Protein S Released From Stimulated Platelets Has Anticoagulant Activity Independent of Activated Protein C and Tissue Factor Pathway Inhibitor (TFPI).

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1137-1137
Author(s):  
Mary J. Heeb ◽  
Erning Duan
Keyword(s):  
Plasma Protein ◽  
Neutralizing Antibodies ◽  
Protein C ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Platelet Protein ◽  
Presence And Absence

Abstract Abstract 1137 Background: Platelets contain in their alpha granules ∼2.5% of the protein S in blood. It has been suggested that this protein S supports the anticoagulant activity of exogenous activated protein C (APC), but it is not known whether protein S that is released from stimulated platelets can exert anticoagulant activity that is independent of APC and TFPI. We recently showed that at least some of the anticoagulant activity of plasma protein S is independent of APC and TFPI, although data suggested that plasma protein S may also have TFPI-dependent activity. Objective and methods: To determine if platelet protein S has anticoagulant activity that is independent of APC and TFPI, prothrombinase and extrinsic FXase reactions were initiated on the surface of fresh stimulated or unstimulated washed platelets in the presence and absence of blocking antibodies against APC, TFPI, and/or protein S, or in the presence and absence of purified plasma-derived protein S. Platelets were adjusted to a concentration of 0.7 to 2 × 10e8/ml, which contained 2.3–6.5 nM total platelet protein S. The last platelet wash contained negligible amounts of plasma protein S. Results: Neutralizing anti-protein S antibodies allowed up to 5.7-fold (mean: 2.1 ± 1.5 –fold, n=13) more thrombin generation on calcium ionophore-stimulated platelets following supplementation with 50–500 pM FXa and 600 nM prothrombin, and allowed up to 2.5-fold (mean: 1.7 ± 0.7 –fold, n=11) more thrombin generation on platelets that were not ionophore-stimulated but were gradually stimulated following FXa and prothrombin supplementation. Anti-protein S antibodies had no effect on thrombin generation on platelets that were treated with prostaglandin E1 (PGE1) to suppress platelet activation and then supplemented with procoagulants. This implies that platelet protein S is released from stimulated platelets and downregulates thrombin generation on platelets, and that neutralizing anti-protein S antibodies block this activity of protein S. Anti-protein S antibodies allowed up to 1.8-fold (mean 1.5 ± 0.2 –fold, n=8) more FXa generation on the surface of stimulated platelets supplemented with 5 pM TF, 100 pM FVIIa, and 160 nM FX, but anti-protein S antibodies had no effect on FXa generation on platelets treated with PGE1. Most of these experiments were performed in the presence of neutralizing antibodies against TFPI and APC, but thrombin and FXa generation on platelets under the varying conditions described were unaffected by the presence of these neutralizing antibodies. Purified plasma-derived zinc-containing protein S downregulated thrombin and FXa generation on platelets (IC50 = 6–18 nM PS) and in plasma >10-fold more potently than zinc-deficient protein S. We could not demonstrate a synergistic anticoagulant effect when TFPI was combined with zinc-deficient protein S in the presence of stimulated platelets and procoagulant proteins. Conclusion: Protein S that is released from stimulated platelets exerts anticoagulant activity that is independent of TFPI and APC. Disclosures: No relevant conflicts of interest to declare.

Protein S Regulates Factor IXa/VIIIa Activity Independent of Activated Protein C

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2196-2196
Author(s):  
Rinku Majumder
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Activated Protein C ◽  
Protein S ◽  
Clotting Time ◽  
Independent Manner ◽  
Factor Ixa ◽  
New Findings ◽  
Presence And Absence

