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PubMed1989

Interaction of type 1 plasminogen activator inhibitor with the enzymes of the contact activation system.

The interaction between type 1 plasminogen activator inhibitor (PAI-1), a serine protease inhibitor, and the three serine proteases generated during contact activation of plasma was studied using functional and immunologic approaches. Incubation of Factor XIIa, Factor XIa, and plasma kallikrein with either purified PAI-1 or platelet-derived PAI-1 resulted in the formation of sodium dodecyl sulfate-stable complexes as revealed by immunoblotting techniques. Functional assays indicated that Factor XIa and, to a lesser extent, Factor XIIa and plasma kallikrein neutralized the ability of purified PAI-1 to bind to immobilized tissue-type plasminogen activator (t-PA). Immunoblotting demonstrated that these enzymes also neutralized the ability of PAI-1 to form complexes with fluid-phase t-PA. Clot lysis assays employing purified proteins and 125I-fibrinogen were used to investigate the profibrinolytic effect of these contact activation enzymes. At enzyme concentrations that did not result in direct activation of plasminogen, only Factor XIa was capable of stimulating the lysis of clots supplemented with both t-PA and PAI-1. As a consequence of their interactions with PAI-1, the amidolytic activity of Factor XIIa, Factor XIa, and plasma kallikrein was neutralized by this inhibitor in a time-dependent and concentration-dependent manner. Minimum values estimated for the apparent second-order rate constant of inhibition were 1.6 x 10(4), 2.1 x 10(5), and 6.0 x 10(4) M-1 s-1 for Factor XIIa, Factor XIa, and plasma kallikrein, respectively. These data define new reactions between coagulation and fibrinolysis proteins and suggest that a major mechanism for stimulation of the intrinsic fibrinolytic pathway may involve neutralization of PAI-1 by Factor XIa.

Blotting, Western↗
PubMed2004

Beta 2-Glycoprotein I binds factor XI and inhibits its activation by thrombin and factor XIIa: loss of inhibition by clipped beta 2-glycoprotein I.

Activation of factor XI (FXI) by thrombin in vivo plays a role in coagulation by providing an important positive feedback mechanism for additional thrombin generation. FXI is activated in vitro by thrombin, or FXIIa in the presence of dextran sulfate. In this report, we investigated the effect of beta(2)-glycoprotein I (beta(2)GPI) on the activation of FXI. beta(2)GPI bound FXI in vitro and inhibited its activation to FXIa by thrombin and FXIIa. The affinity of the interaction between beta(2)GPI and FXI was equivalent to the interaction between FXI and high molecular weight kininogen. Inhibition of FXI activation occurred with lower concentrations of beta(2)GPI than found in human plasma. Proteolytic clipping of beta(2)GPI by plasmin abolished its inhibition of FXI activation. The results suggest a mechanism of regulation whereby physiological concentrations of beta(2)GPI may attenuate thrombin generation in vivo by inhibition of FXI activation. Plasmin cleavage of beta(2)GPI provides a negative feedback that counteracts its inhibition of FXI activation.

Antiphospholipid Syndrome↗
PubMed1989

Coagulation factor XII (Hageman factor) Washington D.C.: inactive factor XIIa results from Cys-571----Ser substitution.

Structural studies on a congenital abnormal coagulation factor XII (Hageman factor), factor XII Washington D.C., have been performed to identify the defect responsible for its lack of procoagulant activity. Amino acid sequence analysis of a tryptic peptide isolated from the abnormal factor XII indicated that Cys-571 (equivalent to Cys-220 in the chymotrypsin numbering system) had been replaced by serine. No other substitutions in the active-site triad--namely, His-393, Asp-442, and Ser-544--were found. We propose that the Cys-571----Ser replacement found in this factor XII variant destroys the formation of the disulfide linkage between Cys-540 and Cys-571, giving rise to an altered conformation of the active-site serine residue or the secondary substrate-binding site and, thus, leads to the loss of enzyme activity.

Amino Acid Sequence↗
PubMed1992

Simultaneous diagnosis of coagulation factor XIIA (F13A) and plasminogen (PLG) phenotypes by polyacrylamide gel isoelectric focusing.

