Plasmin-mediated activation and inactivation of thrombin-activatable fibrinolysis inhibitor

Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates the fibrin cofactor function of tissue-type plasminogen activator-mediated plasmin formation and subsequently fibrin degradation. In the present study, we focused on the role of plasmin in the regulation of TAFIa activity. Upon incubation with plasmin, TAFIa activity was generated, which was unstable at 37 degrees C. Analysis of the cleavage pattern showed that TAFI was cleaved at Arg(92), releasing the activation peptide from the 35.8-kDa catalytic domain. The presence of the 35.8-kDa fragment paralleled the time course of generation and loss of TAFIa activity. This suggested that, in the presence of plasmin, TAFIa is probably inactivated by proteolysis rather than by conformational instability. TAFI was also cleaved at Arg(302), Lys(327), and Arg(330), resulting in a approximately 44.3-kDa fragment and several smaller fragments. The 44.3-kDa fragment is no longer activatable since it lacks part of the catalytic center. We concluded that plasmin can cleave at several sites in TAFI and that this contributes to the regulation of TAFI and TAFIa.     

The role of the membrane in the inactivation of factor va by plasmin. Amino acid region 307-348 of factor V plays a critical role in factor Va cofactor function

The mechanism of inactivation of bovine factor Va by plasmin was studied in the presence and absence of phospholipid vesicles (PCPS vesicles). Following 60-min incubation with plasmin (4 nm) membrane-bound factor Va (400 nm) is completely inactive, whereas in the absence of phospholipid vesicles following a 1-h incubation period, the cofactor retains 90% of its initial cofactor activity. Amino acid sequencing of the fragments deriving from cleavage of factor Va by plasmin demonstrated that while both chains of factor Va are cleaved by plasmin, only cleavage of the heavy chain correlates with inactivation of the cofactor. In the presence of a membrane surface the heavy chain of the bovine cofactor is first cleaved at Arg(348) to generate a fragment of M(r) 47,000 containing the NH(2)-terminal part of the cofactor (amino acid residues 1-348) and a M(r) 42,000 fragment (amino acid residues 349-713). This cleavage is associated with minimal loss in cofactor activity. Complete loss of activity of the membrane-bound cofactor coincides with three cleavages at the COOH-terminal portion of the M(r) 47,000 fragment: Lys(309), Lys(310), and Arg(313). These cleavages result in the release of the COOH terminus of the molecule and the production of a M(r) 40,000 fragment containing the NH(2)-terminal portion of the factor Va molecule. Factor Va was treated with plasmin in the absence of phospholipid vesicles followed by the addition of PCPS vesicles and activated protein C (APC). A rapid inactivation of the cofactor was observed as a result of cleavage of the M(r) 47,000 fragment at Arg(306) by APC and appearance of a M(r) 39,000 fragment. These data suggest a critical role of the amino acid sequence 307-348 of factor Va. A 42-amino acid peptide encompassing the region 307-348 of human factor Va (N42R) was found to be a good inhibitor of factor Va clotting activity with an IC(50) of approximately 1.3 microm. These data suggest that plasmin is a potent inactivator of factor Va and that region 307-348 of the cofactor plays a critical role in cofactor function and may be responsible for the interaction of the cofactor with factor Xa and/or prothrombin.     

Insecticidal activity and processing in larval gut juices of genetically engineered 130-kDa proteins of Bacillus thuringiensis subsp. aizawai.

The 130-kDa insecticidal protein (IP) of Bacillus thuringiensis subsp. aizawai is proteolytically processed in the gut juice of susceptible insect larvae to yield an insecticidally active 60-kDa fragment. Twenty-seven mutant IP genes with the replacement of codons for Arg and Lys with codons for Gln in the active fragment and its adjacent regions of the 130-kDa IP were constructed by site-directed mutagenesis and expressed in Escherichia coli cells. The produced mutant IPs at Arg87, Arg131, Arg198, Arg311, Arg368, Arg402, Arg458, Arg502, Arg512, Arg524, Arg526, Arg528, and Arg601 had reduced insecticidal activity against Spodoptera litura larvae. The mutant at Arg601 was sensitive to proteolytic digestion in the gut juice of S. litura larvae. Although the mutants at Arg619, Lys622, and Lys637 had nearly the same activity as that of the wild type, the mutant with the triple replacement at Arg619, Lys622, and Lys637 was 2.5 times more active against S. litura larvae than the wild type. This triple mutant showed a slightly different processing profile in the gut juice than that of the wild type.     

