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PubMed2004

Factor VIIa-mediated tenase function on activated platelets under flow.

BACKGROUND: Tissue factor (TF) and/or active factor (F)VIIa may be stored inside resting platelets. OBJECTIVES: The objective of this study was to examine if platelets, following activation of GPVI, could support tenase and prothrombinase activity without any exogenously added tissue factor. METHODS: Thrombin (IIa) formation on gel-filtered platelets with added factors or the clotting of platelet-free plasma (PFP) or platelet-rich plasma (PRP) supplemented with corn trypsin inhibitor (CTI) (to inhibit factor XIIa) was studied in well plate assays with a fluorogenic thrombin substrate or in flow assays by fibrin visualization. RESULTS: Pretreatment of convulxin (CVX)-stimulated, fibrinogen-adherent, gel-filtered platelets with anti-TF, anti-FVII/VIIa, or 1 nm PPACK [inhibitor of FVIIa, factor XIa and factor (F)IIa] delayed fibrin deposition on platelets perfused with PFP/CTI at 62.5 s(-1). Anti-TF or anti-FVII/VIIa also attenuated thrombin generation in plate assays using recalcified PRP/CTI treated with CVX. Anti-TF or anti-FVII/VIIa (but not inhibited factor IXa) delayed the burst in thrombin production by gel-filtered platelets suspended in prothrombin and CVX by 14 min and 40 min, respectively. Anti-FVII/VIIa completely eliminated thrombin generation on fibrinogen-adherent, gel-filtered platelets pretreated with 10 micro m PPACK and 10 micro m EGR-CK [inhibitor of factor (F)Xa], rinsed, and then supplemented with CVX, prothrombin, and FX. Addition of anionic phospholipid to PFP/CTI or to a mixture of prothrombin, FX, and recVIIa was not sufficient to generate detectable tenase activity. Lastly, isolated, unactivated neutrophils suspended in FX, FII and recVIIa supported a very low level of thrombin generation sensitive to antagonism of P-selectin, CD18, and TF. CONCLUSIONS: Activated platelets supported tenase and prothrombinase activity by elevating the function or level of FVIIa and exposing active FVIIa or FVIIa-cofactor(s), distinct from anionic lipid, that may be, in part, TF.

Animals↗
PubMed1980

New fluorogenic peptide substrates for plasmin.

Fluorogenic peptides, peptidyl-4-methylcoumaryl-7-amides (MCA), containing COOH-terminal lysine residues, were newly synthesized and tested as substrates for plasmin. Among six peptidyl-MCA's, Boc-Val-Leu-Lys-MCA and Boc-Glu-Lys-Lys-MCA were found to be useful for the specific and sensitive assay of plasmin. The Km values estimated from Line-weaver-Burk plots for these substrates using human and bovine plasmins were in the region of 10(-4) M. Boc-Glu-Lys-Lys-MCA was slightly hydrolyzed by bovine plasma kallikrein, and Boc-Val-Leu-Lys-MCA was slightly hydrolyzed by human and hog urinary kallikreins and hog pancreatic kallikrein. However, both of the fluorogenic peptides were essentially unaffected by urokinase, alpha-thrombin, Factor Xa, Factor IXa, Factor XIa, and Factor XIIa. It was confirmed that plasmin hydrolyzed Boc-Glu-Lys-Lys-MCA, cleaving the lysyl-MCA bond, but not the lysyl-lysyl bond. These fluorogenic peptides were resistant to human plasmin activated by streptokinase. Boc-Glu-Lys-Lys-MCA was not hydrolyzed by human plasmin or plasminogen in the presence of more than a 5-fold molar excess of streptokinase. The sensitivity of Boc-Val-Leu-Lys- of more than a 5-fold molar excess of streptokinase. The sensitivity of Boc-Val-Leu-Lys-MCA to human plasmin was also reduced, but plasmin retained 35% of the maximum activity even in the presence of a 20-fold molar excess of streptokinase. These results suggest that streptokinase-plasmin complex has essentially no activity towards Boc-Glu-Lys-Lys-MCA.

