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Thromb Haemost 1996; 76(06): 1090-1095
DOI: 10.1055/s-0038-1650711
Original Article
Schattauer GmbH Stuttgart

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

C Ravanat
1   NSERM U.311, Etablissement de Transfusion Sanguine de Strasbourg, Strasbourg
,
M Freund
1   NSERM U.311, Etablissement de Transfusion Sanguine de Strasbourg, Strasbourg
2   Laboratoire Centeon, Lingolsheim, France
,
S Schuhler
1   NSERM U.311, Etablissement de Transfusion Sanguine de Strasbourg, Strasbourg
,
P Grunert
1   NSERM U.311, Etablissement de Transfusion Sanguine de Strasbourg, Strasbourg
,
L Meyer
2   Laboratoire Centeon, Lingolsheim, France
,
J-P Cazenave
1   NSERM U.311, Etablissement de Transfusion Sanguine de Strasbourg, Strasbourg
› Author Affiliations
Further Information

Publication History

Received 29 April 1996

Accepted after rvision 07 August 1996

Publication Date:
11 July 2018 (online)

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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
    PubMedGoogle Scholar
  • 2 Mannucci PM, Giangrande PLF. Detection of the prethrombotic state due to procoagulant imbalance. Eur J Haematol 1992; 48: 65-69
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    Article in Thieme ConnectPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    Article in Thieme ConnectPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 16 Drummond O, McLaughlin LF, MacGregor IR. Development of a radioimmunoassay for rat fibrinopeptide A. Br J Haematol 1991; 77: 59
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    Article in Thieme ConnectPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    Article in Thieme ConnectPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 29 Carson SD, Brozna JP. The role of tissue factor in the production of thrombin. Blood Coag Fibrinol 1993; 4: 281-292
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    Article in Thieme ConnectPubMedGoogle Scholar
  • 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
    Article in Thieme ConnectPubMedGoogle Scholar
  • 33 Herbert JM, Bemart A, Maffrand JP. Importance of platelets in experimental venous thrombosis in rat. Blood 1992; 80: 2281-2286
    PubMedGoogle Scholar
  • 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
    Article in Thieme ConnectPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    Article in Thieme ConnectPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar