Protein C Inhibitor Secreted from Activated Platelets Efficiently Inhibits Activated Protein C on Phosphatidylethanolamine of Platelet Membrane and Microvesicles*

Junji NishiokaDagger , Ma Ning§, Tatsuya HayashiDagger , and Koji SuzukiDagger

From the Dagger  Department of Molecular Pathobiology and the § Second Department of Anatomy, Mie University School of Medicine, Tsu-city, Mie 514, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Protein C inhibitor (PCI) was detected in human platelets (2.9 ng/109 cells) and megakaryocytic cells (1.5 ng/106 cells). PCI mRNA was also detected in both platelets and megakaryocytic cells using nested polymerase chain reaction. PCI was found to be located in the alpha -granules of resting platelets. Approximately 30% of the total amount of PCI in platelets was released after stimulation with ADP, collagen, adrenalin, thrombin, or thrombin receptor-activating peptide. Secreted PCI was detected on the surface of activated platelets and phospholipid microvesicles. PCI secreted from thrombin receptor-activating peptide-stimulated platelets inhibited activated protein C (APC) efficiently. PCI significantly inhibited APC in the presence of phospholipid vesicles prepared using rabbit brain cephalin (RBC) or a mixture of 40% phosphatidylethanolamine (PE), 20% phosphatidylserine (PS), and 40% phosphatidylcholine (PC) with a second order rate constant of 1.0 × 106 M-1·min-1. Of these phospholipids, PE was critical for this inhibition. The dissociation constants of the binding of APC or PCI to solid phase phospholipids showed that APC binds more preferably to PE than to RBC or PS, and PCI to PE or RBC than to PS or PC. PCI binding to solid phase phospholipids depended on the presence of PE. RBC- or PE-bound PCI inhibited APC significantly but only weakly the gamma -carboxyglutamic acid domainless APC. The gamma -carboxyglutamic acid fragment of protein C suppressed the PCI-mediated inhibition of APC on solid phase RBC or PE. Most of the APC·PCI complex formed on solid phase RBC or PE was released into the soluble phase. These findings suggest that PCI secreted from activated platelets binds preferably to PE of platelet membrane and microvesicles and that it inhibits phospholipid-bound APC efficiently.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Protein C inhibitor (PCI),1 a member of the plasma serine protease inhibitor (serpin) family, was originally identified in plasma as an inhibitor of activated protein C (APC), the main proteolytic enzyme of the protein C anticoagulant pathway (1-3). During the PCI-mediated inhibition of APC, PCI forms an acyl-bond complex with APC (2). This complex formation is enhanced by heparin and negatively charged dextran sulfate (1, 2). Patients with venous thrombosis or disseminated intravascular coagulation show increased plasma levels of APC·PCI complex (4-7). PCI is the main inhibitor of APC, but when its plasma concentration becomes limited in conditions associated with increased activation of protein C, alpha 1-antitrypsin and alpha 2-macroglobulin also form complexes with APC (8, 9). PCI also inhibits plasma serine proteases that are involved in blood coagulation and fibrinolysis such as thrombin (2, 10), factor Xa (2, 10), factor XIa (10, 11), urokinase (12), tissue plasminogen activator (12), and plasma kallikrein (10, 11). Recently, PCI was also found to be a potent inhibitor of the thrombin-thrombomodulin complex (13), giving further support to the important role that it plays in the regulation of the anticoagulant protein C pathway. Plasma PCI seems to be produced mainly in the liver because the plasma level of PCI decreased in patients with severe liver diseases (14). Recently, PCI was detected in human seminal plasma, Graaf follicular fluid, testis, epididymis, prostate, seminal vesicle (15), and the preacrosomal region of human spermatozoa (16, 17). PCI was found previously to inhibit acrosin, a serine protease localized in the acrosome of spermatozoa (17, 18). However, the physiological significance of PCI in the reproductive organs has not been established.

Platelets play a crucial role in hemostasis. Platelets contain several blood coagulation factors, such as von Willebrand factor (19), fibrinogen (20), factor V (21), factor IX (22), factor X (23), factor XIII (24), and thrombospondin (25). Clotting factors derived from platelets promote blood coagulation and wound healing (26). Platelets also provide a surface for binding factor Va and factor Xa, which leads to increased prothrombin activation (27). In addition to their procoagulant activity, platelets may also modulate the activation of the coagulation and complement systems. Protein S secreted from platelets serves as a cofactor for APC-catalyzed inactivation of factor Va and factor VIIIa on the platelet surface (28). Thrombomodulin on the platelet surface may facilitate the thrombin-catalyzed activation of protein C (29). C1 inhibitor, a member of the plasma serpin family secreted by platelets, may modulate the activation of the complement and intrinsic coagulation systems at inflammatory sites (30).

