Affinity For Fibrinogen Research Articles

Interaction between thrombin mutant W215A/E217A and direct thrombin inhibitor

Thrombin mutant W215A/E217A acts as an anticoagulant when administered intravenously by activating plasma protein C in concert with endothelial thrombomodulin [1,2]. The double mutation in the catalytic domain of the W215A/E217A molecule leads to compromised interactions with fibrinogen and chromogenic thrombin substrates [1,3]. Among direct thrombin inhibitors (DTIs), argatroban exerts antithrombotic activity by a rapid binding to the catalytic domain of thrombin, while lepirudin and bivalirudin occupy both exosite I and catalytic domains [4]. We hereby report on the changes in DTI-interaction with W215A/E217A, using thrombin substrate hydrolysis [1,3], activated partial thromboplastin time (aPTT) and the Thrombinoscope methods (Thrombinoscope BV, Maastricht, Netherlands) [5]. Based on the inhibition of H-D-Phe-Pro-Arg-p-nitroanilide hydrolysis, argatroban and lepirudin interact with the catalytic domain of W215A/E217A with a 7000-fold reduced affinity relative to wild-type thrombin (Table 1) [6]. This reduction coincides with the 19 000-fold decrease in affinity for fibrinogen [1]. On the contrary, lack of inhibition of the substrate hydrolysis with bivalirudin suggests that W215A/E217A binds to bivalirudin utilizing exosite I without blocking the active site. This finding was confirmed in studies looking at the competitive inhibitory effect of bivalirudin on the activation of protein C by W215A/E217A in the presence of thrombomodulin (Table 1). Table 1 Binding of wild-type and mutant thrombin to thrombin inhibitors and substrates Although the direct interaction between W215A/E217A and DTIs seems to be weak, addition of W215A/E217A in DTI-treated plasma corrected prolongation of aPTT toward normal (Fig. 1). Prolonged aPTT with DTIs was not reversed with active site mutant S195A [7] or active-site blocked thrombin (wild-type thrombin inactivated with H-D-Phe-Pro-Arg-CH2Cl; FPR-WT). Inactive thrombin species, S195A and FPR-wild-type, occupy thrombin binding sites on fibrin(ogen) and cause dose-dependent inhibition of clotting [8]. It is thus speculated that the catalytic domain of W215A/E217A exerts some procoagulant function through activation of factor V [9]. Anticoagulant effects of DTIs have been shown to be mitigated in the presence of factor Va [10,11]. Fig. 1 Activated partial thromboplastin time, measured in normal volunteer plasma, spiked with a direct thrombin inhibitor (n = 8). Mean values (±SD) of activated partial thromboplastin time(aPTT) (in seconds). Note aPTT values were shortened by W215A/E217A ... Using the Thrombinoscope system that continuously monitors thrombin generation based on the hydrolysis of fluorogenic substrate, Z-Gly-Gly-AMC (Bachem Bioscience, King of Prussia, Pennsylvania, USA), we showed that in plasma the combination of W215A/E217A (5µg/ml~135 nmol/l) and thrombomodulin (0.75µg/ml; Asahi Kasei Pharma, Oh-hito, Japan) [12], but not W215A/E217A alone exerted anticoagulant effects based on the reduced peak levels of thrombin generation (Fig. 2a). The addition of W215A/E217A was found to reverse prolonged thrombin generation lag time for all DTIs (Fig. 2). Reduced peak thrombin levels were not significantly recovered with W215A/E217A at therapeutic levels (0.5–1µg/ml) of argatroban (Fig. 2b) [13]. Our present data are in agreement with the reversal of melagatran-prolonged lag time in thrombin generation with factor Va [10]. For bivalent inhibitors, it is plausible that bivalirudin and lepirudin reduce the interaction of factor V with W215A/E217A by binding to exosite I [9]. FPR-wild-type (30µg/ml) reversed delayed onset of thrombin generation with bivalirudin, but clotting was extensively inhibited due to occupation of thrombin binding sites of fibrin with FPR-wild-type (Fig 1 and Fig 2c) [8]. Lepirudin obliterated thrombin generation, but this was restored toward normal after the addition of W215A/E217A, especially at therapeutic level (1µg/ml) of lepirudin (Fig. 2d). Fig. 2 Effects of W215A/E217A on thrombin generation. Panel a: a representative series of thrombin generation curves. The peak thrombin generation is slightly decreased by the addition of W215A/E217A, 5µg/ml, and modestly decreased by recombinant human ... In summary, the catalytic-site double mutant, W215A/E217A, shortens DTI-prolonged aPTT and the onset of endogenous thrombin generation in vitro. Further modifications of the catalytic domain or exosites of thrombin molecule or both may lead to a development of antidotes for DTI-related bleeding complications [10].

