A Study of the Protection of Plasmin from Antiplasmin Inhibition within an Intact Fibrin Clot during the Course of Clot Lysis

Previous work using soluble fibrin surrogates or very dilute fibrin indicate that inhibition of plasmin by antiplasmin is attenuated by fibrin surrogates; however, this phenomenon has not been quantified within intact fibrin clots. Therefore, a novel system was designed to measure plasmin inhibition by antiplasmin in real time within an intact clot during fibrinolysis. This was accomplished by including the plasmin substrate S2251 and a recombinant fluorescent derivative of plasminogen (S741C-fluorescein) into clots formed from purified components. Steady state plasmin levels were estimated from the rates of S2251 hydrolysis, the rates of plasminogen activation were estimated by fluorescence decrease over time, and residual antiplasmin was deduced from residual fluorescence. From these measurements, the second order rate constant could be inferred at any time during fibrinolysis. Immediately after clot formation, the rate constant for inhibition decreased 3-fold from 9.6 × 106m–1 s–1 measured in a soluble buffer system to 3.2 × 106m–1 s–1 in an intact fibrin clot. As the clot continued to lyse, the rate constant for inhibition continued to decrease by 38-fold at maximum. To determine whether this protection was the result of plasmin exposure of carboxyl-terminal lysine residues, clots were formed in the presence of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). In the presence of TAFIa, the initial protective effect associated with clot formation occurred; however, the secondary protective effect associated with lysine residue exposure was delayed in a TAFIa concentration-dependent manner. This latter effect represents another mechanism whereby TAFIa attenuates fibrinolysis. Previous work using soluble fibrin surrogates or very dilute fibrin indicate that inhibition of plasmin by antiplasmin is attenuated by fibrin surrogates; however, this phenomenon has not been quantified within intact fibrin clots. Therefore, a novel system was designed to measure plasmin inhibition by antiplasmin in real time within an intact clot during fibrinolysis. This was accomplished by including the plasmin substrate S2251 and a recombinant fluorescent derivative of plasminogen (S741C-fluorescein) into clots formed from purified components. Steady state plasmin levels were estimated from the rates of S2251 hydrolysis, the rates of plasminogen activation were estimated by fluorescence decrease over time, and residual antiplasmin was deduced from residual fluorescence. From these measurements, the second order rate constant could be inferred at any time during fibrinolysis. Immediately after clot formation, the rate constant for inhibition decreased 3-fold from 9.6 × 106m–1 s–1 measured in a soluble buffer system to 3.2 × 106m–1 s–1 in an intact fibrin clot. As the clot continued to lyse, the rate constant for inhibition continued to decrease by 38-fold at maximum. To determine whether this protection was the result of plasmin exposure of carboxyl-terminal lysine residues, clots were formed in the presence of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). In the presence of TAFIa, the initial protective effect associated with clot formation occurred; however, the secondary protective effect associated with lysine residue exposure was delayed in a TAFIa concentration-dependent manner. This latter effect represents another mechanism whereby TAFIa attenuates fibrinolysis. As a fibrin clot forms in response to a vascular injury, it regulates its own degradation by serving as cofactor for plasminogen (Pgn) 1The abbreviations used are: Pgn, plasminogen; Pn, plasmin; tPA, tissue-type plasminogen activator; AP, antiplasmin; TAFI, thrombin-activatable fibrinolysis inhibitor; TAFIa, activated TAFI; TAFI-T, TAFI variant with Thr-325; TAFI-I, TAFI variant with Ile-325; 5IAF-Pgn, fluorescein-labeled recombinant (S741C) Pgn; 5IAF-Pn, fluorescein-labeled recombinant (S741C) Pn; rform, rate of formation; rinh, rate of inhibition; dF, change in fluorescence; dt, change in time. activation to plasmin (Pn) (1Lucas M.A. Fretto L.J. McKee P.A. J. Biol. Chem. 1983; 258: 4249-45256Google Scholar). Once formed, Pn directly catalyzes clot breakdown by cleaving fibrin. In the process, carboxyl-terminal lysine residues are exposed that serve to enhance Pgn activation through a positive feedback mechanism for Pn formation (2de Vries C. Veerman H. Pannekoek H. J. Biol. Chem. 1989; 264: 12604-12610Google Scholar). Once formed, Pn is quickly inhibited by the potent serine protease inhibitor antiplasmin (AP), which forms a tight nearly irreversible complex with Pn. Therefore, Pn levels and consequently the rate of fibrinolysis are determined by the balance between the kinetics of Pn formation and inhibition. