Biomedical subjects

Syed S Ahmad

Publications and source records attributed to Syed S Ahmad.

6 recordsLinked to original sources

PubMed2005

Role of the C2 domain of factor VIIIa in the assembly of factor-X activating complex on the platelet membrane.

Optimal rates of factor X (FX) activation require binding of factor IXa (FIXa), factor VIII(a) [FVIII(a)], and FX to activated platelet receptors. To define the FVIIIa domains that mediate platelet interactions, albumin density gradient washed, gel-filtered platelets (3.5 x 10(8)/mL) activated by the thrombin receptor peptide, SFLLRN (25 microM), were incubated with 125I-labeled FVIII C2 domain, or 125I-FVIIIa, or 125I-FVIII((LC)), or peptides from the C2 domain region, with or without anti-C2 domain monoclonal antibodies (MoAb), ESH4 or ESH8. FVIIIa (Kd approximately 1.7 nM), FVIII((LC)) (Kd approximately 3 nM), and the C2 domain (Kd approximately 16 nM) all interacted with approximately 700-800 binding sites/platelet. Unlike FVIIIa, the C2 domain did not respond to the presence of excess EGR-FIXa (45 nM) and FX (1.5 microM) with enhanced binding stoichiometry and affinity. Both the MoAb ESH4 and a synthetic peptide corresponding to FVIII residues 2303-2332 (epitope for FVIII MoAb, ESH4) inhibited FVIIIa binding to platelets, whereas MoAb ESH8 and a C2 domain peptide corresponding to residues 2248-2285 (epitope for the FVIII MoAb, ESH8) failed to inhibit FVIIIa binding. Thus, a major platelet-binding site resides within residues 2303-2332 in the C2 domain of FVIIIa, and an additional site within residues 2248-2285 increases the stoichiometry and affinity of FVIIIa binding to activated platelets only in the presence of FIXa and FX but does not directly mediate FVIIIa binding to the platelet surface.

Amino Acid Sequence↗

PubMed2005

A Glu113Ala mutation within a factor VIII Ca2+-binding site enhances cofactor interactions in factor Xase.

We recently identified an acidic-rich segment in the A1 domain of factor VIII (residues 110-126) that functions in the coordination of Ca(2+), an ion necessary for cofactor activity [Wakabayashi et al. (2004) J. Biol. Chem. 279, 12677-12684]. Mutagenesis studies showed that replacement of residue Glu113 with Ala (E113A) yielded a factor VIII point mutant possessing increased specific activity as determined by a one-stage clotting assay. Mutagenesis at this site suggested that substitution with relatively small, nonpolar residues was well tolerated, whereas replacement with a number of polar or charged residues appeared detrimental to activity. Ala substitution resulted in the greatest enhancement, yielding an approximately 2-fold increased specific activity. Time course experiments following reaction with thrombin revealed similar rates of activation and inactivation of E113A as observed for the wild type. Results from factor Xa generation assays showed minimal differences in kinetic parameters and factor IXa affinity for E113A and wild-type factor VIIIa when run in the presence of synthetic phospholipid vesicles, whereas factor VIIIa E113A displayed an approximately 4-fold greater affinity for factor IXa compared with factor VIIIa wild type in reactions run on the platelet membrane surface. This latter effect may be attributed, in part, to a 2-fold increased affinity of factor VIIIa E113A for the platelet membrane. Considering that low levels of factors VIIIa and IXa are generated during clotting in plasma, the increased cofactor specific activity observed for E113A factor VIII may result from its enhanced affinity for factor IXa on the physiological membrane.

Alanine↗

PubMed2002

Coordinate binding studies of the substrate (factor X) with the cofactor (factor VIII) in the assembly of the factor X activating complex on the activated platelet surface.

The assembly of the factor X activating complex on the platelet surface requires the occupancy of three receptors: (1) enzyme factor IXa, (2) cofactor factor VIII(a), and (3) substrate factor X. To further evaluate this three-receptor model, simultaneous binding isotherms of (125)I-factor X and (131)I-factor VIII(a) to activated platelets were determined as a function of time and also as a function of the concentrations of both ligands in the presence of active site-inhibited factor IXa (45 nM) and 5 mM CaCl(2). In the presence of active site-inhibited factor IXa and factor VIIIa there are two independent factor X binding sites: (1) low affinity, high capacity (approximately 9000 sites/platelet; K(d) approximately 380 nM) and (2) low capacity, high affinity (1700 sites/platelet; K(d) approximately 30 nM). A single specific and selective factor X binding site was expressed (1200 sites/platelet; K(d) approximately 9 nM) when the shared factor X/factor II site was blocked by excess factor II (4 microM). In the presence of active site-inhibited factor IXa (4 nM) and factor II (4 microM), factor X binds to 3-fold more platelet sites than procofactor VIII with relatively low affinity (K(d) approximately 250 nM). The activation of procofactor VIII to factor VIIIa increases the affinity of binding to platelets of both factor VIIIa ( approximately 4-fold to K(d) approximately 0.8-1.5 nM) and factor X ( approximately 25-50-fold to K(d) approximately 5-9 nM). In the presence of excess zymogen factor IX, which blocks the shared factor IX/factor IXa binding site, the substrate, factor X, and the active cofactor, factor VIIIa, form a 1:1 stoichiometric complex. These coordinate binding studies support the conclusion that factor X initially binds to a high-capacity, low-affinity platelet binding site shared with prothrombin, which then presents factor X to a specific high-affinity site consisting of factor VIIIa bound to a high-affinity, low-capacity receptor on activated platelets.

Binding Sites↗

PubMed2002

Proteases in blood clotting.

