Vitamin K-dependent Plasma Protein Research Articles

Protein Z measurement in acute myeloid leukemia patients

Background Protein Z (PZ) is a vitamin K-dependent plasma protein. The role of PZ in the pathogenesis of hemostatic disorders remains to be established. However, a significant association was found between low PZ levels and arterial vascular diseases, pregnancy complications and disseminated intravascular coagulation, stroke and antiphospholipid syndrome. On the other hand, it has been suggested that PZ deficiency is associated with hemorrhagic disorders, perhaps related to capillary fragility.Aim Measurement of the level of PZ in newly diagnosed acute myeloid leukemia (AML) patients and correlate it with bleeding tendency or thrombosis.Patients and methods This study was held on 90 participants divided into 60 adult (AML) patients and 30 healthy controls of matched age and sex from the Internal Medicine Department, Ain Shams University Hospital. Enzyme-linked immunosorbent assay kits are used for the estimation of PZ levels after taking informed consent.Results A low level of PZ was detected in AML patients compared with controls (P<0.001). A statistical correlation was observed between bleeding attacks and PZ level (P=0.008). The best cutoff value for PZ as a predictor for bleeding was more than 350 ng/l by the receiver operating characteristic curve.Conclusion Low PZ level is a cofactor of bleeding in AML patients.

A critical role of the Gas6-Mer axis in endothelial dysfunction contributing to TA-TMA associated with GVHD

AbstractEndothelial dysfunction in the early phases of hematopoietic stem cell transplantation (HSCT) contributes to a common pathology between transplant-associated thrombotic microangiopathy (TA-TMA) and graft-versus-host disease (GVHD), which are serious complications of HSCT. Growth arrest-specific (Gas) 6 structurally belongs to the family of plasma vitamin K-dependent proteins working as a cofactor for activated protein C, and has growth factor-like properties through its interaction with receptor tyrosine kinases of the TAM family: Tyro3, Axl, and Mer. Serum Gas6 levels were significantly increased in HSCT patients with grade II to IV acute GVHD (aGVHD), and Gas6 and Mer expression levels were upregulated in aGVHD lesions of the large intestine and skin. The increased serum Gas6 levels were also correlated with elevated lactate dehydrogenase, d-dimer, and plasmin inhibitor complex values in HSCT patients with aGVHD. In human umbilical vein endothelial cells (ECs), exogenous Gas6 or the exposure of sera isolated from patients with grade III aGVHD to ECs induced the downregulation of thrombomodulin and the upregulation of PAI-1, as well as the upregulation of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, which were inhibited by UNC2250, a selective Mer tyrosine kinase inhibitor. In mouse HSCT models, we observed hepatic GVHD with hepatocellular apoptosis, necrosis, and fibrosis, as well as TA-TMA, which is characterized pathologically by thrombosis formation in the microvasculature of the liver and kidney. Of note, intravenous administration of UNC2250 markedly suppressed GVHD and TA-TMA in these mouse HSCT models. Our findings suggest that the Gas6-Mer axis is a promising target for TA-TMA after GVHD.

A Critical Role of Growth Arrest-Specific Gene 6-Mer Axis in the Pathogenesis of Endothelial Damage Contributing to Thrombotic Microangiopathy Associated with Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