Animals↗
PubMed1977

Demonstration and mode of action of an inhibitor for activated Hageman factor (factor XIIa) of the intrinsic blood coagulation pathway from Schistosoma mansoni.

An anticoagulant activity from adult Schistosoma mansoni whole worm homogenate is described. The inhibitor appears to be specific for the contact activation step of the intrinsic pathway. Experiments with both human and mouse plasmas have defined the specificity of the inhibitor as follows: (1) It lengthens the partial thromboplastin time of normal plasma. (2) It has no effect on the prothombin time and Russell's viper venom time of normal plasma. (3) Preactivation of normal plasma by a contact activator such as Celite eliminates essentially all inhibitory activity. (4) The inhibitor appears to be heat stable and can be precipitated by centrifugation above 27,000 g. (5) The inhibitor has no effect on the activation of factor XII by Celite. (6) The activation of factor XI by factor XIIa, however, is inhibited by the schistosomal inhibitor. The above data are consistent with the view that S. mansoni adults possess an anticoagulant that is capable of specifically inhibiting the conversion of factor XI to factor XIa by factor XIIa.

Animals↗
PubMed1990

Mutations in hemophilia Bm occur at the Arg180-Val activation site or in the catalytic domain of factor IX.

Hemophilia Bm is characterized by a strikingly prolonged plasma ox brain prothrombin time. In an attempt to find an explanation for this phenomenon we have analyzed various aspects of the Bm variants factor IX Deventer, factor IX Milano, factor IX Novara, and factor IX Bergamo. Proteolytic cleavage by factor XIa was normal in two Bm variants, but absent at the Arg180-Val bond in the other two. In the latter variants Arg180 was replaced by either Trp or Gln, whereas Val181----Phe and Pro368----Thr replacements have occurred in the variants that were normally cleaved by factor XIa. In all four variants the Bm effect could be neutralized with a single monoclonal antibody against factor IX. Also, after treatment with factor XIa, none of the Bm variants reacted with antithrombin III (in contrast to normal factor IXa). Purified factor IX Deventer (one of the variants with a replacement of Arg181), either with or without pretreatment with factor XIa, was found to be a more effective competitive inhibitor of the factor VIIa-tissue factor-induced factor X activation than similarly treated normal factor IX. In addition, this inhibitory effect was much more pronounced when bovine tissue factor was used instead of human tissue factor. We propose that the normal activation of factor IX not only produces a conformational change around the active site serine that allows efficient substrate binding and catalysis, but that the same conformational change is instrumental in effectively dissociating factor IXa from the activating factor VIIa-tissue factor complex. Amino acid replacements that disrupt this conformational transition directly (e.g. Pro368----Thr near the catalytic center) or indirectly (mutations at the Arg180-Val activation site) therefore lead to a combination of 1) the loss of coagulant activity and 2) an inhibitory effect in the ox brain prothrombin time assay.

Amino Acid Sequence↗
PubMed2000

Coagulation factor XI is a contaminant in intravenous immunoglobulin preparations.

A small number of thromboembolic events, including deep venous thrombosis and myocardial infarction, have been reported in patients receiving IVIG. These events have primarily occurred in patients receiving high-dose IVIG and have been attributed to an increase in blood viscosity. To test the hypothesis that a procoagulant might be present in IgG preparations, twenty-nine samples of intravenous immunoglobulin (IVIG) from eight different manufacturers were assayed for procoagulant activity. Twenty-six of these samples shortened the clotting time of factor XI-deficient plasma. Of these, fourteen samples had factor XI activities greater than 0.001 U/ml of normal pooled plasma. The remaining samples possessed less than 0. 001 U/ml of normal plasma activity. The procoagulant activity in these samples could be inhibited by an anti-factor XI polyclonal antibody, suggesting that the procoagulant activity was factor XI. The procoagulant activity increased in two samples after storage at 4 degrees C for 4 weeks, likely as a result of factor XIa autoactivation. Additionally, activity in some IVIG samples was able to directly activate factor IX, indicating that activated factor XI was present in these samples. Finally, the degree of factor XI(a) contamination in the samples was correlated with the manufacturer, suggesting that variations in the manufacturing process or source plasma affect the level of factor XI in the IVIG product. Because addition of small amounts of factor XIa to plasma can lead to production of significant amounts of thrombin, we suggest that factor XIa present in some IVIG preparations could contribute to the in vivo risk of thrombosis after IVIG therapy.