In this paper, we report a simple rapid method for simultaneous determination of Coagulation Factor XIIIA (F13A) and plasminogen (PLG) phenotypes by PAGIF with a nominal pH range of 3.5 to 10, followed by immunofixation and silver stain. Critical considerations concerning the conditions of molecular separation and detection strategies are also presented.

Genetic Markers↗
PubMed2004

Crystallization, data collection and phasing of infestin 4, a factor XIIa inhibitor.

Infestin is a protein from Triatoma infestans (kissing bug) composed of seven Kazal-type domains that is further processed to yield several serine protease inhibitors with varying specificities. Infestins 3 and 4 are the last two domains of the infestin gene and are found in vivo in the insect's anterior midgut. The last domain, infestin 4, has been cloned, expressed and purified. Here, the crystallization of infestin 4 using the sitting-drop vapour-diffusion method with PEG 8000 as precipitant is described. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 25.89, b = 45.64, c = 57.41 A. X-ray diffraction data were collected to a maximum resolution of 1.8 A using a synchrotron-radiation source. Initial phases were calculated by molecular replacement using an edited rhodniin molecule as the search model. Structure refinement is in progress.

Animals↗
PubMed1984

Active-site mapping of bovine and human blood coagulation serine proteases using synthetic peptide 4-nitroanilide and thio ester substrates.

A series of 14 tripeptide 4-nitroanilide substrates of the type Z-AA-Gly-Arg-NA and Z-AA-Phe-Arg-NA where AA = Ala, Asn, Glu, Lys, Phe, Pro, or Ser were used to map the S3 subsite of several serine proteases involved in blood coagulation. The enzymes studied included bovine thrombin, factor IXa, factor Xa, factor XIa, human beta-factor XIIa (factor XIIa fragment), and activated bovine and human protein C. Kinetic constants (kcat, KM, and kcat/KM) for the enzymatic hydrolysis of the substrates by each enzyme were determined and used to compare the relative reactivities of the individual enzymes. Most of the enzymes reacted with all the substrates, although a few showed considerable specificity. Human beta-factor XIIa showed the highest reactivity of all the coagulation proteases studied and was also very substrate specific (kcat/KM ranged over 470-fold). The best substrate was Z-Lys-Phe-Arg-NA with kcat/KM = 140 000 M-1 s-1. Activated bovine protein C (best substrate = Z-Ser-Phe-Arg-NA), factor Xa (best substrate = Z-Glu-Gly-Arg-NA), and thrombin (best substrate = Z-Lys-Gly-Arg-NA) were the group of enzymes that showed next highest reactivity toward the substrates. Activated bovine protein C, thrombin, and factor Xa displayed relatively little substrate specificity. Activated human protein C (best substrate = Z-Ser-Phe-Arg-NA) and factor XIa (best substrate = Z-Glu-Gly-Arg-NA) are moderately reactive enzymes. Activated human protein C is an extremely specific enzyme since it has such a large range of kcat/KM values.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Kinetics of inhibition of human plasma kallikrein by a site-specific modified inhibitor Arg15-aprotinin: evaluation using a microplate system and comparison with other proteases.

Human plasma kallikrein, a product of contact-activated plasma proteolysis, is moderately inhibited by aprotinin, a small polypeptide from bovine lung that has been used as an experimental drug in human disease states. Aprotinin has a Lys residue in the P1 (reactive center) position occupying residue 15. Since kallikrein is an arginine-directed serine protease, we hypothesized that an altered form of aprotinin, Arg15-aprotinin, might be a better inhibitor. Kinetic evaluations were performed in 96-well microplates. We found that the KL (loose or Michaelis-Menten complex) was unchanged by the modification. However, the association rate constant was increased from 1.14 X 10(4) (mol/L)-1s-1 to 1.5 X 10(5) (mol/L)-1s1, thus indicating that the inhibition rate was increased 14-fold for the modified protein. The Ki (at equilibrium) was decreased from 3.2 X 10(-7) mol/L to 1.5 X 10(-8) mol/L after substituting Arg for Lys in the P1 position. Therefore, the modified inhibitor binds to plasma kallikrein more tightly than the natural protein. We also investigated the effect of Arg15-aprotinin on tissue kallikrein, plasmin, factor XIIa, factor XIa, and thrombin and found that the Ki slightly decreased from 5.1 X 10(-7) mol/L to 1.2 X 10(-7) mol/L for tissue kallikrein and slightly decreased from 2 X 10(-8) mol/L to 1 X 10(-8) mol/L for plasmin. Arg15-aprotinin did not inhibit thrombin or factor XIIa, even though both enzymes are arginine-directed serine proteases. However, factor XIa, although it was not inhibited by aprotinin, had a Ki of 3.4 X 10(-8) mol/L for Arg15-aprotinin. Therefore, Arg15-aprotinin is a more effective inhibitor of plasma kallikrein as well as factor XIa but shows minimal preference for plasmin and tissue kallikrein. This study also indicates that it is possible and practical to perform kinetic analyses directly in microplates.