In this report, we describe the presence of PCI in the alpha -granules of human platelets and its secretion and function in the APC inhibition on the surface of phospholipid membrane.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Sepharose CL-2B was obtained from LKB Biotechnology. Bovine serum albumin (BSA), rabbit brain cephalin (RBC), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and heparin (160 units/mg) were from Sigma. RBC is composed of 35-40% PE, 30-35% PC, and 10-15% PS (31). Monoclonal murine anti-human glycoprotein (GP)Ib (CD42b) IgG and FITC-labeled anti-human GPIb IgG were from DAKO A/S (Glostrup, Denmark). Goat anti-mouse IgG conjugated to 15 nm of colloidal gold was from Zymed Labs. Inc. (San Francisco). The synthetic substrates for APC, Boc-Leu-Ser-Thr-Arg-methylcoumaryl amide (MCA), and S-2366 (Glu-Pro-Arg-p-nitroaniline) were from Protein Research (Osaka, Japan) and Chromogenix (Möndal, Sweden), respectively. The thrombin receptor-activating peptide (TRAP), Ser-Phe-Leu-Leu-Arg-Asn, was synthesized by a solid phase method using an Applied Biosystems model 431A peptide synthesizer. PermeaFixTM was from Ortho Diagnostic (Raritan, NJ), a Maxisorp 96-well microtiter plate was from Nunc (Roskilde, Denmark), and L. R. White acrylic resin was from London Resin (Basingstoke Hampshire, U. K.). All other chemicals were of the highest grade commercially available.

Preparation of Proteins, Antibodies, and Phospholipids-- PCI (1) and protein C (32) were purified from fresh frozen human plasma as described. Gla-domainless protein C, Gla-domainless APC, and the Gla fragment of protein C were prepared as described by Esmon et al. (33). APC·PCI complex was prepared as described (34). Thrombin (1,800 units/mg) was prepared from human prothrombin as described by Mann (35). The concentrations of proteins were determined at 280 nm absorbance using the following extinction coefficients (E280 nm[1%]): 14.5 for protein C and APC (36) and 14.1 for PCI (1). The concentration of the Gla fragment of protein C was determined as described (36). Monoclonal murine anti-human protein C IgG (MFC-9) (37), anti-human PCI IgG (MFCI-10) (34), and anti-human factor V IgG (38) were prepared as described previously. FITC-labeled anti-PCI IgG was prepared as described (39), the molar ratio of FITC to IgG being 4:1. Phospholipid microvesicles containing RBC or a mixture of various ratios of phospholipids (PE, PS, and/or PC) in Hepes-buffered saline (20 mM Hepes, 100 mM NaCl, pH 7.4) were prepared as described by Bloom et al. (40). Immobilization of phospholipids in microplate wells was carried out as follows. Appropriate concentrations of phospholipid suspension were prepared in 100 mM NaHCO3 buffer, pH 9.3, and incubated in microplate wells at 4 °C overnight. The concentration of phospholipids was determined by an elemental phosphorus analysis as described previously (41).

Preparations of Gel-filtered Platelets and Megakaryocytic Cells and Cell Lysate-- Gel-filtered platelets were prepared from sodium citrate-treated blood of healthy volunteers by using Sepharose CL-2B in Hepes-buffered saline containing 4 mM KCl and 0.1% glucose as described previously (42). CMK cells were kindly provided by Dr. T. Sato, Department of Microbiology, Chiba University School of Medicine, Chiba, Japan. This CMK cell line, which is derived from human megakaryocytic leukemia, contains proteins and granules typical of platelets (platelet peroxidase, membrane GPIb, GPIIb/IIIa, and alpha -granules) (43). Lysates of gel-filtered platelets or CMK cells were obtained by treating each cell pellet with Tris-buffered saline (50 mM Tris-HCl, 100 mM NaCl, pH 7.5), containing 0.5% Triton X-100, for 15 min at room temperature.

Determination of PCI and APC·PCI Complex-- The concentration of PCI antigen was determined by an enzyme-linked immunosorbent assay (ELISA) using peroxidase-coupled polyclonal anti-PCI IgG and tetramethylbenzidine as described previously (44). PCI activity was assayed by determining the inhibitory effect of PCI on the amidolytic activity of APC on specific substrates (S-2366 or Boc-Leu-Ser-Thr-Arg-MCA) in wells of a microtiter plate as described previously (2). The concentration of APC·PCI complex was determined by an ELISA using monoclonal anti-PCI IgG and peroxidase-coupled monoclonal anti-protein C IgG by a method described previously (44).

