Hypercysteinemia promotes atherosclerosis by reducing protein S-nitrosylation.

Protein S-nitrosylation plays important role in the regulation of cardiovascular functions in nitric oxide (NO) Pathway. Hypercysteinemia (HHcy) is an independently risk factor for atherosclerosis. We hypothesized that HHcy promotes atherosclerosis by reducing level of vascular protein S-nitrosylation. The aim of present study is to investigate effect of HHcy on vascular protein S-nitrosylation. A total of 45 male apoE-/- mice were randomly divided into three groups. The control group was fed a Western-type diet. The HHcy group was fed a diet containing 4.4% L-methionine, and the HHcy+NONOate group was fed a diet containing 4.4% L-methionine and administrated NONOate (ip). Human umbilical vein endothelial cells were performed for in vitro experiment. Plasma lipids were measured every 4 weeks. After 12 weeks, aortic atherosclerotic lesion areas were detected as well as cellular components. The levels of plasma homocysteine (Hcy) and NO were measured. S-nitrosylation was detected using immunofluorescence, and further confirmed by biotin switch method. We found that compared with the control group, Hcy levels, and atherosclerotic plaque, and content of vascular smooth muscle cells and macrophages in lesions significantly increased, and levels of NO significantly decreased in the HHcy group. However, NONOate reverses this effect. In addition, Hcy significantly reduced protein S-nitrosylation in human umbilical vein endothelial cells. This reduction of protein S-nitrosylation was accompanied by reduced levels of NO. Our results suggested that Hcy promoted atherosclerosis by inhibiting vascular protein S-nitrosylation.

Acquired protein S deficiency leads to obliterative portal venopathy and to compensatory nodular regenerative hyperplasia in HIV-infected patients.

OBJECTIVE: To identify the mechanism of nodular regenerative hyperplasia in HIV-infected patients. DESIGN: Case-control study. SETTING: The hepatology and the infectious disease units of two tertiary care centers in France. PATIENTS: We compared 13 consecutive HIV-positive patients with unexplained nodular regenerative hyperplasia to 16 consecutive HIV-positive patients without nodular regenerative hyperplasia, to eight HIV-negative patients with nodular regenerative hyperplasia from an identified cause and to 10 anonymous healthy blood donors. MAIN OUTCOME MEASURE: Patients and controls were screened for diminished protein S activity and antiprotein S immunoglobulin G (IgG) antibodies. The antiprotein S activity of purified IgG from patients and controls was assessed in a functional test of activation of protein C in which protein S serves as a cofactor. A full liver CT portography was realized on the liver explant of a case patient. RESULTS: The CT portography disclosed diffuse obliterative portal venopathy. Levels of protein S activity were lower among patients with HIV-associated nodular regenerative hyperplasia when compared with HIV-positive patients without nodular regenerative hyperplasia and when compared with HIV-negative patients with nodular regenerative hyperplasia (P < 0.005 for all comparisons). HIV-positive patients with nodular regenerative hyperplasia had significantly higher levels of antiprotein S IgG than HIV-positive patients without nodular regenerative hyperplasia and healthy controls. Purified IgG from patients with HIV-associated nodular regenerative hyperplasia specifically inhibited the protein S-dependent protein C activation. CONCLUSION: Acquired autoimmune protein S paucity and secondary thrombophilia appear to be causes of obliterative portal venopathy and compensatory nodular regenerative hyperplasia in HIV-positive patients.

Effect of phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine on the protein C/protein S anticoagulation system.

Phosphatidylserine is known to significantly accelerate the blood coagulation reaction. In a previous communication submitted for publication, we demonstrated that phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine showed effects on the blood coagulation reaction using the factor Xa-prothrombin reaction system, and discuss a new function of membrane phospholipids. The present study examined the role of phospholipids in the blood coagulation regulatory reaction (anticoagulation system), by studying the effects of phospholipids on the protein C/protein S reaction. We have established quantitative methods for measuring activated protein C activity and protein S activity, and used them to measure their activity after the addition of liposomes with different phospholipid compositions. We found that phosphatidylcholine inhibited activated protein C and protein S activities in a dose-dependent manner, as in the factor Xa-prothrombin reaction system. On the other hand, phosphatidylethanolamine and lysophosphatidylcholine showed no effect on activated protein C activity. Phosphatidylethanolamine inhibited and lysophosphatidylcholine accelerated coagulation activity in the factor Xa-prothrombin system, but such effects were not observed in the protein C/protein S reaction system. The coagulation and anticoagulation reactions are exquisitely balanced by thrombin, with a role both as a procoagulant and anticoagulant. Therefore, it is understandable that phosphatidylethanolamine and lysophosphatidylcholine show different effects in the factor Xa-prothrombin and protein C/protein S reaction systems. It appears that coagulation and anticoagulation reactions are co-ordinated and controlled by changes in phospholipid composition of the cellular membrane where the coagulation reaction takes place.

