Prothrombinase Components Can Accelerate Tissue Plasminogen Activator-catalyzed Plasminogen Activation

The enzymatic and cofactor subunits of human prothrombinase, factor Xa (FXa) and factor Va (FVa), respectively, were evaluated as modulators of Glu- and Lys-plasminogen (Pg) activation by tissue plasminogen activator (tPA). The data revealed that both FXa and FVa could accelerate tPA activity by as much as 60-fold for Lys-Pg and >150-fold for Glu-Pg. This function of FVa depended on pretreatment with plasmin (Pn), whereas the FXa fibrinolytic cofactor activity was endogenous. In the native state, FVa was observed to inhibit the acceleration of Pn generation by FXa. These effects were dependent on Ca2+ and procoagulant phospholipid. Interactions between plasminogen and prothrombinase components were quantified. The apparent Kd for binding to FXa was 35 nM. Strikingly, the affinity between FVa and Pg was increased by approximately 2 orders of magnitude when the FVa was Pn-pretreated (Kd = 0.1 μM). These data cumulatively suggest a mechanism by which Pn production is coordinated with coagulation and localized to sites where procoagulant phospholipid is exposed on a cell surface. The enzymatic and cofactor subunits of human prothrombinase, factor Xa (FXa) and factor Va (FVa), respectively, were evaluated as modulators of Glu- and Lys-plasminogen (Pg) activation by tissue plasminogen activator (tPA). The data revealed that both FXa and FVa could accelerate tPA activity by as much as 60-fold for Lys-Pg and >150-fold for Glu-Pg. This function of FVa depended on pretreatment with plasmin (Pn), whereas the FXa fibrinolytic cofactor activity was endogenous. In the native state, FVa was observed to inhibit the acceleration of Pn generation by FXa. These effects were dependent on Ca2+ and procoagulant phospholipid. Interactions between plasminogen and prothrombinase components were quantified. The apparent Kd for binding to FXa was 35 nM. Strikingly, the affinity between FVa and Pg was increased by approximately 2 orders of magnitude when the FVa was Pn-pretreated (Kd = 0.1 μM). These data cumulatively suggest a mechanism by which Pn production is coordinated with coagulation and localized to sites where procoagulant phospholipid is exposed on a cell surface. Solubilization and clearance of fibrin clot requires the generation of plasmin (Pn) 1The abbreviations used are: PnplasminPgplasminogentPAtissue plasminogen activatorPSphosphatidylserinePCphosphatidylcholineBSAbovine serum albuminEGRckGlu-Gly-Arg chloromethyl ketoneCPBcarboxypeptidase BGEMSA2-guanidinoethylmercaptosuccinic acidFXafactor XaEGR-FXaEGRck-inactivated FXaFVafactor Va. 1The abbreviations used are: PnplasminPgplasminogentPAtissue plasminogen activatorPSphosphatidylserinePCphosphatidylcholineBSAbovine serum albuminEGRckGlu-Gly-Arg chloromethyl ketoneCPBcarboxypeptidase BGEMSA2-guanidinoethylmercaptosuccinic acidFXafactor XaEGR-FXaEGRck-inactivated FXaFVafactor Va.1 from its inactive precursor, plasminogen (Pg) (32Lijnen H.R. Collen D. Methods Enzymol. 1994; 223: 197-206Google Scholar; 52Robbins K.C. Colman R.W. Hirsh J. Marder V.J. Salzman E.W. Hemostasis and Thrombosis: Basic Principles and Clinical Applications. J. B. Lippincott Company, Toronto1982: 623-639Google Scholar). One of the proteases that mediates Pg activation is tissue plasminogen activator (tPA), which has relatively poor intrinsic reactivity toward Pg. Thus, a fundamental regulatory step in fibrinolysis is the acceleration of tPA through the assembly of a ternary complex between tPA, Pg, and a protein cofactor such as fibrin or a cell surface receptor. The colocalization of Pg and tPA functions to increase the rate of catalysis by 1-2 logs through a Km effect and to restrict tPA activity to sites of fibrin deposition. Additionally, the association of Pg with endothelial cell receptors is known to accelerate the conversion of the native NH2-terminal Glu-Pg to the truncated Lys-Pg species (18Hajjar K.A. Nachman R.L. J. Clin. Invest. 