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K G Mann

Publications and source records attributed to K G Mann.

At least 127 records · Page 7Linked to original sources
PubMed1993

Factor V.

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Animals↗
PubMed1993

Assembly of prothrombinase complex.

The approaches described in this article have resulted in an increased understanding of the reaction steps involved in the stabilization and assembly of the prothrombinase complex. Because prothrombinase is considered an archetype for some of the other coagulation complexes, the quantitative information derived from these studies (Table I) provides the framework for future studies of prothrombinase and suggests experimental approaches for studies of the other analogous coagulation reactions.

Animals↗
PubMed1993

The assembly of the prothrombinase complex on adherent platelets.

Prothrombinase complex assembly, in real time, on platelets adherent to immobilized von Willebrand Factor (vWf) was examined by total internal reflection fluorescence spectroscopy (TIRFS). Electron microscopy showed that the platelets adhered to vWf in a largely unactivated state and could be activated by thrombin. Antibody binding to glycoprotein (GP) Ib and functional GPIIb-IIIa receptor molecules on adherent platelet membranes monitored by TIRFS also indicated that platelets adhered in a largely unactivated state. Maximal expression of the receptor form of GPIIb-IIIa detected by antibody binding was seen only after thrombin stimulation of the adherent platelets. Antibody binding to GPIb was detected on adherent platelets. A reduction in antibody binding was observed after thrombin stimulation of the platelets, indicating a change in GPIb as a consequence of thrombin stimulation of the platelets. The binding of the protein components of the prothrombinase complex to adherent and thrombin-stimulated adherent platelets was then studied individually. Factor Va bound to adherent and thrombin-stimulated adherent platelets was then studied individually. Factor Va bound to adherent and thrombin-stimulated adherent platelets with an estimated Kd of 58 nmol/L. Minimal factor Xa binding was observed on adherent platelets before thrombin stimulation. Factor Xa binding was, however, readily observed on thrombin-stimulated adherent platelets. This factor Xa binding was not saturable, and no Kd value could be estimated. Direct measurement of prothrombinase complex assembly was demonstrated by using an energy transfer phenomenon between fluorescein-labeled factor Va and rhodamine-labeled factor Xa. Prothrombinase complex assembly was observed on both adherent and thrombin-stimulated adherent platelets. The estimated Kd for the factor Va/factor Xa interaction was 4 nmol/L. TIRFS demonstrated that adherent platelets have the ability to support prothrombinase complex assembly, as shown by a direct energy transfer reaction between fluorescently labeled factors Va and Xa.

Animals↗
PubMed1993

Intact platelet membranes, not platelet-released microvesicles, support the procoagulant activity of adherent platelets.

The possibility that platelets release microvesicles on adherence to either von Willebrand factor (vWf) or collagen was examined by flow cytometry analysis of the supernatant above layers of adherent platelets. No microvesicle release was detected as a result of adherence to vWf or to collagen, a known platelet agonist. Approximately 8% of the total platelet mass was released as microvesicles after thrombin stimulation of the vWf- or collagen-adherent platelets. A larger portion of the vWf-adherent platelet membranes (approximately 21%) was released as microvesicles subsequent to platelet stimulation with the nonphysiological agonist calcium ionophore A23187. Calpeptin, a calpain inhibitor, had no effect on microvesicle release, suggesting that calpain proteolysis of platelet cytoskeletal proteins was not responsible for microvesicle shedding under the conditions studied. Examination of the vWf-adherent platelets by scanning electron microscopy showed that virtually no microvesicles bound to exposed vWf multimers. No microvesicle binding to the adherent platelets was observed, indicating that the majority of the microvesicles were shed from the platelet and vWf surface on platelet activation. The ability of the microvesicle population to support procoagulant activity was measured with a prothrombinase activity assay and was compared with the activity supported by the adherent platelet membranes. More than 85% of the total prothrombinase activity remained associated with the adherent platelet membranes, both for unstimulated platelets and platelets stimulated with physiological agonists. Furthermore, the residual activity found in the buffer fraction containing detached platelets and any released microvesicles could be attributed to the detached platelets. No activity could be attributed to the microvesicles, as thrombin stimulation of either vWf-or collagen-adherent platelets did not promote increased procoagulant activity relative to the unstimulated adherent platelets, even though microvesicle release was detected as a result of agonist addition. Neither full platelet activation nor microvesicle shedding played an essential role in generating procoagulant activity in the adherent platelet system.

Blood Coagulation↗
PubMed1992

Role of the membrane surface in the activation of human coagulation factor X.