Drug Contamination↗
PubMed2000

Characterization of the protein Z-dependent protease inhibitor.

Protein Z-dependent protease inhibitor (ZPI) is a 72-kd member of the serpin superfamily of proteinase inhibitors that produces rapid inhibition of factor Xa in the presence of protein Z (PZ), procoagulant phospholipids, and Ca(++) (t(1/2) less than 10 seconds). The rate of factor Xa inhibition by ZPI is reduced more than 1000-fold in the absence of PZ. The factor Xa-ZPI complex is not stable to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but is detectable by alkaline-polyacrylamide gel electrophoresis. The combination of PZ and ZPI dramatically delays the initiation and reduces the ultimate rate of thrombin generation in mixtures containing prothrombin, factor V, phospholipids, and Ca(++). In similar mixtures containing factor Va, however, PZ and ZPI do not inhibit thrombin generation. Thus, the major effect of PZ and ZPI is to dampen the coagulation response prior to the formation of the prothrombinase complex. Besides factor Xa, ZPI also inhibits factor XIa in the absence of PZ, phospholipids, and Ca(++). Heparin (0.2 U/mL) enhances the rate (t(1/2) = 25 seconds vs 50 seconds) and the extent (99% vs 93% at 30 minutes) of factor XIa inhibition by ZPI. During its inhibitory interaction with factor Xa and factor XIa, ZPI is proteolytically cleaved with the release of a 4.2-kd peptide. The N-terminal amino acid sequence of this peptide (SMPPVIKVDRPF) establishes Y387 as the P(1) residue at the reactive center of ZPI. ZPI activity is consumed during the in vitro coagulation of plasma through a proteolytic process that involves the actions of factor Xa with PZ and factor XIa.

Blood Coagulation↗
PubMed1994

Surface independent factor XI activation by thrombin in the presence of high molecular weight kininogen.

A deficiency of one of the proteins of the contact system of blood coagulation does not result in a bleeding disorder. For this reason activation of blood coagulation via this system is believed to be an in vitro artefact. However, patients deficient in factor XI do suffer from variable bleeding abnormalities. Recently, an alternative pathway for factor XI activation has been described. Factor XI was found to be activated by thrombin in the presence of dextran sulfate as a surface. However, high molecular weight kininogen (HK), to which factor XI is bound in plasma, and fibrinogen were shown to block this activation suggesting it to be an in vitro phenomenon. We investigated the thrombin-mediated factor XI activation using an amplified detection system consisting of factors IX, VIII and X, which was shown to be very sensitive for factor XIa activity. This assay is approximately 4 to 5 orders of magnitude more sensitive than the normal factor XIa activity assay using a chromogenic substrate. With this assay we found that factor XI activation by thrombin could take place in the absence of dextran sulfate. The initial activation rate was approximately 0.3 pM/min (using 25 nM factor XI and 10 nM thrombin). The presence of dextran sulfate enhanced this rate about 8500-fold. A very rapid and complete factor X activation was observed in the presence of dextran sulfate. Although only minute amounts of factor XIa were formed in the absence of dextran sulfate, significant activation of factor X was detected in the amplification assay within a few minutes.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Phenomena↗
PubMed1983

Cleavage and inactivation of Factor IX by granulocyte elastase.