1988; 82: 1769-1778Google Scholar). The outcome is that Lys-Pg is activated by tPA approximately 10 times more efficiently than Glu-Pg (20Holaerts M. Rijken D.C. Lijnen H.R. Collen D. J. Biol. Chem. 1982; 257: 2912-2919Google Scholar). plasmin plasminogen tissue plasminogen activator phosphatidylserine phosphatidylcholine bovine serum albumin Glu-Gly-Arg chloromethyl ketone carboxypeptidase B 2-guanidinoethylmercaptosuccinic acid factor Xa EGRck-inactivated FXa factor Va. plasmin plasminogen tissue plasminogen activator phosphatidylserine phosphatidylcholine bovine serum albumin Glu-Gly-Arg chloromethyl ketone carboxypeptidase B 2-guanidinoethylmercaptosuccinic acid factor Xa EGRck-inactivated FXa factor Va. Several proteins have been identified as Pg receptors, including: annexin II (6Cesarman G.M. Guevara C.A. Hajjar K.A. J. Biol. Chem. 1994; 269: 21198-21203Google Scholar; 19Hajjar K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Google Scholar), a novel 45-kDa endothelial protein (9Dudani A.K. Cummings C. Hashemi S. Ganz P.R. Thromb. Res. 1993; 69: 185-196Google Scholar, 10Dudani A.K. Pluskota A. Ganz P.R. Biochem. Cell Biol. 1994; 72: 126-131Google Scholar), α-enolase (40Miles L.A. Dahlberg C.M. Plescia J. Felez J. Kato K. Plow E.F. Biochemistry. 1991; 30: 1682-1691Google Scholar), osteonectin (24Kelm Jr., R.J. Swords N.A. Orfeo T. Mann K.G. J. Biol. Chem. 1994; 269: 30147-30153Google Scholar), and complement component C7 (51Reinartz J. Hansch G.M. Kramer M.D. J. Immunol. 1995; 154: 844-850Google Scholar). Through the use of lysine analogues and carboxypeptidase B, these proteins have been shown to have in common a COOH-terminal lysine that may be the only necessary structural feature for binding to Pg (40Miles L.A. Dahlberg C.M. Plescia J. Felez J. Kato K. Plow E.F. Biochemistry. 1991; 30: 1682-1691Google Scholar). Most of the Pg receptors have been shown to accelerate tPA-mediated activation of Pg (6Cesarman G.M. Guevara C.A. Hajjar K.A. J. Biol. Chem. 1994; 269: 21198-21203Google Scholar; 24Kelm Jr., R.J. Swords N.A. Orfeo T. Mann K.G. J. Biol. Chem. 1994; 269: 30147-30153Google Scholar; 25Krishnaswamy S. Church W.R. Nesheim M.E. Mann K.G. J. Biol. Chem. 1987; 262: 3291-3299Google Scholar) or to bind to tPA and Pg, which is presumed to form the necessary ternary complex for rapid Pn generation (9Dudani A.K. Cummings C. Hashemi S. Ganz P.R. Thromb. Res. 1993; 69: 185-196Google Scholar, 10Dudani A.K. Pluskota A. Ganz P.R. Biochem. Cell Biol. 1994; 72: 126-131Google Scholar). In the current study, the human blood coagulation factor Xa (FXa), which is known from its cDNA sequence to possess the necessary lysyl moiety (14Fung M.R. Hay C.W. MacGillivray R.T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3591-3595Google Scholar; 39Messier T.L. Pittman D.D. Long G.L. Kaufman R.J. Church W.R. Gene (Amst.). 1991; 99: 291-294Google Scholar), was evaluated as a Pg-binding protein and accelerator of Pn generation by tPA. The established function of the serine protease FXa in hemostasis is to produce the final enzyme in the coagulation pathway, thrombin (28Krishnaswamy S. Nesheim M.E. Pryzdial E.L.G. Mann K.G. Methods Enzymol. 1994; 222: 260-280Google Scholar; 28Krishnaswamy S. Nesheim M.E. Pryzdial E.L.G. Mann K.G. Methods Enzymol. 