A predictive model for prostate cancer incorporating PSA molecular forms and age.

The diagnostic specificity of prostate specific antigen (PSA) is limited. We aimed to characterize eight anti-PSA monoclonal antibodies (mAbs) to assess the prostate cancer (PCa) diagnostic utility of different PSA molecular forms, total (t) and free (f) PSA and PSA complexed to α1-antichymotrypsin (complexed PSA). MAbs were obtained by immunization with PSA and characterized by competition studies, ELISAs and immunoblotting. With them, we developed sensitive and specific ELISAs for these PSA molecular forms and measured them in 301 PCa patients and 764 patients with benign prostate hyperplasia, and analyzed their effectiveness to discriminate both groups using ROC curves. The free-to-total (FPR) and the complexed-to-total PSA (CPR) ratios significantly increased the diagnostic yield of tPSA. Moreover, based on model selection, we constructed a multivariable logistic regression model to predictive PCa that includes tPSA, fPSA, and age as predictors, which reached an optimism-corrected area under the ROC curve (AUC) of 0.86. Our model outperforms the predictive ability of tPSA (AUC 0.71), used in clinical practice. In conclusion, The FPR and CPR showed better diagnostic yield than tPSA. In addition, the PCa predictive model including age, fPSA and complexed PSA, outperformed tPSA detection efficacy. Our model may avoid unnecessary biopsies, preventing harmful side effects and reducing health expenses.

Plasma protein S residues 37-50 mediate its binding to factor Va and inhibition of blood coagulation.

Protein S (PS) is an anticoagulant plasma protein whose deficiency is associated with increased risk of venous thrombosis. PS directly inhibits thrombin generation by the blood coagulation pathways by several mechanisms, including by binding coagulation factors (F) Va and Xa. To identify PS sequences that mediate inhibition of FVa activity, antibodies and synthetic peptides based on PS sequence were prepared and employed in plasma coagulation assays, purified component prothrombinase assays, binding assays, and immunoblots. In the absence of activated protein C, monoclonal antibody (Mab) S4 shortened FXa-induced clotting in normal plasma but not in PS-depleted plasma. Mab S4 also blocked PS inhibition of FVa-dependent prothrombinase activity in purified component assays in the absence or presence of phospholipids and inhibited binding of PS to immobilised FVa. Epitope mapping identified N-terminal region residues 37-67 of PS as this antibody's epitope. A peptide representing PS residues 37-50 inhibited FVa-dependent prothrombinase activity in a non-competitive manner, with 50% inhibition observed at 11 µM peptide, whereas a peptide with a D-amino acid sequence of 37-50 was ineffective. FVa, but not FXa, bound specifically to the immobilised peptide representing residues 37-50, and the peptide inhibited binding of FVa to immobilised PS. These data implicate PS residues 37-50 as a binding site for FVa that mediates, at least in part, the direct inhibition of FVa-dependent procoagulant activity by PS.

Phosphorylation of protein S by platelet kinases enhances its activated protein C cofactor activity.

Protein S (PS) is a multifunctional plasma protein of the hemostatic and inflammatory pathways, although mechanisms for its regulation are poorly understood. Since certain plasma proteins are regulated through extracellular phosphorylation, we investigated whether the anticoagulant activity of PS is regulated through phosphorylation by platelet-secreted kinases. PS was phosphorylated on exposure to activated platelets or their releasates, as judged by immunoblotting for phospho-amino acids and PS. PS phosphorylation was reduced by specific inhibitors of casein kinase 1 (CK1) and casein kinase 2 (CK2) (10 µM D4476, 100 µM CK2-inhibitory peptide YNLKSKSSEDIDESS). Involvement of CKs in PS phosphorylation was confirmed using purified CK1/CK2. Phosphorylation of PS by purified CK1 did not affect its activated protein C (APC) cofactor activity in activated partial thromboplastin time assays in PS-depleted plasma. However, phosphorylation of PS by CK2 or by CK1/CK2 increased PS cofactor activity ∼1.5-fold (158.7±4.8%, P<0.01) or ∼2-fold (191.5±6.4%, P<0.0001), respectively. The APC cofactor activity of PS in PS-depleted plasma exposed to platelet-secreted kinases was enhanced, while CK2 but not CK1 inhibitors reduced APC cofactor activity. Mass spectrometry revealed a phosphorylated CK2 site at Thr37 within the N-terminal Gla-domain. Thus, platelet-mediated extracellular phosphorylation of PS is a potential mechanism by which its activity is regulated.

Platelet protein S directly inhibits procoagulant activity on platelets and microparticles.

Anticoagulant 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 PS-direct that was similar to or greater than that of Zn(2+)-containing pdPS and much greater than that of Zn(2+)-deficient pdPS. Findings were confirmed using purified pltPS. Platelet-bound pltPS and microparticle-bound 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.

Species-specific anticoagulant and mitogenic activities of murine protein S.

