scholarly journals Prothrombotic Phenotype of a Protein S Mutant Lacking Anticoagulant Activity

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2098-2098
Author(s):  
Stephanie Springborn ◽  
Sourav Ghosh ◽  
Marie L Schulte ◽  
Carla Rothlin ◽  
Brian R Branchford
Keyword(s):  
Platelet Activation ◽  
Tyrosine Kinases ◽  
Protein C ◽  
Receptor Tyrosine Kinases ◽  
Anticoagulant Activity ◽  
Protein S ◽  
Mutant Form ◽  
Wild Type ◽  
Significant Difference

Abstract Protein S is a vitamin K-dependent protein that plays an important role in balancing pro- and anti-thrombotic responses to vascular injury. On one hand, it circulates bound to activated protein C, functioning as an anticoagulant complex that downregulates the activities of coagulation factors V and VIII. On the other hand, protein S is a ligand for platelet TYRO3 and MERTK, which, along with the third paralog AXL, constitute the TAM family of receptor tyrosine kinases that functions to potentiate the action of platelet-activating agonists, ultimately resulting in activation of the α IIbβ3 integrin. The relative importance of these two activities in vivo, however, is not known. To better understand the importance of the TYRO3/MERTK-stimulating ability of protein S and thus gain additional insight into its role in platelet activation, we used CRISPR/Cas9 technology to generate mice with a D136A mutant form of protein S that lacks the ability to bind protein C and function as an anticoagulant, while retaining its ability to bind platelet-activating TAM receptors - hereafter termed Pros1 D136A mice. Since homozygosity for this variant was embryonically lethal, all assays were carried out in wild-type animals and their heterozygous littermates comprising a wild-type Pros1 allele and an allele encoding Pros1 D136A. Though there was no significant difference between Pros1 D136A mice and their wild-type littermates in either a collagen/epinephrine-induced pulmonary embolism model or in tail vein bleeding times, platelets from Pros1 D136A mice accumulated at the injury site to a significantly greater degree following in vivo laser injury to the cremaster muscle microvasculature. Taken together with the embryonic phenotype of Pros1 D136A homozygous mice, these data support the notion that protein S can function to augment platelet responsiveness. Further studies on this and other ligands for the TAM family of receptor tyrosine kinases should provide additional insights into their roles in physiological platelet activation. Disclosures Branchford: Bio Products Laboratory: Honoraria; Novo Nordisk: Honoraria.

Platelet-mediated proteolytic down regulation of the anticoagulant activity of protein S in individuals with haematological malignancies

2012 ◽  
Vol 107 (03) ◽  
pp. 468-476 ◽  
Author(s):  
Ilze Dienava-Verdoold ◽  
Marina R. Marchetti ◽  
Liane C. J. te Boome ◽  
Laura Russo ◽  
Anna Falanga ◽  
...  
Keyword(s):  
Protein C ◽  
Normal Values ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Intact Protein ◽  
The Impact ◽  
Independent Protein

SummaryThe natural anticoagulant protein S contains a so-called thrombin-sensitive region (TSR), which is susceptible to proteolytic cleavage. We have previously shown that a platelet-associated protease is able to cleave protein S under physiological plasma conditions in vitro. The aim of the present study was to investigate the relation between platelet-associated protein S cleaving activity and in vivo protein S cleavage, and to evaluate the impact of in vivo protein S cleavage on its anticoagulant activity. Protein S cleavage in healthy subjects and in thrombocytopenic and thrombocythaemic patients was evaluated by immunological techniques. Concentration of cleaved and intact protein S was correlated to levels of activated protein C (APC)-dependent and APC-independent protein S anticoagulant activity. In plasma from healthy volunteers 25% of protein S is cleaved in the TSR. While in plasma there was a clear positive correlation between levels of intact protein S and both APC-dependent and APC-independent protein S anticoagulant activities, these correlations were absent for cleaved protein S. Protein S cleavage was significantly increased in patients with essential thrombocythaemia (ET) and significantly reduced in patients with chemotherapy-induced thrombocytopenia. In ET patients on cytoreductive therapy, both platelet count and protein S cleavage returned to normal values. Accordingly, platelet transfusion restored cleavage of protein S to normal values in patients with chemotherapy-induced thrombocytopenia. In conclusion, proteases from platelets seem to contribute to the presence of cleaved protein S in the circulation and may enhance the coagulation response in vivo by down regulating the anticoagulant activity of protein S.