Fibrinogen synthesized by cancer cells augments the proliferative effect of fibroblast growth factor-2 (FGF-2).

Fibroblast growth factor (FGF)-2 is a critical growth factor in normal and malignant cell proliferation and tumor-associated angiogenesis. Fibrinogen and fibrin bind to FGF-2 and modulate FGF-2 functions. Furthermore, we have shown that extrahepatic epithelial cells are capable of endogenous production of fibrinogen. Herein we examined the role of fibrinogen and FGF-2 interactions on prostate and lung adenocarcinoma cell growth in vitro. Cell proliferation was measured by (3)H-thymidine uptake and the specificity of FGF-2-fibrinogen interactions was measured using wild-type and mutant FGF-2s, fibrinogen gamma-chain (FGG) RNAi and co-immunoprecipitation. Metabolic labeling, immunopurification and fluorography demonstrated de novo fibrinogen production. FGF-2 stimulated DU-145 cell proliferation, whereas neither FGF-2 nor fibrinogen affected the growth of PC-3 or A549 cells. Fibrinogen augmented the proliferative effect of FGF-2 on DU-145 cells. The role of fibrinogen in FGF-2-enhanced DNA synthesis was confirmed using an FGF-2 mutant that exhibits no binding affinity for fibrinogen. FGG transcripts were present in PC-3, A549 and DU-145 cells, but only PC-3 and A549 cells produced detectable levels of intact protein. RNAi-mediated knockdown of FGG expression resulted in decreased production of fibrinogen protein and inhibited (3)H-thymidine uptake in A549 and PC-3 cells by 60%, which was restored by exogenously added fibrinogen. FGF-2 and fibrinogen secreted by the cells were present in the medium as a soluble complex, as determined by coimmunoprecipitation studies. These data indicate that endogenously synthesized fibrinogen promotes the growth of lung and prostate cancer cells through interaction with FGF-2.

Charaterization of Human Primitive Megakaryocytes and “Platelets”.

Primitive megakaryocytes (PMs) have been first observed in murine embroynal yolk sac [Xu et al. Blood . 2001 Apr 1; 97(7):2016–22.]. The biological characteristics and functional aspects of human primitive megakaryocytes are not well established even though they can be transiently observed by bone marrow stromal cell (OP9) co-culture methodology [Gaur, M et al J Thromb Haemost. 2006 Feb; 4(2):436–42.; Eto K et al. Proc Natl Acad Sci U S A. 2002 Oct 1; 99(20):12819–24.]. The goal of this study is to charaterize PMs' unique biological and functional features. To obtain a pure population of human primitive megakaryocytes, human embryonic stem (hES) cells were cultured to form embryoid bodies.CD34+/CD31+/VE-Cadherin+/Flk-1+ hemangioblasts on day 12 were isolated and further cultured for 10–12 days in media containing thrombopoietin (TPO), interleukin 3 (IL3), stem cell factor (SCF). 30% of the non-adherent cells were strongly positive for CD31/CD41/CD61/CD42b, and weakly positive for CD42a/CD45/beta-2-microglobulin (B2M) but negative for CD117 or HLA-DR. The DNA ploidies are exclusively 2N–8N confirming the primitive status of PMs. Morphologically when compared to bone marrow derived megakaryocytes, PMs readily produce long processes with chains of “proplatelet” like structures. By live videomicroscopy, these “proplatelet” structures can actively recruit mitochondria. Interestingly, “platelet-like” fragment shedding from megakaryocytes is significantly stimulated in the presence of nitric oxide and human plasma. The “platelet-like” fragments and megakaryocytes respond to strong agonists, including thrombin and TRAP, with aggregation, alpha granule release and increased affinity for fibrinogen. In contrast, weaker agonists such as ADP only generate minimal response. This study provides preliminary evidence that primitive megakaryocytes have acquired some basic machinery of hemostasis even when the blood flow, liver function and coagulation factors are not present at the early phase of embryonal development. [We thank Dr.William L. Nichols at Mayo Clinic for his critical reviews of this abstract.]

