Activated Platelets but Not Endothelial Cells Participate in the Initiation of the Consolidation Phase of Blood Coagulation*

To address the question of whether initiation of the consolidation phase of coagulation occurs on platelets or on endothelium, we have examined the interaction of coagulation factor XI with human umbilical vein endothelial cells (HUVEC) and with platelets. In microtiter wells factor XI binds to more sites in the absence of HUVEC (1.8 × 1010 sites/well,K D = 2.6 nm) than in their presence (1.3 × 1010 sites/well, K D = 12 nm) when high molecular weight kininogen (HK) and zinc are present. Binding was volume-dependent and abrogated by HUVEC or Chinese hamster ovary cells and was a function of nonspecific binding of HK to the artificial plastic surface. Factor XI did not bind to HUVEC or to HEK293 cell monolayers anchored to microcarrier beads. Activation of HUVEC resulted in von Willebrand's factor secretion, but factor XI binding was not observed. Only activated platelets supported factor XI binding in the presence of HK and zinc (K D = 8 nm, Bmax = 1319 sites/cell). Activation of factor XI was observed in plasma in the presence of platelets activated by the thrombin receptor activation peptide but not with activated HUVEC. These results support the concept that activated platelets, but not endothelial cells, expose a procoagulant surface for binding and activating factor XI, thereby initiating the consolidation phase of coagulation.

It is well known that plasma proteins and blood and vascular cells play a pivotal role in hemostasis. However, under normal physiological conditions blood is fluid, and yet a clot can form locally at the site of vascular injury. Fluidity of blood is maintained by the anti-thrombotic nature of the vascular endothelium, and thrombus formation is mediated in part by the activation of platelets. Contrary to the anticoagulant paradigm of endothelial function, it has been reported that quiescent endothelial cells can bind coagulation factor XI (FXI) 1 and promote its activation and expression of its enzymatic activity in vitro (1,2). The present study examines the role of both human platelets and human umbilical vein endothelial cells (HUVEC) in the binding and activation of FXI, which participates in the initiation of the intrinsic or consolidation pathway of blood coagulation.
FXI is a 160-kDa homodimeric protein found in plasma at a concentration of ϳ30 nM complexed with high molecular weight kininogen (HK) (3,4). FXIIa, thrombin, and FXIa have been shown to activate FXI to FXIa in the presence of an artificial negatively charged surface (3,5,6). Two potentially physiologically relevant cellular surfaces that might promote blood coagulation within the vasculature are the platelet and the endothelial cell. FXI binds to activated platelets with high affinity (K D ϭ 10 nM, B max ϭ 1,500 sites/platelet) in the presence of HK (45 nM) and zinc ions (25 M) (7) or in the presence of prothrombin (1 M) and calcium ions (2 mM) (8), but this only occurs after the platelet has been pretreated with an agonist such as thrombin (7,8). Activated platelets also promote the activation of platelet-bound FXI by thrombin, FXIIa, and FXIa with thrombin demonstrated to be the preferred activator (9,10). Thus, the platelet exists in the blood stream in a dormant form that exposes a surface for assembly of coagulation complexes only after appropriate stimulation.
The negative charge associated with the endothelium comes in the form of constitutively expressed heparan sulfate and chondroitan sulfate glycosaminoglycans (11). Thus, the endothelium is constantly exposing a negative charge to plasma, yet coagulation does not ubiquitously occur. This anti-thrombotic property of the endothelium has been attributed to the potentiation of inhibitors of plasma serine proteases by glycosaminoglycans (12). It has been reported that FXI binds to HUVEC with a high affinity (K D ϭ 4.5-6.9 nM, B max ϭ 2.7-13 ϫ 10 6 sites/cell) requiring HK and zinc ions (1,2). HUVEC-bound FXI was activated to FXIa by FXIIa, and HUVEC-bound FXIa was able to generate FIXa (1). This suggests that an otherwise phenotypically anticoagulant cell type is capable of generating procoagulant activity.
We have examined the binding of FXI to endothelial cells and have found a discrepancy between the experimental results reported previously and our data. Using several assays with various controls, and under several assay conditions, we have shown that FXI does not associate with HUVEC but does bind specifically to high affinity receptors on activated platelets. Although we were able to reproduce the experimental results previously published (1, 2), we have shown that they represent an artifact of the binding assay utilized (13)(14)(15). Our studies of endothelial cell and platelet interactions with FXI suggest that the activated platelet, not the endothelium, is the preferred surface for FXI binding and consequent activation by thrombin.

