Formation of meizothrombin as intermediate in factor Xa-catalyzed prothrombin activation on endothelial cells. The influence of thrombin on the reaction mechanism

Incubation of prothrombin on cultured human umbilical vein endothelial cells with factor Xa and calcium ions induced the activation of prothrombin. The mechanism of prothrombin activation was analyzed on sodium dodecyl sulfate gels using immuno- and amido-blotting techniques. It was demonstrated that meizothrombin was formed as an intermediate in prothrombin activation on the endothelial cell surface. In addition, considerable amounts of meizothrombin des-fragment-1 accumulated during prothrombin activation and were not further converted to thrombin. Although preincubation of the endothelial cells with thrombin did not influence the formation of meizothrombin, addition of hirudin to the prothrombin activation mixture inhibited the formation of meizothrombin and meizothrombin des-fragment-1 almost completely. This indicated that the activity of endogenously formed thrombin influenced the formation of meizothrombin via a feedback mechanism. The increased formation of meizothrombin and accumulation of meizothrombin des-fragment-1 in a latter phase of prothrombin activation points to a regulatory mechanism in hemostasis which subdues the formation of the procoagulant alpha-thrombin.     

Meizothrombin formation during factor Xa-catalyzed prothrombin activation. Formation in a purified system and in plasma

Meizothrombin and thrombin formation were quantitated during factor Xa-catalyzed activation of human prothrombin in reaction systems containing purified proteins and in plasma. In the purified system considerable amounts of meizothrombin accumulated when prothrombin was activated by factor Xa (with or without accessory components) under initial steady state conditions. The ratio of the rates of meizothrombin and thrombin formation was not influenced by variation of the pH, temperature, or ionic strength of the reaction medium. When 2 microM prothrombin was activated by the complete prothrombinase complex (factor Xa, factor Va, Ca2+, and phospholipid) 80-90% of the initially formed reaction product was meizothrombin. Lowering the prothrombin concentration from 2 to 0.03 microM caused a gradual decrease in the ratio of meizothrombin/thrombin formation from 5 to 0.6. When the phosphatidylserine content of the phospholipid vesicles was varied between 20 and 1 mol % and prothrombin activation was analyzed at 2 microM prothrombin the relative amount of meizothrombin formed decreased from 85 to 55%. With platelets, cephalin, or thromboplastin as procoagulant lipid, thrombin was the major reaction product and only 30-40% of the activation product was meizothrombin. We also analyzed complete time courses of prothrombin activation both with purified proteins and in plasma. In reaction systems with purified proteins substantial amounts of meizothrombin accumulated under a wide variety of experimental conditions. However, little or no meizothrombin was detected in plasma in which coagulation was initiated via the extrinsic pathway with thromboplastin or via the intrinsic pathway with kaolin plus phospholipid (cephalin, platelets, or phosphatidylserine-containing vesicles). Thus, thrombin was the only active prothrombin activation product that accumulated during ex vivo coagulation experiments in plasma.     

The prothrombinase-catalyzed activation of prothrombin proceeds through the intermediate meizothrombin in an ordered, sequential reaction

The activation of bovine prothrombin by prothrombinase (Factor Xa, Factor Va, synthetic phospholipid vesicles, and calcium ion) was studied in the presence of the fluorescent, reversible thrombin inhibitor dansylarginine-N-(3-ethyl-1,5-pentanediyl) amide (DAPA). Recordings of fluorescence intensity during prothrombin activation exhibited maxima that decreased to stable limiting values. These data suggested the transient appearance of the meizothrombin-DAPA complex, which exhibits fluorescence with 1.5-fold greater intensity than the thrombin-DAPA complex. At substrate concentrations well below Km, progress curves could be fitted by equations describing an ordered, sequential conversion of prothrombin to thrombin through the intermediate meizothrombin via two pseudo-first order steps. The pseudo-first order rate constants for both steps varied linearly with enzyme concentration, indicating that both steps are catalyzed by prothrombinase. The progress of the reaction was also monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry analyses of aliquots removed at intervals spanning the reaction. These analyses confirmed both the existence of meizothrombin and its time course as predicted from the equations used to analyze fluorescence intensity profiles. Meizothrombin levels peaked at about 0.3 mol/mol initial prothrombin under the conditions typically studied. In addition, prethrombin 2, which is the intermediate expected from cleavages occurring in the order opposite that required to form meizothrombin, was not observed under any of the conditions examined. These data indicate that prothrombin activation catalyzed by the fully assembled prothrombinase complex proceeds via an ordered, sequential reaction with meizothrombin as the sole intermediate.     