Evaluation of PCI RNA Expression in Platelets, CMK, and HepG2 Cells-- Gel-filtered platelets (3 × 108), CMK cells (1 × 104), or hepatocellular carcinoma cell HepG2 cells (1 × 105) were used to prepare total RNA by using guanidine isothiocyanate and CsCl gradient centrifugation (45). Reverse transcription of the total RNA (1 µg) from platelets, CMK cells, or HepG2 cells and polymerase chain reaction (PCR) amplification of the cDNA were performed according to methods described previously (46). The 5'-sense primer (20-mer) and the 3'-antisense primer (20-mer) used for PCI cDNA amplification correspond to nucleotides 76-95 (ATGCAGCTCTTCCTCCTCTT) and 281-300 (GACACTCGTAGAGGTACTCG) of a human PCI cDNA sequence, respectively; these primers produced a 225-base pair fragment (3). An SmaI-cleaved PCI cDNA was used as a PCR control (3). Each cycle of PCR consisted of 2 min of denaturation at 94 °C, 1 min of annealing at 55 °C, and 3 min of polymerization at 72 °C. An aliquot of the PCR product was run on a 1% agarose gel, which was then stained with ethidium bromide.

Flow Cytometric Analysis-- For cytometric analysis of activated platelets, 100 µl of gel-filtered platelets (2.5 × 108/ml) suspended in Hepes-buffered saline, containing 2 mM CaCl2 and 0.1% BSA, was incubated with TRAP (100 µM final concentration) without stirring at 37 °C for 30 min. Platelets were then washed with the same buffer and incubated with FITC-labeled anti-PCI IgG (5 µg/ml) or with FITC-labeled anti-GPIb IgG in the same buffer containing 0.02% NaN3 for 1 h in the dark. After washing, platelet-bound anti-PCI IgG was analyzed using FACScan (Becton Dickinson) with a fluorescence-1 channel. For analysis of intracellular PCI in resting platelets, gel-filtered platelets were treated with PermeaFix in Hepes-buffered saline containing 0.1% BSA and 0.02% NaN3 for 45 min in the dark. After washing, the platelets were incubated with 5 µg/ml FITC-labeled anti-PCI IgG for 1 h in the dark. After appropriate washing, platelet-bound anti-PCI IgG was determined as described above.

Immunoelectron Microscopic Analysis-- Ultrathin sections of resting platelets or TRAP-stimulated platelets (fixed for 5 min after activation with 100 µM TRAP in Hepes-buffered saline containing 2 mM CaCl2) were prepared as follows. Platelets were fixed with 4% paraformaldehyde and 1% glutaraldehyde in 100 mM phosphate buffer, pH 7.5, at 4 °C for 3 h; after dehydrating, the blocks were embedded in L. R. White acrylic resin. The resin-embedded platelets were cut onto nickel 200-mesh grids. For labeling studies, the grids were incubated with 10 µg/ml anti-PCI IgG in 100 mM phosphate buffer overnight in a moist chamber and then conjugated with goat anti-mouse IgG to 15-nm colloidal gold for 1.5 h. After washing and drying the grids, they were stained with uranyl acetate and lead citrate. For controls, the sections were incubated with anti-GPIb IgG, anti-factor V IgG, or normal mouse IgG.

Assay for PCI-mediated Inhibition of APC in the Presence of Phospholipid Vesicles-- APC (42 nM final concentration) was incubated with PCI (90 or 180 nM final concentration) in 50 µl of Hepes-buffered saline containing 0.1% BSA and 2 mM CaCl2 in the presence of phospholipid vesicles (125 µg/ml) prepared with RBC or with various concentrations of PE, PS, and PC or in the presence of heparin (5 units/ml). After 10 min of incubation, the amidolytic activity of the residual APC was determined by adding 2 ml of 100 µM Boc-Leu-Ser-Thr-Arg-MCA in 50 mM Tris-HCl, pH 8.0, containing 100 mM CsCl and 2 mM CaCl2 to the reaction mixture. The release of 7-amino-4-methylcoumarin from the substrate by APC was determined using a fluorospectrophotometer (Shimadzu RF-510) with excitation at 380 nm and emission at 440 nm. Second order rate constants of the inhibition of APC by PCI were calculated according to the method of Cooper et al. (47) using the equation k'/[I] = (-lna)/t[I], where a is the residual protease activity in relation to an uninhibited control, t is the time, and [I] is the PCI concentration. Each reaction was performed twice in triplicate, and averages were calculated. Relative k'/[I] values were calculated by dividing the k'/[I] value in the presence of phospholipid vesicles by the value in the absence of phospholipids under the same assay conditions.