Study of protein C, protein S, and antithrombin III in hypoxic newborns.

OBJECTIVE: The aim of this study was to clarify the effect of hypoxia on the physiologic inhibition system of coagulation including protein S, protein C, and antithrombin III and to study their effect on thromboembolic accidents of hypoxic newborns. DESIGN: Clinical study including ten hypoxic-ischemic neonates and ten normal neonates as a control group. DATA SOURCES: MEDLINE, pediatric textbooks, neonatal intensive care unit, Department of Paediatrics, Faculty of Medicine, Cairo University. RESULTS: The results of this study revealed a marked decrease in the level of the physiologic inhibition system of coagulation including antithrombin III, protein C, and protein S in 100% of the hypoxic-ischemic neonates compared with the control group (p <.001) before the occurrence of thromboembolic complications. Fifty percent of the hypoxic-ischemic neonates developed disseminated intravascular coagulation and died, 40% developed necrotizing enterocolitis and rectal bleeding, 20% developed hematuria, 30% developed hematemesis, 20% developed intracranial hemorrhage, and 100% had convulsions. CONCLUSIONS: In this study, we evaluated the effect of asphyxia on the physiologic inhibition system of coagulation in neonates. Care providers should suspect hypoxia resulting from any obstructed labor and perform the necessary laboratory investigations for coagulation, including antithrombin III, protein C, and protein S levels, to help prevent thromboembolic accidents in asphyxiated neonates, including disseminated intravascular coagulation, necrotizing enterocolitis, and intracranial hemorrhage. Based on the development of antithrombin III and protein C concentrates, which are commercially available, require minimal monitoring, and have very few side effects, the time is ripe for evaluation of optimal treatment for thromboembolic accidents after neonatal asphyxia. This could be even more important if successful neuroprotectant strategies are also developed.

Vitamin K-dependent protein S localizing complement regulator C4b-binding protein to the surface of apoptotic cells.

Apoptosis is characterized by a lack of inflammatory reaction in surrounding tissues, suggesting local control of complement activation. During the initial stage of apoptosis, cells expose negatively charged phospholipid phosphatidylserine on their surfaces. The vitamin K-dependent protein S has a high affinity for this type of phospholipid. In human plasma, 60-70% of protein S circulates in complex with C4b-binding protein (C4BP). The reason why protein S and C4BP form a high-affinity complex in plasma is not known. However, C4BP is an important regulator of the classical pathway of the complement system where it acts as a cofactor in degradation of complement protein C4b. Using Jurkat cells as a model system for apoptosis, we now show protein S to bind to apoptotic cells. We further demonstrate protein S-mediated binding of C4BP to apoptotic cells. Binding of the C4BP-protein S complex to apoptotic cells was calcium-dependent and could be blocked with Abs directed against the phospholipid-binding domain in protein S. Annexin V, which binds to exposed phosphatidylserine on the apoptotic cell surface, could inhibit the binding of protein S. The C4BP that was bound via protein S to the apoptotic cells was able to interact with the complement protein C4b, supporting a physiological role of the C4BP/protein S complex in regulation of complement on the surface of apoptotic cells.

Alpha 2-macroglobulin enhances prothrombin activation and thrombin potential by inhibiting the anticoagulant protein C/protein S system in cord and adult plasma.

Protein S (PS) is a vitamin K-dependent plasma protein and serves as a cofactor for the anticoagulant activities of activated protein C (APC). We investigated the effects of different PS concentrations on prothrombin activation and thrombin generation in cord and adult plasma containing APC and different amounts of alpha 2-macroglobulin (a2-M). Prothrombin activation was assessed by monitoring the time-course of prothrombin fragment 1+2 (F1+2) generation. Thrombin generation curves were determined by means of a subsampling technique using the chromogenic substrate S-2238. We demonstrate a dose-dependent inhibition of the anticoagulant action of PS by a2-M: suppression of F1+2 and thrombin generation due to addition of PS was stronger in plasma containing low amounts of a2-M than in plasma with elevated a2-M levels. Since no complex formation between a2-M and PS was observed by means of SDS-PAGE, we attribute decreased anticoagulant action of PS at high a2-M levels to enhanced complex formation between APC and a2-M. Thereby, APC is subtracted from its cofactor PS, resulting in suppressed formation of the anticoagulant APC/PS complex. Thus, our data suggest that a2-M, besides its well-known anticoagulant effects, also acts as a procoagulant by suppressing the formation of the anticoagulant APC/PS complex. Our findings have implications particularly on thrombin generation and inhibition in cord plasma, since a2-M levels in newborns are elevated over adult values and the antithrombotic APC/PS pathway is up-regulated at birth. Therefore, elevated levels of a2-M might restrict the up-regulation of the APC/PS pathway.

