Factor XI, but Not Prekallikrein, Blocks High Molecular Weight Kininogen Binding to Human Umbilical Vein Endothelial Cells*

T. Regan Baird {ddagger} § and Peter N. Walsh {ddagger} § ¶ ||

From the {ddagger}The Sol Sherry Thrombosis Research Center, the §Department of Biochemistry, and the Department of Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

Received for publication, January 8, 2003 , and in revised form, March 24, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies on the interaction of high molecular weight kininogen (HK) with endothelial cells have reported a large number of binding sites (106-107 sites/cell) with differing relative affinities (KD = 7–130 nM) and have implicated various receptors or receptor complexes. In this study, we examined the binding of HK to human umbilical vein endothelial cells (HUVEC) with a novel assay system utilizing HUVEC immobilized on microcarrier beads, which eliminates the detection of the high affinity binding sites found nonspecifically in conventional microtiter well assays. We report that HK binds to 8.5 x 104 high affinity (KD = 21 nM) sites per HUVEC, i.e. 10–100-fold fewer than previously reported. Although HK binding is unaffected by the presence of a physiological concentration of prekallikrein, factor XI abrogates HK binding to HUVEC in a concentration-dependent manner. Disruption of the naturally occurring complex between factor XI and HK by the addition of a 31-amino acid peptide mimicking the factor XI-binding site on HK restored HK binding to HUVEC. Furthermore, HK inhibited thrombin-stimulated von Willebrand factor release by HUVEC but not thrombin receptor activation peptide (SFLLRN-amide)-stimulated von Willebrand factor release. Factor XI restored the ability of thrombin to stimulate von Willebrand factor release in the presence of low HK concentrations. These results suggest that free HK, or HK in complex with prekallikrein but not in complex with factor XI, interacts with the endothelium and can maintain endothelial cell quiescence by preventing endothelial stimulation by thrombin.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
High molecular weight kininogen (HK)1 is a multifunctional protein that serves as a cofactor in blood coagulation, fibrinolysis, and the complement system. Cryptic within the fourth kininogen domain is the vasoactive peptide bradykinin (BK), which binds to the B1 and B2 receptors on endothelial cells leading to changes in blood pressure and elevation in endothelial nitric oxide and prostacyclin synthesis (1, 2, 3, 4, 5). BK is released from HK primarily through the action of kallikrein; however, factor XIIa (FXIIa) and FXIa have also been shown to excise BK in vitro (2, 6, 7). It is suggested that HK must remain in close proximity to the BK receptor in order for BK to function due to its short half-life (t1/2 = 15 s) (8, 9).

HK contains the anti-angiogenic domain 5, kininostatin, which inhibits endothelial cell proliferation and stimulates apoptosis through an anti-adhesive mechanism that involves the displacement of endothelial cells from vitronectin (10). Other HK domains interact with platelets and inhibit calpain-related platelet aggregation via domain 2 and prevent thrombin binding to glycoprotein Ib-IX-V (GPIb-IX-V) via domain 3 or protease activated receptor-1 activation via domain 4 (11, 12, 13). Both FXI (30 nM) and prekallikrein (PK) (490 nM) circulate in plasma in high-affinity (KD ~ 108 M) complexes with HK (670 nM) (14, 15, 16, 17, 18). Thus, virtually all the FXI and all the PK in plasma are found in non-covalent complexes with HK. A 31-amino acid and 56-amino acid overlapping region of HK domain 6 interacts with the Apple 1, 2, and 4 domains of either PK or FXI, respectively (19, 20, 21, 22, 23, 24). According to the current model, HK binding to FXI evokes a conformational change exposing the Apple 3 domain of FXI, which mediates binding to activated platelets (25). This specific, saturable, high-affinity interaction of FXI with platelets requires platelet activation and is essential for promoting a 5,000- to 10,000-fold acceleration of the rate of FXI-activation by thrombin (14, 26, 27, 28).

The putative endothelial cell receptor for HK is a matter of some controversy because several proteins have been identified including GPIb-IX-V (29). However, HK has been reported to bind to Bernard-Soulier platelets, which lack this receptor complex (30). Affinity chromatography identified cytokeratin 1 and gC1qR as putative HK receptors, and it has been suggested that these proteins along with the urokinase plasminogen activator receptor form a multiprotein receptor complex on the endothelial cell surface (30, 31, 32, 33, 34). However, gC1qR is a mitochondrial membrane protein not conclusively identified on the endothelial plasma membrane (35, 36, 37, 38). Furthermore, these proteins are not found on endothelium in sufficient quantities to accommodate the millions of endothelial cell-binding sites reported for HK (39, 40, 41, 42, 43). Alternatively, the heparan sulfate moieties found in vast quantities on the endothelium were identified as an HK receptor using confocal microscopy studies; however, binding was localized only to the cell-cell junctions (44). Also, Mac-1 integrin (CD11b/18) was identified as the HK receptor on neutrophils because antibodies against Mac-1 blocked HK binding although the Mac-1 antibodies may have simultaneously down-regulated urokinase plasminogen activator receptor expression (34, 45).

It had been proposed that FXI and FXIa interacted specifically with endothelial cells in an HK-dependent manner (39, 46). However, we have shown that FXI does not bind to endothelial cells, and although FXIa does bind to endothelial cells, physiological concentrations of HK block this interaction (47, 48). Our studies differed from those performed previously in that we utilized endothelial cells grown on microcarrier beads, allowing us to eliminate the high-affinity, nonspecific interactions of HK with the artificial surfaces contained within microtiter wells. These nonspecific interactions will appear to augment the number of HK receptors and thus FXI- or FXIa-binding sites. In the following study we report that HK does bind to human umbilical vein endothelial cells (HUVEC) and that the number of HK-binding sites on washed confluent HUVEC-coated microcarrier bead cultures is 10–100 times lower than reported in assays employing HUVEC monolayers in microtiter wells. Physiological concentrations of FXI inhibit low levels of HK from binding to HUVEC, whereas physiological concentrations of PK have no effect on HK binding.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Proteins—FXI was purchased from Hematologic Technologies, Inc. (Essex Junction, VT). HK, PK, and thrombin were purchased from Enzyme Research Laboratories (South Bend, IN). The thrombin receptor activation peptide (TRAP) (SFLLRN-amide) and the HK-derived peptide comprising a portion of the FXI-binding site referred to as the 31-mer HK peptide (SDDDWIPMDIQTDPNGLSFNPISDFAPDTTSPK-amide) were synthesized at the Protein Chemistry Laboratory at the University of Pennsylvania (Dr. John Lambris, Director).

Radiolabeling with Iodine—HK was radiolabeled with 125I using the Iodogen method (49). Generally >98% radioactivity was bound to the protein, and the specific radioactivity was 1.1 x 1018 CPM/mol. The radiolabeled HK had a specific biological activity of 12.75 units/mg, i.e. 102% of the coagulation activity of unlabeled HK.

Cell Culture—HUVECs were purchased from Cascade Biologicals (Portland, OR) and cultured in EGM-2 complete media (2% fetal bovine serum, BioWhittaker, Rockland, ME) in a humidified atmosphere of 5% CO2 at 37 °C as previously described (48). HUVEC (1 x 106 cells/ml) was added to 5 ml (1.5 x 105 beads) of swelled Cytodex-3 collagen-coated microcarrier beads (Amersham Biosciences) that were washed in complete endothelial cell media. After 30 min at room temperature the beads and cells were transferred to a spinner flask (Wheaton Scientific, Millville, NJ) and incubated under constant stirring (60 RPM) in a humidified atmosphere of 5% CO2 at 37 °C. Transformed human embryonic kidney cells (HEK293) were obtained from American Type Culture Collection (CRL1573) and were similarly grown to confluence on Cytodex-3 collagen-coated microcarrier beads. Microcarrier bead cultures (0.5 ml) were mixed with 0.5 ml trypan blue (Invitrogen) to determine the extent of confluence, because the microcarrier beads but not cell-coated beads retained trypan blue staining (48).

