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We determined anticoagulant parameters that depend on protein S function in plasma, i.e. the APC-independent anticoagulant activity of protein S (expressed as pSR) and APC resistance determined with thrombin generation-based tests (expressed as APCsr) as well as plasma levels of total and free protein S and prothrombin in men, women not using oral contraceptives (OC), and in women using second or third generation OC. Thrombin generation in the APC resistance assays was initiated either with factor Xa (Xa-APCsr) or tissue factor (TF-APCsr). The APC-independent anticoagulant activity of protein S was highest in men (pSR=1.69) and gradually decreased from women not using OC (pSR=1.49) via women using second generation (pSR=1.35) to women using third generation OC (pSR=1.27). The pSR correlated inversely with nAPCsr determined with the tissue factor-based APC resistance test (TF-APCsr) but not with nAPCsr determined with the factor Xa-based assay (Xa-APCsr). Multiple linear regression analysis in which sex, OC use, and protein S and prothrombin levels were included as independent variables and the pSR, TF-APCsr or Xa-APCsr as dependent variables indicated that plasma protein S levels poorly predict the pSR and the TF-APCsr, but are the main determinant of the Xa-APCsr. This indicates that OC use alters the expression of protein S activity. This phenomenon can be caused by differences in modulation of the activity of protein S by other plasma proteins that change during OC use or by OC-induced changes in the protein S molecule that impair its anticoagulant activity. Functional impairment of protein S as a result of hormonal influence may, at least in part, contribute to the thrombotic risk of OC users.

The chemokines platelet factor 4 (PF4) and RANTES (regulated on activation normal T cell expressed and secreted) are secreted by activated platelets and influence multiple cell types and biologic processes. For instance, PF4 inhibits progenitor cell proliferation and angiogenesis, while platelet-derived RANTES is involved in vascular recruitment of monocytes. However, little is known about functional interactions of PF4 and RANTES. Here we show that the presence of PF4 enhanced the arrest of RANTES-stimulated monocytes and monocytic cells on activated endothelial cells under flow conditions, while binding of PF4 to the monocyte surface was increased by RANTES. Both RANTES-triggered arrest and PF4 binding involved monocytic chondroitin sulfate. Ligand blots and surface plasmon resonance revealed a robust heterophilic interaction of PF4 with RANTES but not with RANTES variants defective in higher order oligomerization. The tetrameric mutant E26A bound to the monocyte surface without increasing PF4 binding, and monocyte arrest induced by E26A-RANTES was not enhanced by PF4. Stimulation of monocytes with supernatants of activated platelets triggered arrest involving RANTES and PF4, as shown by inhibition studies. Our results suggest that heterophilic interactions with PF4 require structural motifs important in RANTES oligomerization and amplify RANTES-triggered effects on monocyte adhesion. This may have implications for the modulation of inflammatory recruitment by platelet-derived chemokines.

[structure: see text] A bimodal target-specific contrast agent based on a cyclic peptide containing the target-specific NGR sequence, gadolinium(III) diethylenetriaminepentaacetic acid (Gd(III)DTPA), and Oregon Green 488 (OG488) suitable for both MR imaging and optical imaging of angiogenesis is developed. The synthetic strategy for this target-specific contrast agent exploits the use of highly efficient, chemoselective reactions, such as native chemical ligation, and gives a straightforward approach for double labeling of peptides in general.

The activated protein C (APC)-independent anticoagulant activity of protein S on tissue factor-induced thrombin generation was quantified in plasma. In absence of APC, protein S significantly decreased the endogenous thrombin potential (ETP) in a concentration-dependent manner. The APC-independent anticoagulant activity of protein S in plasma was not affected by phospholipid concentrations but strongly depended on tissue factor concentrations: protein S inhibited the ETP from 6% at 140 pM tissue factor to 74% at 1.4 pM tissue factor. Plasma with both 60% protein S and 140% prothrombin showed an ETP of 240% compared to normal plasma, suggesting an APC-independent protective role of protein S in the development of thrombosis as a result of protein S deficiency and the prothrombin-G20210A mutation. At high tissue-factor concentrations, protein S hardly expressed APC-independent anticoagulant activity but exerted potent APC-cofactor activity when thrombomodulin or APC were added to plasma. Neutralization of protein S under these conditions resulted in a 20-fold reduction of the anticoagulant activity of APC. The present study shows that protein S effectively regulates coagulation at 2 levels: at low procoagulant stimuli, protein S maintains the hemostatic balance by directly inhibiting thrombin formation, and at high procoagulant stimuli, protein S restores the hemostatic balance via its APC-cofactor activity.

Protein S is a vitamin K-dependent plasma protein that functions as an APC-cofactor, but also exhibits anticoagulant activity in the absence of APC. The Heerlen polymorphism of protein S is characterized by a Ser460Pro substitution and lacks glycosylation at Asn458. It is associated with decreased protein S levels due to selective deficiency of free protein S Heerlen. To understand the lack of thrombotic complications associated with the protein S Heerlen mutation, we compared recombinant protein S Heerlen, wild type (wt) protein S and plasma-derived protein S. wt-Protein S and protein S Heerlen each bound 1:1 to C4BP with dissociation constants of 0.27 and 0.33 nM, respectively. Both wt-protein S and protein S Heerlen, either free or in complex with C4BP, were equally active as prothrombinase inhibitors in the absence of APC. All three protein S preparations stimulated APC-catalyzed inactivation of normal FVa, FVa Leiden and FVIIIa to the same extent. If extrapolated to plasma, it is not likely that the decreased free protein S levels in carriers of the protein S Heerlen mutation are compensated by an increased anticoagulant activity of protein S Heerlen-C4BP complexes. It is possible that an unrecognized plasma factor selectively enhances the anticoagulant activity of protein S Heerlen. If not, the reduction of free protein S levels in heterozygous protein S Heerlen-carriers combined with (low) normal total protein S levels apparently minimally affects the total anticoagulant activity of protein S (APC-cofactor and APC-independent activity) and hence is not associated with increased risk of venous thrombosis.

Protein S exhibits anticoagulant activity independent of activated protein C (APC). An automated factor Xa-based one-stage clotting assay was developed that enables quantification of the APC-independent activity of protein S in plasma from the ratio of clotting times (protein S ratio [pSR]) determined in the absence and presence of neutralizing antibodies against protein S. The pSR was 1.62 +/- 0.16 (mean +/- SD) in a healthy population (n = 60), independent of plasma levels of factors V, VIII, IX, and X; protein C; and antithrombin, and not affected by the presence of factor V Leiden. The pSR strongly correlates with the plasma level of protein S and is modulated by the plasma prothrombin concentration. In a group of 16 heterozygous protein S-deficient patients, the observed mean pSR (1.31 +/- 0.09) was significantly lower than the mean pSR of the healthy population, as was the pSR of plasma from carriers of the prothrombin G20210A mutation (1.47 +/- 0.21; n = 46). We propose that the decreased APC-independent anticoagulant activity of protein S in plasma with elevated prothrombin levels may contribute to the thrombotic risk associated with the prothrombin G20210A mutation.

Hemostasis governs two essential processes of human life in that it maintains the fluidity of blood under physiological conditions and prevents excessive blood loss after injury. Hemostasis is regulated by components of the vessel wall and blood cells and by humoral coagulation factors. Under normal conditions, these components are involved in an active equilibrium through activation, propagation, and termination of the hemostatic pathways. This equilibrium is disturbed upon vascular injury, leading to a procoagulant response when needed. Unfortunately, pathological disturbances can occur as a result of inherited or acquired coagulation factor deficiencies that may lead to bleeding or thrombotic disorders.