EXPERIMENTAL PROCEDURES
Proteins-FXI was purchased from Hematologic Technologies, Inc. (Essex Junction, VT). HK and thrombin were purchased from Enzyme Research Laboratories (South Bend, IN). The thrombin receptor activation peptide (SFLLRN-amide) and an HK-derived peptide comprising a portion of the FXI binding site referred to as the 31-mer HK peptide (SDDDWIPMDIQTDPNGLSFNPISDFAPDTTSPK) were synthesized at the Protein Chemistry Laboratory at the University of Pennsylvania (Dr. John Lambris, Director).
Radiolabeling with 125 I-FXI was radiolabeled with 125 I using the iodogen method (16). Briefly, ϳ100 g of protein was incubated with 1 mCi of carrier-free Na 125 I (Amersham Biosciences) for 20 min in an iodogen-coated vial and centrifuged through a 1-cc Sephadex G-50 packed spin column at 200 ϫ g for 5 min to separate 125 I-bound protein from free 125 I.
Measurement of Specific Radioactivity-Radiolabeled protein (1 l) was added to 99 l of 0.5% bovine serum albumin (BSA) in Hepesbuffered saline and 100 l of 40% trichloroacetic acid. This was vortexed and incubated in an ice bath for 5 min. The solution was centrifuged at 14,000 ϫ g for 5 min, and half of the supernatant was removed and placed in a separate vial. Both vials, 100 l of the supernatant (S) and 100 l of the pellet (P), were measured for ␥-emission in a Wallace Wizard 1470 automatic gamma counter. The following equations were used to determine percent radioactivity bound (Equation 1) and specific radioactivity (Equation 2; SRA).
Generally Ͼ98% radioactivity was bound to FXI, and the specific activity was 0.6 -1.4 ϫ 10 18 cpm/mol. Measurement of Specific Biological Activity-Each of the following components (50 l) was mixed and incubated at 37°C in an Amelung KC 4A microcoagulometer: FXI-deficient plasma, extracted phosphatidylserine/phosphatidylcholine phospholipid vesicles (0.1 mg/ml), kaolin (5 mg/ml), 0.15 M NaCl, and 125 I-FXI. After 5 min, 50 l of 40 mM CaCl 2 was added, and the clotting time was measured. A plot of FXI concentration versus clotting time was compared with a standard curve generated from normal pooled plasma or from non-labeled FXI from the same lot number. Radiolabeled FXI retained Ͼ90% of the activity of non-labeled FXI, or 213-248 units/mg.
Measurement of Amidolytic Activity-The substrate PyroGlu-Pro-Arg-pNA (S-2366, Chromogenix, Môlndal, Sweden) was used to determine FXIa activity. The rate of cleavage of S-2366 (250 M) by a 1:320 dilution of 125 I-FXI was determined by the absorbance at 405 nm and compared with a standard curve generated by non-labeled FXIa. Contaminating enzyme in the zymogen preparations was removed by incubation with a benzamidine-coated agarose (Sigma) slurry for 1 h at 37°C. The beads were removed by centrifugation, and the supernatant was analyzed by SDS-PAGE analysis for the disappearance of the 30and 50-kDa enzyme bands. No detectable (less than 0.1%) FXIa contaminated the radiolabeled zymogen preparation.
Effect of Platelets or HUVEC on Generation of Amidolytic Activity in Whole Plasma-HUVEC or HEK293 cells were grown to confluence in fibronectin-coated, silicone-treated microtiter wells. Plasmas (1:100 final dilution, George King Biomedical, Overland Park, KS) were added to these cell-containing wells or to empty silicone-treated wells. Platelets (1 ϫ 10 7 cells) were added to some of these empty wells. Protease nexin 2 (PNII) (10 nM, gift from Dr. William van Nostrand, State University of New York, Stony Brook, NY), corn trypsin inhibitor (100 nM, Hematological Technologies, Essex Junction, VT), hirudin (10 units/ml, Sigma), or Glu-Gly-Arg-chloromethylketone (100 nM, Bachem, San Carlos, CA) was added to the reaction in some cases. The chromogenic substrate, S-2366 (250 M), and calcium ions (4 mM) were added to the reaction, and A 405 was measured continuously for 1 h.