Measurement of von Willebrand Factor (vWF) Release by HUVEC— The amount of von Willebrand factor released by HUVEC after stimulation, a marker of activation, was measured by sandwich ELISA using a goat anti-human vWF IgG primary antibody and horseradish peroxidase-conjugated goat anti-human vWF IgG secondary antibody performed according to manufacturer's instructions (Enzyme Research Laboratories, South Bend, IN). Because these experiments could not be performed in the absence of microtiter wells, HUVECs were grown to confluence in 12-well gelatin-coated microtiter plates and treated with agonists or incubation buffer alone (0.5% bovine serum albumin). Supernatants (100 µl) were removed and analyzed by ELISA. The optical density (OD490) was measured, and vWF concentrations were calculated by comparison to a standard curve determined in the presence of diluted normal pooled human plasma (George King Biomedical, Overland Park, KS) where 100% plasma contains 10 ng/ml vWF.

Equilibrium Binding Experiments Using Cells in Suspension— HUVECs were placed into suspension on microcarrier beads, and binding was measured using a procedure similar to that previously described for platelets (50). The HUVEC-coated microcarrier beads were washed and resuspended in incubation buffer to a density of 106 cells/ml. Silicone oil (15 µl, DC500/DC200, 4:1) was centrifuged to the bottom of a 200 µl microcentrifuge tube with a narrow bore tip (Sarstedt, Inc., Princeton, NJ). The microcarrier bead suspension was added to the radiolabeled protein mixture (50 µl each) and incubated at 37 °C for 1 h with shaking by hand every 15 min. To separate the bound from free protein, tubes were centrifuged at 12,000 x g for 5 min in a vertical centrifuge (model B, Beckman Instruments, Inc., Cedar Grove, NJ). The tubes were placed in dry ice/ethanol slurry for 5 min, and the tips of the microtubes were amputated. Both the pellets and 10 µl of the supernatant were measured for {gamma}-emission.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
HK Binding to HUVEC on Microcarrier Beads—Saturable and specific HK binding to HUVEC has been demonstrated previously (39, 40, 41, 42, 43); however, all these experiments were performed in microtiter well assays. We observed a high level of nonspecific background binding of FXI when HK was utilized as a cofactor in empty microtiter wells (48). High levels of nonspecific binding of 125I-HK to empty microtiter wells were also observed (data not shown). Attempts to prevent this background binding using different blocking agents (i.e. gelatin, polyethylene glycol, Irish cream), different coating agents (i.e. gelatin, fibronectin, or silicone treatment), or different brands of microtiter plates, did not result in a lower number of binding sites in the absence of cells. Therefore, we carried out 125I-HK binding studies with HUVEC-coated microcarrier beads in suspension rather than with HUVEC monolayers in microtiter wells. Cell-coated beads were pelleted by centrifugation through silicone oil after incubation with the protein mixture. This method allows for the detection of specific cellular binding sites without the high background determined in microtiter wells because only radioactivity associated with the cellular pellets rather than associated with a washed microtiter well was measured. Cytodex-3 microcarrier beads on HEK293 cells were grown to confluence, demonstrated very low level, nonsaturable binding of HK (data not shown). In contrast, when HUVECs were grown to confluence, it was shown that HK binds saturably, specifically, and reversibly to 8.5 ± 0.3 x 104 sites per cell with a KD = 21 ± 2.6 nM (Fig. 1).



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FIG. 1.
HK binding to HUVEC-coated microcarrier Beads. 125I-HK was added to HUVEC and incubated for 30 min in the presence of 25 µM zinc ions (•) before separation through silicone oil. Other results were obtained in the presence of 25 µM ZnCl2 in addition to 500 nM prekallikrein ({square}); or 30 nM FXI ({circ}); or both 500 nM PK and 30 nM FXI ({blacksquare}). Results shown in the inset represent the same data plotted on an expanded abscissa (HK, 0–100 nM). The specific binding, or the difference between the total and non-displaceable binding, is shown. Results represent the means (±S.E.) of three experiments performed in triplicate.

 

HK circulates at a physiological concentration of ~650 nM, 90% in a 1:1 complex with plasma PK (~490 nM), and ~10% in a 2:1 complex with dimeric FXI (~30 nM) (17, 51). Thus, very little if any unbound HK is found in plasma. Therefore, we also performed 125I-HK binding studies in the presence of either a physiological concentration of PK or FXI, or with both proteins present. Whereas PK had no effect on the binding of HK (Fig. 1), the presence of 30 nM FXI (i.e. 60 nM FXI monomer) inhibited concentrations of HK <100 nM from binding to HUVEC. As the concentration of HK increased, it was able to overcome the inhibitory effect of FXI. Under conditions when both PK and FXI were added together at physiological concentrations, HK binding to HUVEC was detected even at the lower HK concentrations.

