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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2765-2775.)
© 1997 American Heart Association, Inc.

Articles

Protein C Activation and Factor Va Inactivation on Human Umbilical Vein Endothelial Cells

Matthew F. Hockin; Michael Kalafatis; Marie Shatos; ; Kenneth G. Mann

From the College of Medicine, Department of Biochemistry, University of Vermont, Burlington.

Correspondence to Kenneth G. Mann, College of Medicine, Department of Biochemistry, University of Vermont, Burlington, VT 05405-0068. E-mail kmann{at}protein.med.uvm.edu

	Abstract

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Abstract The inactivation of factor Va was examined on primary cultures of human umbilical vein endothelial cells (HUVECs), either after addition of activated protein C (APC) or after addition of -thrombin and protein C (PC) zymogen. Factor Va proteolysis was visualized by Western blot analysis using a monoclonal antibody (HVa_HC No. 17) to the factor Va heavy chain (HC), and cofactor activity was followed both in a clotting assay using factor V–deficient plasma and by quantitation of prothrombinase function. APC generation was monitored using the substrate 6-(D-VPR)amino-1-naphthalenebutylsulfonamide (D-VPR-ANSNHC₄H₉), which permits quantitation of APC at 10 pmol/L. Addition of APC (5 nmol/L) to an adherent HUVEC monolayer (3.5x10⁵ cells per well) resulted in a 75% inactivation of factor Va (20 nmol/L) within 10 minutes, with complete loss of cofactor activity within 2 hours. Measurements of the rate of cleavage at Arg⁵⁰⁶ and Arg³⁰⁶ in the presence and absence of the HUVEC monolayer indicated that the APC-dependent cleavage of the factor Va HC at Arg⁵⁰⁶ was accelerated in the presence of HUVECs, while cleavage at Arg³⁰⁶ was dependent on the presence of the HUVEC surface. Factor Va inactivation proceeded with initial cleavage of the factor Va HC at Arg⁵⁰⁶, generating an M_r 75 000 species. Further proteolysis at Arg³⁰⁶ generated an M_r 30 000 product. When protein C (0.5 µmol/L), -thrombin (1 nmol/L), and factor Va (20 nmol/L) were added to HUVECs an APC generation rate of 1.56±0.11x10^-14 mol/min per cell was observed. With APC generated in situ, cleavage at Arg⁵⁰⁶ on the HUVEC surface is followed by cleavage at Arg³⁰⁶, generating M_r 75 000 and M_r 30 000 fragments, respectively. In addition, the appearance of two novel products derived from the factor Va HC are observed when thrombin is present on the HUVEC surface: the HC is processed through limited thrombin proteolysis to generate an M_r 97 000 fragment, which is further processed by APC to generate an M_r 43 000 fragment. NH₂-terminal sequence analysis of the M_r 97 000 fragment revealed that the thrombin cleavage occurs in the COOH-terminus of the intact factor Va HC since both the intact HC as well as the M_r 97 000 fragment have the same sequence. Our data demonstrate that the inactivation of factor Va on the HUVEC surface, initiated either by APC addition or PC activation, follows a mechanism whereby cleavage is observed first at Arg⁵⁰⁶ followed by a second cleavage at Arg³⁰⁶. The latter cleavage is dependent on the availability of the HUVEC surface. This mechanism of inactivation of factor Va is similar to that observed on synthetic phospholipid vesicles.

Key Words: factor Va • protein C • endothelial cells • thrombomodulin

	Introduction

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Regulation of the blood coagulation process involves a large array of proteins, vascular and perivascular cells, platelets, cytokines, and growth factors. The combined interaction of these components serves as a biological "switch" that regulates the production of thrombin by the prothrombinase complex. Prothrombinase is composed of the serine protease factor Xa and its cofactor factor Va, assembled on a membrane surface in the presence of Ca²⁺.^{1 2 3 4} The assembly of prothrombinase is regulated through surface expression and availability of the activated protein and membrane components. The downregulation of prothrombinase is, in part, mediated by limited proteolysis of factor Va by APC.^{5 6 7 8 9 10 11} PC activation in vivo occurs via the PC pathway, in which thrombin binds to the endothelial transmembrane protein TM, which acts as a cofactor in accelerating PC activation by thrombin. Thus, the inactivation of factor Va by APC represents a feedback mechanism for prothrombinase regulation.^{12 13 14} The role that APC plays in maintaining hemostasis is illustrated through two congenital thrombotic disorders, APC resistance (the mutation of an APC cleavage site in factor Va [factor V^LEIDEN]), and congenital PC deficiency.^{14 15 16 17 18 19 20}

Factor V circulates as an M_r 330 000 procofactor that is proteolytically activated through -thrombin cleavages at Arg 709, Arg 1018, and Arg 1545.^{21 22 23 24} The activated cofactor, factor Va, is composed of heavy (M_r 105 000) and light (M_r 75 000) chains noncovalently associated through a divalent metal ion–dependent process. Factor Va serves as a receptor for factor Xa and influences both the recognition of substrate and rate of cleavage. The inactivation of factor Va by APC requires phospholipid and proceeds via three cleavages of the HC.^{9 10} In a phospholipid system composed of 25% phosphatidylserine and 75% phosphatidylcholine, factor Va is cleaved at Arg⁵⁰⁶, yielding a partially active molecule (1-506, 507-709, and 1546-2196). This species is further cleaved at Arg³⁰⁶ and Arg⁶⁷⁹ to produce the inactive product (1-306, 307-506, 507-679, 680-709, and 1546-2196).¹⁰ Using platelet factor Va on the platelet surface, initial cleavage is observed at both Arg⁵⁰⁶ and Arg³⁰⁶, generating a mixture of products (1-306/1-506 306-709/506-709); extended incubation with APC does not completely inactivate factor Va in contrast to what has been observed on anionic phospholipid membrane vesicles.²⁵

PC is the vitamin K–dependent zymogen precursor of APC. The activation of PC requires only -thrombin; however, in the presence of an anionic membrane, Ca²⁺, and TM, the rate of PC activation is accelerated by three orders of magnitude.²⁶ TM is constitutively expressed on the luminal surface of endothelial cell membranes in most vascular beds.²⁶ Other receptors involved in localizing PC or APC onto the endothelial surface independent of TM have also been reported.^{27 28} The endothelial cell PC receptor has been reported to play a role in recruitment of PC to the HUVEC surface and thus acts to enhance the activation of PC by thrombin-TM.²⁹

The physiological surface available for assembly of the prothrombinase complex is most likely the activated platelet surface localized to the site of injury. The vascular endothelium may also provide a surface for prothrombinase assembly when it is activated through cytokine stimulation. Examination of the PC pathway and its terminal function (factor Va inactivation) on the endothelial surface has been approached through segmental analysis of the individual components and their functions. Previous work has shown that both bovine aortic endothelial cells and HUVECs support APC inactivation of factor Va.^{30 31}

In the present study, the inactivation of factor Va by either direct addition of APC or in situ–generated APC is investigated on the HUVEC surface. The use of the presumed physiological surface in this study provides insights into the mechanism of inactivation of factor Va HC and demonstrates two new products derived from factor Va HC.