Graft-versus-host disease (GVHD) and thrombotic microangiopathy (TMA) are severe complications of allogeneic hematopoietic stem cell transplantation (allo-SCT). Host endothelial cells are targets of alloreactive donor cytotoxic T lymphocytes or various proinflammatory cytokines following allo-SCT, and endothelial damage plays an important role in the pathogenic mechanism(s) of TMA associated with GVHD. However, the detailed mechanism(s) of TMA as well as GVHD have not yet been fully elucidated. Growth arrest-specific (Gas) 6 structurally belongs to the family of plasma vitamin K-dependent proteins working as a cofactor for activated protein C, and has growth factor-like properties through its interaction with receptor tyrosine kinases of the TAM family; Tyro3, Axl, and Mer. Gas6 and the TAM receptor tyrosine kinases have been reported to be associated with systemic inflammatory and autoimmune disorders, as well as hemostatic abnormalities. We hypothesized that Gas6-TAM pathway signaling contributed to endothelial dysfunction in the pathogenesis of TMA associated with GVHD. ELISA showed that the serum levels of Gas6 were markedly increased in the all-SCT patients with grades 2 or 3 GVHD at 21-35 days after stem-cell engraftment. The increased serum levels of Gas6 were also correlated with the elevated D-dimer and plasmin-α2 plasmin inhibitor complex values in the allo-SCT patients. Immunostaining showed that the expression of Gas6 and Mer was significantly upregulated in the intestinal acute GVHD lesion. The platelet aggregation test showed that UNC2250 (5 μM), a selective Mer tyrosine kinase inhibitor markedly suppressed increased platelet aggregation caused by exogenous Gas6 and collagen. Using Western blot analysis, exposure of human umbilical vein endothelial cells (ECs) to, Gas6 or 25-50% (vol/vol) serum isolated from patients with GVHD caused the downregulation of thrombomodulin (TM) and plasminogen activator inhibitor type-1 (PAI-1) as well as the upregulation of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), as assessed by Western blot analyses, which were inhibited by UNC2250 or Mer siRNA. Furthermore, exogenous Gas6 or the addition of serum from patients with GVHD to ECs induced apoptosis of ECs with activation of caspase-3 and poly (ADP-ribose) polymerase, which were inhibited by UNC2250 or Mer siRNA. These results indicate that the Gas6-Mer axis may be crucial for platelet activation, endothelial damage and subsequent hypercoagulation in the pathogenesis of TMA associated with GVHD after allo-SCT. Furthermore, in the murine models of allo-SCT, we observed hepatic GVHD with hepatocellular apoptosis, necrosis and fibrosis and TMA characterized by thrombosis formation in the microvasculature of liver and kidney. The intravenous administration of 3 mg/kg UNC2250 markedly inhibited hepatic GVHD and TMA formations of liver and kidney in murine models of allo-SCT at 21 days after allo-SCT. The present study provides new evidence that the Gas6-Mer axis may be involved in the mechanism(s) underlying the progression of the pathogenesis of TMA associated with GVHD after all-SCT. We suggest that Gas6-Mer axis may be an attractive therapeutic target for treating TMA associated with GVHD caused by endothelial damage. DisclosuresNo relevant conflicts of interest to declare.

Coagulation Factor X Regulated by CASC2c Recruited Macrophages and Induced M2 Polarization in Glioblastoma Multiforme

Tumor-associated macrophages (TAMs) constitute a major component of inflammatory cells in the glioblastoma multiforme (GBM) tumor microenvironment. TAMs have been implicated in GBM angiogenesis, invasion, local tumor recurrence, and immunosuppression. Coagulation factor X (FX) is a vitamin K-dependent plasma protein that plays a role in the regulation of blood coagulation. In this study, we first found that FX was highly expressed and positively correlated with TAM density in human GBM. FX exhibited a potent chemotactic capacity to recruit macrophages and promoted macrophages toward M2 subtype polarization, accelerating GBM growth. FX bound to extracellular signal-related kinase (ERK)1/2 and inhibited p-ERK1/2 in GBM cells. FX was secreted in the tumor microenvironment and increased the phosphorylation and activation of ERK1/2 and AKT in macrophages, which may have been responsible for the M2 subtype macrophage polarization. Moreover, although the lncRNA CASC2c has been verified to function as a miR-101 competing endogenous RNA (ceRNA) to promote miR-101 target genes in GBM cells, we first confirmed that CASC2c did not function as a miR-338-3p ceRNA to promote FX expression, and that FX was a target gene of miR-338-3p. CASC2c interacted with and reciprocally repressed miR-338-3p. Both CASC2c and miR-388-3p bound to FX and commonly inhibited its expression and secretion. CASC2c repressed M2 subtype macrophage polarization. Taken together, our findings revealed a novel mechanism highlighting CASC2c and FX as potential therapeutic targets to improve GBM patients by altering the GBM microenvironment.

Low Protein Z levels in patients with plasma cell neoplasms are inversely correlated with IL-6 levels

Patients with multiple myeloma (MM) have an increased thrombotic risk, but pathogenesis remains uncertain. Low levels of Protein Z (PZ), a vitamin K-dependent plasma protein, are associated with venous as well as arterial thrombosis. The purpose of this study was to analyze PZ levels in patients with plasma cell neoplasms. Patients and methodsThe study consisted of 64 plasma cells neoplasm patients and 42 healthy individuals. Clinical investigations included measurement of plasma PZ and IL-6 levels. ResultsPZ levels in patients with plasma cell neoplasms were significantly lower compared to healthy controls in the entire cohort (1392±659 vs.2010±603ng/mL, P<0.01), as well as in specific disease subgroups; symptomatic MM (1428±652ng/mL, p<0.01), smoldering MM (1437±883ng/mL, p=0.045) and monoclonal gammopathy of undetermined significance (MGUS) (1247±593ng/mL, p=0.01). PZ was negatively correlated with IL-6 levels in MM patients (r=−0.7, P<0.01). There was no significant difference in PZ levels between patients with or without thrombotic event. ConclusionPlasma cell neoplasm patients have low levels of PZ. This is presumably related to the increased IL-6 production by the bone marrow microenvironment, and could have a potential role in the increased thrombotic tendency in those patients.