Assay for Binding of APC, PCI, or APC·PCI Complex to Solid Phase Phospholipids-- Assays for binding APC, PCI, or APC·PCI complex to solid phase phospholipids were carried out based on methods described previously (48). Microtiter plate wells were coated with RBC, PE, PS, or PC (200 µg/ml) or with a mixture of various PE:PC, PE:PS, or PS:PC ratios (0:100, 25:75, 50:50, 75:25 or 100:0) (200 µg/ml) in 100 µl of 100 mM NaHCO3 buffer, pH 9.3. After 2 h of incubation, the wells were blocked for 2 h with 5% BSA in 250 µl of 100 mM phosphate buffer, pH 7.5, containing 100 mM NaCl. After washing the wells with Hepes-buffered saline containing 0.1% BSA and 2 mM CaCl2, 100 µl of various concentrations of APC, PCI, or APC·PCI complex in the same buffer was added to the wells. After 2 h of incubation, the wells were washed, and well-bound APC, PCI, or APC·PCI complex was determined. Bound APC was determined using 300 µM S-2366 in 100 µl of 50 mM Tris-HCl, pH 8.0, containing 100 mM CsCl and 2 mM CaCl2 after incubating at 37 °C for 45 min. The release of p-nitroaniline from the substrate by APC was determined at 405 nm using a microplate reader. For the determination of bound PCI, 100 µl of peroxidase-coupled anti-PCI IgG in Tris-buffered saline was added to the wells and incubated for 2 h. After washing the wells, 100 µl of freshly prepared tetramethylbenzidine (0.1 mg/ml) and 0.005% H2O2 in 100 mM sodium acetate buffer, pH 5.5, was added to each well. The enzyme reaction was terminated after 15 min by adding 100 µl of 1 M sulfuric acid, and absorbance at 450 nm was determined using a microplate reader. For measuring bound APC·PCI complex, 100 µl of peroxidase-coupled anti-protein C IgG (MFC-9), which binds equally to both free APC and APC·PCI complex (37), was added to the wells and incubated for 2 h. After washing the wells, bound peroxidase-coupled anti-protein C IgG was determined as described above for bound PCI. The apparent dissociation constants (Kd values) of the binding of APC, PCI, or APC·PCI complex to solid phase phospholipids were determined by the double-reciprocal plot analysis as described previously (48). Each binding assay was performed three times in duplicate, and the average was calculated.

Assay for Inhibition of APC by PCI Bound to Solid Phase Phospholipids-- Microtiter plate wells were coated with RBC (200 µg/ml) or PE (80 µg/ml) in 100 µl of 100 mM NaHCO3 buffer, pH 9.3. After 2 h of incubation, the wells were blocked for 2 h with 5% BSA in 250 µl of 100 mM phosphate buffer, pH 7.5, containing 100 mM NaCl. After washing with Hepes-buffered saline containing 0.1% BSA and 2 mM CaCl2, 100 µl of various concentrations of PCI in the same buffer was added to each well and incubated for 2 h. The wells were washed three times with the same buffer, and then 100 µl of APC or 0.8 nM Gla-domainless APC dissolved in the same buffer was added to each well and incubated for 30 min. Thereafter, the residual amidolytic activity of APC was determined by adding to the wells 50 µl of 600 µM S-2366 in 150 mM Tris-HCl, pH 8.0, containing 300 mM CsCl and 6 mM CaCl2 and incubated at 37 °C for 2 h. The release of p-nitroaniline from the substrate by APC was determined at 405 nm using a microplate reader. The effect of the Gla fragment of protein C on the inhibition of APC by PCI bound to solid phase RBC or PE was examined as follows. 100 µl of a mixture of 0.8 nM APC and various concentrations of Gla fragment was added to RBC- or PE-coated wells pretreated with PCI. After incubation for 30 min, the residual amidolytic activity of APC was determined as described above.

Assay for Complex Formation of APC with PCI Bound to Solid Phase Phospholipids or Heparin-- RBC- or PE-coated microtiter plate wells were prepared as described above. Heparin-coated microtiter wells were prepared by incubating overnight 100 µl of 1 mg/ml heparin in 100 mM NaHCO3 buffer, pH 9.3, in the microtiter wells. The assay for complex formation of APC with PCI bound to solid phase phospholipids or heparin was performed as follows. 100 µl of 50 nM PCI in Hepes-buffered saline containing 0.1% BSA and 2 mM CaCl2 was added to the wells and incubated for 2 h. After washing three times, 100 µl of various concentrations of APC in the same buffer was added to each well and incubated for 30 min. To determine the formation of APC·PCI complex in the supernatant of the wells, aliquots of the supernatant were transferred to other microtiter plate wells coated with anti-PCI IgG and incubated for 2 h. After washing the wells with Tris-buffered saline containing 0.05% Tween 20, APC·PCI complex was determined by ELISA using peroxidase-coupled anti-protein C IgG and tetramethylbenzidine as described above. To determine the amount of APC·PCI complex bound to solid phase RBC, PE, or heparin, 100 µl of Tris-buffered saline containing 0.5% Triton X-100 was added to RBC-, PE-, or heparin-coated wells and incubated for 15 min with agitation. Then APC·PCI complex in solubilized samples was determined by ELISA as described above.

