METHODS OF MEASURING INHIBITION OF PLATELET AGGREGATION BY THROMBIN RECEPTOR ANTAGONISTS
A method is provided for measuring inhibition of platelet aggregation by a thrombin receptor antagonist. First, a blood sample is obtained from a patient treated with a thrombin receptor antagonist. The blood sample is mixed in combination with particles including an immobilized GPIIb/IIIa receptor ligand and a thrombin receptor activator. The combination is then incubated under conditions suitable for agglutinating the particles, and platelet-mediated agglutination is assessed in the mixture. The absence of agglutination indicates that the patient has reduced ability to form platelet thrombi in response to the thrombin receptor antagonist treatment. Also provided is a kit for measuring inhibition of platelet aggregation by a thrombin receptor antagonist that includes a GPIIb/IIIa receptor ligand immobilized on a particle, a thrombin receptor activator, an anticoagulant, and a buffer to maintain the anticoagulated blood in a condition suitable for platelet aggregation.
1 . A method for measuring inhibition of platelet aggregation by a thrombin receptor antagonist, comprising the steps of:
a) providing a platelet containing blood sample from an individual treated with a thrombin receptor antagonist;
b) contacting said platelet containing blood sample with a thrombin receptor activator under conditions suitable for activating platelet aggregation in said platelet containing blood sample; and
c) assessing platelet aggregation in said platelet containing blood sample to determine the presence, absence and/or degree of inhibition of platelet aggregation by said thrombin receptor antagonist in said individual, wherein absence or a reduction of said platelet aggregation indicates that said individual has reduced ability to form platelet aggregation in response to said thrombin receptor antagonist treatment.
2 . The method of claim 1 , wherein the thrombin receptor is PAR-1 or PAR-4.
3 . The method of claim 1 , wherein the thrombin receptor activator comprises a substance selected from the group consisting of a thrombin, a PAR-1 thrombin receptor activating peptide (TRAP-1) and a PAR-4 thrombin receptor activating peptide (TRAP-4).
4 . The method of claim 3 , wherein the thrombin has a final concentration of about 0.01 U/ml to about 0.5 U/ml.
5 . The method of claim 3 , wherein the TRAP-1 has a final concentration of about 0.5 μM to about 10 μM.
6 . The method of claim 3 , wherein the TRAP-4 has a final concentration of about 50 M to about 1 mM.
7 . The method of claim 1 , wherein the thrombin receptor activator comprises TRAP-1.
8 . The method of claim 1 , wherein the platelet containing blood sample is a whole blood sample.
9 . The method of claim 1 , wherein the platelet containing blood sample is a plasma sample.
10 . The method of claim 9 , wherein the plasma sample is a platelet rich plasma (PRP) sample.
11 . The method of claim 1 , wherein the thrombin receptor antagonist comprises a substance selected from the group consisting of an antibody for the thrombin binding domain of a thrombin receptor, a peptide derivative of TRAP-1, a peptide derivative of TRAP-4, a peptidomimetic of a TRAP-1 derivative, a peptidomimetic of a TRAP-4 derivative, E5555 and SCH 530348.
12 . The method of claim 11 , wherein the thrombin receptor antagonist comprises E5555.
13 . The method of claim 11 , wherein the thrombin receptor antagonist comprises SCH 530348.
14 . The method of claim 1 , wherein the thrombin receptor activator is contained in an assay medium having a peak absorption at about 800 nm.
15 . The method of claim 1 , which is conducted at a temperature ranging from 30° C. to 40° C., and the total time of the readings from the time of the contact between the platelet containing blood sample and the thrombin receptor activator ranges from about 10 seconds to about 10 minutes.
16 . The method of claim 1 , wherein said platelet containing blood sample is contacted with said thrombin receptor activator in a single use assay device.
17 . A method for measuring inhibition of platelet aggregation by a thrombin receptor antagonist, comprising the steps of:
a) providing a platelet containing blood sample from an individual treated with a thrombin receptor antagonist;
b) contacting said platelet containing blood sample with particles comprising a GPIIb/IIIa receptor ligand immobilized thereon and a thrombin receptor activator under conditions suitable for agglutination of said particles mediated by said platelet in said blood sample; and
c) assessing said agglutination to determine the presence, absence and/or degree of inhibition of platelet aggregation by said thrombin receptor antagonist in said individual, wherein absence or a reduction of said agglutination indicates that said individual has reduced ability to form platelet aggregation in response to said thrombin receptor antagonist treatment.
18 . The method of claim 17 , wherein the thrombin receptor is PAR-1 or PAR-4.