1994; 222: 260-280Google Scholar). To do so, FXa must associate in the presence of Ca2+ with the cofactor Va (FVa) and procoagulant phospholipid (e.g. phosphatidylserine), which synergistically accelerate FXa activity by 5 orders of magnitude (43Nesheim M.E. Taswell J.B. Mann K.G. J. Biol. Chem. 1979; 254: 10952-10962Google Scholar). Procoagulant phospholipid is accessible only on the surface of activated cells and therefore restricts prothrombinase assembly to sites of vascular damage (46Nesheim M.E. Tracy R.P. Tracy P.B. Boskovic D.S. Mann K.G. Methods Enzymol. 1992; 215: 316-328Google Scholar; 59Zwaal R.F.A. Comfurius P. Bevers E.M. Biochem. Soc. Trans. 1993; 21: 248-253Google Scholar). Since FVa stabilizes the interaction of FXa with biological membranes, a role for FVa in fibrinolysis was also evaluated. We now report that both FXa and FVa can participate as cofactors for tPA-mediated Pg activation. HEPES, EDTA (Sigma), Glu-Gly-Arg chloromethyl ketone (EGRck), 2-guanidinoethylmercaptosuccinic acid (GEMSA), aprotonin, porcine carboxypeptidase B (CPB, Calbiochem), benzoyl-Ile-Glu-(piperidyl)-Gly-Arg-p-nitroanilide (S-2337), Glu-Pro-Arg-p-nitroanilide (S-2366, Helena Laboratories) were obtained commercially. Human coagulation factor X was purified from fresh frozen plasma (3Bajaj S.P. Rapaport S.I. Prodanos C. Prep. Biochem. 1981; 11: 387-412Google Scholar) or from prothrombin complex concentrate diluted to 1 unit/ml factor X clotting activity (obtained as generous gifts from the Canadian Red Cross Society, Ottawa Collection Centre or from Miles Therapeutics, Inc., respectively). Factor Xa (approximately 1000 units/mg) was generated from factor X (21Jesty J. Nemerson Y. Methods Enzymol. 1976; 45: 95-107Google Scholar; 25Krishnaswamy S. Church W.R. Nesheim M.E. Mann K.G. J. Biol. Chem. 1987; 262: 3291-3299Google Scholar) by treatment with the purified activator from Russell's viper venom (Haematologic Technologies) (21Jesty J. Nemerson Y. Methods Enzymol. 1976; 45: 95-107Google Scholar), followed by affinity chromatography using benzamidine-Sepharose (Pharmacia Biotech Inc.) to remove the factor X activation peptide, Russell's viper venom, and residual inactivated factor X (25Krishnaswamy S. Church W.R. Nesheim M.E. Mann K.G. J. Biol. Chem. 1987; 262: 3291-3299Google Scholar). The irreversible inhibition of factor Xa was accomplished by treatment with a 4-fold molar excess of EGRck (to produce EGR-factor Xa) (49Pryzdial E.L.G. Mann K.G. J. Biol. Chem. 1991; 266: 8969-8977Google Scholar). Complete inhibition of factor Xa (1.5 μM) was confirmed by the lack of detectable conversion of the chromogenic substrate S-2337 (200 μM) over a 15-min period using a kinetic microplate reader (Vmax, Molecular Devices) with full scale set to 0.03 OD. Excess EGRck was removed by Sephadex G-25 gel filtration chromatography (Pharmacia). Human Glu-plasminogen and Lys-plasminogen were purified as described (45Nesheim M. Fredenburgh J.C. Larsen G.R. J. Biol. Chem. 1990; 265: Scholar) and over benzamidine-Sepharose to remove of serine protease was using and to remove Human factor Va was Technologies) to established D.S. C. Mann K.G. Biochemistry. Scholar; Nesheim M.E. Mann K.G. Proc. Natl. Acad. Sci. U. S. A. 1981; Scholar). The of proteins was by Scholar) and where by using and human plasmin tPA and human were To the interaction between prothrombinase proteins and of a by M. J. C.M. Mann K.G. Biochemistry. 