Measurement of blood coagulation Factor XIIIa formation in plasma containing glycyl-L-prolyl-L-arginyl-L-proline.
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 mM calcium chloride, and 0.25 mM 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 microliter 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-arginyl-L-proline was directly measured using the assay.
Miraglia
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Greenberg
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D001780:Blood Coagulation Tests; D002384:Catalysis; D005176:Factor XIII / Q000096:biosynthesis* / Q000378:metabolism; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D066298:In Vitro Techniques; D009842:Oligopeptides / Q000097:blood*; D013917:Thrombin / Q000502:physiology; D011503:Transglutaminases
D009842:Oligopeptides; C034585:glycyl-prolyl-arginyl-proline; D005337:Fibrin; D005340:Fibrinogen; D005176:Factor XIII; D011503:Transglutaminases; D013917:Thrombin