Biosynthesis of a novel recombinant peptide derived from hPTH(1-34)

A recombinant human parathyroid hormone fragment, Pro-Pro-[Arg(11)]hPTH(1-34)-Pro-Pro (MW=4550 Da), was developed by substituting Arg for Leu at position 11 and adding Pro-Pro at the carboxyl terminus. Following a single injection (0.5-13.5 μg/bird) of the rhPTH fragment, the serum calcium level in chickens increased 12-42% (P<0.05) after 1h as determined by the Parson's Chicken Assay. The functional activity of Pro-Pro-[Arg(11)]hPTH(1-34)-Pro-Pro may be due to removal of the N-terminus Pro-Pro- by X-prolyl dipeptidyl peptidase IV (DPPIV) in vivo, increasing its activity compared to Pro-Pro-hPTH(1-34). This artificial rhPTH fragment could be used to increase calcium mobilization and potentially improve bone health.     

Glucagon is processed to the (19-29) fragment at the level of the hepatocyte membrane

Upon incubation with hepatic plasma membranes, glucagon is processed into its (19-29) C-terminal fragment. This suggests that, in physiological conditions, glucagon is processed in a target tissue at the level of its Arg17-Arg18 basic doublet, leading to the production of a fragment which is known to display an original biological specificity, namely the modulation of the calcium pump present in hepatocyte plasma membrane.     

Conformational study of the neurotensin and substance P fragments: Pro-Arg-Arg-Pro and Arg-Pro-Lys-Pro

The conformational behaviour of the basic hydrophilic Pro-Arg-Arg-Pro and Arg-Pro-Lys-Pro peptides, neurotensin (NT) and Substance P fragments, has been taken up by semi-empirical calculations. The presence of two Pro residues prevents these peptides from giving any folded structure (alpha helix, beta turn . . .). In both peptides the most stable conformations are essentially relative to more or less stretched structures; structures involving one or more residues in a gamma turn form are often encountered in Pro-Arg-Arg-Pro peptide while mixed structures involving residues in very different conformations are found for the Arg-Pro-Lys-Pro-peptide. In both peptides, positively charged Lys and Arg side-chains most often point in opposite directions. The Pro-Arg-Arg-Pro peptide is part of the active NT (7-13) fragment where both Arg residues are necessary to the activity. A tentative study shows that the hydrophilic tetrapeptide induces NT (7-13) stretched conformations.     

Identification of the collagen-binding site of the von Willebrand factor A3-domain

Von Willebrand factor (vWF) is a multimeric glycoprotein that mediates platelet adhesion and thrombus formation at sites of vascular injury. vWF functions as a molecular bridge between collagen and platelet receptor glycoprotein Ib. The major collagen-binding site of vWF is contained within the A3 domain, but its precise location is unknown. To localize the collagen-binding site, we determined the crystal structure of A3 in complex with an Fab fragment of antibody RU5 that inhibits collagen binding. The structure shows that RU5 recognizes a nonlinear epitope consisting of residues 962-966, 981-997, and 1022-1026. Alanine mutants were constructed of residues Arg(963), Glu(987), His(990), Arg(1016), and His(1023), located in or close to the epitope. Mutants were expressed as fully processed multimeric vWF. Mutation of His(1023) abolished collagen binding, whereas mutation of Arg(963) and Arg(1016) reduced collagen binding by 25-35%. These residues are part of loops alpha3beta4 and alpha1beta2 and alpha-helix 3, respectively, and lie near the bottom face of the domain. His(1023) and flanking residues display multiple conformations in available A3-crystal structures, suggesting that binding of A3 to collagen involves an induced-fit mechanism. The collagen-binding site of A3 is located distant from the top face of the domain where collagen-binding sites are found in homologous integrin I domains.     

