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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Antithrombin I. Inhibition of thrombin generation in plasma by fibrin formation.

Thrombin substrate binding is mediated through fibrinogen recognition "exosite 1" in thrombin, resulting in fibrinopeptide cleavage to form fibrin. In addition, thrombin exhibits "non-substrate" binding to fibrin, an activity termed "Antithrombin I". Antithrombin I (AT-I) is characterized by two classes of thrombin binding sites, the first of "low affinity" in the fibrin E domain, and the other of high affinity, that is situated between C-terminal residues 414 and 427 of a variant gamma chain termed gamma'(1-427L), Plasma fibrinogen molecules containing gamma' chains ("fibrinogen 2") are virtually all heterodimers containing one gamma(A) chain (platelet-binding) and one gamma' chain.

Fibrinogen assembly and crosslinking on a fibrin fragment E template.

There is an ongoing controversy concerning whether crosslinked gamma chains in fibrin are oriented "transversely" between fibril strands or "end-to-end" along fibril strands. From the latter viewpoint, Veklich et al. [Proc Natl Acad Sci (USA) 95: 1438, 1998] observed that fibrinogen fibrils that had been assembled on a fibrin fragment E template, cross-linked with factor XIIIa, and then dissociated in acetic acid solution, were aligned end-to-end.