We tested the possibility that immune complexes formed following platelet factor 4 (PF4/CXCL4) binding to anti-PF4 antibody can stimulate neutrophil activation, similar to previous reports with platelets. Monoclonal Abs against PF4 and IgG from a heparin-induced thrombocytopenia (HIT) patient were applied. We observed that although PF4 or anti-PF4 antibody alone did not alter neutrophil function, costimulation with both reagents IntroductionPlatelet factor 4 (PF4/CXCL4) is an ELR Ϫ tetrameric, cationic chemokine that constitutes 25% of the protein in platelet ␣-granules. 1,2 It is also found bound to the luminal vascular endothelial surface. 3,4 Although platelets represent the primary source of PF4, a recent report 5 also suggests that the protein is expressed at lower levels in other cells of the immune system including cultured T cells, monocytes, and endothelial and smooth muscle cells. Treatment of patients with heparin results in PF4-heparin complex formation, and a dramatic increase in blood concentration of PF4. Heparin-PF4 binding is facilitated by the multivalent nature of both heparin and PF4. Whereas PF4 binds heparin with relatively high apparent affinity (K d , ϳ 4-20 nM), 6 binding interactions with glycosaminoglycans including those on the neutrophil surface are weaker (K d , ϳ 650 nM). 7 PF4 is typically at less than 1 nM in normal serum, and this level rises to 0.4 to 2.5 M upon platelet activation. 1,2 One to five percent of patients receiving unfractionated heparin suffer from heparin-induced thrombocytopenia (HIT). 8,9 In these cases, PF4-heparin complexes trigger an immune response and the generation of anti-PF4 antibodies. Macromolecular antibodyheparin-PF4 immune complexes then bind Fc␥RIIa (CD32a) on platelets and induce platelet activation. 4,10,11 Using chondroitinase ABC to cleave cell surface glycosaminoglycans, Rauova et al 12 suggest a role for platelet chondroitin sulfates in cell activation. Platelet activation in turn results in more PF4 release into circulation, enhanced immune-complex formation, exaggerated platelet activation, and cell clearance.Ten to fifty percent of HIT patients experience thrombosis in the arterial and venular circulation. This can lead to limb-and life-threatening complications. Although the precise mechanism(s) of thrombosis is yet unestablished, evidence in literature supports a role for activated platelets, resulting procoagulants, and microparticles. 13 Immune complexes also bind to endothelial cells 3,4 and they may up-regulate adhesion molecules that are typically associated with inflammatory ailments including E-/P-selectin, VCAM-1, and ICAM-1. 14 The release of tissue factor and interleukin-8 by monocytes is an additional feature that is thought to contribute to thrombosis. 15,16 Finally, plasma from HIT patients has been shown to activate neutrophils and enhance platelet-neutrophil adhesion via yet unidentified mechanisms. 17 In the current paper, we examined the effect of PF4, heparin, and anti-PF4 antibodies (monoclonals and polyclonal HIT pa...

The function of the mechanosensitive, multi-meric blood protein von Willebrand factor (VWF) is dependent on its size. We tested the hypothesis that VWF may self-associate on the platelet glycoprotein Ib (GpIb) receptor under hydrodynamic shear. Consistent with this proposition, whereas Alexa-488-conjugated VWF (VWF-488) bound platelets at modest levels, addition of unla-beled VWF enhanced the extent of VWF-488 binding. Recombinant VWF lacking the A1-domain was conjugated with Alexa-488 to produce A1-488. Although A1-488 alone did not bind platelets under shear, this protein bound GpIb on addition of either purified plasma VWF or recombinant full-length VWF. The extent of self-association increased with applied shear stress more than 60 to 70 dyne/cm 2. A1-488 bound plate-lets in the milieu of plasma. On application of fluid shear to whole blood, half of the activated platelets had A1-488 bound, suggesting that VWF self-association may be necessary for cell activation. Shearing plate-lets with 6-m beads bearing either immobilized VWF or anti-GpIb mAb resulted in cell activation at shear stress down to 2 to 5 dyne/cm 2. Taken together, the data suggest that fluid shear in circulation can increase the effective size of VWF bound to platelet GpIb via protein self-association. This can trigger mechanotransduction and cell activation by enhancing the drag force applied on the cell-surface receptor. (Blood. 2010;116(19):3990-3998) Introduction von Willebrand factor (VWF) is a large, multidomain glycoprotein found in normal blood at concentrations of approximately 10 g/mL. 1 The protein plays an important role in hemostasis by both carrying the coagulation protein factor VIII (FVIII) in circulation and by regulating the adhesion of platelets to sites of vascular injury. Whereas the DD3 domain of VWF binds FVIII, the A1 and C1 domains engage platelet receptors glycoprotein Ib (GPIb) and IIb 3 (GPIIb-IIIa), respectively. Monomeric VWF has a molecular mass of approximately 250 kDa. This unit further polymerizes, via disulfide linkage formation in the endoplasmic reticulum and Golgi of endothelial cells and megakaryocytes. Multimeric VWF size ranges from 0.5 to 20 MDa. 2 Ultra/unusually-large VWF is secreted from the Weibel-Palade bodies of endothe-lial cells on stimulation with a variety of agonists associated with inflammation and thrombosis, including thrombin, histamine, and tumor necrosis factor-. The hemostatic potential of VWF increases with protein size and the magnitude of the applied hydrodynamic shear. 3,4 Ultra/ unusually-large VWF secreted from endothelial cells under shear is extended in the form of strings or bundles on the vessel wall. 5,6 Shear-mediated extension enhances cleavage of the cryptic Y 1605-M 1606 bond within the VWF-A2 domain by the constitutively active blood metalloprotease, ADAMTS13. In addition to cleavage when immobilized on the endothelium, VWF subjected to fluid shear in flowing blood 7 and on platelets 8 is also susceptible to proteolysis by ADAMTS13. Together, these mechanisms reduce a...