	Methods

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Materials
The -thrombin inhibitor hirudin was obtained from American Diagnostica. Ovalbumin and HEPES were purchased from Sigma. The inhibitors Phe-Pro-Arg-ck, DAPA, and human factor Xa were gifts from Haematologic Technologies (Essex Junction, Vt). Simplastin Excel, a thromboplastin reagent was purchased from Organon Teknika. Goat anti-mouse IgG peroxidase was purchased from Southern Biotechnology and the chemiluminescent reagent Renaissance, from DuPont chemicals. The APC substrate D-VPR-ANSNHC₄H₉ was synthesized by Dr Saulius Butenas in our laboratory. All other chemicals were purchased through J.T. Baker. Transfer blotting systems were from Bio-Rad. The mouse monoclonal antibody hTM-531, which blocks the thrombin/TM interaction, was the gift of Dr John Morser of BERLEX (South San Francisco, Calif). The mouse monoclonal antibody HVa_HC No. 17 to the region encompassing amino acids 307-506 of the human factor Va HC (Fig 1) was provided by the monoclonal antibody facility, University of Vermont, Department of Biochemistry. This antibody was obtained from the same fusion as the no longer available antibody HV_aHC No. 6, which has been described previously.^{19 25 32} Prothrombin was isolated from fresh frozen human plasma according to the methods of Bajaj et al,³³ and further purified by passing over both anti-factor X and anti-PC immunoaffinity columns. -Thrombin was obtained through prothrombinase activation of prothrombin and purified as described.^{34 35} PC was purified from fresh frozen plasma. Fractions containing PC activity from the flow-through of DEAE Sepharose chromatography were applied to a heparin-Sepharose column that was developed by a linear NaCl gradient. The resulting PC was applied to an anti-PC column (2.5 mmx25 mm) to remove traces of factor X and prothrombin. The purified PC was treated with the chloromethyl ketone Phe-Pro-Arg-ck, dialyzed, and stored at -20° as a 50% glycerol/water solution. Before activation, PC was dialyzed extensively into HBS, 5 mmol/L CaCl₂, and APC generated by the addition of 30 nmol/L -thrombin (3 NIH U/mL) to purified PC (1 µmol/L) with incubation at 37° for 30 minutes. APC was purified as described.³⁶ Human factor V was purified as described.^{22 37} All purified proteins were stored at -20° in 50% glycerol.

View larger version (29K): [in this window] [in a new window]   Figure 1. HVaHC No. 17 epitope recognition. This schematic diagram represents the epitope of HVaHC No. 17 and its fate during APC-catalyzed inactivation of factor Va. The epitope is located between amino acid residues 307 and 506 of the factor Va HC and is retained throughout APC-catalyzed proteolysis of factor Va. This antibody recognizes factor V Mr 330 000 species, factor Va HC Mr 105 000 species, factor Va1-506 Mr 75 000 species (APC cleavage at Arg506), and factor Va307-506 Mr 30 000 species (APC cleavage at Arg506/Arg306). The antibody also recognizes a factor Va307-709 Mr 62 000 species representing initial cleavage at Arg306.19 In addition, it will recognize the fragment containing 307 through 506 of any proteolytic alterations of factor Va HC outside of the region 307 through 506.

Western Blotting
During the time course of the experiment, samples were quenched with 62.5 mm Tris-HCl, 2% SDS, 10% glycerol, 2% ß-mercaptoethanol, and 0.001% bromphenol blue, pH 6.8; the samples were then heated at 95°C for 4 minutes and stored at -20°C. Thawed samples were applied to a 4% to 12% gradient SDS-PAGE gel,³⁸ transferred to nitrocellulose, and immunoreactive fragments were detected by the monoclonal antibody HVa_HC No. 17.

HUVEC Cell Culture
Primary cultures of HUVECs were isolated using collagenase digestion as described.³⁹ Cells were seeded at a density of 200 000 cells per 2.9 cm² well, in serum supplemented (10% fetal calf serum) medium M-199, and grown to confluence. Before experimentation, cells were rinsed 3 times with HBS CaCl₂, pH 7.4, and were maintained at 37°C by suspension in a 2 mmol/L water bath during the experiment. Primary HUVEC cultures were routinely characterized using the following pattern of identification of immunohistochemical markers, positive for perinuclear von Willebrand Factor, positive for ß-actin (non–smooth muscle), and negative for -actin as reported.³⁹

Assay Measuring Thrombin Formation
Factor Va activity was monitored either in a factor V–deficient clotting assay or a prothrombinase assay using purified components.⁹ The factor Va clotting assay was standardized to serial dilutions of normal pooled plasma (12 donors).²² In a typical assay, 50 µL of factor V–deficient plasma (immunodepleted) was combined with an equal volume of the analyte. To start the assay, 100 µL of the diluted PT reagent (Simplastin Excel) was added while rocking the tube at 37°C by suspension in a water bath. The assay end point was determined by visualization of the fibrin strands. A standard curve encompassing the range 0.125 to 0.000976 U/mL factor V activity was established daily (dilution of normal plasma from 1:4 to 1:1024). The standard curves were linear when plotted as log clot time versus log U/mL; these plots were fit to a single exponential expression (R² correlation of 0.9) that was used in determination of unknown sample activity. Unknown samples were diluted such that the assay end point fell between 22 and 50 seconds; this procedure eliminated the variation found in the determination of clot times outside of this range. The prothrombinase assay involves addition of the factor Va species of interest at 0.5 nmol/L to a solution (DAPA mix) containing prothrombin (1.4 µmol/L), DAPA (3.0 µmol/L), PC/PS (20 µmol/L; 75% phosphatidylcholine/25% phosphatidylserine) in 20 mmol/L HEPES, 150 mmol/L NaCl, 5 mmol/L CaCl₂. After addition of factor Va, the solution was incubated for 1 minute at room temperature. On addition of factor Xa (2.5 nmol/L or 10.0 nmol/L) the fluorescence was monitored over time in an SLM 8000 fluorometer equipped with a 450 W xenon arc bulb. The monochromators were set at 280 nm (excitation wavelength) and 565 nm (emission wavelength), while emission light was filtered with a long pass (KV 500) filter. Two different factor Xa concentrations were used: 2.5 nmol/L and 10.0 nmol/L. The conditions used in each assay are stated in the "Results" section for the corresponding experiments.