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a recently identified plasma zymogen (3Bajzar L. Manuel R. Nesheim M.E. J. Biol. Chem. 1995; 270: 14477-14484Google Scholar, 4Tan A.K. Eaton D.L. Biochemistry. 1995; 34: 5811-5816Google Scholar, 5Wang W. Hendriks D.F. Scharpe S.S. J. Biol. Chem. 1994; 269: 15937-15944Google Scholar) that can be activated to the carboxypeptidase B-like enzyme TAFIa by thrombin/thrombomodulin (6Bajzar L. Morser J. Nesheim M. J. Biol. Chem. 1996; 271: 16603-16608Google Scholar), free thrombin (3Bajzar L. Manuel R. Nesheim M.E. J. Biol. Chem. 1995; 270: 14477-14484Google Scholar, 7Eaton D.L. Malloy B.E. Tsai S.P. Henzel W. Drayna D. J. Biol. Chem. 1991; 266: 21833-21838Google Scholar), or plasmin (3Bajzar L. Manuel R. Nesheim M.E. J. Biol. Chem. 1995; 270: 14477-14484Google Scholar, 8Mao S.S. Cooper C.M. Wood T. Shafer J.A. Gardell S.J. J. Biol. Chem. 1999; 274: 35046-35052Google Scholar). In the context of a fibrin clot, TAFIa removes carboxyl-terminal lysine residues from Pn-modified fibrin (9Wang W. Boffa M.B. Bajzar L. Nesheim M.E. J. Biol. Chem. 1998; 273: 2127-2135Google Scholar). Although no naturally occurring inhibitors of TAFIa have been identified in human plasma, TAFIa is thermally unstable and this instability is probably how TAFIa is regulated once it is activated (10Boffa M.B. Bell R. Stevens W.K. Nesheim M.E. J. Biol. Chem. 2000; 275: 12868-12878Google Scholar, 11Marx P.F. Hackeng T.M. Dawson P.E. Griffin J.H. Meijers J.C.M. Bouma B.N. J. Biol. Chem. 2000; 275: 12410-12415Google Scholar). Previously, a naturally occurring polymorphism at amino acid position 325 (Thr/Ile) was identified that substantially affects the thermal stability of TAFIa (12Schneider M. Boffa M. Stewart R. Rahman M. Koschinsky M. Nesheim M. J. Biol. Chem. 2002; 277: 1021-1030Google Scholar). The more stable TAFI variant with Ile-325 (TAFI-I) had a half-life of 15 min at 37 °C, whereas the less stable TAFI variant with Thr-325 (TAFI-T) had a half-life of 8 min at 37 °C. When studied using an in vitro plasma clot lysis assay, this increased stability also corresponded to a more potent clot-stabilizing effect. TAFIa-T can prolong clot lysis time by 2.7-fold at saturation, whereas TAFIa-I can prolong clot lysis by 4-fold (12Schneider M. Boffa M. Stewart R. Rahman M. Koschinsky M. Nesheim M. J. Biol. Chem. 2002; 277: 1021-1030Google Scholar). Therefore, the carboxypeptidase B-like activity of TAFIa stabilizes fibrin clots and thermal stability is a potent regulator of this antifibrinolytic potential. Carboxyl-terminal lysine residues are important to the regulation of fibrinolysis because many key members of the fibrinolytic cascade, such as Pgn, Pn, and tissue-type plasminogen activator (tPA), contain kringle domains that mediate binding to the fibrin surface through exposed lysine residues (1Lucas M.A. Fretto L.J. McKee P.A. J. Biol. Chem. 1983; 258: 4249-45256Google Scholar, 2de Vries C. Veerman H. Pannekoek H. J. Biol. Chem. 1989; 264: 12604-12610Google Scholar, 13Ny T. Elgh F. Lund B. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 5355-5359Google Scholar, 14Hoylaerts M. Rijken R.C. Lignen H.R. Collen D. J. Biol. Chem. 1982; 257: 2912-2919Google Scholar). Previously, it was shown that TAFIa removal of these carboxyl-terminal lysine residues from fibrin removes the positive feedback for Pgn activation on a Pn modified fibrin surface (9Wang W. Boffa M.B. Bajzar L. Nesheim M.E. J. Biol. Chem. 1998; 273: 2127-2135Google Scholar). Thus, TAFIa modulates fibrinolysis, in part at least, by regulating Pn levels through attenuation of Pgn activation. Studies using soluble fibrin derivatives have demonstrated that when Pn is bound to fibrin, it is much less susceptible to inhibition by AP (15Wiman B. Collen D. Eur. J. Biochem. 