Pablo García de Frutos

Deficiency or inhibition of Gas6 causes platelet dysfunction and protects mice against thrombosis.

The growth arrest-specific gene 6 product (Gas6) is a secreted protein related to the anticoagulant protein S but its role in hemostasis is unknown. Here we show that inactivation of the Gas6 gene prevented venous and arterial thrombosis in mice, and protected against fatal collagen/epinephrine-induced thrombo embolism. Gas6−/− mice did not, however, suffer spontaneous bleeding and had normal bleeding after tail clipping. In addition, we found that Gas6 antibodies inhibited platelet aggregation in vitro and protected mice against fatal thrombo embolism without causing bleeding in vivo. Gas6 amplified platelet aggregation and secretion in response to known agonists. Platelet dysfunction in Gas6−/− mice resembled that of patients with platelet signaling transduction defects. Thus, Gas6 is a platelet-response amplifier that plays a significant role in thrombosis. These findings warrant further evaluation of the possible therapeutic use of Gas6 inhibition for prevention of thrombosis.

Molecular basis of protein S deficiency.

Protein S deficiency (PSD) has been the most difficult to study among the classical inherited thrombophilic factors. This is in part due to the peculiar biology of protein S (PS), which has an anticoagulant role but no enzymatic activity, and because it interacts with plasma components that function in both haemostasis and inflammation. Clinically, it also has been difficult to define and standardise valuable assays to determine PS status and implication in thrombosis. Despite these drawbacks, at present heterozygous PS deficiency is well established as an autosomal dominant trait associated with an increased risk of thrombosis from data on familial and population studies. Almost two-hundred mutations have been characterised in PROS1, and approximately 30% of them have been characterised in vitro, clarifying the mechanisms leading to PSD. Furthermore, recent studies on the presence of large deletions in PROS1 have increased the number of PSD associated to PROS1 mutations. Finally, the discovery of new functions for PS, both in the anticoagulant system as well as in the interaction with cellular components through receptor tyrosine kinases, is broadening the importance of this molecule in the context of biomedicine.

Nonanticoagulant heparin prevents histone-mediated cytotoxicity in vitro and improves survival in sepsis

Extracellular histones are considered to be major mediators of death in sepsis. Although sepsis is a condition that may benefit from low-dose heparin administration, medical doctors need to take into consideration the potential bleeding risk in sepsis patients who are already at increased risk of bleeding due to a consumption coagulopathy. Here, we show that mechanisms that are independent of the anticoagulant properties of heparin may contribute to the observed beneficial effects of heparin in the treatment of sepsis patients. We show that nonanticoagulant heparin, purified from clinical grade heparin, binds histones and prevents histone-mediated cytotoxicity in vitro and reduces mortality from sterile inflammation and sepsis in mouse models without increasing the risk of bleeding. Our results demonstrate that administration of nonanticoagulant heparin is a novel and promising approach that may be further developed to treat patients suffering from sepsis.

Inhibition of Alzheimer beta-peptide fibril formation by serum amyloid P component.

A 39-43-amino acid residue-long fragment (β-peptide) from the amyloid precursor protein is the predominant component of amyloid deposits in the brain of individuals with Alzheimers disease. Serum amyloid P component (SAP) is present in all types of amyloid, including that of Alzheimers disease. We have used an in vitro model to study the effects of purified SAP on the fibril formation of synthetic Alzheimer β-peptide 1-42. SAP was found to inhibit fibril formation and to increase the solubility of the peptide in a dose-dependent manner. At a 5:1 molar ratio of Aβ1-42 peptide to SAP, fibril formation was completely inhibited, and approximately 80% of the peptide remained in solution even after 4 days of incubation. At lower SAP concentrations, e.g. at peptide to SAP ratio of 1000:1, short fibrillar like structures, lacking amyloid characteristics, were formed. These structures frequently contained associated SAP molecules, suggesting that SAP binds to the polymerizing peptide in a reaction which prevented further fibril formation.

Serum Amyloid P Component Binding to C4b-binding Protein

Human C4b-binding protein (C4BP), which is a regulator of the classical complement pathway C3 convertase, forms high affinity complexes with anticoagulant protein S and with the pentraxin serum amyloid P component (SAP). SAP is a plasma protein present in all amyloid deposits. Recently, SAP was shown to inhibit the complement regulatory functions of C4BP. In this investigation, we have studied the structural requirements for the C4BP-SAP interaction. C4BP was subjected to chymotrypsin digestion, which yielded two major fragments corresponding to the central core (160 kDa) and to the cleaved-off tentacles (48 kDa). SAP-Sepharose specifically bound the 160-kDa fragment, suggesting that the central core of C4BP contains the binding site for SAP. In a quantitative affinity chromatography assay, the dissociation constants for binding of intact C4BP and of the 160-kDa central core fragment to SAP were found to be 30 and 70 nM, respectively. Recombinant C4BP composed of only α-chains bound SAP with similar affinity (K= 22 nM), whereas nonglycosylated recombinant α-chain C4BP (synthesized in the presence of tunicamycin) bound SAP with lower affinity (K = 126 nM). This suggests that the carbohydrate moiety of the central core of C4BP is important for binding of C4BP to SAP in contrast to the C4BP β-chain, which is not required. EDTA, heparin, and phosphorylethanolamine as well as a peptide comprising amino acids 27-39 of SAP were found to completely displace C4BP from the SAP matrix. Moreover, the immobilized SAP peptide bound C4BP in a reaction that, in contrast to the C4BP-SAP interaction, was not dependent on calcium.

Growth arrest-specific gene 6 (GAS6) - An outline of its role in haemostasis and inflammation

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.

