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PubMed1992

Purification of factor XIa inhibitor from human platelets.

An inhibitor of activated factor XI (FXIa) in human platelets was recently identified as an amyloid beta-protein precursor (APP). We purified an FXIa inhibitor (XIaI) from the supernatant of activated human platelets, and assessed its inhibitor activity toward FXIa amidolytic activity. Approximately 90 micrograms of XIaI that cross-reacted with anti-APP antibody was obtained from two hundred units of platelet suspension by employing a six-step column chromatography procedure. The molecular weight of the purified XIaI was 94,000. The Ki value of XIaI to factor XIa was 526 +/- 120 pM, and the inhibition was enhanced by the addition of ZnCl2. The amino-terminal sequence of XIaI was L-E-V-P-T-D-G-N-A-, which is identical to that for the leucine (N18) to alanine (N26) sequence of APP751 and the amino-terminal sequence of protease nexin-2.

Amino Acid Sequence↗
PubMed1991

Activation of human blood coagulation factor XI independent of factor XII. Factor XI is activated by thrombin and factor XIa in the presence of negatively charged surfaces.

Human blood coagulation factor XI was activated by either autoactivation or thrombin. These reactions occurred only in the presence of negatively charged materials, such as dextran sulfate (approximately Mr 500,000), sulfatide, and heparin. During the activation, factor XI was cleaved at a single Arg-Ile bond by thrombin or factor XIa to produce an amino-terminal 50-kDa heavy chain and a carboxyl-terminal 35-kDa light chain. This activation pattern is identical to that produced by factor XIIa. The addition of a small amount of thrombin and sulfatide to factor XII-deficient plasma produced shorter clotting times than when these agents were added to factor XI/factor XII combined-deficient plasma. These results suggest that the activation of factor XI by thrombin and possibly the autoactivation of factor XI proceed in plasma to lead fibrin clot formation. These reactions may have a role on an appropriate negatively charged surface in normal hemostasis.

Anions↗
PubMed2005

Structural and mutational analyses of the molecular interactions between the catalytic domain of factor XIa and the Kunitz protease inhibitor domain of protease nexin 2.

Factor XIa (FXIa) is a serine protease important for initiating the intrinsic pathway of blood coagulation. Protease nexin 2 (PN2) is a Kunitz-type protease inhibitor secreted by activated platelets and a physiologically important inhibitor of FXIa. Inhibition of FXIa by PN2 requires interactions between the two proteins that are confined to the catalytic domain of the enzyme and the Kunitz protease inhibitor (KPI) domain of PN2. Recombinant PN2KPI and a mutant form of the FXI catalytic domain (FXIac) were expressed in yeast, purified to homogeneity, co-crystallized, and the structure of the complex was solved at 2.6 angstroms (Protein Data Bank code 1ZJD). In this complex, PN2KPI has a characteristic, disulfide-stabilized double loop structure that fits into the FXIac active site. To determine the contributions of residues within PN2KPI to its inhibitory activity, selected point mutations in PN2KPI loop 1 11TGPCRAMISR20 and loop 2 34FYGGC38 were tested for their ability to inhibit FXIa. The P1 site mutation R15A completely abolished its ability to inhibit FXIa. IC50 values for the wild type protein and the remaining mutants were as follows: PN2KPI WT, 1.28 nM; P13A, 5.92 nM; M17A, 1.62 nM; S19A, 1.86 nM; R20A, 5.67 nM; F34A, 9.85 nM. The IC50 values for the M17A and S19A mutants were not significantly different from those obtained with wild type PN2KPI. These functional studies and activated partial thromboplastin time analysis validate predictions made from the PN2KPI-FXIac co-crystal structure and implicate PN2KPI residues, in descending order of importance, Arg15, Phe34, Pro13, and Arg20 in FXIa inhibition by PN2KPI.

Alanine↗
PubMed1986

Interaction of factor XIa and antithrombin in the presence and absence of heparin.