APC Activity Assay
PC activation on the cell surface was determined by a fluorogenic microassay for APC.⁴⁰ Aliquots were withdrawn from the supernatant above the cell surface and used to assay APC activity. These aliquots were brought to 100 µL in a solution containing hirudin (40 nmol/L), PEG-4000 (0.1%), in HBS pH 7.4 (no calcium) and combined with 100 µmol/L D-VPR-ANSNHC₄H₉ in the same buffer. After a 25-minute incubation of the substrate with APC, sample fluorescence was determined at excitation 360 nm, emission 465 nm (16-nm slit width), in a 200-µL quartz cuvette. The assay was standardized to serial dilutions of purified APC from 2.0 to 0.005 nmol/L. The fluorescence response was linear from 5 pmol/L to 1 nmol/L APC over a 25-minute incubation. Calibration of the measurement is obtained through simple linear least-squares fitting and is typified by an R² coefficient of greater than 0.9984.

APC Inactivation of Factor Va on the HUVECs
A solution containing 20 nmol/L human factor V, 0.1% ovalbumin in HBS, pH 7.4 (5 mmol/L CaCl₂), was activated by a 5-minute incubation with 0.1 nmol/L -thrombin (0.01 NIH U/mL), and then -thrombin was inhibited by addition of a 20-fold molar excess of hirudin (2 nmol/L). For activity comparisons, the activity after activation was set to 100% (typically 700 U/mg). Four hundred microliters of the factor Va solution was added to prewashed HUVECs and incubated for 15 minutes at 37°. After 15 minutes, APC (5 nmol/L) was added to the factor Va solution above the HUVEC surface. At all times, the solution was mixed constantly by gentle mechanical rocking of the culture plate to provide for adequate surface exchange of the products and reactants. Samples were withdrawn for both Western blot and activity assessment by both clotting assay and prothrombinase assay (2.5 nmol/L factor Xa).

APC Inactivation of Factor Va and Influence of HUVEC Surface on Proteolytic Cleavage
A solution containing factor Va (20 nmol/L), ovalbumin (0.1%) in HBS (5 mmol/L CaCl₂) was added to either a confluent HUVEC monolayer or a media-treated blank culture well. After a 5-minute incubation, APC (5 nmol/L) was added to both wells, and time-point samples were taken for Western blot analysis. The relative rates of cleavage in the factor Va HC at Arg⁵⁰⁶ and Arg³⁰⁶ (±HUVECs) are estimated from densitometric analysis of product formation as detected on Western blots using the monoclonal antibody HVa_HC No. 17.

Activity Assessment of Factor Va HC Species 1-709, 1-506+507-709, and 1-306+307-506+507-709
Factor V was dialyzed into HBS (2 mmol/L CaCl₂). After dialysis, factor V (630 nmol/L) was activated by treatment with -thrombin (10 nmol/L)for 10 minutes at 37°C. The -thrombin was inhibited by addition of 40 nmol/L hirudin. APC (36 nmol/L) was added to the factor Va (630 nmol/L) and activity assessed at various time points by both clotting and prothrombinase assays (10 nmol/L Xa). After a 60-minutes incubation with APC, PC/PS (20 µmol/L) was added to the Va/APC solution and activity assessed at various time points. Samples containing 12 µg Va were withdrawn for Coomassie blue staining of SDS-PAGE. Samples containing 100 ng were withdrawn for Western blot analysis (hfVa_HC No. 17).

Correlation of HUVEC/IIa Activation of PC and APC Inactivation of Factor Va
The procedure for APC inactivation of factor Va was modified as follows. After activation of factor V, the factor Va/-thrombin solution was added directly to the HUVECs without addition of hirudin. On addition to the cell surface, the -thrombin concentration was brought to 1 nmol/L. After 10 minutes of incubation on the HUVECs (Va/IIa), 500 nmol/L PC was added and the rate of PC activation and factor Va inactivation (clotting assay) were determined as described previously. The K_m for PC of the -thrombin/TM complex has been reported to be 700 nmol/L.⁴¹ To assure efficient activation of PC by the -thrombin/TM complex, we chose 500 nmol/L PC for these experiments.

Scanning and Image Processing
The film exposure of a Western blot was scanned using a bright field scanner, (Microscan 1000 scanning densitometer, TRI, Inc). Scanner image files were transferred to a Macintosh PowerBook 5300 cs, and were imported into NIH Image (a public domain program developed at the US National Institutes of Health. It is available from the Internet at zippy.nimh.nih.gov or through part number PB-95-500195GEI at the National Technical Information Service). NIH Image was employed to crop the image and standardize image density. These images were photographically reproduced.

NH₂ Amino Acid Sequencing
Factor V (200 nmol/L) in HBS Ca²⁺ (5 mmol/L) was treated with -thrombin (20 nmol/L) for 60 minutes at 37°C. The factor Va species resulting from -thrombin digestion were analyzed (reduced and nonreduced) on 4% to 12% gradient SDS-PAGE gels and transferred to a polyvinyl difluoride membrane using a method previously described.⁹ After transfer, the membrane was stained as described.⁹ The NH₂ terminal sequence of the peptides derived from -thrombin digestion were determined by automated Edman degradation on an Applied Biosystem 475A protein sequencing system equipped with a blot cartridge in the laboratory of Dr Alex Kurosky (University of Texas, Medical Branch, at Galveston).

	Results

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Influence of Factor Xa Concentration in the Assessment of Factor Va Activity
Factor Va functions through association with factor Xa on a lipid surface. The various assays used to assess factor Va activity are dependent on this association and the subsequent conversion of prothrombin to -thrombin. In the clotting assay, factor Xa is generated through the tissue factor pathway. When 5 nmol/L -thrombin (from 1.4 µmol/L prothrombin) is produced through the activity of 5 pmol/L prothrombinase, fibrinogen is cleaved to form a fibrin clot representing the assay end point.⁴² Under these conditions factor Xa is the limiting component of prothrombinase assembly.⁴² The quantitative assessment of prothrombinase concentration uses the fluorescent thrombin active site probe DAPA to measure thrombin formation by continuous functional analyses. (In this assay, fixed concentrations of factor Xa are used [10 nmol/L] such that the limiting component in prothrombinase assembly is factor Va [0.5 nmol/L]). While these two assay systems are each dependent on the ability of factor Va to bind factor Xa on a lipid surface and activate prothrombin, they can yield quite different factor Va assessments due to the different concentrations of factor Xa available.