1978; 84: 573-578Google Scholar, 16Wiman B. Boman L. Collen D. Eur. J. Biochem. 1978; 87: 143-146Google Scholar, 17Wang H. Yu A. Wiman B. Pap S. Eur. J. Biochem. 2003; 270: 2023-2039Google Scholar, 18Anonick P.K. Gonias S.L. Biochem. J. 1991; 275: 53-59Google Scholar, 19Lee A.Y.Y. Fredenburgh J.C. Stewart R.J. Rischke J.A. Weitz J.I. Thromb. Haemostasis. 2001; 85: 502-508Google Scholar). Although Pn protection from AP is potentially an important mechanism for regulating Pn levels, previous work has been hampered by the gel-like nature of native fibrin. In this study, we have designed a novel system to study protection within an intact fibrin clot in real time. To do this, identical clots were formed in the presence of the chromogenic Pn substrate S2251 and in the presence of a fluorescent Pgn derivative in which the active site serine was mutated to cysteine and labeled with fluorescein (5IAF-Pgn) (20Horrevoets A.J.G. Pannekoek H. Nesheim M.E. J. Biol. Chem. 1996; 272: 2183-2191Google Scholar, 21Horrevoets A.J.G. Pannekoek H. Nesheim M.E. J. Biol. Chem. 1997; 272: 2176-2182Google Scholar). One clot was monitored by fluorescence, which can be used to estimate the rate of Pn formation and the residual concentration of AP. The other clot was monitored by absorbance, which can be used to estimate the concentration of Pn at any time. By knowing the rate of Pn formation and the concentrations of Pn and AP, the second order rate constant for Pn inhibition by AP can be calculated during fibrinolysis. With this model, it was found that modification of fibrin by Pn dramatically increased the ability of fibrin to protect Pn from inhibition. This protective effect was further characterized by forming clots in the presence of TAFIa, and it was found that TAFIa could effectively attenuate protection in a manner that depends on the concentration and thermal stability of the TAFIa isoform used. Therefore, for the first time, Pn protection from AP was measured in an intact clot and the of intact fibrin, Pn-modified fibrin, or TAFIa fibrin were thrombin was from as L. Fredenburgh J.C. Nesheim M.E. J. Biol. Chem. Scholar, Nesheim M.E. J. Biol. Chem. 1999; 274: Scholar). Pgn was from human plasma as of which was used to human Pn Nesheim M.E. J. Biol. Chem. 1999; 274: Scholar). and the were a from of human antiplasmin was in and as (3Bajzar L. Manuel R. Nesheim M.E. J. Biol. Chem. 1995; 270: 14477-14484Google Scholar). The recombinant human Pgn (S741C) by (20Horrevoets A.J.G. Pannekoek H. Nesheim M.E. J. Biol. Chem. 1996; 272: 2183-2191Google Scholar, 21Horrevoets A.J.G. Pannekoek H. Nesheim M.E. J. Biol. Chem. 1997; 272: 2176-2182Google Scholar) was active with to the fluorescent Pgn a soluble derivative of was a from human was a from and were from of from was from human plasma using the by Nesheim M.E. J. Biol. Chem. 1999; 274: Scholar) with the of a to the a concentration of was used. was into and at TAFI and of human TAFI, the less stable variant with a at position and the more stable variant with an at position were in using the as D.L. Malloy B.E. Tsai S.P. Henzel W. Drayna D. J. Biol. Chem. 1991; 266: 21833-21838Google Scholar, M.B. W. Bajzar L. Nesheim M.E. J. Biol. Chem. 1998; 273: Scholar). was and the TAFI were using (12Schneider M. Boffa M. Stewart R. Rahman M. Koschinsky M. Nesheim M. J. Biol. Chem. 2002; 277: 1021-1030Google Scholar), and at To TAFIa, or was with thrombin and for 15 min at °C. The was on to the thermal of the of the to for Pn during this study, a novel system was designed for the second order rate constant for Pn inhibition by AP within an intact fibrin clot during the of clot To do this, identical fibrin clots were formed from purified in the of a and in the of an In clots were formed by thrombin and to AP Pgn S2251 and formed in the were monitored by in a at and and from these measurements, the to state Pn concentration in the clot can be inferred from the rate of S2251 during clot the time, clots formed in the were monitored by fluorescence of the recombinant derivative using and with a in a to by in a of fluorescence this derivative can be used to estimate the to rates of Pn formation and the residual AP concentration during fibrinolysis. of the associated with clot formation and clot was estimated within the intact clot to clot formation and to the of