Purification and characterization of a lectin from endophytic fungus Fusarium solani having complex sugar specificity

A lectin from the mycelial extract of an endophytic strain of Fusarium solani was purified. Its hemagglutinating activity was inhibited by glycoproteins possessing N-linked as well as O-linked glycans. The thermodynamics and kinetics of binding of glycans and glycoproteins to F. solani lectin was studied using surface plasmon resonance. The lectin showed high affinity for asialofetuin, asialomucin, asialofibrinogen, and thyroglobulin; and comparatively low affinity for mucin, fetuin, fibrinogen, and holotransferrin. Glycoproteins showed several fold higher affinity than their corresponding glycans with significant contribution from enthalpy and positive entropy, suggesting the involvement of non-polar protein–protein interaction. Moreover, the higher affinity of the glycoproteins was due to their faster association rates and low activation energy.

The protein-binding and drug release properties of macromolecular conjugates containing daptomycin and dextran

Prototype daptomycin–dextran macromolecular conjugates were prepared in an attempt to modify the biodistribution and protein-binding properties of daptomycin. Synthesis of daptomycin macromolecular conjugates involved dextran activation, daptomycin–dextran coupling, and purification. The reaction mixtures were separated on a Sephadex G-100 column using 10% acetronitrile in water as a mobile phase. UV and fluorescence characteristics of high molecular weight fractions demonstrated imine product formation while the lower molecular weight fractions contained free daptomycin, imine, and anilide products. Daptomycin macromolecular conjugates were characterized by drug loading, drug release, and binding affinity for fibrinogen using HPLC analysis and surface plasmon resonance. Drug loading was calculated to be 160 mg of daptomycin per gram of macromolecule. Approximately 9% of the conjugated daptomycin was released from the macromolecular conjugates in aqueous media in the pH range of 1–7.4. The conjugates possessed higher affinity for fibrinogen than that of daptomycin.

Protein resistant surfaces: Comparison of acrylate graft polymers bearing oligo-ethylene oxide and phosphorylcholine side chains

The objective of this work was to compare poly(ethylene glycol) (PEG) and phosphorylcholine (PC) moieties as surface modifiers with respect to their ability to inhibit protein adsorption. Surfaces were prepared by graft polymerization of the methacrylate monomers oligo(ethylene glycol) methyl ether methacrylate (OEGMA, MW 300, PEG side chains of length n=4.5) and 2-methacryloyloxyethyl phosphorylcholine (MPC, MW 295). The grafted polymers thus contained short PEG chains and PC, respectively, as side groups. Grafting on silicon was carried out using surface-initiated atom transfer radical polymerization (ATRP). Graft density was controlled via the surface density of the ATRP initiator, and chain length of the grafts was controlled via the ratio of monomer to sacrificial initiator. The grafted surfaces were characterized by water contact angle, x-ray photoelectron spectroscopy, and atomic force microscopy. The effect of graft density and chain length on fibrinogen adsorption from buffer was investigated using radio labeling methods. Adsorption to both MPC- and OEGMA-grafted surfaces was found to decrease with increasing graft density and chain length. Adsorption on the MPC and OEGMA surfaces for a given chain length and density was essentially the same. Very low adsorption levels of the order of 7 ngcm(2) were seen on the most resistant surfaces. The effect of protein size on resistance to adsorption was studied using binary solutions of lysozyme (MW 14 600) and fibrinogen (MW 340 000). Adsorption levels in these experiments were also greatly reduced on the grafted surfaces compared to the control surfaces. It was concluded that at the lowest graft density, both proteins had unrestricted access to the substrate, and the relative affinities of the proteins for the substrate (higher affinity of fibrinogen) determined the composition of the layer. At the highest graft density also, where the adsorption of both proteins was very low, no preference for one or the other protein was evident, suggesting that adsorption did not involve penetration of the grafts and was occurring at the outer surface of the graft layer. It thus seems likely that preference among different proteins based on ability to penetrate the graft layer would occur, if at all, at a grafting density intermediate between 0.1 and 0.39 cm(2). Again the MPC and OEGMA surfaces behaved similarly. It is suggested that the main determinant of the protein resistance of these surfaces is the "water barrier layer" resulting from their hydrophilic character. In turn the efficacy of the water barrier depends on the monomer density in the graft layer.