To analyze the relative assay dependence on factor Xa concentration, experiments were performed in a phospholipid system. On addition of APC (36 nmol/L) to factor Va (630 nmol/L) in the absence of phospholipid, a rapid decrease in activity is observed in the clotting assay (Fig 2A, ), yielding 10% of initial activity after 15 minutes. This activity remains stable over the subsequent 45 minutes. Activity assessed by the quantitative prothrombinase assay (at 10 nmol/L Xa) rapidly decreases to 60% of initial in 15 minutes and remains stable during a subsequent 45-minute incubation (Fig 2A, ). Western blot analysis reveals that the factor Va HC is rapidly cleaved at Arg⁵⁰⁶ generating an M_r 75 000 product (Fig 2D). On the Coomassie blue–stained gel, the M_r 75 000 product runs coincident with the factor Va light chain (Fig 2C). Densitometric analysis of the factor Va HC (Coomassie blue) reveals a rapid decrease in density during the initial 15 minutes (Fig 2B). The loss of HC from the gel parallels the decrease in clotting activity observed during the initial 15 minutes. During the subsequent 45 minutes, both the factor Va clotting activity and HC density are stable at 10% of their respective initial values (compare Fig 2A, with Fig 2B, ).

View larger version (46K): [in this window] [in a new window]   Figure 2. Activity assessment of factor Va species and factor Xa dependence of activity. Factor V (630 nmol/L) was activated with thrombin and treated with APC (36 nmol/L) as described in the "Methods" section. A, Factor Va cofactor activity as assessed by either clotting () or prothrombinase function (). The arrow indicates the addition of lipid vesicles. B, Densitometric analysis of Coomassie blue–stained factor Va fragments shown in C. HC density as a function of maximum density is shown by (). The appearance of the Mr 30 000 fragment is shown by (). C, Coomassie blue-stained gel of factor Va fragments present at the time points indicated below the gel lanes. Apparent molecular weight by comparison with standards is shown on the right edge. D, Western blot of the same time-point samples shown in C.

On addition of phospholipid (20 µmol/L PC/PS) at 64 minutes, the clotting activity immediately decreases to less than 1% of initial and is below detectable levels at 84 minutes (Fig 2A). The activity assessed in the prothrombinase assay rapidly decreases from 60% to less than 1% within 6 minutes and is below detectable levels at 84 minutes (Fig 2A). Western blot analyses show that after phospholipid addition, the M_r 75 000 intermediate is cleaved at Arg³⁰⁶, generating an M_r 30 000 product that is stable (Fig 2D). The Coomassie blue–stained gel shows rapid consumption of the M_r 75 000 intermediate, generating products at M_r 45 000 (1-306) and M_r 30 000 (307-506) (Fig 2C). Densitometric analyses of the M_r 30 000 product reveal a rapid increase in density, which plateaus at 70 minutes (Fig 2B). The rapid decrease in activity on lipid addition, observed in the quantitative prothrombinase assay, is directly related to the formation of the M_r 30 000 fragment. A comparison of panel A and B shows a reciprocal relationship between product formation (M_r 30 000 density) and factor Va activity.

APC Inactivation of Factor Va on the HUVEC Monolayer
Cofactor activity was measured in a clotting assay and in the prothrombinase assay at 2.5 nmol/L factor Xa. The factor Xa concentration was picked such that intact factor Va (K_d=0.5 nmol/L) would be saturated with respect to factor Xa in prothrombinase formation. On addition of APC to the HUVECs, a rapid decrease in cofactor activity is observed; 85% of the initial activity is lost within 6 minutes (clotting assay, ), while the activity assessed by the prothrombinase assay is 50% of initial at 6 minutes (Fig 3, ). Western blot analysis of the initial 6 minutes (Fig 4, lanes 4 through 6) indicates that initial cleavage occurs only at Arg⁵⁰⁶, and small amounts of the M_r 30 000 product begin to accumulate due to cleavage at Arg³⁰⁶ within the M_r 75 000 intermediate. The activity differences, 15% versus 50% at 6 minutes (clotting versus continuous assay), suggest that while factor Va cleaved at Arg⁵⁰⁶ displays little clotting activity (ie, at 5 pmol/L factor Xa), at 2.5 nmol/L factor Xa, this same species displays 50% of its activity. This corresponds well to the data derived from the phospholipid system (Fig 2). After 4 minutes, an M_r 30 000 fragment begins to appear (Fig 4) and accumulates over time. This is due to cleavage at Arg³⁰⁶ in the M_r 75 000 fragment of the HC (Fig 4, lanes 6 through 13). At 12 minutes, the clotting activity is 10% of initial, while the quantitative prothrombinase assay activity (at 2.5 nmol/L factor Xa) is 32% of initial (Fig 3). The clotting activity decreases below measurable levels in 60 minutes (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 3. Inactivation of factor Va by APC on the surface of HUVECs and cofactor activity data. Factor Va cofactor activity was assessed by both a DAPA () and clotting () assay. At the specified time intervals, aliquots were assessed for cofactor activity in both assay systems. The activity of factor Va (20 nmol/L) on the HUVEC surface was assessed as the zero time point. On addition of APC (5 nmol/L), the percent of remaining cofactor activity is shown on the y axis, while the time from APC addition is shown on the x axis. The proteolytic fate of factor Va in this system is shown in Fig 4, which presents a replicate experiment.

View larger version (77K): [in this window] [in a new window]   Figure 4. Inactivation of factor Va by APC on the HUVEC surface and proteolytic processing of the HC, by Western blot. Samples were taken during factor Va (20 nmol/L) incubation on the HUVECs at 0, 5, and 15 minutes. APC (5 nmol/L) was then added, and samples were taken at 2, 4, 6, 10, 15, 20, 30, 60, 90, and 120 minutes. These samples were analyzed by SDS-PAGE under reducing conditions. After transfer to nitrocellulose, the immunoreactive fragments were detected by using HVaHC No. 17. The lane number is shown across the top of the gel, molecular weight markers along the left edge, factor Va species identification on the right edge, and time point of gel sample on the bottom edge. The arrow between lanes 3 and 4 represents the point of APC addition.