Structural investigation of C4b-binding protein by molecular modeling: Localization of putative binding sites

C4b‐binding protein (C4BP) contributes to the regulation of the classical pathway of the complement system and plays an important role in blood coagulation. The main human C4BP isoform is composed of one β‐chain and seven α‐chains essentially built from three and eight complement control protein (CCP) modules, respectively, followed by a nonrepeat carboxy‐terminal region involved in polymerization of the chains. C4BP is known to interact with heparin, C4b, complement factor I, serum amyloid P component, streptococcal Arp and Sir proteins, and factor VIII/VIIIa via its α‐chains and with protein S through its β‐chain. The principal aim of the present study was to localize regions of C4BP involved in the interaction with C4b, Arp, and heparin. For this purpose, a computer model of the 8 CCP modules of C4BP α‐chain was constructed, taking into account data from previous electron microscopy (EM) studies. This structure was investigated in the context of known and/or new experimental data. Analysis of the α‐chain model, together with monoclonal antibody studies and heparin binding experiments, suggests that a patch of positively charged residues, at the interface between the first and second CCP modules, plays an important role in the interaction between C4BP and C4b/Arp/Sir/heparin. Putative binding sites, secondary‐structure prediction for the central core, and an overall reevaluation of the size of the C4BP molecule are also presented. An understanding of these intermolecular interactions should contribute to the rational design of potential therapeutic agents aiming at interfering specifically some of these protein–protein interactions. Proteins 31:391–405, 1998.

Association of specific haplotypes of GAS6 gene with stroke

The product of the growth arrest-specific gene 6 (GAS6), a ligand for tyrosine kinase receptors, is a vitamin K-dependent protein, structurally related to anticoagulant protein S. Gas6-deficient mice are protected against thrombosis, demonstrating the importance of this protein in the cardiovascular system. In a preliminary study on GAS6 polymorphisms and atherothrombotic disease we found an association between the AA genotype of the c.834 + 7G > A GAS6 polymorphism and stroke. In order to further explore this association by considering GAS6 haplotypes and the main stroke subtypes, 457 patients with ischemic stroke, 199 with hemorrhagic stroke and 150 asymptomatic controls were genotyped for eight GAS6 polymorphisms and other genetic markers in the same genome region. Association was measured by logistic regression analysis. The THESIAS program was used to measure linkage disequilibrium and haplotype frequencies. In univariate analysis, the GAS6 c.834 + 7AA genotype was found associated with decreased risk for stroke (OR: 0.59; 95%CI: 0.37-0.93). After adjustment for vascular risk factors, association was maintained when stroke subtypes affecting the microvasculature such as lacunar stroke and deep haemorrhage, were grouped together (OR: 0.44; 95%CI: 0.21-0.90). Furthermore, haplotype analysis revealed that association was even stronger when the c.834 + 7A allele was present in a specific haplotype (CACA) of four GAS6 polymorphisms. From these results we conclude that the A allele of the GAS6 c.834 + 7G > A polymorphism and more specifically, the CACA haplotype, is less prevalent in patients with stroke, suggesting a protective role for stroke of this haplotype.

The SHBG‐like region of protein S is crucial for factor V‐dependent APC‐cofactor function

Activated protein C (APC) regulates blood coagulation by degrading factor Va (FVa) and factor VIIIa (FVIIIa). Protein S is a cofactor to APC in the FVa degradation, whereas FVIIIa degradation is potentiated by the synergistic APC‐cofactor activity of protein S and factor V (FV). To elucidate the importance of the sex‐hormone‐binding globulin (SHBG)‐like region in protein S for expression of anticoagulant activity, a recombinant protein S/Gas6 chimera was constructed. It comprised the amino‐terminal half of protein S and the SHBG‐like region of Gas6, a structurally similar protein having no known anticoagulant properties. The protein S/Gas6 chimera expressed 40–50% APC‐cofactor activity in plasma as compared to wild‐type protein S. In the degradation of FVa by APC, the protein S/Gas6 chimera was only slightly less efficient than wild‐type protein S. In contrast, the protein S/Gas6 chimera expressed no FV‐dependent APC‐cofactor activity in a FVIIIa‐degradation system. This demonstrates the SHBG‐like region to be important for expression of APC‐cofactor activity of protein S and suggests that the SHBG‐like region of protein S interacts with FV during the APC‐mediated inactivation of FVIIIa.

SHBG region of the anticoagulant cofactor protein S: Secondary structure prediction, circular dichroism spectroscopy, and analysis of naturally occurring mutations

Protein S (PS) and growth arrest specific factor 6 (GAS6) are vitamin K‐dependent proteins with similar structures. They are mosaic proteins possessing a carboxyl‐terminal region presenting sequence similarity with plasma sex hormone binding globulin (plasma SHBG), although apparently not involved in steroid binding. The SHBG‐like modules have sequence similarity with the G repeats of the chain A of laminin. Laminin G repeats have been reported to contain mainly β‐strands (about 40–50%) but no or little α structure by circular dichroism (CD) spectroscopy. Secondary structure predictions carried out in the present work unexpectedly showed a 20 to 27% helices content in the SHBG region of PS/GAS6 (about 100 residues), while plasma SHBG and laminin G repeats had around 10% helices. CD measurements for human PS indicated also that its SHBG region had about 100 residues in α‐helical structure. These data suggest that the SHBG region of PS/GAS6 on the one hand, and the laminin G repeats and possibly plasma SHBG on the other hand, could present important structural differences. Previously reported polymorphisms and point mutations leading to PS deficiency and thrombophilia have been analyzed with our structural predictions. We found a good agreement between these structural predictions, CD measurements, experimental and clinical data. This information allows us to gain insights into the three‐dimensional structure of PS that will be helpful for the design of new experiments and future clinical investigations. Proteins 29:478–491, 1997.