Cell Culture-HUVEC were isolated from fresh umbilical cords using techniques described elsewhere (17). Isolated cells were seeded into dishes coated with 1% gelatin and grown to confluence in EGM-2 complete medium (2% fetal bovine serum, BioWhittaker, Rockland, ME) in a humidified atmosphere of 5% CO 2 at 37°C. The medium was changed after 24 h and then on alternate days until the cells reached confluence. Cells were passaged and were no longer used after the fifth passage. HEK293 and CHO cells were cultured similarly in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics.
HUVEC or HEK293 cells (1 ϫ 10 6 cells/ml) were added to 5 ml (1.5 ϫ 10 5 beads) of swelled Cytodex-3 collagen-coated microcarrier beads (Amersham Biosciences) that were washed in complete endothelial cell medium. 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% CO 2 at 37°C. Microcarrier bead cultures (0.5 ml) were mixed with 0.5 ml of trypan blue (Invitrogen) to determine the level of confluence because the microcarrier beads but not the cell-coated beads retained trypan blue staining.
Platelet Isolation-Platelets were prepared as described (18). Briefly, platelets were obtained from platelet-rich plasma after centrifugation of whole blood collected into acid-citrate-dextrose (ACD) anticoagulant. Platelet-rich plasma was centrifuged through a BSA gradient and gelfiltered through a 50-ml Sepharose 2B column. Platelet eluates were counted electronically using a particle counter (Coulter Electronics, LOC, Hialeah, FL) and then washed once in 10 mM Hepes containing 5 mM EDTA and three times in 10 mM Hepes buffer without EDTA. Activation of platelets was measured by shape change, aggregation, and release of ADP using a Lumi-Aggregometer (Chrono-Log Corp., Havertown, PA).
Measurement of von Willebrand's Factor Release by HUVEC-The amount of von Willebrand's factor (vWF) released by HUVEC after stimulation, a marker of endothelial cell activation, was measured by sandwich enzyme-linked immunosorbent assay using a goat anti-human vWF IgG primary antibody and horseradish peroxidase-conjugated goat anti-human vWF IgG secondary antibody performed according the to manufacturer's instructions (Enzyme Research Laboratories). The absorbance at 490 nm 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), where 100% plasma contains 10 ng/ml vWF.
Equilibrium Binding Experiments Using HUVEC in Microtiter Wells-Binding studies were performed as described previously (1) except that HUVEC or CHO cells were grown to Ͼ90% confluence in 98-well fibronectin-coated plates instead of 48-well plates. 125 I-FXI in incubation buffer was added to cells at varying concentrations for 1 h at 37°C. In some experiments 100 nM HK, 1 M prothrombin, or 8 M 31-mer HK peptide was added. In other experiments, 1% gelatin, 1% non-fat dry milk, 1% polyethylene glycol, or 1% Irish cream was substituted for BSA. Also fibronectin-coated 96-well plates were substituted with 96-well non-coated Primeria plates (BD PharMingen) or fibronectin-coated filter bottom plates in which the filter of individual wells could be removed and measured for radioactivity.
Equilibrium Binding Experiments Using Cells in Suspension-To eliminate the effect of FXI binding to an artificial surface, HUVEC were placed into suspension non-enzymatically or cultured in suspension on microcarrier beads, and binding was measured using a procedure similar to that described previously for platelets (19). HUVEC were grown to confluence on non-coated plates and detached using 5 mM EDTA in phosphate-buffered saline for 5 min at 37°C. Cells (10 6 ) were triturated, washed, placed in a 15-ml conical flask, centrifuged for 5 min at 1,500 ϫ g, and resuspended in 5 ml of incubation buffer. In other cases, HUVEC or HEK293 cells grown to confluence on microcarrier beads were washed and resuspended in incubation buffer to a density of 10 6 cells/ml. Silicone oil (15 l, DC500:DC200, 4:1) was centrifuged to the bottom of a 200-l microcentrifuge tube with a narrow bore tip (Sarstedt, Inc., Princeton, NJ). The cell suspension (washed platelets, HUVEC or HEK293 cells) or microcarrier bead suspension (HUVEC or HEK293 cells) was added to the radiolabeled protein mixture (50 l, each) and incubated at 37°C for 1 h with shaking by hand every 15 min. To separate the bound from the free protein, tubes were centrifuged at 12,000 ϫ g for 5 min in a vertical centrifuge (Model B, Beckman Instruments, Inc., Cedar Grove, NJ). The tubes were placed in a dry ice/ethanol slurry for 5 min, and the tips of the microtubes were amputated. Both the pellets (cell-bound proteins) and 10 l of the supernatant (free protein) were measured for ␥-emission.