C4b-binding protein inhibits the factor V-dependent but not the factor V-independent cofactor activity of protein S in the activated protein C-mediated inactivation of factor VIIIa.

Activated protein C (APC) is an important inactivator of coagulation factors Va and VIIIa. In the inactivation of factors Va and VIIIa, protein S serves as a cofactor to APC. Protein S can bind to C4b-binding protein (C4BP), and thereby loses its cofactor activity to APC. By modulating free protein S levels, C4BP is an important regulator of protein S cofactor activity. In the factor VIIIa inactivation, protein S and factor V act as synergistic cofactors to APC. We investigated the effect of C4BP on both the factor V-independent and factor V-dependent cofactor activity of protein S in the factor VIIIa inactivation using a purified system. Protein S increased the APC-mediated inactivation of factor VIIIa to 60% and in synergy with protein S, factor V at equimolar concentrations increased this effect further to 90%. The protein S/factor V synergistic effect was inhibited by preincubation of protein S and factor V with a four-fold molar excess of C4BP. However, C4BP did not inhibit the factor V-independent protein S cofactor activity in the purified system whereas it inhibited the cofactor activity in plasma. We conclude that C4BP-bound protein S retains its cofactor activity to APC in the factor VIIIa inactivation.

The interaction between anticoagulant protein S and complement regulatory C4b-binding protein (C4BP).

An important mechanism of regulation of blood coagulation is the anticoagulant protein C pathway. In this pathway, the anticoagulant activity of activated protein C is increased by its cofactor protein S. The cofactor activity of protein S can be regulated by binding to complement regulatory C4b-binding protein (C4BP). The sites of interaction of protein S and C4BP are discussed.

A plasma coagulation assay for an activated protein C-independent anticoagulant activity of protein S.

To study the physiological importance of the activated protein C (APC)-independent anticoagulant activity of protein S, we developed an assay specific for this activity. The ability of protein S to prolong the clotting time in an APC-independent way was expressed as the ratio of the clotting time in a plasma sample divided by the clotting time in the presence of a polyclonal antibody against human protein S (both after 1:1 dilution in protein S-C4BP deficient plasma). The mean protein S-dependent anticoagulant ratio (PSdAR) was 1.53+/-0.18 in 34 healthy controls, and was significantly lower in 16 heterozygous protein S deficient patients (PSdAR=1.15+/-0.09). In plasmas from patients under oral anticoagulant therapy the mean PSdAR was within the range of the control population (1.50+/-0.18). The mean total protein S antigen level in these plasmas was 58%, suggesting a higher specific APC-independent anticoagulant activity of protein S in these patients than in normals. This functional protein S assay can be used for the laboratory diagnosis of protein S deficiency, and to study the mechanism of the APC-independent anticoagulant activity in plasma.

Human protein S cleavage and inactivation by coagulation factor Xa.

Human factor Xa specifically cleaves the anticoagulant protein S within the thrombin-sensitive domain. Amino-terminal amino acid sequencing of the heavy chain cleavage product indicates cleavage of protein S by factor Xa at Arg60, a site that is distinct from those utilized by alpha-thrombin. Cleavage by factor Xa is unaffected by the presence of hirudin and is completely blocked by tick-anticoagulant-peptide and D-Glu-Gly-Arg-chloromethyl ketone, the latter two being specific inhibitors of factor Xa. The cleavage requires the presence of phospholipid and Ca2+, and is markedly inhibited by the presence of factor Va. Factor Xa-cleaved protein S no longer possesses its activated protein C-dependent or -independent anticoagulant activity, as measured in a factor VIII-based activated partial thromboplastin time clot assay. The apparent binding constant for protein S binding to phospholipid (Kd approximately 4 nM +/- 1.0) is unaffected by factor Xa or thrombin cleavage, suggesting that the loss of anticoagulant activity resulting from cleavage is not primarily due to the loss of membrane binding ability. Cleavage and inactivation of protein S by factor Xa may be an additional way in which factor Xa exerts its procoagulant effect, after the initial stages of clot formation.

