Regulation of endothelial cell barrier function by antibody-driven affinity modulation of platelet endothelial cell adhesion molecule-1 (PECAM-1).

PECAM-1 is a 130-kDa member of the immunoglobulin (Ig) superfamily that is expressed on the surface of platelets and leukocytes, and at the intracellular junctions of confluent endothelial cell monolayers. Previous studies have shown that PECAM-1/PECAM-1 homophilic interactions play a key role in leukocyte transendothelial migration, in allowing PECAM-1 to serve as a mechanosensory complex in endothelial cells, in its ability to confer cytoprotection to proapoptotic stimuli, and in maintaining endothelial cell junctional integrity. To examine the adhesive properties of full-length PECAM-1 in a native lipid environment, we purified it from platelets and assembled it into phospholipid nanodiscs. PECAM-1-containing nanodiscs retained not only their ability to bind homophilically to PECAM-1-expressing cells, but exhibited regulatable adhesive interactions that could be modulated by ligands that bind membrane- proximal Ig Domain 6. This property was exploited to enhance the rate of barrier restoration in endothelial cell monolayers subjected to inflammatory challenge. The finding that the adhesive properties of PECAM-1 are regulatable suggests novel approaches for controlling endothelial cell migration and barrier function in a variety of vascular permeability disorders.

Nanoscale probing reveals that reduced stiffness of clots from fibrinogen lacking 42 N-terminal Bbeta-chain residues is due to the formation of abnormal oligomers.

Removal of Bbetal-42 from fibrinogen by Crotalus atrox venom results in a molecule lacking fibrinopeptide B and part of a thrombin binding site. We investigated the mechanism of polymerization of desBbeta1-42 fibrin. Fibrinogen trinodular structure was clearly observed using high resolution noncontact atomic force microscopy. E-regions were smaller in desBbeta1-42 than normal fibrinogen (1.2 nm +/- 0.3 vs. 1.5 nm +/- 0.2), whereas there were no differences between the D-regions (1.7 nm +/- 0.4 vs. 1.7 nm +/- 0.3). Polymerization rate for desBbeta1-42 was slower than normal, resulting in clots with thinner fibers. Differences in oligomers were found, with predominantly lateral associations for desBbeta1-42 and longitudinal associations for normal fibrin. Clot elasticity as measured by magnetic tweezers showed a G' of approximately 1 Pa for desBbeta1-42 compared with approximately 8 Pa for normal fibrin. Spring constants of early stage desBbeta1-42 single fibers determined by atomic force microscopy were approximately 3 times less than normal fibers of comparable dimensions and development. We conclude that Bbeta1-42 plays an important role in fibrin oligomer formation. Absence of Bbeta1-42 influences oligomer structure, affects the structure and properties of the final clot, and markedly reduces stiffness of the whole clot as well as individual fibrin fibers.

Caveolin-1-dependent apoptosis induced by fibrin degradation products.

In mice lacking the blood coagulation regulator thrombomodulin, fibrinolytic degradation products (FDP) of fibrin induce apoptotic cell death of a specialized cell type in the placenta, polyploid trophoblast giant cells. Here, we document that this bioactivity of FDP is conserved in human FDP, is not limited to trophoblast cells, and is associated with an Aalpha-chain segment of fibrin fragment E (FnE). The majority of proapoptotic activity is arginine-glycine-aspartic acid (RGD)-independent and requires caveolin-1-dependent cellular internalization of FnE. Internalization through caveoli is mediated by an epitope contained within Aalpha52-81 that is necessary and sufficient for cellular uptake of FnE. Aalpha52-81 does not cause apoptosis itself, and competitively inhibits FnE internalization and apoptosis induction. Apoptotic activity per se resides within Aalpha17-37 and requires the N-terminal neoepitope generated by release of fibrinopeptide A. Cellular internalization of FnE elicits depression of mitochondrial function and consequent apoptosis that is strictly dependent on the activity of caspases 9 and 3. These findings describe the molecular details of a novel mechanism linking fibrin degradation to cell death in the placenta, which may also contribute to pathologic alterations in nonplacental vascular beds that are associated with fibrinolysis.

Fibrinogen Columbus: a novel gamma Gly200Val mutation causing hypofibrinogenemia in a family with associated thrombophilia.

Fibrinogen is an essential component of the coagulation cascade and the acute phase response. The native 340 kDa molecule has a symmetrical trinodular structure composed of a central E-domain connected to outer D-domains by triple helical coiled-coils.1 Several mutations known to cause hypofibrinogenemia occur within the C-terminal gammaD-domain and have helped to elucidate the structurally and functionally important areas of this domain.2-5 Here we report the identification of a novel point mutation gammaG200V (fibrinogen Columbus) causing hypofibrinogenemia and co-segregating with three genetic thrombophilia risk factors.

