Biomedical subjects

A S Wolberg

Publications and source records attributed to A S Wolberg.

10 recordsLinked to original sources

PubMed2003

Elevated plasma homocysteine leads to alterations in fibrin clot structure and stability: implications for the mechanism of thrombosis in hyperhomocysteinemia.

Elevated plasma homocysteine is associated with an increased risk of atherosclerosis and thrombosis. However, the mechanisms by which homocysteine might cause these events are not understood. We hypothesized that hyperhomocysteinemia might lead to modification of fibrinogen in vivo, thereby causing altered fibrin clot structure. New Zealand White rabbits were injected intraperitoneally (i.p.) every 12 h through an indwelling catheter with homocysteine or buffer for 8 weeks. This treatment raised the plasma homocysteine levels to about 30 micro mol L(-1) compared with 13.5 micro mol L(-1) in control rabbits by the end of the treatment period. The fibrinogen levels were 3.2 +/- 0.6 in homocysteine-treated and 2.5 +/- 1.1 mg mL(-1) in control rabbits. The reptilase time was prolonged to 363 +/- 88 for plasma from homocysteine-treated rabbits compared with 194 +/- 48 s for controls (P < 0.01). The thrombin clotting time (TCT) for the homocysteine-treated rabbits was significantly shorter, 7.5 +/- 1.7 compared with 28.6 +/- 18 s for the controls (P < 0.05). The calcium dependence of the thrombin clotting time was also different in homocysteinemic and control plasmas. Clots from plasma or fibrinogen of homocysteinemic rabbits were composed of thinner fibers than control clots. The clots formed from purified fibrinogen from homocysteine-treated rabbits were lyzed more slowly by plasmin than comparable clots from control fibrinogen. Congenital dysfibrinogenemias have been described that are associated with fibrin clots composed of thin, tightly packed fibers that are abnormally resistant to fibrinolysis, and recurrent thrombosis. Our results suggest that elevated plasma homocysteine leads to a similar acquired dysfibrinogenemia. The formation of clots that are abnormally resistant to fibrinolysis could directly contribute to the increased risk of thrombosis in hyperhomocysteinemia.

Animals↗

PubMed2002

Analyzing fibrin clot structure using a microplate reader.

Fibrin clot structure studies are often performed using optical methods. For example, the clot's fiber structure can be assessed by measuring light scattering as a function of wavelength. From these measurements, one can calculate the mass/length ratio (mu), a relative measure of fibrin thickness. Fiber thickness has important functional correlates in terms of clot stability and resistance to fibrinolysis. Typically, measurements to calculate mass/length ratios are carried out on high-end spectrophotometers. However, limitations of this instrument include the large sample volume required and the inability to read multiple samples at one time. To circumvent these limitations, a plate-reading spectrophotometer is more commonly used to monitor clot formation; increases in absorbance indicate clot formation, while decreases indicate clot lysis. However, it is unclear whether plate-reading spectrophotometers can be used to quantitatively evaluate fibrin fiber structure. In the current study, we compared spectrophotometric analysis of fibrin gels on single-sample and plate-reading spectrophotometers. Results show that a plate-reading spectrophotometer does not give accurate measurements of the fiber mass/length ratio. However, the plate-reading spectrophotometer can provide a qualitative measure of fiber structure for both purified fibrinogen and plasma. We suggest that plate-reading spectrophotometers can provide a convenient, rapid, and inexpensive means of analyzing fibrin clot structure.

Calcium↗

PubMed2000

Deencryption of cellular tissue factor is independent of its cytoplasmic domain.

Tissue factor (TF) is a transmembrane molecule that, when exposed to plasma, is the key initiator of coagulation. Cellular TF activity is normally "encrypted", but treating cells with calcium ionophore (i.e. , ionomycin or A23187) increases ("deencrypts") TF activity without increasing TF mRNA or antigen expression. Deencryption results from both plasma membrane phosphatidylserine (PS)-dependent and -independent mechanisms; however, the nature of the PS-independent component is unclear. Since deencryption has been suggested to result from release of TF dimers on the cell surface, and since TF's cytoplasmic domain binds to actin-binding protein 280 and interacts with the cytoskeleton, we hypothesized that interactions with the cytoskeleton, through the cytoplasmic domain, play a role in mediating encryption/deencryption. We examined TF deencryption and the role of the cytoplasmic domain in the PS-independent component using baby hamster kidney (BHK) cells expressing full length TF (BHK-TF) or TF lacking its cytoplasmic domain (BHK-descyt) (Sorensen et al. (1999) J. Biol. Chem. 274, 21349). Both BHK-TF and BHK-descyt cells exhibited a dose-dependent, 1.5- to 10-fold increase in TF activity upon treatment with calcium ionophore, and this increase in activity was only partially blocked by annexin V. These results indicate that deencryption is not restricted to cells which naturally express TF and that the PS-independent component of deencryption is intact on cells transfected with either full length or truncated TF. Our results clearly indicate that deencryption is not dependent on an intact cytoplasmic domain in transfected BHK cells.