Abstract 548: Molecular Mechanism Behind Protein S Deficiency in Obese Patients

Introduction: Protein S is a vitamin K-dependent plasma protein, produced mainly in the liver; Protein S circulates in the blood at a concentration of 450 nM. Protein S is an anticoagulant, serving as a cofactor for APC and TFPI, and as an inhibitor of Factor IX (FIXa). Protein S deficiency causes deep vein thrombosis (DVT), increased risk of inflammation and, because DVT is a complication commonly observed in obese individuals, Protein S deficiency might be associated with obesity. Aim: To identify a correlation between Protein S deficiency and obesity, and identify the probable molecular mechanism behind the Protein S deficiency in the obese subjects. Methods: Immunoblots, ELISA, EMSA, CHIP, aPTT assay, and thrombin generation assay. Results: By ELISA, we measured a decrease in Protein S level in obese mice compared with wild type mice. In obesity, the liver becomes hypoxic, thus, we hypothesized that hypoxia and hypoxia inducible factor 1 alpha (HIF1α) may regulate Protein S expression in obesity. We found that a high fat diet induced HIF1α stability in mice. HIF1α levels were inversely proportional to Protein S levels, suggesting that HIF1α is a negative regulator of Protein S expression. We further identified a putative HIF1α binding site in the Protein S promoter, and, by using in vitro and in vivo assays, we demonstrated that HIF1α binds directly to the Protein S promoter and suppresses transcription. We further confirmed HIF1α-mediated Protein S transcriptional regulation in vivo, Plasma Protein S levels are increased in the liver-specific HIF1α knockout mouse whereas, liver-specific overexpression of HIF1α reduced the concentration of Protein S in the plasma. Conclusion: We conclude that HIF1α regulates Protein S expression in mouse liver and in obesity. Inhibition of HIF1α or intravenous injection of Protein S may reduce the occurrence of DVT in obese individuals.

Circulating protein Z concentration, PROZ variants, and unexplained cerebral infarction in young and middle-aged adults.

Protein Z (PZ) is a vitamin K-dependent plasma protein that exhibits both pro- and anticoagulant properties. Both low and high PZ levels have been linked to ischaemic stroke. Although PZ-lowering gene variants have been found to be less common in ischaemic stroke, the relationship remains unclear. We investigated PZ levels and PROZ variants in a multi-ethnic case-control study of unexplained stroke in participants aged 18 to 64. Plasma PZ was measured in cases (≥2 months post-stroke) and controls. PZ polymorphisms G79A (rs3024735) and A13G (2273971) were genotyped. A combined genetic score (0-4 minor alleles) was created assuming additive effects. A total of 715 individuals (1:1.4 cases:controls) was included. Analyses revealed evidence of a non-linear association. After adjusting for demographic and clinical covariates, PZ levels >2.5 µg/ml (90th %ile) were significantly associated with cryptogenic stroke (OR 2.41 [95 % CI 1.34, 4.34]) as compared with lower levels. Higher genetic score was related to progressively lower levels of PZ, and the presence of four minor alleles was associated with lower odds of stroke (adjusted OR 0.26 [95 % CI 0.07, 0.96]) versus 0 minor alleles. In this multi-ethnic study of young and middle-aged adults, there was evidence of a non-linear positive association between PZ level and unexplained stroke, with a directionally consistent association for genetic variants related to PZ levels and cryptogenic stroke. These findings support elevated PZ levels as a risk factor for cryptogenic stroke.

Characterization of porcine GAS6 cDNA gene and its expression analysis in weaned piglets.

The growth arrest-specific 6 gene (GAS6) is a member of the family of plasma vitamin K-dependent proteins, which are able to bind to phospholipids using an N-terminal gamma-carboxyglutamic acid domain. A recent report has demonstrated that the GAS6 gene can promote fat deposition and is associated with an increased number of fat cells in mice. In order to investigate whether GAS6 expression is associated with meat quality in pigs, a 2382-bp cDNA sequence of the porcine GAS6 gene (GenBank accession No. KC526197) was first obtained using rapid amplification of cDNA ends from porcine longissimus dorsi tissue. One A/G single nucleotide polymorphism anchored in exon 12 was genotyped using the marker PCR-RFLP-BglI, and the G allele was dominant in the pig breeds tested. Quantitative real-time polymerase chain reaction showed that the porcine GAS6 gene was expressed in all tissues examined in weaned male Shaziling (SZL) and Yorkshire (YS) weanling piglets, and mRNA expression of GAS6 in the longissimus dorsi tissue of SZL piglets was significantly higher than that in YS piglets (P < 0.05). The GAS6 protein was likely to be a membrane protein and was detected in longissimus dorsi tissue from SZL and YS piglets using immunohistochemistry, with an abundant protein expression index (P > 0.05). The results imply that the GAS6 gene can be considered a potential candidate for meat quality trait selection and fat deposition in pigs.