To determine the influence of the HUVEC surface on APC cleavages, factor Va was added to either the HUVEC surface or to a serum-treated blank well, and samples were removed over time for Western blot analysis. The cleavages at Arg⁵⁰⁶ and Arg³⁰⁶ are both accelerated in the presence of the HUVECs (Fig 5; compare the HUVECs, lanes 3, 5, 7, and 9 with the blank well, lanes 4, 6, 8, and 10). Cleavage at Arg³⁰⁶ occurs only in the presence of the HUVECs (Fig 5; compare lanes 5, 7, 9, 11, and 13 with 6, 8, 10, 12, and 14). The relative reactivities of the antibody and the transfer efficiencies of each species are not equivalent; thus, comparisons of density in the vertical dimension are not possible. However, comparison of density in the horizontal direction is a dependable relative quantitation method.⁴² Densitometric analyses of product formation with time are presented in Fig 6 as percentages of the maximum intensities observed for each product (M_r 75 000 and 30 000). In the presence of the HUVECs, the M_r 75 000 intermediate is present within 1 minute and accumulates to a "steady state" value in 8 minutes (Fig 6A, ); in contrast (in the serum-treated well), the M_r 75 000 product is observed only after 3 minutes and increases in a linear fashion (Fig 6A, ). After 8 minutes, the M_r 75 000 fragment density in the blank well is 34% of that observed on the HUVECs, illustrating enhanced cleavage at Arg⁵⁰⁶ in the presence of the HUVECs (Fig 6A, ). The plateau in concentration of the M_r 75 0000 product on the HUVECs reflects the competing kinetic processes that respectively form (cleavage at Arg⁵⁰⁶) and deplete (cleavage at Arg³⁰⁶) this species. Analysis of the formation of the M_r 30 000 product on the HUVECs shows a linear increase in time (Fig 6B, ). No M_r 30 000 fragment is seen in the absence of the HUVECs (Figs 5 and 6, ), illustrating the dependence of cleavage at Arg³⁰⁶ on the HUVEC surface. At no time in either case is the product corresponding to initial cleavage at Arg³⁰⁶ (M_r 62 000) observed.

View larger version (73K): [in this window] [in a new window]   Figure 5. Inactivation of factor Va in the presence or absence of the HUVEC surface and a comparison of rates. Samples were taken during a time course of APC (5 nmol/L) inactivation of factor Va (20 nmol/L) in the presence or absence of the HUVEC surface. A zero time point was taken subsequent to a 5-minute incubation on the cell surface. On the addition of APC (5 nmol/L), samples were taken at 1, 3, 5, 8, 11, and 15 minutes. The samples were analyzed on 4% to 12% SDS-PAGE gels under reducing conditions. After transfer to nitrocellulose, immunoreactive fragments were detected with the monoclonal antibody HVaHC No. 17. The gel was loaded such that identical time points from experiments in which the HUVEC surface was present (numbers in parentheses) or absent are next to each other. The position of the molecular-weight markers is indicated on the left, gel lane number along the top, and time point of the gel sample along the bottom; (minutes) indicates the presence of the HUVEC surface. Product identification is shown along the right edge.

View larger version (16K): [in this window] [in a new window]   Figure 6. Densitometric analysis of product formation in APC inactivation of factor Va in the presence or absence of the HUVEC surface. The Western blot shown in Fig 5 was used for densitometric analysis of product formation. The band indicated by fVa 1-506 corresponding to the Mr 75 000 product was quantified by densitometric analysis. Identical analysis was accomplished across the band labeled fVa 307-506, corresponding to the Mr 30 000 product. A, After determination of the maximum intensity obtained for the fVa product 1-506, the percent of maximum density obtained is plotted vs time for each point in the experiment in which the HUVECs was present () or absent (). B, After determination of the maximum intensity obtained for the product fVa 307-506, the percent of maximum density is plotted vs time for each point in the experiment in which the HUVECs was present () or absent ().

Correlation of HUVEC/IIa Activation of PC With APC Inactivation of Factor Va
The rates of both PC activation and factor Va inactivation on the cell surface were monitored simultaneously. Factor Va is stable on the HUVEC surface during the 10-minute incubation before the addition of PC (Fig 7 and Fig 8, lanes 2 through 4). On addition of PC (500 nmol/L), PC activation occurs at a rate of 1.56±0.11x10^-14 mol/min per cell (n=3) (Table 1). Factor Va clotting activity decreases rapidly to 10% of initial in 10 minutes (Fig 7, ). Western blot analyses show rapid cleavage at Arg⁵⁰⁶, generating a transient M_r 75 000 fragment that is cleaved (Arg²⁰⁶) to generate the M_r 30 000 product (Fig 8, lanes 6 through 12). The APC concentration in this experiment continuously increases, and 104 nmol/L APC is formed in 60 minutes (Fig 7, ). Plasma PC concentrations are near 70 nmol/L; thus, under physiological conditions, 104 nmol/L APC could never be obtained. Initial cleavage by APC at Arg³⁰⁶ in the intact HC would generate an M_r 62 000 fragment; however, no fragment corresponding to this molecular weight is observed, although the APC concentration is 104 nmol/L at 60 minutes (Figs 7 and 8).

View larger version (21K): [in this window] [in a new window]   Figure 7. Inactivation of factor Va by in situ–generated APC on the surface of HUVECs. Factor Va cofactor activity was monitored by a factor V–deficient clotting assay. APC concentration was monitored by the fluorogenic assay described in "Methods." The cofactor activity data is presented as a percentage of maximum relative cofactor activity and is shown on the left axis. The APC concentration (nmol/L per 100 000 cells) is shown on the right axis. On addition of factor V (20 nmol/L) and IIa (1 nmol/L) to the HUVECs, 0-, 5-, and 15-minute samples were taken; PC (500 nmol/L) was then added, and at 2, 4, 6, 10, 15, 20, 30, 60, and 90 minutes, samples were taken. All time-point samples were assayed for APC activity () and for factor Va cofactor activity (). The line drawn through the data points () represents a linear fit using a least-squares fitting algorithm, yielding an APC generation rate of 1.56x10-14 mol/min per cell. The numbers represent the mean for three replicate experiments. Inset, Cofactor activity () during the initial 10 minutes after PC addition. At the specified time intervals, aliquots were taken and analyzed by SDS-PAGE under reducing conditions. After transfer to nitrocellulose, immunoreactive fragments were detected with HVaHC No. 17 as shown in Fig 8. The inset details the initial 10 minutes of the reaction.

