Mary J. Heeb

Lack of Protein S in mice causes embryonic lethal coagulopathy and vascular dysgenesis

Protein S (ProS) is a blood anticoagulant encoded by the Pros1 gene, and ProS deficiencies are associated with venous thrombosis, stroke, and autoimmunity. These associations notwithstanding, the relative risk that reduced ProS expression confers in different disease settings has been difficult to assess without an animal model. We have now described a mouse model of ProS deficiency and shown that all Pros1-/- mice die in utero,from a fulminant coagulopathy and associated hemorrhages. Although ProS is known to act as a cofactor for activated Protein C (aPC), plasma from Pros1+/- heterozygous mice exhibited accelerated thrombin generation independent of aPC, and Pros1 mutants displayed defects in vessel development and function not seen in mice lacking protein C. Similar vascular defects appeared in mice in which Pros1 was conditionally deleted in vascular smooth muscle cells. Mutants in which Pros1 was deleted specifically in hepatocytes, which are thought to be the major source of ProS in the blood, were viable as adults and displayed less-severe coagulopathy without vascular dysgenesis. Finally, analysis of mutants in which Pros1 was deleted in endothelial cells indicated that these cells make a substantial contribution to circulating ProS. These results demonstrate that ProS is a pleiotropic anticoagulant with aPC-independent activities and highlight new roles for ProS in vascular development and homeostasis.

Gas6, a ligand for the receptor protein-tyrosine kinase Tyro-3, is widely expressed in the central nervous system.

Gas6 (growth arrest specific gene-6) is a ligand for members of the Axl subfamily of receptor protein-tyrosine kinases. One of these receptors, Tyro-3, is widely expressed in the central nervous system. We have used biochemical and histological techniques, including in situ hybridization, to determine the expression patterns of Gas6 mRNA and protein during development. Gas6 is widely expressed in the rat central nervous system (CNS) beginning at late embryonic stages and its levels remain high in the adult. Gas6 is detected as a single 85 kDa protein, which is encoded by a single 2.5 kb mRNA species. At embryonic day 14 it is detected in the heart, blood vessels, testes, choroid plexus, and in the ventral spinal cord. In the adult, Gas6 is expressed in the cerebral cortex, (predominantly in layer V), the piriform cortex, and the hippocampus (areas CA1, CA3 and the dentate gyrus). It is also expressed in thalamic and hypothalamic structures, the midbrain, and in a subset of motor and trigeminal nuclei. In the cerebellum, it is expressed in Purkinje neurons and deep cerebellar nuclei. Protein S, a protein related to Gas6, is only detected at low levels in the CNS. The spatial and temporal profiles of Gas6 expression suggest that it could potentially serve as the physiologically relevant ligand for Tyro-3 in the postnatal rat nervous system.

Reversible Regulation of Tissue Factor–Induced Coagulation by Glycosyl Phosphatidylinositol–Anchored Tissue Factor Pathway Inhibitor

Endothelial and tumor cells synthesize tissue factor pathway inhibitor (TFPI-1), which regulates tissue factor (TF) function by TF. VIIa. Xa. TFPI-1 quaternary complex formation (where VIIa and Xa are coagulation factors) and by translocation of these complexes into glycosphingolipid-rich microdomains of the cell membrane. Recombinant TFPI-1 added exogenously to cells is targeted to a degradation pathway. This study analyzes whether quaternary complex formation with endogenous TFPI-1 results also in internalization and degradation. We demonstrate that endogenous TFPI-1 and recombinant TFPI-1 differ in their distribution on the cell surface. Recombinant TFPI-1 is found in phospholipid- and glycosphingolipid-rich membrane domains, whereas endogenous TFPI-1 preferentially localizes to glycosphingolipid-rich microdomains. On quaternary complex formation, endogenous TFPI-1 remains protease sensitive and accessible for antibodies on intact cells, demonstrating that it is not appreciably internalized. Rather, regulation of TF by TFPI-1 is restored within 12 hours, consistent with dissociation of quaternary complexes on the cell surface. Endogenous TFPI-1 can be released from the cell surface by phospholipase treatment, indicating that TFPI-1 either is a glycosyl phosphatidylinositol (GPI)-anchored protein or binds to a GPI-linked receptor. We demonstrate that expression of a recombinant GPI-anchored form of TFPI-1 targets TF. VIIa complexes to glycosphingolipid-rich membrane fractions. Thus, GPI anchoring of TFPI-1 is sufficient for regulation of TF. VIIa complex function by a pathway of reversible inhibition rather than internalization and degradation.