Radioiodinated Factor IX was cleaved by a crude sonicate from leukocytes. In the absence of calcium, fragments of less than 15,000 mol wt were seen from reduced samples on gel electrophoresis. After digestion in 2 mM calcium, however, electrophoresis of reduced samples showed, in addition to low molecular weight fragments, protein bands corresponding in size to heavy and light chains of Factor XIa-activated Factor IX. The cleaving activity in leukocyte sonicates was inhibited by soybean trypsin inhibitor, but only to a small extent by aprotinin. Granulocyte elastase was isolated from purified polymorphonuclear leukocyte granules by affinity chromatography on soybean trypsin inhibitor-agarose and further chromatography on carboxymethyl cellulose. The purified fraction contained two isozymes on acidic gels which cleaved both an ester sensitive to elastase and radiolabeled Factor IX. These two activities were inhibited by elastase-specific chloromethyl ketone. The isolated protease fraction rapidly inactivated apparent Factor IX activity in a coagulant assay system. The degree of inactivation correlated with the amount of intact, radiolabeled Factor IX cleaved. As with the crude sonicate, generation of the larger heavy and light chain-sized fragments was dependent upon calcium. To assess directly the effect of elastase on Factor IX, an immunospecific, active site-directed assay was developed. In this assay, the sample was incubated with solid-phase antibody to Factor IX and the amount of activated product was detected as that which had complexed with radioiodinated antithrombin III. In this system, exposure of Factor IX to Factor XIa showed progressive increase in the ability to bind antithrombin III, whereas after elastase, Factor XIa was unable to generate antithrombin III binding. The elastase-degraded Factor IX did not inhibit activation of additional Factor IX in clotting assays. When Factor IXa was incubated with elastase, binding of antithrombin III was decreased, corresponding to appearance of low molecular weight fragments on parallel samples that were reduced and electrophoresed. These data are consistent with elastase inactivating Factor IX by cleaving bonds near, but distinct from, bonds cleaved by Factor XIa.

Amino Acid Chloromethyl Ketones↗
PubMed1983

Isolation and functional characterization of the active light chain of activated human blood coagulation factor XI.

Human blood coagulation Factor XIa was reduced and alkylated under mild conditions. The mixture containing alkylated heavy and light chains was subjected to affinity chromatography on high Mr kininogen-Sepharose. Alkylation experiments using [14C]iodoacetamide showed that a single disulfide bridge between the light and heavy chains was broken to release the light chain. The alkylated light chain (Mr = 35,000) did not bind to high Mr kininogen-Sepharose while the heavy chain (Mr = 48,000), like Factors XI and XIa, bound with high affinity. The isolated light chain retained the specific amidolytic activity of native Factor XIa against the oligopeptide substrate, pyroGlu-Pro-Arg-p-nitroanilide. Km and kcat values for this substrate were 0.56 mM and 350 s-1 for both Factor XIa and its light chain, and the amidolytic assay was not affected by CaCl2. However, in clotting assays using Factor XI-deficient plasma in the presence of kaolin, the light chain was only 1% as active as native Factor XIa. Human coagulation Factor IX was purified and labeled with sodium [3H]borohydride on its carbohydrate moieties. When this radiolabeled Factor IX was mixed with Factor XIa, an excellent correlation was observed between the appearance of Factor IXa clotting activity and tritiated activation peptide that was soluble in cold trichloroacetic acid. Factor XIa in the presence of 5 mM CaCl2 activated 3H-Factor IX 600 times faster than Factor XIa in the presence of EDTA. In the absence of calcium, Factor XIa and its light chain were equally active in activating 3H-Factor IX. In contrast to Factor XIa, the light chain in this reaction was inhibited by calcium ions such that, in the presence of 5 mM CaCl2, Factor XIa was 2000 times more effective than its light chain. Neither phospholipid nor high Mr kininogen and kaolin affected the activity of Factor XIa or its light chain in the activation of 3H-Factor IX. These observations show that the light chain region of Factor XIa contains the entire enzymatic active site. The heavy chain region contains the high affinity binding site for high Mr kininogen. Furthermore the heavy chain region of Factor XIa plays a major role in the calcium-dependent mechanisms that contribute to the activation of Factor IX.