Activated protein C-catalyzed inactivation of human factor VIII and factor VIIIa. Identification of cleavage sites and correlation of proteolysis with cofactor activity

Human factor VIII and factor VIIIa were proteolytically inactivated by activated protein C. Cleavages occurred within the heavy chain (contiguous A1-A2-B domains) of factor VIII and in the heavy chain-derived A1 and A2 subunits of factor VIIIa, whereas no proteolysis was observed in the light chain or light chain-derived A3-C1-C2 subunit. Reactivity to an anti-A2 domain monoclonal antibody and NH2-terminal sequence analysis of three terminal digest fragments from factor VIII allowed ordering of fragments and identification of cleavage sites. Fragment A1 was derived from the NH2 terminus and resulted from cleavage at Arg336-Met337. The A2 domain was bisected following cleavage at Arg562-Gly563 and yielded fragments designated A2N and A2C. A third cleavage site is proposed at the A2-B junction (Arg740-Ser741) since fragment A2C was of equivalent size when derived either from factor VIII or factor VIIIa. The site at Arg562 was preferentially cleaved first in factor VIII(alpha) compared with the site at Arg336, and it was this initial cleavage that most closely correlated with the loss of cofactor activity. Factor VIIIa was inactivated 5-fold faster than factor VIII, possibly as a result of increased protease utilization of the site at Arg562 when the A2 subunit is not contiguous with the A1 domain. When initial cleavage occurred at Arg336, it appeared to preclude subsequent cleavage at Arg562, possibly by promoting dissociation of the A2 domain (subunit) from the A1/light chain dimer. This conclusion was supported by the failure of protease treated A1/A3-C1-C2 dimer to bind A2 subunit and gel filtration analysis that showed dissociation of the A2 domain-derived fragments, A2N and A2C, from the A1 fragment/light chain dimer. These results suggest a mechanism for activated protein C-catalyzed inactivation of factor VIII(alpha) involving both covalent alteration and fragment dissociation.     

Cell-mediated cleavage of Pseudomonas exotoxin between Arg279 and Gly280 generates the enzymatically active fragment which translocates to the cytosol.

Pseudomonas exotoxin (PE) is a three-domain toxin which is cleaved by a cellular protease within cells and then reduced to generate two prominent fragments (Ogata, M., Chaudhary, V. K., Pastan, I., and FitzGerald, D. J. (1990) J. Biol. Chem. 265, 20678-20685). The N-terminal fragment is 28 kDa in size and contains the binding domain. The 37-kDa C-terminal fragment, which translocates to the cytosol, contains the translocation domain and the ADP-ribosylation domain. Cleavage followed by reduction is essential for toxicity since mutant forms of the toxin that cannot be cleaved by cells are nontoxic. Previous results with these mutants suggest that cleavage occurred in an arginine-rich (arginine residues are at positions 274, 276, and 279) disulfide loop near the beginning of the translocation domain, but the exact site of cleavage was not determined. Since very few molecules of the 37-kDa fragment are generated within cells it was not possible to determine the site of cleavage by performing a conventional N-terminal sequence analysis of the 37-kDa fragment. Two experimental approaches were used to overcome this limitation. First, existing amino acids near the cleavage sites were replaced with methionine residues; this was followed by the addition of [35S]methionine-labeled versions of these toxins to cells. The pattern of radioactive toxin fragments recovered from the cells indicated that the toxin was cleaved either just before or just after Arg279. Second, [3H]leucine-labeled toxin was produced and added to the cells. Sequential Edman degradations were performed on the small amount of radioactive 37-kDa fragment that could be recovered from toxin-treated cells. A peak of radioactivity in the fifth fraction indicated that leucine was the 5th amino acid on the C-terminal side of the cleavage site. This result confirmed that cleavage was between Arg279 and Gly280.     