Abstract Abstract 2196 Protein S (PS) is a vitamin-K-dependent plasma protein that plays an important role in the feedback regulation of thrombin generation. PS is a well recognized anticoagulant protein, the absence of which causes venous and arterial thrombosis. While PS has been studied for over three decades, the precise role this protein plays in attenuating the hemostatic response is far from clear. PS was initially identified as a cofactor for activated protein C (APC). Interestingly, recent reports suggest PS may function in an APC-independent manner as it has been suggested to inhibit prothrombinase complex formation and act as a stimulator of factor Xa-TFPI (tissue factor pathway inhibitor) complex. Based on these new data, we have begun to examine possible roles of PS outside of the PC pathway which contribute to the regulation of thrombin generation. We recently obtained exciting preliminary data which suggests that PS plays an important role in the inhibition of Xa generation by the intrinsic Xase complex. Our central hypothesis is that PS binds to the enzyme FIXa in the presence of Phosphatidyl serine (PhosSer) containing membranes and inhibits FXa generation in the presence and absence of VIIIa. We did clotting assays with FIX deficient plasma both with and without anti PS antibody. Clotting initiated with 0.1 nM FIXa shows a must faster clotting in the presence of anti PS antibody (57 seconds compared to 107 seconds without the incubation with anti PS antibody) (see Table 1). However, there is no difference in clotting times when the clotting is initiated with FVIIa in the presence and absence of anti PS antibody. Our results showing PS is only able to alter FIXa initiated clotting indicates that PS inhibits the FXa generation by FIXa/FVIIIa in plasma. Table 1: Clotting of FIX deficient plasma initiated with FIXa (A) & initiated with FVIIa (B) (A) Conc of FVIIa nM Clotting time in seconds - AntiPS Antibody Clotting time in seconds + AntiPS Antibody 0.1 107 57 1 46 31 5 31 14.7 10 30 14.4 (B) Conc of FVIIa nM Clotting time in seconds - AntiPS Antibody Clotting time in seconds + AntiPS Antibody 0.1 113 112 1 56 54 5 41 42 10 36 35 We determined the binding of PS to factor IXa by determining the change in anisotropy and fluorescence of DEGR(5 (dimethylamino)1-naphthalenesulfonyl]-glutamylglycylarginyl chloromethyl ketone)-IXa. The Kd was ~ 1 nM in both cases suggesting a tight binding of PS with factor IXa. We also determined the activation of FX by FIXa in the presence of FVIIIa and 50 μM Phosphatidylserine membrane in the presence of PS. We used physiological concentrations of FX, PS, 0.1 nM FIXa, 5 nM FVIIIa and monitored the rate of FXa generation by using S2222 (chromogenic substrate). There was ~ 95 % inhibition in the rate of FXa generation by FIXa in the 0.2 presence of FVIIIa. Our results clearly indicate that PS inhibits FXa generation by factors IXa/VIIIa. We believe that these new findings have major implications for better understanding how FXa generation is regulated during the initial phases of coagulation and may shed light on a major outstanding question in the field relating to how a key anticoagulant, PS contributes to the down regulation of the haemostatic response. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Protein S Coordinates the Inhibition of Prothrombinase By Tfpiα and Activated Protein C

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2386-2386
Author(s):  
Xian Li ◽  
Sara J Bidarian ◽  
Martha Sim ◽  
Xiaohong Song ◽  
Jeremy P. Wood
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Complement Factor ◽  
Protein Assay ◽  
The Impact