FREE AND COMPLEXED PROSTATE SPECIFIC ANTIGEN IN THE DIFFERENTIATION OF BENIGN PROSTATIC HYPERPLASIA AND PROSTATE CANCER: STUDIES IN SERUM AND PLASMA SAMPLES

PURPOSE We prospectively evaluated serum and plasma concentrations of total and free prostate specific antigen (PSA), and PSA complexed to alpha1-antichymotrypsin in 170 patients who underwent biopsy, including 59 with prostate cancer and 111 with benign prostatic hyperplasia. We compared the usefulness of the ratios of free-to-total and complexed-to-total PSA for distinguishing between prostate cancer and benign prostatic hyperplasia, and studied the influence of blood clotting on the ratios. MATERIALS AND METHODS Blood samples were processed to generate serum and citrated plasma. To calculate complexed-to-total and free-to-total PSA we assayed plasma and serum samples for total and complexed PSA using homemade immunoassays, and total and free PSA using the Immulite assay. The 2 total PSA assays were compared using the Tandem-E PSA assay. Receiver operating characteristics curves were constructed for the total population, and for 2 to 20, 4 to 20, 2 to 10 and 4 to 10 ng./ml. total PSA. RESULTS In all groups complexed-to-total PSA had higher specificity than free-to-total and total PSA, especially at 90 to 100% sensitivity. Generally citrated plasma samples provided higher specificity than serum samples for all sensitivity values. The best performance for complexed-to-total and free-to-total PSA was obtained in the subset of patients in whom total PSA was 2 to 10 ng./ml. CONCLUSIONS Our results indicate that the complexed-to-total PSA ratio performed better for classifying disease status than the free-to-total PSA ratio in the whole patient population and in the diagnostic gray zone of 2 to 10 ng./ml. In addition, plasma samples should be used to calculate the complexed-to-total and free-to-total PSA ratio.

Binding Site for Blood Coagulation Factor Xa Involving Residues 311–325 in Factor Va

Factor Va inactivation by activated protein C is associated with cleavages at Arg306, Arg506, and Arg679 with Arg306cleavage causing the major activity loss. To study functional roles of the Arg306 region, overlapping 15-mer peptides representing the sequence of factor Va residues 271–345 were synthesized and screened for anticoagulant activities. The peptide containing residues 311–325 (VP311) noncompetitively inhibited prothrombin activation by factor Xa, but only in the presence of factor Va. Fluorescence studies showed that VP311 bound to fluorescence-labeled 5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg factor Xa in solution with a K d of 70 μm. Diisopropylphosphoryl factor Xa and factor Xa but not factor VII/VIIa or prothrombin bound to immobilized VP311 with relatively high affinity. These results support the hypothesis that residues 311–325, which are positioned between the A1 and A2 domains of factor Va and likely exposed to solvent, contribute to the binding of factor Xa by factor Va. Based on this hypothesis, it is suggested that cleavage by activated protein C at Arg306 in factor Va not only severs the covalent connection between the A1 and A2 domains but also disrupts the environment and structure of residues 311–325, thereby down-regulating the binding of factor Xa to factor Va.

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 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(K_{d}^{\mathrm{app}}\) \end{document} 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.

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 Villa. 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 Zn2+ that is required for PS‐direct and that is lost during certain purification procedures. immunoaffinity‐purified PS contained 1.4 ± 0.6 Zn2+/mol, whereas MonoQ‐purified and commercial PS contained 0.15 ± 0.15 Zn2+/mol. This may explain the controversy regarding the validity of PS‐direct. Zn2+ content correlated positively with PS‐direct in prothrombinase assays and clotting assays, but APC‐cofactor activity of PS was indepen‐dent of Zn2+ content. PS‐direct and Zn2+ were restored to inactive PS under mildly denaturing conditions. Conversely, o‐phenanthroline reversibly impaired the PS‐direct of active PS. Zn2+‐containing PS bound FXa more efficiently (Xdapp = 9.3 nM) than Zn2+‐deficient PS (Xdapp=110 nM). PS bound TFPI efficiently, independently of Zn2+ content (Xdapp = 21 nM). Antibodies that block PS‐direct preferentially recognized Zn2+‐containing PS, suggesting conformation differences at or near the interface of 2 laminin G‐like domains near the PS C terminus. Thus, Zn2+ is required for PS‐direct and efficient FXa binding and may play a role in stabilizing PS conformation.—Heeb, M. J., Prashun, D., Griffin, J. H., Bouma, B. N. 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. FASEB J. 23, 2244–2253 (2009)