1994; was In these were with of a of phosphatidylserine and phosphatidylcholine or phospholipid in of by in a the to for a were for 2 with of 5 bovine serum albumin and with HEPES, 2 with 1 In of or a of the proteins was with for 1 In the prothrombinase proteins were for 1 and times with of and were to inhibit Pn activity and by the were times with and the was quantified. To the effect of Pn on the binding of FXa or FVa to the FXa and FVa were for with Pn and by of a excess of the the The tPA activation of Pg μM) was followed in using a chromogenic 5 of the was with of chromogenic substrate μM) and the rate of was using a kinetic reader (Vmax, Molecular tPA was the final component to the The effect of FXa and FVa 0.1 μM) was evaluated by these proteins to the Pg. In the FXa and FVa μM) were with Pn for The Pn was with a excess of To be the with Pn were and were in the presence of phospholipid of phosphatidylcholine and phosphatidylserine μM) (28Krishnaswamy S. Nesheim M.E. Pryzdial E.L.G. Mann K.G. Methods Enzymol. 1994; 222: 260-280Google to the known effect on Pn of FVa Mann K.G. J. Biol. Chem. 1987; 262: Scholar) and for in prothrombinase assembly G.L. Biochemistry. Scholar; K.G. S. Tracy P.B. Nesheim M.E. in and Inc., Scholar; M.E. Tracy R.P. Tracy P.B. Boskovic D.S. Mann K.G. Methods Enzymol. 1992; 215: 316-328Google Scholar). To the with the chromogenic activation was followed by were using to the Pg from on the were to using and The Pn were from the gel to using a To for a from that to a on the was and from of to a of FXa in was to the interaction of to prothrombinase components that were to binding of was the was dependent on the presence of both FXa and whereas in the FXa or the Since FVa is known to the interaction of FXa with is the of the in the between the is that to the in B. This may be the of inhibition by FXa. that binding is dependent on and The association of was by of a molar excess of Lys-Pg to the observed with these with FXa as a between Pg and that is by that FXa is of as a for Pg, FXa FVa can accelerate the activation of Lys-Pg by tPA. the chromogenic substrate used in these is as for Pn as the substrate was to increase the by FXa and FVa were used 0.1 which the Kd for Pg binding to receptors P.R. D. A.K. Hashemi S. Biochem. Cell Biol. 1991; 69: Scholar; 19Hajjar K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Google Scholar). shown in FXa the rate of Lys-Pg activation. was from the to the and to the of Pn activity in the of FXa which a 60-fold increase in the rate of tPA catalysis by FXa. of of FVa to the FXa the a was a to a fibrinolytic the effect of a to that of the FVa was observed to have approximately of the effect of FXa. The of the on tPA and Pg was confirmed by the lack of chromogenic substrate were in the presence or of only FVa was in the Lys-Pg activation the of Pn generated was the as tPA over the the rate was by Since the of FVa by Pn has been Mann K.G. Scholar; Mann K.G. J. Biol. Chem. 1987; 262: Scholar), that the FVa may be fibrinolytic cofactor activity the by a To the FXa FVa were with The effect of treatment was to the observed in the when FVa was in the The FVa fibrinolytic activity the as the Pn pretreatment was the Pn with Pn was also observed to the effect of FXa on tPA This was as as observed for FVa and in approximately of the FXa To for a on FXa has been described for observed in We a of the Pn pretreatment the in FXa fibrinolytic activity The effect of Pn-pretreated FXa and FVa on tPA activity was approximately the of the The for Pn production was to that of which may that the tPA is these and the Pn pretreatment of the to its tPA cofactor Since was to the effect of Pn on the or FVa can be that is the for To chromogenic followed conversion of to by These data with the from FXa and a of accelerate