Background: Protein S (PS), a vitamin K-dependent plasma glycoprotein, functions as a cofactor for the anticoagulants activated protein C (APC) and tissue factor (TF) pathway inhibitor alpha (TFPIa), which inhibit factors Va (FVa) and Xa (FXa), respectively. Although it is unclear which of these functions is/are important in vivo, homozygous deficiency of PS is associated with life-threatening thrombosis shortly after birth. FVa and FXa form the prothrombinase complex, which generates thrombin, suggesting that PS has a role in the direct inhibition of thrombin production. However, neither the PS/APC nor PS/TFPIα system alone is effective at inhibiting thrombin generation by prothrombinase. In addition to its role in regulating coagulation, PS also functions as an inhibitor of the complement system. Approximately 60% of plasma PS circulates bound to complement factor C4bp, which blocks its anticoagulant activity. We sought to determine the impact of PS/APC on TFPIα function, and vice versa, using purified protein and plasma-based systems. Methods and Results: To assess the effect of the combined PS/APC and PS/TFPIα systems on thrombin generation, we supplemented plasma with thrombomodulin, which promotes APC activation. In the absence of thrombomodulin, 5nM TFPIα decreased peak thrombin by 55.1% (33.1±1.9 nM in the presence of TFPIα vs. 73.7±39.9 nM in the absence) and endogenous thrombin potential (ETP) by 35.4% (475±42 nM*min vs. 735±189nM*min). In the presence of thrombomodulin, TFPIα decreased these parameters by 65.7% (11.4 ± 2.6 nM) and 77.5% (107±22 nM*min), respectively, suggesting that APC makes TFPIα a more potent inhibitor of thrombin generation. We next sought to study each of these PS functions in a purified protein system and in plasma. To study the effect of PS/APC on TFPIα function, we produced a recombinant protein consisting of the first two epidermal growth factor-like domains of PS (EGF1-2), which contain the putative APC binding sites. In a purified protein assay, APC inhibited the rate of thrombin activation by prothrombinase by 19.62±0.01% in the absence of PS and by 34.96±0.02% in the presence of 50nM PS. EGF1-2 dose-dependently reversed the effect of PS, with 75% reversal achieved with the addition of 200nM EGF1-2. Unexpectedly, EGF1-2 had the opposite effect in plasma thrombin generation assays and potently inhibited TF-initiated thrombin generation either in the presence or absence of thrombomodulin. We hypothesize that the EGF1-2 domains also form part of the C4bp binding site, and that addition of EGF1-2 protein resulted in release of PS from C4bp and an increase in the anticoagulant PS pool. In support of this hypothesis, EGF1-2 had no effect on thrombin generation in PS-depleted plasma, which is also depleted of C4bp. When the PS-depleted plasma was supplemented with 150nM PS, EGF1-2 had the expected procoagulant activity (increasing peak thrombin from 50.4±19.9 nM to 90.4±6.0 nM). Notably, even with a saturating concentration of EGF1-2, thrombomodulin and PS significantly decreased thrombin generation, suggesting that PS-TFPIα-mediated FXa inhibition promotes APC-mediated FVa degradation, even if PS cannot directly bind APC. We similarly assessed the impact of the PS-TFPI function, using a protein from the saliva of black flies, "black fly protease inhibitor" (BFPI), which contains the TFPIα domain that inhibits FXa but lacks the domain that binds PS. BFPI inhibits free FXa similarly to TFPIα, but PS does not promote this inhibition. Like TFPIα, BFPI is a poor inhibitor of thrombin generation by prothrombinase containing thrombin-activated FVa (5 nM BFPI had no impact on thrombin generation in the presence or absence of PS). However, in the presence of APC and PS, 5nM BFPI decreased the maximum rate of thrombin generation by 17.3±3.3%. These data suggest that PS/APC-mediated degradation of FVa promotes TFPIα-mediated inhibition of FXa, regardless of whether PS is able to bind TFPIα. Conclusions: Our data suggest that the PS-APC and PS-TFPIα systems cooperatively regulate thrombin generation by prothrombinase. While maximal inhibition requires that PS act as a cofactor for both APC and TFPIα, PS-APC independently promotes TFPIα function, and PS-TFPIα separately promotes APC. Based on these data, we propose a model in which PS-APC-mediated inhibition of FVa renders FXa susceptible to TFPIα and vice versa. Disclosures No relevant conflicts of interest to declare.

scholarly journals Platelet protein S directly inhibits procoagulant activity on platelets and microparticles

2013 ◽  
Vol 109 (02) ◽  
pp. 229-237 ◽  
Author(s):  
Fabian Stavenuiter ◽  
Nicole Davis ◽  
Erning Duan ◽  
Andrew Gale ◽  
Mary Heeb
Keyword(s):  
Electrophoretic Mobility ◽  
Western Blotting ◽  
Plasma Protein ◽  
Protein C ◽  
Thrombin Generation ◽  
Activated Protein C ◽  
Protein S ◽  
Neutralising Antibodies ◽  
Platelet Protein ◽  
Cofactor Activity

