Identification and characterization of novel mutations implicated in congenital fibrinogen disorders.

INTRODUCTION: Fibrinogen is a complex molecule comprised of two sets of Aα, Bß, and γ chains. Fibrinogen deficiencies can lead to the development of bleeding or thromboembolic events. The objective of this study was to perform DNA sequence analysis of patients with clinical fibrinogen abnormalities, and to perform genotype-phenotype correlations. MATERIALS AND METHODS: DNA from 31 patients was sequenced to evaluate disease-causing mutations in the three fibrinogen genes: FGA,FGB, and FGG. Clinical data were extracted from medical records or from consultation with referring hematologists. Fibrinogen antigen and functional (Clauss method) assays, as well as reptilase time (RT) and thrombin time (TT) were obtained for each patient. Molecular modeling was used to simulate the functional impact of specific missense variants on the overall protein structure. RESULTS: Seventeen mutations, including six novel mutations, were identified in the three fibrinogen genes. There was little correlation between genotype and phenotype. Molecular modeling predicted a substantial conformational change for a novel variant, FGG p.Ala289Asp, leading to a more rigid molecule in a region critical for polymerization and alignment of the fibrin monomers. This mutation is associated with both bleeding and clotting in the two affected individuals. CONCLUSIONS: Robust genotype-phenotype correlations are difficult to establish for fibrinogen disorders. Molecular modeling might represent a valuable tool for understanding the function of certain missense fibrinogen mutations but those should be followed by functional studies. It is likely that genetic and environmental modifiers account for the incomplete penetrance and variable expressivity that characterize fibrinogen disorders.

Effects of anti-ß2GPI antibodies on VWF release from human umbilical vein endothelial cells and ADAMTS13 activity.

BACKGROUND: Antiphospholipid syndrome (APS) is characterized by recurrent thromboembolic events in the setting of pathologic autoantibodies, some of which are directed to ß2-Glycoprotein 1 (ß2GPI). The mechanisms of thrombosis in APS appear to be multifactorial and likely include a component of endothelial activation. Among other things, activated endothelium secretes von Willebrand factor, a hemostatic protein that in excess can increase the risk of thrombosis. OBJECTIVE: We hypothesized that anti-ß2GPI antibodies could regulate the release and modulation of VWF from endothelial cells. PATIENTS/METHODS: Isolated anti-ß2GPI antibodies from patients with APS were assayed for their ability to induced VWF release from HUVECs and modulate the effects of ADAMTS13 in a shear-dependent assay. RESULTS: We observed that anti-ß2GPI antibodies from some patients with APS induced VWF release from human endothelial cells but did not induce formation of cell-anchored VWF-platelet strings. Finally, we also determined that one of the Anti-ß2GPI antibodies tested can inhibit the function of ADAMTS13, the main modulator of extracellular VWF. CONCLUSIONS: These results suggest that VWF and ADAMTS13 may play a role in the prothrombotic phenotype of APS.

Mutations in the D'D3 region of VWF traditionally associated with type 1 VWD lead to quantitative and qualitative deficiencies of VWF.

Type 1 von Willebrand disease (VWD) is characterized by low plasma levels of von Willebrand factor (VWF) and clinical bleeding. Several mechanisms have been described that cause a decrease in plasma VWF levels in VWD, and the goal of this study was to elucidate the pathogenic origins of VWD for a group of mutations in the VWF D'D3 region traditionally associated with type 1 VWD. Varying ratios of mutant-to-wild-type VWF were expressed in two cell lines in order to study the intracellular location, multimer assembly, secretion and function of VWF. We identified four mutants (M771I, Y1146C, T1156M, R782Q) that caused defective intracellular packaging and markedly reduced VWF secretion. Consistent with previous reports, Y1146C and T1156M VWF led to a loss of high molecular weight multimers. In a functional analysis, Y1146C demonstrated a novel FVIII binding defect. Mutations R924W and I1094T were processed normally and did not show abnormal FVIII binding suggesting that other mechanisms such as plasma clearance or platelet binding defects may contribute to the pathogenicity of these mutants.