tPA activity to approximately the in the presence of FVa Pn generation a the rate is Pn pretreatment of FVa the the FXa effect is by Pn pretreatment and the of Pn-pretreated has the rate of Lys-Pg conversion as FXa 5 that FXa and FVa also the rate of Glu-Pg activation by tPA. FXa has tPA cofactor activity FVa a in the FXa effect was much more to Lys-Pg Pn pretreatment of the FVa a tPA Pn pretreatment the FXa cofactor and Pn-pretreated FXa and FVa to increase the rate of Pn in the Lys-Pg is more to Pn than Glu-Pg by tPA. We observed in the of prothrombinase where over of Glu-Pg activation was a the of FVa by Pn was observed using Glu-Pg Since a was observed in the rate that Pn production when FVa was the that by Pn the FVa was a in is the effect of the FVa on Pn generation from Lys-Pg a of FXa. The data that the of the is dependent on the of FVa and is to the of the a rapid rate of Lys-Pg activation is which is to by both FXa and FVa fibrinolytic This that the prothrombinase the of FXa as a fibrinolytic cofactor is the FVa is by established that FXa and FVa can participate in the acceleration of Pn production by tPA, the of the FXa serine protease and procoagulant phospholipid were evaluated. and the effect of the of FXa with a ketone In to native tPA cofactor activity and Since Pg is to with the of known receptors (6Cesarman G.M. Guevara C.A. Hajjar K.A. J. Biol. Chem. 1994; 269: 21198-21203Google Scholar; A.K. Cummings C. Hashemi S. Ganz P.R. Thromb. Res. 1993; 69: 185-196Google Scholar; 24Kelm Jr., R.J. Swords N.A. Orfeo T. Mann K.G. J. Biol. Chem. 1994; 269: 30147-30153Google Scholar; L.A. Dahlberg C.M. Plescia J. Felez J. Kato K. Plow E.F. Biochemistry. 1991; 30: 1682-1691Google Scholar), the effect of the of FXa may be on tPA. Pn pretreatment between and FVa was This effect was apparent Pn pretreatment which that FXa may function to accelerate the of FVa by To the role of Ca2+ in the tPA cofactor function of FXa and excess EDTA was to the The data that Pn treatment the cofactor effects of FXa and FVa were in the of The data also suggest that the association of FXa with is necessary for tPA cofactor when the FXa and FVa were with Pn in the presence of Ca2+ the of EDTA a of the tPA approximately of the FVa effect on tPA the was in the presence of was and FXa FVa on the rate of Lys-Pg activation of a Pn pretreatment step was The data that Pn is of the of FVa and to a of FXa in Pg activation by tPA. To may be effect on the of FVa and FXa to with Pg, the were quantified. The binding shown in were obtained by the and prothrombinase binding was by the of to from the to The apparent and of were by the data to a which a of sites the from these data is that Pn treatment of FVa the affinity for Lys-Pg by approximately 2 orders of This may for the of tPA cofactor activity by FVa when with the in the tPA cofactor activity of FXa Pn treatment may be by the observed in affinity for with the kinetic is that to Pn FVa the binding of Lys-Pg to FXa. Pn the FXa and FVa were shown to have a effect on Pg activation by tPA FVa effect on Lys-Pg binding to FXa. These may be by effects on tPA that have to be in a function for the FXa COOH-terminal was by the FXa with The data revealed that the tPA cofactor function of FXa is to which the of a COOH-terminal in FXa. To for the that the or residual activity a effect on Pn generation by tPA, the FXa was with and in the to pretreatment of FXa was was to the in FXa cofactor activity when with were when Glu-Pg was used of Lys-Pg as a substrate II in a and fibrinolysis To the activation of procoagulant and of a of between these must the of to the fibrinolytic by as accelerator of Pn generation (32Lijnen H.R. Collen D. Methods Enzymol. 