SummaryAnticoagulant plasma protein S (PS) is essential for maintaining haemostatic balance. About 2.5% of PS is stored in platelets and released upon platelet stimulation. So far, little is known about the functionality and importance of platelet (plt)PS. A platelet-associated protease cleaves plasma-derived (pd)PS and pltPS in the “thrombin-sensitive region”, abolishing activated protein C (APC) cofactor activity. However, we showed that cleaved PS retains APC-independent anticoagulant activities (“PS-direct”). To investigate whether pltPS or pdPS exert PS-direct on platelets or platelet-shed microparticles, thrombin and factor (F)Xa generation on unstimulated or stimulated washed platelets and microparticles were measured. Western blotting revealed that pltPS and pdPS bound to washed, stimulated platelets and microparticles, and that pltPS had slower electrophoretic mobility than pdPS. Platelet stimulation in the presence of inhibitory anti-PS antibodies resulted in 2.6 ± 1.6-fold (p<0.0004, n=20) more thrombin generation upon addition of FXa and prothrombin. PltPS exerted PSdirect that was similar to or greater than that of Zn2+-containing pdPS and much greater than that of Zn2+-deficient pdPS. Findings were confirmed using purified pltPS. Platelet-bound pltPS and microparticlebound pltPS had similar PS-direct. Finally, platelet stimulation in the presence of inhibitory anti-PS antibodies resulted in 1.5 ± 0.2-fold (p<0.0001, n=11) more FXa generation upon addition of TF/FVIIa and FX. Thus, pltPS inhibits both prothrombinase and extrinsic FXase activities. Neutralising antibodies against APC and TFPI had no effect on the PS-direct of pltPS or pdPS on platelets. This study indicates that pltPS may be an essential pool of PS that counterbalances procoagulant activities on platelets.

scholarly journals Identification and quantitation of protein S in human platelets

Blood ◽  
1985 ◽  
Vol 66 (6) ◽  
pp. 1452-1455 ◽  
Author(s):  
HP Schwarz ◽  
MJ Heeb ◽  
JD Wencel-Drake ◽  
JH Griffin
Keyword(s):  
Plasma Protein ◽  
Anticoagulant Activity ◽  
Limited Proteolysis ◽  
Activated Protein C ◽  
Protein S ◽  
Human Platelets ◽  
Immunofluorescent Staining ◽  
Metabolic Inhibitors ◽  
Platelet Surface ◽  
Platelet Protein

Gel filtered human platelets contaminated with less than 0.02% of plasma protein S contained 490 ng of protein S antigen per 3 X 10(8) platelets, equivalent to 2.5% of protein S in whole blood. Three patients with heterozygous plasma protein S deficiency, a congenital disorder associated with venous thrombotic disease, had platelet protein S antigen levels that were 40% of the mean platelet level in ten normal volunteers. In immunoblotting analysis, platelet protein S was indistinguishable from plasma protein S. Thrombin stimulation of platelets caused release of 63% of total protein S antigen and this release was abolished when platelets were preincubated with metabolic inhibitors. Thrombin effected limited proteolysis of platelet protein S and this reaction was inhibited by calcium ions. Immunofluorescent staining of platelets using protein S antibodies demonstrated that protein S colocalized with fibrinogen, an established alpha-granule protein. Thus, human platelets contain protein S in alpha granules that can be released by thrombin stimulation. The released protein S may bind to stimulated platelets and thereby promote and localize the anticoagulant activity of activated protein C on the platelet surface.

scholarly journals Dissociation of Activated Protein C Functions by Elimination of Protein S Cofactor Enhancement

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 21-21
Author(s):  
Roger JS Preston ◽  
Shona Harmon ◽  
Fionnuala B Ni Ainle ◽  
Jennifer A Johnson ◽  
Moya Cunningham ◽  
...  
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Single Amino Acid ◽  
Wild Type ◽  
Maximal Inhibition ◽  
Gla Domain ◽  
Anticoagulant Function

