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K G Mann

Publications and source records attributed to K G Mann.

At least 109 records · Page 6Linked to original sources
PubMed1994

A model for the tissue factor pathway to thrombin. II. A mathematical simulation.

A mathematical simulation of the tissue factor pathway to the generation of thrombin has been developed using a combination of empirical, estimated, and deduced rate constants for reactions involving the activation of factor IX, X, V, and VIII, in the formation of thrombin, as well as rate constants for the assembly of the coagulation enzyme complexes which involve factor VIIIa-factor IXa (intrinsic tenase) and factor Va-Xa (prothrombinase) assembled on phospholipid membrane. Differential equations describing the fate of each species in the reaction were developed and solved using an interactive procedure based upon the Runge-Kutta technique. In addition to the theoretical considerations involving the reactions of the tissue factor pathway, a physical constraint associated with the stability of the factor VIIIa-factor IXa complex has been incorporated into the model based upon the empirical observations associated with the stability of this complex. The model system provides a realistic accounting of the fates of each of the proteins in the coagulation reaction through a range of initiator (factor VIIa-tissue factor) concentrations ranging from 5 pM to 5 nM. The model is responsive to alterations in the concentrations of factor VIII, factor V, and their respective activated species, factor VIIIa and factor Va, and overall provides a reasonable approximation of empirical data. The computer model permits the assessment of the reaction over a broad range of conditions and provides a useful tool for the development and management of reaction studies.

Blood Coagulation Factors↗
PubMed1994

Structure of membrane-bound human factor Va.

Coagulation factor Va is an essential cofactor which combines with the serine protease factor Xa on a phospholipid surface to form the prothrombinase complex. In the present study, the structure of factor Va interacting with lipid surfaces containing phosphatidylserine was studied by electron microscopy. Two-dimensional crystals of factor Va were obtained on planar lipid films under quasi-physiological conditions. The two-dimensional projected structure of factor Va was calculated at a resolution of 2 nm, revealing dimers of factor Va arranged on the surface lattice with the symmetry of the plane group p2. Average unit cell dimensions are a = 14.4 nm, b = 8.8 nm, gamma = 107 degrees. Each factor Va molecule presents two distinct domains of protein density consisting of one small domain, of 3 nm in diameter, connected to a larger domain of about 6 nm x 4.5 nm. The projected structure of factor Va covers an area equivalent to about fifty phospholipid molecules. In addition, edge-on views of factor Va molecules bound to liposomes reveal a globular structure connected through a thin stem to the liposome surface. A three-dimensional model of membrane-bound factor Va is proposed.

Cell Membrane↗
PubMed1994

Loss of membrane-dependent factor Va cleavage: a mechanistic interpretation of the pathology of protein CVermont.

Clinical manifestations of arterial and venous thrombosis in a family with protein C deficiency was associated with two mutations in the light chain of protein C: Glu20-->Ala and Val34-->Met. Further studies showed that the mutation Glu20-->Ala which eliminated a gamma-carboxylation site was exclusively responsible for the anticoagulant defect of activated protein C (APC). Membrane-bound human factor Va is inactivated by APC after two sequential cleavages of the heavy chain at Arg506 and Arg306. Human factor Va inactivation by human recombinant APC (rAPC) and a mutant molecule with an alanine instead of a glutamic acid at position 20 (rAPC(gamma 20A)) was investigated in the presence and absence of phospholipid vesicles. During a 2-hour incubation period of the cofactor with either rAPC or rAPC(gamma 20A). In the absence of a membrane surface, factor Va is cleaved quantitatively at Arg506 and retains approximately 60% of its initial cofactor activity. After a 2-hour incubation period with rAPC membrane-bound factor Va has no cofactor activity, whereas in the presence of a membrane surface and rAPC(gamma 20A) factor Va retains 60% of its initial cofactor activity. The completed loss in factor Va cofactor activity upon incubation of the membrane-bound cofactor with phospholipid vesicles and rAPC is associated with cleavages at Arg506 and Arg306, whereas membrane-bound factor Va cleavage at Arg306 by rAPC(gamma 20A) is impaired, resulting in a cofactor that is cleaved at Arg506. Slow cleavage at Arg306 occurs when membrane-bound factor Va is incubated with rAPC(gamma 20A) and only small amounts of fragments of M(r) = 45,000 and 30,000 are noticed. Our data show that the genetic defect which leads to the absence of a gamma-carboxylation site at Glu20 impairs membrane binding of human APC, which in turn is required for cleavage of factor Va at Arg306 and inactivation of the cofactor. The consequence of impaired membrane-dependent cleavage at Arg306 is manifested in vivo by venous and arterial thrombosis.