clot of the were to and were at °C. a state Pn within a fibrin clot over time In the presence of Pgn, AP, and tPA, Pn is formed on the fibrin and by AP. Therefore, the rate of change in the concentration of Pn at any time during clot lysis is a result of the between the rate of formation and the rate of inhibition by AP, which is by Therefore, the rate of change in Pn concentration within a clot over time is as shown in depends on the concentrations of AP and Pn, the can be as shown in In the the concentration of Pn within the clot is and can be as in the second order rate constant for Pn inhibition in of the rform, and residual AP concentration as shown in rform, and can be measured during the of fibrinolysis, can be calculated in real time within an intact fibrin clot. of the Steady of fibrin clot, monitored by absorbance, in the measured at because of S2251 and associated with in clot as it Therefore, to determine the change in to S2251 hydrolysis, the of the clot was measured at and and the change in at was used to the rate of S2251 from the of the clot. measured at was found to be measured at To determine the rate of change at to substrate hydrolysis, the rate of change measured at was by the and from the rate of change measured at To be to the rate of S2251 to the Pn within an clot at any time, a was by forming clots in the presence of concentrations of Pn and in the of AP. To do this, a of clots were formed in a by thrombin and to S2251 and concentrations of purified human Pn The clots were monitored by at and and the rates of S2251 were found by the of the change in change in time at over a to clot The rate of change in was calculated as and time. The of the could be used to the from the rate of substrate at any time in a fibrin clot. In the assay, Pn concentrations were calculated at time The at was by of of the from from The that the were nearly By the calculated to the the rates of S2251 can be used to determine the in a fibrin clot at any time. can be used to estimate the from the estimated from the is the constant for the of Pn and The of the have been that the change in substrate concentration was no more over the of fibrinolysis, and the concentration of S2251 was to be Although the was in a system of purified thrombin was another serine protease that could S2251 and the when was Pn Therefore, for of a clot was formed by thrombin and to and S2251 in the of to the substrate by of the of Pn clot monitored by fluorescence a decrease associated with of to In a fibrin clot, the rate of the change in fluorescence change in time was measured The at was by of of the from The that the were nearly The measured can be used to as is the initial fluorescence and is the initial concentration as shown in Pgn and are with the can be used to estimate to the rate of Pn formation and the rate of of the of residual fluorescence can also be used to estimate the of AP of the to activation of the Pgn to Pn. Once formed, the Pn is quickly inhibited by AP and of the Pgn that has been activated have been Therefore, the residual fluorescence can be measured at any time to the of AP as shown in The of TAFIa on within TAFIa-T or TAFIa-I was activated as and on of fibrin clots were formed as in the presence of concentrations of TAFIa-T and or TAFIa-I and clots were monitored in by and fluorescence, and the rform, and were measured within the intact clots at as were calculated during the of clot of the study was the on the concentrations of the the concentrations of and TAFIa that were By the nature of the was very Pn during the initial part of the and in the presence of TAFIa, was Therefore, substrate at this was an concentration of activator was used. increased concentrations of the initial rates of Pn formation to measure Pgn was also in rates the of the Therefore, the concentration and TAFIa concentrations were by that determined that of of the during the of the in the presence and of was for this that of the concentration of TAFIa-I with TAFIa-T was used in the concentrations of TAFIa variant TAFIa-T and in the presence of the in initial rates of substrate that were very and to measure The concentrations were to the of within an intact clot to be measured and the effect of TAFIa to be measured over a concentration that is to be lysis a effect at To Pn inhibition by AP, clots