Measurement of blood coagulation Factor XIIIa formation in plasma containing glycyl- l-prolyl- l-arginyl- l-proline

Abstract

A method to directly measure the formation of blood coagulation Factor XIIIa in platelet-poor plasma unmodified by heat is described. The synthetic peptide glycyl- l-prolyl- l-arginyl- l-proline, a fibrin-polymerization inhibitor, was used to prevent clotting of platelet-poor plasma. Plasma was diluted to a final concentration of 2.5% ( v v ) in 0.1 m Tris-HCl, pH 8.5, buffer containing 25% glycerol, 5 m m calcium chloride, and 0.25 m m glycyl- l-prolyl- l-arginyl- l-proline and then activated by thrombin (20 U/ml) for 15 min. The Factor XIIIa-catalyzed incorporation of [ 3H]putrescine into Hammersten casein was used to measure Factor XIIIa formation. The assay detected Factor XIIIa in 2.5 to 50 μl of thrombin-treated plasma. When purified Factor XIII was added to Factor XIII-deficient plasma, there was complete recovery of the Factor XIII added. Glycyl- l-prolyl- l-arginyl- l-proline did not inhibit Factor XIIIa activity in thrombin-treated plasma or purified platelet Factor XIIIa. Glycerol stabilized Factor XIIIa activity in thrombin-treated plasma and buffer for 60 min. The presence of fibrinogen in plasma did not modify the assay results. The time course of thrombin-catalyzed Factor XIIIa formation in platelet-poor plasma containing glycyl- l-prolyl- l-proline was directly measured using the assay.