1-Carboxyglutamic Acid↗
PubMed1994

Contribution of the heavy and light chains of factor Va to the interaction with factor Xa.

The interactions of the isolated heavy and light chains of factor Va with factor Xa were evaluated using active-site-modified factor Xa [(carboxytetramethyl)rhodamine-Glu-Gly- Arg-factor Xa (ctr-EGR-Xa)]. The Kd for the factor Va heavy-chain interaction with ctr-EGR-Xa was 60 microM. A series of monoclonal antibodies directed against bovine factor Va were tested for their ability to inhibit thrombin formation in an assay using the fluorescent thrombin inhibitor dansylarginine N,N-(3-ethyl-1,5-pentanediyl)amide (DAPA). Monoclonal antibody alpha BFV-4, which recognizes the light chain of the cofactor, was found to inhibit the formation of thrombin. Similarly, monoclonal antibody alpha BFV-5, which is directed against the heavy chain of the cofactor, was found to inhibit thrombin formation. In contrast, monoclonal antibody alpha BFV-1, also directed against the heavy chain of the cofactor, did not inhibit thrombin generation by the prothrombinase complex. Monoclonal antibodies alpha BFV-4 and alpha BFV-5 inhibited the interaction of active-site-modified radiolabeled factor Xa (125I-Xa-EGR) with factor Va bound to PC/PS-coated microtiter wells, whereas nonimmune mouse IgG did not have any effect on the 125I-Xa-EGR.membrane-bound factor Va interaction. The antibodies effect upon the phospholipid-independent interaction between the cofactor and ctr-EGR-Xa was evaluated by analytical ultracentrifugation. Both alpha BFV-4 and alpha BFV-5 inhibited the phospholipid-independent interaction between factor Va and ctr-EGR-Xa.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Platelet coagulation factor Va: the major secretory platelet phosphoprotein.

Platelet-derived coagulation factor Va is the primary secreted substrate for a thrombin-stimulation-dependent platelet kinase. Human platelet factor Va, consisting of a molecular weight (M(r)) 105,000 heavy chain and an M(r) 74,000 light chain, incorporates phosphate in at least two sites on the light chain. Phosphorylated factor Va represents 50% of the secreted protein-associated phosphate. This modification occurs exclusively at serine residues and is inhibited by H-7 and staurosporine, which suggests a protein kinase C (PKC)-mediated event. Purified plasma factor V and Va are phosphorylated in the light chain region by rat brain PKC. The activity of platelet factor Va in prothrombinase on platelets is not altered when phosphorylation is inhibited by staurosporine. Plasma-derived factor Va in the presence of thrombin stimulated platelets is phosphorylated on both the heavy chain and the light chain. Plasma factor V and factor Va heavy chain phosphorylation occurs without light chain phosphorylation in the presence of added 32P gamma-ATP and non-stimulated or collagen-stimulated platelets or casein kinase II. This differential phosphorylation of factor Va heavy and light chain shows two independent platelet kinase activities that act on factor Va. The heavy chain factor V/Va kinase activity is similar to casein kinase II, which we have demonstrated previously to act on factor Va and accelerate activated protein C inactivation of the cofactor. Our data show platelet-dependent phosphorylation of platelet and plasma factor V and Va resulting in significant covalent modifications of the cofactor. These modifications may play a role in directing the extracellular distribution of factor V and factor Va.

Blood Platelets↗
PubMed1994

Cooperative interaction of divalent metal ions, substrate, and tissue factor with factor VIIa.