Cleavage of Factor V at Arg 506 by Activated Protein C and the Expression of Anticoagulant Activity of Factor V

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2552-2558 ◽  
Author(s):  
Elisabeth Thorelli ◽  
Randal J. Kaufman ◽  
Björn Dahlbäck
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Factor V ◽  
Wild Type ◽  
Apc Resistance ◽  
S 100 ◽  
Negative Effect ◽  
Cofactor Activity

Activated protein C (APC) inhibits coagulation by cleaving and inactivating procoagulant factor Va (FVa) and factor VIIIa (FVIIIa). FV, in addition to being the precursor of FVa, has anticoagulant properties; functioning in synergy with protein S as a cofactor of APC in the inhibition of the FVIIIa-factor IXa (FIXa) complex. FV:Q506 isolated from an individual homozygous for APC-resistance is less efficient as an APC-cofactor than normal FV (FV:R506). To investigate the importance of the three APC cleavage sites in FV (Arg-306, Arg-506, and Arg-679) for expression of its APC-cofactor activity, four recombinant FV mutants (FV:Q306, FV:Q306/Q506, FV:Q506, and FV:Q679) were tested. FV mutants with Gln (Q) at position 506 instead of Arg (R) were found to be poor APC-cofactors, whereas Arg to Gln mutations at positions 306 or 679 had no negative effect on the APC-cofactor activity of FV. The loss of APC-cofactor activity as a result of the Arg-506 to Gln mutation suggested that APC-cleavage at Arg-506 in FV is important for the ability of FV to function as an APC-cofactor. Using Western blotting, it was shown that both wild-type FV and mutant FV was cleaved by APC during the FVIIIa inhibition. At optimum concentrations of wild-type FV (11 nmol/L) and protein S (100 nmol/L), FVIIIa was found to be highly sensitive to APC with maximum inhibition occurring at less than 1 nmol/L APC. FV:Q506 was inactive as an APC-cofactor at APC-concentrations ≤ 1 nmol/L and only partially active at higher APC concentrations. Our results show that increased expression of FV anticoagulant activity correlates with APC-mediated cleavage at Arg-506 in FV, but not with cleavage at Arg-306 nor at Arg-679.

Optimisation of EGFR TKI efficiency in the therapeutic scheme of EGFR wild-type lung cancer.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e18532-e18532
Author(s):  
Mathilde Cabart ◽  
Judith Raimbourg ◽  
Lisenn Lalier ◽  
Jaafar Bennouna ◽  
Francois Vallette
Keyword(s):  
Lung Cancer ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Wild Type ◽  
Short Term ◽  
Egfr Ligands ◽  
Egfr Mutated ◽  
Egfr Tki

e18532 Background: EGFR tyrosine kinase inhibitors (EGFR TKI) have improved the therapeutic care of lung cancer patients but only a small sub-population of patients, namely those harboring EGFR-mutated tumors, benefit from this therapy. The observation that EGFR TKI enhance prognosis and quality of life in all patients when used as second line or maintenance treatment impelled us into the search of potential markers of the optimal introduction kinetics of EGFR TKI in the therapeutic scheme. Methods: We used lung cancer cell lines harboring either wild-type or mutated EGFR (NCI-H1650, NCI-H1975) to study the consequences of cisplatin treatment in vitro on the consecutive sensitivity to erlotinib. Results: Sub-lethal cisplatin pretreatment (3µM) enhances erlotinib toxicity in EGFR wild-type, but not EGFR mutated cells (A549 IC50 drops from 28 to 15µM for short-term or 12µM for long-term exposure). This correlates with EGFR activation following short-term or prolonged cisplatin treatment through the secretion of EGFR ligands. This activation of EGFR is concomitant to the decrease in other receptor tyrosine kinases phosphorylation including Met. Conclusions: The sensitivity of EGFR wild-type lung cancer cells to erlotinib in vitro is enhanced by cisplatin pretreatment. We identified potential markers of this sensitization, namely EGFR ligands, which serum level might be predicitive of the optimal efficiency of EGFR TKI. In vivo validation of these markers is under investigation. The concomitant decrease in other receptor tyrosine kinases phosphorylation suggests that the targeting of other receptor tyrosine kinases might potentiate EGFR TKI efficiency.

scholarly journals Timing of neuronal death in trkA, trkB and trkC mutant embryos reveals developmental changes in sensory neuron dependence on Trk signalling

Development ◽  
1996 ◽  
Vol 122 (10) ◽  
pp. 3255-3261 ◽  
Author(s):  
L.G. Pinon ◽  
L. Minichiello ◽  
R. Klein ◽  
A.M. Davies
Keyword(s):  
Neuronal Death ◽  
Tyrosine Kinases ◽  
Marked Decrease ◽  
Trigeminal Ganglia ◽  
Wild Type ◽  
Embryonic Mouse ◽  
Target Field ◽  
Significant Difference