Fate of membrane-bound reactants and products during the activation of human prothrombin by prothrombinase

Membrane binding by prothrombin, mediated by its N-terminal fragment 1 (F1) domain, plays an essential role in its proteolytic activation by prothrombinase. Thrombin is produced in two cleavage reactions. One at Arg(320) yields the proteinase meizothrombin that retains membrane binding properties. The second, at Arg(271), yields thrombin and severs covalent linkage with the N-terminal fragment 1.2 (F12) region. Covalent linkage with the membrane binding domain is also lost when prethrombin 2 (P2) and F12 are produced following initial cleavage at Arg(271). We show that at the physiological concentration of prothrombin, thrombin formation results in rapid release of the proteinase into solution. Product release from the surface can be explained by the weak interaction between the proteinase and F12 domains. In contrast, the zymogen intermediate P2, formed following cleavage at Arg(271), accumulates on the surface because of a approximately 20-fold higher affinity for F12. By kinetic studies, we show that this enhanced binding adequately explains the ability of unexpectedly low concentrations of F12 to greatly enhance the conversion of P2 to thrombin. Thus, the utilization of all three possible substrate species by prothrombinase is regulated by their ability to bind membranes regardless of whether covalent linkage to the F12 region is maintained. The product, thrombin, interacts with sufficiently poor affinity with F12 so that it is rapidly released from its site of production to participate in its numerous hemostatic functions.     

X-linked dominant Charcot-Marie-Tooth neuropathy (CMTX): new mutations in the connexin32 gene

X-linked dominant Charcot-Marie-Tooth (CMTX) neuropathy has been mapped to the Xq13 region. Subsequently, several mutations that could account for CMTX have been detected in the coding part of the connexin32 (Cx32) gene, which is located within this region. In order to develop more specific diagnostic tools, we have begun a systematic screening of families with dominant CMTX for mutations in the coding region of the Cx32 gene. This report describes a study of ten families and different mutations segregating with the disease were detected in five of them. In addition to the previously reported Arg22stop and Arg215Trp substitutions, three novel mutations are described, including two different missense mutations at codon Arg22 (Arg22Pro and Arg22Gly), and a nonsense mutation at codon Trp133. The identification of new CMTX-causing mutations is a critical step for carrier detection and presymptomatic diagnosis, and should provide essential information on the structure-function relationship of Cx32 in vitro as well as in vivo. Received: 11 January 1996 / Revised: 29 March 1996     

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.     

A novel apolipoprotein A-1 variant, Arg173Pro, associated with cardiac and cutaneous amyloidosis

An American kindred was found to have hereditary amyloidosis with cutaneous and cardiac involvement. Characterization of fibrils isolated from skin identified the amyloid protein as the N-terminal 90 to 100 residues of apolipoprotein A-1. Sequence of the apolipoprotein A-1 gene was normal except for a G/C transversion at position 1638 which predicts an Arg to Pro substitution at residue 173. This mutation, unlike previously described amyloidogenic mutations is not in the N-terminal fragment which is incorporated into the fibril. The mutation is at the same residue as in apolipoprotein A-1 Milano (Arg173Cys) which does not result in amyloid formation. Decreased plasma HDL cholesterol levels in carriers of the Arg173Pro mutation suggest an increased rate of catabolism as has been shown for the amyloidogenic Gly26Arg mutation. This suggests that altered metabolism caused by the mutation may be a significant factor in apolipoprotein A-1 fibrillogenesis.     

Minimum stable structure of the receptor for advanced glycation end product possesses multi ligand binding ability

The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor involved in the development of diabetic complications. Although the soluble form of the extracellular domain maintains the ability to bind multi-ligands, it is unstable and degrades into several peptide species during storage. Proteolysis with thrombin or factor Xa revealed several protease sensitive sites. Most sensitive site is located between Arg228 and Val229, and peptide bond next to Arg216, Arg116, Arg114 and Trp271 are also cleaved. Seven truncated extracellular domains of RAGE were engineered in order to obtain a stable soluble fragment. RAGE 143 (Ala23-Thr143) is not only protease resistant but also shows the same ligand-binding ability as that of the full-length extracellular domain. The resultant minimum RAGE 143 works as a stable recognition devise to detect advanced glycation end products (AGEs).     

