Kits for screening for blood coagulation disorders

• US 20040235078A1
• Filed: 05/28/2004
• Published: 11/25/2004
• Est. Priority Date: 03/19/1998
• Status: Active Grant

• Current Assignee: Instrumentation Lab SPA (Instrumentation Lab)
• Original Assignee: Instrumentation Lab Co (Instrumentation Lab)
• Inventors: Rosen, Bert Steffen, Hall, Christina Maria Yvonne
• Granted Patent: US 7,118,880 B2
• Time in Patent Office: Days
• US Class Current: 435/13
• CPC Class Codes: C12Q 1/56 involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
• CPC Class Codes: G01N 33/86 involving blood coagulating time or factors, or their receptors
• CPC Class Codes: Y10S 435/975 Kit

Abstract

In vitro methods for qualitative screening and/or quantitative determination of the functional activity of components of the Protein C anticoagulant pathway of blood coagulation are described. The methods entail measuring the conversion rate of a substrate by an enzyme, the activity of which is related to the Protein C anticoagulant activity, in a blood sample of a human comprising coagulation factors and said substrate, after at least partial activation of coagulation through the intrinsic, extrinsic or common pathway and triggering coagulation by adding calcium ions; and comparing said conversion rate with the conversion rate of a normal human blood sample determined in the same way. The methods include the addition of additional metal ions to the sample to enhance activity, sensitivity and resolution. Kits and reagents for use in the methods are also provided.

Claims

1-34. -34. (canceled)

35. A kit for determining the activity of one or more components of a Protein C anticoagulant pathway, the kit comprising the following components:

(a) procoagulant reagent;

(b) an exogenous synthetic substrate for an enzyme influenced by Protein C anticoagulant activity

(c) calcium ions; and

(d) at least one metal ion that increases anticoagulant activity of the Protein C anticoagulant pathway, the metal ion selected from the group consisting of Mg⁺², Mn⁺², Zn⁺², Ni⁺², Sr⁺², Cu⁺², and Cu⁺;

wherein the components are in separate containers and/or in containers comprising mixtures of at least two of the components.

45. The kit according to claim 35, wherein the metal ion comprises Mg⁺².

46. The kit according to claim 35, wherein the metal ion comprises at least one of Mn⁺², Zn⁺², Ni⁺², Sr⁺², Cu⁺², and Cu⁺.

47. The kit according to claim 35, wherein the exogenous substrate comprises a substrate for a component of the coagulation cascade selected from the group consisting of Factor Xa and thrombin.

48. The kit according to claim 47, wherein the substrate is selected from the group consisting of Benzoyl-Ile-Glu-Gly-Arg-pNA, N-a-Z-D-Arg-Gly-Arg-pNA, CH₃SO₂-D-Leu-Gly-Arg-pNA, and MeO-CO-D-CHG-Gly-Arg-pNA.

49. The kit according to claim 47, wherein the substrate is selected from the group consisting of H-D-Phe-Pip-Arg-pNA, pyroGlu-Pro-Arg-pNA, H-D-Ala-Pro-Arg-pNA, Z-D-Arg-Sarc-Arg-pNA, AcOH*H-D-CHG-But-Arg-pNA, and H-D-HHT-Ala-Arg-pNA.

50. The kit according to claim 35, wherein the exogenous substrate comprises a photometrically measurable leaving group.

51. The kit according to claim 50, wherein the photometrically measurable leaving group is selected from the group consisting of a chromophore, a fluorophore, and a luminophore.

52. The kit according to claim 51, wherein the chromophore comprises a p-nitroaniline group (pNA).

53. The method according to claim 51, wherein the fluorophore comprises a naphthylamine or coumarine derivative group.

54. The kit according to claim 56, wherein the luminophore comprises an isoluminolamide group.

55. The kit according to claim 35, wherein the procoagulant reagent comprises at least one of a phospholipid and a contact activator.

56. The kit according to claim 35, wherein the procoagulant reagent comprises at least one phospholipid and at least one intrinsic pathway factor selected from the group consisting of Factor IXa, Factor XIIa, and Factor XIa.

57. The kit according to claim 35, wherein the procoagulant reagent comprises at least one phospholipid, at least one contact activator, and at least one intrinsic pathway factor selected from the group consisting of Factor IXa, Factor XIIa, and Factor XIa.