Factor VIIa-tissue factor (TF) complex formation in the presence of EDTA or divalent cations (Me2+) was investigated. The influence of Me2+ on the amidolytic activity of factor VIIa and factor VIIa-TF complex was evaluated using low molecular weight synthetic substrates possessing substituted aminonaphthalenesulfonamides as detecting groups. Factor VIIa expressed low amidolytic activity in the presence of EDTA. In the presence of EDTA and saturating concentrations of TF, the amidolytic activity of factor VIIa was increased approximately 90-fold. Gel electrophoresis and sedimentation velocity studies demonstrated complex formation between factor VIIa and TF in the presence of EDTA. Substrate titration curves obtained at fixed factor VIIa and TF concentrations gave sigmoidal shapes, indicating that substrates influenced factor VIIa amidolytic activity in the presence of TF. In the absence of Me2+, the KD,app of the factor VIIa-TF complex was influenced by substrate structure and varied from 3.9 to 34 nM. All Me2+ used increased the amidolytic activity of factor VIIa approximately 8-fold compared with experiments in the presence of EDTA. The KD,app values for factor VIIa-Ca2+ complex and factor VIIa-Mn2+ complex were independent of substrate and were 270 and 40 microM, respectively. The KD,app for factor VIIa-Mg2+ complex varied from 3 to 12 mM and was substrate structure dependent. The presence of TF had no influence upon the KD,app for the factor VIIa-Ca2+ complex. The amidolytic activity of factor VIIa was enhanced by TF significantly in the presence of Ca2+, and similar results were obtained with Mg2+ and Mn2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1994

Electrostatic and hydrophobic interactions are involved in factor Va binding to membranes containing acidic phospholipids.

The interaction of factor Va with phospholipid monolayers was studied using alternating current polarography. For these studies a hanging mercury drop electrode is positioned in contact with the monolayer at the air-monolayer interface. Factor Va introduced into the aqueous subphase beneath the monolayer caused alterations in the capacitance of the circuit. When factor Va was introduced beneath monolayers containing 13-25% mole fraction phosphatidylserine (PS), 87-75% mole fraction phosphatidylcholine (PC), a significant capacitance increase occurred, which is interpreted to be the result of the penetration of the factor Va molecules through the monolayer. No penetration or associated capacitance changes were observed with monolayers composed of pure PC or pure PS. Polarography experiments were also conducted with the electrode placed in the aqueous subphase to measure the binding of factor Va to pure PS monolayers. This approach has allowed detection of factor Va (at nM concentrations) adsorption to a pure PS monolayer. Thus the membrane adsorption process can be distinguished from factor Va penetration through the monolayer. These results suggest that there are two types of interaction of the factor Va molecule with PS-containing monolayers. The interaction with the PS component of the monolayer is essential for binding the factor Va (at nM concentrations) that occurs by surface adsorption. For mixed PC/PS monolayers, PS adsorption is followed by penetration of the protein through the membrane.

Animals↗
PubMed1994

Factor Va-membrane interaction is mediated by two regions located on the light chain of the cofactor.

Factor Va was incubated with 1-azidopyrene, a fluorescent lipophilic probe, in the presence of phospholipid vesicles composed of various proportions of phosphatidylcholine (PC) and phosphatidylserine (PS). The majority of the label was associated with the light chain of factor Va. The light chain was found to be labeled in the presence of phospholipid vesicles containing either 100% PC or 100% PS. After cleavage by factor Xa and incubation with PC/PS vesicles composed of 75% PC and 25% PS, label was found both on the M(r) = 30,000 fragment, derived from the NH2-terminal portion of the bovine factor Va light chain (residues 1537-1752), and on the M(r) = 46,000/48,000 carboxyl-terminal fragment of the factor Va light chain (residues 1753-2183). The M(r) = 46,000/48,000 fragment incorporated 1-azidopyrene independent of the phospholipid composition, while label incorporation into the M(r) = 30,000 fragment required phospholipid vesicles containing PC. No labeling of the M(r) = 30,000 fragment was observed with phospholipid vesicles composed of 100% PS. The label incorporation into the two portions of the molecule was found to be independent of the ionic strength in the presence of phospholipid vesicles containing 75% PC and 25% PS. In contrast, the labeling of the M(r) = 46,000/48,000 fragment with phospholipid vesicles composed of 100% PS was ionic strength dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

The isomorphous structures of prethrombin2, hirugen-, and PPACK-thrombin: changes accompanying activation and exosite binding to thrombin.