FXI and PK Titrations—Because FXI but not PK appears to inhibit HK binding to HUVEC (Fig. 1), HK binding was examined at various concentrations of FXI or PK or with both proteins present. The binding of 125I-HK (500 nM) to HUVEC-coated microtiter beads was unaffected by PK even at supraphysiological concentrations (1,400 nM) either in the absence or presence of 30 nM FXI (Fig. 2A). In contrast, FXI potently (IC50 ~30 nM) and completely inhibited the binding of 125I-HK at a subphysiological concentration (100 nM; Fig. 2B, inset). At an HK concentration (500 nM) approximating that in plasma, FXI completely inhibited HK binding, and the presence of PK (500 nM) abrogated the inhibitory effect of FXI (Fig. 2), a result consistent with that observed in Fig. 1.



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FIG. 2.
The effect of FXI and prekallikrein on HK binding to HUVEC. A, an increasing concentration of PK was incubated with 500 nM 125I-HK and HUVEC-coated microcarrier beads in the presence of zinc ions (25 µM) in the absence ({blacksquare}) or in the presence ({square}) of FXI (30 nM) for 30 min before separation through silicone oil. B, 125I-HK (500 or 100 nM, inset) binding to HUVEC was titrated with increasing concentrations of FXI and incubated for 30 min in the presence of zinc ions (25 µM) in the absence (•) or in the presence ({circ}) of PK (500 nM) before separation through silicone oil. Results represent the means (± S.E.) of three experiments performed in triplicate.

 

FXI·HK Complex Formation Is Required for Inhibition of HK Binding to HUVEC—FXI and PK bind to an overlapping region of domain 6 on HK (22, 23); however, only FXI inhibits HK binding to HUVEC, whereas PK does not. To determine whether FXI binding to domain 6 is required for the inhibition by FXI of HK binding to HUVEC we incubated HK with the 31-amino acid peptide that mimics the FXI- and PK-binding site on HK (31-amino acid HK peptide) in the presence or absence of FXI prior to incubation with HUVEC. The 31-amino acid HK peptide utilized at a concentration that blocks FXI from binding to HK (10 µM) as determined by surface plasmon resonance (52), has no effect on the binding of HK to HUVEC (Fig. 3), suggesting that this portion of domain 6 does not mediate HK binding to HUVEC. However, also shown in Fig. 3, the 31-amino acid HK peptide reverses the ability of FXI to inhibit HK binding to HUVEC observed in Fig. 1 by preventing HK binding to FXI, suggesting that FXI·HK complex formation is required for the inhibition by FXI of HK binding to HUVEC.



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FIG. 3.
Complex formation between FXI and HK is required for inhibition. Increasing concentrations of 125I-HK were incubated with 31-amino acid HK peptide (10 µM) and zinc ions (25 µM) and HUVEC-coated microcarrier beads in the presence ({circ}) or absence (•) of FXI (30 nM). Results represent the mean (± S.E.) of three experiments performed in triplicate.

 

Activation of HUVEC—Activation of HUVEC by various agonists is characterized by vWF release. Release of vWF was measured by ELISA and quantitated using a standard curve derived using normal pooled plasma. Both thrombin (1 nM) and TRAP (25 µM) stimulated maximum vWF release within 30–60 min (data not shown), and endotoxin (lipopolysaccharide) induced maximal vWF release after 6 h. HK, FXI, or buffer alone was unable to stimulate significant amounts of vWF release after 6 h (data not shown).

Thrombin (1 nM) was able to stimulate HUVEC monolayers to release 66 ng per 1 x 105 HUVEC (Fig. 4). HK and FXI were unable to stimulate vWF release. However, either physiological concentrations of HK (500 nM) or sub-physiological concentrations (100 nM) inhibited the ability of thrombin to stimulate HUVEC to release vWF. FXI (30 nM) blocked the ability of lower HK concentrations to inhibit thrombin-stimulation of HUVEC (Fig. 4, inset), but not when HK was added at the higher concentration (500 nM; Fig. 4). PK did not prevent the inhibitory effect of HK on thrombin-stimulated vWF release (Fig. 4, inset). However, the presence of PK reversed the capacity of FXI to prevent the inhibition by HK of thrombin-induced vWF release from HUVEC (Fig. 4, inset). HK did not inhibit TRAP-stimulated vWF release (data not shown). These results complement those obtained in the binding studies because although PK and FXI bind to a similar site on HK (22, 23), FXI, but not PK, prevents HK binding to HUVEC and thus the inhibition of thrombin-mediated vWF release from HUVEC by HK.



