Analytical Biochemistry 272, 64–70 (1999) Article ID abio.1999.4148, available online at http://www.idealibrary.com on Acetylated Prothrombin as a Substrate in the Measurement of the Procoagulant Activity of Platelets: Elimination of the Feedback Activation of Platelets by Thrombin Jolyon Jesty* and Danny Bluestein† †Program in Biomedical Engineering and *Division of Hematology, Schools of Engineering and Medicine, State University of New York, Stony Brook, New York 11794 Received February 2, 1999 and to what extent by other causes, such as exposure Human prothrombin was acetylated to produce a to shear stress (1, 2). It is known, however, that modified prothrombin that upon activation by plate- mechanical stress can activate platelets, and this is let-bound prothrombinase generates a form of throm- particularly relevant to studies of the effects of bio- bin that does not activate platelets but retains its medical devices—chiefly prosthetic valves—on amidolytic activity on a chromogenic peptide sub- platelets and the increased thrombotic risk in pa- strate. If normal prothrombin is used in such an assay, tients with such valves (3, 4). To measure the effect the thrombin that is generated activates the platelets of these devices on platelets in vitro, platelet aggre- in a feedback manner, accelerating the rate of throm- gation has often been used, but this represents a bin generation and thereby preventing accurate mea- final event in platelet activation, and it is difficult to surement of the initial platelet procoagulant activity. quantify. Acetylation of prothrombin was carried out over a Platelet activation causes the exposure of two crit- range of concentrations of sulfo-N-succinimidyl ace- ical factors involved in the formation of thrombin. tate (SNSA). Acetylation by 3 mM SNSA at room tem- (i) Anionic phospholipid—mainly phosphatidylser- perature for 30 min at pH 8.2 in the absence of metal ine—is transferred from the inner leaflet of the cell ions produced a modified prothrombin that has <0.1% membrane to the outer leaflet and there supports the clotting activity (by specific prothrombin clotting as- binding and activation of the vitamin K-dependent say), but it is activated by factor Xa (in the presence of proteins of coagulation: factors VII, IX, and X and either activated platelets or factor Va 1 anionic phos- pholipid) to produce thrombin activity that is measur- prothrombin (see Ref. 5 for a general review of the able with a chromogenic substrate. Because the feed- coagulation pathways). (ii) Factor V, which is a back action on the platelets is blocked, thrombin present in the -granules of the platelets, is concom- generation is linear, allowing quantitative measure- itantly activated and the resulting factor Va, which ment of the initial platelet activation state. © 1999 is a required cofactor for prothrombin activation by Academic Press factor Xa, is expressed on the membrane surface. Key Words: platelets; platelet activation; cell activa- Thus, activated platelets provide both the major co- tion; prothrombin. factors required for prothrombin activation by factor Xa. The complete complex, of factor Xa 1 Va 1 anionic phospholipid, has been called the prothrom- binase complex. A number of investigators, led by Platelets are a major contributor to the pathology Rosing and Zwaal and their colleagues (6, 7), have of thrombosis, particularly arterial thrombosis, taken advantage of the platelets’ providing these where they form the bulk of thromboembolic occlu- activities in using the kinetics of prothrombin acti- sions. It is not clear to what extent such platelets are vation in the presence of platelets as a measure of activated in vivo by the ordinary hemostatic path- their “procoagulant” activity and by extension of way and initiated by exposure of the subendothelium their activation state. However, the use of this assay 64 0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. ACETYLATED PROTHROMBIN AND PLATELET ACTIVITY 65 is problematic because the enzyme generated and MATERIALS AND METHODS measured in the assay—thrombin—is a potent plate- Materials. Bovine serum albumin (BSA1; fatty acid let activator. As the platelets become activated, or free, fraction V), calcium ionophore A23187, phospha- further activated, during the assay, the thrombin tidylserine, phosphatidylcholine, and common reagent- generation rate increases, and measurement of the grade chemicals were obtained from Sigma Chemical initial rate of thrombin generation is often unreli- (St. Louis, MO). N-Tris(hydroxymethyl)methylglycine able. (Tricine) and N-2-hydroxyethylpiperazine-N9-2-eth- We need here a prothrombin species that is acti- anesulfonic acid (Hepes) were from Calbiochem-Beh- vated by prothrombinase, but