We have studied the interaction between purified human factor XIa and antithrombin in the presence and absence of well-characterized preparations of heparin. The concentrations of hemostatic enzyme, protease inhibitor, and mucopolysaccharide were 5.76 X 10(-8) mol/L, 5.76 X 10(6) mol/L, and either 5.88 X 10(6) mol/L or 0, respectively. Kinetic investigation of this process using a tritiated factor IX substrate demonstrated that the pseudo first-order rate constants of this reaction in the presence and absence of heparin are approximately 1.0 min-1 and approximately 0.025 min-1, respectively. Thus, the rate of hemostatic enzyme-protease inhibitor complex formation is accelerated by about 40-fold in the presence of saturating levels of the mucopolysaccharide. These results were confirmed in a qualitative manner by directly monitoring the generation of factor XIa-antithrombin interaction product with sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis using an antibody population specific for the protease inhibitor.

Antithrombins↗
PubMed1995

Coagulation factor XIa cleaves the RHDS sequence and abolishes the cell adhesive properties of the amyloid beta-protein.

Amyloid beta-protein (A beta) is the major constituent of senile plaques and cerebrovascular amyloid deposits in Alzheimer's disease and is proteolytically derived from its transmembrane parent protein the amyloid beta-protein precursor (A beta PP). Although the physiological role(s) of secreted A beta PPs are not fully understood, several potential functions have been described including the regulation of hemostatic enzymes factors XIa and IXa and a role in cell adhesion. In the present study, we investigated the proteolytic processing of A beta PP by factor XIa (FXIa). Incubation of the human glioblastoma cell line U138 stably transfected to overexpress the 695 isoform of A beta PP with FXIa (2.5-5 nM) resulted in proteolytic cleavage of secreted A beta PP. Higher concentrations of FXIa (> 25 nM) resulted in loss in cell adherence. Coincubation of FXIa with purified, recombinant Kunitz protease inhibitor domain of A beta PP blocked both the proteolytic processing of A beta PP and the loss of cell adhesion. The RHDS cell adhesion site of A beta PP resides within residues 5-8 of the A beta domain. Incubation of synthetic A beta 1-40 peptide with increasing concentrations of FXIa resulted in cleavage of A beta between Arg5 and His6 within the cell adhesion domain of the peptide. FXIa-digested A beta 1-40 or A beta PP695 lost their abilities to serve as cell adhesion substrates consistent with cleavage through this cell adhesion site. Together, these results suggest a new potential biological function for FXIa in the modulation of cell adhesion. In addition, we have shown that FXIa can proteolytically alter A beta and therefore possibly modify its physiological and perhaps pathological properties.

Amino Acid Sequence↗
PubMed2001

Model for a factor IX activation complex on blood platelets: dimeric conformation of factor XIa is essential.

Human coagulation factor XI (FXI) is a plasma serine protease composed of 2 identical 80-kd polypeptides connected by a disulfide bond. This dimeric structure is unique among blood coagulation enzymes. The hypothesis was tested that dimeric conformation is required for normal FXI function by generating a monomeric version of FXI (FXI/PKA4) and comparing it to wild-type FXI in assays requiring factor IX activation by activated FXI (FXIa). FXI/PKA4 was made by replacing the FXI A4 domain with the A4 domain from prekallikrein (PK). A dimeric version of FXI/PKA4 (FXI/PKA4-Gly326) was prepared as a control. Activated FXI/PKA4 and FXI/PKA4-Gly326 activate factor IX with kinetic parameters similar to those of FXIa. In kaolin-triggered plasma clotting assays containing purified phospholipid, FXI/PKA4 and FXI/PKA4-Gly326 have coagulant activity similar to FXI. The surface of activated platelets is likely to be a physiologic site for reactions involving FXI/FXIa. In competition binding assays FXI/PKA4, FXI/PKA4-Gly326, and FXI have similar affinities for activated platelets (K(i) = 12-16 nM). In clotting assays in which phospholipid is replaced by activated platelets, the dimeric proteins FXI and FXI/PKA4-Gly326 promote coagulation similarly; however, monomeric FXI/PKA4 has greatly reduced activity. Western immunoblot analysis confirmed that activated monomeric FXI/PKA4 activates factor IX poorly in the presence of activated platelets. These findings demonstrate the importance of the dimeric state to FXI activity and suggest a novel model for factor IX activation in which FXIa binds to activated platelets by one chain of the dimer, while binding to factor IX through the other.