Growth arrest-specific gene 6 (gas6) and vascular hemostasis.

Gas6 (growth arrest-specific 6) belongs structurally to the family of plasma vitamin K-dependent proteins. Gas6 has a high structural homology with the natural anticoagulant protein S, sharing the same modular composition. Interestingly, despite the presence of a γ-carboxyglutamic acid domain in its structure, no role in the coagulation cascade has been identified for gas6. Gas6 has been shown to be involved in vascular homeostasis and more precisely is involved in proliferation, apoptosis, efferocytosis, leukocyte migration, and sequestration and platelet aggregation. It is also involved in the activation of different cell types, from platelets to endothelial and vascular smooth muscle cells. Thus, it has been shown to play a role in several pathophysiological processes such as atherosclerosis, cancer, and thrombosis. Interestingly, studies using gas6 null mice highlighted that gas6 may represent a novel potential target for anticoagulant therapy, because these animals are protected from lethal venous thromboembolism without excessive bleeding. However, the mechanism in thrombus occurrence remains to be further explored. In the present review, we will focus on the role of gas6 in innate immunity, atherosclerosis, thrombosis, and cancer-related events.

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

Abstract 1197Coagulation 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.

Protein Z is present in human breast cancer tissue

Thromboembolic complications are common findings in breast cancer patients [1, 2]. Hemostatic system components play a role in tumor progression, independent of their role in hemostasis [1, 2]. Since the report of multifactorial activation of factor X in cancer patients [1], inhibitors which control the activity of this factor have attracted attention. Protein Z (PZ), a vitamin K-dependent plasma nonpeptidase glycoprotein [3, 4], is involved in one mechanism responsible for the direct inhibition of factor Xa. PZ circulates in plasma complexed with PZ-dependent protease inhibitor (ZPI) and enhances the rate of ZPImediated factor Xa inhibition [5, 6]. The presence of ZPI in breast cancer tissue was previously reported [7], while information on PZ in tumor malignant tissue is lacking. The purpose of the present study was to evaluate the localization of PZ (both protein and mRNA) at the primary site of human breast cancer. Tumor fragments were obtained during the surgical treatment of 13 previously untreated breast cancer patients (clinical stage T1-2N0M0). Studies were performed on 4 cases of G2 and 9 cases of G3 invasive ductal carcinomas. Immunohistochemical studies (IHC) were performed (Vectastain Kits, Vector Laboratories, Burlingame, CA, USA) [7] employing a polyclonal antibody against homogeneous, plasma-derived human PZ (prepared in rabbits, and purified from immune sera by protein A-Sepharose chromatography). The antibody did not cross-react with all other purified human plasma vitamin K-dependent proteins including prothrombin, protein C, protein S, factor VII, factor X, and factor IX by ELISA. Controls consisted of omission of the primary antibody from the procedure. The control fragments of normal breast tissues obtained from neoplasm-free surgical margins were processed simultaneously as well. Antigen staining was detected by the dark brown reaction product. A semiquantitative analysis of PZ IHC expression in cancer cells (based on both the percentage of cancer cells with PZ positive staining and the intensity of staining) was performed [7]. Immunoreactive score (IRS) values of 1–4 were interpreted as weak, 5–8 as medium, and 9–12 as strong PZ expression. In situ hybridization (ISH) method employed a biotin-labeled 25-nucleotide single-stranded DNA probe (probe sequence: 50 Biotin-CGTCATACCGCATGTGCA CATGGAC-Biotin 30) specific for PZ mRNA. The probe was synthesized by Sigma-Aldrich, Poznan, Poland. The ISH protocol of R&D Systems (R&D Systems, Minneapolis, MN, USA) was followed. Hybrids were detected with rabbit anti-biotin monoclonal antibodies according to the IHC ABC technique associated with ImmunoMax amplification technique described in detail elsewhere [7]. Negative controls included hybridization without addition of the molecular probe and incubation of slides in a RNase A solution (R&D Systems, Minneapolis, MN, USA) before hybridization. Reaction results appeared as dark brown staining. E. Sierko M. Z. Wojtukiewicz (&) Department of Oncology, Medical University, 12 Ogrodowa St, Bialystok, Poland e-mail: mzwojtukiewicz@gmail.com

Down-regulation of PROS1 Gene Expression by 17β-Estradiol via Estrogen Receptor α (ERα)-Sp1 Interaction Recruiting Receptor-interacting Protein 140 and the Corepressor-HDAC3 Complex