Coagulation factor X is activated by the extrinsic Xase complex composed of factor VIIa associated with the integral membrane protein tissue factor. The kinetics of human factor X activation was studied following reconstitution of this reaction system using purified human proteins and synthetic phospholipid vesicles composed of phosphatidylcholine and phosphatidylserine (PCPS) or phosphatidylcholine alone (PC). Factor X activation was evaluated by discontinuous measurements of the amidolytic activity of the product, factor Xa, or continuously monitored using the fluorescent serine protease inhibitor 4-aminobenzamidine. The results of both techniques were verified by direct physical measurements of zymogen activation using SDS-polyacrylamide gel electrophoresis. The rate of factor X activation with PC vesicles was less than 5% of that observed with PCPS vesicles. Since factor X does not bind to vesicles containing only PC, these data suggested an important role for the substrate-membrane interaction in the catalytic cycle. The importance of the substrate-membrane interaction in the activation process was investigated by using membrane-binding proteins to compete with the substrate for combining sites on PCPS vesicles. Prothrombin fragment 1 was an inhibitor of factor X activation. The dependence of inhibition by fragment 1 on PCPS and factor X was consistent with a significant reduction in initial velocity due to the displacement of factor X from the membrane surface. The inhibition data also suggested that the membrane-bound pool of factor X was the preferred substrate for the human extrinsic Xase complex. The influence of PCPS concentrations on the rate of factor X activation was systematically investigated. Increasing concentrations of PCPS resulted in a modest change in the Km,app and a dramatic change in the Vmax,app for the reaction. The initial velocity data could be globally analyzed according to the preferential utilization of membrane-bound factor X with the intrinsic kinetic constants: Km approximately equal to 1 microM and kcat = 37 s-1 at saturating PCPS. In addition, the equilibrium parameters for the factor X-membrane interaction inferred from these studies were in excellent agreement with the directly determined values. Collectively, the data suggest that the substrate-membrane interaction must precede catalysis for the efficient activation of human factor X by the extrinsic Xase complex.

Animals↗
PubMed1992

Characterization of human osteoblast and megakaryocyte-derived osteonectin (SPARC).

Osteonectin is an adhesive, cell, and extracellular matrix-binding glycoprotein found primarily in the matrix of bone and in blood platelets in vivo. Osteonectins isolated from these two sources differ with respect to the complexity of their constituent N-linked oligosaccharide. In this study, osteonectin synthesized by bone-forming cells (osteoblasts) and platelet-producing cells (megakaryocytes) in vitro was analyzed to determine if the proteins produced were analogous in terms of glycosylation to those isolated from bone and platelets, respectively. Immunoblot analyses of osteonectin produced by the osteoblast-like cell lines, SaOS-2 and MG-63, indicated that secreted and intracellular forms of the molecule are structurally distinct. Endoglycosidase treatment and immunoblotting of osteonectin secreted from SaOS-2 and MG-63 cells, under serum-deprived conditions, suggested that the molecule possessed a complex type oligosaccharide unlike the high-mannose moiety found on bone matrix-derived osteonectin. Biosynthetic labeling of SaOS-2 cells and human megakaryocytes indicated that both cell types synthesize osteonectin de novo. Electrophoretic and glycosidase sensitivity analyses of [35S]-osteonectin isolated from lysates of metabolically labeled SaOS-2 cells and megakaryocytes indicated that these two cell types synthesize osteonectin molecules that are identical in oligosaccharide structure to the isolated bone and platelet proteins. These data suggest that the intracellular form of the osteonectin molecule is glycosylated differently in SaOS-2 cells and megakaryocytes but that the extracellular form which is secreted from platelets in vivo and osteoblasts in vitro is characterized by the presence of a complex type N-linked oligosaccharide.

Adult↗
PubMed1992

Aminonaphthalenesulfonamides, a new class of modifiable fluorescent detecting groups and their use in substrates for serine protease enzymes.