    RESULTS
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Procedures
Results
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References

Secretion of PCI by Activated Platelets-- PCI was detected in human platelets. The amount of PCI (mean ± S.D.) in gel-filtered platelets from healthy volunteers (n = 15, platelet counts: 23 ± 7 × 104 cells/µl blood) was approximately 2.9 ± 0.7 ng of PCI/1 × 109 platelets. In a preliminary examination, the amount of PCI contained in platelets decreased after treating them with thrombin or collagen, suggesting that PCI is secreted from platelets during its activation. To confirm this, different numbers of platelets suspended in Hepes-buffered saline were stimulated with thrombin, and the platelets were removed by centrifugation at 10,000 × g for 5 min. The amount of PCI in the supernatant of the incubation mixture containing resting platelets was scarce, whereas that found in the supernatant of thrombin-stimulated platelets increased significantly in proportion to the number of platelets. Secretion of PCI by platelets was also observed when they were stimulated with TRAP or with other platelet activators such as ADP, collagen, or adrenalin. Table I shows the amount of PCI secreted by platelets after stimulation with several activators. The amount of PCI secreted by stimulated platelets increased 1.5-2 fold in the presence of Ca2+. Although not all PCI was secreted by platelets after being stimulated with their activators, 24~32% of the total amount of PCI in platelets was secreted upon stimulation.

                              
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Table I
Secretion of PCI from platelets after stimulation with various activators

PCI and Its mRNA Expression in CMK Cells-- PCI was also detected in the cell lysate of megakaryocytic CMK cells (1.5 ng of PCI/106 cells). Nested PCR revealed that platelets, CMK cells, and HepG2 cells express mRNA fragments of the same size (225 base pairs) corresponding to -19 to +56 residues of the NH2-terminal region of PCI precursor (data not shown). These results suggest that megakaryocytes express PCI mRNA, synthesize PCI, and that this is stored in platelets.

Flow Cytometric Localization of PCI in Resting or Activated Platelets-- To localize PCI in resting platelets, flow cytometric analysis was carried out using FITC-labeled anti-PCI IgG or anti-GPIb IgG as a control. As shown in Fig. 1A, FITC-labeled anti-GPIb IgG specifically bound to resting platelets (white peak); however, the binding of FITC-labeled anti-PCI IgG was nonspecific (black peak). Resting platelets were then fixed with PermeaFix, which enhances permeability of extracellular high molecular weight materials (e.g. immunoglobulins) and subjected to flow cytometric analysis. This study showed that PermeaFix-treated platelets were markedly reactive to FITC-labeled anti-PCI IgG (data not shown), suggesting that PCI is located only in the intracellular space of resting platelets.


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Fig. 1.   Flow cytometric localization of PCI secreted from platelets after activation with TRAP. Panel A, detection of GPIb (white peak) but not PCI (black peak) on the surface of resting platelets. Panel B, detection of PCI (black peak) and GPIb (white peak) on the surface of TRAP-activated platelets. Panel C, detection of PCI (black peak) and GPIb (white peak) on microvesicles derived from TRAP-activated platelets. Platelet-derived phospholipid microvesicles were determined according to a method reported previously (49).

To localize PCI in activated platelets, TRAP-stimulated platelets were subjected to flow cytometric analysis. Analysis of the distribution of cellular size showed that TRAP-treated platelets, in addition to the area occupied by activated platelets, also distributed in an area containing much smaller sized particles (this would be recognized as phospholipid membrane-derived microvesicles) (data not shown) (49). The area of resting platelets, TRAP-stimulated platelets, or the area containing microvesicles released from stimulated platelets was analyzed by flow cytometry using FITC-labeled anti-PCI IgG or anti-GPIb IgG as a control. Both FITC-labeled anti-PCI IgG and anti-GPIb IgG specifically bound to the surface of TRAP-stimulated platelets (Fig. 1B) and to platelet-derived microvesicles (Fig. 1C). These data indicate that PCI secreted by activated platelets is present on the surface of platelets, platelet-derived microvesicles, and/or in the solution phase.