Stimulation of Sky tyrosine phosphorylation by bovine protein S--domains involved in the receptor-ligand interaction.

Protein S is an anticoagulant vitamin-K-dependent plasma glycoprotein, which acts as a cofactor to activated protein C in the degradation of coagulation factors Va and VIIIa. It has been proposed that protein S has an additional function as a growth factor. Protein S and a structurally similar protein, Gas6, have been found to stimulate members of the Axl/Sky family of receptor tyrosine kinases. Human Gas6 is able to activate Axl and Sky. In contrast, while bovine protein S activates human Sky and its murine homologue, human protein S activates murine Sky but not the human receptor. In the present investigation, we studied the structural background of this species difference. Using protein S chimeras with domains from human and bovine origin, we found that only those chimeras with the steroid-hormone-binding globulin-like (SHBG) region from bovine protein S activate human Sky, indicating that the SHBG region is essential for the interaction. This observation was confirmed by inhibition of Sky phosphorylation by C4b-binding protein, a plasma protein that interacts tightly with the SHBG region of protein S. Another chimeric molecule, composed of the N-terminal 4-carboxyglutamic-acid-containing domain (Gla domain) and the two epidermal-growth-factor-like domains of human factor IX, and the SHBG region of bovine protein S, stimulated the receptor less efficiently. Antibodies directed against the Gla domain of protein S, inhibited the activation of human Sky by bovine protein S. These results indicate that the N-terminal domains of protein S are not essential for activation of the receptor, but contribute to the affinity of the interaction. Our data suggest that protein S might be a ligand of Sky in some species despite the lack of activity of human protein S on human Sky. The bovine/human protein S species difference will be a useful model to establish the structural requirements for the interaction between Sky and its ligands.

Binding sites for blood coagulation factor Xa and protein S involving residues 493-506 in factor Va.

Inactivation due to cleavage of Factor Va (FVa) at Arg 506 by activated protein C (APC) helps to downregulate blood coagulation. To identify potential functional roles of amino acids near Arg 506, synthetic overlapping pentadecapeptides comprising FVa heavy chain residues 481-525 were tested for their ability to inhibit prothrombin activation by prothrombinase complexes [Factor Xa (FXa):FVa:phospholipids:Ca2+]. The most potent inhibition was observed for peptide VP493 (residues 493-506), with 50% inhibition at 2.5 microM. VP493 also inhibited FXa in plasma in FXa-1-stage clotting assays by 50% at 3 microM. When the C-terminal carboxamide group of VP493 was replaced by a carboxyl group, most prothrombinase inhibitory activity was lost. VP493 preincubated with FXa inhibited prothrombinase with a pattern of mixed inhibition. Homologous peptides from Factor VIII sequences did not inhibit prothrombinase. Affinity-purified antibodies to VP493 inhibited prothrombinase activity and prolonged FXa-1-stage clotting times. VP493 also blocked the ability of protein S to inhibit prothrombinase independently of APC. Immobilized VP493 bound specifically with similar affinity to both FXa and protein S (Kd approximately 40 nM), but did not measurably bind prothrombin or APC. These studies suggest that FVa residues 493-506 contribute to binding sites for both FXa and protein S, providing a rationale for the ability of protein S to negate the protective effect of FXa toward APC cleavage of FVa. Possible loss of this FVa binding site for FXa due to cleavage at Arg 506 by APC may help explain why this cleavage causes 40% decrease in FVa activity and facilitates inactivation of FVa.

Acquired free protein S deficiency associated with multiple myeloma: a case report.

Investigation of recurrent venous thromboembolic events in a 46-year-old man with progressive IgG kappa (total serum IgG, 74.3 mg/ml) multiple myeloma revealed profound reductions in free protein S (PS) antigen (<0.l U/ml) and PS activity (0.33 U/ml). Total PS antigen, protein C, antithrombin III, and C4b-binding protein levels were within normal limits. The patient had no family history suggestive of a congenital PS deficiency and no history of thrombosis predating the diagnosis of his plasma cell dyscrasia. Patient IgG was isolated from serum using a protein A-sepharose affinity column and characterized. PS-dependent clotting assays (Staclot Protein S, Diagnostica Stago, Asnieres sur-Seine, France) performed on normal pooled plasma mixed with dilutions of patient IgG (0.0-33.0 mg/ml) revealed a dose-dependent neutralization of PS activity by 43%. Total and free PS antigen levels were measured using Laurell rocket electroimmunodiffusion (Assera-Plate Protein S, Diagnostica Stago), which revealed a similar dose-dependent reduction in free PS antigen but preserved normal total PS antigen. Free PS antigen was reduced by 77% to 0.23 U/ml using an IgG concentration (16.5 mg/ml) less than one-fourth of that of the patient at time of serum collection. Specific binding of the patient IgG to commercially available purified human PS was demonstrated by Western immunoblot analysis. Whereas acquired free PS deficiency has been previously reported in association with nephrotic syndrome, inflammatory bowel disease, HIV infection, and varicella infection, this is the first reported case of a hypercoagulable syndrome associated with acquired free PS deficiency and multiple myeloma.