generates an abnormal ring (San Diego, CA). The chromogenic amide thrombin that (i) does not activate platelets but (ii) substrate for thrombin was Chromozym-TH (tosyl-Gly- can still be detected. Such prothrombin species do L-Pro-L-Arg-p-nitroanilide), from Boehringer-Mann- exist, both as very rare congenitally mutant proteins heim (IN). BioGel A-15M was obtained from Bio-Rad and as molecules produced by chemical modification Laboratories (Richmond, CA). Sulfo-N-succinimidyl ac- of prothrombin. Landaburu and Seegers (8) demon- etate (SNSA) was from Pierce Chemical (Rockford, IL). strated nearly 40 years ago that acetylation of pro- Phosphatidylserine and phosphatidylcholine were pre- thrombin produces a protein that can be activated by pared as a sonicated 30:70 (PS:PC) mixture at a stock prothrombinase, but the acetylated thrombin that is concentration of 2 mg/ml in 0.1 M NaCl/0.05 M Tris/ generated is inactive on fibrinogen, even though it HCl, pH 7.5. retains activity on small substrates like amino acid Prothrombin-deficient plasma for clotting assays or peptide esters and amides. White and colleagues was prepared by the following method. (The older (9) chemically modified a small number of the lysine method of BaSO4 adsorption of oxalated plasma is not residues of human a-thrombin using pyridoxal 59- applicable to modern clotting instruments, because the phosphate and showed that the modified enzyme is calcium oxalate precipitate formed upon addition of defective not only in fibrinogen cleavage but also in CaCl2 in the assay interferes with the optical detection platelet aggregation. Similarly, Morrison (10) ob- system; hence, this preparation based on citrated plas- served that reaction of prothrombin with a nucleo- ma.) Blood was collected into 1/100 vol 40% sodium philic reagent (dichlorotriazinylaminofluorescein) citrate and centrifuged at 2500g for 15 min to yield produced a molecule that was activated normally, plasma. One-twentieth volume of 1.5 M BaCl2 was but generated a defective thrombin with ,5% activ- added to the plasma on ice, the mixture was stirred for ity on fibrinogen. Two congenital mutants of pro- 15 min, and the barium citrate precipitate was re- thrombin with very similar properties were de- moved by centrifugation (15,000g for 10 min). To re- 21 scribed over the same period, the prothrombins Metz move the excess free Ba in the plasma, Na2SO4 was (11) and Quick I (12, 13). Both can be activated by added to a concentration of 70 mM and stirred for 10 the prothrombinase complex, but the thrombin spe- min on ice. The resulting BaSO4 was removed by cen- cies produced, just as with chemically modified pro- trifugation. Finally, sodium citrate was added to a tein, are defective in their action on both fibrinogen concentration of 10 mM to mimic the free citrate con- and platelets. Interestingly, a chemical-modification centration in ordinary congenitally deficient plasma. study of another clotting zymogen has shown that Human factor X was prepared by the method of this behavior is not unique to prothrombin: acetyla- Morrison and Jesty (16), and human factor Xa by the tion of factor X under mild conditions produces a method of Jesty and Nemerson (17). The concentration molecule that is activated normally, but the acety- of factor Xa was determined by reference to a standard lated factor Xa generated has no activity on two factor Xa that had been assayed by titration with pure macromolecular substrates that were tested, pro- bovine antithrombin III. Bovine factor Va was pre- thrombin and factor VIII (14, 15). pared as described by Martin and Jesty (18). Human a-thrombin was prepared by a modification of the We demonstrate in this report that acetylated pro- method of Fenton et al. (19) using cation-exchange thrombin is activated by the prothrombinase complex chromatography on CM-Sephadex. to yield an enzyme that is inactive on fibrinogen, but m retains activity on a peptide p-nitroanilide substrate. Acetylation. Prothrombin (10 M) was dialyzed Moreover, thrombin generation from acetylated pro- against 0.1 M NaHCO3 (pH 8.2) in the absence of thrombin by platelet-bound prothrombinase is linear divalent metal ions and then acetylated with SNSA at with time, strongly suggesting that the acetylated room temperature for time courses up to 90 min, using thrombin produced upon activation is also inactive on platelets, allowing the quantitative measurement of 1 Abbreviations used: BSA, bovine serum albumin; Tricine, N- initial prothrombinase activity—and hence the activa- tris(hydroxymethyl)methylglycine; SNSA, sulfo-N-succinimidyl ace- tion state—of the platelets provided. tate; PS, phosphatidylserine; PC, phosphatidylcholine. 66 JESTY AND BLUESTEIN reagent concentrations of 0.1, 0.3, 1, 3, and