Abstract Activated protein C (APC) plays a critical anticoagulant role by inactivating factor Va (FVa) and factor VIIIa (FVIIIa) and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor (EPCR) can initiate PAR-1 mediated cytoprotective signalling. Although protein S constitutes a critical cofactor for APC anticoagulant function, the molecular basis through which protein S interacts with APC is not fully understood. In this study, we employed a site-directed mutagenesis strategy to characterise the effects of four single amino acid substitutions (D35T, D36A, L38D and A39V) within a region of the APC Gla domain important for protein S cofactor enhancement. To maintain Gla domain structural integrity, each residue was substituted with the corresponding residue of the human prothrombin Gla domain. Protein C variants were expressed in HEK 293 cells and purified by ion-exchange chromatography. Upon activation, the amidolytic activity of each recombinant APC variant was identical to that of wild type APC. The anticoagulant function of recombinant wild type and variant APC was compared in a tissue factor-initiated thrombin generation assay using protein C-deficient plasma. Wild type APC diminished thrombin generation in a concentration-dependent manner as expected. Variants APC-D35T, APC-D36A and APC-A39V exhibited only mildly impaired (&lt;2-fold) anticoagulant activity compared to wild type APC. The anticoagulant activity of APC-L38D, however, was severely impaired. APC-L38D was unable to achieve half-maximal inhibition of endogenous thrombin potential (ETP) at APC concentrations as high as 150nM, compared to wild type APC, which achieved half-maximal inhibition at 7.2nM APC. To clarify the role of Leu-38 in facilitating APC anticoagulant function, we further studied the ability of APC-L38D to be stimulated in protein S-deficient plasma reconstituted with plasma-purified protein S. Co-incubation of wild type APC with increasing protein S concentration (12.5–200nM) caused a corresponding reduction in ETP (IC50 = 24nM protein S). In contrast, APC-L38D was unresponsive to protein S. In the presence of APC-L38D, ETP was reduced only 22% at 1.5μM protein S (10-fold higher than plasma free protein S). In a phospholipid-dependent FVa proteolysis time course assay, both wild type APC and APC-L38D rapidly reduced FVa cofactor activity, indicating that the observed impaired plasma anticoagulant activity of APC-L38D is not mediated by impaired interaction with anionic phospholipids or FVa. In a modified version of this assay, wild type APC-mediated FVa proteolysis was rapidly enhanced by added protein S, with half-maximal inhibition observed at 5nM protein S. In contrast, APC-L38D exhibited no protein S-enhanced FVa proteolysis. Cumulatively, these data confirm that Leu-38 mediates APC anticoagulant function in plasma by facilitating critical protein S cofactor enhancement of FVa proteolysis. Previous studies have shown that APC Gla domain mutations can influence EPCR binding, a pre-requisite for PAR-1 mediated cytoprotective signalling. Consequently, we assessed APC binding to sEPCR using surface plasmon resonance. Binding affinities of APC-L38D and wild type APC were very similar (KD 112±25nM versus 117±36nM). Furthermore, the ability of APC-L38D to protect EAhy926 cells from staurosporine-induced apoptosis was also investigated using RT-PCR quantification of pro- (bax) and anti- (bcl-2) apoptotic gene expression. Pre-incubation with APC-L38D significantly reduced the bax/bcl-2 ratio to the same extent as wild type APC. The EPCR-dependence of these anti-apoptotic activities was confirmed using RCR-252, (an inhibitory anti-EPCR antibody) which ablated the cytoprotective effect of both APC species. In conclusion, we demonstrate that a single amino acid substitution (L38D) can significantly impair APC anticoagulant activity due to elimination of protein S cofactor enhancement. However, despite the location of Leu-38 in the Gla domain, APC-L38D retains its ability to bind EPCR, and trigger PAR-1 mediated cytoprotective signalling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to dissociate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.

Protein S Regulates Factor IXa in the Absence and Presence of Factor VIIIa Independently of Activated Protein C

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1197-1197
Author(s):  
Rinku Majumder ◽  
Rima Chattopadhyay ◽  
Tanusree Sengupta
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Activity Measurement ◽  
Clotting Time ◽  
Thrombotic Risk ◽  
Increased Risk