Pregnant women show a low level of protein S (PS) in plasma, which is known to be a risk for deep venous thrombosis. 17Beta-estradiol (E(2)), an estrogen that increases in concentration in the late stages of pregnancy, regulates the expression of various genes via the estrogen receptor (ER). Here, we investigated the molecular mechanisms behind the reduction in PS levels caused by E(2) in HepG2-ERalpha cells, which stably express ERalpha, and also the genomic ER signaling pathway, which modulates the ligand-dependent repression of the PSalpha gene (PROS1). We observed that E(2) repressed the production of mRNA and antigen of PS. A luciferase reporter assay revealed that E(2) down-regulated PROS1 promoter activity and that this E(2)-dependent repression disappeared upon the deletion or mutation of two adjacent GC-rich motifs in the promoter. An electrophoretic mobility shift assay and DNA pulldown assay revealed that the GC-rich motifs were associated with Sp1, Sp3, and ERalpha. In a chromatin immunoprecipitation assay, we found ERalpha-Sp protein-promoter interaction involved in the E(2)-dependent repression of PROS1 transcription. Furthermore, we demonstrated that E(2) treatment recruited RIP140 and the NCoR-SMRT-HDAC3 complex to the PROS1 promoter, which hypoacetylated chromatin. Taken together, this suggested that E(2) might repress PROS1 transcription depending upon ERalpha-Sp1 recruiting transcriptional repressors in HepG2-ERalpha cells and, consequently, that high levels of E(2) leading to reduced levels of plasma PS would be a risk for deep venous thrombosis in pregnant women.

Molecular Basis for A Thrombophilic Patient's Combined Deficiencies in Proteins C and S.

Abstract 4198 Background/objectivesProteins C and S (PC and PS) are vitamin K-dependent plasma proteins with anticoagulant properties. Protein S functions as a non-enzymatic cofactor for activated protein C (APC). APC proteolytically degrades coagulation factors Va and VIIIa, thereby diminishing the activities of the prothrombinase and tenase complexes, respectively. The human PC gene (PROC) is located on the long arm of chromosome 2 (2q13-q14) and contains 9 exons which code for 461 amino acid residues. The human PS (PROS1) gene resides on chromosome 3 (3p11.1-q11.2) and contains 15 exons coding for 636 amino acid residues. Hereditary PS and PC deficiencies are both autosomal dominant disorders in which patients have diminished functional levels of the respective protein (usually ∼50% relative to normal controls). Clinically, this results in increased propensity toward thromboembolic disease, also known as thrombophilia. In this study, we describe a unique thrombophilic patient who has combined deficiencies in both proteins C and S. The objective of the study was to elucidate the precise genetic defect(s) causing these deficiencies. MethodsFollowing purification of the patient's DNA from peripheral blood leukocytes, PCR amplifications were performed using oligonucleotide primers flanking all exons of the PROS1 and PROC genes. In addition, the 400bp region upstream of the first exon of PROS1 (corresponding to the promoter region) was also amplified by PCR. The PCR products were purified and their DNA sequences determined in both forward and reverse directions using the dye-terminator method. The resultant nucleotide sequences were compared with the PROS1 and PROC reference sequences [web address]. ResultsThe patient was found to be heterozygous for a novel missense PROC gene mutation in exon 8, which codes in part for the proteolytic domain of protein C. The resulting ValàGly substitution of residue 221 is in close proximity to the catalytic triad, which could abrogate its enzymatic activity. Although no mutations were present in any of the patient's PROS1 exons, there was a novel heterozygous CàG nucleotide substitution in the PROS1 promoter region. This substitution is present within an Sp1 transcription factor binding site that is highly conserved among mammals. Therefore, this mutation could significantly diminish expression of the otherwise normal PROS1 gene, leading to the observed decreased protein S level. Both mutations are unreported in the literature and are not listed in the PROC and PROS1 mutation databases. ConclusionsWe have successfully identified two novel genetic defects leading to deficiencies of the anticoagulant proteins C and S in a patient with thrombophilia. Further studies are underway to confirm the suggested biochemical effects of the mutation on protein C and protein S gene expression. Disclosures:No relevant conflicts of interest to declare.

Platelets Contribute to Protein S Cleavage In Vivo.