Purification and characterization of a recombinant anti-angiogenic kringle fragment expressed in Escherichia coli: Purification and characterization of a tri-kringle fragment from human apolipoprotein (a) (kringle IV (9)–kringle IV (10)–kringle V)

A kringle fragment (type IV (9)–IV (10)–V) from human apolipoprotein (a) (called LK68) was expressed in an inclusion body in Escherichia coli. The LK68 in this inclusion body was rendered soluble with urea, and efficiently refolded via oxidation in the presence of re-dox couple. The refolded LK68 was then purified via two steps of ion exchange chromatography, concentrated via preparative reversed-phase chromatography, and freeze-dried, at a final yield of approximately 30%. The purified LK68 exhibited profound affinity for lysine and fibrinogen, which suggests the proper folding of the kringle fragment, and also indicates that the native characteristics of apolipoprotein (a) were preserved. The purified LK68 was determined to be highly homogeneous upon reversed-phase HPLC analysis and size-exclusion HPLC analysis, in the presence of 20% (v/v) acetonitrile. However, on size-exclusion HPLC analysis without acetonitrile, it was determined to be somewhat heterogeneous, and this was corroborated by native analyses, including native PAGE and IEF.

Characterization of the fibrinogen-binding surface protein Fbl of Staphylococcus lugdunensis

The fbl gene of Staphylococcus lugdunensis encodes a protein Fbl that is 58 % identical to the clumping factor A (ClfA) of Staphylococcus aureus. The fbl gene was present in eight clinical isolates of S. lugdunensis. When Fbl was expressed on the surface of Lactococcus lactis it promoted adherence to immobilized fibrinogen and cell clumping in a fibrinogen solution. Purified recombinant Fbl region A bound to immobilized fibrinogen in a dose-dependent manner and inhibited the adherence of both Fbl-expressing and ClfA-expressing strains of L. lactis to fibrinogen. Adherence of S. lugdunensis and L. lactis Fbl(+) to immobilized fibrinogen was also inhibited by rabbit anti-Fbl region A antibodies and rabbit anti-ClfA region A antibodies, as well as by human immunoglobulin with a high level of anti-ClfA antibodies. Alignment of the A domains of CflA and Fbl revealed that all of the ClfA residues implicated in binding to the gamma-chain of fibrinogen are conserved in Fbl. Nevertheless Fbl had a tenfold lower affinity for fibrinogen, suggesting that sequence differences that occur elsewhere in the protein, possibly in beta-strand E of domain N2, affect ligand binding.

Primary coronary intervention for acute myocardial infarction

DESPITE DRAMATIC IMPROVEments in the treatment of acute ST-segment elevation myocardia l infarct ion (STEMI) during the past decade, approximately 1 in 10 patients still die of this disease. Three critical factors in the immediate management of patients with STEMI result in reduced mortality: prompt diagnosis, immediate treatment with aspirin, and rapid reestablishment of blood flow in the infarctrelated artery. The latter aim may be achieved either pharmacologically, with administration of thrombolytic therapy, or mechanically, with percutaneous coronary intervention (PCI). Primary PCI refers to the strategy of emergent angiography followed by mechanical recanalization of the occluded artery with a balloon catheter, without prior administration of thrombolytic therapy. In its early years, the data regarding primary PCI were limited to observational studies from specialized centers. With the publication of randomized controlled trials (RCTs)comparingPCIwith thrombolytic therapy, however, primary PCI has become accepted as part of the standard armamentarium in the treatment of STEMI. The most recent RCTs on this topic have begun to examine the role of primary PCI in specific subsets of patients with STEMI and the role of adjunctive therapies in patients undergoing primary PCI. Pathophysiology of STEMI ST-segment elevation myocardial infarction is the clinical correlate of fullthickness ischemia and infarction of myocardium and is the result of sudden thrombotic occlusion of its blood supply. The transition from a diseased but patent coronary artery to one that is occluded by thrombus begins with either rupture or erosion of a coronary atherosclerotic plaque. The cellular and molecular events within the plaque that lead to its disruption remain incompletely understood but are clearly unrelated to the severity of the preexisting luminal stenosis. In fact, the majority of STEMI evolve from mild to moderate stenoses. Plaque disruption results in exudation of its lipid-rich core into the lumen and adherence of platelets to the arterial subendothelium. The platelets become activated and develop high affinity for fibrinogen, causing their crosslinking and degranulation. Simultaneously, the release of tissue factor from the lipid-rich core results in activation of the coagulation cascade and generation of thrombin. The result is a luminal thrombus consisting of aggregated platelets, cross-linked fibrin strands, and entrapped red blood cells (FIGURE 1). The enlarging thrombus can interrupt blood flow and lead to an imbalance between oxygen supply and demand that, if severe and persistent, causes transmural infarction of the myocardium.