Animals↗

PubMed2000

Coagulation factor XI is a contaminant in intravenous immunoglobulin preparations.

A small number of thromboembolic events, including deep venous thrombosis and myocardial infarction, have been reported in patients receiving IVIG. These events have primarily occurred in patients receiving high-dose IVIG and have been attributed to an increase in blood viscosity. To test the hypothesis that a procoagulant might be present in IgG preparations, twenty-nine samples of intravenous immunoglobulin (IVIG) from eight different manufacturers were assayed for procoagulant activity. Twenty-six of these samples shortened the clotting time of factor XI-deficient plasma. Of these, fourteen samples had factor XI activities greater than 0.001 U/ml of normal pooled plasma. The remaining samples possessed less than 0. 001 U/ml of normal plasma activity. The procoagulant activity in these samples could be inhibited by an anti-factor XI polyclonal antibody, suggesting that the procoagulant activity was factor XI. The procoagulant activity increased in two samples after storage at 4 degrees C for 4 weeks, likely as a result of factor XIa autoactivation. Additionally, activity in some IVIG samples was able to directly activate factor IX, indicating that activated factor XI was present in these samples. Finally, the degree of factor XI(a) contamination in the samples was correlated with the manufacturer, suggesting that variations in the manufacturing process or source plasma affect the level of factor XI in the IVIG product. Because addition of small amounts of factor XIa to plasma can lead to production of significant amounts of thrombin, we suggest that factor XIa present in some IVIG preparations could contribute to the in vivo risk of thrombosis after IVIG therapy.

Drug Contamination↗

PubMed1999

Tissue factor de-encryption: ionophore treatment induces changes in tissue factor activity by phosphatidylserine-dependent and -independent mechanisms.

Coagulation is initiated on tissue-factor-bearing cells when factor VIIa complexes with membrane-bound tissue factor and activates factors X and IX. Cellular tissue factor activity does not correlate with tissue factor antigen; treatment with calcium ionophore rapidly increases tissue factor activity without increasing tissue factor antigen. Our study examined the effect of calcium ionophore A23187 on tissue factor activity of freshly isolated, lipopolysaccharide-stimulated monocytes and non-transformed human dermal fibroblasts. A23187 increased tissue factor activity on monocytes and fibroblasts in a dose-dependent fashion between 0.1 and 50 micromol/l ionophore. This increase in activity was proportional to an increase in intracellular calcium in monocytes. The increase in tissue factor activity was partially attributable to an increase in phosphatidylserine expression, as measured by increased prothrombinase activity (1.1- to 4-fold) on ionophore-treated cells. The phosphatidylserine-binding protein annexin V decreased tissue factor activity on both ionophore-treated and untreated cells, reflecting the role of phosphatidylserine in tissue factor activity. However, even in the presence of saturating concentrations of annexin V, the tissue factor activity of ionophore-treated cells was 1.3- to 11.3-fold higher than that of untreated cells, indicating that the increase in tissue factor activity did not result solely from increased expression of phosphatidylserine. A23187 increased tissue-factor-dependent activation of factors IX and X 1.4- to 7-fold on both cell types, indicating that ionophore treatment did not alter factor VIIa/tissue factor substrate specificity. We conclude that the mechanism by which calcium ionophore increases tissue factor activity is not unique to monocytoid or transformed cells. Furthermore, the ionophore-induced increase in activity is not solely the result of increased exposure to phosphatidylserine. Finally, tissue factor de-encryption by A23187 does not alter factor VIIa/tissue factor substrate specificity.

Calcimycin↗

PubMed1997

Human factor IX binds to specific sites on the collagenous domain of collagen IV.

The primary region of factor IX that mediates binding to bovine aortic endothelial cells resides in residues 3-11 of the N-terminal region known as the Gla domain. Recently, it was proposed that the observed binding to endothelial cells is actually a measure of the interaction between factor IX and collagen IV (Cheung, W. F., van den Born, J., Kuhn, K., Kjellen, L., Hudson, B. G., and Stafford, D. W. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11068-11073). To confirm that factor IX binds to collagen IV and to examine the specificity of this interaction, we used scanning force microscopy to examine factor IX binding to collagen IV. We imaged collagen IV in the presence and the absence of factor IX and observed specific interactions between factor IX and collagen IV. Our results demonstrate that factor IX binds to collagen IV at specific sites in the collagenous domain approximately 98 and approximately 50 nm from the C-terminal pepsin-cleaved end.

Animals↗

PubMed1997

Factor IX activation by factor XIa proceeds without release of a free intermediate.