The X-ray crystal structure of prethrombin2 (pre2), the immediate inactive precursor of alpha-thrombin, has been determined at 2.0 A resolution complexed with hirugen. The structure has been refined to a final R-value of 0.169 using 14,211 observed reflections in the resolution range 8.0-2.0 A. A total of 202 water molecules have also been located in the structure. Comparison with the hirugen-thrombin complex showed that, apart from the flexible beginning and terminal regions of the molecule, there are 4 polypeptide segments in pre2 differing in conformation from the active enzyme (Pro 186-Asp 194, Gly 216-Gly 223, Gly 142-Pro 152, and the Arg 15-Ile 16 cleavage region). The formation of the Ile 16-Asp 194 ion pair and the specificity pocket are characteristic of serine protease activation with the conformation of the catalytic triad being conserved. With the determination of isomorphous structures of hirugen-thrombin and D-Phe-Pro-Arg chloromethyl ketone (PPACK)-thrombin, the changes that occur in the active site that affect the kinetics of chromogenic substrate hydrolysis on binding to the fibrinogen recognition exosite have been determined. The backbone of the Ala 190-Gly 197 segment in the active site has an average RMS difference of 0.55 A between the 2 structures (about 3.7 sigma compared to the bulk structure). This segment has 2 type II beta-bends, the first bend showing the largest shift due to hirugen binding. Another important feature was the 2 different conformations of the side chain of Glu 192. The side chain extends to solvent in hirugen-thrombin, which is compatible with the binding of substrates having an acidic residue in the P3 position (protein-C, thrombin platelet receptor). In PPACK-thrombin, the side chain of Asp 189 and the segment Arg 221A-Gly 223 move to provide space for the inhibitor, whereas in hirugen-thrombin, the Ala 190-Gly 197 movement expands the active site region. Although 8 water molecules are expelled from the active site with PPACK binding, the inhibitor complex is resolvated with 5 other water molecules.

Amino Acid Chloromethyl Ketones↗
PubMed1994

The clinical use of flow cytometry for assessing platelet activation in acute coronary syndromes. TIMI-III Thrombosis and Anticoagulation Group.

BACKGROUND: Unstable angina signals a sudden transition from stable to unstable atherosclerotic coronary artery disease. Although assessment of platelet activity has been shown to provide diagnostic and prognostic information, most available methods lack sufficient sensitivity or specificity for them to be useful in clinical practice. In recent years, fluorescently labeled antibodies and flow cytometry have permitted the detection of activation proteins on individual platelets. The purpose of the present study was to assess the ability of flow cytometry to detect platelet activation in patients with unstable angina and non-Q-wave myocardial infarction. METHODS: Platelet activation before treatment was determined from whole-blood samples in 19 patients participating in the Thrombolysis in Myocardial Ischemia (TIMI)-IIIB study and in nine healthy volunteers (total of 24 samples) using flow cytometry and a monoclonal antibody (clone 1E3) to the activation-dependent, surface-expressed protein P-Selectin. RESULTS: Among healthy volunteers, a small percentage of platelets were activated (2.0 +/- 2.5%). In contrast, 15 out of 19 (79%) patients with suspected myocardial ischemia at rest had 10.4 +/- 11.1% activated platelets (95% confidence interval 4.9-15.9%; P < 0.01). Seven patients (37%) were found to have more than 10% activated platelets. The degree of platelet activation did not differ significantly between patients with unstable angina and those with elevated creatine kinase levels (non-Q-wave myocardial infarction). CONCLUSION: Whole-blood flow cytometric techniques can minimize the artifactual platelet activation often seen with other techniques and therefore offer distinct advantages in the investigation of patients with thrombotic coronary syndromes. In TIMI-III, patients with unstable angina and non-Q-wave myocardial infarction had clear evidence of platelet activation. An ongoing study will examine the correlation between platelet activation assessed using flow cytometry and clinical events in a larger patient cohort.

Acute Disease↗
PubMed1993

Role of the membrane in the inactivation of factor Va by activated protein C.

