. The relevance of these new assays and of the commercially available ELISA kit for thrombin-antithrombin III (TAT) complexes was demonstrated in a rat model of a prethrombotic state induced by intravenous infusion of varying doses of thromboplastin (90 to 2400 μl/kg/h). In this model, the immunoassays allowed simultaneous detection of low levels of rFPA and rPF4 which were correlated with fibrinogen and platelet consumption and TAT generation and further proved to be of higher sensitivity than the classical methods of platelet count or measurement of fibrinogen levels. Plasma concentrations of rFPA, rPF4 and TAT were dependent on infusion time and thromboplastin dose, while hirudin (1 mg/kg) prevented their appearance. Thus the new specific immunoassays for rPF4 and rFPA and the commercial human TAT assay represent useful tools for pathophysiological studies or the screening of antithrombotic drugs in rats.

• References

• 1 Bauer KA, Rosenberg RD. The pathophysiology of the prethrombotic state in humans: insights gained from studies using markers of hemostatic system activation. Blood 1987; 70: 343-350
• 2 Mannucci PM, Giangrande PLF. Detection of the prethrombotic state due to procoagulant imbalance. Eur J Haematol 1992; 48: 65-69
• 3 Boisclair MD, Philippou H, Lane DA. Thrombogenic mechanisms in the human: fresh insights obtained by immunodiagnostic studies of coagulation markers. Blood Coag Fibrinol 1993; 4: 1007-1021
• 4 Nossel HL, Yudelman I, Canfield RE, Butler VP, Spanondis K, Winer GD, Qureshi GD. Measurement of fibrinopeptide A in human blood. J Clin Invest 1974; 54: 43-53
• 5 Amiral J, Walenga JM, Fareed J. Development and performance characteristics of a competitive enzyme immunoassay for fibrinopeptide A. Semin Thromb Haemost 1984; 10: 228-242
• 6 Lau HK, Rosenberg JS, Beeler DL, Rosenberg RD. The isolation and characterization of a specific antibody population directed against the prothrombin activation fragment F2 and F 1+2. J Biol Chem 1979; 254: 8751-8761
• 7 Pelzer H, Schwarz A, Heimburger N. Determination of human thrombin-antithrombin III complex in plasma with enzyme-linked immunosorbent assay. Thromb Haemost 1988; 59: 101-106
• 8 Files JC, Malpass TW, Yee EK, Ritchie JL, Harker LA. Studies of human platelet α,-granule release in vivo. Blood 1981; 58: 607-618
• 9 Kaplan KL, Owen J. Plasma levels of β,-thromboglobulin and platelet factor 4 as indices of platelet activation in vivo. Blood 1988; 57: 199-202
• 10 Amiral J, Adam M, Mimilla F, Larrivaz I, Chambrette B, Boffa MC. Design and validation of a new immunoassay for soluble forms of thrombomodulin and studies on plasma. Hybridoma 1994; 13: 205-213
• 11 Bounameaux H, de Moerloose P, Perrier A, Reber G. Plasma measurement of D-dimers as diagnostic aid in suspected venous thromboembolism: an overview. Thromb Haemost 1994; 71: 1-6
• 12 Ravanat C, Freund M, Herbert JM, Dol F, Maffrand JP, Boneu B, Cadroy Y, Drouet L, Roussi J, Legrand C, Incardona F, Cazenave JP. Cross-reactivity of human molecular markers for detection of prethrombotic states in various animal species. Blood Coag Fibrinol 1995; 6: 446-455
• 13 Shigeta O, Lu W, Holt JC, Edmunds LH, Niewiarowski S. Ovine platelet factor 4 : purification, amino acid sequence, radioimmunoassay and comparison with platelet factor 4 of other species. Thromb Res 1991; 64: 509-520
• 14 Uchiyama H, Ohtani H, Hiraishi S, Horie S, Ishii H, Kazawa M. Changes in plasma thrombomodulin antigen in rabbit developing endotoxin-induced disseminated intravascular coagulation and the effect of heparin. Thromb Res 1992; 65: 593-604
• 15 Fredrich M, Zierz R, Morser J, Wydro R, Witt W. A new animal model of venous thrombosis in rats with low flow conditions in the venous blood stream. Blood Coag Fibrinol 1994; 5: 243-248
• 16 Drummond O, McLaughlin LF, MacGregor IR. Development of a radioimmunoassay for rat fibrinopeptide A. Br J Haematol 1991; 77: 59
• 17 Chen JP, Marsh LC, Schroeder EC. Haemostatic derangements associated with arenavirus infection in the guinea-pig: radioimmunoassay of fibrinopeptide A to assess thrombin action in infected animals. Blood Coag Fibrinol 1993; 4: 165-172
• 18 MacGregor IR, Ferguson JF, Dawes J, McLaughlin L, Prowse CV. Thrombogenicity of a factor IX concentrate quantitated in a canine model. Blood Coag Fibrinol 1990; 1: 23-30