Abstract Abstract 1197 Coagulation is a finely tuned process. During thrombin formation, several anticoagulant reactions are initiated to prevent systematic activation of coagulation, and impairment of anticoagulant activity causes an increased risk of venous thrombosis. One such anticoagulant factor is protein S, deficiencies of which have been linked to venous and arterial thrombosis. While protein S has been studied for over three decades, the precise role this protein plays in attenuating the hemostatic response is far from clear. Protein S is a vitamin K-dependent plasma protein that functions in feedback regulation of thrombin generation. Protein S was initially identified as a cofactor for activated protein C (APC) but later it was observed that there is only a 3–10 fold increase in APC activity in the presence of protein S. Plasma coagulation assays in the absence of APC suggest that protein S may have other anticoagulant role(s). We report here an anticoagulant activity of Protein S mediated by inhibition of fIXa in the absence and presence of fVIIIa independent of APC. Although an APC-independent anticoagulant activity has been reported for protein S interacting with fVIIIa, no study has shown that the inhibitory effect of protein S is mediated through its interaction with fIXa, thus making our observations novel and significant. Moreover, previous studies that reported an interaction between fVIIIa and protein S were performed with low amounts of phospholipid, a condition that produces activity measurement artifacts due to the presence of active protein S multimers. We used both ex vivo (plasma studies) and in vitro methods at high phospholipid (100–200 micro molar) concentration to determine whether and how the intrinsic pathway of blood coagulation is regulated by protein S. We obtained the following results: 1) activated partial thromboplastin time (aPTT) assays with protein S-supplemented plasma confirmed that protein S prolongs clotting time, and a normal clotting time was restored with addition of anti-protein S antibody, 2) a modified aPPT assay with fIX-deficient plasma confirmed that protein S affects fIX-initiated clotting time, 3) thrombin generation assay through fIXa/fVIIIa pathway, initiated with a limiting amount of tissue factor (TF), was regulated by protein S, 4) in vitro studies with fIXa/fVIIIa and protein S in the presence of phosphatidylserine (PS) vesicles showed ∼40% and ∼65% inhibition in the activity of fIXa in the absence and presence of fVIIIa, respectively, and 5) protein S altered only the KM for fX activation by fIXa but altered both kcat and KM for fX activation by fIXa and fVIIIa. Our findings underscore the central role of protein S in regulation of coagulation. We anticipate these results will unravel important implications for the evaluation of thrombotic risk associated with protein S-deficiency. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Plasma protein S deficiency in familial thrombotic disease

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1297-1300 ◽  
Author(s):  
HP Schwarz ◽  
M Fischer ◽  
P Hopmeier ◽  
MA Batard ◽  
JH Griffin
Keyword(s):  
Plasma Protein ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Oral Anticoagulant Therapy ◽  
Activated Protein C ◽  
Protein S ◽  
Factor X ◽  
Protein C Deficiency ◽  
Thrombotic Disease ◽  
Disease Protein

Abstract A family with a history of severe recurrent venous thromboembolic disease was studied to determine if a plasma protein deficiency could account for observed disease. Protein S levels in plasma were determined immunologically using the Laurell rocket technique. The propositus, his mother, his aunt, and his cousin who were clinically affected had 17% to 65% of the control levels of protein S antigen (normal range, 71% to 147%). Since three of these patients were receiving oral anticoagulant therapy, the ratios of protein S to prothrombin, factor X, and protein C in these patients were compared with values for a group of orally anticoagulated controls. These results suggested that protein S is half-normal in all family members with thrombotic disease. Other proteins known to be associated with familial thrombotic disease, including antithrombin III, plasminogen, fibrinogen, and protein C, were normal. Because plasma protein S serves as a cofactor for the anticoagulant activity of activated protein C and because protein C deficiency is associated with recurrent thrombotic disease, it is suggested that recurrent thrombotic disease in this family is the result of an inherited deficiency of protein S.

A Single Amino Acid Substitution (I18V) in the Gla Domain of Activated Protein C Markedly Impairs Ability of PE and GlyCer to Enhance Anticoagulant Activity

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3071-3071
Author(s):  
Jennifer Johnson ◽  
Fionnuala Ni Ainle ◽  
Shona Harmon ◽  
James S O’Donnell ◽  
Roger JS Preston
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Phospholipid Vesicles ◽  
Wild Type ◽  
Thrombin Generation Assay ◽  
Increased Sensitivity ◽  
Gla Domain