Abstract 2124Poster Board II-101 Background:Protein S, a vitamin K-dependent plasma protein, is one of the molecules involved in down-regulation of the coagulation process. Protein S serves as a cofactor of activated protein C (APC) in the proteolytic inactivation of activated factor V and VIII. Protein S is also able to exert its anticoagulant activity independent of APC, e.g. by supporting the anticoagulant activity of tissue factor pathway inhibitor. In plasma, protein S circulates in two forms: a single chain molecule with full anticoagulant activity and a two-chain form with reduced anticoagulant activity. This two-chain variant is the result of cleavage in the protease sensitive loop, the so-called thrombin sensitive region (TSR). The enzyme responsible for the presence of cleaved protein S in the circulation is not known yet. In vitro, thrombin and factor Xa, both enzymes of the coagulation cascade, are able to cleave the TSR. In a recent study (Brinkman et al, J Thromb Haemost 2005; 3: 2712-2720), it was shown that these enzymes do not cleave protein S under physiological conditions. Instead, a platelet protease was found to cleave protein S in plasma during tissue factor induced clotting in vitro. We hypothesize that this platelet protease may be responsible for the presence of cleaved protein S in the circulation. Objective:To correlate cleaved protein S levels in the circulation with the activity of platelet-associated protein S cleaving protease and to evaluate the impact of protein S cleavage on its anticoagulant activity in terms of total protein S activity. Method:Protein S cleavage was evaluated by immunological methods employing a monoclonal antibody specific for uncleaved, single chain protein S. The total anticoagulant activity of protein S (APC-independent and APC-dependent) was assessed employing thrombography. Results:We observed in the in vitro thrombin generation test massive generation of thrombin in protein S deficient plasma that was dose-dependently inhibited by intact, single chain protein S. TSR-cleaved protein S had totally lost its anticoagulant activity. In plasma from healthy volunteers, cleaved protein S expressed as % of the total amount of protein S ranged between 8 and 51% with an average normal value of 26 ± 9% (mean ± SD, n=46). Levels of intact protein S correlated well with total protein S activity in the individual plasma samples, while this correlation was not observed for levels of TSR-cleaved protein S. These data suggest that not only in vitro but also in vivo, cleavage of protein S has an impact on its anticoagulant activity. A significant reduction of levels of cleaved protein S was observed in chemotherapy induced thrombocytopenia in hematological patients (see Figure). At a platelet count < 50, the average protein S cleavage was dropped to 16 ± 7% (mean ± SD, n=15). Upon platelet transfusion, cleavage of protein S dramatically increased to 36 ± 11% (mean ± SD, n=11). Levels of total protein S were similar in healthy volunteers and thrombocytopenic patients before and after platelet transfusion. It thus appears that platelets contribute to protein S cleavage in vivo. However, in healthy individuals showing a wide range of protein S cleavage (see Figure), no correlation was observed between platelet count (200-400 .109/l) and protein S cleavage. This suggests additional sources of protease that cleave protein S in vivo. We therefore examined lysates of a variety of cell types for protein S proteolytic activity. Protein S proteolytic activity was absent in lysates of erytrocytes, vascular smooth muscle cells and fibroblasts. Lysates of HUVEC did show protein S proteolytic activity similar to platelets. [Display omitted] Conclusion:Our results strongly suggest that platelets contribute to the proteolytic modification of protein S in vivo resulting in an abolished anticoagulant activity. An additional source of protein S proteolytic activity may be the endothelium. Cleavage of protein S provoked by platelet infusion may contribute to the beneficial effect of platelet transfusion in the treatment of bleeding episodes. Disclosures:No relevant conflicts of interest to declare.

Differential Anticoagulant Effects of Protein S on Vascular Cells and Platelets.