View larger version (77K): [in this window] [in a new window]   Figure 8. Inactivation of factor Va by in situ–generated APC and proteolytic processing of the HC by Western blot. During the activity assay shown in Fig 7, aliquots were removed at 0, 5, and 10 minutes; PC (500 nmol/L) was then added, and at 2, 4, 6, 10, 15, 20, 30, 60, and 90 minutes, samples were taken and run on a 4% to 12% SDS-PAGE gel under reducing conditions, and immunoreactive fragments were detected by Western blot by using HVaHC No. 17. The lane number is shown across the top of the gel, molecular-weight markers along the left edge, factor Va species identification on the right edge, and time point of gel sample on the bottom edge. The arrow between lanes 3 and 4 represents the point of PC addition. The open arrow at Mr 97 000 indicates the factor Va HC derived from thrombin proteolysis of the Mr 105 000 factor Va HC. The asterisk at the position of Mr 43 000 indicates the product most probably derived from cleavage at Arg306 in the Mr 97 000 HC species.

View this table: [in this window] [in a new window]   Table 1. APC Generation Rates for All PC Addition Experiments

Products of M_r 97 000 and M_r 43 000 are also observed and accumulate over the time course (Fig 8). The M_r 97 000 band appears to be the result of an -thrombin–related cleavage near the COOH terminus of the factor Va HC (detailed in experiments described later). The M_r 43 000 band appears to represent the product derived from cleavage at Arg³⁰⁶ in the precursor M_r 97 000 fragment. Cleavage at Arg⁵⁰⁶ in the M_r 97 000 HC fragment would generate an HC fragment 1-506 (M_r 75 000), which is indistinguishable from that derived from cleavage at Arg⁵⁰⁶ in the intact HC.

To determine whether PC activation is dependent on the cofactor activity of -thrombin/TM, the rate of PC activation was determined under a variety of conditions in which one or more components required for this interaction are inhibited. Thrombin catalyzed activation of PC in the absence of the HUVECs (1 nmol/L IIa/500 nmol/L PC) does not occur at a detectable rate during a 20-minute incubation. In the presence of HUVECs, 30 nmol/L APC is generated over this time interval.

The level of PC activation on the HUVECs when -thrombin is inhibited was determined by inhibiting -thrombin (1 nmol/L) with hirudin (10 nmol/L). The PC activation rate under these conditions is 4.0x10^-17 mol/min per cell (Table 1 and Fig 9, ), which is <1% (0.26%) that of the rate obtained with active -thrombin. Factor Va cofactor activity (clotting assay) remains at 82% of initial after 90 minutes (Fig 9, ). Western blot analyses show a faint band of M_r 75 000 late in the time course; this product is indicative of cleavage at Arg⁵⁰⁶ in the factor Va HC by the low levels of APC that are generated (Fig 10A). In a control experiment in which hirudin was omitted, both factor Va inactivation and PC activation occurred at rates comparable to those outlined previously.

View larger version (18K): [in this window] [in a new window]   Figure 9. Stability of factor Va during inhibition of in situ PC activation on the surface of HUVECs. Factor Va cofactor activity was monitored by a factor V–deficient clotting assay. APC concentration was monitored by the fluorogenic assay described in "Methods." Cofactor activity is presented as a percentage of maximum relative activity and is shown on the left axis. The APC concentration (nmol/L per 100 000 cells) is shown on the right axis. On addition of factor Va (20 nmol/L), IIa (1 nmol/L), and either hirudin (40 nmol/L) or hTM-531 (600 nmol/L) to the HUVECs, 0-, 5-, 10-, and 20-minute samples were taken; PC (500 nmol/L) was then added, and at 2, 4, 6, 10, 15, 20, 30, 60, 90, and 120 minutes, samples were taken. All time-point samples were assayed for APC activity and for factor Va cofactor activity. (), Cofactor activity when hirudin was added with the PC. (), Cofactor activity when hTM-531 was added with the PC. (), APC concentration over the HUVEC surface when hirudin was added with PC. (), APC concentration over the HUVEC surface when hTM-531 was added with PC. At the specified time intervals, aliquots were taken and analyzed by SDS-PAGE under reducing conditions. After transfer to nitrocellulose, immunoreactive fragments were detected with HVaHC No. 17 as shown in Fig 10.

View larger version (73K): [in this window] [in a new window]   Figure 10. Factor Va HC stability when PC activation is inhibited on the HUVECs, by Western blot. During the experiment shown in Fig 9, samples were taken over time. The lane number is shown across the top of the gel, molecular-weight markers (x10-3) along the left edge, factor Va species identification on the right edge, and time point of gel sample on the bottom edge. The vertical arrow between lanes 3 and 4 in A and lanes 5 and 6 in B represents the addition of PC to the incubation. A, On addition of factor Va (20 nmol/L), IIa (1 nmol/L), and hirudin (40 nmol/L) to the HUVECs, samples were taken and run on 4% to 12% SDS-PAGE gels under reducing conditions. After transfer to nitrocellulose, immunoreactive fragments were detected with monoclonal antibody HVaHC No. 17. B, On addition of factor Va (20 nmol/L), IIa (1 nmol/L), and hTM-531 (600 nmol/L) to the HUVECs, samples were taken and run on 4% to 12% SDS-PAGE gels under reducing conditions. After transfer to nitrocellulose, fragments were detected with monoclonal antibody HVaHC No. 17. The IgGHC at the right of B shows the HC of IgG hTM-531, which is recognized by the secondary goat anti-mouse IgG. Similarly, the IgGLC shown to the right of B indicates the light chain of IgG hTM-531, which is slightly cross-reactive with our secondary antibody. The open arrow indicates the Mr 97 000 band, which is derived through -thrombin proteolysis of the factor Va HC. Note its absence in A in which -thrombin is inhibited.

The rate of PC activation on the HUVECs when the -thrombin/TM interaction is blocked with 60 nmol/L of the antibody hTM-531 is 1.84x10^-15 mol/min per cell. Addition of 600 nmol/L hTM-531 decreased the rate to 4.63x10^-17 mol/min per cell (Table 1 and Fig 9, ). When 600 nmol/L hTM-531 is used, the factor Va activity (clotting) is 80% of initial at 60 minutes (Fig 9, ). Western blot analyses revealed that cleavage at Arg⁵⁰⁶ in the factor Va HC occurs late in the time course (Fig 10B) due to the low levels of APC generated (0.75 nmol/L in 30 minutes [Fig 9 and Table 1]). In addition, a product of M_r 97 000 is seen to accumulate over time in this experiment (Fig 10B), suggesting that -thrombin is catalyzing a cleavage in the HC of factor Va (Figs 8 and 10B). Catalytically active -thrombin appears to be necessary for generation of the M_r 97 000 HC fragment (compare Fig 10A with 10B). This fragment is observed only under reducing conditions. In a control experiment in which the antibody was omitted, both factor Va inactivation and PC activation occurred, as previously described.