Acetamides↗
PubMed1996

A mathematical model for the spatio-temporal dynamics of intrinsic pathway of blood coagulation. II. Results.

This paper continues our study (see Part I) where we modeled the spatio-temporal dynamics of the intrinsic pathway of blood coagulation. Here, we analyzed this model and showed that it describes the threshold behavior of coagulation. When activation is subthreshold (which produces not more than 0.07 nM factor XIa at saturating free calcium concentrations of 2 mM or higher), the concentration of generated thrombin remains below 0.01 nM. At the abovethreshold activation corresponding to factor XIa exceeding 0.07 nM, the concentration of thrombin explosively increases and then abruptly decreases. The peak concentration of thrombin reaches hundreds nM. With respect to free calcium concentration, the system also behaves in a threshold manner. For activation corresponding to 0.3 nM factor XIa, the threshold concentration of free calcium where the outburst of explosive thrombin generation occur is equal to 0.21 mM. The model simulations are in a good agreement with the experimentally recorded kinetics of thrombin generation at different concentrations of free calcium (1). Analysis of the spatial dynamics of coagulation showed that if activation exceeded the threshold level at a certain point, the concentration wave of thrombin arises and propagates at a high speed from the activation zone. The parameters of this wave depends mainly on the efficiency of the feedback loops. The feedback loops through the backbone factors of the intrinsic pathway (autoactivation of factor X or activation of factor XI by thrombin) has a potential for the unlimited propagation of the thrombin wave. With increasing activity of activated protein C (the effect equivalent to that of thrombomodulin), oscillating regimes arise in the model. The first thrombin wave is followed by several secondary running waves. The amplitudes of secondary waves increases to the periphery of the clot consolidating its surface layer.

Blood Coagulation↗
PubMed1989

Factor IX Kawachinagano: impaired function of the Gla-domain caused by attached propeptide region due to substitution of arginine by glutamine at position -4.

Factor IX Kawachinagano (KWC) is a mutant factor IX protein initially recognized in a patient with severe haemophilia B, who had 46% of normal factor IX antigen and no detectable clotting activity. Previous studies indicated that factor IX KWC was not activated by factor XIa in the presence of Ca ions. In the present study, we purified and analysed factor IX KWC at a structural level in an attempt to clarify the nature of the impaired reaction with factor XIa. Kinetic studies showed that activation of factor IX KWC by factor X activator from Russell's viper venom (RVV-X) was normal, whereas activation by factor XIa was defective. Amino acid sequence analysis of tryptic peptides and direct analysis of the NH2-terminal sequence of factor IX KWC demonstrated that this mutant factor IX retained the propeptide region of 18 amino acids due to a substitution of arginine-(-4) by glutamine. These data suggested that the Gla-domain of factor IX KWC was dysfunctional, although the total gamma-Gla content, measured by alkaline-hydrolysis, was normal. We assumed that this attached propeptide region of the molecule directly interferes with the adjacent NH2-terminus and prevents the metal-induced conformational changes which are essential for biological activity of normal factor IX.

1-Carboxyglutamic Acid↗
PubMed1989

Purification and characterization of plasma protein C inhibitor.