Background: Protein S is a vitamin K-dependent plasma protein and involved in down-regulation of the coagulation process. Protein S serves as a cofactor of activated protein C (APC) in the proteolytic inactivation of activated factor V and VIII. Protein S is also able to exert its anticoagulant activity independent of APC, e.g. by supporting the anticoagulant activity of tissue factor pathway inhibitor (TFPI). The anticoagulant properties of protein S have been thoroughly characterized by in vitro methods. However, fewer studies focus on protein S function on vascular cells. These studies are limited to model systems employing purified coagulation factors. The aim of this study was to investigate the role of protein S in plasma that is in contact with natural cell membranes, including endothelial cells, smooth muscle cells and platelets.Method: We employed thrombography to evaluate protein S function in 50 % v/v recalcified citrated plasma in the presence of washed platelets, cultured umbilical vein endothelial cells or cultured umbilical artery smooth muscle cells. Since we aimed at a comparison between different cellular membranes, micro-particle free plasma was used. As a reference, we also examined synthetic phospholipid membranes composed of phosphatidyl serine, phosphatidyl choline and phosphatidyl ethanolamine in a 2/6/2 molar ratio. Thrombin activity was measured employing the fluorogenic substrate z-Gly-Gly- Arg-AMC. Protein S activity was probed with CLB-PS13, an antibody directed against the protein S Gla-domain. The APC-independent activity of protein S was assayed in the presence of an inhibitory antibody against protein C. In studies employing phospholipids, thrombin generation was triggered with relipidated tissue factor (TF). Expression of TF on endothelial cells was induced during a 6-hour preincubation with PMA.Results: In the presence of CLB-PS13, the APC-independent activity of protein S became apparent as an increase in peak height in the thrombogram. Lag time, time to peak and area under the curve remained essentially unaffected. Peak height was increased two-fold when examining phospholipids at standard conditions (4 μM lipids and 1 pM TF). This increase in peak height by CLB-PS13 was concentration dependent and complete at 10 μg/ml IgG. Increasing the TF concentration from 1 to 5 pM resulted in loss of the APC-independent activity of protein S on these membranes. APC cofactor activity was assessed in the presence of APC. Addition of APC resulted in inhibition of the thrombin formation on phospholipids with an IC50 of 0.4 nM. CLB-PS13 completely abolished this decrease in thrombin generation up to 50 nM APC, irrespective whether 1 or 5 pM TF was present. Our results are compatible with the view that at high procoagulant stimuli the TFPI-cofactor activity of protein S is abolished. Furthermore, these results show that in plasma APC is completely dependent on protein S. Protein S activity on platelets was studied in the presence of 1 pM TF. As for synthetic lipid membranes and in the absence of APC, CLB-PS13 increased the peak height in the thrombogram. APC inhibited platelet mediated thrombin generation (IC50 = 19 nM), and this inhibition was completely abolished by CLB-PS13. These observations suggest that platelets support both APC-dependent and APC-independent anticoagulant activities of protein S. Thrombography on TF-expressing endothelial cells and smooth muscle cells revealed massive thrombin generation that could not be enhanced with CLB-PS13, indicating that protein S does not contribute to regulation of thrombin formation under these conditions. The APC-dependent activity of protein S became apparent as an abolished inhibition by APC in the presence of CLB-PS13. However, APC proved relatively inefficient in inhibiting thrombin generation on TF-expressing vascular cells (IC50 > 50 nM). Inhibition of TF restored the APC-independent protein S activity.Conclusion: Our results indicate that, in plasma, vascular cells and platelets are able to support the APC-dependent as well as the APC-independent anticoagulant activities of protein S. The APC-independent activity on vascular cells is abolished upon increasing TF expression, while the APC-dependent activity of protein S is limited by the relatively low anticoagulant activity of APC on these cell surfaces. We conclude that protein S activity on cells require relatively high levels of APC or low expression of TF.

Role of the PROS1 gene in thrombosis: lessons and controversies

The PROS1 gene codes for the vitaminK-dependent plasma protein, protein S (PS), an essential anticoagulant and multifunctional protein. The association of PS with thrombosis [1] is well established, but the particular anticoagulant activity or activities of PS that are compromised in thrombosis are not entirely clear and the effects of PS deficiency on more recently discovered functions of PS in inflamma tion, apoptosis and receptor activation are unknown. Lessons can be learned from the phenotypes of reported mutations and polymorphisms in the PROS1 gene, but many questions and controversies remain. For greater depth, the reader is referred to extensive databases of PS mutations [2,3], recent reviews [4,5] and molecular models that rationalize PS mutations [6,7]. PS cofactor activity for activated protein C

Protein Z-dependent Protease Inhibitor Binds to the C-terminal Domain of Protein Z

Protein Z (PZ) is a multidomain vitamin K-dependent plasma protein that functions as a cofactor to promote the inactivation of factor Xa (fXa) by PZ-dependent protease inhibitor (ZPI) by three orders of magnitude. To understand the mechanism by which PZ improves the reactivity of fXa with ZPI, we expressed wild-type PZ, PZ lacking the gamma-carboxyglutamic acid domain (GD-PZ), and a chimeric PZ mutant in which both Gla and EGF-like domains of the molecule were substituted with identical domains of fXa. The ZPI binding and the cofactor function of the PZ derivatives were characterized in both binding and kinetic assays. The binding assay indicated that all PZ derivatives interact with ZPI with a similar dissociation constant (K(D)) of approximately 7 nm. However, the apparent K(D) for the chimeric PZ-mediated ZPI inhibition of fXa was elevated 6-fold on PC/PS vesicles and its capacity to function as a cofactor to accelerate the ZPI inhibition of fXa was also decreased 6-fold. The cofactor activity of GD-PZ was dramatically impaired; however, the deletion mutant exhibited a normal cofactor function in solution. A chimeric activated protein C mutant containing the Gla domain of fXa was susceptible to inhibition by ZPI in the presence of PZ. These results suggest that: (i) the ZPI interactive site of PZ is located within the C-terminal domain of the cofactor and (ii) a specific interaction between the Gla domains of PZ and fXa contributes approximately 6-fold to the acceleration of the ZPI inhibition of fXa on phospholipid membranes.