Update on antithrombin I (fibrin).

Antithrombin I (fibrin) is an important inhibitor of thrombin generation that functions by sequestering thrombin in the forming fibrin clot, and also by reducing the catalytic activity of fibrinbound thrombin. Thrombin binding to fibrin takes place at two classes of non-substrate sites: 1) in the fibrin E domain (two per molecule) through interaction with thrombin exosite 1; 2) at a single site on each gamma' chain through interaction with thrombin exosite 2. The latter reaction results in allosteric changes that down-regulate thrombin catalytic activity. Antithrombin I deficiency (afibrinogenemia), defective thrombin binding to fibrin (antithrombin I defect) found in certain dysfibrinogenemias (e.g. fibrinogen Naples 1), or a reduced plasma gamma' chain content (reduced antithrombin I activity), predispose to intravascular thrombosis.

Crystal structure of thrombin in complex with fibrinogen gamma' peptide.

Elevated levels of heterodimeric gamma(A)/gamma' fibrinogen 2 have been associated with an increased incidence of coronary artery disease, whereas a lowered content of gamma' chains is associated with an increased risk of venous thrombosis. Both situations may be related to the unique features of thrombin binding to variant gamma' chains. The gamma' peptide is an anionic fragment that binds thrombin with high affinity without interfering directly with substrate binding. Here we report the crystal structure of thrombin bound to the gamma' peptide, solved at 2.4 A resolution. The complex reveals extensive interactions between thrombin and the gamma' peptide mediated by electrostatic contacts with residues of exosite II and hydrophobic interactions with a pocket in close proximity to the Na(+) binding site. In its binding mode, the gamma' peptide completely overlaps with heparin bound to exosite II. These findings are consistent with functional data and broaden our understanding of how thrombin interacts with fibrinogen at the molecular level.

Structural basis for sequential cleavage of fibrinopeptides upon fibrin assembly.

Nonsubstrate interaction of thrombin with fibrinogen promotes sequential cleavage of fibrinopeptides A and B (fpA and fpB, respectively) from the latter, resulting in its conversion into fibrin. The recently established crystal structure of human thrombin in complex with the central part of human fibrin clarified the mechanism of this interaction. Here, we reveal new details of the structure and present the results of molecular modeling of the fpA- and fpB-containing portions of the Aalpha and Bbeta chains, not identified in the complex, in both fibrinogen and protofibrils. The analysis of the results reveals that in fibrinogen the fpA-containing portions are in a more favorable position to bind in the active site cleft of bound thrombin. Surface plasmon resonance experiments establish that the fpB-containing portions interact with the fibrin-derived dimeric D-D fragment, suggesting that in protofibrils they bind to the newly formed DD regions bringing fpB into the vicinity of bound thrombin. These findings provide a coherent rationale for the preferential removal of fpA from fibrinogen at the first stage of fibrin assembly and the accelerated cleavage of fpB from protofibrils and/or fibrils at the second stage.

Fibrinogen Saint-Germain II: hypofibrinogenemia due to heterozygous gamma N345S mutation.

We have identified a novel heterozygous fibrinogen gamma chain mutation, gammaN345S (Fibrinogen Saint-Germain II), in a subject with hypofibrinogenemia. There was no evidence by mass spectrometry of plasma fibrinogen containing the mutant chain. The hypofibrinogenemia was discovered in a 26-year-old man who experienced extensive deep venous thrombosis of the left leg associated with pulmonary embolism. Investigation of potential thromboembolic risk factors revealed heterozygosity of the factor V R506Q mutation (factor V Leiden) and heterozygosity of the prothrombin gene G20210A mutation. The hypofibrinogenemia may be contributory to the thrombophilic manifestations.

Studies on the basis for the properties of fibrin produced from fibrinogen-containing gamma' chains.

Human fibrinogen 1 is homodimeric with respect to its gamma chains (gammaA-gammaA'), whereas fibrinogen 2 molecules each contain one gammaA (gammaA1-411V) and one gamma' chain, which differ by containing a unique C-terminal sequence from gamma'408 to 427L that binds thrombin and factor XIII. We investigated the structural and functional features of these fibrins and made several observations. First, thrombin-treated fibrinogen 2 produced finer, more branched clot networks than did fibrin 1. These known differences in network structure were attributable to delayed release of fibrinopeptide (FP) A from fibrinogen 2 by thrombin, which in turn was likely caused by allosteric changes at the thrombin catalytic site induced by thrombin exosite 2 binding to the gamma' chains. Second, cross-linking of fibrin gamma chains was virtually the same for both types of fibrin. Third, the acceleratory effect of fibrin on thrombin-mediated XIII activation was more prominent with fibrin 1 than with fibrin 2, and this was also attributable to allosteric changes at the catalytic site induced by thrombin binding to gamma' chains. Fourth, fibrinolysis of fibrin 2 was delayed compared with fibrin 1. Altogether, differences between the structure and function of fibrins 1 and 2 are attributable to the effects of thrombin binding to gamma' chains.

