Hemostasis assay
AssigneeStichting Katholieke Universiteit
InventorWaander Laurens Van Heerde
Life of this patent
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Abstract
Provided is a hemostasis assay comprising a reaction mixture comprising a blood product to be tested, a trigger molecule for inducing thrombin generation, a thrombin-specific substrate which, upon cleavage by thrombin, produces a measurable thrombin-specific signal, a trigger molecule for inducing plasmin generation, a plasmin-specific substrate which, upon cleavage by plasmin, produces a measurable plasmin-specific signal, a phospholipid-containing surface, and calcium ions. The assay allows determination of the amount of thrombin and the amount of plasmin generated in the reaction mixture in time, starting at t=0, by measuring the thrombin-specific and plasmin-specific signals.
Claims27
An in vitro method for determining thrombin potential and plasmin potential generated in a single enzymatic reaction mixture in a single well, the method comprising:
- adding (i) a trigger molecule for inducing thrombin generation;
- (ii) a thrombin-specific substrate, which, upon cleavage by thrombin, produces a measurable, thrombin-specific signal;
- (iii) a trigger molecule for inducing plasmin generation;
- (iv) a plasmin-specific substrate, which, upon cleavage by plasmin, produces a measurable, plasmin-specific signal;
- (v) a phospholipid-containing surface;
- and
- (vi) calcium ions;
- to a test sample comprising a blood product, thereby generating the single enzymatic reaction mixture;
- simultaneously measuring thrombin-specific and plasmin-specific signals in the single enzymatic reaction mixture in the single well;
- and
- determining the thrombin potential and the plasmin potential generated in the single enzymatic reaction mixture in the single well.
An in vitro method for determining the effect of a drug, protein, cell or other additive on thrombin potential and plasmin potential in a single enzymatic reaction mixture in a single well, the method comprising:
- a) adding (i) a trigger molecule for inducing thrombin generation;
- (ii) a thrombin-specific substrate, which, upon cleavage by thrombin, produces a measurable, thrombin-specific signal;
- (iii) a trigger molecule for inducing plasmin generation;
- (iv) a plasmin-specific substrate, which, upon cleavage by plasmin, produces a measurable, plasmin-specific signal;
- (v) a phospholipid-containing surface;
- and
- (vi) calcium ions;
- to a test sample comprising a blood product, thereby generating a first single enzymatic reaction mixture;
- simultaneously measuring thrombin-specific and plasmin-specific signals in the first single enzymatic reaction mixture in a single well;
- b) adding (i) a trigger molecule for inducing thrombin generation;
- (ii) a thrombin-specific substrate, which, upon cleavage by thrombin, produces a measurable, thrombin-specific signal;
- (iii) a trigger molecule for inducing plasmin generation;
- (iv) a plasmin-specific substrate, which, upon cleavage by plasmin, produces a measurable, plasmin-specific signal;
- (v) a phospholipid-containing surface;
- (vi) calcium ions;
- and
- (vii) the drug, protein, cell or other additive
- to a test sample comprising a blood product, thereby generating a second single enzymatic reaction mixture;
- simultaneously measuring thrombin-specific and plasmin-specific signals in the second single enzymatic reaction mixture in a single well;
- and
- c) comparing the thrombin-specific and plasmin-specific signals in the first single enzymatic reaction mixture with the thrombin-specific and plasmin-specific signals in the second single enzymatic reaction mixture;
- thereby determining the effect of the drug, protein, cell or other additive on thrombin potential and plasmin potential in the second reaction mixture.
A kit for performing an in vitro hemostasis assay for measuring thrombin potential and plasmin potential comprising a single container comprising:
- a trigger molecule for inducing thrombin generation, a thrombin-specific substrate which, upon cleavage by thrombin, produces a measurable thrombin-specific signal, a plasmin-specific substrate which, upon cleavage by plasmin, produces a measurable plasmin-specific signal, a trigger molecule for inducing plasmin generation, a phospholipid-containing surface, and calcium ions.
Dependencies are inferred from the claim text.