Identification of a binding site on human FGF-2 for fibrinogen

AbstractEndothelial cell adhesive interactions are mediated by both fibrinogen and fibrin, and growth is stimulated by fibroblast growth factor 2 (FGF-2). We have shown previously that FGF-2 binds specifically and with high affinity to fibrinogen and fibrin and that fibrinogen potentiates the proliferative capacity of FGF-2 and also protects it from proteolytic degradation. To further characterize this interaction we have performed FGF-2 mutagenesis to identify the interactive site. Because FGF-1 has a similar structure to FGF-2 but does not bind to fibrinogen, we used a strategy of cassette and site-directed mutagenesis, exchanging residues from FGF-1 and FGF-2 and correlating structural changes with fibrinogen binding. Two cassette interchange mutants, 2212 and 2211, contained either the third cassette or both the third and fourth cassettes from FGF-1, and neither exhibited any affinity for fibrinogen. Exchange of 5 residues (Phe95, Ser100, Asn102, Arg107, and Arg109) from FGF-2 into the corresponding sites in the third cassette of FGF-1 imparted high-affinity binding with apparent dissociation constants (Kd) of 5.3 nM and 8.6 nM, respectively, compared with 1.3 nM for wild-type FGF-2. We conclude that these 5 residues define a high-affinity binding site in FGF-2 for fibrinogen.

Primary coronary intervention for acute myocardial infarction.

DESPITE DRAMATIC IMPROVEments in the treatment of acute ST-segment elevation myocardia l infarct ion (STEMI) during the past decade, approximately 1 in 10 patients still die of this disease. Three critical factors in the immediate management of patients with STEMI result in reduced mortality: prompt diagnosis, immediate treatment with aspirin, and rapid reestablishment of blood flow in the infarctrelated artery. The latter aim may be achieved either pharmacologically, with administration of thrombolytic therapy, or mechanically, with percutaneous coronary intervention (PCI). Primary PCI refers to the strategy of emergent angiography followed by mechanical recanalization of the occluded artery with a balloon catheter, without prior administration of thrombolytic therapy. In its early years, the data regarding primary PCI were limited to observational studies from specialized centers. With the publication of randomized controlled trials (RCTs)comparingPCIwith thrombolytic therapy, however, primary PCI has become accepted as part of the standard armamentarium in the treatment of STEMI. The most recent RCTs on this topic have begun to examine the role of primary PCI in specific subsets of patients with STEMI and the role of adjunctive therapies in patients undergoing primary PCI. Pathophysiology of STEMI ST-segment elevation myocardial infarction is the clinical correlate of fullthickness ischemia and infarction of myocardium and is the result of sudden thrombotic occlusion of its blood supply. The transition from a diseased but patent coronary artery to one that is occluded by thrombus begins with either rupture or erosion of a coronary atherosclerotic plaque. The cellular and molecular events within the plaque that lead to its disruption remain incompletely understood but are clearly unrelated to the severity of the preexisting luminal stenosis. In fact, the majority of STEMI evolve from mild to moderate stenoses. Plaque disruption results in exudation of its lipid-rich core into the lumen and adherence of platelets to the arterial subendothelium. The platelets become activated and develop high affinity for fibrinogen, causing their crosslinking and degranulation. Simultaneously, the release of tissue factor from the lipid-rich core results in activation of the coagulation cascade and generation of thrombin. The result is a luminal thrombus consisting of aggregated platelets, cross-linked fibrin strands, and entrapped red blood cells (FIGURE 1). The enlarging thrombus can interrupt blood flow and lead to an imbalance between oxygen supply and demand that, if severe and persistent, causes transmural infarction of the myocardium.

The role of gingipains in the pathogenesis of periodontal disease.

