DSpace Academic/Research Unit: Haematology

Quantification of unfractionated heparin in human plasma and whole blood by means of novel fluorogenic anti-FXa assays

2011-10-05T14:20:52Z

Title: Quantification of unfractionated heparin in human plasma and whole blood by means of novel fluorogenic anti-FXa assays Author: O'DONNELL, JAMES Abstract: Novel and sensitive plate-based fluorogenic anti-factor Xa (FXa) assays were investigated to quantify unfractionated heparin (UFH) in human plasma and whole blood within the therapeutic ranges of 0-1.6 U/mL and 0–0.8 U/mL, respectively. Two fluorogenic anti-FXa assay methods were defined for low (0-0.6 U/mL) and high (0.6-1.2 U/mL) pharmacologically relevant UFH concentration ranges in pooled human plasma. In both cases significant differences were observed at intervals of 0.2 U/mL (P < 0.05). The semi-logarithmic plots of the calibration curves in the low and high UFH range were both fitted to linear regressions with correlation coefficients of 0.96 and >0.99, respectively. The assay was also optimized for whole blood which was capable of differentiating UFH concentrations at intervals of 0.2 U/mL (P < 0.05) in the range of 0-0.4 U/mL. The statistically different results were fitted to a linear regression with a correlation coefficient of >0.99. The results obtained in this study could assist diagnostic laboratories towards improved monitoring of UFH therapy. Description: PUBLISHED

Multifunctional specificity of the protein C/activated protein C Gla domain.

2010-11-13T03:02:26Z

Title: Multifunctional specificity of the protein C/activated protein C Gla domain. Author: PRESTON, ROGER Abstract: Activated protein C (APC) has potent anticoagulant and anti-inflammatory properties that are mediated in part by its interactions with its cofactor protein S and the endothelial cell protein C receptor (EPCR). The protein C/APC Gla domain is implicated in both interactions. We sought to identify how the protein C Gla domain enables specific protein-protein interactions in addition to its conserved role in phospholipid binding. The human prothrombin Gla domain, which cannot bind EPCR or support protein S cofactor activity, has 22/45 residues that are not shared with the human protein C Gla domain. We hypothesized that the unique protein C/APC Gla domain residues were responsible for mediating the specific interactions. To assess this, we generated 13 recombinant protein C/APC variants incorporating the prothrombin residue substitutions. Despite anticoagulant activity similar to wild-type APC in the absence of protein S, APC variants APC(PT33-39) (N33S/V34S/D35T/D36A/L38D/A39V) and APC(PT36/38/39) (D36A/L38D/A39V) were not stimulated by protein S, whereas APC(PT35/36) (D35T/D36A) exhibited reduced protein S sensitivity. Moreover, PC(PT8/10) (L8V/H10K) displayed negligible EPCR affinity, despite normal binding to anionic phospholipid vesicles and factor Va proteolysis in the presence and absence of protein S. A single residue variant, PC(PT8), also failed to bind EPCR. Factor VIIa, which also possesses Leu-8, bound soluble EPCR with similar affinity to wild-type protein C, collectively confirming Leu-8 as the critical residue for EPCR recognition. These results reveal the specific Gla domain residues responsible for mediating protein C/APC molecular recognition with both its cofactor and receptor and further illustrate the multifunctional potential of Gla domains. Description: PUBLISHED

ADAMTS13 substrate recognition of von Willebrand factor A2 domain.