BACKGROUND: The protein C pathway down-regulates thrombin generation and promotes cytoprotection during inflammation and stress. In preclinical studies using models of murine injury (e.g., sepsis and ischemic stroke), murine protein S may be required because of restrictive species specificity. DESIGN AND METHODS: We prepared and characterized recombinant murine protein S using novel coagulation assays, immunoassays, and cell proliferation assays. RESULTS: Purified murine protein S had good anticoagulant co-factor activity for murine activated protein C, but not for human activated protein C, in mouse or rat plasma. In human plasma, murine protein S was a poor co-factor for murine activated protein C and had no anticoagulant effect with human activated protein C, suggesting protein S species specificity for factor V in addition to activated protein C. We estimated that mouse plasma contains 22+/-1 microg/mL protein S and developed assays to measure activated protein C co-factor activity of the protein S in murine plasma. Activated protein C-independent anticoagulant activity of murine protein S was demonstrable and quantifiable in mouse plasma, and this activity was enhanced by exogenous murine protein S. Murine protein S promoted the proliferation of mouse and human smooth muscle cells. The potency of murine protein S was higher for mouse cells than for human cells and similarly, human protein S was more potent for human cells than for mouse cells. CONCLUSIONS: The spectrum of bioactivities of recombinant murine protein S with mouse plasma and smooth muscle cells is similar to that of human protein S. However, in vitro and in vivo studies of the protein C pathway in murine disease models are more appropriately performed using murine protein S. This study extends previous observations regarding the remarkable species specificity of protein S to the mouse.

Plasma protein S contains zinc essential for efficient activated protein C-independent anticoagulant activity and binding to factor Xa, but not for efficient binding to tissue factor pathway inhibitor.

Protein S (PS) is a cofactor for activated protein C (APC), which inactivates coagulation factors (F) Va and VIIIa. Deficiency of protein C or PS is associated with risk of thrombosis. We found that PS also has APC-independent anticoagulant activity (PS-direct) and directly inhibits thrombin generated by FXa/FVa (prothrombinase complex). Here we report that PS contains Zn(2+) that is required for PS-direct and that is lost during certain purification procedures. Immunoaffinity-purified PS contained 1.4 +/- 0.6 Zn(2+)/mol, whereas MonoQ-purified and commercial PS contained 0.15 +/- 0.15 Zn(2+)/mol. This may explain the controversy regarding the validity of PS-direct. Zn(2+) content correlated positively with PS-direct in prothrombinase assays and clotting assays, but APC-cofactor activity of PS was independent of Zn(2+) content. PS-direct and Zn(2+) were restored to inactive PS under mildly denaturing conditions. Conversely, o-phenanthroline reversibly impaired the PS-direct of active PS. Zn(2+)-containing PS bound FXa more efficiently (K(d)(app)=9.3 nM) than Zn(2+)-deficient PS (K(d)(app)=110 nM). PS bound TFPI efficiently, independently of Zn(2+) content (K(d)(app)=21 nM). Antibodies that block PS-direct preferentially recognized Zn(2+)-containing PS, suggesting conformation differences at or near the interface of 2 laminin G-like domains near the PS C terminus. Thus, Zn(2+) is required for PS-direct and efficient FXa binding and may play a role in stabilizing PS conformation.

Factor Va residues 311-325 represent an activated protein C binding region.

Activated protein C (APC) inactivates factor Va (fVa) by proteolytically cleaving fVa heavy chain at Arg(506), Arg(306), and Arg(679). Factor Xa (fXa) protects fVa from inactivation by APC. To test the hypothesis that fXa and APC share overlapping fVa binding sites, 15 amino acid-overlapping peptides representing the heavy chain (residues 1-709) of fVa were screened for inhibition of fVa inactivation by APC. As reported, VP311-325, a peptide comprising residues 311-325 in fVa, dose-dependently and potently inhibited fVa-dependent prothrombin activation by fXa in the absence of APC. This peptide also inhibited the inactivation of fVa by APC, suggesting that this region of fVa interacts with APC. The peptide inhibited the APC-dependent cleavage of both Arg(506) and Arg(306) because inhibition was observed with plasma-derived fVa and recombinant R506Q and RR306/679QQ fVa. VP311-325 altered the fluorescence emission of dansyl-active site-labeled APC(i) but not a dansyl-active site-labeled thrombin control, showing that the peptide binds to APC(i). This peptide also inhibited the resonance energy transfer between membrane-bound fluorescein-labeled fVa (donor) and rhodamine-active site-labeled S360C-APC (acceptor). These data suggest that peptide VP311-325 represents both an APC and fXa binding region in fVa.

Down-regulation of factor IXa in the factor Xase complex by protein Z-dependent protease inhibitor.