Studies of the role of factor Va in the factor Xa-catalyzed activation of prothrombin, fragment 1.2-prethrombin-2, and dansyl-L-glutamyl-glycyl-L-arginine-meizothrombin in the absence of phospholipid

In order to specifically evaluate the role of Factor Va in the prothrombinase complex, studies of the activation of prothrombin, Fragment 1.2-prethrombin-2, and active-site-blocked meizothrombin were carried out, both in the absence of phospholipid and at concentrations of substrates and Factor Va sufficient to approach saturation in all components. Km values were independent of Factor Va concentrations, whereas kcat (apparent) values approached saturation with respect to Factor Va concentrations. The three respective substrates exhibited the following parameters of kinetics (Km, microM; kcat, s-1 at saturating [Factor Va]): prothrombin (9.0 +/- 0.4; 31 +/- 1); Fragment 1.2-prethrombin-2 (5.4 +/- 0.4; 13 +/- 2); and meizothrombin (3.6 +/- 0.3; 51 +/- 5). Models of kinetics were constructed to interpret the results, and two of these were formally consistent with experimental results. Both models indicated that the variation of kcat(app) with concentrations of Factor Va reflects the formation of a Factor Va-Factor Xa binary complex. Analysis of kinetics indicated Kd values for this interaction of 1.3 +/- 0.1, 3.0 +/- 0.5, and 1.0 +/- 0.1 microM for the three respective substrates. The models differed in the interpretation of Km. One indicated that Km reflects a binary interaction between Factor Xa and prothrombin, whereas the other indicated a binary interaction between Factor Va and prothrombin. Both indicated that two of the three possible binary interactions between the three components would be reflected in Km and kcat values but not the third. To distinguish these models, the binary interactions were studied by extrinsic fluorescence (Va.Xa), light-scattering (Factor Va.prothrombin), and competition kinetics (Xa.II). The first two interactions were detected and were characterized by Kd values of 2.7 +/- 0.1 microM (Va.Xa) and 8.8 +/- 0.8 microM (Factor Va.prothrombin). No active-site-dependent interaction between prothrombin and Factor Xa could be detected in the absence of Factor Va. The results of these studies suggest that Factor Va interacts with both Factor Xa and prothrombin and effectively presents one to the other in the formation of a ternary enzyme-substrate-cofactor complex. In addition, a comparison of the parameters of kinetics of conversion of prothrombin and its intermediates indicates that meizothrombin is the major intermediate of prothrombin activation in the absence, as well as in the presence of phospholipid.     

Mutations in autolytic loop-2 and at Asp554 of human prothrombin that enhance protein C activation by meizothrombin

Thrombin acts on many protein substrates during the hemostatic process. Its specificity for these substrates is modulated through interactions at regions remote from the active site of the thrombin molecule, designated exosites. Exosite interactions can be with the substrate, cofactors such as thrombomodulin, or fragments from prothrombin. The relative activity of alpha-thrombin for fibrinogen is 10 times greater than that for protein C. However, the relative activity of meizothrombin for protein C is 14 times greater than that for fibrinogen. Modulation of thrombin specificity is linked to its Na(+)-binding site and residues in autolytic loop-2 that interact with the Na(+)-binding site. Recombinant prothrombins that yield recombinant meizothrombin (rMT) and rMT des-fragment 1 (rMT(desF1)) enable comparisons of the effects of mutations at the Na(+)-binding residue (Asp(554)) and deletion of loop-2 (Glu(466)-Thr(469)) on the relative activity of meizothrombin for several substrates. Hydrolysis of t-butoxycarbonyl-VPR-p-nitroanilide by alpha-thrombin, recombinant alpha-thrombin, or rMT(desF1) was almost identical, but that by rMT was only 40% of that by alpha-thrombin. Clotting of fibrinogen by rMT and rMT(desF1) was 12-16% of that by alpha-thrombin, as already known. Strikingly, however, although meizothrombins modified by substitution of Asp(554) with either Ala or Leu or by deletion of loop-2 had 6-8 and <1%, respectively, of the clotting activity of alpha-thrombin, the activity of these meizothrombins for protein C was increased to >10 times that of alpha-thrombin. It is proposed that interactions within thrombin that involve autolytic loop-2 and the Na(+)-binding site primarily enhance thrombin action on fibrinogen, but impair thrombin action on protein C.     