Gingipains are trypsin-like cysteine proteinases produced by Porphyromonas gingivalis, a major causative bacterium of adult periodontitis. HRgpA (95 kDa) and RgpB (50 kDa), products of 2 distinct but related genes, rgpA and rgpB, respectively, are specific for Arg-Xaa peptide bonds. Kgp, a product of the kgp gene, is specific for Lys-Xaa bonds. HRgpA and Kgp are non-covalent complexes containing separate catalytic and adhesion/ hemagglutinin domains, while RgpB has only a catalytic domain with a primary structure essentially identical to that of the catalytic subunit of HRgp. HRgpA and RgpB induce vascular permeability enhancement through activation of the kallikrein/kinin pathway and activate the blood coagulation system, which, respectively, are potentially associated with gingival crevicular fluid production and progression of inflammation leading to alveolar bone loss in the periodontitis site. Kgp is the most potent fibrinogen/fibrin degrading enzyme of the 3 gingipains in human plasma and is involved in the bleeding tendency at the diseased gingiva. HRgpA activates coagulation factors and degrades fibrinogen/fibrin more efficiently than RgpB due to the adhesion/hemagglutinin domains, which have affinity for phospholipids and fibrinogen. Gingipains degrade macrophage CD14, thus inhibiting activation of the leukocytes through the lipopolysaccharide (LPS) receptor, and thereby facilitating sustained colonization of P. gingivalis. Gingipains play a role in bacterial housekeeping and infection, including amino acid uptake from host proteins and fimbriae maturation. Based on the important activities of gingipains in the bacterial infection and the pathogenesis of periodontitis, the bacterial proteinases can be targets for periodontal disease therapy. Immunization with RgpB, HRgpA, or a portion of HRgpA catalytic domain attenuated P. gingivalis induced disorders in mice. In addition, a trypsin-like proteinase inhibitor retarded P. gingivalis growth specifically. Gingipains are potent virulence factors of P. gingivalis, and are likely to be associated with the development of periodontitis. It is, therefore, suggested that gingipain inhibition by vaccination and gingipain-specific inhibitors is a useful therapy for adult periodontitis caused by P. gingivalis infection.

Albumin adsorption on cibacron blue F3G-A immobilized onto oligo(ethylene glycol)-terminated self-assembled monolayers.

Self-assembled monolayers can be tailored with specific ligands to a certain protein and at the same time prevent the non-specific adsorption of other proteins. Cibacron Blue F3G-A (CB-thiol) was successfully immobilized onto tetra(ethylene glycol)-terminated alkanethiol (CB-thiol). The affinity of human serum albumin (HSA) to immobilized Cibacron Blue F3G-A was studied using mixed thiolate self-assembled monolayers on gold with different n-alkyl chain lengths and functional terminal groups (CH(3)-; OH- and tetra(ethylene glycol)). Surfaces were characterized using X-ray photoelectron spectroscopy and water contact angle measurements. Albumin adsorption and exchangeability of the adsorbed albumin molecules with other albumin molecules in solution were evaluated using (125)I-radiolabeled HSA. Competitive adsorption between albumin and fibrinogen to the different self-assembled monolayers (SAMs) was also investigated. Results showed that the incorporation of CB-thiol on the monolayers does not increase the HSA adsorption and reversibility on the SAMs. However, although specific adsorption of HSA to the immobilized Cibacron Blue F3G-A was not demonstrated, the presence of CB-thiol decreases the affinity of fibrinogen to the OH-terminated SAMs.

A novel variant of the immunoglobulin fold in surface adhesins of Staphylococcus aureus: crystal structure of the fibrinogen-binding MSCRAMM, clumping factor A.

We report here the crystal structure of the minimal ligand-binding segment of the Staphylococcus aureus MSCRAMM, clumping factor A. This fibrinogen-binding segment contains two similarly folded domains. The fold observed is a new variant of the immunoglobulin motif that we have called DE-variant or the DEv-IgG fold. This subgroup includes the ligand-binding domain of the collagen-binding S.aureus MSCRAMM CNA, and many other structures previously classified as jelly rolls. Structure predictions suggest that the four fibrinogen-binding S.aureus MSCRAMMs identified so far would also contain the same DEv-IgG fold. A systematic docking search using the C-terminal region of the fibrinogen gamma-chain as a probe suggested that a hydrophobic pocket formed between the two DEv-IgG domains of the clumping factor as the ligand-binding site. Mutagenic substitution of residues Tyr256, Pro336, Tyr338 and Lys389 in the clumping factor, which are proposed to contact the terminal residues (408)AGDV(411) of the gamma-chain, resulted in proteins with no or markedly reduced affinity for fibrinogen.