1994; 223: 197-206Google Scholar). tPA activity is clot has In the current study, that the protein components of prothrombinase, FXa and also of fibrinolysis by acceleration of tPA. that the between fibrinolysis and prothrombinase is in In Pn was to the FXa cofactor role of FVa in thrombin production Mann K.G. Scholar; Mann K.G. J. Biol. Chem. 1987; 262: Scholar). We now report that FVa tPA cofactor in the presence or of FXa. of FVa with Pn also with the of a Pg binding The apparent Kd of 0.1 is the plasma of Pg μM) and may be a for the of FVa in a effect on tPA also FVa is by Pn is Pn a in FVa from a procoagulant in the native to a The FXa moiety of prothrombinase was also observed to accelerate Pn generation by tPA. the tPA cofactor activity of FXa was endogenous. that FVa the FXa fibrinolytic activity was obtained from that of native FVa a the acceleration of Pn generation was Pn the FVa was to is that may the interaction between FXa and FVa in a to the inhibition of FVa by activated protein J. Biol. Chem. Scholar; E.L.G. Mann K.G. J. Biol. Chem. 1991; 266: 8969-8977Google Scholar). The from these data is that the intrinsic fibrinolytic of FXa through Since that FVa the binding affinity of Pg to regulatory mechanism is to effect on tPA. The association of Pg with FXa was to be approximately Kd = 35 than Pg binding to This may FXa a In have to the of tPA acceleration by FXa or to the of the prothrombinase components to known tPA have the of acceleration to tPA a of In the presence of FXa or Pn-pretreated observed approximately 60-fold for the rate of Lys-Pg activation. This is the as the known tPA annexin II (6Cesarman G.M. Guevara C.A. Hajjar K.A. J. Biol. Chem. 1994; 269: 21198-21203Google Scholar) and fibrin (32Lijnen H.R. Collen D. Methods Enzymol. 1994; 223: 197-206Google Scholar). Glu-Pg was used as a the generation of Pn in the presence of tPA over the of the which the of the increase in observed rate to FXa to be >150-fold and for Pn-pretreated These than for Pg receptors (24Kelm Jr., R.J. Swords N.A. Orfeo T. Mann K.G. J. Biol. Chem. 1994; 269: 30147-30153Google Scholar; J. Hansch G.M. Kramer M.D. J. Immunol. 1995; 154: 844-850Google Scholar) and fibrin (32Lijnen H.R. Collen D. Methods Enzymol. 1994; 223: 197-206Google Scholar). of the must be to these The role of procoagulant phospholipid as prothrombinase cofactor and as a to thrombin production has been E.M. Comfurius P. R.F.A. J. Biochem. 1982; Scholar, E.M. Comfurius P. R.F.A. Scholar; Mann K.G. J. Biol. Chem. 1987; 262: Scholar; and Scholar). is also for prothrombinase assembly and functions by the interaction between FVa subunits J. Biol. Chem. 1979; 254: Scholar; S. Mann K.G. J. Biol. Chem. Scholar), the binding of FXa to J. Biol. Chem. 1976; Scholar; M.E. C. Mann K.G. J. Biol. Chem. 1981; Scholar) and the binding of FXa to FVa (49Pryzdial E.L.G. Mann K.G. J. Biol. Chem. 1991; 266: 8969-8977Google Scholar). In the current the acceleration of tPA by FXa and FVa was also to be dependent on and This may be by the known increased rate of FVa in the presence of Mann K.G. J. Biol. Chem. 1987; 262: Scholar). the that was (approximately for the of tPA acceleration by Pn-pretreated FVa in the of when Pn of FVa was in the presence of and and EDTA to for tPA activity with of the FVa cofactor activity these data that interaction between FVa and is necessary for FVa to tPA cofactor function and the association of the of the FVa with may be In the of observed that acceleration of tPA was only when a complex could The interaction of FXa with Ca2+ W.R. T.L. Mann