Cooperative roles of factor V(a) and phosphatidylserine-containing membranes as cofactors in prothrombin activation

Activation of prothrombin, as catalyzed by the prothrombinase complex (factor X(a), enzyme; factor V(a) and phosphatidylserine (PS)-containing membranes, cofactors), involves production and subsequent proteolysis of two possible intermediates, meizothrombin (MzII(a)) and prethrombin 2 plus fragment 1.2 (Pre2 & F1.2). V(max), K(m), or V(max)/K(m) for all four proteolytic steps was determined as a function of membrane-phospholipid concentration. Proteolysis was monitored using a fluorescent thrombin inhibitor, a chromogenic substrate, and SDS-PAGE. The kinetic constants for the conversion of MzII(a) and Pre2 & F1.2 to thrombin were determined directly. Pre2 & F1.2 conversion was linear in substrate concentration up to 4 microm, whereas MzII(a) proteolysis was saturable. First order rate constants for formation of MzII(a) and Pre2 & F1.2 could not be determined directly and were determined from global fitting of the data to a parallel, sequential model, each step of which was treated by the Michaelis-Menten formalism. The rate of direct conversion to thrombin without release of intermediates from the membrane-V(a)-X(a) complex (i.e. "channeling") also was adjusted because both the membranes and factor V(a) have been shown to cause channeling. k(cat), K(m), or k(cat)/K(m) values were reported for one lipid concentration, for which all X(a) was likely incorporated into a X(a)-V(a) complex on a PS membrane. Comparing previous results, which were obtained either with factor V(a) (Boskovic, D. S., Bajzar, L. S., and Nesheim, M. E. (2001) J. Biol. Chem. 276, 28686-28693) or with membranes individually (Wu, J. R., Zhou, C., Majumder, R., Powers, D. D., Weinreb, G., and Lentz, B. R. (2002) Biochemistry 41, 935-949), with results presented here we conclude that both factor V(a) and PS-containing membranes induce similar rate increases and pathway changes. Moreover, we have determined: 1) factor V(a) has the greatest effect in enhancing rates of individual proteolytic events; 2) PS-containing membranes have the greatest role in increasing the preference for the MzII(a) versus Pre2 pathway; and 3) PS membranes cause approximately 50% of the substrate to be activated via channeling at 50 microm membrane concentration, but factor V(a) extends the range of efficient channeling to much lower or higher membrane concentrations.     

Interactions between heparin and factor Xa inhibition of prothrombin activation

The effects of heparin on prothrombin activation have been examined. Heparin was found to inhibit the rate of prothrombin activation by Factor Xa, calcium and phospholipid. In the absence of phospholipid, heparin had no effect on the rate of prothrombin activation. In contrast, heparin was found to increase the rate of activation of prethrombin-1 and prethrombin-2. Initial velocity studies indicated that heparin blocks lipid stimulation of prothrombin activation. In accord with this, binding studies demonstrated that heparin could displace Factor Xa, and in separate experiments, prothrombin, from phospholipid vesicles.     

Role of proexosite I in factor Va-dependent substrate interactions of prothrombin activation

Regulatory exosite I of thrombin is present on prothrombin in a precursor state (proexosite I) that specifically binds the Tyr(63)-sulfated peptide, hirudin(54-65) (Hir(54-65)(SO(3)(-))) and the nonsulfated analog. The role of proexosite I in the mechanism of factor Va acceleration of prothrombin activation was investigated in kinetic studies of the effects of peptide binding. The initial rate of human prothrombin activation by factor Xa was inhibited by the peptides in the presence of factor Va but not in the absence of the cofactor. Factor Xa and factor Va did not bind the peptide with significant affinity compared with prothrombin. Maximum inhibition reduced the factor Va-accelerated rate to a level indistinguishable from the rate in the absence of the cofactor. The effect of Hir(54-65)(SO(3)(-)) on the kinetics of prothrombin activation obeyed a model in which binding of the peptide to proexosite I prevented productive prothrombin interactions with the factor Xa-factor Va complex. Comparison of human and bovine prothrombin as substrates demonstrated a similar correlation between peptide binding and inhibition of factor Va acceleration. Inhibition of prothrombin activation by hirudin peptides was opposed by assembly on phospholipid vesicles of the membrane-bound factor Xa-factor-Va-prothrombin complex. Factor Va interactions of human and bovine prothrombin activation are concluded to share a common mechanism in which proexosite I participates in productive interactions of prothrombin as the substrate of the factor Xa-factor Va complex, possibly by directly mediating productive prothrombin-factor Va binding.     