Evidence that the Platelet Integrin αIIbβ3 Is Regulated by the Integrin-Linked Kinase, ILK, in a PI3-Kinase Dependent Pathway

Platelet aggregation is mediated by the integrin alphaIIb beta3 which is activated by intracellular signals during platelet activation. We have attempted to determine if ILK ("Integrin-Linked Kinase") is involved in the regulation of alphaIIb beta3 function. ILK co-immunoprecipitated with beta3 in stimulated platelets. Using confocal microscopy, ILK was detected in the cytoplasm of resting platelets. ADP or PMA stimulation led to its translocation to the plasma membrane. In parallel, there was a transient increase in ILK kinase activity, association with and phosphorylation of beta3. Inhibition of PI3-kinase by two unrelated inhibitors (wortmannin and LY294002) prevented ILK-related functions. However, it did not prevent the conformational change in alphaIIb beta3 (shown by PAC-1 binding), although integrin affinity for fibrinogen was decreased as measured using FITC-fibrinogen. Furthermore, aggregate formation was reduced. Thus ILK transiently associates with and phosphorylates beta3 in a PI3-kinase dependent manner suggesting that it participates at an intermediate stage in a critical mechanism for assuring large stable aggregates.

Structural Organization of the Fibrinogen-binding Region of the Clumping Factor B MSCRAMM of Staphylococcus aureus

The clumping factor B (ClfB) of Staphylococcus aureus is a surface protein that binds to fibrinogen (Ni Eidhin, D., Perkins, S., Francois, P., Vaudaux, P., Hook, M., and Foster, T. J., 1998 Mol. Microbiol. 30, 245-257). The ligand-binding activity is located in the approximately 500-residue A-region (residues 44-542), which represents the N-terminal half of the MSCRAMM protein. We now hypothesize that the ClfB A-region is composed of three subdomains, which we have named N1, N2, and N3, respectively. To examine this hypothesis, we expressed recombinant forms of the individual putative subdomains, the tandem motifs N12 and N23, and the full-length A-region N123. Far UV circular dichroism spectra showed that each subdomain is composed mainly of beta-sheets with little or no discernible alpha-helices. Heat-induced unfolding of individual subdomains occurred with a single state transition and was reversible, indicating that the subdomains can fold as discreet units. Gel permeation chromatography indicated that N2, N3, and N23 are globular. In contrast, domain N1 appeared to be elongated and conferred a somewhat elongated structure on segments containing this subdomain (i.e. N12 or N123). N123, N12, and N23 all bound to fibrinogen, but N23 had a higher affinity for fibrinogen than that observed for the full-length A-region; N123 or for N12. However, an extended N terminus of N23 was required for ligand binding. A form of N23 that was generated by proteolytic processing and lacked the N-terminal extension was unable to bind fibrinogen. Recombinant forms of individual subdomains did not bind fibrinogen. The addition of recombinant N23 effectively inhibited ClfB-mediated bacterial adherence to fibrinogen, and N123 caused some reduction in bacterial attachment, whereas N12 was essentially inactive. Antibodies raised against the central N2 domain of the A-region were the most effective at inhibiting bacterial adhesion to immobilized fibrinogen, although anti-N3 or anti-N1 antibodies also caused some reduction in ClfB-mediated adherence to fibrinogen.

Vascular biology of thrombosis: the role of platelet-vessel wall adhesion.

Arterial thrombosis is initiated after atherosclerotic plaque rupture. The ruptured plaque triggers a cascade of platelet-mediated events that result in the formation of a platelet-rich thrombus. Thrombus development ultimately leads to vessel occlusion, the precipitating event in most myocardial infarctions and many strokes. Arterial thrombosis involves a process of platelet adhesion, activation, and aggregation. Platelets first form a monolayer by adhering to blood vessel subendothelial matrix proteins (collagen and von Willebrand factor) via interactions with platelet membrane glycoprotein (GP) receptors. This interaction produces an intracellular signal that activates platelets to increase expression of a specific GP receptor, GPIIb/IIIa, with a high affinity for fibrinogen and other adhesion molecules. Binding of fibrinogen to adjacent platelets results in irreversible platelet aggregation and the formation of the platelet aggregate. Understanding of this process has led to considerable research into potential antiplatelet compounds. A number of effective antiplatelet drugs are now available, which affect different stages along the platelet adhesion/activation/aggregation pathway. These include aspirin, dipyridamole, and the adenosine diphosphate antagonists. Much effort is now focused on development of thrombin receptor antagonists. However, this is proving to be difficult. Considerable interest has surrounded a new class of platelet drugs, the GPIIb/IIIa receptor antagonists. These drugs have proved effective in short-term infusion trials. However, long-term studies of oral GPIIb/IIIa receptor antagonists have shown increases in mortality. It is believed that these antagonists may act as partial agonists and may increase the risk for thrombosis.