C-terminal Residues 621–635 of Protein S Are Essential for Binding to Factor Va

Protein S is anticoagulant in the absence of activated protein C because of direct interactions with coagulation Factors Xa and Va. Synthetic peptides corresponding to amino acid sequences of protein S were tested for their ability to inhibit prothrombinase activity. The peptide containing the C-terminal sequence of protein S, residues 621–635 (PSP14), reversibly inhibited prothrombinase activity in the presence but not in the absence of Factor Va (K i ∼2 μm). PSP14 inhibition of prothrombinase was independent of phospholipids but could be competitively overcome by increasing Factor Xa concentrations, suggesting that the C-terminal region of protein S may compete for a Factor Xa binding site on Factor Va. Studies using peptides with amino acid substitutions suggested that lysines 630, 631, and 633 were critical residues. PSP14 inhibited Factor Va activity in Factor Xa-one-stage clotting assays. PSP14 inhibited protein S binding to immobilized Factor Va. When preincubated with protein S, antibodies raised against PSP14 inhibited binding of protein S to Factor Va and blocked inhibition of prothrombinase activity by protein S. These results show that the C-terminal region of protein S containing residues 621–635 is essential for binding of protein S to Factor Va and that this interaction contributes to anticoagulant action.

Direct anticoagulant activity of protein S‐C4b binding protein complex in Heerlen heterozygotes and normals*

Summary.  Background: Plasma protein S normally circulates free (40%) or complexed with C4b‐binding protein (PS‐C4BP); only free protein S is a cofactor for activated protein C during factor (F) Va inactivation. Protein S‐Heerlen lacks a carbohydrate group, leading to low plasma free protein S levels, but normal levels of PS‐C4BP. Objectives: Because protein S‐Heerlen is not associated with thrombosis, we investigated whether PS‐C4BP is directly anticoagulant in plasma and whether PS‐Heerlen‐C4BP has enhanced direct anticoagulant activity. Methods: An assay for protein S direct activity was applied to Heerlen‐heterozygous plasmas. Free and complexed protein S were repeatedly isolated from normal and Heerlen‐heterozygous plasmas and tested for direct anticoagulant activity in prothrombinase assays and in plasma. Results: Heerlen‐heterozygous plasmas were deficient in free and total protein S antigen but had normal to high protein S direct anticoagulant activity. Purified Heerlen‐heterozygous PS‐C4BP was 7‐fold more potent than normal PS‐C4BP in inhibiting full prothrombinase activity, and 22‐fold more potent in inhibiting prothrombin activation in the absence of FVa; it also specifically prolonged plasma clotting times 14‐fold more than normal PS‐C4BP. Heerlen‐heterozygous PS‐C4BP did not compete for limiting phospholipids any better than normal PS‐C4BP. However, ligand blots and surface plasmon resonance studies showed that Heerlen‐heterozygous PS‐C4BP bound more avidly to FXa than did normal PS‐C4BP (apparent Kd = 4.3 nm vs. 82 nm). Conclusions: Plasma‐derived PS‐C4BP has direct anticoagulant activity in plasma and in purified systems. Enhanced direct activity of PS‐Heerlen‐C4BP may compensate for low free protein S levels and low cofactor activity in individuals with protein S‐Heerlen.

Species-specific anticoagulant and mitogenic activities of murine protein S

Mice and other animals have proved extremely useful testing grounds for hypotheses and for potential therapies. However, significant interspecies differences can confound correct interpretation and attempts to extrapolate results to man. In this study Fernandez and co-workers demonstrate that the human and murine protein C-protein S systems function in a similar way but do not interact efficiently with one another. 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 μg/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.