Human plasma lipoproteins as accelerators of prothrombin activation.

The activation rate of bovine prothrombin by Factor Xa and Ca2+ has long been known to be greatly enhanced by addition of phospholipid. Upon substitution of human plasma lipoproteins for phospholipid (cephalin) in this activation system, only very low density lipoprotein enhances prothrombin activation. Low density lipoprotein and high density lipoprotein have no stimulatory effect on prothrombin activation. On the other hand, the sonicated lipid extracts from very low, low, and high density lipoproteins all can substitute for phospholipid in potentiating prothrombin activation. The efficiency of each lipid extract, in this regard, depends upon its source of extraction, and is greatest for the lipid extract of very low density lipoprotein.     

人凝血酶原复合物生产过程中凝血因子活化分析

目的通过比较以组分Ⅲ沉淀和血浆为原料制备人凝血酶原复合物(Prothrombin complex concentrates,PCC)过程中凝血因子活化情况,为选择最适PCC制备原料提供数据支持。方法分别对以组分Ⅲ沉淀和血浆为原料制备PCC过程中中间品的活化的凝血因子活性和人凝血酶活性两个项目进行检定,分析凝血因子的活化情况。观察以组分Ⅲ沉淀为原料制备PCC过程中添加肝素能否抑制PCC中凝血因子的活化。结果以组分Ⅲ沉淀为原料制备的PCC中间品活化的凝血因子活性和人凝血酶活性两个项目均不合格。以组分Ⅲ沉淀为原料制备PCC生产过程中添加肝素后,PCC中间品的活化的凝血因子活性和人凝血酶活性均不合格。以血浆为原料制备的PCC中间品活化的凝血因子活性和人凝血酶活性两个项目均合格。结论组分Ⅲ沉淀为原料制备PCC会增加凝血因子活化的风险,新鲜冰冻血浆可作为制备PCC的原料。     

Structural comparisons of meizothrombin and its precursor prothrombin in the presence or absence of procoagulant membranes.

A stable form of meizothrombin derived from an active-site (Ser528----Ala) mutant of recombinant bovine prothrombin [Pei et al. (1991) J. Biol. Chem. 266, 9598-9604] has been used to determine the physical properties and conformation of meizothrombin both in solution and when bound to a procoagulant membrane. As determined with quasi-elastic light scattering, meizothrombin and prothrombin had similar molecular dimensions normal to a membrane (9.4 +/- 1.0 nm) and similar binding affinities to procoagulant membranes (1.8 +/- 0.2 microM at 0.4 M NaCl). However, meizothrombin had a greater tendency to form oligomers or aggregates in solution. The enhanced oligomerization of meizothrombin was also evidenced by a high apparent z-weighted molecular weight in equilibrium sedimentation experiments at low spin speeds. However, velocity sedimentation experiments performed at high spin speeds demonstrated the same sedimentation coefficient for meizothrombin (s20,w(0) = 4.7 +/- 0.2 S) as for prothrombin (s20,w(0) = 4.7 +/- 0.15 S). Circular dichroism measurements revealed minor differences in protein secondary structure between meizothrombin and prothrombin either in the presence or in the absence of phospholipid membranes, as reflected in an increased theta 222/theta 208 ratio in meizothrombin relative to prothrombin. The main endotherm of the meizothrombin thermal denaturation profile in a Ca(2+)-containing buffer, as determined by differential scanning calorimetry, was indistinguishable from that of prothrombin. However, in the presence of phosphatidylserine-containing membranes, the peak temperatures of denaturation profiles of meizothrombin were distinct from those of prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mathematical model of the activation of prothrombin by factor X a and factor V t

A mathematical model of prothrombin activation is being proposed which includes the feedback mechanism of thrombin and the alteration of factor V by thrombin. This model is in good agreement with experimental data for the dependence of the rate of thrombin formation on the concentrations of factors V and X _a . In particular, it correctly predicts the existence and location of a maximum in both of these cases.     

Interactions between heparin and factor Xa. Inhibition of prothrombin activation.