The fibrin-binding site of human plasminogen. Arginines 32 and 34 are essential for fibrin affinity of the kringle 1 domain

Kringle 1 (Tyr 79/Leu 80-His 167 and Tyr 79/Leu 80-Tyr 173), a chymotryptic fragment of human plasminogen that has high affinity for fibrin and omega-aminocarboxylic acids, has been subjected to modification with 1,2-cyclohexanedione to identify arginine residues essential for ligand binding. Reaction of 1,2-cyclohexanedione with kringle 1 was found to rapidly abolish the fibrin-Sepharose affinity of the fragment, whereas the affinity for lysine-Sepharose was lost at a significantly slower rate. Successive affinity chromatography of modified kringle 1 on fibrin- and lysine-Sepharose was used to separate kringle 1 that lost affinity for fibrin-, but retained affinity for lysine-Sepharose from kringle 1 that lost affinity for both affinants. The modified proteins were subjected to structural studies in order to locate the labeled arginine residues in kringle 1. These studies have revealed that modification of Arg 34 leads to the loss of both the fibrin- and lysine-Sepharose affinities of kringle 1, whereas reaction of Arg 32 abolishes fibrin affinity but leaves lysine-Sepharose affinity unaltered. The results suggest that Arg 32 and Arg 34 are both involved in fibrin binding and that Arg 34 is also involved in binding omega-aminocarboxylic acids. Previous NMR studies on kringles have indeed shown that the segment containing residue 34 is in the proximity of and interacts with the omega-aminocarboxylic acid-binding site. This interaction may explain the influence of omega-aminocarboxylic acids on fibrin binding by kringle 1.     

Structural changes in the cytoplasmic domain of phospholamban by phosphorylation at Ser16: a molecular dynamics study

Phospholamban is a 52-residue integral membrane protein that regulates the activity of the sarcoplasmic reticulum calcium pump in cardiac muscle. Its inhibitory action is relieved when phospholamban is phosphorylated at Ser16 by cAMP-dependent protein kinase. To computationally explore all possible conformations of the phosphorylated form, and thereby to understand the structural effects of phosphorylation, replica-exchange molecular dynamics (REMD) was applied to the cytoplasmic domain that includes Ser16. The simulations showed that (i) without phosphorylation, the region from Lys3 to Ser16 takes all alpha-helical conformations; (ii) when phosphorylated, the alpha-helix is partially unwound in the C-terminal part (from Ser10 to Ala15) resulting in less extended conformations; (iii) the phosphate at Ser16 forms salt bridges with Arg9, Arg13, and/or Arg14; and (iv) the salt bridges with Arg13 and Arg14 distort the alpha-helix and induce unwinding of the C-terminal part. We then applied conventional all-atom molecular dynamics simulations to the full-length phospholamban in the phospholipid bilayer. The results were consistent with those obtained with REMD simulations, suggesting that the transmembrane part of phospholamban and the lipid bilayer itself have only minor effects on the conformational changes in the cytoplasmic domain. The distortions caused by the salt bridges involving the phosphate at Ser16 readily explain the relief of the inhibitory effect of phospholamban by phosphorylation, as they will substantially reduce the population of all helical conformations, which are presumably required for the binding to the calcium pump. This will also be the mechanism for releasing the phosphorylated phospholamban from kinase.     

Analysis of the transforming growth factor-beta1 (TGF-beta1) codon 25 gene polymorphism by LightCycler-analysis in patients with chronic hepatitis C infection

The polymorphism at position 25 of the gene encoding transforming growth factor-beta1 (TGF-beta1), which changes the amino acid sequence of the signal peptide sequence (arginine to proline), is causing a variation in TGF-beta1 production. The homozygous genotype (Arg25Arg) is associated with higher TGF-beta1 production than the heterozygous (Arg25Pro) genotype. Therefore, the possible involvement of this genetic variation in the TGF-beta1 gene for induction and progression of various diseases is under close investigation. At present, several labor-intensive established assays ranging from amplification refractory mutation system (ARMS)-PCR methodologies, sequence specific oligonucleotide probing (SSOP), restriction fragment length polymorphism (RFLP) analysis, 5' nuclease assays, and specialized fingerprinting protocols are applied to analyze the polymorphism in question. We developed a novel approach for analyzing this polymorphism in a LightCycler system and determined the allele frequency distributions between patients with different degrees of hepatic fibrosis induced by chronic hepatitis C virus infection. In patients with severe hepatic fibrosis (METAVIR-score 3-4), the Pro25 allele was twice as frequent compared to patients with mild fibrosis (METAVIR-score 0-2). However, we found no association of necroinflammatory activity and genotype distribution. This suggests that the stage of hepatic fibrosis, rather than the grade (inflammation), is influenced by the presence of proline at codon 25 in patients with chronic hepatitis C.     