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FIG. 4.
HK inhibits thrombin stimulation of HUVEC. A confluent monolayer of HUVEC was incubated with a combination of 0.1 units/ml thrombin (TB), 500 nM HK (100 nM, inset), 30 nM FXI, and/or 500 nM PK for one hour. The amount of HUVEC stimulation was determined by vWF release measured using an ELISA (see "Experimental Procedures"). Results were compared with a buffer control and to cells that were incubated with a lysis buffer in the absence of agonist (total) for an additional 10 min. Note the ability of HK to inhibit thrombin-stimulated vWF release in the absence of FXI. However, in the presence of physiological concentrations of FXI inhibition by HK (at 100 nM) is negated (inset). Results represent the means (±S.E.) of three experiments each performed in quadruplicate.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Although we have confirmed the observation that HK binds to saturable, high-affinity sites on HUVEC, the number of binding sites determined in our present study (85,000 sites/HUVEC, KD = 21 nM) is 10–100-fold less than previously reported (1–10 x 106 sites/HUVEC, KD = 7–130 nM) (39, 40, 41, 42, 43). Because our previous results suggested and our present studies confirm that HK binds nonspecifically to the wells of microtiter plates, HK binding studies were carried out using HUVEC-coated microcarrier beads (48). Using this suspension assay, we have confirmed the conclusion that HK binds to HUVEC (Fig. 1).

HK may bind to the endothelium for the purpose of releasing BK resulting in local stimulation of endothelium (40). Proposed HK receptors include the urokinase plasminogen activator receptor, C1qR, cytokeratin-1 receptor complex, the GPIb-IX-V receptor complex, and heparan sulfate glycosaminoglycans located at cell junctions (29, 31, 32, 44, 53). Because HK is the pro-hormone form of BK released by kallikrein, and BK has a very short half-life (15 s), the pro-hormone must be within close proximity of its receptor on endothelial cells (8, 9). HK normally circulates in a complex with PK or FXI. The fraction of HK in complex with PK binds to HUVEC (Fig. 1), whereas the fraction of HK in complex with FXI does not bind to HUVEC (Figs. 1 and 2). In contrast, we have previously shown (48) that FXI binding to activated platelets requires the presence of HK and Zn2+ ions. The activation of FXI by thrombin on the activated platelet surface also requires the presence of HK and Zn2+ ions (28), or prothrombin and Ca2+ ions (14, 26, 28), whereas there is no cofactor requirement for thrombin-catalyzed FXI-activation in the presence of dextran sulfate (54).

Under more relevant conditions, i.e. when physiological concentrations of both PK and FXI are present, the displacement of HK by FXI is not observed and HK binding to HUVEC is maintained. This is a consequence of the fact that the FXI concentration in plasma (30 nM dimer or 60 nM monomer), utilized in the present experiments, is ~6% (or ~12% monomer) of that of PK (500 nM). Because the affinity of PK and FXI for HK is similar (KD = 10 nM), the association of HK with PK and not FXI will be favored given their relative concentrations. Thus, HK binds to HUVEC in the presence of a physiological concentration of both proteins. The percentage of HK associated with FXI, which does not bind to HUVEC, is a minor proportion of the total number of HK molecules and has a minor effect on the HK-binding isotherms examined at saturating, physiological HK concentrations. Therefore, we conclude that in plasma the 10–12% of HK complexed with FXI does not bind to HUVEC, whereas the majority of HK (~90%), either free or in complex with PK, is available to associate with the endothelium.