Characterization of collagen thin films for von Willebrand factor binding and platelet adhesion.

Von Willebrand factor (VWF) binding and platelet adhesion to subendothelial collagens are initial events in thrombus formation at sites of vascular injury. These events are often studied in vitro using flow assays designed to mimic vascular hemodynamics. Flow assays commonly employ collagen-functionalized substrates, but a lack of standardized methods of surface ligation limits their widespread use as a clinical diagnostic. Here, we report the use of collagen thin films (CTF) in flow assays. Thin films were grown on hydrophobic substrates from type I collagen solutions of increasing concentration (10, 100, and 1000 µg/mL). We found that the corresponding increase in fiber surface area determined the amount of VWF binding and platelet adhesion. The association rate constant (k(a)) of plasma VWF binding at a wall shear stress of 45 dyn/cm(2) was 0.3 × 10(5), 1.8 × 10(5), and 1.6 × 10(5) M(-1) s(-1) for CTF grown from 10, 100, and 1000 µg/mL solutions, respectively. We observed a 5-fold increase in VWF binding capacity with each 10-fold increase in collagen solution concentration. The association rates of Ser1731Thr and His1786Asp VWF mutants with collagen binding deficiencies were 9% and 22%, respectively, of wild-type rates. Using microfluidic devices for blood flow assays, we observed that CTF supported platelet adhesion at a wall shear rate of 1000 s(-1). CTF grown from 10 and 100 µg/mL solutions had variable levels of platelet surface coverage between multiple normal donors. However, CTF substrates grown from 1000 µg/mL solutions had reproducible surface coverage levels (74 ± 17%) between normal donors, and there was significantly diminished surface coverage from two type 1 von Willebrand disease patients (8.0% and 24%). These results demonstrate that collagen thin films are homogeneous and reproducible substrates that can measure dysfunctions in VWF binding and platelet adhesion under flow in a clinical microfluidic assay format.

Characterization of human platelet binding of recombinant T cell receptor ligand.

BACKGROUND: Recombinant T cell receptor ligands (RTLs) are bio-engineered molecules that may serve as novel therapeutic agents for the treatment of neuroinflammatory conditions such as multiple sclerosis (MS). RTLs contain membrane distal α1 plus ß1 domains of class II major histocompatibility complex linked covalently to specific peptides that can be used to regulate T cell responses and inhibit experimental autoimmune encephalomyelitis (EAE). The mechanisms by which RTLs impede local recruitment and retention of inflammatory cells in the CNS, however, are not completely understood. METHODS: We have recently shown that RTLs bind strongly to B cells, macrophages, and dendritic cells, but not to T cells, in an antigenic-independent manner, raising the question whether peripheral blood cells express a distinct RTL-receptor. Our study was designed to characterize the molecular mechanisms by which RTLs bind human blood platelets, and the ability of RTL to modulate platelet function. RESULTS: Our data demonstrate that human blood platelets support binding of RTL. Immobilized RTL initiated platelet intracellular calcium mobilization and lamellipodia formation through a pathway dependent upon Src and PI3 kinases signaling. The presence of RTL in solution reduced platelet aggregation by collagen, while treatment of whole blood with RTL prolonged occlusive thrombus formation on collagen. CONCLUSIONS: Platelets, well-known regulators of hemostasis and thrombosis, have been implicated in playing a major role in inflammation and immunity. This study provides the first evidence that blood platelets express a functional RTL-receptor with a putative role in modulating pathways of neuroinflammation.

A role for factor XIIa-mediated factor XI activation in thrombus formation in vivo.