The serine proteases, cofactors and cell-receptor molecules that comprise the haemostatic mechanism are highly conserved modular proteins that have evolved to participate in biochemical reactions in blood coagulation, anticoagulation and fibrinolysis. Blood coagulation is initiated by exposure of tissue factor, which forms a complex with factor VIIa and factor X, which results in the generation of small quantities of thrombin and is rapidly shutdown by the tissue factor pathway inhibitor. The generation of these small quantities of thrombin then activates factor XI, resulting in a sequence of events that lead to the activation of factor IX, factor X and prothrombin. Sufficient thrombin is generated to effect normal haemostasis by converting fibrinogen into fibrin. The anticoagulant pathways that regulate blood coagulation include the protein C anticoagulant mechanism, the serine protease inhibitors in plasma, and the Kunitz-like inhibitors, tissue factor pathway inhibitor and protease nexin 2. Finally, the fibrinolytic mechanism that comprises the activation of plasminogen into plasmin prevents excessive fibrin accumulation by promoting local dissolution of thrombi and promoting wound healing by reestablishment of blood flow.

Animals↗

PubMed2001

The factor IXa second epidermal growth factor (EGF2) domain mediates platelet binding and assembly of the factor X activating complex.

Previously we have determined that residues 88-109 (but not Arg(94)) in the second epidermal growth factor (EGF2)-like domain of factor IXa (FIXa) are important for assembly of the factor X (FX) activating complex on phospholipid vesicles (Wilkinson, F. H., London, F. S., and Walsh, P. N. (2002) J. Biol. Chem. 277, 5725-5733). Here we report that these residues are important for platelet binding affinity, stoichiometry, and assembly of the FX activating complex. We prepared several chimeric FIXa proteins using homologous sequences from factor VII (FVII): FIXa(FVIIEGF2) (FIX Delta 88-124,inverted Delta FVII91-127), FIXa(loop1) (FIX Delta 88-99,inverted Delta FVII91-102), FIXa(loop2) (FIX Delta 95-109,inverted Delta FVII98-112), and FIXa(loop3) (FIX Delta 111-124,inverted Delta FVII114-127) and tested their ability to bind to thrombin-activated platelets. Binding affinities (K(d) values in 10(-9) m) for the proteins were as follows in the presence and absence of FVIIIa, respectively: FIXa(N) (0.55 +/- 0.06, 2.9 +/- 0.45), FIXa(WT) (0.80 +/- 0.08, 3.5 +/- 0.5), FIXa(loop1) (19 +/- 4.0, 27 +/- 5.0), FIXa(loop2) (35 +/- 9.0, 65 +/- 12.0), and FIXa(loop3) (1.1 +/- 0.09, 5.0 +/- 0.90). These K(d) values are in good agreement with K((d)(app)) values (in 10(-9) m) determined from the activation of FX (in the presence and absence of FVIIIa, respectively): FIXa(N) (0.46 +/- 0.05, 1.40 +/- 0.14), FIXa(WT) (0.72 +/- 0.08, 3.8 +/- 0.08), FIXa(loop1) (3.2 +/- 0.72, 14.0 +/- 1.60), FIXa(loop2) (18.4 +/- 1.60, 26.3 +/- 3.40), and FIXa(loop3) (0.7 +/- 0.05, 3.0 +/- 0.15). Moreover, the stoichiometry of binding (sites/platelet) showed an agreement with V(max) of FX activation and was reduced in those proteins that also showed a decreased platelet binding affinity. A peptide corresponding to the FIX EGF2 domain (Leu(84)-Val(128)) was an effective inhibitor of FIXa binding to platelets in both the presence (K(i) = 0.7 x 10(-6) m) and the absence (K(i) = 1.5 x 10(-6) m) of FVIIIa and FX. We conclude that residues 88-109 of the FIXa EGF2 domain mediate binding to platelets and assembly of the FX activating complex.ut not Ar

Amino Acid Sequence↗

PubMedDate not supplied

Dactinomycin impairs cellular respiration and reduces accompanying ATP formation.

The effect of dactinomycin on cellular respiration and accompanying ATP formation was investigated in Jurkat and HL-60 cells. Cellular mitochondrial oxygen consumption (measured by a homemade phosphorescence analyzer) and ATP content (measured by the luciferin-luciferase bioluminescence system) were determined as functions of time t during continuous exposure to the drug. The rate of respiration, k, was the negative of the slope of [O2] versus t. Oxygen consumption and ATP content were diminished by cyanide, confirming that both processes involved oxidations in the mitochondrial respiratory chain. In the presence of dactinomycin, k decreased gradually with t, the decrease being more pronounced at higher drug concentrations. Cellular ATP remained constant for 5 h in untreated cells, but in the presence of 20 microM dactinomycin it decreased gradually (to one-tenth the value at 5 h for untreated cells). The drug-induced inhibition of respiration and decrease in ATP were blocked by the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethyl ketone (zVAD-fmk). A rapid but temporary decrease in cellular ATP observed on the addition of zVAD-fmk was shown to be due to DMSO (added with zVAD-fmk). The effect of dactinomycin on respiration differed from that of doxorubicin. Plots of [O2] versus t were curved for dactinomycin so that k decreased gradually with t. The corresponding plots for doxorubicin were well fit by two straight lines; so k was constant for approximately 150 min, at which time k decreased, remaining constant at a lower level thereafter. The results for cells treated with mixtures of the two drugs indicated that the drugs acted synergistically. These results show the onset and severity of mitochondrial dysfunction in cells undergoing apoptosis induced by dactinomycin.

Adenosine Triphosphate↗