The ability of FXI to interfere with HK binding to the surface of HUVEC was also observed when HUVECs were pretreated with contact proteins prior to addition of thrombin. HK (100 nM) inhibited thrombin-stimulated vWF release by HUVEC, but this inhibitory effect of low concentrations of HK (100 nM) was abrogated in the presence of FXI (Fig. 4), most likely because FXI forms a complex with HK and prevents HK binding to the endothelium. When higher concentrations of HK were used (500 nM), or in the presence of PK (500 nM), the inhibition of thrombin-stimulated vWF release was restored, most likely because PK does not inhibit HK binding to HUVEC (Fig. 2A). HK has also been shown to inhibit platelet activation by thrombin at a high concentration (IC50 = 2 µM), four times greater than the highest concentrations utilized in the present study (55). This inhibitory effect has been attributed to the binding of HK to the GPIb-IX-V complex and its capacity to displace thrombin on platelets (55). It is possible that the inhibition by HK of thrombin-induced secretion of vWF by HUVEC is mediated by a similar mechanism, i.e. competitive binding of HK and thrombin to GPIb-IX-V on endothelial cells. In contrast, HK was unable to inhibit vWF release from HUVEC activated through PAR-1 by TRAP (Fig. 4) (56). As expected, neither HK nor FXI alone promoted vWF release from HUVEC.

It is interesting that two structurally related proteins, FXI and PK, which bind to HK on an overlapping region within domain 6 with nearly identical affinities, have markedly different effects on the interaction of HK with HUVEC. However, there are major differences between PK and FXI. Most prominent is the fact that FXI is a homodimeric molecule whereas PK is monomeric, resulting in twice as many HK-binding sites on FXI compared with PK. This fact by itself does not explain the differential effects of FXI and PK on HK binding unless the quaternary structure of the HK·FXI complex is vastly different from that of the HK·PK complex. In fact, the HK·PK complex does not bind to activated platelets, whereas the HK·FXI complex does (48).

We conclude that HK serves as a divergence point for inflammation and fibrinolysis versus coagulation, by trafficking two structurally related proteins, FXI and PK, to different compartments of the vasculature. PK is directed toward the endothelium where activation to kallikrein results in the release from HK of BK, which in turn stimulates the endothelial cell (9). On the other hand, HK in complex with FXI is inhibited from binding to the large number of HK receptors (85,000 sites/HUVEC) that are found within the vasculature. The inhibition of HK binding to HUVEC by FXI may be mediated by an allosteric mechanism because FXI itself does not bind to endothelial cells and therefore cannot compete with HK for HUVEC-binding sites (48). FXI must form a complex with HK to block HK binding because when this interaction is disrupted by the 31-amino acid HK peptide, the inhibitory effect is abrogated. The 31-amino acid HK peptide itself has no effect on HK binding to HUVEC; therefore, this region does not mediate HK binding to HUVEC. Thus, FXI is maintained in a conformation suitable for the amplification of the coagulation cascade at the site of vascular injury, where platelets become activated and expose binding sites for the activation by thrombin of FXI in complex with HK (28).

In conclusion, the present results support an intriguing and novel hypothesis that HK aids in the maintenance of quiescent endothelium by preventing activation by thrombin. The biological relevance of the inhibition by HK of thrombin-induced vWF release from endothelium requires further experimentation. However, this hypothesis is entirely consistent with a large body of accumulating evidence (reviewed in Ref. 57) to support the concept that one of the major physiological functions of the proteins of the contact activation pathway, including HK, is anti-coagulant and anti-thrombotic.


    FOOTNOTES
 
* This study was supported in part by Research Grants HL46213, HL64943, HL70683, and HL56914 (to P. N. W.) from the National Institutes of Heath and Grant 9910069U from the American Heart Association (to T. R. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, 3400 N. Broad Street, Philadelphia, PA 19140. Tel.: 215-707-4375; Fax: 215-707-3005; E-mail: pnw{at}temple.edu.

1 The abbreviations used are: HK, high molecular weight kininogen; HUVEC, human umbilical vein endothelial cells; BK, bradykinin; FXIIa, factor XIIa; GPIb-IX-V, glycoprotein Ib-IX-V; PK, prekallikrein; TRAP, thrombin receptor activation peptide; vWF, von Willebrand factor; ELISA, enzyme-linked immunosorbent assay. Back


    ACKNOWLEDGMENTS
 
We are grateful to Patricia Pileggi for her expertise in manuscript preparation.



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 ABSTRACT
 INTRODUCTION
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 DISCUSSION
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