NH₂-Terminal Analyses of M_r 97 000 HC Fragment
Production of the M_r 97 000 HC fragment was accomplished by extended -thrombin treatment of factor Va in the absence of the HUVECs. NH₂-terminal sequence of the factor Va HC (under reducing and nonreducing conditions) and the M_r 97 000 fragment (apparent only under reducing conditions) is shown in Table 2, as background-corrected picomolar yields of the amino acid phenylthiohydantoin derivatives. The NH₂-terminal sequence of both the intact factor Va HC and the M_r 97 000 fragment were identical to that of the predicted sequence for the factor Va HC NH₂ terminus obtained from cDNA analysis.²⁴

View this table: [in this window] [in a new window]   Table 2. Sequence of NH2-Terminal Amino Acid Residues of Fragments Derived From -Thrombin Treatment of Factor Va HC

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Our data demonstrate that (1) the inactivation of factor Va on the HUVECs occurs in a sequential fashion, with initial cleavage at Arg⁵⁰⁶ followed by cleavage at Arg³⁰⁶; (2) the HUVEC surface accelerates the cleavage at Arg⁵⁰⁶ and promotes cleavage at Arg³⁰⁶, which is required for inactivation; (3) the cleavages are dependent on the presence of PC, factor IIa, and TM; (4) -thrombin bound to the HUVEC surface induces cleavages of factor Va HC within the COOH-terminal -loop to generate an M_r 97 000 fragment; and, (5) the observed cofactor activity of APC-cleaved factor Va is dependent on the concentration of factor Xa available and thus on the assay used to measure cofactor activity.

Although product-precursor quantitation relationships based on blot density are not presently possible, the data in Figs 4 and 5 provide semiquantitative support to the argument for a sequential cleavage process occurring on the HUVECs. In such a mechanism, product formation proceeds according to:

(1)

where A is factor Va HC (1-709) and B and C are the M_r 75 000 (1-506) and M_r 30 000 (307-506) fragments, respectively. Clearly, k₂ must be rate limiting, otherwise no M_r 75 000 fragment could be seen to accumulate. On HUVECs (Fig 5A), this intermediate appears to reach its steady state maximum concentration (Fig 5A), defined by:

(2)

Furthermore, no significant lag is apparent for formation of the M_r 75 000 product (B) on HUVECs. In contrast, the lag in formation of the M_r 30 000 product (C) suggests its accumulation must follow the accumulation of the M_r 75 000 product (B), whereupon at steady state, the rate of formation of C is given by:

(3)

which predicts a linear rate of generation of the M_r 30 000 product (C) after a lag period, which is the process we observed (Fig 5B). The rate constants reported for the catalysis at Arg⁵⁰⁶ and Arg³⁰⁶ of 4.3x10⁷ mol/L^-1 s^-1 and 2.3x10⁶ mol/L^-1 s^-1, respectively,⁴³ in a random process would predict product formation derived from initial cleavage at either Arg⁵⁰⁶ or Arg³⁰⁶. Overall, the data do not fit a random cleavage process on the HUVECs.⁴⁴

The factor Va species derived after APC cleavage at Arg⁵⁰⁶ has an apparent K_d for factor Xa within prothrombinase of 3.9 nmol/L.⁴³ Intact factor Va has been shown to posses an apparent K_d for factor Xa in the range of 0.2 to 1 nmol/L.^{43 44} Our data show that factor Va_APC/506 displays little cofactor activity in a clotting assay (ie, in the presence of limiting factor Xa) and yet displays 60% of its initial activity when assessed at 10 nmol/L Xa. In contrast, fully cleaved factor Va (Va_506/306/679) displays no cofactor activity, even at 10 nmol/L factor Xa. These data demonstrate that the consequences of factor Va proteolysis by APC at Arg⁵⁰⁶ are dependent on the assay used to measure cofactor activity. In these studies, 630 nmol/L factor Va was used to allow quantitative physical analyses such as Coomassie blue staining of SDS-PAGE. Nicolaes et al⁴⁴ came to a similar conclusion regarding factor Va species' apparent cofactor activity and factor Xa concentration. However, their conclusions were drawn from two contrasting experiments using different concentrations of factor Va, factor Xa, and APC. The rate of factor Va inactivation in these two experiments cannot have been the same, and thus it becomes difficult to assign measured activity/factor Xa concentration correlations when the nature of the species analyzed, as well as the experimental protocol, is different. The significant differences in the literature concerning factor Va inactivation by APC will only be reconciled through comparison of assays performed under identical experimental conditions. The comparison of factor Va species activity assessed under dissimilar protocols can lead to apparently large discrepancies between laboratories. These differences most probably arise due to the change in the physical binding parameters of factor Va (for factor Xa) on APC cleavage. Dissimilar assay conditions may or may not satisfy the binding parameters for each factor Va species, such that the true maximal activity is measured.

The mechanism of inactivation of factor Va by APC has been the subject of considerable controversy. The position of the APC cleavages in factor Va have been well established^{9 10} ; however, confusion over the rate and order of cleavage, the effects on activity of individual cleavages, and the contribution of membrane composition to the inactivation of factor Va have led to a variety of reports.^{9 25 30 32 43 44 45 46} Several studies using anionic phospholipid vesicles as a surface for factor Va inactivation have reported ordered sequential cleavage of factor Va,^{9 10 44} whereas another study reported a random cleavage order.⁴³ The lipid dependence of cleavage at Arg³⁰⁶ has been shown numerous times^{9 10 19} ; however, at high APC concentrations, lipid-independent cleavage at Arg³⁰⁶ has also been reported.⁴³ The inclusion of phosphatidylethanolamine into vesicles has been reported to accelerate lipid-dependent cleavages in the inactivation of factor Va; however, no information is available on the nature of specific bond cleavage rates.⁴⁶ Additionally, the inactivation of (platelet) factor Va on the platelet surface is reported to be different in mechanism and rate to that observed for plasma factor Va on synthetic vesicles. Platelet factor Va inactivation on platelets is characterized by an initial cleavage at either Arg⁵⁰⁶ or Arg³⁰⁶; furthermore, extended incubation with APC does not result in complete cofactor inactivation (platelet factor Va).²⁵ Thus, it is apparent that membrane composition and/or expression plays an important role in the reaction.