Factor IX activation by factor XIa is thought to proceed through the singly-cleaved free intermediate, factor IX alpha. However, we observed no intermediate development during factor IX activation by factor XIa when using a low substrate to enzyme ratio (44:1 mol/mol). This result can be explained by one of two mechanisms: (1) factor XIa-catalyzed activation proceeds via a singly-cleaved free intermediate with a much higher efficiency of cleavage than factor IX zymogen, or (2) the reaction occurs without free intermediate generation, whereby factor XIa makes both proteolytic cleavages in a single substrate molecule before releasing the final product (processive mechanism). We compared the factor XIa cleavage rates of free factor IX alpha and factor IXa alpha with that of factor IX zymogen. In contrast to the requirements of mechanism (1), the cleavage rate constants of factor IX zymogen, factor IX alpha, and factor IXa alpha were similar: 0.38 +/- 0.02 s(-1), 0.34 +/- 0.05 s(-1), and 0.27 +/- 0.01 s(-1), respectively. It seems likely that factor XIa-generated intermediates observed under some reaction conditions are produced through the occasional failure of a processive mechanism. Indeed, in reactions using a high substrate to enzyme ratio (1900:1 mol/mol), we observed some factor IX alpha development; however, the pattern of intermediate and product development over time was inconsistent with a mechanism involving an obligate intermediate. Rather, it corresponded to behavior expected from a processive mechanism undergoing a consistent low failure. We conclude that factor XIa-catalyzed activation of factor IX proceeds via a processive mechanism without release of a free intermediate.

Antibodies, Monoclonal↗

PubMed1996

Characterization of gamma-carboxyglutamic acid residue 21 of human factor IX.

We investigated the functional role of gamma-carboxyglutamic acid (Gla) residue 21 of human factor IX, using site-directed mutagenesis to change the glutamic acid residue to aspartic acid (FIX21D). FIX21D had reduced activity in an activated partial thromboplastin time (aPTT) assay and was activated by factor XIa more slowly than wild-type factor IX (FIXwt). FIX21D underwent normal, two-stage calcium-dependent intrinsic fluorescence quenching, indicating that a folding event similar to that seen in FIXwt occurred upon the addition of calcium ions. Antibody A-7, which recognizes factor IX-specific residues at positions 33-40, bound FIX21D as well as FIXwt; however, the calcium-specific monoclonal antibody, JK-IX-2, whose epitope includes residues 1 and 22, did not recognize FIX21D. FIX21D bound phosphatidylserine/phosphatidylcholine (PS/PC) vesicles with Kd approximately 10-fold greater than FIXwt, as measured by a fluorescence light scattering assay. Finally, although FIXwt binds endothelial cells with a Kd of 2.8 nM, FIX21D did not bind endothelial cells. Molecular modeling simulations of FIXwt and FIX21D indicate that mutating Gla 21 to Asp causes structural changes in residues 3-5 and 8-10, as well as in two exposed calcium ions, consistent with the reduced function of FIX21D. Immunological and intrinsic fluorescence quenching assays and the molecular dynamics simulations suggest normal folding in the C-terminal region of the Gla domain. Thus we hypothesize the FIX21D has reduced JK-IX-2 and phospholipid and endothelial cell binding due to localized structural changes in residues 3-10 and the exposed calcium ions. Our study suggests that the Gla 21 to Asp mutation disrupts function in the N-terminal region of the Gla domain without affecting structure in the C-terminal Gla domain region.

1-Carboxyglutamic Acid↗

PubMed1995

Localization of a metal-dependent epitope to the amino terminal residues 33-40 of human factor IX.

Metal binding sites within the Gla domain of vitamin K-dependent coagulation factors have been divided into nonspecific metal sites and calcium-specific sites. We demonstrate here that five residues within the Gla domain of factor IX are responsible for the reactivity with the metal-dependent factor IX monoclonal antibody, A-7. First we demonstrate that modifying any one of three residues within this site in factor IX abolishes the binding of A-7. To confirm the specificity of the antibody, the Gla domain of factor VII was changed at residues 32, 33, 34, 38 and 39 to the homologous residues of human factor IX. These changes were sufficient to generate a factor VII Gla domain with an A-7 binding site of the same affinity as that in factor IX. The site identified is one of the two major surfaces of the Gla domain and may represent the metal-dependent binding site.

1-Carboxyglutamic Acid↗

PubMed1993

The transcriptional control region of the human carcinoembryonic antigen gene: DNA sequence and homology studies.

Phage clones containing human carcinoembryonic antigen (CEA) 5' flanking sequences were isolated from a chromosome 19 genomic library. The clones were confirmed to contain CEA sequences flanking the CEA transcriptional start site by restriction endonuclease mapping, PCR analysis, and sequence determination. Restriction endonuclease mapping determined that the clones spanned approximately 26 kbp of contiguous sequence from 14 kbp 5' to 12 kbp 3' of the CEA transcriptional start. The DNA sequence of a 11,288 bp Hind III/Sau 3A restriction endonuclease fragment was determined. This sequence extends from 10.7 kbp 5' to 0.6 kbp 3' of the transcriptional start. This sequence was analyzed for the presence of consensus transcriptional regulatory sequences, repetitive sequences, and other features. Several transcriptional regulatory consensus sequences were identified. These consensus sequences may have significance for the transcriptional regulation of CEA.

Base Sequence↗