Increased prothrombin activation in protein S-deficient plasma under flow conditions on endothelial cell matrix: an independent anticoagulant function of protein S in plasma.

Protein S is a vitamin K-dependent nonenzymatic coagulation factor involved in the regulation of activated protein C (aPC). In this study, we report an aPC-independent anticoagulant function of protein S in plasma under flow conditions. Plasma, anticoagulated with low-molecular-weight heparin allowing tissue factor-dependent prothrombin activation, was perfused at a wall shear rate of 100 s-1 over tissue factor containing matrices of stimulated endothelial cells placed in a perfusion chamber. Fractions were collected in time at the outlet and prothrombin activation was determined by measuring the activation fragment F1+2 of prothrombin. In normal plasma, a time-dependent prothrombin activation was detected by the generation of fragment1+2. Prothrombin activation had ceased after 12 minutes perfusion, independent of the amount of tissue factor present in the matrix. Depletion of protein S from plasma or inhibition of protein S in plasma by monoclonal antibodies induced a 5- to 25-fold increase of prothrombin activation on the procoagulant endothelial cell matrix. A prolonged prothrombin activation was detected in protein S-depleted plasma up to 20 minutes after onset of the thrombin generation. The increased prothrombin activation in protein S-depleted plasma could not be explained by the absence of the cofactor function of protein S for aPC because depletion of protein C from plasma did not result in increased prothrombin activation. These data provide further evidence for a strong anticoagulant function of protein S in plasma independent from activated protein C.

Identification of residues 413-433 of plasma protein S as essential for binding to C4b-binding protein.

Reversible association of protein S with C4b-binding protein (C4BP) in plasma down-regulates protein S activity, since free protein S but not the protein S.C4BP complex is an anticoagulant cofactor for activated protein C. To identify regions on the surface of protein S that mediate complex formation with C4BP, a number of nonoverlapping synthetic pentadecapeptides comprising protein S sequences were prepared and tested for their ability to inhibit complex formation. The most potent pentadecapeptide, residues 420-434 (PSP-420) (SGIKEIIQEKQNKHC), gave half-maximal effect at 20 microM. A peptide with the reverse sequence, 434-420, did not inhibit. A peptide containing the sequence of protein S residues 408-434 inhibited complex formation by > 95% with 50% inhibition at 5 microM peptide. Biotinylated C4BP bound specifically to plates coated with PSP-420 but not with the 434-420 peptide; and biotinylated PSP-420 bound to plates coated with C4BP. Rabbit antibodies were raised against several keyhole limpet hemocyanin-conjugated peptides, and each was tested for ability to inhibit complex formation. Anti-PSP-420 antibody potently inhibited complex formation with half-maximal effect at 25 nM IgG. A monoclonal antibody (LJ-56) made against PSP-420 showed high affinity for protein S and inhibited complex formation; this monoclonal antibody specifically recognized free protein S but not the protein S.C4BP complex. These results imply that the PSP-420 sequence is surface-exposed, capable of binding to C4BP, and essential for protein S binding to C4BP.

Cathepsin G inactivates human protein S in vitro.

Human polymorphonuclear leukocyte cathepsin G was used in vitro to digest human protein S. While clotting assays indicated that the proteinase induced a rapid decrease in activity, polyacrylamide gel electrophoresis-sodium dodecyl sulphate indicated the removal of a peptide of low molecular mass product protein S (des 1-40). The loss in activity was the result of cathepsin G cleaving position Phe40-Tyr41 and removing the calcium-binding region. Calcium ions almost totally protected the cofactor from the action of cathepsin G in vitro.

Effects of annexin V on the activity of the anticoagulant proteins C and S.

The effects of annexin V on the anticoagulant activity of activated protein C (APC) and protein S were examined. Although annexin V did not influence the amidolytic potential of APC, it inhibited both APC and protein S function in a factor Va inactivation assay. Competition experiments demonstrated that annexin V inhibits protein S binding to phospholipid vesicles in a dose-dependent manner. These results demonstrate that annexin V effectively interferes with the anticoagulant arm of the hemostatic system.