The endothelial cell surface is presumably the site of PC activation and factor Va inactivation in vivo. Platelets may contribute to this process; however, they have been shown to be relatively ineffective in factor Va inactivation. The HUVEC acceleration of cleavage at Arg⁵⁰⁶ shown in Figs 5 and 6 appears to be similar to the anionic phospholipid effect reported in the bovine system using purified components.⁹ The endothelial membrane at confluence is comprised of (in EA.hy 926 cells) 55% phosphatidylcholine, 23% sphingomyelin, 14% phosphatidylethanolamine, 7% phosphatidylinositol, and 3% phosphatidylserine.⁴⁷ The asymmetric surface expression of these components is not well defined; however, the relative phospholipid composition of endothelial membranes appears to be consistent with their ability to support factor Va inactivation in a manner that appears analogous to a model system. In our experiments, cleavage at Arg³⁰⁶ is totally dependent on the HUVEC surface (Figs 2, 5, and 6). The HUVEC monolayer in our system presents a surface area equivalent to that expressed by only 550 nmol/L phospholipid vesicles. Thus, cleavage at Arg³⁰⁶ on the HUVEC surface, although dependent on its presence, is relatively slow compared with that observed using 20 µmol/L phospholipids (compare Fig 2D with Fig 4). On the HUVEC surface, it is difficult to directly assign activity values to factor Va species due to continuous proteolysis. However, the activity product profiles shown in Figs 3 and 4 (2.5 nmol/L Xa prothrombinase and clotting assay) are qualitatively similar to those seen on phospholipid vesicles comprised of 25% phosphatidylserine and 75% phosphatidylcholine. In the experiments conducted on the HUVEC surface, 20 nmol/L factor Va was used. Previous studies have shown factor V to be completely activated during the process of blood clotting,⁴² thus 20 nmol/L factor Va (mean plasma V concentration) would appear to be a physiologically relevant level of "challenge" to the PC pathway.

A number of recent studies have reported PC/APC binding sites on the HUVECs that are independent of TM. The reported apparent K_d of PC/APC for these receptors varies from 6 to 30 nmol/L.^{27 28} The HUVEC monolayer provides TM at a concentration such that PC activation occurs at a rate of 1.6x10^-14 mol/min per cell in our system. Under similar conditions, a rate of 3.0x10^-14 mol/min per cell was reported.⁴⁸ To satisfy the K_m for PC/thrombin-TM, we used 500 nmol/L PC in our experiments. The mean plasma concentration of PC is 70 nmol/L; thus, the APC levels obtained in this system are higher than that expected physiologically. However, most of the "excess" APC generated in our system occurs after most of the factor Va inactivation has already occurred (ie, at 20 minutes, only 20 nmol/L APC is generated, while most of the factor Va is completely cleaved). The effective TM concentration in a capillary bed is quite high in comparison with that expressed on our planar culture well; thus, activation of PC at 70 nmol/L, although well below the K_m, is probably efficient. Although our measurements of factor Va activity were all made from fluid-phase sampling, we have shown the reaction to be dependent on the HUVEC surface; thus, changes in the composition of fluid-phase factor Va are direct measures of events mediated by the HUVEC surface and the APC generated on it. The relative dissociation/association rates of factor Va for a membrane surface are mediated through the light chain of the factor Va molecule.⁴⁹ Since APC does not cleave this portion of factor Va (in the human system), we do not expect significant "sequestering" of intermediate species to occur. Hirudin inhibition of -thrombin reduced the rate of PC activation on the HUVECs to <1% that of control, whereas a dose-dependent inhibition of PC activation was observed in the presence of an anti-TM monoclonal antibody (hTM-531) that inhibits the TM/-thrombin interaction. These results show that PC activation on the HUVEC surface is due primarily to the cofactor activity of TM, as had been previously reported.^{12 13 14 26} Thus, the vasculature is primed to rapidly activate PC, and the APC that is generated subsequently uses the surface provided to cleave factor Va at Arg⁵⁰⁶, rapidly generating a cofactor that, under low factor Xa concentrations, is ineffective in prothrombinase formation. Ultimately, the proteolytic fate of factor Va once PC is activated appears to be dependent on the vascular surface (platelets or HUVECs) to which it is bound.

During experiments in which -thrombin was present on the HUVEC surface, an M_r 97 000 band accumulated over time (Fig 10B). A species with similar reduced SDS-PAGE migration characteristics has been noted previously.^{32 50 51} In the bovine system, generation of a fragment with similar characteristics of M_r 90 000 (intact bovine HC M_r 94 000) was dependent on a platelet membrane–associated protease that was expressed on activation of the platelets. The M_r 97 000 fragment we report appears to be dependent on the presence of catalytically active -thrombin and is most efficiently generated on the HUVECs. NH₂-terminal sequence analysis determined that this fragment possesses an identical NH₂ sequence to that of intact factor Va HC. Disulfide mapping of the bovine factor Va HC indicated the existence of a disulfide bond between residues 579 and 660, which in the human system would be 585 to 654 by homology.⁵² Thus, we speculate that the cleavage occurs between residues 586 and 654. Although we cannot yet assign a cleavage site, analysis of the sequence between residues 586 and 654 revealed a consensus sequence for -thrombin cleavage (X-P-R) at residues 640 to 643 (S-P-R). -Thrombin cleavage at arginine 643 would produce a peptide containing residues 644 through 709 in a disulfide interaction with the remaining HC and on reduction would yield an apparent mobility shift of approximately M_r 8000.

In conclusion, our data demonstrate that the inactivation of factor Va on the HUVEC surface proceeds through sequential cleavages of the HC. The first cleavage generates an intermediate species with a cofactor activity dependent on the factor Xa concentration present in the assay. On the HUVEC surface, a COOH-truncated M_r 97 000 HC fragment is generated in the presence of -thrombin, which appears to be similar to a fragment noted previously in the bovine platelet system.

	Selected Abbreviations and Acronyms

 

APC = activated PC DAPA = dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide HBS = HEPES-buffered saline HC = heavy chain (factor Va) HUVEC = human umbilical vein endothelial cell PAGE = polyacrylamide gel electrophoresis PC = protein C PC/PS = phosphatidylcholine/phosphatidylserine vesicles TM = thrombomodulin

	Acknowledgments

 
This work was supported by NIH merit award No. HL34575. We wish to thank Dr John Morser of BERLEX Inc for the generous gift of the monoclonal antibody hTM-531, Dr W. Church from the University of Vermont for the providing the monoclonal antibody HVaHC No. 17, Dr Richard Jenny of Haematologic Technologies for providing factor Xa and DAPA, the staff of the birthing ward at the Fletcher Allen University Health Care Center for their eager assistance in collecting source material for our HUVEC isolation, and Dr Alex Kurosky and Steve Smith of the University of Texas for amino acid sequencing.

	Footnotes

 
Presented in part at the 37th annual American Society for Hematology meeting, December 1-5, 1995, Seattle, Wash.

Received March 12, 1997; accepted June 5, 1997.

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