Blood Coagulation↗
PubMed1996

Inactivation of factor Xia in vivo: studies in chimpanzees and in humans.

C1-inhibitor (C1Inh), antithrombin III (ATIII), alpha 1-antitrypsin (a1AT), and alpha 2-antiplasmin (a2AP) are known inhibitors of factor XIa (FXIa). However, their precise contribution to FXIa inactivation in vivo is not known. We investigated FXIa inactivation in chimpanzees and assessed the contribution of these inhibitors to FXIa inactivation in patients with presumed FXI activation. Chimpanzees were infused with FXIa and the various FXIa-FXIa inhibitor complexes formed were measured. Most of FXIa was complexed to C1Inh (68%), followed by a2AP (13%), a1AT (10%), and ATIII (9%). Analysis of the plasma elimination kinetics revealed a half-life time of clearance (t1/2) for the FXIa-FXIa inhibitor complexes of 95 to 104 min, except for FXIa-a1AT, which had a t1/2 of 349 min. Due to this long t1/2, FXIa-a1AT complexes were predicted to show the highest levels in plasma samples from patients with activation of FXI. This was indeed shown in patients with disseminated intravascular coagulation, recent myocardial infarction or unstable angina pectoris. We conclude from this study that in vivo C1Inh is the predominant inhibitor of FXIa, but that FXIa-a1AT complexes due to their relatively long t 1/2 may be the best parameter to assess FXI activation in clinical samples.

Angina, Unstable↗
PubMed2006

Contributions of basic amino acids in the autolysis loop of factor XIa to serpin specificity.

The autolysis loops (amino acids 143-154, chymotrypsinogen numbering) of plasma serine proteases play key roles in determining the specificity of protease inhibition by plasma serpins. We studied the importance of four basic residues (Arg-144, Lys-145, Arg-147, and Lys-149) in the autolysis loop of the coagulation protease factor XIa (fXIa) for inhibition by serpins. Recombinant fXIa mutants, in which these residues were replaced individually or in combination with alanine, were prepared. The proteases were compared to wild-type fXIa (fXIa-WT) with respect to their ability to activate factor IX in a plasma clotting assay, to hydrolyze the chromogenic substrate S2366, and to undergo inhibition by the C1-inhibitor (C1-INH), protein Z dependent protease inhibitor (ZPI), antithrombin (AT), and alpha(1)-protease inhibitor (alpha(1)-PI). All mutants exhibited normal activity in plasma and hydrolyzed S2366 with catalytic efficiencies similar to that of fXIa-WT. Inhibition of mutants by C1-INH was increased to varying degrees relative to that of fXIa-WT, with the mutant containing alanine replacements for all four basic residues (fXIa-144-149A) exhibiting an approximately 15-fold higher rate of inhibition. In contrast, the inhibition by ZPI was impaired 2-3-fold for single amino acid substitutions, and fXIa-144-149A was essentially resistant to inhibition by ZPI. Alanine substitution for Arg-147 impaired inhibition by AT approximately 7-fold; however, other substitutions did not affect it or slightly enhanced inhibition. Arg-147 was also required for inhibition by alpha(1)-PI. Cumulatively, the results demonstrate that basic amino acids in the autolysis loop of fXIa are important determinants of serpin specificity.

Amino Acid Sequence↗
PubMed1995

Zinc (II) selectively enhances the inhibition of coagulation factor XIa by protease nexin-2/amyloid beta-protein precursor.