Low protein Z levels but not the protein Z gene G79A polymorphism are a risk factor for ischemic stroke

Background Protein Z (PZ) is a vitamin K-dependent plasma protein that plays a role in both pro-and anticoagulant pathways, but its exact physiological function remains unclear. The aim of this study was to determine the association between the G79A PZ gene polymorphism in intron F, PZ levels and the occurrence of ischemic stroke. Methods We performed a case–control study in 118 Caucasian patients with first ever ischemic stroke or TIA confirmed by CT, and 113 age-and sex-matched population controls. Venous blood samples for PZ levels were collected 7 to 14 days and 3 months after stroke onset. Estimates of relative risk (odds ratios) were adjusted for vascular risk factors. Results The adjusted relative risk of ischemic stroke associated with PZ levels in the lowest quartile versus the highest quartile was 3.0 (95% CI: 1.1–8.7) at 7–14 days, and 5.1 (95% CI: 1.2–21.9) at 3 months after the stroke. PZ levels in the convalescent sample were significantly lower than in the acute sample. In the convalescent sample, odds ratios increased with lower quartiles of protein Z level (test for trend p = 0.02). Thirty-nine patients (33%) and 32 (28%) controls were heterozygous for the G79A PZ gene polymorphism and 4 (3%) patients and 4 (4%) controls had the AA-genotype. The PZ levels were significantly lower in subjects with the AA-genotype and intermediate in heterozygote subjects. The odds ratio of ischemic stroke associated with A-allele carriers versus GG-homozygotes was 1.2 (95% CI: 0.7–2.1). Conclusion No association between the G79A PZ gene polymorphism and the occurrence of stroke was observed. However, low PZ levels are independently associated with an increased risk of ischemic stroke.

Growth arrest-specific gene 6 (GAS6)

GAS6 (growth arrest-specific 6) belongs structurally to the family of plasma vitamin K-dependent proteins. GAS6 has a high structural homology with the natural anticoagulant protein S, sharing the same modular composition and having 40% sequence identity. Despite this, the low concentration of GAS6 in plasma and the pattern of tissue expression of GAS6 suggest a distinct function among vitamin-K dependent proteins. Indeed, GAS6 has growth factor-like properties through its interaction with receptor tyrosine kinases of the TAM family; Tyro3, Axl and MerTK. GAS6 employs a unique mechanism of action, interacting through its vitamin K-dependent GLA (gamma-carboxyglutamic acid) module with phosphatidylserine-containing membranes and through its carboxy-terminal LamG domains with the TAM membrane receptors. During the last years there has been a considerable expansion of our knowledge of the biology of TAM receptors that has lead to a clear picture of their importance in inflammation, haemostasis and cancer, making this system an interesting target in biomedicine. The innate immune response and the coagulation cascade have been shown to be interconnected. Mediators of inflammation are essential in the initiation and propagation of the coagulation cascade, while natural anticoagulants have important anti-inflammatory functions. GAS6 represents a new player in this context, while protein S seems to have new functions beyond its anticoagulant role through its interaction with TAM receptors.

Activated protein C.

Protein C is a vitamin K-dependent plasma protein zymogen whose genetic mild or severe deficiencies are linked with risk for venous thrombosis or neonatal purpura fulminans, respectively. Studies over past decades showed that activated protein C (APC) inactivates factors (F) Va and VIIIa to down-regulate thrombin generation. More recent basic and preclinical research on APC has characterized the direct cytoprotective effects of APC that involve gene expression profile alterations, anti-inflammatory and anti-apoptotic activities and endothelial barrier stabilization. These actions generally require endothelial cell protein C receptor (EPCR) and protease activated receptor-1. Because of these direct cytoprotective actions, APC reduces mortality in murine endotoxemia and severe sepsis models and provides neuroprotective benefits in murine ischemic stroke models. Furthermore, APC reduces mortality in patients with severe sepsis (PROWESS clinical trial). Although much remains to be clarified about mechanisms for APC's direct effects on various cell types, it is clear that APC's molecular features that determine its antithrombotic action are partially distinct from those providing cytoprotective actions because we have engineered recombinant APC variants with selective reduction or retention of either anticoagulant or cytoprotective activities. Such APC variants can provide relatively enhanced levels of either cytoprotective or anticoagulant activities for various therapeutic applications. We speculate that APC variants with reduced anticoagulant action but normal cytoprotective actions hold the promise of reducing bleeding risk because of attenuated anticoagulant activity while reducing mortality based on direct cytoprotective effects on cells.