Assay for detection of selective Protein C inhibition by patients

A method for testing a patient plasma sample for indications of a propensity for thrombotic disease, comprising the steps of:

• (a) measuring a first clotting time for a first aqueous reaction mixture comprising a first aliquot of a citrated patient plasma sample in the presence of activated Protein C, a volume of normal plasma in an amount about equal to said aliquot of a citrated plasma sample, and a membrane source comprising a phospholipid component comprising an effective amount of phosphatidylethanolamine to allow clotting differences to be detected between normal plasma and plasma from patients with a thrombotic propensity and an effective amount of phosphatidylserine to complement the function of said phosphatidylethanolamine in a clotting assay;
• (b) measuring a second clotting time for a second aqueous reaction mixture comprising a second aliquot of said citrated patient plasma sample, in the presence of a membrane source comprising a phospholipid component comprising an effective amount of phosphatidylethanolamine to allow clotting differences to be detected between normal plasma and plasma from patients with a thrombotic propensity and an effective amount of phosphatidylserine to complement the function of said phosphatidylethanolamine in a clotting assay, and a volume of normal plasma in an amount about equal to said aliquot of a citrated plasma sample, and in the absence of activated Protein C;
• (c) measuring a third clotting time for a third aqueous reaction mixture comprising a first aliquot of a control plasma sample in the presence of activated Protein C, and a membrane source comprising a phospholipid component comprising an effective amount of phosphatidylethanolamine to allow clotting differences to be detected between normal plasma and plasma from patients with a thrombotic propensity and an effective amount of phosphatidylserine to complement the function of said phosphatidylethanolamine in a clotting assay;
• (d) measuring a fourth clotting time for a fourth aqueous reaction mixture comprising a second aliquot of said control plasma in the presence of a membrane source comprising a phospholipid component comprising an effective amount of phosphatidylethanolamine to allow clotting differences to be detected between normal plasma and plasma from patients with a thrombotic propensity and an effective amount of phosphatidylserine to complement the function of said phosphatidylethanolamine in a clotting assay, and in the absence of activated Protein C;
• (e) comparing said first, second, third and fourth clotting times and denoting said patient sample to be at risk for thrombosis if said first clotting time is less than said third clotting time, and said second clotting time is greater than or equal to said fourth clotting time, at an optimal phospholipid concentration.

A method for monitoring the effectiveness of antiinflammatory drug or anticoagulant therapy in lupus anticoagulant patients or patients with antiphospholipid antibodies, comprising the steps of:

• (a) forming a first citrated plasma sample from a blood sample obtained from said patients at a first time;
• (b) testing said first citrated plasma sample for coagulation properties in the presence and absence of activated Protein C and in the presence of a membrane source comprising a phospholipid component comprising an effective amount of phosphatidylethanolamine to allow clotting difference to be observed between normal plasma and patients with thrombotic propensities and an effective amount of phosphatidylserine to complement the function of said phosphatidylethanolamine in a clotting assay, and comparing said coagulation properties of said first citrated plasma sample to control plasma to obtain a first result;
• (c) repeating step (b) on a second sample of citrated plasma obtained from said patient at a second time to obtain a second result;
• (d) determining said therapy to be providing a desired effect if, in the presence of activated protein C, said second result indicates said second citrated plasma sample takes more time to clot than said first citrated plasma sample and if, in the absence of activated protein C, said second citrated plasma sample takes less than or equal time to clot compared to said first citrated plasma sample.

A method for testing a patient plasma sample for indications of a propensity for thrombotic disease, comprising the steps of:

• (a) mixing in a first reaction chamber a phospholipid membrane source comprising at least 10% phosphatidylethanolamine and at least 5% phosphatidylserine, activated Protein C, a coagulation initiator, and a patient citrated plasma sample;
• (b) mixing in a second reaction chamber a phospholipid membrane source comprising at least 10% phosphatidylethanolamine and at least 5% phosphatidylserine, activated Protein C, a coagulation activator, and a normal control plasma sample;
• (c) mixing in a third reaction chamber a phospholipid membrane source comprising at least 10% phosphatidylethanolamine and at least 5% phosphatidylserine, a coagulation activator, and a patient citrated plasma sample;
• (d) mixing in a fourth reaction chamber a phospholipid membrane source comprising at least 10% phosphatidylethanolamine and at least 5% phosphatidylserine, a coagulation activator, and a normal control plasma sample;
• and
• (e) adding an effective amount of Ca.sup.2+ to each of said first, second, third and fourth reaction chamber to initiate clotting;
• and
• (f) observing the amount of time it takes for the mixture in each of said first, second, third and fourth reaction chambers to form a clot, and if the time for said first mixture to clot is less than the time for said second mixture to clot and the time for said third mixture to clot is greater than or equal to the time for said fourth mixture to clot, concluding that said patient plasma sample is indicative of a propensity for thrombic disease.