The sensory neurons of the embryonic mouse trigeminal ganglion are supported in culture by different neurotrophins at successive stages of development. Initially the neurons survive in response to BDNF and NT3 and later switch to becoming NGF-dependent (Buchman, V. I. and Davies, A. M. (1993), Development 118, 989–1001). To determine if this in vitro switch in neurotrophin responsiveness is physiologically relevant, we studied the timing of neuronal death in the trigeminal ganglia of embryos that are homozygous for null mutations in the trkA, trkB and trkC genes, which encode receptor tyrosine kinases for NGF, BDNF and NT3, respectively. In wild-type embryos, the number of pyknotic nuclei increased from E11 to peak between E13 and E14, and decreased gradually at later ages, becoming negligible by birth. Neuronal death in the trigeminal ganglia of trkA−/− embryos also peaked between E13 and E14, but was almost threefold greater than in wild-type embryos at this stage. Whereas there was no significant difference between the number of pyknotic nuclei in trkA−/− and wild-type embryos at E11 and E12, there was a substantial increase in the number of pyknotic nuclei in the trigeminal ganglia of trkB−/− at these earlier stages. Counts of the total number of neurons in E13 trigeminal ganglia revealed a marked decrease in trkB−/− but not trkA−/− or trkC−/− embryos. Consistent with the later onset of excessive neuronal death in trkA−/− embryos, there was a marked decrease in the neuronal complement of the trigeminal ganglia of trkA−/− embryos at E15. These results demonstrate that TrkB signalling is required for the in vivo survival of many trigeminal neurons during the early stages of target field innervation before they become NGF-dependent.

Prevention of thrombosis following deep arterial injury in rats by bovine activated protein C requiring co-administration of bovine protein S

2003 ◽  
Vol 90 (08) ◽  
pp. 227-234 ◽  
Author(s):  
Björn Dahlbäck ◽  
Björn Arnljots ◽  
Karl Malm
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Arterial Injury ◽  
Control Group ◽  
Clotting Time ◽  
Antithrombotic Effect

SummaryThe antithrombotic effect of bovine activated protein C (bAPC) given with or without bovine protein S (bPS) was investigated in a rat model of deep arterial injury. A segment of the left common carotid artery was isolated between vascular clamps and opened longitudinally. An endarterectomy was performed and the arteriotomy was closed with a running suture, whereafter the vessel was reperfused by removing the clamps. The antithrombotic effect (vascular patency rates 31 minutes after reperfusion) and the arteriotomy bleeding were measured. Ten treatment groups each containing 10 rats and a control group of 20 animals were in a blind random fashion given intravenous bolus injections of increasing doses of activated protein C, with or without co-administration of protein S. The groups received either bAPC alone (0.8, 0.4, 0.2 or 0.1 mg/kg), bAPC (0.8, 0.4, 0.2, 0.1 or 0.05 mg/kg) combined with bPS (0.6 mg/kg), or bPS alone (0.6 mg/kg) whereas the control group received vehicle only. Administered alone, bAPC or bPS had no antithrombotic effect, regardless of dosage. In contrast, all groups that were treated with bAPC in combination with bPS demonstrated a significant antithrombotic effect, as compared to controls. Neither bAPC, bPS, nor the combination of bAPC and bPS increased the arteriotomy bleeding significantly compared to controls. In vitro clotting assays using bAPC or bPS alone yielded only minor prolongation of clotting time, whereas bAPC combined with bPS prolonged the clotting time considerably, demonstrating the dependence on the APC-cofactor activity of bPS for expression of anticoagulant activity by bAPC. In conclusion, our study shows the in vivo significance of protein S as a cofactor to activated protein C, and that potent anti-thrombotic effect can be achieved by low doses of bAPC combined with bPS, without producing hemorrhagic side effects.

