EVALUATION OF COMMERCIAL PROTEIN C ASSAYS

Plasma protein C inactivates the activated coagulation factors V and VIII. The assay of protein C is important, because a protein C deficiency is associated with a thrombotic tendency. We therefore evaluated 5 commercial assays in 49 normal volunteers, 48 patients suspected of deep vein thrombosis (DVT) of the leg but with negative impedance plethysmography (IPG), and 52 patients with DVT proven by IPG. The assays were rocket electrophoresis (Merz and Dade antibody), ELISA (Boehringer Mannheim), 2 chromogenic activity assays (Behringwerke and Kabi) and a clotting assay (Behringwerke). Coumarin therapy was used by 13 DVT positive, and 3 DVT negative patients. Results are presented in the table.TABLE: In correlation 1 and 2, the assay results all non-coumarin treated individuals (n = 133) were compared with rocket electrophoresis and ELISA resp.In the non-coumarin treated patients, both in the DVT positive and in the DVT negative patient group one protein C deficiency was detected by all assays.Based upon the large assay VC (ROCKET) and normal range (B. CLOT), and poor correlation of the assays with the ELISA (ROCKET, B. CLOT) we conclude that the ELISA, B.CHROM and K.CHROM are to be preferred. However, as B.CHROM does not need a plasma absorption step it is somewhat preferable for activity assays.

Identification of glycoprotein Ib as a target for autoantibody in idiopathic (autoimmune) thrombocytopenic purpura

An antibody (DIL) from a patient with idiopathic thrombocytopenic purpura (ITP) was shown to have autospecificity on the basis of reactions with autologous platelets that were identical to those obtained with platelets from normal subjects. DIL antibody also reacted strongly in an immunofluorescence test with platelets from a patient with Glanzmann's thrombasthenia, but failed to react with platelets from a patient with the Bernard-Soulier syndrome who was known to be deficient in glycoprotein Ib (GPIb). Purified GPIb and control platelets, but not Bernard-Soulier platelets, inhibited the lytic activity of DIL. Using the GPIb-specific monoclonal antibody AP1 and one-dimensional rocket electrophoresis into gels containing rabbit antihuman platelet membrane antibody, it was shown that staphylococcal protein A-Sepharose beads coated with DIL antibody selectively remove GPIb from solubilized platelet preparations. By crossed immunoelectrophoresis it was found that DIL recognizes a determinant on GPIb on the membrane side of the cleavage site of the platelet calcium- activated protease (calpain). These studies provide direct evidence for binding of a platelet autoantibody to a determinant on GPIb relatively close to the site of insertion of this protein into the platelet membrane.

Identification of glycoprotein Ib as a target for autoantibody in idiopathic (autoimmune) thrombocytopenic purpura

An antibody (DIL) from a patient with idiopathic thrombocytopenic purpura (ITP) was shown to have autospecificity on the basis of reactions with autologous platelets that were identical to those obtained with platelets from normal subjects. DIL antibody also reacted strongly in an immunofluorescence test with platelets from a patient with Glanzmann's thrombasthenia, but failed to react with platelets from a patient with the Bernard-Soulier syndrome who was known to be deficient in glycoprotein Ib (GPIb). Purified GPIb and control platelets, but not Bernard-Soulier platelets, inhibited the lytic activity of DIL. Using the GPIb-specific monoclonal antibody AP1 and one-dimensional rocket electrophoresis into gels containing rabbit antihuman platelet membrane antibody, it was shown that staphylococcal protein A-Sepharose beads coated with DIL antibody selectively remove GPIb from solubilized platelet preparations. By crossed immunoelectrophoresis it was found that DIL recognizes a determinant on GPIb on the membrane side of the cleavage site of the platelet calcium- activated protease (calpain). These studies provide direct evidence for binding of a platelet autoantibody to a determinant on GPIb relatively close to the site of insertion of this protein into the platelet membrane.