The effects of heparin on prothrombin activation have been examined. Heparin was found to inhibit the rate of prothrombin activation by Factor Xa, calcium and phospholipid. In the absence of phospholipid, heparin had no effect on the rate of prothrombin activation. In contrast, heparin was found to increase the rate of activation of prethrombin-1 and prethrombin-2. Initial velocity studies indicated that heparin blocks lipid stimulation of prothrombin activation. In accord with this, binding studies demonstrated that heparin could displace Factor Xa, and in separate experiments, prothrombin, from phospholipid vesicles.     

Mechanisms of factor Xa-catalyzed cleavage of the factor VIIIa A1 subunit resulting in cofactor inactivation

Activation of factor VIII by factor Xa is followed by proteolytic inactivation resulting from cleavage within the A1 subunit (residues 1-372) of factor VIIIa. Factor Xa attacks two sites in A1, Arg(336), which precedes the highly acidic C-terminal region, and a recently identified site at Lys(36). By using isolated A1 subunit as substrate for proteolysis, production of the terminal fragment, A1(37-336), was shown to proceed via two pathways identified by the intermediates A1(1-336) and A1(37-372) and generated by initial cleavage at Arg(336) and Lys(36), respectively. Appearance of the terminal product by the former pathway was 7-8-fold slower than the product obtained by the latter pathway. The isolated A1 subunit was cleaved slowly, independent of the presence of phospholipid. The A1/A3-C1-C2 dimer demonstrated an approximately 3-fold increased cleavage rate constant, and inclusion of phospholipid further enhanced this value by approximately 2-fold. Although association of A1 or A1(37-372) with A3-C1-C2 enhanced the rate of cleavage at Arg(336), inclusion of A3-C1-C2 did not affect the cleavage at Lys(36) in A1(1-336). A synthetic peptide 337-372 blocked the cleavage at Lys(36) (IC(50) = 230 microm) while showing little if any effect on cleavage at Arg(336). Proteolysis at Lys(36), and to a lesser extent Arg(336), was inhibited in a dose-dependent manner by heparin. These results suggest that inactivating cleavages catalyzed by factor Xa at Lys(36) and Arg(336) are regulated in part by the A3-C1-C2 subunit. Furthermore, cleavage at Lys(36) appears to be selectively modulated by the C-terminal acidic region of A1, a region that may interact with factor Xa via its heparin-binding exosite.     

Deuterium solvent isotope effect and proton-inventory studies of factor Xa-catalyzed reactions

Kinetic solvent isotope effects (KSIEs) for the factor Xa (FXa)-catalyzed activation of prothrombin in the presence and absence of factor Va (FVa) and 5.0 x 10(-5) M phospholipid vesicles are slightly inverse, 0.82-0.93, when substrate concentrations are at 0.2 Km. This is consistent with the rate-determining association of the enzyme-prothrombin assembly, rather than the rate-limiting chemical transformation. FVa is known to effect a major conformational change to expose the first scissile bond in prothrombin, which is the likely event triggering a major solvent rearrangement. At prothrombin concentrations > 5 Km, the KSIE is 1.6 +/- 0.3, when FXa is in a 1:1 ratio with FVa but becomes increasingly inverse, 0.30 +/- 0.05 and 0.19 +/- 0.04, when FXa/FVa is 1:4, with an increasing FXa and substrate concentration. The rate-determining step changes with the conditions, but the chemical step is not limiting under any circumstance. This corroborates the proposed predominance of the meizothrombin pathway when FXa is well-saturated with the prothrombin complex. In contrast, the FXa-catalyzed hydrolysis of N-alpha-Z-D-Arg-Gly-Arg-pNA.2HCl (S-2765) and H-D-Ile-L-Pro-L-Arg-pNA.HCl (S-2288) is most consistent with two-proton bridges forming at the transition state between Ser195 OgammaH and His57 N(epsilon)2 and His57 Ndelta1 and Asp102 COObeta- at the active site, with transition-state fractionation factors of phi1 = phi2 = 0.57 +/- 0.07 and phiS = 0.78 +/- 0.16 for solvent rearrangement for S-2765 and phi1 = phi2 = 0.674 +/- 0.001 for S-2288 under enzyme saturation with the substrate at pH 8.40 and 25.0 +/- 0.1 degrees C. The rate-determining step(s) in these reactions is most likely the cleavage of the C-N bond and departure of the leaving group.