Equilibrium Binding Studies Performed in the Microtiter
Well Assay-In order to determine the affinity and stoichiometry of FXI binding to the HUVEC surface, cells were grown to confluence in microtiter wells, washed, and then incubated with 125 I-labeled FXI for 1 h at 37°C under the conditions reported previously (1). These experiments were conducted in the presence of zinc ions (100 M) and in the presence or absence of HK (100 nM, Fig. 1). In the absence of HK no evidence of saturable binding was detected at a concentration as high as 100 nM added FXI, i.e. ϳ3ϫ the physiological concentration. The small amount of detectable binding was not displaceable by an excess of non-labeled FXI. Non-saturable binding also occurred in the absence of cells.
In the presence of HK, saturable binding was achieved by 100 nM added 125 I-FXI both in the presence and absence of HUVEC ( . This result suggests that the FXI binding detected was a property of the plastic surface of the microtiter wells and that binding was abrogated by the presence of the cells. Because the vertical sides of the microtiter wells themselves appeared to be playing a significant role in the amount of FXI bound, the number of binding sites detected may be influenced by volume rather than protein concentration. The number of binding sites increased by varying the volume within the range of 50 -200 l in the presence of zinc, 30 nM FXI, and 100 nM HK (Fig. 2). A volume-dependent increase in the number of binding sites was detected when HUVEC, CHO cells, or empty wells were incubated with the reaction mixture ( Fig. 2) but not in the absence of HK (data not shown). The significant amount of nonspecific background binding determined in the presence of HK may explain both the similarities and the discrepancies between our results and those described previously.
To optimize the microtiter well assay, several experimental conditions were examined in order to block the binding of FXI/HK to the microtiter well in the absence of HUVEC. BSA was replaced with gelatin, Irish cream, polyethylene glycol, or non-fat dry milk. None of these blocking agents prevented the binding of FXI/HK to the microtiter wells; in fact BSA appeared to be the best blocking agent (data not shown). Furthermore, the microtiter plates themselves were replaced with Primeria brand microtiter plates, which allow for the growth of endothelial cells without precoating with a matrix protein. Binding of FXI in the presence of HK was still detected when these plates were used, suggesting that matrix coatings were not playing a role. Other cofactors known to mediate the binding of FXI to platelets (8,9) were then substituted for HK; however, neither prothrombin nor an HK-derived peptide consisting of the 31amino acid residue region responsible for the HK/FXI interac-tion was able to mediate FXI binding to the HUVEC surface in microtiter wells (data not shown).
FXI Binding Studies with Cells on Microcarrier Beads-HUVEC were grown to confluence on Cytodex-3 collagencoated microcarrier beads, and binding assays were conducted on cells in suspension similarly to the platelet binding experiments (20). Microcarrier beads allow for a monolayer of anchorage-dependent cells to be manipulated in suspension. HUVECcoated beads did not release vWF until pretreated with agonists (data not shown). Accordingly, HUVEC-coated beads either untreated or pretreated with thrombin receptor activation peptide (TRAP) were assayed in suspension (Fig. 3A). Only trace amounts of non-saturable, nonspecific, and non-displaceable FXI binding were observed in either case. Also HEK293 cells were grown to confluence on the beads and used in the suspension assay. No detectable FXI binding to HEK293 cellcoated beads was observed, and the results with HEK293 cells were similar to those with HUVEC grown on beads (Fig. 3A). On the other hand, saturable HK binding was detected to HUVEC-coated microcarrier beads but not to HEK293 cellcoated beads. 2 A titration with HK in the presence of zinc ions did not promote FXI binding to HUVEC or HEK293 cells at any concentration (data not shown). These results indicate that neither HUVEC nor HEK293 cells immobilized on microcarrier beads participate in FXI binding reactions. Also, HUVEC that had been dissociated from tissue culture dishes non-enzymatically did not support FXI binding in suspension (data not shown).