58. The kit according to any one of claims 57, wherein the at least one phospholipid is selected from the group consisting of a synthetic phospholipid, a purified phospholipid, and a crude extract of a phospholipid derived from a biological source.

59. The kit according to claim 57, wherein the at least one contact activator is selected from the group consisting of ellagic acid, collagen, a collagen-related substance, and silica.

60. The kit according to claim 59, wherein the silica is selected from the group consisting of micronized silica, colloidal silica, and kaolin.

61. The kit according to claim 35, wherein the procoagulant reagent comprises at least one material selected from the group consisting of native human tissue factor, recombinant human tissue factor, native non-human tissue factor, recombinant non-human tissue factor, native human Factor VII/Factor VIIa, recombinant human Factor VII/Factor VIIa, native non-human Factor VII/Factor VIIa, and recombinant non-human Factor VII/Factor VIIa.

62. The kit according to claim 57, wherein the at least one phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylserine, and sphingomyelin.

63. The kit according to claim 35, wherein the procoagulant reagent comprises a material selected from the group consisting of exogenous Factor Xa, exogenous Factor X and an exogenous activator of Factor X, and an exogenous activator for endogenous Factor X.

64. The kit according to claim 63, wherein the exogenous activator for Factor X comprises snake venom enzyme.

65. The kit according to claim 64, wherein the snake venom enzyme comprises Russelli Viperii snake venom enzyme.

66. The kit according to claim 35, the kit further comprising at least one component selected from the group consisting of Protein C, activated Protein C, Protein S, Factor V, and Factor Va.

67. The kit according to claim 35, the kit further comprising a fibrin polymerization inhibitor is added to the blood sample.

68. The kit according to claim 67, wherein the fibrin polymerization inhibitor comprises Gly-Pro-Arg-Pro.

69. The kit according to claim 35, wherein the procoagulant reagent comprises at least one material selected from the group consisting of Factor VIII/Factor VIIIa, Factor X, and prothrombin.

70. The kit according to claim 35, the kit further comprising exogenous activated Protein C.

71. The kit according to claim 35, the kit further comprising an activator of Protein C.

72. The kit according to claim 35, the kit further comprising an exogenous Protein C and an activator of Protein C.

73. The kit according to claim 71, wherein the activator of Protein C comprises at least one of Protein C activating snake venom enzyme and thrombin.

74. The kit according to claim 71, wherein the activator of Protein C comprises thrombomodulin and thrombin.

75. The kit according to claim 71, wherein the activator of Protein C is recombinant.

76. The kit according to claim 75, wherein the snake venom enzyme is derived from the Agkistrodon family.

77. The kit according to claim 75, wherein the snake venom enzyme is derived from Agkistrodon contortrix contortrix.

78. The kit according to claim 75, wherein the snake venom enzyme comprises crude snake venom enzyme.

79. The method according to claim 75, wherein the snake venom enzyme comprises purified snake venom enzyme.

80. The kit according to claim 35, wherein the procoagulant reagent comprises Factor VIIa and at least one phospholipid.

81. The kit according to claim 35, wherein the procoagulant reagent comprises at least one material selected from the group consisting of prothrombin, Factor V/Va, Factor IX, and Factor X.

82. The kit according to claim 35, the kit further comprising a plasma deficient in at least one component selected from the group consisting of Protein C, Protein S, and Factor V.

83. The kit according to claim 35, the kit further comprising a plasma deficient in all components of the Protein C anticoagulant pathway.

84. The kit according to claim 35, the kit further comprising a plasma deficient in the Protein C anticoagulant pathway component to be measured.

36. (canceled)

37. A composition for determining the activity of one or more components of a Protein C anticoagulant pathway comprising

(a) a procoagulant reagent;

(b) an exogenous synthetic substrate for an enzyme influenced by Protein C anticoagulant activity

(c) calcium ions; and

(d) at least one metal ion in a concentration that increases anticoagulant activity of the Protein C anticoagulant pathway, the metal ion selected from the group consisting of Mg⁺², Mn⁺², Zn⁺², Ni⁺², Sr⁺², Cu⁺²,

38-44. -44. (canceled)