Abstract Phosphatidylethanolamine (PE) and glucosylceramide (GlyCer) are cell surface lipids that preferentially enhance anticoagulant, rather than procoagulant pathways. In particular, both PE and GlyCer enhance the anticoagulant activity of activated protein C (APC). Previous studies have indicated that specific APC Gla domain residues may mediate APC interaction with PE and GlyCer. To investigate whether specific APC residues mediate PE and GlyCer enhanced APC anticoagulant activity, we expressed a series of APC variants in which APC Gla domain residues not shared with the human prothrombin Gla domain were substituted with their prothrombin amino acid equivalent. The anticoagulant activity of each APC Gla domain variant was assessed in a tissue factor-initiated thrombin generation assay containing phospholipid vesicles of differing composition (80% PC/20% PS); or PC/PS/PE (60%/20%/20%); or PC/PS/GlyCer (60%/20%/20%). For each of these lipid mixtures, thrombin generation (endogenous thrombin potential, ETP) was not significantly different in the absence of APC. In the presence of PC/PS vesicles, APC reduced thrombin generation by 63±3% at the highest APC concentration tested (6nM). However, APC impairment of thrombin generation was enhanced 3-fold in the presence of PC/PS/PE compared with vesicles containing PC/PS alone, and in the presence of PC/PS/GlyCer was enhanced 4.3-fold. Enhancement of anticoagulant function by PE and GlyCer was similar for the majority of the APC Gla domain variants tested. Interestingly, one APC variant (APC-I18V) exhibited similar anticoagulant activity to that of wild type APC with PC/PS vesicles, but was not enhanced by the presence of PE- or GlyCer-containing vesicles. Phospholipid vesicles containing PE or GlyCer have been previously described to enhance protein S cofactor enhancement of APC. Therefore, to further characterize APC-I18V, we assessed the ability of wild type APC and APC-I18V to be enhanced by protein S in the presence of PC/PS, PC/PS/PE or PC/PS/GlyCer using a protein S-sensitive thrombin generation assay. In the presence of PC/PS, increasing protein S concentration in protein S-deficient plasma resulted in an APC-mediated slow decrease in thrombin generation, irrespective of whether wild type or APC-I18V was used (IC50 for protein S-mediated APC inhibition of thrombin generation with PC/PS, 130nM). However, in the presence of PC/PS/PE or PC/PS/GlyCer, thrombin generation was impaired by wild type APC at 3–4-fold lower protein S concentration than that observed when PC/PS vesicles alone were used (IC50, PC/PS/PE=31.5nM and PC/PS/GlyCer=37.5nM). APC-I18V, however, did not exhibit a similarly increased sensitivity to protein S in the presence of PE or GlyCer, as the anticoagulant activity of this variant was the same as when only PC/PS was included. To investigate whether the loss of specific neutral lipid enhancement in APC-I18V affected its ability to initiate cytoprotective signaling via EPCR-PAR-1 on endothelial cells, the capacity of APC-I18V to inhibit thrombin-induced endothelial cell barrier permeability was assessed. When cells were pre-treated with either wild type APC or APC-I18V, there was a significant enhancement in barrier integrity and attenuation of thrombin-induced permeability (P&lt;0.05), demonstrating that loss of PE/GlyCer enhancement of APC anticoagulant activity does not adversely affect EPCR binding and EPCR/PAR-1 cytoprotective signaling. Collectively, these results suggest PE and GlyCer enhancement of APC anticoagulant activity is mediated by increased sensitivity to protein S, and that Ile-18 in the APC Gla domain is critical for mediating APC-specific functional enhancement by PE/GlyCer.

scholarly journals Identification and quantitation of protein S in human platelets

Blood ◽  
1985 ◽  
Vol 66 (6) ◽  
pp. 1452-1455 ◽  
Author(s):  
HP Schwarz ◽  
MJ Heeb ◽  
JD Wencel-Drake ◽  
JH Griffin
Keyword(s):  
Plasma Protein ◽  
Anticoagulant Activity ◽  
Limited Proteolysis ◽  
Activated Protein C ◽  
Protein S ◽  
Human Platelets ◽  
Immunofluorescent Staining ◽  
Metabolic Inhibitors ◽  
Platelet Surface ◽  
Platelet Protein

Abstract Gel filtered human platelets contaminated with less than 0.02% of plasma protein S contained 490 ng of protein S antigen per 3 X 10(8) platelets, equivalent to 2.5% of protein S in whole blood. Three patients with heterozygous plasma protein S deficiency, a congenital disorder associated with venous thrombotic disease, had platelet protein S antigen levels that were 40% of the mean platelet level in ten normal volunteers. In immunoblotting analysis, platelet protein S was indistinguishable from plasma protein S. Thrombin stimulation of platelets caused release of 63% of total protein S antigen and this release was abolished when platelets were preincubated with metabolic inhibitors. Thrombin effected limited proteolysis of platelet protein S and this reaction was inhibited by calcium ions. Immunofluorescent staining of platelets using protein S antibodies demonstrated that protein S colocalized with fibrinogen, an established alpha-granule protein. Thus, human platelets contain protein S in alpha granules that can be released by thrombin stimulation. The released protein S may bind to stimulated platelets and thereby promote and localize the anticoagulant activity of activated protein C on the platelet surface.

Sign in / Sign up

Export Citation Format

Share Document