Bovine factor Va inactivation by activated protein C (APC) was evaluated in the presence and absence of phospholipid vesicles and protein S. Following a 30-min incubation with APC (10 nM), membrane-bound factor Va (200 nM) is completely inactivated, whereas in the absence of phospholipid vesicles, after a 2-h incubation, the cofactor retains 60% of its initial cofactor activity. The complete loss of activity of membrane-bound factor Va is associated with the appearance of M(r) 40,000, 28,000, and 20,000 fragments derived from the heavy chain of the cofactor which correspond to cleavage at Arg306, Arg505, and Arg662. In the absence of a lipid bilayer, cleavage at Arg505 and Arg662 results in a cofactor with reduced activity. No difference is observed in the cleavage of the light chain of the cofactor by APC in the presence or absence of phospholipid vesicles. The rate of the cleavage of factor Va heavy chain at Arg306, Arg505, and Arg662 as well as the rate of the membrane-bound cofactor inactivation by APC were enhanced in the presence of protein S. Our data demonstrate that the anionic lipid-dependent cleavage of factor Va by APC at Arg306 is required for the complete inactivation of the cofactor.

Amino Acid Sequence↗
PubMed1993

Synthetic substrates for human factor VIIa and factor VIIa-tissue factor.

A series of 100 tripeptide fluorogenic substrates has been synthesized. These substrates contain Arg in the P1 position, various amino acids in the P2 and P3 positions, and different 6-amino-1-naphthalenesulfonamides (ANSN) as the detecting group (P'). The 38 compounds possessing the highest initial rates of factor VIIa hydrolysis were evaluated for substrate kinetic parameters in the presence and absence of tissue factor (TF) and by factor Xa. Most of these substrates had a higher kcat/KM (keff) value for the factor VIIa-TF complex than for factor Xa. Substitution of different amino acids in the P2 position showed that substrates with bulkier amino acids such as Leu, Pro, and Val have higher values for KM and kcat than those with smaller amino acids (Gly or Ser). The highest second-order rate constants were found for substrates with Val or Pro in the P2 position. A decrease or increase in volume of the P2 substituent (Gly, Ser, or Leu) resulted in a decrease in this constant. Substrates with the highest keff values have Phe in the P3 position. As the hydrophobicity and volume of the amino acid in the P3 position decreased, the keff was reduced. The efficiency of substrates for hydrolysis by factor VIIa was enhanced by an increase of hydrophobicity in the P' structure. TF enhanced the amidolytic activity of the "family" of 38 substrates with ANSN in the P' position on an average of 58-fold.

Amino Acid Sequence↗
PubMed1993

Determination of the disulfide bridges in factor Va light chain.

The 74-kDa light chain of bovine factor Va is composed of three domains: the NH2-terminal A3 domain and the COOH-terminal C1 and C2 domains. In total, the light chain has eight cysteines: two in the A3 domain and three in each C domain. To determine the locations of the disulfide bridges, peptides were obtained from factor Va and iodo[1-14C]acetamide-labeled factor Va light chains by digestion with trypsin, activated protein C, lysylendopeptidase, and V8 protease. After HPLC purification, amino acid sequence and composition analyses showed that each domain of bovine Va light chain possesses a disulfide bond. The sites are Cys1684-Cys1710 (A3), Cys1866-Cys2020 (C1), and Cys2025-Cys2180 (C2). One free cysteine is located in each C domain, i.e., Cys1953 and Cys2100. The locations of the disulfide bonds in human Va and VIIIa light chains are anticipated to be similar to those of bovine Va light chain, because the cysteines involved are conserved.

Amino Acid Sequence↗
PubMed1993

Phosphorylation of factor Va and factor VIIIa by activated platelets.