To ensure that our inability to detect FXI binding to HUVEC was a consequence of the absence of specific FXI binding sites on HUVEC and not the result of a deficiency in the binding assay, cell-coated microcarrier beads were replaced with platelets. The binding of 125 I-FXI to platelets (Fig. 3B) was specific, saturable, and reversible (K D ϭ 8 nM, B max ϭ 1319 sites/ platelet), which is similar to previously published work (7,21). The difference in the ordinate between Fig. 3, A (fmol/10 5 cells) and B (pmol/10 8 platelets) should be noted.
Rate of FXIa Formation in Plasmas-A critical reaction in the initiation of the intrinsic or consolidation pathway of blood coagulation is the activation of FXI to FXIa. It has been shown that both activated platelets (9) and the HUVEC surface (1) facilitate the activation of FXI in purified systems. To investigate the role of platelets, HUVEC, HEK293 cells, and kaolin in the FXI-dependent initiation of blood coagulation in a plasmabased assay, we utilized the chromogenic substrate S-2366 to detect generation of enzymatic activity in the wells of microtiter plates with normal or coagulation factor-deficient plasma in 2 T. R. Baird and P. N. Walsh, unpublished results. the absence or presence of various protease inhibitors. This chromogenic substrate, S-2366, is cleaved by FXIa (K m ϳ250 M) and thrombin (K m ϳ150 M) but not by other activated coagulation factors such as FXIIa, kallikrein, FIXa, FXa, or FVIIa. Therefore, the generation of FXIa and thrombin was monitored as the change in absorbance at 405 nm over a period of 1 h, and the results are presented in Fig. 4 as mOD/min. Neither incubation with HUVEC (4.2 mOD/min) nor HEK293 cells (3.4 mOD/min) resulted in the generation of significant amounts of amidolytic activity in normal plasma as measured by S-2366 hydrolysis. TRAP-activated platelets (43 mOD/min) but not unactivated platelets (8.2 mOD/min, Fig. 4A, inset) resulted in the generation of amidolytic activity, which was inhibited completely by PNII (3.3 mOD/min, a potent FXIa inhibitor) and partially by hirudin (16.3 mOD/min, a thrombin inhibitor) but not by corn trypsin inhibitor (CTI, 38 mOD/min, a FXIIa inhibitor). Kaolin, a contact pathway activating surface, also supported an increase in the rate of S-2366 hydrolysis (56 mOD/min), which was inhibited by PNII (3.6 mOD/min) and CTI (0.73 mOD/min) but not by hirudin (41 mOD/min). Rates were reduced to Ͻ1 mOD/min in the presence of Glu-Gly-Arg-chloromethylketone, a nonspecific serine protease inhibitor, in all cases.
To detect the generation of amidolytic activity caused by the generation of enzymes other than FXIa, clotting factor-deficient plasmas were used in the presence of TRAP-activated platelets (Fig. 4B). Amidolytic activity was reduced from 43 mOD/min in normal plasma (Fig. 4A) to 15 mOD/min in FXIdeficient plasma (Fig. 4B) and was completely inhibited by hirudin (3.7 mOD/min). It is possible that this residual plateletdependent amidolytic activity in FXI-deficient plasma is a consequence of platelet FXI (23) because this activity is also inhibited by PNII (Fig. 4B). FXII-and HK-deficient plasmas showed no reduction in the rate of hydrolysis unless PNII or hirudin were present, consistent with the conclusion that thrombin can hydrolyze S-2366 and preferentially activates FXI on the platelet surface (8,9). The results of the experiments carried out in HK-deficient plasma, which were similar to those with normal plasma, suggest that the use of microtiter wells is valid in this assay. DISCUSSION The results presented in this paper demonstrate that FXI interacts with specific, saturable, high affinity binding sites exposed on TRAP-activated platelets where it is activated by thrombin to initiate the consolidation phase of blood coagulation. FXI does not bind to either resting HUVEC or HUVEC activated with TRAP to release vWF even in the presence of HK and zinc ions when coated on microcarrier beads, contrary to previously published results performed in microtiter wells (1,2). Using a microtiter well assay, we were able to reproduce the results published previously (1, 2) for FXI binding in the presence of HK and zinc ions, i.e. saturable, specific, reversible high affinity (K D ϭ 12 nM) binding of FXI to microtiter wells containing HUVEC (1.3 ϫ 10 10 sites/well ϭ 1.3 Ϯ 0.2 ϫ 10 6 sites/cell). However, we found a higher number of FXI binding

FIG. 4. Activation of FXI on cellular surfaces in plasma.