were formed from purified in the presence of the Pn substrate S2251 and the fluorescent plasminogen By identical clots by and fluorescence in rform, and the could be measured within an intact clot from this, could be calculated in real time using of the by the change in of the clot at and is shown in is the in the of TAFIa and is the in the presence of In clot formation was associated with a in that can be at and In the first as the fibrin clot formed, it was to measure S2251 the change in as the clot was a decrease in which is in after Therefore, to measure of the inhibition were to the time to clot formation and to clot of the for clots monitored by fluorescence is shown in As the fluorescent plasminogen derivative was to the fluorescent Pn derivative 5IAF-Pn, was a decrease in fluorescence that corresponded to the the of in the of TAFIa-T to the in the presence of TAFIa it is that TAFIa the rate of Pgn by the positive feedback associated with Pn modification of the fibrin clot (9Wang W. Boffa M.B. Bajzar L. Nesheim M.E. J. Biol. Chem. 1998; 273: 2127-2135Google Scholar). From the fluorescence the and the residual can be estimated at any time as of Pn from AP within an the shown in and the fluorescence shown in rform, and were determined during the of clot are shown in The from were also used to in real time using as This is shown as In the of TAFIa, to clot formation, was found to be 3.2 × 106m–1 The in the of fibrin was also measured in a soluble buffer system and was found to 9.6 × 106m–1 s–1 not which is very to measured for this (15Wiman B. Collen D. Eur. J. Biochem. 1978; 84: 573-578Google Scholar, 16Wiman B. Boman L. Collen D. Eur. J. Biochem. 1978; 87: 143-146Google Scholar, 17Wang H. Yu A. Wiman B. Pap S. Eur. J. Biochem. 2003; 270: 2023-2039Google Scholar, C. Biochemistry. 1991; Scholar, U. S. 1996; Scholar). Therefore, intact fibrin, to modification by Pn, a 3-fold protective effect. As clot lysis continued to decrease to × 106m–1 to a 38-fold protective effect. Therefore, intact fibrin a protective Pn-modified fibrin is a potent of Pn, dramatically the rate of inhibition. The of in the presence of TAFIa-T is very clot formation, the is estimated to be × to clots formed in the of however, in the presence of the in the protective effect associated with Pn modification of the clot was as Pn exposed carboxyl-terminal lysine residues, TAFIa-T and the protective of this TAFIa fibrin was to fibrin to Pn Therefore, within an intact fibrin clot, TAFIa not the rate of Pn formation also the fibrin surface less to protect Pn from AP. As this attenuation of the protective of Pn-modified fibrin not at °C, TAFIa-T was thermally unstable and the enzyme had a half-life of the attenuation of protection was and a was the concentration of active enzyme was not to of the lysine residues exposed by Pn. this to decrease and the protection of Pn by fibrin increased to a to the clots in the of of and that as the TAFIa activity was the state Pn levels to in the clot, probably to the increased cofactor activity of the fibrin surface and protection of Pn from inhibition. The of TAFIa-T and TAFIa-I on Pn by thermal stability of TAFIa has been shown to its antifibrinolytic effect when studied using in vitro clot lysis (3Bajzar L. Manuel R. Nesheim M.E. J. Biol. Chem. 1995; 270: 14477-14484Google Scholar, M. Boffa M. Stewart R. Rahman M. Koschinsky M. Nesheim M. J. Biol. Chem. 2002; 277: 1021-1030Google Scholar, 14Hoylaerts M. Rijken R.C. Lignen H.R. Collen D. J. Biol. Chem. 1982; 257: 2912-2919Google Scholar). Therefore, with to the protective of Pn modified fibrin, the ability of TAFIa on the concentration and the thermal stability of the TAFIa variant used. To study this a of were with concentrations of the less stable TAFIa-T variant that had a half-life of min at and the more stable TAFIa-I variant that had a half-life of min at (12Schneider M. Boffa M. Stewart R. Rahman M. Koschinsky M. Nesheim M. J. Biol. Chem. 2002; 277: 1021-1030Google Scholar). the effect that TAFIa had on the within fibrin clots. is to that and of is as much of the more stable TAFIa-I variant used to mediate nearly the effect as the less stable TAFIa-T Therefore, TAFIa Pn formation in fibrin clots in a manner that on the activity of the TAFIa TAFIa or TAFIa with a half-life was more to from the were also