Protein Z-dependent protease inhibitor (ZPI) is a serpin inhibitor of coagulation factor (F) Xa dependent on protein Z, Ca2+, and phospholipids. In new studies, ZPI inhibited FIXa in the FXase complex. Since this observation could merely represent inhibition of the FXa product whose activity was measured, inhibition of FIXa was investigated five ways. 1) FXase incubation mixtures with/without ZPI/protein Z were diluted in EDTA; FXa activity was measured after reversal of its inhibition. 2) FXase incubation mixtures were immunoblotted for FXa product. 3) FX activation peptide region was 3H-labeled; release of 3H was used to measure FXase activity. 4) Activity was monitored in a FIXa-based clotting assay. 5) FIXa amidolytic activity was measured. In all cases, FIXa was inhibited by subphysiologic levels of ZPI. Unlike inhibition of FXa, inhibition of FIXa did not strictly require protein Z. Low concentrations of FVIIIa increased the efficiency of ZPI inhibition of FIXa; FVIIIa in molar excess was not protective of FIXa unless FIXa/FVIIIa interacted prior to ZPI exposure. Unusual time courses were observed for inhibition of both FIXa in the FXase complex and FXa in the prothrombinase complex. Activity loss stabilized in <100 s at a level dependent on ZPI concentration, suggesting equilibrium interactions rather than typical covalent serpin-protease interactions. Surface plasmon resonance binding experiments revealed binding and dissociation of ZPI/FIXa with Kd (app) of 9-12 nm, similar to the concentration of ZPI needed for 50% inhibition. ZPI may be an unusual physiologic regulator of both the intrinsic FXase and the prothrombinase complexes.

Both protein S and Gas6 stimulate outer segment phagocytosis by cultured rat retinal pigment epithelial cells.

Survival of the retina requires the daily phagocytosis of photoreceptor outer segments (OS) by the overlying retinal pigment epithelium (RPE). OS phagocytosis by cultured RPE requires serum and we have recently shown that the vitamin K-dependent serum protein, Gas6, can completely replace serum in this process. Surprisingly, however, we show here that 4-month-old Gas6 knockout mice have normal appearing retinas, except for a reduced ratio of outer segment to inner segment length. We also show that removal of Gas6 from serum does not abrogate the ability of serum to support OS phagocytosis by rat RPE. Both of these findings suggest the presence of an additional serum ligand that is able to support OS phagocytosis by RPE cells. Protein S (PS) is a vitamin K-dependent serum protein with a high degree of structural similarity to Gas6, and a well characterized role in blood coagulation. We report here that recombinant rat PS is able to stimulate OS phagocytosis, and similar to Gas6, it does so through a Mer-dependent mechanism. This is the first demonstration of a common role for Gas6 and PS in any biological process. The existence of redundant ligands for Mer-dependent OS phagocytosis underscores the critical role of this process in the maintenance of retinal function.

Low protein Z levels and risk of ischemic stroke: differences by diabetic status and gender.

Protein Z was recently shown to act as an essential cofactor for protein Z-dependent protease inhibitor, a potent downregulator of coagulation Factor Xa. Thus, deficiency of protein Z is hypothesized to lead to a prothrombotic state, but two publications reported opposing results for the relationship of protein Z levels with ischemic stroke in young European subjects (mean age 33-40 years). We performed a study of stroke in a different ethnic population of greater mean age (57 years) to further clarify this issue. An ELISA was developed to measure protein Z antigen in 154 patients with ischemic stroke and in 206 controls in a largely Hispanic population. Low plasma protein Z values were significantly associated with ischemic stroke except in diabetic subjects and females. The mean protein Z value was significantly lower in stroke cases than in controls for nondiabetic subjects [1.78 +/- 0.77 (S.D.) versus 2.28 +/- 0.88 microg/ml, P < 0.0001] and for males (1.90 +/- 0.90 versus 2.42 +/- 0.99 microg/ml, P = 0.0004). Stroke risk was higher in subjects with protein Z levels at or below the fifteenth percentile of controls (

Activated protein C-dependent and -independent anticoagulant activities of protein S have different structural requirements.

Plasma protein S exhibits multiple anticoagulant activities. About 20% of protein S normally circulates in a form that is cleaved in its thrombin-sensitive region (TSR, residues 47-72) and this cleaved protein S is inactive as a cofactor for activated protein C (APC). To clarify whether the same cleavage(s) in the TSR neutralizes both APC-cofactor and APC-independent direct anticoagulant activities, protein S was treated with several proteases, and activities and cleavages were monitored. Thrombin cleaved protein S first at Arg49, which abolished protein S APC-cofactor activity, but not APC-independent activity. A slower second thrombin cleavage at Arg70 abolished the direct prothrombinase inhibitory activity of protein S and its ability to bind phospholipids. Factor Xa cleaved protein S only at Arg60 and abolished APC-cofactor activity but not APC-independent anticoagulant activity. The snake venom enzyme Protac C efficiently cleaved protein S at two sites in the TSR, which impaired both types of protein S anticoagulant activity in the presence of phospholipids. Protac C-cleaved protein S did not compete with Factor Xa for limiting phospholipid surfaces but could still inhibit prothrombinase activity in the absence of phospholipids. Thus, the APC-cofactor activity protein S is significantly more sensitive to structural changes in the TSR than is the APC-independent activity of protein S.