In Vivo Anti-Thrombotic Potency of Engineered Activated Protein C Variants.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2704-2704
Author(s):  
Laurent O. Mosnier ◽  
Jose A. Fernandez ◽  
Antonella Zampolli ◽  
Xia V. Yang ◽  
Zaverio M. Ruggeri ◽  
...  
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Dependent Manner ◽  
Thrombosis Model ◽  
Factor Va ◽  
Cofactor Activity

Abstract Activated protein C (APC) has both anticoagulant activity via inactivation of factors Va and VIIIa and cytoprotective activities on cells that include anti-apoptotic and anti-inflammatory activities, alterations of gene expression profiles and protection of endothelial barrier function. The relative importance of APC’s anticoagulant activity vs. APC’s direct cytoprotective effects on cells for reduction of mortality in severe sepsis patients and protective effects in animal injury models is not entirely clear. In this current study, genetically engineered APC variants with different activity spectra were tested for in vivo anti-thrombotic potency. Recently we made a non-anticoagulant APC variant, 5A-APC (RR229/230AA and KKK191-193AAA), that retains normal in vitro cytoprotective effects and an ability to reduce mortality in murine sepsis models (Kerschen et al, ASH2006, J Exper Med, 2007). In contrast to 5A-APC, mutation of E149 to A in APC increased anticoagulant activity in clotting assays while diminishing cytoprotective effects on cells. Murine APC variants, E149A-APC and 5A-APC (KKK192-194AAA + RR230/231AA) were used to determine in vivo anti-thrombotic potency in an acute carotid artery thrombosis model in mice, using FeCl3-induced injury. Under the conditions employed, first occlusion occurred within 3.5 min (mean: 171 sec; range 150-200 sec) in the absence of APC. Murine wild type (wt)-APC effectively delayed time to first occlusion in a dose-dependent manner (0 to 1.8 mg/kg wt-APC; mean: 561 sec; range 400-960 sec). The E149A-APC variant exhibited potent in vivo anti-thrombotic activity (1.8 mg/kg; mean: 1020 sec; range 540- >1600 sec) and was superior to wt-APC as evident by the absence of appreciable occlusion in 2/6 E149A-APC vs. 0/6 wt-APC treated animals. Thus E149A-APC was hyperactive in plasma clotting assays as well as hyperactive in an acute FeCl3-induced arterial thrombosis model. To test the hypothesis that an increased protein S cofactor activity contributed to its enhanced anticoagulant activity, E149A-APC anticoagulant activity was tested in normal and protein S deficient plasma. Compared to wt-APC, E149A-APC showed 3-fold increased anticoagulant activity in normal plasma but not in protein S deficient plasma. In studies with purified proteins, protein S concentrations required for half-maximal stimulation of factor Va inactivation by E149A-APC were 3-fold lower compared to wt-APC, whereas factor Va inactivation rates were indistinguishable in the absence of protein S. These data support our hypothesis that increased protein S cofactor activity is, at least partially, responsible for the observed hyper anticoagulant and anti-thrombotic potency in vitro and in vivo. In contrast to E149A-APC, 5A-APC was severely deficient in anti-thrombotic activity in vivo. Even at concentrations up to 8 mg/kg, 5A-APC (mean: 245 sec; range 172-300 sec) failed to delay significantly time to first occlusion compared to no APC. These data highlight important distinctions between structural requirements for APC’s anticoagulant, anti-thrombotic and cytoprotective functions. Engineered APC variants with differentially altered activities (e.g. cytoprotective vs. anticoagulant) may lead to safer or better therapeutic APC variants for a variety of indications including sepsis, ischemic stroke or other pathologies.

Platelet Factor 4 Mediates Activated Protein C Resistance by Impairment of Protein S Cofactor Enhancement

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 20-20
Author(s):  
Roger JS Preston ◽  
Jennifer A Johnson ◽  
Fionnuala Ni Ainle ◽  
Shona Harmon ◽  
Owen P. Smith ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Protein S ◽  
Endothelial Barrier ◽  
Platelet Factor ◽  
Barrier Permeability ◽  
Gla Domain ◽  
Anti Inflammatory