Protease nexin-2 (PN-2) is the secreted isoform of the Alzheimer's Amyloid beta-Protein Precursor (A beta PP) that contains the Kunitz-type protease inhibitor (KPI) domain. PN-2/A beta PP is a potent inhibitor of coagulation factor XIa (FXIa) and is secreted in large quantities by activated platelets suggesting a normal function in regulating this protease at sites of vascular injury. In the present study, the effect of Zn2+ on the protease inhibitory properties of PN-2/A beta PP was quantitatively investigated. Zn2+ (1 microM to 1 mM) had no effect on the inhibition of trypsin or chymotrypsin by PN-2/A beta PP. In contrast, Zn2+ at concentrations > 1 microM increased the inhibition of FXIa by PN-2/A beta PP. Enhancement of FXIa inhibition was virtually saturated at approximately 100 microM Zn2+ resulting in a final Ki approximately 6.0 x 10(-11) M. Zn2+ had no effect on the inhibition of FXIa by a purified, recombinant KPI domain of PN-2/A beta PP indicating that the native protein is required for the potentiation of FXIa inhibition. Heparin and Zn2+ were found to further augment each other's ability to stimulate the inhibition of FXIa by PN-2/A beta PP. Together, these findings suggest that the interaction of Zn2+ with PN-2/A beta PP may be important for optimal inhibition of FXIa.

Amyloid beta-Protein Precursor↗
PubMed2002

The interaction of factor XIa with activated platelets but not endothelial cells promotes the activation of factor IX in the consolidation phase of blood coagulation.

We have previously shown that the zymogen factor XI (FXI) binds to activated platelets but not to human umbilical vein endothelial cells (HUVEC), a conclusion that is in conflict with previous reports stating that FXI binds to 2.7-13 x 10(6) high affinity sites per HUVEC (Berrettini, M., Schleef, R. R., Heeb, M. J., Hopmeier, P., and Griffin, J. H. (1992) J. Biol. Chem. 267, 19833-19839; Shariat-Madar, Z., Mahdi, F., and Schmaier, A. H. (2001) Thromb. Haemostasis 85, 544-551). It has also been reported that activated FXI (FXIa) binds to 1.5 x 10(6) sites per HUVEC and promotes the activation of factor IX by cell bound FXIa (Berrettini, M., Schleef, R. R., Heeb, M. J., Hopmeier, P., and Griffin, J. H. (1992) J. Biol. Chem. 267, 19833-19839). Therefore, the binding of FXIa to activated platelets was compared with FXIa binding to HUVEC and HEK293 cells immobilized on microcarrier beads. Specific and saturable zinc-dependent FXIa binding was demonstrated to 250 +/- 48 sites per activated platelet (K(D) = 1.7 +/- 0.78 nm) and 6.5 +/- 0.4 x 10(4) sites per HUVEC (K(D) = 2.4 +/- 0.5 nm), whereas no binding to HEK293 cells was detected. A titration with high molecular weight kininogen had no effect on FXIa binding to platelets, but revealed a concentration-dependent decrease in the amount of FXIa bound to HUVEC. The rate of factor IXa generation catalyzed by FXIa was unaffected by the presence of surfaces; however only the activated platelet surface protected FXIa from inhibition by protease nexin 2. The results presented here confirm the conclusion that activated platelets are procoagulant while unstimulated endothelial cells are not.

Amyloid beta-Protein Precursor↗
PubMed2000

Fibrillar amyloid beta-protein binds protease nexin-2/amyloid beta-protein precursor: stimulation of its inhibition of coagulation factor XIa.

Cerebrovascular deposition of fibrillar 39-42 amino acid amyloid beta-protein (Abeta), a condition known as cerebral amyloid angiopathy (CAA), is a key pathological feature of Alzheimer's disease and related disorders including hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). Severe cases of CAA, particularly in HCHWA-D, lead to recurrent and often fatal hemorrhagic strokes. Although the reasons for this pathological consequence remain unclear, alterations in proteolytic hemostasis mechanisms have been implicated. For example, the Abeta parent molecule protease nexin-2/amyloid beta-protein precursor (PN-2/AbetaPP), which is elevated in HCHWA-D cerebral vessels with Abeta deposits, is a potent inhibitor of coagulation factor XIa (FXIa). Here we show that fibrillar HCHWA-D Abeta binds PN-2/AbetaPP, but not its isolated Kunitz-type proteinase inhibitor (KPI) domain, in a saturable, dose-dependent manner with a K(d) of approximately 28 nM. Neither PN-2/AbetaPP nor its KPI domain bound to nonfibrillar HCHWA-D Abeta. The fibrillar Abeta binding domain on PN-2/AbetaPP was localized to residues 18-119. PN-2/AbetaPP that bound to fibrillar HCHWA-D Abeta immobilized either in plastic wells or on the surface of cultured cerebrovascular smooth muscle cells was active in inhibiting FXIa. Quantitative kinetic measurements revealed that fibrillar HCHWA-D Abeta caused a >5-fold enhancement of FXIa inhibition by PN-2/AbetaPP. Similar stimulatory effects on FXIa inhibition by PN-2/AbetaPP were also observed with fibrillar wild-type Abeta. However, fibrillar Abeta had no effect on the inhibition of trypsin by PN-2/AbetaPP. These findings suggest that fibrillar Abeta deposits in cerebral vessels can effectively localize and enhance the anticoagulant functions of PN-2/AbetaPP, thereby contributing to a microenvironment conducive to hemorrhaging.