Platelet activation leads to the incorporation of 32[PO4(2-)] into bovine coagulation factor Va and recombinant human factor VIII. In the presence of the soluble fraction from thrombin-activated platelets and (gamma-32P) adenosine triphosphate, radioactivity is incorporated exclusively into the M(r) = 94,000 heavy chain (H94) of factor Va and into the M(r) = 210,000 to 90,000 heavy chains as well into the M(r) = 80,000 light chain of factor VIII. Proteolysis of the purified phosphorylated M(r) = 94,000 factor Va heavy chain by activated protein C (APC) gave products of M(r) = 70,000, 24,000, and 20,000. Only the intermediate M(r) = 24,000 fragment contained radioactivity. Because the difference between the M(r) = 24,000 and M(r) = 20,000 fragments is located on the COOH-terminal end of the bovine heavy chain, phosphorylation of H94 must occur within the M(r) = 4,000 peptide derived from the carboxyl-terminal end of H94 (residues 663 through 713). Exposure of the radioactive factor VIII molecule to thrombin ultimately resulted in a nonradioactive light chain and an M(r) = 24,000 radioactive fragment that corresponds to the carboxyl-terminal segment of the A1 domain of factor VIII. Based on the known sequence of human factor VIII, phosphorylation of factor VIII by the platelet kinase probably occurs within the acidic regions 337 through 372 and 1649 through 1689 of the procofactor. These acidic regions are highly homologous to sequences known to be phosphorylated by casein kinase II. Results obtained using purified casein kinase II gave a maximum observed stoichiometry of 0.6 mol of 32[PO4(2-)]/mol of factor Va heavy chain and 0.35 mol of 32[PO4(2-)]/mol of factor VIII. Phosphoamino acid analysis of phosphorylated factor Va by casein kinase II or by the platelet kinase showed only the presence of phosphoserine while phosphoamino acid analysis of phosphorylated factor VIII by casein kinase II showed the presence of phosphothreonine as well as small amounts of phosphoserine. The platelet kinase responsible for the phosphorylation of the two cofactors was found to be inhibited by several synthetic protein kinase inhibitors. Finally, partially phosphorylated factor Va was found to be more sensitive to APC inactivation than its native counterpart. Our findings suggest that phosphorylation of factors Va and VIIIa by a platelet casein kinase II-like kinase may downregulate the activity of the two cofactors.

Amino Acid Sequence↗
PubMed1993

Complex-dependent inhibition of factor VIIa by antithrombin III and heparin.

The regulation of the factor VIIa-tissue factor complex is essential for control of the hemostatic response. However, the role of the inhibitor antithrombin III in the regulation of factor VIIa has remained in question. The inhibition of factor VIIa activity by antithrombin III and heparin in the presence and absence of tissue factor was evaluated using the fluorescent substrate m-LGR-nds. Our data show that the activity of recombinant human factor VIIa is inhibited by antithrombin III in the presence of heparin at a rate of 1.7 x 10(2) M-1 s-1. In the presence of tissue factor, the rate constant for this reaction increases to 5.6 x 10(3) M-1 s-1. A 1:1 stoichiometric complex between factor VIIa and antithrombin III, with an apparent molecular weight of 110,000, was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A heterogeneous mixture of factor VIIa products with molecular weights between 50,000 and 80,000, most likely representing proteolytically degraded factor VIIa-antithrombin III complexes, was also observed.

Antithrombin III↗
PubMed1993

Immunolocalization of noncollagenous bone matrix proteins in lumbar vertebrae from intact and surgically menopausal cynomolgus monkeys.

The noncollagenous matrix proteins, composing about 10% of the organic matrix of bone, are considered important for cell matrix organization and regulation of mineralization in bone. In the present study, seven of the major noncollagenous bone matrix proteins were localized immunohistochemically in serial sections of lumbar vertebrae from 24 (12 intact and 12 ovariectomized) adult female cynomolgus monkeys (Macaca fascicularis). Osteocalcin was the only protein restricted to bone cells and mineralized bone matrix. Bone sialoprotein was present in both bone and calcified cartilage, and all the other proteins were distributed in soft tissues as well as bone. Staining for both osteocalcin and bone sialoprotein was present diffusely throughout the bone matrix, but osteonectin, osteopontin, matrix gla protein, decorin, and biglycan staining was concentrated along bone surfaces. Osteoid was negative for osteocalcin and bone sialoprotein, but all other proteins had areas of positive immunostaining within osteoid. All proteins except biglycan exhibited strong immunostaining of a subset of active osteoblasts, suggesting that they may be markers of osteoblast maturity or state of activation. The pattern of immunostaining in intact and surgically menopausal monkeys was similar, except that staining for matrix proteins concentrated along bone surfaces appeared to be more widely distributed in the surgically menopausal monkeys, probably due to the higher rate of bone formation in these animals.

Animals↗