19 . The method of claim 17 , wherein the thrombin receptor activator comprises a substance selected from the group consisting of a thrombin, a PAR-1 thrombin receptor activating peptide (TRAP-1) and a PAR-4 thrombin receptor activating peptide (TRAP-4).
20 . The method of claim 19 , wherein the thrombin has a final concentration of about 0.01 U/ml to about 0.5 U/ml.
21 . The method of claim 19 , wherein the TRAP-1 has a final concentration of about 0.5 μM to about 10 μM.
22 . The method of claim 19 , wherein the TRAP-4 has a final concentration of about 50 μM to about 1 mM.
23 . The method of claim 17 , wherein the thrombin receptor activator comprises TRAP-1.
24 . The method of claim 17 , wherein the platelet containing blood sample is a whole blood sample.
25 . The method of claim 17 , wherein the platelet containing blood sample is a plasma sample.
26 . The method of claim 25 , wherein the plasma sample is a platelet rich plasma (PRP) sample.
27 . The method of claim 17 , wherein the thrombin receptor antagonist comprises a substance selected from the group consisting of an antibody for the thrombin binding domain of a thrombin receptor, a peptide derivative of TRAP-1, a peptide derivative of TRAP-4, a peptidomimetic of a TRAP-1 derivative, a peptidomimetic of a TRAP-4 derivative, E5555 and SCH 530348.
28 . The method of claim 27 , wherein the thrombin receptor antagonist comprises E5555.
29 . The method of claim 27 , wherein the thrombin receptor antagonist comprises SCH 530348.
30 . The method of claim 17 , wherein the particles comprise polystyrene or latex.
31 . The method of claim 17 , wherein the particle has a diameter from about 1 micron to about 8 microns.
32 . The method of claim 17 , wherein the particles comprise an infrared dye, the contact between the platelet containing blood sample and the particles forms an assay mixture, the agglutination of the particles mediated by the platelet is assessed by irradiating the assay mixture with a light in the infrared spectrum, and assessing the transmission of infrared light from the assay mixture.
33 . The method of claim 17 , wherein the GPIIb/IIIa receptor ligand comprises a substance selected from the group consisting of fibrinogen, monoclonal antibody 10E5, monoclonal antibody c7E3, von Willebrand factor, fibronectin, vitronectin, a ligand that has an arginine glycine-aspartic acid (RGD) sequence and a peptide or a peptidomimetic that mimics RGD sequence.
34 . The method of claim 17 , wherein the GPIIb/IIIa receptor ligand comprises fibrinogen.
35 . The method of claim 17 , wherein the particles and the thrombin receptor activator are contained in an assay medium having a peak absorption at about 800 nm.
36 . The method of claim 17 , which is conducted at a temperature ranging from 30° C. to 40° C., and the total time of the readings from the time of the contact among the platelet containing blood sample, the particles comprising an attached GPIIb/IIIa receptor ligand, and the thrombin receptor activator ranges from about 10 seconds to about 10 minutes.
37 . The method of claim 17 , wherein said platelet containing blood sample is contacted with said particles comprising said GPIIb/IIIa receptor ligand and said thrombin receptor activator in a single use assay device.
38 . A kit for measuring inhibition of platelet aggregation by a thrombin receptor antagonist, comprising a GPIIb/IIIa receptor ligand immobilized on a particle and a thrombin receptor activator.
39 . The kit of claim 38 , further comprising an anticoagulant and a buffer to maintain the pH and salt concentration of the anticoagulated blood within a range suitable for platelet aggregation.
40 . The kit of claim 38 , wherein the GPIIb/IIIa receptor ligand comprises a substance selected from the group consisting of fibrinogen, monoclonal antibody 10E5, monoclonal antibody c7E3, von Willebrand factor, fibronectin, vitronectin, a ligand that has an arginine glycine-aspartic acid (RGD) sequence and a peptide or a peptidomimetic that mimics RGD sequence.
41 . The kit of claim 38 , wherein the GPIIb/IIIa receptor ligand comprises fibrinogen.
42 . The kit of claim 38 , wherein the thrombin receptor activator comprises a substance selected from the group consisting of a thrombin, a PAR-1 thrombin receptor activating peptide (TRAP-1) and a PAR-4 thrombin receptor activating peptide (TRAP-4).
43 . The kit of claim 38 , wherein the thrombin receptor activator comprises TRAP-1.
44 . The kit of claim 38 , wherein the particle comprises polystyrene or latex.
45 . The kit of claim 38 , wherein the particle has a diameter ranging from about 1 micron to about 8 microns.
46 . The kit of claim 38 , wherein said kit comprises a single use assay device.