A series of new compounds, 6-amino-1-naphthalenesulfonamides (ANSN), were used as fluorescent detecting groups for substrates of amidases. These compounds have a high quantum fluorescent yield, and the sulfonyl moiety permits a large range of chemical modification. Fifteen ANSN substrates with the structure (N alpha-Z)Arg-ANSNR1R2 were synthesized and evaluated for their reactivity with 8 proteases involved in blood coagulation and fibrinolysis. Thrombin, activated protein C, and urokinase rapidly hydrolyzed substrates with monosubstituted sulfonamide moieties (R1 = H). The maximum rate of substrate homologue). The hydrolysis rates for substrates with branched substituents were slower than their linear analogues. Monosubstituted (N alpha-Z)Arg-ANSNR1R2 possessing cyclohexyl or benzyl groups in the sulfonamide moiety were hydrolyzed by these three enzymes at rates similar to that of the n-butyl homologue (except the cyclohexyl compound for u-PA). Factor Xa rapidly hydrolyzed substrates with short alkyl chains, especially when R1 = R2 = CH3 or C2H5. Lys-plasmin and rt-PA demonstrated low activity with these compounds, and the best results were accomplished for monosubstituted compounds when R2 = benzyl (for both enzymes). Factor VIIa and factor IXa beta exhibited no activity with these substrates. A series of 14 peptidyl ANSN substrates were synthesized, and their reactivity for the same 8 enzymes was evaluated. Thrombin, factor Xa, APC, and Lys-plasmin hydrolyzed all of the substrates investigated. Urokinase, rt-PA, and factor IXa beta exhibited reactivity with a more limited group of substrates, and factor VIIa hydrolyzed only one compound (MesD-LGR-ANSN(C2H5)2). The substrate ZGGRR-ANSNH (cyclo-C6H11) showed considerable specificity for APC in comparison with other enzymes (kcat/KM = 19,300 M-1 s-1 for APC, 1560 for factor IIa, and 180 for factor Xa). This kinetic advantage in substrate hydrolysis was utilized to evaluate the activation of protein C by thrombin in a continuous assay format. Substrate (D-LPR-ANSNHC3H7) was used to evaluate factor IX activation by the factor VIIa/tissue factor enzymatic complex in a discontinuous assay. A comparison between the commercially available substrate chromozyme TH (p-nitroanilide) and the ANSN substrate with the same peptide sequence (TosGPR) demonstrated that aminonaphthalenesulfonamide increased the specificity (kcat/KM) of substrate hydrolysis by thrombin more than 30 times, with respect to factor Xa substrate hydrolysis.

Chemical Phenomena↗
PubMed1992

The evaluation of complex-dependent alterations in human factor VIIa.

Factor VIIa is a plasma glycoprotein which, when bound to the integral membrane glycoprotein tissue factor, forms an enzymatic complex that is essential for normal hemostasis. We have developed a fluorescent substrate (6-(Mes-D-Leu-Gly-Arg)amino-1-naphthalenediethylsulfamide) which can be used to directly measure the enzymatic activity of factor VIIa in the presence and absence of tissue factor and phospholipid. The sensitivity of this substrate allows for detection of factor VIIa at concentrations below 10(-9) M. The kinetics of substrate hydrolysis by factor VIIa were evaluated and it was observed that the binding of factor VIIa to tissue factor increases the catalytic efficiency (kcat/Km) of factor VIIa substrate hydrolysis greater than 100-fold. The increase in enzymatic efficiency of factor VIIa, when complexed to tissue factor, is mediated primarily by an increase in kcat. These data suggest that tissue factor induces an alteration in the catalytic site of factor VIIa, which allows for more efficient hydrolysis of the small fluorescent substrate. Measurements conducted using various phospholipids and detergents demonstrated that the increase in catalytic efficiency of factor VIIa, when complexed to tissue factor, is independent of the supporting surface. The differential rate of substrate hydrolysis when factor VIIa is complexed to tissue factor was used to estimate the binding of factor VIIa to tissue factor. From these data an apparent dissociation constant for factor VIIa binding to tissue factor was calculated to be between 1.1 and 2.1 nM with a binding stoichiometry of 1.04:1 (factor VIIa:tissue factor). When the reactivity of this small fluorescent substrate toward single-chain factor VII was investigated, both in the presence and absence of tissue factor, no substrate hydrolysis was observed.

Calcium↗
PubMed1992

The complete cDNA sequence of bovine coagulation factor V.

Lack of availability of a primary structure for bovine factor V has hindered detailed analysis of a vast majority of structure-function correlations on this molecule. To determine the primary structure of bovine factor V, we used liver mRNA as a template for the synthesis of three cDNA libraries. The sequences of seven overlapping cDNA clones infer two bovine factor V variants. Variant 1 results in a 6910-basepair (bp) cDNA including 103 bp of 5'-untranslated sequence, 6633 bp of coding sequence and 171 bp of 3'-untranslated sequence with a putative polyadenylation site. Variant 2 differs only in the size of the coding sequence (6618 bp). The open reading frame translates to factor V consisting of 2211 (or 2206) amino acids including a 28-amino acid signal peptide. Comparison of the amino acid sequences with human factor Va reveals 84% identity for the heavy and 86% for the light chains. In contrast, the B domain (connecting region) exhibits only 59% identity relative to the human molecule. The bovine B domain contains two repeats of a 14-amino acid structure that is contained only once in the human sequence. Bovine factor V lacks one of the nine amino acid repeats and one of the 17 amino acid repeats present in the human B domain. Factor V has little homology to the factor VIII molecule in the B domain. The 17-amino acid repeat missing in bovine factor V allows identification of an 18-amino acid sequence that is homologous to the B domain of human factor VIII. These 18 amino acids may either constitute the unique vestige of a divergent evolution between the B domains of factors V and VIII or reveal the convergent evolution toward a critical epitope involved in the activation of both procofactors.