Plasma protein C inhibitor (PCI) was purified to homogeneity (greater than 95%) with good recovery (greater than 25%) and reproducibility, and the inhibition of a number of blood clotting and fibrinolytic enzymes by purified PCI was studied. PCI inhibited activated protein C (APC), two-chain urokinase (2c-uPA), two-chain tissue plasminogen activator (2c-tPA), thrombin, factor Xa, plasma kallikrein and factor XIa, and this inhibition was accelerated by heparin. The inhibition of each enzyme was accompanied by formation of enzyme inhibitor complexes and by degradation of the inhibitor to lower molecular weight derivatives. Plasma kallikrein and factor XIa cleaved PCI of native Mr = 57,000 into two products with Mr = 54,000 and 52,000 whereas the other enzymes converted the PCI to a product with Mr = 54,000. PCI did not detectably inhibit alpha-factor XIIa or plasmin. Kinetic studies using PCI yielded the following second-order rate constants for inhibition of human APC, 2c-uPA, 2c-tPA, thrombin, factor Xa, kallikrein and factor XIa respectively: 0.65 x 10(4), 0.22 x 10(4), 0.08 x 10(4), 0.61 x 10(4), 2.01 x 10(4), 6.50 x 10(4), and 9.03 x 10(4) M-1s-1 in the absence of heparin and 1.58 x 10(6), 0.43 x 10(6), 0.03 x 10(6), 0.52 x 10(6), 0.09 x 10(6), 0.18 x 10(6) and 0.74 x 10(6) M-1s-1 in the presence of optimal concentrations of heparin. The rate constants for the inhibition of factor XIa and 2c-uPA by PCI suggest a possible role of PCI in the physiologic regulation of these enzymes. The second order rate constants for inhibition of bovine APC and Gla-domainless bovine APC by human PCI were 0.61 x 10(4) and 0.26 x 10(4) M-1s-1 in the absence of heparin and 0.54 x 10(6) and 0.71 x 10(6) M-1s-1 in the presence of heparin, respectively. Calcium ions (0.05 to 4 mM) did not affect these rate constants. The results obtained with normal and Gla-domainless APC indicate that the Gla domain of APC is not required for inactivation by PGI and is not essential for the heparin stimulation of this reaction.

Blood Coagulation Factors↗
PubMed1990

Factor IX is activated in vivo by the tissue factor mechanism.

Despite significant progress in elucidating the biochemistry of the hemostatic mechanism, the process of blood coagulation in vivo remains poorly understood. Factor IX is a vitamin K-dependent glycoprotein that can be activated by factor XIa or the factor VII-tissue factor complex in vitro. To investigate the role of these two pathways in factor IX activation in humans, we have developed a sensitive procedure for quantifying the peptide that is liberated with the generation of factor IXa. The antibody population used for the immunoassay was raised in rabbits and chromatographed on a factor IX-agarose immunoadsorbent to obtain antibody populations with minimal intrinsic reactivity toward factor IX. We determined that the mean level of the factor IX activation peptide (FIXP) in normal individuals under the age of 40 years was 203 pmol/L and that levels increased significantly with advancing age. The mean concentration of FIXP was markedly reduced to 22.7 pmol/L in nine patients with hereditary factor VII deficiency (factor VII coagulant activity less than 7%) but was not significantly different from normal controls in nine subjects with factor XI deficiency (factor XI coagulant activity less than 8%). These data indicate that factor IXa generation in vivo results mainly from the activity of the tissue factor mechanism rather than the contact system (factor XII, prekallikrein, high molecular-weight kininogen, factor XI). Our results may also help to explain the absence of a bleeding diathesis in many patients with deficiencies of the contact factors of coagulation.

Adult↗
PubMed1994

Membrane binding properties of the factor IX gamma-carboxyglutamic acid-rich domain prepared by chemical synthesis.