Noncovalent association between the von Willebrand factor (VWF) propeptide (VWFpp) and mature VWF aids N-terminal multimerization and protein compartmentalization in storage granules. This association is currently thought to dissipate after secretion into blood. In the present study, we examined this proposition by quantifying the affinity and kinetics of VWFpp binding to mature VWF using surface plasmon resonance and by developing novel anti-VWF D'D3 mAbs. Our results show that the only binding site for VWFpp in mature VWF is in its D'D3 domain. At pH 6.2 and 10mM Ca(2+), conditions mimicking intracellular compartments, VWFpp-VWF binding occurs with high affinity (K(D) = 0.2nM, k(off) = 8 × 10(-5) s(-1)). Significant, albeit weaker, binding (K(D) = 25nM, k(off) = 4 × 10(-3) s(-1)) occurs under physiologic conditions of pH 7.4 and 2.5mM Ca(2+). This interaction was also observed in human plasma (K(D) = 50nM). The addition of recombinant VWFpp in both flow-chamber-based platelet adhesion assays and viscometer-based shear-induced platelet aggregation and activation studies reduced platelet adhesion and activation partially. Anti-D'D3 mAb DD3.1, which blocks VWFpp binding to VWF-D'D3, also abrogated platelet adhesion, as shown by shear-induced platelet aggregation and activation studies. Our data demonstrate that VWFpp binding to mature VWF occurs in the circulation, which can regulate the hemostatic potential of VWF by reducing VWF binding to platelet GpIbα.

BackgroundVon Willebrand Factor (VWF) A1‐domain binding to platelet receptor GpIbα is an important fluid‐shear dependent interaction that regulates both soluble VWF binding to platelets, and platelet tethering onto immobilized VWF. We evaluated the roles of different structural elements at the N‐terminus of the A1‐domain in regulating shear dependent platelet binding. Specifically, the focus was on the VWF D′D3‐domain, A1‐domain N‐terminal flanking peptide (NFP), and O‐glycans on this peptide.Methods and ResultsFull‐length dimeric VWF (ΔPro‐VWF), dimeric VWF lacking the D′D3 domain (ΔD′D3‐VWF), and ΔD′D3‐VWF variants lacking either the NFP (ΔD′D3NFP─‐VWF) or just O‐glycans on this peptide (ΔD′D3OG─‐VWF) were expressed. Monomeric VWF‐A1 and D′D3‐A1 were also produced. In ELISA, the apparent dissociation constant (KD) of soluble ΔPro‐VWF binding to immobilized GpIbα (KD≈100 nmol/L) was 50‐ to 100‐fold higher than other proteins lacking the D′D3 domain (KD~0.7 to 2.5 nmol/L). Additionally, in surface plasmon resonance studies, the on‐rate of D′D3‐A1 binding to immobilized GpIbα (kon=1.8±0.4×104 (mol/L)−1·s−1; KD=1.7 μmol/L) was reduced compared with the single VWF‐A1 domain (kon=5.1±0.4×104 (mol/L)−1·s−1; KD=1.2 μmol/L). Thus, VWF‐D′D3 primarily controls soluble VWF binding to GpIbα. In contrast, upon VWF immobilization, all molecular features regulated A1‐GpIbα binding. Here, in ELISA, the number of apparent A1‐domain sites available for binding GpIbα on ΔPro‐VWF was ≈50% that of the ΔD′D3‐VWF variants. In microfluidics based platelet adhesion measurements on immobilized VWF and thrombus formation assays on collagen, human platelet recruitment varied as ΔPro‐VWF<ΔD′D3‐VWF<ΔD′D3NFP─‐VWF<ΔD′D3OG─‐VWF.ConclusionsWhereas VWF‐D′D3 is the major regulator of soluble VWF binding to platelet GpIbα, both the D′D3‐domain and N‐terminal peptide regulate platelet translocation and thrombus formation.

The aim of our study was to estimate the uncultured eubacterial diversity of a soil sample collected below a dead seal, Cape Evans, McMurdo, Antarctica by an SSU rDNA gene library approach. Our study by sequencing of clones from SSU rDNA gene library approach revealed high diversity in the soil sample from Antarctica. More than 50% of clones showed homology to Cytophaga-Flavobacterium-Bacteroides group; sequences also belonged to alpha, beta, gamma proteobacteria, Thermus-Deinococcus and high GC gram-positive group; Phylogenetic analysis of the SSU rDNA clones showed the presence of species belonging to Cytophaga spp., Vitellibacter vladivostokensis, Aequorivita lipolytica, Aequorivita crocea, Flavobacterium spp., Flexibacter sp., Subsaxibacter broadyi, Bacteroidetes, Roseobacter sp., Sphingomonas baekryungensis, Nitrosospira sp., Nitrosomonas cryotolerans, Psychrobacter spp., Chromohalobacter sp., Psychrobacter okhotskensis, Psychrobacter fozii, Psychrobacter urativorans, Rubrobacter radiotolerans, Marinobacter sp., Rubrobacteridae, Desulfotomaculum aeronauticum and Deinococcus sp. The presence of ammonia oxidizing bacteria in Antarctica soil was confirmed by the presence of the amoA gene. Phylogenetic analysis revealed grouping of clones with their respective groups.