Mice lacking factor XII (fXII) or factor XI (fXI) are resistant to experimentally-induced thrombosis, suggesting fXIIa activation of fXI contributes to thrombus formation in vivo. It is not clear whether this reaction has relevance for thrombosis in pri mates. In 2 carotid artery injury models (FeCl(3) and Rose Bengal/laser), fXII-deficient mice are more resistant to thrombosis than fXI- or factor IX (fIX)-deficient mice, raising the possibility that fXII and fXI function in distinct pathways. Antibody 14E11 binds fXI from a variety of mammals and interferes with fXI activation by fXIIa in vitro. In mice, 14E11 prevented arterial occlusion induced by FeCl(3) to a similar degree to total fXI deficiency. 14E11 also had a modest beneficial effect in a tissue factor-induced pulmonary embolism model, indicating fXI and fXII contribute to thrombus formation even when factor VIIa/tissue factor initiates thrombosis. In baboons, 14E11 reduced platelet-rich thrombus growth in collagen-coated grafts inserted into an arteriovenous shunt. These data support the hypothesis that fXIIa-mediated fXI activation contributes to thrombus formation in rodents and primates. Since fXII deficiency does not impair hemostasis, targeted inhibition of fXI activation by fXIIa may be a useful antithrombotic strategy associated with a low risk of bleeding complications.

Identification of coagulation factor XI as a ligand for platelet apolipoprotein E receptor 2 (ApoER2).

OBJECTIVE: Factor XI (FXI) promotes hemostasis and thrombosis through enhancement of thrombin generation and has been shown to play a critical role in the formation of occlusive thrombi in arterial injury models. The aim of this study was to investigate the mechanisms governing interactions between FXI and platelets. METHODS AND RESULTS: Platelet adhesion to immobilized FXI was abrogated in the presence of the low-density lipoprotein (LDL) receptor antagonist, receptor-associated protein (RAP), soluble recombinant apolipoprotein E receptor 2 (ApoER2), or the LDL-binding domain 1 or 2 of ApoER2. FXI supported wild-type murine platelet binding; in contrast, ApoER2-deficient murine platelets did not adhere to FXI. In the presence of shear, platelet aggregates formed on FXI or activated FXI (FXIa) surfaces, whereas the presence of RAP, binding domain 1 of ApoER2, or an anti-GPIb alpha mAb blocked platelet adhesion to FXI or FXIa under shear. Soluble FXI bound to immobilized ApoER2' with an affinity of 61 nmol/L. CONCLUSIONS: This study has identified apolipoprotein E receptor 2 (ApoER2, LRP8), a member of the LDL receptor family, as a platelet receptor for FXI. The interaction of FXI with other cell types that express ApoER2 remains to be explored.

Activated protein C ligation of ApoER2 (LRP8) causes Dab1-dependent signaling in U937 cells.

Binding of activated protein C (APC) to cells triggers multiple beneficial cytoprotective activities that suppress apoptosis, inflammation, and endothelial barrier breakdown. One paradigm for APC's signaling emphasizes its binding to endothelial cell protein C receptor (EPCR) and subsequent protease activated receptor (PAR)-1 activation. Here we used human monocytic-like U937 cells to evaluate apolipoprotein E receptor 2 (ApoER2)-dependent signaling by APC and found that APC initiated rapid phosphorylation of Tyr-220 in the adaptor protein disabled-1 (Dab1) and of Ser-473 in Akt. APC also induced phosphorylation of Ser-9 in glycogen synthase kinase 3beta (GSK3beta), which was blocked by the PI3K inhibitor LY294002. Receptor-associated protein (RAP), a general antagonist for binding of ligands to LDL receptor family members, inhibited APC-induced phosphorylation of Dab1 and GSK3beta, whereas anti-EPCR or anti-PAR1 blocking antibodies did not. Knocking down ApoER2 by using siRNA-ablated APC induced Dab1 phosphorylation, suggesting that RAP-sensitive APC-induced signaling requires ApoER2. In surface plasmon resonance equilibrium binding studies, APC bound with high affinity to soluble (s) ApoER2 (apparent K(d), approximately 30 nM) but not to soluble very low density lipoprotein receptor. RAP blocked APC binding to sApoER2 but not to sEPCR. RAP blocked binding of U937 cells to immobilized APC. RAP also blocked APC's ability to inhibit endotoxin-induced tissue factor pro-coagulant activity of U937 cells. Thus, we propose that ligation of ApoER2 by APC signals via Dab1 phosphorylation and subsequent activation of PI3K and Akt and inactivation of GSK3beta, thereby contributing to APC's beneficial effects on cells.