Quantification of Hydrophobicity of Heparin-Treated Polyvinylchloride Tubing

It is understood that the biocompatibility of a material depends upon the proteins that bind to its surface. It is this protein layer that interacts directly with components of the coagulation and inflammatory systems. Fibrinogen, capable of platelet binding and activation, is a particularly important protein in determining a material’s biocompatibility. Hydrophobic materials tend to have a greater affinity for fibrinogen, making them less biocompatible than hydrophilic materials. We compared the hydrophobicity of three different heparin-coated polyvinylchloride (PVC) tubing preparations with uncoated PVC tubing. We also determined if there would be a difference in the applied solution; water, an ionic solution (saline), and a protein solution (fresh frozen plasma). Hydrophobicity was quantified with five separate measurements of contact angles of water, saline, and plasma, and droplet spread diameter of water and saline. We found that Duraflo II displayed hydrophobicity similar to that of uncoated PVC tubing (initial contact angles of water were 78.0 ± 1.1 and 79.6 ± 0.6 degrees, respectively). Carmeda and 3M heparin-coated tubings displayed significantly (p < .01) less hydrophobicity (initial contact angles of water were 59.8 ± 2.1 and 39.6 ± 1.9, respectively). Three minutes after initial contact, the 3M heparin coating was the only preparation that remained significantly (p < .01) less hydrophobic than the uncoated PVC. These data suggest that 3M heparin-coated PVC is the most biocompatible of the tubings we examined, followed by Carmeda, Duraflo II, and the untreated PVC tubing.

Platelet glycoprotein IIb/IIIa receptors and Glanzmann's thrombasthenia.

Platelet aggregation and fibrin formation are essential for the maintenance of normal hemostasis, a system designed to act quickly and effectively to arrest hemorrhage. This system is also triggered by pathogenic events, such as the rupture of an atherosclerotic plaque, which can lead to thrombotic vaso-occlusion, ischemia, and infarction. Platelets are a major contributor to these damaging and life-threatening thrombotic phenomena because of their adhesive properties, which result in the release of soluble mediators, platelet aggregation, and enhancement of thrombin generation.1 The platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptor is a key component in the pathway to platelet aggregation; consequently, this receptor has become the target for therapeutic intervention. A paradigm of this antiplatelet treatment modality is found naturally in the inherited disorder Glanzmann’s thrombasthenia. A key feature of this disease is that patients present with mucocutaneous bleeding but only rarely demonstrate spontaneous central nervous system hemorrhage,2 a feared complication of anticoagulant and antiplatelet therapy. All of the mutations that have been identified in patients with Glanzmann’s thrombasthenia result in a functional deficiency of GPIIb/IIIa receptors,2 3 and a hallmark of this disease is the absence of agonist-induced platelet aggregation. The molecular characterization of mutations causing Glanzmann’s thrombasthenia has provided a wealth of information on structure-function relations of the GPIIb/IIIa receptor. This review will briefly summarize those mutations that affect ligand-binding domains and receptor activation and present them in the context of predicted structures. More comprehensive coverage can be found in reviews discussing the structure and function of the GPIIb/IIIa receptor complex4 5 and the clinical and molecular basis of Glanzmann’s thrombasthenia.2 3 Platelets are the first line of defense in preventing blood loss from injured blood vessels via recognition and adhesion to components of the subendothelial matrix. This event is followed by formation of a platelet …

Fibrinogen Caracas V, an Abnormal Fibrinogen with an Aα 532 Ser→Cys Substitution Associated with Thrombosis

A new dysfibrinogenemia associated with thrombophilia has been identified in a Venezuelan kindred. Thrombin and Reptilase times were prolonged and the accelerating capacity of the patient's fibrin on the t-PA-induced plasminogen activation was decreased. In addition the affinity of fibrinogen for plasminogen was diminished. Permeability and electron microscopy studies revealed that the abnormal clot was made up of thin and densely packed fibres giving rise to a reduced fibrin gel porosity. This was confirmed by turbidity studies showing a decreased fibre mass/length ratio. Affected members were heterozygous for an Aalpha 532 Ser-->Cys mutation as demonstrated by genetic analyses. This abnormal fibrinogen has been designated as Fibrinogen Caracas V. The family study showed a convincing association between the mutation and thrombotic manifestations. The thrombotic tendency may be ascribed to lack of accelerating capacity of fibrin to induce fibrinolysis caused by an abnormal clot structure with thin fibres and reduced porosity.