A, plasma was added to empty wells, platelets, HUVEC, HEK293 cells, or kaolin in the presence or absence (inset) of TRAP. The rate of S-2366 hydrolysis was measured for 1 h in the presence of 4 mM calcium and PNII, CTI, or hirudin. B, factor-deficient plasmas were added to TRAPactivated platelets, and S-2366 hydrolysis was measured in the presence of protease inhibitors. Results represent the mean of two observations. sites in the absence of cells (1.8 Ϯ 0.1 ϫ 10 10 sites/well) with high affinity binding (K D ϭ 2.4 nM, Fig. 1). This suggests that saturable, specific, high affinity binding of FXI observed in the presence of HK is a property of the artificial surface within the microtiter wells. In the absence of HK there was no detectable binding, similar to earlier reports (1).
We recognize that the properties of cellular membranes can be vastly affected by minor manipulations, which can significantly differ between laboratories, thereby limiting comparisons between our results and those established previously. However, the large number of high affinity binding sites reported previously for FXI on the unactivated HUVEC surface (K D ϭ 4.5-6.9 nM, B max ϭ 2.7-13 ϫ 10 6 sites/cell) (1, 2) suggests that the entire pool of plasma FXI should be bound under normal physiological conditions as determined by the following calculations. There are ϳ7.23-9.03 ϫ 10 16 molecules of FXI physiologically present in circulation (30 nM ϫ 4 -5 liters ϫ 6.023 ϫ 10 23 molecules/mol). Since there are ϳ1-6 ϫ 10 13 endothelial cells per adult vascular system (24), there would be 2.7-78 ϫ 10 19 high affinity endothelial sites for FXI per adult, i.e. 373-8,637 more receptors than FXI molecules. Thus, FXI always would be associated with the endothelium, and none would be found free in plasma.
Furthermore, a single endothelial cell could not accommodate 2.7-13 million molecules of FXI. The surface area of the HUVEC luminal membrane is ϳ100 m 2 , assuming a flat rectangular shape. If it is assumed that 2.7 ϫ 10 6 FXI molecules occupy 100% of the cellular surface, 37 nm 2 per binding site (100 m 2 /2.7 ϫ 10 6 sites) would be available, each with a radius of 3.4 nm ((37 nm 2 /) Ϫ2 ). Thus, the diameter of the widest dimension of FXI when bound to its receptor would be 6.8 nm. The same calculation can be performed assuming 13 million sites/cell, providing a binding contact of 7.7 nm 2 with a cylindrical maximum diameter of 3.2 nm. Therefore, the diameter of the FXI molecule interacting with the entire exposed endothelial cell surface would have to be ϳ3.2-6.8 nm, assuming 100% occupancy and precluding cellular interaction with any other molecules. According to crystallographic data, the smallest dimension of the FVIIa (50 kDa) unit cell is 10 nm, FXa (58.8 kDa) is 5.5 nm, and FIXa (56 kDa) is 9 nm (25)(26)(27). The size of the FVIIa/tissue factor complex (90 kDa) is 4 -5 nm in diameter, and the smallest dimension of the unit cell is 7.11 nM (28). These coagulation factors are all globular and elongated with molecular sizes at least three times lower than that of FXI (160 kDa). Therefore, it is unreasonable for a single endothelial cell to bind as many as 13 million molecules of FXI.