2010-11-13T03:02:26Z

Title: ADAMTS13 substrate recognition of von Willebrand factor A2 domain. Author: PRESTON, ROGER Abstract: ADAMTS13 controls the multimeric size of circulating von Willebrand factor (VWF) by cleaving the Tyr1605–Met1606 bond in theA2 domain. To examine substrate recognition, we expressed in bacteria and purified three A2 (VWF76-(1593–1668), VWF115-(1554–1668), VWFA2-(1473–1668)) and one A2-A3 (VWF115-A3-(1554–1874)) domain fragments. Using high pressure liquid chromatography analysis, the initial rates of VWF115 cleavage by ADAMTS13 at different substrate concentrations were determined, and from this the kinetic constants were derived (Km 1.61 μm; kcat 0.14 s–1), from which the specificity constant kcat/Km was calculated, 8.70 × 104 m–1 s–1. Similar values of the specificity constant were obtained for VWF76 and VWF115-A3. To identify residues important for recognition and proteolysis of VWF115, we introduced certain type 2A von Willebrand disease mutations by site-directed mutagenesis. Although most were cleaved normally, one (D1614G) was cleaved ∼8-fold slower. Mutagenesis of additional charged residues predicted to be in close proximity to Asp1614on the surface of the A2 domain (R1583A, D1587A, D1614A, E1615A, K1617A, E1638A, E1640A) revealed up to 13-fold reduction in kcat/Km for D1587A, D1614A, E1615A, and K1617A mutants. When introduced into the intact VWFA2 domain, proteolysis of the D1587A, D1614A, and E1615A mutants was also slowed, particularly in the presence of urea. Surface plasmon resonance demonstrated appreciable reduction in binding affinity between ADAMTS13 and VWF115 mutants (KD up to ∼1.3 μm), compared with VWF115 (KD 20 nm). These results demonstrate an important role for Asp1614 and surrounding charged residues in the binding and cleavage of the VWFA2 domain by ADAMTS13. Description: PUBLISHED

Dissociation of activated protein C functions by elimination of protein S cofactor enhancement.

2010-11-13T03:02:24Z

Title: Dissociation of activated protein C functions by elimination of protein S cofactor enhancement. Author: HARMON, SHONA; PRESTON, ROGER; O'DONNELL, JAMES; NI AINLE, FIONNUALA; JOHNSON, JENNIFER Abstract: Activated protein C (APC) plays a critical anticoagulant role in vivo by inactivating procoagulant factor Va and factor VIIIa and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor can initiate protease-activated receptor-1 (PAR-1)-mediated cytoprotective signaling. Protein S constitutes a critical cofactor for the anticoagulant function of APC but is not known to be involved in regulating APC-mediated protective PAR-1 signaling. In this study we utilized a site-directed mutagenesis strategy to characterize a putative protein S binding region within the APC Gla domain. Three single amino acid substitutions within the APC Gla domain (D35T, D36A, and A39V) were found to mildly impair protein S-dependent anticoagulant activity (<2-fold) but retained entirely normal cytoprotective activity. However, a single amino acid substitution (L38D) ablated the ability of protein S to function as a cofactor for this APC variant. Consequently, in assays of protein S-dependent factor Va proteolysis using purified proteins or in the plasma milieu, APC-L38D variant exhibited minimal residual anticoagulant activity compared with wild type APC. Despite the location of Leu-38 in the Gla domain, APC-L38D interacted normally with endothelial cell protein C receptor and retained its ability to trigger PAR-1 mediated cytoprotective signaling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to separate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain. Description: PUBLISHED

Platelet factor 4 impairs the anticoagulant activity of activated protein C.

2010-11-12T03:01:51Z

Title: Platelet factor 4 impairs the anticoagulant activity of activated protein C. Author: HARMON, SHONA; PRESTON, ROGER; JOHNSON, JENNIFER; O'DONNELL, JAMES; NI AINLE, FIONNUALA Abstract: Platelet factor 4 (PF4) is an abundant platelet -granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the -carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anticoagulant activity. PF4 inhibited both protein S-dependentAPC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q/ R679Q and FVa-R306Q/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg306 by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activation Description: PUBLISHED

Severe Plasmodium falciparum malaria is associated with circulating ultra-large von Willebrand multimers and ADAMTS13 inhibition.