Amino Acid Sequence↗
PubMed1992

Growth on type I collagen promotes expression of the osteoblastic phenotype in human osteosarcoma MG-63 cells.

Using MG-63 cells as a model system capable of partial osteoblastic differentiation, we have examined the effect of growth on extracellular matrix. MG-63 cell matrix and purified type I collagen induced a morphological change characterized by long cytoplasmic processes reminiscent of those seen in osteocytes. Concurrent biochemical changes involving bone marker proteins included increased specific activity of cell-associated alkaline phosphatase and increased secretion of osteonectin (up to 2.5-fold for each protein); all changes occurred without alterations in the growth kinetics of the MG-63 cells. The increase in alkaline phosphatase activity was maximal on days 6-8 following seeding; increased osteonectin secretion was most prominent immediately following seeding; all changes decreased as cells reached confluence. Growing cells on type I collagen resulted in an increased induction of alkaline phosphatase activity by 1,25(OH)2D3 (with little change in the 1,25(OH)2D3 induction of osteonectin and osteocalcin secretion), and increased TGF-beta induction of alkaline phosphatase activity as well (both TGF-beta 1 and TGF-beta 2). Both the 1,25(OH)2D3 and TGF-beta effects appeared to be synergistic with growth on type I collagen. These studies support the hypothesis that bone extracellular matrix may play an important role in osteoblastic differentiation and phenotypic expression.

Alkaline Phosphatase↗
PubMed1992

Platelet factor Xa receptor.

The assembly and function of the prothrombinase complex on the bovine and human platelet membrane is mediated through binding interactions in which factor Va bound to the platelet surface forms at least part of the "receptor" for factor Xa in a 1:1 stoichiometric complex. A model depicting these binding interactions is shown in Fig. 12. Data from our laboratory indicate that the prothrombinase catalyst assembles in an analogous manner on the surface of monocytes, lymphocytes, neutrophils, and well-defined phospholipid vesicles employed in model systems. The 74,000-Da subunit of factor Va, component E, which mediates the binding of factor Va to either bovine platelets, human monocytes, or phospholipid vesicles, is shown binding to the cell membrane through its putative "receptor." The 94,000-Da subunit of factor Va, component D, is associated with the membrane surface through its metal ion-dependent interaction with component E. Factor Va forms at least part of the receptor that mediates the binding of factor Xa to an appropriate membrane surface, because component E has been shown to contribute significantly to the interaction of factor Xa with either the platelet, monocyte, or vesicle membrane surface. Our data do not preclude the possibility that component D contributes to the binding of factor Xa and the function of the prothrombinase complex. Component D appears to be important for several reasons. Cleavage of component D by activated protein C results in the complete loss of factor Va cofactor activity. An interaction between factor Xa and component D is implied from the observation that factor Xa protects factor Va from activated protein C inactivation. Furthermore, the binding of factor Xa to platelet-bound factor Va results in the time-dependent cleavage of components D and D'. Because component D is not required absolutely for prothrombinase complex assembly, we would speculate that it may be important in mediating prothrombin binding (depicted as a three-domain molecule) and increasing the catalytic efficiency of the enzymatic complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Potential analytes for the diagnosis of thrombosis. An overview.

The coagulation response is a complex interaction involving the vascular surface, blood platelets, and the plasma coagulation factors. These reactions are integrated to give rise to a locally efficient generation of both platelet aggregates and the enzymatic process associated with fibrin formation. Following mechanical, chemical, or biological "damage" to the vascular endothelial surface, coagulation is initiated by a composite of cellular adhesive reactions certainly involving the platelet and potentially also involving other inflammatory cells. The blood coagulation mechanism can be presented as a collection of zymogen-to-enzyme transformations, with each proteases participating with a cofactor protein on a "surface" that gives rise to the competent blood clotting complex. These complexes catalyze the generation of additional enzymes required for succeeding reaction complexes. It is likely that the coagulation reaction system is continuously "on," producing products at some low "idling" rate, with the products of the various reactions being neutralized by the collection of protease inhibitors and cofactor-neutralizing reactions that regulate the blood clotting process. These latter systems include, as principal components, the antithrombin III-heparin anticoagulant and the activated protein C pathway that disables cofactor proteins. Small changes in the concentrations of modulators can cause large effects in response to relatively small inputs. The coagulation process may be regarded as being at an incipient stage, separated from visually observable coagulation by a narrow threshold, which, once crossed, gives rise to the generation of fibrin and other products associated with alpha-thrombin generation.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Coagulation↗