To eliminate the high level of background binding detected in the presence of HK, we attempted to block the microtiter wells using a variety of potential blocking agents and microtiter plates themselves. The 27% decrease in the number of FXI binding sites detected when HUVEC were present compared with that detected in their absence is nearly identical to the 28% of a 96-well microtiter well surface area, including the sides, that is covered by HUVEC when incubated in 100 l of buffer. Furthermore, we report about half as many sites per HUVEC as was reported earlier (1) using smaller microtiter wells with approximately half the surface area. This strongly suggests that cells abrogate the number of binding sites by coating the base of the microtiter well. To test the hypothesis that the microtiter well surface was the cause of the high background binding, we determined the effect of incubation volume on FXI binding, which was predicted to be volume-dependent because an increase in volume would increase the available surface area of the microtiter well without changing the number of cells. As the volume of the reaction mixture was varied while maintaining the FXI (30 nM) and HK (100 nM) concentrations we found that the number of FXI binding sites detected per well increased in direct proportion to the incubation volume regardless of whether HUVEC, CHO cells, or empty wells were studied. The high number of detected binding sites is related not to the presence of cells but to the artificial surface contained within the microtiter wells, and it is a property of HK (13,14), which binds nonspecifically to plastic surfaces.
The inability to detect specific binding sites in the microtiter well assay was suggestive but not conclusive proof of the absence of FXI receptors on HUVEC. Therefore, to reexamine the binding of FXI to HUVEC in comparison to platelets, HUVEC were grown to confluence on microcarrier beads and assayed in suspension. This method eliminates interference by artificial surfaces because only radioactive proteins bound to the cellular pellet are measured. Binding studies performed with HUVEC in suspension directly may have induced anoikis, or cell death caused by lack of surface contacts. However, neither quiescent nor TRAP-pretreated HUVEC-coated microcarrier beads bound FXI in the presence or absence of HK. The amount of FXI detected in the pellets of quiescent or activated HUVEC was no different when substituted with HEK293 cell-coated beads. The amount of FXI bound was negligible, in contrast to the 2.7-13 ϫ 10 6 sites/cell previously reported (1,2). When TRAP-activated platelets were used instead of HUVEC, specific and saturable FXI binding was detected in the presence of HK (45 M) and zinc ions (25 M). Platelets did not bind FXI in the absence of HK, zinc ions, or TRAP stimulation.
The examination of the functional consequences of FXI binding to cellular surfaces supports the conclusion that activated platelets but not endothelial cells participate in the initiation of the consolidation phase of blood coagulation. Our studies were carried out in plasma, providing the advantage of incorporating all blood proteins in one reaction. To elucidate candidate FXI activators and cofactors as well as enzymes that may hydrolyze S-2366, studies were carried out in the presence or absence of various specific inhibitors, coagulation proteins, and cells. As predicted from the binding experiments, neither HUVEC nor HEK293 cells facilitate the activation of FXI. Activated platelets and kaolin support the activation of FXI via separate and distinct mechanisms as shown by the inhibition profile. The ability of hirudin, a specific thrombin inhibitor, to inhibit the rate of substrate hydrolysis in the presence of activated platelets but not kaolin suggests that thrombin is a major activator of FXI on platelets but not on artificial surfaces. However, the incomplete inhibition of amidolytic activity by hirudin on platelets suggests that FXI activation proceeds via another mechanism, possibly FXIIa. Kaolin supports the activation of FXI through FXIIa as seen by the complete inhibition of amidolytic activity in the presence of CTI but not hirudin. These conclusions are confirmed by the factor-deficient plasma studies. FXII-deficient plasma had no effect on the rate of hydrolysis in the presence of activated platelets, but in the presence of hirudin a condition in which FXIIa and thrombin are absent or inactive resulted in a 9-fold reduction in rate. On the other hand, the rate of hydrolysis on kaolin in FXII-deficient plasma was reduced by 4.5-fold and was reduced 18-fold by the combined absence of FXII and inactivation of thrombin by hirudin.
The present studies demonstrate conclusively that activated platelets express saturable and specific high affinity receptors for FXI that are functionally important in promoting the initiation of the consolidation or intrinsic pathway of blood coagulation. In contrast, neither resting nor thrombin-activated HU-VEC have any capacity to interact specifically with FXI or to promote blood coagulation reactions involved in the initiation of the intrinsic pathway. These observations are consistent with the concept that the primary phenotype of resting endothelium is anticoagulant and non-thrombogenic, whereas the major function of activated (but not resting) platelets is to participate in the initiation of the consolidation pathway of blood coagulation, leading to the local explosive generation of thrombin at sites of vascular injury.