Amyloid beta-Peptides↗
PubMed1975

Studies on a circulating anticoagulant in systemic lupus erythematosus: evidence for inhibition of the function of activated plasma thromboplastin antecedent (factor XIa).

Plasma proteins which interfere with blood coagulation have often been described in patients with systemic lupus erythematosus (SLE). The most frequent type interferes with the conversion of prothrombin to thrombin and thus prolongs the prothrombin time. Infrequently, SLE patients exhibit anticoagulants which appear to block the earlier stages of coagulation such as those involving factor VIII or the formation of activated factor XI (factor XIa). The anticoagulant reported here was studied by means of a sequential clotting system utilizing crude coagulation factors and was noted to interfere with the action of activated plasma thromboplastin antecedent (PTA) during the activation of factor IX. This anticoagulant was found in gamma-globulin-rich ethanol fractions of plasma. After gel filtration, it was found principally in fractions containing IgM globulins but also, to a lesser extent, in IgG-rich fractions. In this respect, it is similar to anticoagulants reported in certain other cases of SLE. Attempts to confirm the immunoglobulin nature of the anticoagulant by immunoabsorption were, however, inconclusive

Absorption↗
PubMed1997

Progress curve analysis of the kinetics with which blood coagulation factor XIa is inhibited by protease nexin-2.

Protease nexin-2 (PN-2), a soluble form of amyloid beta-protein precursor (APP) containing a Kunin protease inhibitor domain, has been shown to be a potent, reversible and competitive inhibitor of blood coagulation factor XIa (FXIa). We have analyzed progress curves of the hydrolysis of a sensitive fluorogenic substrate by FXIa in the presence of PN-2 to ascertain the kinetic rate constants governing the inhibition of FXIa by PN-2. The mechanism of this inhibition is best described as a slow equilibration between the free enzyme and inhibitor directly, without prior formation of a loosely-associated complex. The association rate constant (kon) and the dissociation rate constant (koff) were found to be 2.1 +/- 0.2 x 10(6) M-1 s-1 and 8.5 +/- 0.8 x 10(-4) s-1, respectively (n = 23). The inhibition constant calculated from these parameters (Ki) is 400 pM, in good agreement with previous reports. High molecular weight kininogen (HK) and Zn2+ ions exert opposite effects on the inhibition of FXIa by PN-2. HK protects FXIa from inactivation in a dose dependent and saturable manner (EC50 = 61 nM) whereas Zn2+ augments the ability of PN-2 to inhibit FXIa. When both Zn2+ ions and HK are present, only the accessory effect of Zn2+ is observed. PN-2 is known to be an abundant platelet alpha-granule protein (Van Nostrand et al., 1990a; Smith & Broze, 1992). We conducted sensitive measurements of FXIa activity in the presence of human platelets before and after their being activated with the thrombin receptor agonist peptide, SFLLRN-amide. We found that platelet activation, and ostensibly the release of PN-2, limits the lifetime of FXIa activity within the locus of activated platelets. As in the purified system, HK protects FXIa from inactivation and Zn2+ increases the inactivation of FXIa. However, when HK and Zn2+ are both present, it is the protective effect of HK which predominates and prolongs the lifetime of FXIa after platelet activation.

Amyloid beta-Protein Precursor↗