Immunoelectron Microscopic Localization of PCI in Resting and Activated Platelets-- To define the precise location of PCI in resting and activated platelets, immunoelectron microscopic analysis of thin sections of these platelets was performed. As shown in Fig. 2A, most of the PCI was found within the alpha -granules of resting platelets. Quantitative analysis showed that 374 of 442 gold particles were located within the alpha -granules (84.6% of total intracellular PCI). This count was almost the same as that of factor V (267 of 330 gold particles; 80.9% of total intracellular factor V), which was reported to be located within the alpha -granules (42). On the other hand, after treatment with TRAP, PCI was located on the external plasma membrane (Fig. 2B) and partly within the surface-connected cannalicular system of platelets (Fig. 2C). Factor V was also detected on the platelet membrane and in the cannalicular system of activated platelets (data not shown), as reported previously (42). Labeling of alpha -granules in resting platelets and that of the plasma membrane or surface-connected cannalicular system in activated platelets was not significant in negative controls using nonspecific mouse IgG.


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Fig. 2.   Immunogold labeling of PCI in ultrathin sections of resting and TRAP-activated platelets. The scale is shown in each panel. Panel A, in resting platelets labeled using monoclonal murine anti-PCI IgG and goat anti-mouse IgG conjugated to 15-nm colloidal gold. PCI was located within the alpha -granules. Panel B, in TRAP-activated platelets, PCI was partly located on the surface of plasma membrane and/or phospholipid microvesicles released from the platelets. Panel C, in platelets treated with a low concentration of TRAP, PCI was partly located within the surface-connected cannalicular system of platelets.

Complex Formation of Platelet PCI with APC-- To characterize whether PCI secreted from stimulated platelets has the ability to inhibit APC, APC·PCI complex formation was determined in platelet suspension before or after treatment with 100 µM TRAP in the presence of 16 nM APC (final concentration). As shown in Fig. 3, although APC·PCI complex was scarcely formed in a solution containing resting platelets, significant and time-dependent complex formation was observed in a solution containing platelets stimulated with TRAP.


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Fig. 3.   Complex formation of PCI secreted from platelets and exogenous APC after treatment with TRAP. Gel-filtered platelets (6 × 108 cells) in 1 ml of Hepes-buffered saline containing 2 mM CaCl2 and APC (16 nM final concentration) were activated with TRAP (100 µM final concentration) at room temperature. At intervals, aliquots of 100 µl were removed, and to these 1 µl of 1 M diisopropyl fluorophosphate (10 mM final concentration) was added. After solubilization of the platelets in 200 µl of Tris-buffered saline containing 0.5% Triton X-100, the concentration of APC·PCI complex in the samples was determined by ELISA specific for the complex. black-triangle, resting platelets; bullet , TRAP-stimulated platelets.

Inhibition of APC by PCI in the Presence of Phospholipid Vesicles-- Table II shows the second order rate constants of the inhibition of APC by PCI in the presence of phospholipid vesicles (RBC or different ratios of PE, PS, and PC) or heparin. The relative k'/[I] value for inhibition of APC by PCI increased in proportion to the increase of PE ratio in phospholipid vesicles (7-fold increase in the presence of 40% PE in the phospholipids compared with buffer alone), and the second order rate constant of APC inhibition by PCI in the presence of 40% PE in phospholipid vesicles was the same as that observed in the presence of phospholipid vesicles prepared using RBC. Under the same assay conditions, heparin (5 units/ml) increased about 32-fold the relative k'/[I] values.

                              
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Table II
Second order rate constant of inhibition of APC by PCI in the presence or absence of phospholipid vesicles or heparin
The second order rate constants were determined as described under "Experimental Procedures." Relative k'/[I] values were obtained by dividing the k'/[I] value in the presence of phospholipid vesicles by the value in the absence of phospholipid vesicles under the same assay conditions.

Binding of APC and PCI to Solid Phase Phospholipids-- Binding of APC and PCI to solid phase RBC, PE, PS, or PC (this only for PCI) was saturable (data not shown), and then the Kd values were determined. Binding of APC to PC was not saturable, thus its Kd value could not be obtained. As shown in Table III, APC bound more preferably to PE than to RBC and PS. On the other hand, PCI bound more preferably to RBC or PE than to PS or PC.

                              
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Table III
Apparent dissociation constants (Kd values) of the binding of APC and PCI to solid phase RBC, PE, PS, or PC
The amount of APC bound to solid phase phospholipids was determined using S-2366. The amount of PCI bound to solid phase phospholipids was determined using peroxidase-coupled monoclonal anti-PCI IgG and tetramethylbenzidine. The Kd values of the binding of APC or PCI to solid phase phospholipids were determined by the double-reciprocal plot analysis as described previously (48). Details are noted under "Experimental Procedures." NO, not obtained.