The fully gamma-carboxylated peptides based upon the complete and truncated Gla/aromatic amino acid stack domains of human Factor IX were prepared by solid phase peptide synthesis using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. A 47-residue peptide Factor IX-(1-47) and a 42-residue peptide Factor IX-(1-42), both containing 12 residues of L-gamma-carboxyglutamic acid, were purified by high performance liquid chromatography and oxidized to form the disulfide bond. Quantitative gamma-carboxyglutamic acid analysis of Factor IX-(1-47) and Factor IX-(1-42) indicated the presence of 12.1 and 11.2 gamma-carboxyglutamic acid residues/mol of peptide, respectively; no glutamic acid was detected. As monitored by fluorescence quenching, calcium ions induced the prototypical conformational transition in Factor IX-(1-47), but not in Factor IX-(1-42), that is observed with Factor IX. Half-maximal quenching of the intrinsic fluorescence of Factor IX-(1-47) was observed at Ca(II) concentrations of about 50 microM. Factor IX-(1-47) bound to the conformation-specific antibodies, anti-Factor IX:Mg(II) and anti-Factor IX:Ca(II)-specific in the presence of metal ions. Factor IX-(1-47) bound to phospholipid membranes, as monitored by energy transfer from intrinsic fluorophores to dansyl (5-dimethylaminonaphthalene-1-sulfonyl)-phosphatidylethanolamine incorporated into a lipid bilayer composed of phosphatidylserine:phosphatidylcholine. In contrast, Factor IX-(1-42) bound poorly to these same membranes. Factor IX-(1-47) did not inhibit Factor XIa activation of Factor IX but did inhibit the activation of Factor X by Factor IXa bound to Factor VIII in the presence of calcium ions and phospholipid. These results show that phospholipid membrane binding is a property of the Gla/aromatic amino acid stack domain and that the Factor IX-(1-47) peptide, prepared by chemical synthesis, preserves the membrane binding properties and the metal-induced conformational transitions observed in native Factor IX. These results indicate that Factor IX-(1-47) but not Factor IX-(1-42) is a suitable model for structural studies of Factor IX-membrane interaction.

1-Carboxyglutamic Acid↗
PubMed1990

Immunopurification and protease inhibitory properties of protease nexin-2/amyloid beta-protein precursor.

Protease nexin-2 (PN-2) is a protease inhibitor that is synthesized and secreted by a variety of extravascular cells including human fibroblasts. It forms sodium dodecyl sulfate-stable complexes with trypsin, the epidermal growth factor binding protein and the gamma-subunit of nerve growth factor. Recently we reported that PN-2 is the secreted form of the amyloid beta-protein precursor (APP) and is a potent inhibitor of chymotrypsin. Here we describe a two-step procedure to purify PN-2/APP using a monoclonal antibody immunoaffinity column. We also quantitated the protease inhibitory properties of purified PN-2/APP on a number of serine proteases. PN-2/APP was a potent inhibitor of coagulation factor XIa with a Ki = 2.9 x 10(-10). The inhibition of factor XIa by PN-2/APP was augmented by heparin and resulted in a Ki = 5.5 x 10(-11) M. Trypsin and chymotrypsin were also effectively inhibited with a Ki = 4.2 x 10(-10) and 1.6 x 10(-9), respectively. PN-2/APP also inhibited the epidermal growth factor binding protein, the gamma-subunit of nerve growth factor, and chymase and plasmin to a lesser extent. In view of recent findings that PN-2/APP is contained in alpha-granules of platelets and is secreted upon platelet activation, the potent inhibition of factor XIa suggests that PN-2/APP may play a regulatory role in the coagulation pathway at vascular wound sites. In addition, these studies define biochemical activities of PN-2/APP which may be involved in regulating proteases that lead to the generation and deposition of the beta-protein in neurodegenerative lesions associated with Alzheimer's disease and Down's syndrome.

Amino Acid Sequence↗
PubMed1990

The synthesis of arginylfluoroalkanes, their inhibition of trypsin and blood-coagulation serine proteinases and their anticoagulant activity.