used to the of the less stable TAFIa-T variant and the more stable TAFIa-I variant on the during clot In to clot formation, the decreased 3-fold × 106m–1 and in the protection increased at maximum. the time of this decrease on the concentration of TAFIa and the thermal stability of the variant used. TAFIa-T attenuated the in protection whereas TAFIa-I attenuated the protection much more the effect on protection was in with we have used of the concentrations of the more stable TAFIa-I To this TAFIa concentration the time to the protective effect was the TAFIa concentrations in of the of The ability of TAFIa-T or TAFIa-I to attenuate the decrease in was estimated from the shown in The time to protection was estimated and TAFIa concentration for In this study, an intact fibrin clot was designed to study Pn protection from inhibition by AP. to fibrin clot formation, decreased 3-fold from 9.6 × 106m–1 s–1 to × 106m–1 As clot lysis decreased by 38-fold to × 106m–1 To that this in protection was to Pn exposure of carboxyl-terminal lysine residues, were also in the presence of was found that TAFIa can attenuate protection in a fibrin clot, and this attenuation depends on the concentration and thermal stability of the TAFIa variant In the presence of TAFIa, decreased to clots in the of however, TAFIa attenuated the decrease in the the protective of the clot at of intact fibrin. This attenuation of protection was not and protection increased after a that on the concentration of TAFIa TAFIa was thermally activity of the enzyme be over time not be TAFIa activity to the lysine residues as are In of this a of with the more stable TAFIa-I variant with the less stable TAFIa-T variant that TAFIa-I has an ability to attenuate the used in the assay, the protection was found to be which is in with previous work that has found that soluble fibrin surrogates can protect Pn by (15Wiman B. Collen D. Eur. J. Biochem. 1978; 84: 573-578Google Scholar, 16Wiman B. Boman L. Collen D. Eur. J. Biochem. 1978; 87: 143-146Google Scholar, 17Wang H. Yu A. Wiman B. Pap S. Eur. J. Biochem. 2003; 270: 2023-2039Google Scholar, 18Anonick P.K. Gonias S.L. Biochem. J. 1991; 275: 53-59Google Scholar, 19Lee A.Y.Y. Fredenburgh J.C. Stewart R.J. Rischke J.A. Weitz J.I. Thromb. Haemostasis. 2001; 85: 502-508Google Scholar). using fibrin was also used to study however, with this model, the protection was found to be R. J. Biol. Chem. 1994; 269: Scholar), which is more potent the effect measured with the intact clot on the protection of Pn from AP have been on a fibrin of this work is that the result of the balance of Pn formation and inhibition and how this during the of fibrin modification and breakdown can be By the in real time, the that carboxyl-terminal lysine residue exposure has on Pn the used in this assay, the modified fibrin surface is at Pn the by including TAFIa, the effect of lysine residue exposure can be Previous work has demonstrated that AP has a carboxyl-terminal lysine residue that can be by carboxypeptidase and is a for The work on this phenomenon used to the of the AP Biochem. however, more work with recombinant AP that have the carboxyl-terminal lysine mutated much of the ability of the AP to Pn H. Yu A. Wiman B. Pap S. Eur. J. Biochem. 2003; 270: 2023-2039Google Scholar). TAFIa removal of lysine from the of AP were the of AP in this increased concentrations of TAFIa decrease however, the effect was within the intact clots studied TAFIa the system in a decreased protective and the the concentration of TAFIa the this decreased protection can be In the context of this study, TAFIa was not at concentrations to the AP TAFIa have had or no activity AP. In this was not Therefore, this work to the that TAFIa has effect on the of AP, which when the antifibrinolytic of TAFIa is the first time, the protection of Pn from AP within an intact fibrin clot has been characterized and how this dramatically state Pn levels within a clot has been This work that intact fibrin is to protect Pn, modification of fibrin by Pn as a potent protective further the of this feedback mechanism to fibrinolysis. In these have demonstrated that increased protection associated with the exposure of the lysine residues is and a novel by which TAFIa could clot stability has been

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