The effect of different ratios (w/w) of PE:PC, PE:PS, or PS:PC with constant phospholipid concentrations (200 µg/ml, used to coat on microplate wells) on PCI binding (5 nM final concentration) was also determined. As shown in Fig. 4, binding of PCI to solid phase phospholipids depended on the presence of PE. 75% of PE in PE:PS or PE:PC mixtures was the most optimal concentration for maximal PCI binding to phospholipids.


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Fig. 4.   Binding of PCI to phospholipid vesicles containing various ratios of PE:PC (bullet ), PE:PS (open circle ), or PS:PC (black-triangle) coated to microwells. The concentration of phospholipids used for fixation was 200 µg/ml. The abscissa indicates the percent of phospholipid ratios (w/w) of PE/(PE + PC), PE/(PE + PS), or PS/(PS + PC). Bound PCI was determined using peroxidase-coupled monoclonal anti-PCI IgG and tetramethylbenzidine.

Inhibition of APC by Phospholipid-bound PCI-- To investigate further the mechanism of APC inhibition by PCI, inhibition of the amidolytic activity of APC or Gla-domainless APC by solid phase phospholipid-bound PCI was determined. APC was inhibited dose dependently by RBC- or PE-bound PCI, but Gla-domainless APC was inhibited only scarcely by RBC-bound PCI (data not shown). Moreover, the Gla fragment of protein C (0.1~0.8 µM) suppressed dose dependently the inhibition of APC (0.8 nM final concentration) by RBC- or PE-coated microwells pretreated with 50 nM PCI (data not shown). This finding suggests that the (phospholipid-bound) Gla domain of APC is required for APC inhibition by PCI bound to phospholipid surface.

Release of APC·PCI Complex Bound to Solid Phase Phospholipids or Heparin-- To clarify whether the APC·PCI complex is released from or remained bound to phospholipid vesicles, the concentration of the complex in the supernatant of the binding assay of APC to PCI bound to solid phase RBC or PE and also the amount of the complex bound to solid phase RBC or PE were determined. As shown in Fig. 5, the concentration of APC·PCI complex in the supernatant increased in proportion to the increased concentrations of exogenous APC, whereas the amount of APC·PCI complex bound to solid phase RBC or PE increased only a little. About 80% of the total amount of APC·PCI complex was released from solid phase RBC or PE after the addition of APC (16 nM final concentration). Similar results were observed when APC was incubated with PCI bound to heparin-coated microplate wells; in this case, approximately 85% of the total amount of the complex was released from the solid phase heparin (data not shown). These findings suggest that most of the APC·PCI complex formed on phospholipid vesicles (mainly on the PE component) is released from the phospholipid surface. The Kd values of the binding of APC·PCI complex to RBC or PE were 3.1 × 10-8 M and 2.4 × 10-8 M, respectively (data not shown). These values were between the Kd values of the binding of PCI and APC to solid phase RBC or PE (Table III), suggesting that phospholipid-bound PCI is released from the phospholipid surface after forming a complex with APC.


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Fig. 5.   Changes in the concentration of APC·PCI complex in the supernatant and the amount of APC·PCI complex bound to solid phase RBC or PE. PCI bound to solid phase RBC or PE in microwells was incubated with various concentrations of exogenous APC, and then the concentration of APC·PCI complex in the supernatant and the amount of the complex bound to solid phase RBC or PE were determined as described under "Experimental Procedures." bullet , APC·PCI complex in the supernatant released from solid phase RBC; open circle , that bound to solid phase RBC. black-triangle, APC·PCI complex in the supernatant released from solid phase PE; triangle , that bound to solid phase PE.

    DISCUSSION
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Abstract
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Procedures
Results
Discussion
References

We have demonstrated previously the presence of thrombomodulin on platelet surfaces and that this thrombomodulin enhances efficiently the thrombin-catalyzed activation of protein C with the same specific activity as that expressed on endothelial cells (29). APC generated on platelets interacts with protein S on the surface of platelets, inactivates factors Va and VIIIa efficiently (28), and thus inhibits the formation of prothrombinase and tenase complexes (50, 51). However, platelets play a critical role in the process of hemostasis. To evaluate whether platelets themselves also regulate their anticoagulant activity, in the present study we assessed the expression and functional characterization of PCI in platelets. Our results showed that PCI is stored in and secreted from the alpha -granules of platelets and that platelet membrane- and phospholipid microvesicle-bound PCI inhibits efficiently APC. Overall, these findings suggest that platelet PCI modulates the anticoagulant activity of the protein C pathway.