Aprotinin↗
PubMed1982

Preparation of beta-XIIa (Hageman factor fragment) from human plasma.

beta-Factor XIIa (beta-XIIa, Mr approximately 30,000) was isolated from human plasma by a procedure which utilized, as an initial step, the adsorption of Factor XII to celite. Activation of Factor XII and subsequent release of beta-XIIa was brought about by the proteolytic action of co-adsorbed kallikrein. Two successive chromatographic procedures were then used to achieve a final purification of 4,420-fold over plasma and an overall field of 2.3 mg of beta-XIIa per liter.

Adsorption↗
PubMed2006

Functional phenotyping of human plasma using a 361-fluorogenic substrate biosensing microarray.

A microarray presenting glycerol nanodroplets of fluorogenic peptide substrates was used as a biosensor for the detection of multiple enzyme activities within human plasma. Using 10 different plasma proteases (kallikrein, factor XIIa, factor XIa, factor IXa, factor VIIa, factor Xa, thrombin, activated protein C, uPA and plasmin) and a 361-compound fluorogenic substrate library (Ac-Ala-P3-P2-Arg-coumarin for P = all amino acids except Cys), a database was created for deconvoluting the relative activity of each individual enzyme signal in human plasma treated with various activators (calcium, kaolin, or uPA). Three separate deconvolution protocols were tested: searching for "optimal" sensing substrate sequences for a set of 5 enzymes and using these substrates to detect protease signals in plasma; ranking the "optimal" sensing substrates for 10 proteases using local error minimization, resulting in a set of substrates which were bundled via weighted averaging into a super-pixel that had biosensing properties not obtainable by any individual fluorogenic substrate; and treating each 361-element map measured for each plasma preparation as a weighted sum of the 10 maps obtained for the 10 purified enzymes using a global error minimization. The similarity of the results from these latter two protocols indicated that a small subset of <90 substrates contained the majority of biochemical information. The results were consistent with the state of the coagulation cascade expected when treated with the given activators. This method may allow development of future biosensors using minimal and non-specific markers. These substrates can be applied to real-time diagnostic biosensing of complex protease mixtures.

Algorithms↗
PubMed1984

Proteolytic activation of tissue plasminogen activator by plasma and tissue enzymes.

Tissue kallikrein and factor Xa were found to activate tissue plasminogen activator (t-PA) at a rate comparable with that of plasmin. During the activation reaction, the single-chain molecule was converted into a two-chain form. A slight t-PA activating activity was also found in plasma kallikrein. Other activated coagulation factors, factor XIIa, factor XIa, factor IXa, factor VIIa, thrombin and activated protein C had no effect on t-PA activation. t-PA was also activated by a tissue kallikrein-like enzyme that was isolated from the culture medium of melanoma cells. These results indicate that tissue kallikrein and factor Xa may participate in the extrinsic pathway of human fibrinolysis.

Animals↗
PubMed2002

Infestin, a thrombin inhibitor presents in Triatoma infestans midgut, a Chagas' disease vector: gene cloning, expression and characterization of the inhibitor.