John Ferry and the mechanical properties of cross-linked fibrin.

This article describes the role John Ferry played in relating the location of cross-linked gamma-chains in fibrin fibrils to the mechanical properties of fibrin clot.

Regulation of transglutaminase activity in articular chondrocytes through thrombin receptor-mediated factor XIII synthesis.

Transglutaminases are a family of enzymes that catalyze the formation of epsilon-(gamma-glutamyl)lysine isopeptide bonds in proteins, an activity that has been implicated in the pathogenesis of cartilage matrix mineralization in degenerative arthritis. Type II transglutaminase and thrombin-activatable factor XIII have been identified in articular cartilage. Thrombin, a coagulation protease, is found in pathological synovial fluids, and is known to stimulate transglutaminase activity in non-articular tissues. We investigated the effects of thrombin on transglutaminase activity in porcine articular chondrocytes. Direct addition of thrombin to chondrocyte lysates resulted in increased transglutaminase activity due to proteolytic conversion of factor XIII to XIIIa. Thrombin-treated chondrocyte cultures (0.001 to 2.0 U/ml) also showed increased transglutaminase activity. Thrombin treatment of chondrocyte cultures increased transglutaminase activity as early as 15 minutes after addition, an effect that we attributed to factor XIII activation. Additional stimulatory effects of thrombin were observed in cultured chondrocytes at 4 and 24 hours. A thrombin receptor agonist peptide (TRAP) which activates the PAR1 thrombin receptor mimicked these later effects. Thrombin treatment of chondrocyte cultures increased factor XIII mRNA and protein levels, without affecting levels of type II transglutaminase. Thus, thrombin stimulates transglutaminase activity in articular cartilage by directly cleaving factor XIII and by receptor-mediated up-regulation of factor XIII synthesis. Such increases in potential transglutaminase activity may facilitate pathological matrix calcification in degenerative arthritis.

Crystal structure of the complex between thrombin and the central "E" region of fibrin.

Nonsubstrate interactions of thrombin with fibrin play an important role in modulating its procoagulant activity. To establish the structural basis for these interactions, we crystallized d-Phe-Pro-Arg-chloromethyl ketone-inhibited human thrombin in complex with a fragment, E(ht), corresponding to the central region of human fibrin, and solved its structure at 3.65-A resolution. The structure revealed that the complex consists of two thrombin molecules bound to opposite sides of the central part of E(ht) in a way that seems to provide proper orientation of their catalytic triads for cleavage of fibrinogen fibrinopeptides. As expected, binding occurs through thrombin's anion-binding exosite I. However, only part of it is involved in forming an interface with the complementary negatively charged surface of E(ht). Among residues constituting the interface, Phe-34, Ser-36A, Leu-65, Tyr-76, Arg-77A, Ile-82, and Lys-110 of thrombin and the A alpha chain Trp-33, Phe-35, Asp-38, Glu-39, the B beta chain Ala-68 and Asp-69, and the gamma chain Asp-27 and Ser-30 of E(ht) form a net of polar contacts surrounding a well defined hydrophobic interior. Thus, despite the highly charged nature of the interacting surfaces, hydrophobic contacts make a substantial contribution to the interaction.

Cause-effect relation between hyperfibrinogenemia and vascular disease.

Elevated plasma levels of fibrinogen are associated with the presence of cardiovascular disease, but it is controversial whether elevated fibrinogen causally imparts an increased risk, and as such is a true modifier of cardiovascular disease, or is merely associated with disease. By investigating a transgenic mouse model of hyperfibrinogenemia, we show that elevated plasma fibrinogen concentration (1) elicits augmented fibrin deposition in specific organs, (2) interacts with an independent modifier of hemostatic activity to regulate fibrin turnover/deposition, (3) exacerbates neointimal hyperplasia in an experimental model of stasis-induced vascular remodeling, yet (4) may suppress thrombin generation in response to a procoagulant challenge. These findings provide direct experimental evidence that hyperfibrinogenemia is more than a by-product of cardiovascular disease and may function independently or interactively to modulate the severity and/or progression of vascular disease.