Abstract Platelet factor 4 (PF4) is an abundant platelet α-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the γ-carboxyglutamic acid (Gla) domain of protein C to significantly enhance activated protein C (APC) generation by the thrombin-thrombomodulin complex on the surface of endothelial cells. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. The functional consequences of the interaction between the APC Gla domain and PF4 in relation to APC anticoagulant, anti-inflammatory and anti-apoptotic functions have not previously been fully defined. In a tissue factor-initiated thrombin generation assay, APC impaired thrombin generation as previously described. However PF4 inhibited APC anticoagulant activity in a concentration-dependent manner (IC50 for PF4 inhibition of APC anticoagulant function, 11μg/ml). In contrast, addition of two other cationic polypeptides protamine and polybrene, both significantly enhanced APC anticoagulant activity in plasma. To elucidate the mechanism through which PF4 inhibits APC anticoagulant activity, we utilized a phospholipid-dependent FVa proteolysis time course assay. In the absence of protein S, PF4 had no effect upon FVa proteolysis by APC, indicating that PF4 does not influence the ability of APC to interact with either anionic phospholipids or FVa. However, in the presence of protein S, PF4 significantly inhibited APC-mediated FVa proteolysis (3–5 fold). Collectively, these findings demonstrate that in addition to enhancing APC generation, PF4 also significantly attenuates APC anticoagulant activity in plasma by impairing critical protein S cofactor enhancement of FVa proteolysis, and suggest that PF4 contributes to the poorly-understood APC resistance phenotype associated with activated platelets. APC bound to the endothelial cell protein C receptor (EPCR) via its Gla domain can activate PAR-1 on endothelial cells, triggering complex intracellular signaling that result in anti-inflammatory and anti-apoptotic cellular responses. To ascertain whether PF4 interaction with the protein C/APC Gla domain might impair APC-EPCR-PAR-1 cytoprotective signaling, APC protection against thrombin-induced endothelial barrier permeability and staurosporine-induced apoptosis in the presence of PF4 was determined. APC significantly attenuated thrombin-induced endothelial cell barrier permeability, as expected. PF4 alone (up to 1μM) had no independent effect upon endothelial barrier permeability, and did not protect against thrombin-mediated increased permeability. In contrast to its inhibition of APC anticoagulant activity, PF4 did not significantly inhibit the endothelial barrier protective properties of APC. To determine whether PF4 might interfere with APC-mediated cytoprotection, staurosporine-induced apoptosis in EAhy926 cells was assessed by RT-PCR quantification of pro-apoptotic (Bax) to anti-apoptotic (Bcl-2) gene expression. Pre-treatment of EAhy926 cells with APC decreased the Bax/Bcl-2 ratio close to that determined for untreated EAhy926 cells. PF4 alone, or in combination with APC, had no effect upon apoptosis-related gene expression as determined by alteration of Bax/Bcl-2 expression ratios in response to staurosporine. In summary, PF4 inhibits APC anticoagulant function via inhibition of essential protein S cofactor enhancement in plasma, whilst retaining EPCR/PAR-1 mediated cytoprotective signalling on endothelial cells. This provides a rationale for how PF4 can exert prothrombotic effects in vivo, but also mediate enhanced APC generation on the surface of endothelial cells to induce both anti-inflammatory and anti-apoptotic events. Based on these observations, we propose that PF4 acts as a critical regulator of APC generation in vivo, but also targets APC towards cytoprotective, rather than anticoagulant functions at sites of vascular injury with concurrent platelet activation.

Protein S and protein C anticoagulant activity in acute and chronic cardiac ischemic syndromes. Relationship to inflammation, complement activation and in vivo thrombin activity

1994 ◽  
Vol 75 (2) ◽  
pp. 133-142 ◽  
Author(s):  
Armando D'Angelo ◽  
Stefano Gerosa ◽  
Silvana Vigano' D'Angelo ◽  
Alessandra Mailhac ◽  
Alessandro Colombo ◽  
...  
Keyword(s):  
Complement Activation ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Protein S ◽  
Thrombin Activity

Anticoagulant Dysfunction of Human Arg352Trp-Activated Protein C Caused by Defective Factor Va Inactivation

2001 ◽  
Vol 85 (02) ◽  
pp. 274-279 ◽  
Author(s):  
Claudia Rintelen ◽  
Subramanian Yegneswaran ◽  
John Griffin
Keyword(s):  
Venous Thrombosis ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Catalytic Efficiency ◽  
Protein S ◽  
Wild Type ◽  
Factor Va ◽  
Wild Type Protein ◽  
Factor Viiia

SummaryThe dysfunctional mutant R352W-protein C was found in two patients with venous thrombosis. The mutant R352A-protein C was constructed to define the contribution of charge/size of the residue at 352 on protein C (chymotrypsin numbering 187). Compared with wild type-protein C, R352W-protein C showed no difference in activation by thrombin·thrombomodulin or α-thrombin. However, R352W-activated protein C (APC) anticoagulant activity (aPTT assay) was reduced to ~65%. Although the catalytic efficiency of R352W-APC towards the oligopeptide substrate S-2366 was unperturbed, factor Va and R506Q-factor Va were not efficiently inactivated by R352W-APC compared with wild type-APC. R352A-APC showed reduced anticoagulant activity and reduced efficiency in factor Va inactivation and in factor VIIIa-inactivation in the presence of protein S. These observations suggest that the dysfunction of R352W-APC in factor Va inactivation may be one of the mechanisms leading to venous thrombosis in affected patients and that R352 plays an important role in the physiological functioning of APC.

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 (<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.

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