Seven arginylfluoroalkanes ('arginine fluoroalkyl ketones') were synthesized by using a modified Dakin-West procedure. The structure of benzoyl-Arg-CF2CF3 was analysed by 19F-n.m.r. spectroscopy and m.s. and the compound was shown to exist primarily as a hydrate or cyclic carbinolamine. Arginylfluoroalkanes are good inhibitors of blood-coagulation serine proteinases and were found to be slow-binding inhibitors for bovine trypsin with Ki values of 0.2-56 microM. Benzoyl-Arg-CF2CF3 was the best inhibitor for bovine thrombin and human Factor XIa, and inhibited thrombin and Factor XIa competitively with Ki values of 13 microM and 62 microM respectively. The best inhibitor for pig pancreatic kallikrein was p-toluoyl-Arg-CF3, with a Ki value of 35 microM. Benzoyl-Arg-CF3 and benzoyl-Arg-CF2CF3 inhibited human plasma kallikrein competitively, with Ki values of 50 microM. None of the seven arginylfluoroalkanes was a good inhibitor of human factor Xa or of Factor XIIa. The arginylfluoroalkanes were tested in the prothrombin time (PT) and activated partial thromboplastin time (APTT) coagulant assays. Two fluoroketones, benzoyl-Arg-CF2CF3 and 1-naphthoyl-Arg-CF3, had significant anticoagulant activity. Benzoyl-Arg-CF2CF3 was found to prolong the PT 1.8-fold at 120 microM and to prolong the APTT 2.4-fold at 90 microM, whereas 1-naphthoyl-Arg-CF3 only prolonged the APTT 1.7-fold at 100 microM.

Amino Acid Sequence↗
PubMed2000

Plasma lipoproteins enhance tissue factor-independent factor VII activation.

The effect of plasma lipoprotein fractions (large very-low-density lipoprotein, small very-low-density lipoprotein, intermediate-density lipoprotein, and low-density lipoprotein) on initiation of blood coagulation by supporting factor VII activation or by stimulating monocytes to express tissue factor was investigated in vitro. Endotoxin-free preparations of lipoprotein fractions did not induce functional tissue factor in monocytes, whereas all lipoprotein fractions enhanced tissue factor-independent activation of factor VII by factor Xa and by factors Xa/Va. In contrast, no or only slight enhancement of factor IXa-, factor IXa/VIIIa-, factor XIa-, or thrombin-mediated factor VII activation was observed. The effect of small very-low-density lipoprotein was less than that of large very-low-density lipoprotein, and intermediate-density and low-density lipoproteins caused an even lower but still significant increase of factor Xa- and factor Xa/Va-mediated factor VII activation. When the data were normalized for apolipoprotein B-100 content, differences remained between lipoprotein fractions. In contrast, when phospholipid content was used for normalization, differences between lipoprotein fractions in factor Xa- and factor Xa/Va-mediated factor VII activation disappeared, indicating that phospholipids were involved in factor VII activation. This was supported by enhancement of factor Xa-mediated factor VII activation by synthetic phospholipid vesicles containing negatively charged phospholipids.

Blood Coagulation↗
PubMed1983

Identification of the molecular defect in factor IX Chapel Hill: substitution of histidine for arginine at position 145.

Hemophilia B Chapel Hill is a mild hereditary hemorrhagic disorder in which the factor IX antigen is present in normal amounts but factor IX biological activity is markedly reduced. Previous studies have demonstrated that purified factor IX Chapel Hill has 8% of the activity of normal human factor IX and that the activation of factor IX Chapel Hill is defective in that only one of the two peptide bonds hydrolyzed during activation of normal factor IX is cleaved. The tryptic peptides from normal human factor IX and factor IX Chapel Hill were subjected to analysis by high-performance liquid chromatography. Comparison of the elution profile of the peptides obtained from factor IX Chapel Hill and normal factor IX demonstrated that the tripeptide Leu-Thr-Arg, which is derived from the normal molecule (positions 143-145) immediately amino-terminal from the Arg-Ala peptide bond at 145-146 that is cleaved during the activation of factor IX with factor XIa, was absent in the digest obtained from factor factor IX Chapel Hill. The elongated "activation peptide" from factor factor IX Chapel Hill was obtained by further high-performance liquid chromatographic fractionation and subjected to primary structure analysis. The following sequence, corresponding to positions 143-147, was obtained: Leu-Thr-His-Ala-Glu. Thus, the primary molecular defect in factor factor IX Chapel Hill is the substitution of histidine for arginine at position 145. This substitution precludes cleavage by factor XIa at this peptide bond, and the activation peptide region remains associated with the light chain of factor IXa Chapel Hill.

Amino Acid Sequence↗