This work describes the purification, gene cloning and expression of infestin, a thrombin inhibitor from midguts of Triatoma infestans. Infestin is located in the midgut and its purification was performed by anion-exchange and affinity chromatographies. The N-terminal sequence and the sequence of tryptic peptides were determined. Using RT-PCR, total RNA and infestin cDNA information, a DNA fragment was cloned which encodes a multi non-classical Kazal-type serine protease inhibitor. Isolated native infestin has two non-classical Kazal-type domains and shows an apparent molecular mass of 13 kDa, while its gene codes for a protein with four non-classical Kazal-type domains corresponding to an apparent molecular mass of 22 kDa. Two recombinant infestins, r-infestin 1-2 and r-infestin 1-4, were constructed using the vector pVT102U/alpha and expressed in S. cerevisiae. Native and r-infestin 1-2 showed very similar inhibitory activities towards thrombin and trypsin with dissociation constants of 43.5 and 25 pM for thrombin and 2.0 and 3.1 nM for trypsin, respectively. No other serine protease of the blood coagulation cascade was inhibited by the r-infestin 1-2. Surprisingly, r-infestin 1-4 inhibited not only thrombin and trypsin (K(i) of 0.8 and 5.2 nM, respectively), but also factor XIIa, factor Xa and plasmin (K(i) of 78 pM, 59.2 and 1.1 nM, respectively).

Amino Acid Sequence↗
PubMed1999

Elevated plasma fibrinogen and increased fibrin turnover among healthy women who both smoke and use low-dose oral contraceptives--a preliminary report.

Among users of low-dose oral contraceptives (OC), cardiovascular diseases occur mainly in smokers. The mechanisms by which OC and smoking increase the risk for arterial thrombotic risk have not been adequately explained. Epidemiological evidence suggests that changes in blood coagulation and fibrinolysis may play an important role as determinants of thrombotic events. Therefore, we have investigated the associations of OC and smoking with haemostatic variables among 194 premenopausal healthy women. Fourty women were current users of low-dose OC and 62 women were smokers. After adjustment for age and body mass index, mean values of factor XIIa, factor VII activity and antigen, fibrinogen, D-dimer, global fibrinolytic capacity were significantly higher in OC users than in non-users. Mean levels of PAI activity and t-PA antigen were significantly lower in OC users than in non-users. Smokers had significantly higher mean values of fibrinogen than non-smokers. Two-way analysis of variance showed that the differences in mean levels of fibrinogen and D-dimer between OC users and non users were restricted to smokers. The positive and significant interactions between OC use and smoking in their effects on haemostatic variables were consistent with respect to age and type of OC. These preliminary data suggest that elevated plasma levels of fibrinogen and intravascular fibrin deposition may play a role in the pathogenesis of arterial thrombotic disease among women who are both low-dose OC users and smokers.

Adult↗
PubMed1993

Thrombogenic mechanisms in the human: fresh insights obtained by immunodiagnostic studies of coagulation markers.

Although in vitro studies have been invaluable in revealing the complex biochemistry of the blood coagulation system, meaningful in vivo studies of thrombogenic mechanisms have previously been hindered by the absence of suitable assays. This article reviews the recent development and/or contemporary clinical application of plasma-based immunoassays for coagulation markers (factor XIIa, factor IX activation peptide, prothrombin fragment F1 + 2, thrombin-antithrombin complex and fibrinopeptide A) and for the fibrinolytic marker, D-dimer, which have enabled a critical re-appraisal of some long-standing hypotheses. In chronic renal disease the intrinsic coagulation pathway was found to be activated before haemodialysis and increased end-stage coagulation activity was detected during dialysis when heparinization was limiting. No evidence was found to support the generally accepted hypothesis that thrombogenesis in dialysis is triggered by stimulation of the contact system following exposure of blood to the dialyser membrane. Instead, it is postulated that it is a failure of regulation of end-stage coagulation proteinases (owing to the absence of endothelium) which is responsible for increased thrombogenesis in the dialyser circuit. Excessive end-stage coagulation activity was observed during cardiopulmonary bypass (CPB) surgery and in patients undergoing general thoracic surgery. The data did not accord with the hypothesis that the contact system provides the major thrombogenic trigger in CPB surgery. It is proposed that, in general thoracic surgery, a powerful procoagulant stimulus is provided via the tissue factor-factor VIIa pathway and that the same mechanism is also primarily responsible for triggering thrombogenesis during CPB surgery. The established hypothesis of a prethrombotic state in hereditary AT III deficiency is challenged by the inability to detect increased coagulation activity in asymptomatic AT III deficient patients. It is concluded that the AT III concentration in deficient members is sufficient to enable regulation of the coagulation system in the basal state, whereas failure to regulate the coagulation system only occurs following a major procoagulant stimulus, which overwhelms the impaired inhibitory capacity and triggers thrombosis. These findings highlight the advantages of using plasma-based immunoassays to investigate thrombogenic mechanisms in hypercoagulable states and have important implications for the further study and treatment of blood-surface interactions and thrombotic disease.