K.G. J. Biol. Chem. Scholar; J. Biol. Chem. 1976; Scholar; E.L.G. Mann K.G. J. Biol. Chem. 1991; 266: 8969-8977Google Scholar; J. Biol. Chem. Scholar) and with phospholipid S. K.A. Mann K.G. J. Biol. Chem. 1992; Scholar) is known to a that may be for tPA cofactor the effect of FXa and FVa is localized to sites only where of procoagulant phospholipid the generation of II is the only known for Pg that may also be localized to sites where procoagulant phospholipid is This is through the interaction of annexin proteins with procoagulant phospholipid G.M. J. Biol. Chem. 1990; 265: Scholar; P. D. K. D. Biochemistry. 1991; 30: Scholar; D. K. J. Biol. Chem. Scholar). FXa and the fibrinolytic cofactor activity of annexin II on the association with procoagulant phospholipid (6Cesarman G.M. Guevara C.A. Hajjar K.A. J. Biol. Chem. 1994; 269: 21198-21203Google Scholar) and may be as localized as FXa and FXa and FVa can associate with a of cells and may function to Pn these sites or with Pg to the of procoagulant phospholipid B. J. Biochemistry. Scholar; C.M. J. Biol. Chem. Scholar; E.M. Comfurius P. R.F.A. J. Biochem. 1982; Scholar; P.B. Nesheim M.E. Mann K.G. J. Biol. Chem. 1981; Scholar) and a of a prothrombinase is a clot and may participate in with fibrin to accelerate the of FXa and FVa these sites is Since the of fibrin is is that the effect of prothrombinase on fibrinolysis be on cells that by Pg receptors may on annexin II is in association with these cells M. 1993; Scholar). The fibrinolytic activity of annexin II has been identified on the endothelial surface K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Google Scholar), which is also for prothrombinase assembly G.M. 1985; Scholar). annexin and FVa may to fibrinolysis on the is from the that annexin II may on the surface of cells K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Google Scholar), whereas of FXa and FVa requires cell endothelial cell for Pg has been identified (9Dudani A.K. Cummings C. Hashemi S. Ganz P.R. Thromb. Res. 1993; 69: 185-196Google Scholar), the of 45-kDa protein is cell is The of prothrombinase has also been described in association with J. Biol. Chem. 1991; 266: Scholar; P.B. Mann K.G. J. Biol. Chem. 1985; Scholar) and cells T. M. 1990; Scholar). In Pg binding to these cells and generation of Pn on the surface of was observed to be by Pn pretreatment M. S. Biochem. 1991; Scholar). This is to current with cells known to T. M. 1990; Scholar), these to or Pg is In to a fibrinolytic the that FVa and FXa may accelerate Pn generation the surface of or cells a role in tissue Thromb. 1991; Scholar). The interaction of Pg with fibrin (32Lijnen H.R. Collen D. Methods Enzymol. 1994; 223: 197-206Google Scholar), cells (9Dudani A.K. Cummings C. Hashemi S. Ganz P.R. Thromb. Res. 1993; 69: 185-196Google Scholar; G.M. Guevara C.A. Hajjar K.A. J. Biol. Chem. 1994; 269: 21198-21203Google Scholar; M. Rijken D.C. Lijnen H.R. Collen D. J. Biol. Chem. 1982; 257: 2912-2919Google Scholar; L.A. Dahlberg C.M. Plescia J. Felez J. Kato K. Plow E.F. Biochemistry. 1991; 30: 1682-1691Google Scholar), or (24Kelm Jr., R.J. Swords N.A. Orfeo T. Mann K.G. J. Biol. Chem. 1994; 269: 30147-30153Google Scholar) is known to on the of in the Pg receptors, the acceleration of Pg activation by tPA was observed by to a COOH-terminal in FXa. This that in to the of FXa fibrinolytic function by a identified plasma protein carboxypeptidase B activity Nesheim M.E. J. Biol. Chem. 1995; Scholar) may be the of FVa fibrinolytic activity Pn treatment is the of a COOH-terminal is We and Y. for of T. for the and A. for We also S. and P. for of the