2010-06-17T02:02:21Z

Title: Severe Plasmodium falciparum malaria is associated with circulating ultra-large von Willebrand multimers and ADAMTS13 inhibition. Author: JENKINS, P VINCENT; O'DONNELL, JAMES; PRESTON, ROGER Abstract: Plasmodium falciparum infection results in adhesion of infected erythrocytes to blood vessel endothelium, and acute endothelial cell activation, together with sequestration of platelets and leucocytes. We have previously shown that patients with severe infection or fulminant cerebral malaria have significantly increased circulatory levels of the adhesive glycoprotein von Willebrand factor (VWF) and its propeptide, both of which are indices of endothelial cell activation. In this prospective study of patients from Ghana with severe (n = 20) and cerebral (n = 13) P. falciparum malaria, we demonstrate that increased plasma VWF antigen (VWF:Ag) level is associated with disproportionately increased VWF function. VWF collagen binding (VWF:CB) was significantly increased in patients with cerebral malaria and severe malaria (medians 7.6 and 7.0 IU/ml versus 1.9 IU/ml; p<0.005). This increased VWF:CB correlated with the presence of abnormal ultra-large VWF multimers in patient rather than control plasmas. Concomitant with the increase in VWF:Ag and VWF:CB was a significant persistent reduction in the activity of the VWF-specific cleaving protease ADAMTS13 (approximately 55% of normal; p<0.005). Mixing studies were performed using P. falciparum patient plasma and normal pooled plasma, in the presence or absence of exogenous recombinant ADAMTS13. These studies demonstrated that in malarial plasma, ADAMTS13 function was persistently inhibited in a time-dependent manner. Furthermore, this inhibitory effect was not associated with the presence of known inhibitors of ADAMTS13 enzymatic function (interleukin-6, free haemoglobin, factor VIII or thrombospondin-1). These novel findings suggest that severe P. falciparum infection is associated with acute endothelial cell activation, abnormal circulating ULVWF multimers, and a significant reduction in plasma ADAMTS13 function which is mediated at least in part by an unidentified inhibitor. Description: PUBLISHED

A new microtubule-targeting compound PBOX-15 inhibits T-cell migration via post-translational modifications of tubulin.

2010-06-03T15:57:01Z

Title: A new microtubule-targeting compound PBOX-15 inhibits T-cell migration via post-translational modifications of tubulin. Author: MCELLIGOTT, ANTHONY Abstract: The ordered, directional migration of T-lymphocytes is a key process during immune surveillance, immune response, and development. A novel series of pyrrolo-1,5-benzoxazepines have been shown to potently induce apoptosis in variety of human chemotherapy resistant cancer cell lines, indicating their potential in the treatment of both solid tumors and tumors derived from the hemopoietic system. Pyrrolobenzoxazepine 4-acetoxy-5-(1-naphtyl)naphtho[2,3-b]pyrrolo[1,2-d][1,4]-oxazepine (PBOX-15) has been shown to depolymerize tubulin in vitro and in the MCF7 breast cancer cell line. We hypothesized that this may suggest a role for this compound in modulating integrin-induced T-cell migration, which is largely dependent on the microtubule dynamics. Experiments were performed using human T lymphoma cell line Hut78 and peripheral blood T-lymphocytes isolated from healthy donors. We observed that human T-lymphocytes exposed to PBOX-15 have severely impaired ability to polarize and migrate in response to the triggering stimulus generated via cross-linking of integrin lymphocyte function associated antigen-1 receptor. Here, we show that PBOX-15 can dramatically impair microtubule network via destabilization of tubulin resulting in complete loss of the motile phenotype of T-cells. We demonstrate that PBOX-15 inhibitory mechanisms involve decreased tubulin polymerization and its post-translational modifications. Novel microtubule-targeting effects of PBOX-15 can possibly open new horizons in the treatment of overactive inflammatory conditions as well as cancer and cancer metastatic spreading. Description: PUBLISHED