• 19 Spannagl M, Trauner A, Birg A, Frank G, Hoffmann H, Siebeck M, Lill H. Sensitive detection of the activation state of blood coagulation in porcine DIC models by a new fibrin immunoassay. Blood Coag Fibrinol 1993; 4: 103-106
• 20 Winocour PD, Chignier E, Parmentier S, McGregor JL. A member of the selectin family (GMP-140/PADGEM) is expressed on thrombin-stimulated rat platelets in vitro. Comp Biochem Physiol 1992; 102A: 265-271
• 21 Chignier E, Parise M, McGregor L, Delabre C, Faucompret S, McGregor J. A P-selectin/CD62P monoclonal antibody (LYP-20), in tandem with flow cytometry, detects in vivo activated circulating rat platelets in severe vascular trauma. Thromb Haemost 1994; 72: 745-748
• 22 Ravanat C, Schuhler S, Morales M, Cazenave JP, Lanza F. Cloning of the gene for rat platelet GPV, purification of the protein and development of an anti-peptide antibody for the detection of rat thrombin-activated platelets. Thromb Haemost 1995; 73: 1196
• 23 Nawa K, Itani T, Ono M, Sakano K, Marumoto Y, Iwamoto M. The gly-cosaminoglycan of recombinant human soluble thrombomodulin affects antithrombotic activity in a rat model of tissue factor-induced disseminated intravascular coagulation. Thromb Haemost 1992; 67: 366-370
• 24 Dickneite G, Czech J, Keuper H. Formation of fibrin monomers in experimental disseminated intravascular coagulation and its inhibition by recombinant hirudin. Circ Shock 1994; 42: 183-189
• 25 Markwardt F. Hirudin as an inhibitor of thrombin. In: Methods in enzymol-ogy. Vol. 29 Academic Press; New York: 1970. pp 924-932
• 26 Ravanat C, Gachet C, Herbert JM, Schuhler S, Guillemot JC, Uzabiaga F, Picard C, Ferrara P, Freund M, Cazenave JP. Rat platelets contain glycosylated and non glycosylated forms of platelet factor 4. Identification and characterization by mass spectrometry. Eur J Biochem 1994; 223: 203-210
• 27 McKinney MM, Parkinson A. A simple, non-chromatographic procedure to purify immunoglobulins from serum and ascites fluid. J Immunol Methods 1987; 96: 271-278
• 28 Weiss HJ, Witte LD, Kaplan KL, Lages BA, Chemoff A, Nossel HL, Goodman DS, Baumgartner HR. Heterogeneity in storage pool deficiency: Studies on a-granule-bound substances in 18 patients including variants deficient in a-granules, platelet factor 4, β,-thromboglobulin, and platelet-derived growth factor. Blood 1979; 54: 1296-1319
• 29 Carson SD, Brozna JP. The role of tissue factor in the production of thrombin. Blood Coag Fibrinol 1993; 4: 281-292
• 30 MacGregor IR, McLaughlinm LF, Drummond O, Prowse CV, Ferguson JM. In vivo models of thrombogenic potential: usefulness and limitations. Acta Haematol 1995; 94: 18-24
• 31 Freund M, Cazenave JP, Courtney M, Degryse E, Roitsch C, Bemart A, Delebasse D, Defreyn G, Maffrand JP. Inhibition by recombinant hirudins of experimental venous thrombosis and disseminated intravascular coagulation induced by tissue factor in rats. Thromb Haemost 1990; 63: 187-192
• 32 Freund M, Mantz F, Nicolini P, Gachet C, Mulvihill J, Meyer L, Beretz A, Cazenave JP. Experimental thrombosis on a collagen coated arterioarterial shunt in rats: a pharmacological model to study antithrombotic agents inhibiting thrombin formation and platelet deposition. Thromb Haemost 1993; 69: 515-521
• 33 Herbert JM, Bemart A, Maffrand JP. Importance of platelets in experimental venous thrombosis in rat. Blood 1992; 80: 2281-2286
• 34 Dickneite G, Paques EP. Reduction of mortality with antithrombin III in septicemic rats: a study of Klebsiella pneumoniae induced sepsis. Thromb Haemost 1993; 69: 98-102
• 35 Ivanov VS, Suvorova ZK, Tchikin LD, Kozhich AT, Ivanov VT. Effective method for synthetic peptide immobilization that increases the sensitivity and specificity of ELISA procedures. J Immunol Methods 1992; 153: 229-233
• 36 McLaughlin LF, Drummond O, MacGregor IR. A novel rat model of thrombogenicity: its use in evaluation of prothrombin complex concentrates and high purity Factor IX concentrates. Thromb Haemost 1992; 68: 511-515
• 37 Celia G, Myers CJ, Prosdocimi M, Sasahara AA. Human platelet factor 4 and protamine sulphate interaction with glycosaminoglycan in the rabbit. Eur J Clin Invest 1987; 17: 548-554
• 38 Weiss HJ, Witte LD, Kaplan KL, Lages BA, Chemoff A, Nossel HL, Goodman DS, Baumgartner HR. Heterogeneity in storage pool deficiency: Studies on granule-bound substances in 18 patients including variants deficient in a-granules, platelet factor 4, β,-thromboglobulin, and platelet-derived growth factor. Blood 1979; 54: 1296-1319