Structural and functional studies of the interaction of the eukaryotic elongation factor EF-2 with GTP and ribosomes

The structure of the guanosine nucleotide binding site of EF-2 was studied by affinity labelling with the GTP analogue, oxidized GTP (oGTP), and by amino acid sequencing of polypeptides generated after partial degradation with trypsin and N-chlorosuccinimide. Native EF-2 contains two exposed trypsin-sensitive cleavage sites. One site is at Arg66 with a second site at Lys571/Lys572. oGTP was covalently bound to the factor between Arg66 and Lys571. After further cleavage of this fragment with the tryptophan-specific cleavage reagent N-chlorosuccinimide, oGTP was found associated with a polypeptide fragment originating from a cleavage at Trp261 and Trp343. The covalent oGTP . EF-2 complex was capable of forming a high-affinity complex with ribosomes, indicating that oGTP, in this respect, induced a conformation in EF-2 indistinguishable from that produced by GTP. Although GTP could be substituted by non-covalently linked oGTP in the factor and ribosome-dependent GTPase reaction, the factor was unable to utilize the covalently bound oGTP as a substrate. This indicates that the conformational flexibility in EF-2 required for the ribosomal activation of the GTPase was inhibited by the covalent attachment of the nucleotide to the factor. EF-2 cleaved at Arg66 were unable to form the high-affinity complex with ribosomes while retaining the ability to form the low-affinity complex and to hydrolyse GTP. The second cleavage at Lys571/Lys572 was accompanied by a total loss of both the low-affinity binding and the GTPase activity.     

Expression of allosteric linkage between the sodium ion binding site and exosite I of thrombin during prothrombin activation

The specificity of thrombin for procoagulant and anticoagulant substrates is regulated allosterically by Na+. Ordered cleavage of prothrombin (ProT) at Arg320 by the prothrombinase complex generates proteolytically active, meizothrombin (MzT), followed by cleavage at Arg271 to produce thrombin and fragment 1.2. The alternative pathway of initial cleavage at Arg271 produces the inactive zymogen form, the prethrombin 2 (Pre 2).fragment 1.2 complex, which is cleaved subsequently at Arg320. Cleavage at Arg320 of ProT or prethrombin 1 (Pre 1) activates the catalytic site and the precursor form of exosite I (proexosite I). To determine the pathway of expression of Na+-(pro)exosite I linkage during ProT activation, the effects of Na+ on the affinity of fluorescein-labeled hirudin-(54-65) ([5F]Hir-(54-65)(SO-3)) for the zymogens, ProT, Pre 1, and Pre 2, and for the proteinases, MzT and MzT-desfragment 1 (MzT(-F1)) were quantitated. The zymogens showed no significant linkage between proexosite I and Na+, whereas cleavage at Arg320 caused the affinities of MzT and MzT(-F1) for [5F]Hir-(54-65)(SO-3) to be enhanced by Na+ 8- to 10-fold and 5- to 6-fold, respectively. MzT and MzT(-F1) showed kinetically different mechanisms of Na+ enhancement of chromogenic substrate hydrolysis. The results demonstrate for the first time that MzT is regulated allosterically by Na+. The results suggest that the distinctive procoagulant substrate specificity of MzT, in activating factor V and factor VIII on membranes, and the anticoagulant, membrane-modulated activation of protein C by MzT bound to thrombomodulin are regulated by Na+-induced allosteric transition. Further, the Na+ enhancement in MzT activity and exosite I affinity may function in directing the sequential ProT activation pathway by accelerating thrombin formation from the MzT fast form.