Biomarkers↗
PubMed1992

Fibrinogen blocks the autoactivation and thrombin-mediated activation of factor XI on dextran sulfate.

The intrinsic pathway of blood coagulation is activated when factor XIa, one of the three contact-system enzymes, is generated and then activates factor IX. Factor XI has been shown to be efficiently activated in vitro by surface-bound factor XIIa after factor XI is transported to the surface by its cofactor, high molecular weight kininogen (HK). However, individuals lacking any of the three contact-system proteins--namely, factor XII, prekallikrein, and HK--do not suffer from bleeding abnormalities. This mystery has led several investigators to search for an "alternate" activation pathway for factor XI. Recently, factor XI has been reported to be autoactivated on the soluble "surface" dextran sulfate, and thrombin was shown to accelerate the autoactivation. However, it was also reported that HK, the cofactor for factor XIIa-mediated activation of factor XI, actually diminishes the thrombin-catalyzed activation rate of factor XI. Nonetheless, it was suggested that thrombin was a more efficient activator than factor XIIa. In this report we investigated the effect of fibrinogen, the major coagulation protein in plasma, on the activation rate of factor XI. Fibrinogen, the preferred substrate for thrombin in plasma, virtually prevented autoactivation of factor XI as well as the thrombin-mediated activation of factor XI, while having no effect on factor XIIa-catalyzed activation. HK dramatically curtailed the autoactivation of factor XI in addition to the thrombin-mediated activation. These data indicate that factor XI would not be autoactivated in a plasma environment, and thrombin would, therefore, be unlikely to potentiate the activation. We believe that the "missing pathway" for factor XI activation remains an enigma that warrants further investigation.

Dextran Sulfate↗
PubMed1985

Effect of heparin on the inactivation rate of human activated factor XII by antithrombin III.

Human antithrombin III (ATIII) is a plasma inhibitor of several serine proteases of the blood coagulation system. Previous investigations have reported that the presence of heparin has a multifold accelerating effect on the inhibition of factor XIIa and XIIf, the active species derived from factor XII. Recent studies from our laboratories have confirmed that ATIII inactivates factor XIIa and factor XIIf, but only contributes 2% to 3% to the inhibition of activated factor XII species in plasma. The major inhibitor is C1 inhibitor. Therefore, we have reexamined the heparin effect on the rate of inhibition of factor XIIa and factor XIIf in purified systems. We also have studied the effect of heparin on the inactivation of both factor XII-derived active species by various plasmas. Using purified factor XIIa and ATIII, we found that heparin (0.7 to 34.0 U/mL) increased the rate of inhibition of Factor XIIa. However, at heparin concentrations usually achieved during anticoagulant therapy (0.7 U/mL), the inhibition was accelerated only fourfold. This implies only a 6% contribution to the inhibitory effect of plasma. This suggestion was confirmed by the observation that heparin (1.5 U/mL) added to factor XII-deficient plasma and reconstituted with factor XIIa did not produce a detectable enhancement of the rate of inhibition of factor XIIa. Furthermore, using purified factor XIIf and antithrombin III, heparin (3.6 to 57.2 U/mL) increased the inactivation rate constant of factor XIIf by 1.6 to 14.0 times. This small effect was confirmed by the observation that heparin at a concentration greater than that sufficient for anticoagulation (1.4 U/mL) did not modify the inactivation rate of factor XIIf by prekallikrein-deficient plasma, and thus C1 inhibitor remains the major inhibitor even in the presence of heparin. From this study and our previous investigations on the effect of heparin on the inhibition of kallikrein and factor XIa, we conclude that heparin does not significantly affect the protease activity of purified contact activation factors or the activities expressed by these proteases in plasma.

Antithrombin III↗