Approximately 30% of total PCI was secreted from the alpha -granules of platelets after stimulation with several activators, and this PCI secretion was enhanced in the presence of Ca2+. These findings suggest that the release of PCI from platelets occurs in a fashion similar to that of the C1 inhibitor (30) and factor IX (22) from platelets. Immunoelectron microscopic analysis showed that secreted PCI binds to phospholipid microvesicles, to platelet membrane, and to the surface-connected cannalicular system.

The PE component of these phospholipid vesicles was found to be essential for enhancing the PCI inhibition of APC. It was found that APC binds more preferably to PE than to RBC, PS, or PC, and PCI to PE or RBC than to PS or PC. The results obtained in the binding of APC and PCI to solid phase phospholipids containing different ratios of PE, PS, and PC suggest that the bindings of both PCI and APC depend on the presence of PE. Assays on the PCI-mediated inhibition of APC on the surface of solid phase PE or PE-containing RBC showed that the binding of Gla domain of APC and of PCI to phospholipids is required for efficient inhibition of APC by PCI.

Recent studies demonstrated that PE enhances the PS-mediated sensitivity of factor VIIa-tissue factor activity (52) and induces high affinity binding sites for factor VIII on surfaces containing physiologic mole fractions of PS (53), although it exerts almost no influence on prothrombin activation (54). PE has been also reported to regulate the activation of blood coagulation by enhancing the thrombin-thrombomodulin complex-mediated protein C activation (55) and the anticoagulant activity of APC (56). Moreover, PE has been shown to enhance the inhibition of APC by anti-phospholipid antibodies (57). In addition to these facts, the results of the current investigation suggest that the PE component of phospholipids enhances the APC inhibition by PCI. Because PE, in addition to PS, is the major phospholipid component (nearly 40%) of the outer leaflet of activated platelet membrane, PCI probably plays an important role in vivo in the inhibition of APC generated on the surface of activated platelets or platelet-derived phospholipid microvesicles.

Moreover, in the current investigation we found that most of the APC·PCI complex formed on the PE component of the phospholipid membrane is released from this surface. The complex formed on solid phase heparin was also released from heparin. The Kd values for the binding of APC, PCI, or APC·PCI complex to solid phase PE or RBC support the idea that phospholipid-bound PCI is released from the phospholipid surface after forming a complex with APC. It is conceivable that part of the APC·PCI complex which increases in the plasma of patients with intravascular coagulation, such as thrombotic thrombocytopenic purpura (58), disseminated intravascular coagulation (58), acute myocardial infarction (59), and in patients positive for lupus anticoagulant (44), may derive from the complex formed on activated platelet membrane and microvesicles. 1 ml of a normal subject blood contains approximately 2.2 µg of plasma PCI, 1 ng of platelet PCI, and below 0.5 ng of plasma APC·PCI complex. The level of APC·PCI complex increases 10-20-fold in the plasma of patients with intravascular coagulation (58). This remarkable increase in the plasma level of APC·PCI complex suggests that the PCI secreted from platelet may also contribute to the total pool of circulating APC·PCI complex in patients with hypercoagulable states. The PCI levels of gel-filtered platelets from healthy volunteers (n = 15) and those of patients (n = 25) with severe disseminated intravascular coagulation were 2.9 ± 0.7 ng/109 platelets and 0.74 ± 0.17 ng/109 platelets, respectively.2 This suggests that PCI secreted from activated platelets is being used actively during the activation of the blood coagulation system, which is commonly associated with increased APC generation. In conclusion, platelet PCI may play important roles in the down-regulation of the protein C pathway probably by inhibiting thrombin-thrombomodulin complex-mediated protein C activation on platelet surface and also by neutralizing APC through APC·PCI complex formation at sites of platelet activation-associated thrombus formation.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Esteban C. Gabazza and Mariko Ohara for helpful comments.

    FOOTNOTES

* This study was supported by Grants-in-aid 05454622, 06282225, and 06771216 for Scientific Research from the Ministry of Education, Science, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-59-231-5036; Fax: 81-59-231-5209; E-mail: suzuki{at}doc.medic.mie-u.ac.jp.

1 The abbreviations used are: PCI, protein C inhibitor; APC, activated protein C; BSA, bovine serum albumin; RBC, rabbit brain cephalin; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PS, phosphatidylserine; GP, glycoprotein; FITC, fluorescein isothiocyanate; Boc, t-butoxycarbonyl; MCA, 7-amino-4-methylcoumaryl amide; TRAP, thrombin-receptor-activating peptide; Gla, gamma -carboxyglutamic acid; ELISA, enzyme-linked immunosorbent assay; PCR, polymerase chain reaction.

2 J. Nishioka, Ma. Ning, T. Hayashi, and K. Suzuki, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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